2-Phenylaminonaphthoquinones and related compounds: Synthesis, trypanocidal and cytotoxic activities

Article abstract of DOI:10.1016/j.bmc.2014.07.030

A series of new 2-aminonaphthoquinones and related compounds were synthesized and evaluated in vitro as trypanocidal and cytotoxic agents. Some tested compounds inhibited epimastigote growth and trypomastigote viability. Several compounds showed similar or higher activity and selectivity as compared with current trypanocidal drug, nifurtimox. Compound 4l exhibit higher selectivity than nifurtimox against Trypanosoma cruzi in comparison with Vero cells. Some of the synthesized quinones were tested against cancer cells and normal fibroblasts, showing that certain chemical modifications on the naphthoquinone moiety induce and excellent increase the selectivity index of the cytotoxicity (4g and 10). The results presented here show that the anti-T. cruzi activity of 2-aminonaphthoquinones derivatives can be improved by the replacement of the benzene ring by a pyridine moiety. Interestingly, the presence of a chlorine atom at C-3 and a highly lipophilic alkyl group or aromatic ring are newly observed elements that should lead to the discovery of more selective cytotoxic and trypanocidal compounds.

Full text of DOI:10.1016/j.bmc.2014.07.030

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
2-Phenylaminonaphthoquinones and related compounds: Synthesis,
trypanocidal and cytotoxic activities
Ivan Sieveking a, Pablo Thomas a, Juan C. Estévez b, Natalia Quiñones c, Mauricio A. Cuéllar c, Juan Villena d,
Christian Espinosa-Bustos a, Angélica Fierro a, Ricardo A. Tapia a, Juan D. Maya e, Rodrigo López-Muñoz e,
a,
Bruce K. Cassels f, Ramon J. Estévez b, , Cristian O. Salas
⇑
⇑
a Departamento de Química Orgánica, Facultad de Química, Pontificia Universidad Católica de Chile, 702843 Santiago de Chile, Chile
b Centro Singular de Investigación en Química Biológica y Materiales Moleculares and Departamento de Química Orgánica (Facultad de Química), Universidad de Santiago de
Compostela, 15782 Santiago de Compostela, Spain
c Facultad de Farmacia, Universidad de Valparaíso, Av. Gran Bretaña N 1093, Valparaíso, Chile
d Centro de Investigaciones Biomédicas, Escuela de Medicina, Universidad de Valparaíso, Av. Hontaneda N° 2664, Valparaíso, Chile
e Instituto de Ciencias Biomédicas, Programa de Farmacología Molecular y Clínica, Universidad de Chile, PO Box 70000, Santiago, Chile
f Departamento de Química, Facultad de Ciencias, Universidad de Chile, PO Box 653, Santiago, Chile
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new 2-aminonaphthoquinones and related compounds were synthesized and evaluated
in vitro as trypanocidal and cytotoxic agents. Some tested compounds inhibited epimastigote growth
and trypomastigote viability. Several compounds showed similar or higher activity and selectivity as
compared with current trypanocidal drug, nifurtimox. Compound 4l exhibit higher selectivity than nifur-
timox against Trypanosoma cruzi in comparison with Vero cells. Some of the synthesized quinones were
tested against cancer cells and normal fibroblasts, showing that certain chemical modifications on the
naphthoquinone moiety induce and excellent increase the selectivity index of the cytotoxicity (4g and
10). The results presented here show that the anti-T. cruzi activity of 2-aminonaphthoquinones deriva-
tives can be improved by the replacement of the benzene ring by a pyridine moiety. Interestingly, the
presence of a chlorine atom at C-3 and a highly lipophilic alkyl group or aromatic ring are newly observed
elements that should lead to the discovery of more selective cytotoxic and trypanocidal compounds.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 6 May 2014
Revised 8 July 2014
Accepted 17 July 2014
Available online xxxx
Keywords:
2-Phenylaminonaphthoquinones
T. cruzi
Cytotoxicity
Benzocarbazolequinones
Electronic properties
of productivity loss in seven of the countries where it is endemic.4
Although both cancer and Chagas disease have different etiologies,
1. Introduction
Infectious diseases and cancer are responsible for a large num-
ber of worldwide mortality. Cancer is one of the top ten leading
causes of death. It is estimated that 7.6 million people died of can-
cer in 20081,2 and, if current trends continue, this number will rise
to 9 million people by 2015.3 On the other hand, infectious diseases
still constitute a global health problem, causing the death of 8.8
million people in 2008.1 Several neglected diseases are encoun-
tered among those with the highest economic burden. A specific
parasitosis, Chagas disease (American trypanosomiasis, caused by
Trypanosoma cruzi), affects more than 8 million people in Latin
America, and causes approximately 10,000 deaths annually, which
in the Americas is a higher figure than for malaria, and it is respon-
sible for 89% of deaths from tropical-clustered diseases in the
region.1 In addition, Chagas disease causes over US$1.2 billion/year
they share metabolic and pathophysiological features such as glu-
tathione metabolism, some signal transduction pathways, tissue
invasion mechanisms and immune evasion strategies. Thus, the
information obtained from a therapeutic target in any one of these
cell types can be a useful tool for drug research in the other. As a
result, several reports describe the effect of various compounds
from natural extracts or new synthetic molecules against both
T. cruzi and cancer cells.5–12 On the order hand, a considerable num-
ber of natural and synthetic quinones (Fig. 1), have shown interest-
ing biological properties, such as such as antimalarial (calothrixin
A and B),13–16 antibacterial (cribostatin I and streptonigrin),17,18
antitumor (streptonigrin, calothrixin A and B and griffithazanone
A),19–22 and fungicidal activities.23 These compounds have some
important structural features, such as the 2-aminonaphthoquinone
moiety (1), or 2-aminoquinoline- and isoquinoline-5,8-diones, or a
substituted phenylamino group at C-2 (Fig. 1). It is known that the
presence of the nitrogen atom, allows modulation of the
⇑
Corresponding authors.
E-mail addresses: ramon.estevez@usc.es (R.J. Estévez), cosalas@uc.cl (C.O. Salas).
http://dx.doi.org/10.1016/j.bmc.2014.07.030
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Sieveking, I.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.07.030

2
I. Sieveking et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
O
OCH3
O
O
O
O
O
HO2C
OH
O
R1
CH3
NH2
N
NH2
O
R2
N
H3C
HO
NH2
N
H
N
H
O
H3C
1
cribostatin I
streptonigrin
griffithazanone A
O
O
O
O
N
O
R
X
N
N
H
N
N
H
O
H
O
2-aminonaphthoquinones
calothrixin A
calothrixin B
Figure 1. Structures of representative 2-amino-1,4-napthoquinone-type compounds with biological activities.
substituent’s effects on the electronic properties of the quinone
system, as well as modification of the geometry of the quinone
molecules and their reduction intermediates.
was the substitution of the acceptor quinone nucleus (2a–b) by
several aniline derivatives (3a–l) and 1- or 2-naphthylamine,
which were achieved using two different methodologies (i or ii)
reported in the literature.29–31 Using methodology ii, and
CeCl3ꢀ7H2O as Lewis acid catalyst, increased yields of the entire
series were obtained (Fig. 1 and Table 1). In parallel, to study the
importance of the nitrogen atom in the tricyclic system, we
decided to obtain several benzocarbazolequinones, because some
In recent years, we have synthesized several series of 2-amino-
naphthoquinone-type compounds such as benzocarbazolequinon-
es, indazolylnaphthoquinones, benzoquinolinequinones, and
related heterocyclic compounds.24–28 Most of these compounds
showed micromolar IC50 values against several series of tumor cell
lines and trypanosome cultures.25,28 One of the aims of this work
was to analyze the influence of the enlargement of the heteropoly-
cyclic system on their trypanocidal and cytotoxic effects. Since no
data have been reported regarding the influence of the donor-
acceptor and lipophilic properties of 2-phenylaminonaphthoqui-
nones on T. cruzi cultures and their cytotoxic activity on normal
and cancer cells, we synthesized a variety of these compounds to
evaluate their trypanocidal activity and cytotoxic properties
against different morphological stages of T. cruzi and a panel of four
cell lines, including non-tumor dermal human fibroblast (DHF),
and three human-derived tumor cell lines, namely PC-3 (prostate),
MDA-MB231 and MCF-7 (breast).
Table 1
2-Phenylaminonaphthoquinones prepared by amination of quinones 2a–b
Compound
X
R1
R2
R3
R4
Yield (%)
method method
i
ii
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
H
H
H
H
H
H
H
H
H
H
H
OCH3
H
H
H
OCH3
OCH3
H
H
CH3
H
H
H
H
H
OCH3
H
H
H
H
H
H
—
89
67
100
78
—
62
57
49
29
65
OCH3
H
OCH3 OCH3
H
H
H
H
H
H
H
—
—
OCH3
H
O(CH2)5CH3
OH
CH3
H
O(CH2)5CH3
—
—
—
OCH3 50
H
H
H
OCH3
H
—
81
74
86
99
83
92
74
55
2. Results and discussion
2.1. Chemistry
74
—
—
—
—
—
—
Cl OCH3
Cl
H
H
H
—
—
The synthesis of 2-phenylaminonaphthoquinones (4a–n) and
related 5H-benzo[b]carbazole-6,11-dione derivatives (5a–d) fol-
lowed the general pathway outlined in Scheme 1. The first step
—
O
NH2
R3
R4
R4
O
O
O
O
O
O
X
X
R1
R2
X
R3
R2
i or ii
iii or iv
R2
+
R4
N
H
R1
N
H
O
R3
R1
2a
2b
3a-l
X= H
X= Cl
4a-l
5 a-g
v
O
O
R2
ii
O
or
R1
N
H
N
H
N
CH3
4m
4n
O
6a
6b
R1= H; R2 = OCH3
R1= R2 = OCH3
Scheme 1. Reagents and conditions: (i) ethanol, rt, 24–72 h; (ii) aniline 3a–l or
benzoquinone, AcOH, reflux, 12–24 h; (iv) 2–10 mol % Pd(OAc)2, pivalic acid, 140 °C, 12–24 h ; (v) (1) KOH, ethanol, rt, (2) CH3I, acetone, rt, 6 h (85% of yield for 6a and 91% for
6b).
a- or b-naphthylamine, 0.22 equiv CeCl3ꢀ7H2O, ethanol, rt, 12–24 h; (iii) 0.9 equiv Pd(OAc)2,
Please cite this article in press as: Sieveking, I.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.07.030

I. Sieveking et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
of these and other compounds related to 4a–n exhibit antineoplas-
unsuccessful. Other compounds related to 10 have been synthe-
sized by our group (11 and 12)35 and their trypanocidal effect
has not been studied previously. Benzofuranoquinone 15, was pre-
pared by brominating phenol 13 and subsequent oxidation of
dibromophenol 14.36 The reaction of bromobenzoquinone 15 with
2,5-dimethoxyaniline, or with an excess of diazomethane afforded
arylaminobenzofuranquinone 16 or furoindazolequinone 17,
respectively (Scheme 3).
Considering that the synthesis of carbazolediones have some
limitations, our group has reported a new strategy to obtain these
compounds through conversion of 2-hydroxy-3-phenyl-1,4-naph-
thoquinones into the tetracyclic system (Scheme 4).37–39,24 This
methodology started from the coupling of isochroman-1,4-dione
18 with nitrobenzaldehydes 19a,b to give 3-benzylideneisochro-
man-1,4-diones 20a,b. The rearrangement of 20a,b under basic
conditions yielded 3-aryl-2-hydroxy-1,4-naphthoquinones 21a,b,
which were converted into 5H-benzo[b]carbazolequinones 5a
and 5h by reduction with NaBH4 in isopropanol at room
temperature.
tic and antiparasitic activity.25,28 For the biaryl cyclodehydrogena-
tion of
4 leading to benzocarbazolequinones we used the
procedure developed by Luo et al.,31 which is a process catalyzed
by palladium(II) in acetic acid (methodology iii). The use of pivalic
acid as solvent, at a higher temperature, increased the yield in this
step (iv).32 To study the effect of hydrogen bonding involving the
indole nucleus, we alkylated the indole nitrogen position using
classical conditions.33 The structures of the new members of the
series were established on the basis of their spectral properties
(IR, 1H NMR, 13C NMR and HRMS) (Table 2).
To obtain analogs of phenylaminonaphthoquinones and analyze
the consequences of the isosteric replacement of a benzene ring by
pyridine, we synthesized compound 10, using conditions reported
by Yoshida et al.34 Thus, reaction of quinolequinone (8) with ani-
line 9g was regioselective, using nickel(II) (Scheme 2). Attempts
to cyclize 10 under similar conditions to those used for 5a–d were
Table 2
2.2. Bioactivity: trypanocidal effect
Benzocarbazoles
phenylaminonaphthoquinones
prepared
by
cyclodehydrogenation
of
2-
Trypanocidal activity of all the new compounds was tested
against T. cruzi epimastigotes, and those that exhibited higher
activity than nifurtimox, used here as the reference trypanocidal
drug, were also assayed against trypomastigotes. The quinones
were incorporated into the culture medium at different concentra-
tions. Growth inhibition until day 10 was evaluated in comparison
to controls at day 5. To quantify the relative efficacy of the qui-
nones in vitro compared with the standard drug, the IC50 values
were determined. Table 3 shows the effect of the quinones tested
Compound R1
R2
R3
R4
Yield (%)
method iii method iv
5a
5b
5c
5d
5e
5f
H
OCH3
H
H
OCH3
H
H
H
OCH3
H
H
H
H
H
OCH3
H
H
H
H
H
43
67
21
40
50
100
40
80
75
50
—
OCH3 40
H
H
H
O(CH2)5CH3
67
42
5g
H
OCH3 OCH3
O
NH2
O
O
OCH3
OCH3
N
N
OCH3
i
ii
N
N
H
H3CO
OH
O
9
7
8
10
iii
O
O
O
Cl
H3CO
N
N
N
H
NH
O
O
O
N
12
11
OCH3
N
H
Scheme 2. Reagents and conditions: (i) IBDA, CH3CN–H2O, 15 min, rt; (ii) NiCl2, ethanol, rt, 12 h; (iii) Pd(OAc)2, benzoquinone, AcOH, reflux, 12–24 h.
OCH3
OCH3
O
O
O
O
Br
iii
i
ii
O
O
N
H
O
Br
O
Br
OH
OH
13
14
15
16
iv
O
N
O
N
CH3
O
17
Scheme 3. Reagents and conditions: (i) Br2, CHCl3, 0 °C, 1.5 h (83%);36 (ii) CrO3, AcOH–H2O (1:1), rt, 1 h (51%);36 (iii) 2,5-dimethoxyaniline, ethanol, 0.5 equiv CeCl3ꢀ7H2O, rt,
80%; (iv) CH2N2, Et2O, 15 min, 0 °C, 67%.
Please cite this article in press as: Sieveking, I.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.07.030

4
I. Sieveking et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
R2
O
O
R2
O
R2
O
O
O
R1
R1
R1
R1
R2
i
ii
R2
R2
O
R1
R1
O
+
H
NO2
O2N
OH
NO2
O
21a R1= R2= H
20a
R1= R2= H
18a R1= H
19a R2= H
b
R1= R2= OCH3
b R1= R2= OCH3
b
b
R2= OCH3
R1= OCH3
iii
R2
O
O
R2
R1
R1
N
H
5a
R1= R2= H
h R1= R2= OCH3
i
Scheme 4. Reagents and conditions: (i) NaOAc, AcOH, 60 °C, 6 h; (ii) NaOMe, MeOH, , rt, 12 h; (iii) NaBH4, PrOH, rt, 4 h.37,38,40
Table 3
Effect of 2-aminophenylnaphthoquinones and related compounds upon culture growth of T. cruzi and selectivity index versus Vero cells
b
Compound
Epimastigote IC50
(
l
M)a
% trypomastigote mortality
Vero IC50
(l
M)a
Selectivity indexc
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
>25*
—
>10*
>25*
>25*
>25*
>25*
>25*
>50*
—
—
—
—
—
—
—
16
—
—
—
32
0.77
—
3.49 0.3
18.78 1.8
—
—
—
—
—
69.63 4.2
—
—
—
46.30 4.1
86.02 2.6
59.67 7.7
—
>25*
>25*
ꢁ25
>25*
23.30 4.7
1.72 0.3
>25*
27.04 6.1
>25*
>25*
>25*
>100*
>100*
6.04 0.1
9.02 0.2
15.90 1.3
>10*
195.85 26.9
6.97 0.3
>25*
4m
4n
5a
>10*
—
5b
5c
5d
5e
20.86 2.0
12.21 3.0
—
—
—
21.73 5.8
0.95
—
—
—
—
> 25*
>25*
>50*
>50*
>100*
>100*
5f
>100*
—
>100*
5g
5h
6a
6b
10
11
n.d.
—
—
—
—
n.d.
—
—
—
—
6.3
—
>100*
> 100*
>25*
>25*
>100*
>100*
2.45 0.1
32.62 4.6
—
15.43 4.5
>100*
>100*
12
16
17
5.79 0.4
12.35 0.5
2.53 0.6
>100*
58.20 8.4
7.50 2.4
38.57 9.1
—
16.16 1.3
15.54 2.2
13.40 2.1
>100*
2.7
1.3
5.3
—
—
20
21a
21b
Nifurtimox
>100*
—
>100*
21.05 0.1
42.62 3.7
411.13 7.6
n.d.: not determined.
*
Values of IC50 higher than the solubility in the medium.
The results are means of three independent experiments.
Drug concentrations used were the IC50 values on epimastigotes.
a
b
c
Selectivity index: expressed as the ratio of IC50 in Vero cells to IC50 in epimastigotes.
on the growth of Tulahuen strain T. cruzi epimastigotes, at their
respective IC50 concentrations. Some of these compounds showed
remarkable trypanocidal effects. Among them, compound 4h dis-
To validate our hypothesis regarding the 2-phenylaminonaph-
thoquinone structural pattern, several chemical modifications
were carried out on this moiety, as shown in Figure 2. In order to
find out a possible structure–activity relationship, a set of stereo-
electronic properties were calculated (Table 4), and used in an
attempt to understand this biological activity and to contribute
to the design of more effective trypanocidal drugs.
played the most potent inhibitory activity (IC50 = 1.72 0.3
for epimastigotes). However, those compounds with IC50 values
higher than 20 M are equipotent with nifurtimox, and thus rela-
tively devoid of pharmacological interest.
lM
l
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I. Sieveking et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
chlorine on C-3 or
bond C-C (ciclyzation)
substituents on
o, m, p
III
nitrogen or oxygen
heterocycles
O
O
-OCH3
-O(CH2)5CH3
-OH
H
H
-CH3
3
II
I
O
N
Different size of the rings
N
H
N
Figure 2. Main chemical modifications of the 2-aminonaphthoquinone framework for trypanocidal and cytotoxicity studies.
2.2.1. Trypanocidal effect on the epimastigote form
Replacement of the benzene by a pyridine ring (modification I,
Fig. 2), increases the effects on the epimastigote form. For example,
compound 10 is almost ten times more potent than its carbocyclic
analog 4g, with IC50 values of 2.45 and 23.3 lM, respectively. Fur-
thermore, compound 12 is at least four times more active than 4a.
These results are in agreement with previous results from our
group that indicate the importance of nitrogen substitution in
the aromatic ring for the activity of related compounds.25,28 To
understand this behavior we examined the HOMO–LUMO energy
gaps in these compounds (Table 4). These gaps are quite small
for 4g and 10 on one hand, and 4a and 12 on the other, but a dif-
ferent distribution of the HOMO and LUMO can be seen in Figure 3,
where nitrogen substitution in the aromatic ring (4g–10) leads to
more extensive electron flow in the HOMO. This change is associ-
ated with a more planar conformation for 10, which might also
be related to the molecule’s access to a specific biological target.
Although the dihydrofuro derivative 16 was less active than 10,
16 is still more potent than 4g (12.36 vs 23.30 lM) suggesting that
Figure 3. Electrostatic potential and frontier orbitals for 4g and 10.
an extended aromatic system is not a necessary feature. Compound
17, an indazole-furan derivative is equipotent with 10, highlighting
the apparent role of a nitrogen heterocyclic system fused to the
quinone ring. An important result of chemical modification I, is
that these compounds are more active than nifurtimox.
Chemical modification II represents the broadest range of sub-
stitution patterns in all the compounds tested. From Table 3 it is
possible to infer that either increasing volume or lipophilicity of
the arylamine moiety (4h, 4m, 4n) favors the trypanocidal effect,
particularly on the epimastigote form of T. cruzi. Surprisingly, while
methoxy (4c–g), hydroxy (4i) and methyl (4j) substitution at the
benzene ring lead to inactive derivatives, the 20-methoxy
compound (4b) is a remarkable exception (IC50 = 3.49 lM) and is
seven times more potent than nifurtimox. The 20-methoxylated
derivative is probably more easily reduced because its
HOMO–LUMO gap (17.26 eV) is lower than that 4a (17.61 eV).
Compounds 4h, 4m and 4n (IC50 = 1.72, 9.02 and 15.90
lM, respec-
tively) are more potent than nifurtimox (21.05 M). These three
l
very lipophilic compounds are presumably penetrating better
parasite’s plasma membrane by virtue of their high calculated logP
values (5.55 for 4h, 4.38 for 4m and 4.25 for 4n, Table 4).
Table 4
Calculated properties for the studied compounds
Compound
Polar surface area (Å2)
LogP
Dipole (D)
HOMO (eV)
LUMO (eV)
HOMO–LUMO Gap
4a
4b
4g
4h
4k
4l
4m
4n
5b
5e
10
11
12
16
17
36.64
44.08
52.47
44.90
52.51
44.94
36.96
36.31
54.99
38.90
62.16
46.20
46.43
60.39
49.07
84.64
3.06
2.88
2.99
5.55
3.90
6.46
4.38
4.25
1.90
2.06
2.34
2.69
2.91
2.12
0.48
0.89
1.93
1.45
2.11
3.25
2.60
4.58
1.86
1.99
1.30
1.44
2.20
4.78
0.26
1.14
2.00
9.57
ꢂ8.32
ꢂ8.15
ꢂ8.09
ꢂ8.34
ꢂ7.84
ꢂ8.35
ꢂ7.92
ꢂ7.91
ꢂ8.13
ꢂ8.24
ꢂ8.01
ꢂ8.74
ꢂ8.39
ꢂ8.06
ꢂ9.44
ꢂ9.75
9.29
9.11
9.03
9.36
8.57
9.07
8.88
8.91
8.96
9.88
8.95
9.30
9.35
8.97
9.90
10.47
17.61
17.26
17.12
17.70
16.41
17.42
16.80
16.82
17.09
18.82
16.96
18.04
17.74
17.03
19.34
20.22
Nifurtimox
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I. Sieveking et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Figure 4. Geometry analysis of 4g and 4k.
Regarding modification III, replacement of the hydrogen atom
2.2.2. Trypanocidal effect on the trypomastigote form
at C-3 by chlorine afforded 4k and 4l. Both compounds showed
decreased trypanocidal effect compared with their unhalogenated
analogs 4g and 4h. Nevertheless, in 4l an almost four-fold loss of
activity against epimastigotes is compensated by a very significant
decrease in its toxicity toward Vero cells and therefore greater
selectivity than nifurtimox (see an analysis of this aspect in
Section 2.2.3). Fig. 4 shows that substitution of H by Cl at C-3
(4g vs 4k) modifies the overall geometry of the molecule and the
electron distribution around the quinone system. In both cases in
which comparison is possible, chlorination at C-3 is detrimental
for the trypanocidal activity in spite of the concomitant increase
in lipophilicity. The very low activity of compound 11 suggests
that—in contrast to the introduction of nitrogen atom in the
quinone moiety—when the arylamine moiety is heterocyclic, there
is no favorable effect on trypanocidal potency, possibly due to the
sharply reduced lipophilicity.
Cyclization of the 2-phenylaminonaphthoquinone system to
yield the planar, more conjugated benzocarbazole derivatives does
not increase and may actually be detrimental to the trypanocidal
effect (5a–5f). Nevertheless, 5b is still at least as active as nifurti-
mox, corroborating the finding that an o-methoxy group on the
phenylamine moiety is a favorable feature. N-methylation of 5e
and 5f to yield 6a and 6b gave no useful results. As shown in
Figure 5, for 4g and 5e the electronic potential becomes quite
uniform in the benzocarbazole compound although the HOMO
orbital arrangement is similar in both. The MO energies diminish
only slightly on cyclization, but logP and polar surface area
decrease quite considerably from 2.99 to 2.06 and from 52.47 to
38.90 Å2 (Table 4), for 4g and 5e, respectively.
Due to the relevance of the trypomastigote form in the infective
cycle of T. cruzi, the next step in the search for new candidate drugs
against Chagas disease is to evaluate the more active compounds in
the epimastigote assays vs trypomastigotes. With this aim, the per-
centage mortality of trypomastigotes was determined at the IC50
values for the compounds that proved to be more active than nif-
urtimox against epimastigotes (Table 3). The results shown in
Table 3 indicate that compounds 4h, 4l, 4m, 4n and 12 are simi-
larly toxic to both parasite forms (46–87% mortality of trypom-
astigotes at epimastigote IC50). These are interesting results
because they show that these compounds retain the trypanocidal
effect at different stages of the parasite’s life cycle and sometimes
with higher activity against the infective form (4h and 4m). It
seems likely that these results are related to the high lipophilicity
of 4h and 4l–n. However, some compounds with calculated
logP 6 2.9 (4b, 5b, 10, 12, 16 and 17, Table 4), while being at least
as active as nifurtimox against epimastigotes, and with the excep-
tion of 12, are less so against trypomastigotes and no relationship
to their lipophilicity is apparent. This reduced potency might be
related to a lesser ability of these structures to cross the trypomas-
tigote membrane. This membrane is rich in trans-sialidase, and has
a high concentration of sialic acid and an increased negative
charge.41 Therefore, uncharged molecules with substituents that
present more non-bonding electrons or more than two heteroat-
oms might experience electronic repulsion and difficulty in enter-
ing the membrane.
2.2.3. Selectivity of trypanocidal compounds (Selectivity index,
SI)
Finally, the 2-hydroxynaphthoquinones 21a–21b (with a simi-
lar structural pattern to lapachol), starting materials for the syn-
thesis of benzocarbazoles 5e–f, were also evaluated for their
activity versus T. cruzi. Compounds 21a–21b showed very poor
effect, suggesting that the 2-phenylaminonaphthoquinone moiety
is pivotal for the trypanocidal activity.
A potential antichagasic drug must show low toxicity in
mammalian host cells and for this reason, the compounds with
the strongest trypanocidal effects vs epimastigotes, their cyto-
toxic effects in Vero cell cultures were determined. It is well
known that naphthoquinones generate reactive oxygen species
(ROS) and as expected, several of the tested compounds were
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I. Sieveking et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
7
Figure 5. Electronic potential and HOMO of 4g and 5e.
quite cytotoxic. Table 3 shows—for the compounds with higher
2.3. Bioactivity: cytotoxicity in human cell lines
epimastigote toxicity—the IC50 values for Vero cell proliferation
and their selectivity indices as the ratios of the IC50 values in
both assays. These results indicate that most of these com-
pounds are less selective than nifurtimox (SI = 20), although
the potent 4h is almost as selective (SI = 16). Quite unexpectedly
4l, though not the most potent compound of the series, showed
A representative subset of the arylaminonaphthoquinones and
related compounds described here, were tested for their antiprolif-
erative effects against a hormone-independent and a hormone-
responsive breast cancer line (MDA-MB231 and MCF-7), prostate
cancer line (PC-3) and a normal human dermal fibroblast line
(DHF). In the literature quinone/hydroquinone cytotoxicity is usu-
ally attributed to two processes: redox cycling of quinones, result-
ing in the generation of ROS which can damage biomolecules and
inhibition of mitochondrial function and electrophilic arylation of
critical cellular nucleophiles. Both mechanisms can result in oxida-
tive stress and cell death.42,43,29,44
A conventional colorimetric assay was set up to estimate the
IC50 values, which represent the concentration of a drug that is
required for 50% inhibition in vitro after 72 h of continuous expo-
sure to the test compounds. Four serial dilutions (from 12.5 to
100 lM) for each sample were evaluated in triplicate and doxoru-
bicin was used as the reference drug.
rather low toxicity (IC50 = 196 lM and SI = 32), possibly due to
the substitution of hydrogen by halogen at C-2. Interestingly,
4l is as potent as nifurtimox against trypomastigotes at its epi-
mastigote IC50. Consequently, among all the compounds tested
here, 4l is not only three times more potent than nifurtimox
against both the epimastigote and the trypomastigote forms of
T. cruzi, but also exhibits greater selectivity in regard of its tox-
icity toward Vero cells. These two favourable features seem to
stem from the simultaneous presence of the halogen atom on
the quinone ring and the lipophilic hexyloxy group on the aryla-
mino substituent. In contrast, the remaining compounds dis-
played in Table 3 inhibited Vero cell proliferation within the
expected range, and were therefore practically unselective and
therefore lacking of clinical interest.
Table 5 shows the IC50 values for 4b–g, 5b, 5d, 6a–b and 10. In
general, activity of these quinones was scattered among cell
Table 5
Antiproliferative activity of selected arylaminonaphthoquinone derivatives and related compounds against tumor cell lines and normal fibroblasts
Compounds
IC50 (lM)
PC-3
MCF-7
MDA-MB231
DHF
4b
>100
>100
>100
>100
4c
>100
>100
>100
>100
4d
4e
4f
4g
35.4 2.98
10.4 1.01
60.0 8.29
1.03 1.14 (>97)*
>100
>100
22.2 3.02
>100
0.21 0.31(>480)
>100
>100
10.2 1.51
>100
87.5 9.34
>100
65.4 9.48
>100
>100
>100
>100
4h
4l
5b
27.9 4.5
>100
>100
>100
>100
>100
>100
>100
5d
>100
>100
>100
>100
6a
>100
>100
>100
>100
6b
>100
>100
>100
>100
10
0.53 1.01(42)
0.54 0.36 (ꢁ15)
0.12 1.02(188)
0.32 0.24(25)
8.8 1.23
0.31 0.13(25)
22.5 4.87
7.86 1.34
Doxorubicin
n.d.: not determined.
*
In parentheses, the Selectivity index (SI) expressed as the ratio of IC50 in DHF to IC50 in cancer cells.
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I. Sieveking et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
cultures with variable sensitivity. Quinones 4b–g, 5b, 5d, 6a–b lack
4.1.2. Synthesis
cytotoxic effects on cancer cells and together with those com-
4.1.2.1. General synthetic procedures for 2-arylaminonaphtho-
pounds with IC50 values higher than 10
l
M (4d–f), are relatively
quinones (4a–4n).
Method i: In a reaction flask a suspension
uninteresting. Only compounds 4g and 10 showed remarkable
cytotoxicity, with their most potent inhibitory activity on PC-3
and MCF-7 cells. 4g was more selective (SI >97 and 480 for PC-3
and MCF-7, respectively) than 10 (SI = 42 and 188 for PC-3 and
MCF-7, respectively) and both compounds over more selective that
the reference drug. Considering that 4g and 10 are isosteres, these
results are in agreement with the trypanocidal effects showed by
these compounds, where once again the replacement of the
benzene by a pyridine ring increases the cytotoxic effect but not
the selectivity.
A full cytotoxicity analysis for the other naphthoquinones
assayed in T. cruzi is being carried out to understand the structural
requirements for obtaining compounds with selective activity
toward cancer cells. However, the present results seem to suggest
that coplanarity of the tetracyclic quinone ring (5a–b and 6a–b)
and the substitution pattern on the phenyl amino moiety (4b–f)
are related to reduced cytotoxicity and selectivity.
of naphthoquinone 2a (300 mg, 1.89 mmol) in 10 mL EtOH (abs.)
was placed under magnetic stirring at 10 °C until the solid dis-
solved completely. To this solution was added dropwise a solution
of the corresponding amine (0.95 mmol) in 5 mL of EtOH, also at
10 °C. After the addition, the reaction mixture was allowed to reach
room temperature and was stirred for another 24–48 h, after
which the EtOH was removed in a rotary evaporator. The solid res-
idue was purified by column chromatography on silica gel.
Method ii: In a reaction flask was prepared a 10 mL solution of
naphthoquinone 2a (300 mg, 1.89 mmol) or 2b (300 mg,
1.32 mmol), the corresponding amine (0.95 mmol and 0.66 mmol
respectively) and 0.42 mmol of CeCl3*7H2O in 10 mL of EtOH. The
reaction mixture was stirred at room temperature for 12–24 h
until disappearance of the naphthoquinone, and the EtOH was
evaporated. The residue was redissolved in CH2Cl2, the organic
phase was washed successively with 2 M HCl and saturated NaCl
solution, dried with Na2SO4 and concentrated to dryness. The
crude product was purified by column chromatography on silica
gel.
3. Conclusion
4.1.2.1.1. 2-(Phenylamino)naphthalene-1,4-dione (4a). Red
solid. Yield by method ii (192 mg, 89%), mp: 190–192 °C. (lit
A study of mostly new 2-arylaminonaphthoquinone derivatives
(14), related 5H-benzo[b]carbazole-6,11-diones (8) and a few addi-
tional quinone compounds (9), against T. cruzi epi- and trypom-
astigotes, and of some of them against cancer cells and normal
fibroblasts, showed that certain chemical modifications on the
naphthoquinone moiety increase the trypanocidal and cytotoxic
effects. Several of these compounds were more potent than the ref-
erence drug nifurtimox versus both stages of the parasite life cycle,
and exhibited increased selectivity against T. cruzi in comparison
with Vero cells, most notably 4l. Cytotoxicity against prostate
and mammary cancer cells in vitro was also quite selective (vs nor-
mal fibroblasts) in the case of 4g compared with the reference
drug. A preliminary analysis confirms an earlier conclusion that
the replacement of a benzene ring by nitrogen isosteres condensed
to the naphthoquinone core is an important feature to increase
these activities. However, attempts to reveal general structure-
activity relationships using stereoelectronic and lipophilicity prop-
erties were unsuccessful. Nevertheless, the presence of a chlorine
atom at C-3 and a highly lipophilic alkyl group or aromatic ring
are newly observed elements that should lead to the discovery of
more selective cytotoxic and trypanocidal compounds.
189–190 °C)33 1H NMR (400 MHz, CDCl3) d ppm: 6.42 (s, 1H),
.
7.21 (t, J = 7.4 Hz, 1H), 7.27 (d, J = 7.9 Hz, 2H), 7.42 (t, J = 7.9 Hz,
2H), 7.58 (s, 1H), 7.66 (td, J = 1.2, 7.6 Hz, 1H), 7.75 (td, J = 1.2,
7.6 Hz, 1H), 8.11 (m, 2H). 13C NMR (CDCl3) d ppm: 103.4, 122.6
(2C), 125.6, 126.2, 126.5, 129.7 (2C), 130.4, 132.3, 133.2, 134.9,
137.4, 144.7, 182.1, 183.9. IR (KBr, cmꢂ1): 3317, 1668, 1639,
1596. MS (ESI): 250.0 (C16H11NO2 [M+H]+).
4.1.2.1.2. 2-(2-Methoxyphenylamino)naphthalene-1,4-dione
(4b). Red solid. Yield by method i (165 mg, 62%). Yield by method
ii (180 mg, 67%), mp 145–147 °C. (lit 147–148 °C).33 1H NMR
(400 MHz, CDCl3) d ppm: 3.92 (s, 3H), 6.49 (s, 1H), 7.00 (m, 2H),
7.15 (dt, J = 1.1, 7.9 Hz,1H), 7.42 (d, J = 7.9 Hz, 1H), 7.66 (dt,
J = 1.1, 7.9 Hz, 1H), 7.76 (dt, J = 1.2, 7.6 Hz,1H), 7.99 (s, 1H), 8.12
(m, 2H). 13C NMR (CDCl3) d ppm: 13C NMR (101 MHz, CDCl3) d
ppm: 55.77, 103.61, 111.15, 120.88, 121.07, 125.46, 126.10,
126.53, 126.96, 130.53, 132.28, 133.30, 134.77, 143.97, 151.17,
182.12, 183.96. IR (KBr, cmꢂ1): 3307, 1672, 1631, 1597. MS (ESI):
280.0 (C17H13NO3 [M+H]+).
4.1.2.1.3. 2-(3-Methoxyphenylamino)naphthalene-1,4-dione
(4c). Red solid. Yield by method i (152 mg, 57%). Yield by method
ii (266 mg, 100%), mp 157–159 °C (lit 160–163 °C).33 1H NMR
(400 MHz, CDCl3) d ppm: 3.83 (s, 3H), 6.45 (s, 1H), 6.74 (dd,
J = 8.3, 2.0 Hz, 1H), 6.80 (t, J = 2.2 Hz, 1H), 6.86 (dd, J = 7.9, 1.8 Hz,
1H), 7.31 (t, J = 7.6 Hz, 1H), 7.56 (s, 1H), 7.65 (td, J = 7.6, 1.3 Hz,
1H), 7.75 (td, J = 7.6, 1.3 Hz, 1H), 8.10 (dt, J = 7.6, 1.2 Hz, 2H). 13C
NMR (CDCl3) d ppm: 55.4, 103.8, 108.4, 110.9, 114.8, 126.1,
126.5, 130.3, 130.4, 132.3, 133.2, 134.8, 138.6, 144.5, 160.6,
182.0, 183.9. IR (KBr, cmꢂ1): 3227, 1675, 1605, 1594. MS (ESI):
280.0 (C17H13NO3 [M+H]+).
4.1.2.1.4. 2-(4-Methoxyphenylamino)naphthalene-1,4-dione
(4d). Red solid. Yield by method i (130 mg, 49%). Yield by method
ii (207 mg, 78%), mp 145–147 °C. (lit. 155–157 °C).31 1H NMR
(400 MHz, CDCl3) d ppm: 3.83 (s, 3H), 6.22 (s, 1H), 6.94 (d,
J = 8.8 Hz, 2H), 7.20 (d, J = 8.8 Hz, 2H), 7.43 (s, 1H), 7.65 (td,
J = 1.1, 7.6 Hz, 1H), 7.75 (td, J = 1.1, 7.6 Hz, 1H), 8.07–8.10 (m,
2H). 13C NMR (CDCl3) d ppm: 55.6, 102.5, 114.9 (2C), 124.8 (2C),
126.1, 126.4, 130.0, 130.4, 132.2, 133.4, 134.9, 145.7, 157.7,
182.2, 183.7. IR (KBr, cmꢂ1): 3226, 1679, 1621, 1604. MS (ESI):
280.0 (C17H13NO3 [M+H]+).
4. Experimental
4.1. Chemistry
4.1.1. Materials and measurements
Melting points were determined on a Kofler Thermogerate
apparatus and are uncorrected. Infrared spectra were recorded on
a JASCO FT/IR-400 spectrophotometer. Nuclear magnetic reso-
nance spectra were recorded, unless otherwise specified, on a Bru-
ker AM-400 instrument using deuterochloroform solutions
containing tetramethylsilane as internal standard. Mass spectra
were obtained on a HP 5988A mass spectrometer. HPLC-MS exper-
iments were performed on an Exactive Plus Orbitrap MS Thermo
Scientific. Thin layer chromatography (tlc) was performed using
Merck GF-254 type 60 silica gel. Column chromatography was car-
ried out using Merck type 9385 silica gel. Compounds 4a–d, 4f–g,
4i, 4k, 4m, 5a–b, 5d, 5g and 6a were identified by comparing their
spectral properties (Melting point, IR, 1H NMR and 13C NMR) to
those reported for these compounds in the literature.24,26,29,33,41–43
The purity of the compounds was determined by tlc and high-
resolution mass spectrometry (HRMS).
4.1.2.1.5. 2-(3,4-Dimethoxyphenylamino)naphthalene-1,4-dione
(4e). Red solid. Yield by method i (85 mg, 29%), mp 125–127 °C.
1H NMR (400 MHz, CDCl3) d ppm: 3.90 (d, J = 5.1 Hz, 6H), 6.28 (s,
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I. Sieveking et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
9
1H), 6.78 (d, J = 1.9 Hz, 1H), 6.85 (d, J = 8.8 Hz, 1H), 6.90 (d,
J = 8.5 Hz, 1H), 7.44 (s, 1H), 7.66 (t, J = 7.4 Hz, 1H), 7.76 (t,
J = 7.4 Hz, 1H), 8.11 (m, 2H). 13C NMR (CDCl3) d ppm: 56.1, 56.2,
102.8, 107.4, 111.7, 115.6, 126.1, 126.4, 130.3, 130.4, 132.2,
133.4, 134.9, 145.5, 147.3, 149.7, 182.1, 183.7. IR (KBr, cmꢂ1):
3292, 1675, 1596, 1571. MS (ESI): 310.0 (C18H15NO4 [M+H]+).
HRMS for (C18H15NO4 [M]). Calcd: 309.1001. Found: 309.2015.
4.1.2.1.6. 2-(2,4-Dimethoxyphenylamino)naphthalene-1,4-dione
(4f). Red solid. Yield by method i (190 mg, 65%), mp 149–
151 °C. (lit. 150–153 °C).45 1H NMR (400 MHz, CDCl3) d ppm: 3.82
(s, 3H), 3.86 (s, 3H), 6.28 (s, 1H), 6.43–6.59 (m, 2H), 7.29 (d,
J = 8.6 Hz, 1H), 7.52–7.83 (m, 3H), 8.10 (d, J = 7.6 Hz, 2H). 13C
NMR (CDCl3) d ppm: 55.6, 55.8, 99.5, 102.6, 104.0, 119.9, 123.0,
126.0, 126.4, 130.5, 132.1, 133.5, 134.7, 144.8, 152.9, 158.2,
182.2, 183.7. IR (KBr, cmꢂ1): 3306, 1693, 1681, 1576. MS (ESI):
310.0 (C18H15NO4 [M+H]+).
J = 7.6 Hz, 1H). 13C NMR (CDCl3) d ppm: 55.9, 56.1, 110.9, 111.3,
111.4, 115.1, 126.9, 127.0, 127.1, 130.0, 132.6, 132.9, 134.9,
141.7, 146.8, 152.9, 177.4, 180.4. IR (KBr, cmꢂ1): 3314, 1676,
1655, 1576. MS (ESI): 345.0 (C18H14ClNO4 [M+H]+).
4.1.2.1.12. 2-Chloro-3-(4-(hexyloxy)phenylamino) naphthalene-
1,4-dione (4l). Purple solid. Yield by method ii (233 mg, 92%),
mp 130–133 °C. 1H NMR (400 MHz, CDCl3)
d ppm: 0.91 (t,
J = 6.9 Hz, 3H), 1.28–1.41 (m, 4H), 1.46 (m, 2H), 1.73–1.86 (m,
2H), 3.96 (t, J = 6.6 Hz, 2H), 6.85 (d, J = 8.8 Hz, 2H), 7.03 (d,
J = 8.8 Hz, 2H), 7.60–7.70 (m, 2H), 7.74 (td, J = 1.0, 7.6 Hz, 1H),
8.08 (d, J = 7.5 Hz, 1H), 8.16 (d, J = 7.5 Hz, 1H). 13C NMR (CDCl3) d
ppm: 14.0, 22.6, 25.7, 29.2, 31.6, 68.3, 113.4, 126.3, 126.9, 127.0,
127.8, 129.8, 130.1, 132.7, 132.8, 134.7, 135.0, 141.8, 157.4,
177.3, 180.6. IR (KBr, cmꢂ1): 3224, 1677, 1634, 1606. MS (ESI):
385.0 (C22H22ClNO3 [M+H]+). HRMS for (C22H22ClNO3 [M+H]+).
Calcd: 383.1288. Found: 384.1334.
4.1.2.1.7. 2-(2,5-Dimethoxyphenylamino)naphthalene-1,4-dione
(4g). Red solid. Yield by method i (146 mg, 50%). Yield by method
ii (237 mg, 81%), mp 127–129 °C. (lit 128–129 °C).29 1H NMR
(400 MHz, CDCl3) d ppm: 3.80 (s, 3H), 3.87 (s, 3H), 6.53 (s, 1H),
6.65 (dd, J = 1.9, 8.9 Hz, 1H), 6.87 (d, J = 8.9 Hz, 2H), 7.02 (d,
J = 2.8 Hz, 1H), 7,66 (td, J = 1.1, 7.6 Hz, 1H), 7.75 (td, J = 1.1,
7.5 Hz, 1H), 8.02 (s, 1H) 8.08–8.15 (m, 2H). 13C NMR (CDCl3) d
ppm: 55.9, 56.2, 104.0, 107.9, 109.1, 111.7, 126.1, 126.5, 127.7,
130.5, 132.3, 133.2, 134.8, 143.7, 145.4, 153.8, 182.0, 183.9. IR
(KBr, cmꢂ1): 3349, 1673, 1637, 1602. MS (ESI): 310.0 (C18H15NO4
[M+H]+).
4.1.2.1.8. 2-(4-Hexyloxyphenylamino)naphthalene-1,4-dione
(4h). Red solid. Yield by method i (245 mg, 74%). Yield by method
ii (245 mg, 74%), mp 97–101 °C. 1H NMR (400 MHz, CDCl3) d ppm:
0.83 (t, J = 6.0 Hz, 3H), 1.21–132 (m, 4H), 1.36–1.40 (m, 2H), 1.63–
1.77 (m, 2H), 3.86 (t, J = 6.5 Hz, 2H), 6.13 (s, 1H), 6.83 (d, J = 8.6 Hz,
2H), 7.08 (d, J = 8.6 Hz, 2H), 7.38 (s, 1H), 7.54 (t, J = 7.5 Hz, 1H), 7.64
(t, J = 7.5 Hz, 1H) 7.99 (d, J = 7.5 Hz, 2H). 13C NMR (CDCl3) d ppm:
14.0, 22.6, 25.7, 29.2, 31.6, 68.4, 102.4, 115.4 (2C), 124.8 (2C),
126.1, 126.4, 129.8, 130.4, 132.1, 133.4, 134.8, 145.6, 157.2,
182.1, 183.7. IR (KBr, cmꢂ1): 3215, 1678, 1660, 1615. MS (ESI):
350.0 (C22H23NO3 [M+H]+). HRMS for (C22H23NO3 [M+H]+). Calcd:
349.1678. Found: 350.1726.
4.1.2.1.9. 2-(4-Hydroxyphenylamino)naphthalene-1,4-dione
(4i). Brown solid. Yield by method ii (216 mg, 86%), mp 248–
249 °C. (lit. 250–252 °C).31 1H NMR (400 MHz, DMSO) d ppm:
5.88 (s, 1H), 6.83 (d, J = 8.8 Hz, 2H), 7.16 (d, J = 8.8 Hz, 2H), 7.76
(td, J = 1.4, 7.5 Hz 1H), 7.84 (td, J = 1.3, 7.5 Hz, 1H), 7.94 (dd,
J = 1.0, 7.6 Hz, 1H), 8.04 (dd, J = 1.0, 7.6 Hz, 1H), 9.06 (s, 1H), 9.57
(s, 1H). 13C NMR (DMSO) d ppm: 106.0, 121.0 (2C), 130.5, 131.0
(2C), 131.2, 134.2, 135.7, 137.6, 138.1, 140.1, 152.3, 160.6, 186.9,
187.3. IR (KBr, cmꢂ1): 3303, 1671, 1622, 1597. MS (ESI): 265.0
(C16H11NO3 [M+H]+).
4.1.2.1.10. 2-(2,4-Dimethylphenylamino)naphthalene-1,4-dione
(4j). Orange solid. Yield by method ii (260 mg, 99%), mp 155–
158 °C. 1H NMR (400 MHz, CDCl3) d ppm: 2.23 (s, 3H), 2.34 (s,
3H), 5.90 (s, 1H), 7,06 (d, J = 8.0 Hz, 1H), 7.10 (s, 1H), 7.14 (d,
J = 8.0 Hz, 1H), 7.27 (s, 1H), 7.65 (td, J = 1.3, 7.5 Hz, 1H) 7.74 (td,
J = 1.3, 7.5 Hz, 1H) 8.06–8.14 (m, 2H). 13C NMR (CDCl3) d ppm:
12.9, 16.2, 98.2, 120.2, 121.4, 121.6, 122.9, 125.8, 127.3, 127.4,
4.1.2.1.13. 2-(Naphthalen-2-ylamino)naphthalene-1,4-dione (4m).
Orange solid. Yield by method ii (210 mg, 74%), mp 189–191 °C. (lit
191–192 °C).46 1H NMR (400 MHz, CDCl3) d ppm: 6.59 (s, 1H); 7.37
(dd, J = 1.8, 8.7 Hz, 1H), 7.47–7.52 (m, 2H), 7.68 (t, J = 7.5 Hz, 1H)
7.71–7.86 (m, 5H), 7.88 (d, J = 8.7 Hz, 1H) 8.14 (t, J = 6.8 Hz, 2H).
13C NMR (CDCl3) d ppm: 103.8, 119.3, 121.6, 125.9, 126.2, 126.6,
127.1, 127.5, 127.8, 129.8, 130.4, 131.1, 132.4, 133.3, 133.8,
134.9, 144.5, 182.1, 184.0. IR (KBr, cmꢂ1): 3301, 1668, 1627,
1596. MS (ESI): 300.0 (C20H13NO2 [M+H]+).
4.1.2.1.14. 2-(Naphthalen-1-ylamino)naphthalene-1,4-dione
(4n). Red solid. Yield by method ii (156 mg, 55%), mp 156–
158 °C. 1H NMR (400 MHz, CDCl3) d ppm: 6.00 (s, 1H), 7.43–7.61
(m, 4H), 7.66–7.82 (m, 4H), 7.91 (d, J = 3.8 Hz, 2H) 8.09 (d,
J = 7.3 Hz, 1H). 8.17 (d, J = 7.3 Hz, 1H). 13C NMR (CDCl3) d ppm:
103.9, 121.8, 122.4, 125.6, 126.2, 126.5, 126.8, 127.0, 127.4, 128.8
(2C), 130.5, 132.3, 132.9, 133.3, 134.6, 134.9, 146.5, 182.2, 183.8.
IR (KBr, cmꢂ1): 3343, 1669, 1640, 1610. MS (ESI): 299.0
(C20H13NO2 [M+H]+).
4.1.2.2. General synthetic procedure for benzocarbazolequinon-
es 5a–5g.
Method iii: In a 25 mL two-necked flask fitted with a
coil condenser and kept under a dry inert atmosphere, 50 mg
(0.17 mmol) of the corresponding phenylaminonaphthoquinone,
18.4 mg (0.17 mmol) of 1,4-benzoquinone and Pd(OAc)2 (38 mg,
0.15 mmol) were poured into 10 mL of glacial AcOH and the result-
ing suspension was refluxed for about 24 h. The hot suspension
was filtered and the AcOH was removed in rotary evaporator.
The solid residue was purified by recrystallization with CH2Cl2 in
some cases and in others by column chromatography on silica gel.
Method iv: The corresponding naphthoquinone (0.5 mmol),
Pd(OAc)2 (2–10 mol %), K2CO3 (10 mol %) and pivalic acid (PivOH,
450 mg, 0.44 mmol) were mixed in
a test tube which was
immersed in an oil bath at 110–120 °C. The PivOH melted and
the mixture was stirred for 14–48 h at this temperature. The solu-
tion was allowed to cool to room temperature, diluted with CH2Cl2,
washed with saturated Na2CO3 solution, dried with anhydrous
MgSO4, filtered and concentrated under reduced pressure. The
pure product was purified by column chromatography on silica gel.
4.1.2.2.1. 5H-Benzo[b]carbazole-6,11-dione (5a). Orange solid.
Yield by method iii (21 mg, 43%). Yield by method iv (62 mg,
50%), mp 305–306 °C. (lit. 307–310).47 1H NMR (400 MHz, DMSO)
d ppm: 7.36 (t, J = 7.5 Hz, 1H), 7.44 (t, J = 7.6 Hz, 1H), 7.59 (d,
J = 8.2 Hz, 1H), 7.82 (m, 2H), 8.09 (t, J = 7.0 Hz, 2H), 8.20 (d,
J = 7.9 Hz, 1H), 13.04 (s, 1H). 13C NMR (DMSO) d ppm: 114.3,
117.8, 122.8, 124.3, 124.4, 126.4, 126.5, 127.4, 133.1, 133.6,
134.5, 134.7, 137.6, 138.7, 178.0, 180.8. IR (KBr, cmꢂ1): 3254,
1663, 1645, 1621.
128.0, 128.4, 128.7, 130.1, 132.1, 141.4, 177.5, 178.9. IR (KBr, m,
cmꢂ1): 3283, 1681, 1615, 1595. MS (ESI): 278.0 (C18H15NO2
[M+H]+). HRMS for (C18H15NO2 [M+H]+). Calcd: 277.1103. Found:
278.1156.
4.1.2.1.11. 2-Chloro-3-(2,5-dimethoxyphenylamino) naphthalene-
1,4-dione (4k). Purple solid. Yield by method ii (188 mg, 83%), mp
146–149 °C (lit. 146 °C).29 1H NMR (400 MHz, CDCl3) d ppm: 3.77
(s, 3H), 3.81 (s, 3H), 6.57 (d, J = 2.8 Hz, 1H), 6.71 (dd, J = 2.9,
8.9 Hz, 1H), 6.82 (d, J = 8.9 Hz, 1H), 7.61 (s, 1H), 7.68 (t, J = 7.4 Hz,
1H), 7.76 (t, J = 7.5 Hz, 1H), 8.10 (d, J = 7.6 Hz, 1H), 8.19 (d,
4.1.2.2.2. 4-Methoxy-5H-benzo[b]carbazole-6,11-dione (5b). Orange
solid. Yield by method iii (32 mg, 67%). Yield by method iv
(69 mg, 50%), mp 253–255 °C (lit. >250 °C).48 1H NMR (400 MHz,
Please cite this article in press as: Sieveking, I.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.07.030

10
I. Sieveking et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
DMSO) d ppm: 3.97 (s, 3H), 6.98 (d, J = 7.7 Hz, 1H), 7.28 (t,
J = 7.9 Hz, 1H), 7.74–7.88 (m, 3H), 8.09 (ddd, J = 1.6, 5.8, 7.2 Hz,
2H), 13.23 (s, 1H). 13C NMR (DMSO) d ppm: 56.1, 107.2, 114.7,
118.3, 125.4, 125.9, 126.4, 126.5, 129.6, 133.2, 133.7, 134.4,
134.6, 137.4, 147.9, 177.6, 181.0. IR (KBr, cmꢂ1): 3273, 1654, 1267.
4.1.2.2.3. 3-Methoxy-5H-benzo[b]carbazole-6,11-dione (5c). Orange
solid. Yield by method iii (10 mg, 21%). Yield by method iv (55 mg,
40%), mp 294–296 °C. 1H NMR (400 MHz, DMSO) d ppm: 3.86 (s,
3H), 7.09 (dd, J = 2.5, 9.0 Hz, 1H), 7.50 (d, J = 9.0 Hz, 1H), 7.62 (d,
J = 2.4 Hz, 1H), 7.81 (td, J = 1.4, 7.4 Hz, 1H), 7.86 (td, J = 1.5, 7.4 Hz,
1H), 8.10 (td, J = 1.2, 7.7 Hz, 2H), 13.00 (s, 1H). 13C NMR (DMSO) d
ppm: 55.8, 102.6, 115.5, 117.5, 118.9, 125.3, 126.5 (2C), 133.2, 133.6,
134.0, 134.6, 134.7, 137.5 157.4, 177.7, 180.7. IR (KBr, cmꢂ1): 3202,
1664, 1635, 1263. MS (ESI): 278.0 (C17H11NO3 [M+H]+). HRMS for
(C17H11NO3 [M+H]+). Calcd: 277.0739. Found: 278.0793.
4.19 (s, 3H), 6.76 (s, 1H), 7.02 (d, J = 8.2 Hz, 1H), 7.68 (br s, 2H);
8.15 (m, 2H), 8.29 (d, J = 8.7 Hz, 1H). 13C NMR (CDCl3) d ppm:
32.0, 55.6, 92.6, 100.0, 115.7, 118.1, 119.3, 124.7, 126.1, 126.3,
132.8, 133.4, 133.7, 134.0, 134.5, 141.5, 160.2, 178.5, 181.4. IR
(KBr, cmꢂ1): 3448, 1655, 1618, 1513. MS (ESI): 292.0 (C18H13NO3
[M+H]+).
4.1.2.3.2. 2,3-Dimethoxy-5-methyl-5H-benzo[b]carbazole-6,11-dione
(6b). Orange solid. Yield (146 mg, 91%), mp 278–280 °C. 1H
NMR (400 MHz, CDCl3) d ppm: 3.98–4.09 (m, 6H), 4.23 (br s, 3H),
6.79 (d, J = 5.5 Hz, 1H), 7.68 (s, 2H), 7.82 (d, J = 5.5 Hz, 1H), 8.17
(d, J = 6.4 Hz, 2H). 13C NMR (CDCl3) d ppm: 32.2, 56.2, 56.3, 92.2,
102.9, 117.5, 118.9, 120.7, 126.0, 126.3, 132.8, 133.3, 133.6,
134.0, 135.7, 149.3, 151.5, 177.9, 181.6. IR (KBr, cmꢂ1): 3424,
1649, 1590, 1508. MS (ESI): 322.0 (C19H15NO4 [M+H]+).
4.1.2.2.4. 2-Methoxy-5H-benzo[b]carbazole-6,11-dione (5d). Red
solid. Yield by method iii (19 mg, 40%). Yield by method iv
(111 mg, 80%), mp 298–300 °C. (lit. 299–300 °C).49 1H NMR
(400 MHz, CDCl3) d ppm: 3.94 (s, 3H), 7.06 (dd, J = 2.4, 8.8 Hz, 1H),
7.43 (d, J = 8.8 Hz, 1H), 7.13 (dd, J = 1.2, 7.6 Hz, 1H), 7.71 (m, 2H),
7.76 (d, J = 2.4 Hz, 1H), 8.22 (dd, J = 1.2, 7.6 Hz, 1H). 13C NMR (CDCl3)
d ppm: 55.6, 102.6, 114.1, 119.2, 126.1, 126.5, 132.7, 132.9, 133.4,
133.8, 133.9, 134.7, 136.8, 136.9, 157.4, 170.8, 173.8. IR (KBr,
cmꢂ1): 3242, 1667, 1642, 1526. MS (ESI): 278.0 (C17H11NO3 [M+H]+).
4.1.2.4. Synthetic procedure for 7-((2,5-dimethoxyphenyl)am-
ino)quinoline-5,8-dione (10).
A solution of 8 (6.38 mmol)34
in 100 mL EtOH was added to a solution of amine 9 (18.8 mmol)
and NiCl2 (3.14 mmol) in 50 mL EtOH. The reaction mixture was
stirred at 30 °C in the presence of air for 12 h, and was monitored
by thin layer chromatography. After the reaction was completed,
the EtOH was removed under reduced pressure leaving a residue
to which was added a cold aqueous 1% solution of HCl in order
to decompose the Ni complex. The product was extracted repeat-
edly with CHCl3 and then with water, dried with Na2SO4 and the
solvent removed. The product obtained was purified by column
chromatography on silica gel. Purple solid, yield (1.68 g, 85%), mp
70–80 °C. 1H NMR (400 MHz, CDCl3) d ppm: 3.80 (s, 3H), 3.88 (s,
3H), 6.71 (br s, 1H), 6.89 (d, J = 7.2 Hz, 1H), 7.02 (s, 1H), 7.53–
7.71 (m, 2H), 7.98 (S, 1H), 8.45 (d, J = 4.7 Hz, 1H), 9.05 (s, 1H). 13C
NMR (CDCl3) d ppm: 55.9, 56.2, 104.9, 108.1, 109.8, 111.9, 126.4,
127.1, 127.4, 134.4, 143.4, 145.5, 148.9, 153.8, 155.2, 181.8,
182.2. IR (KBr, cmꢂ1): 3328, 1673, 1630, 1570. HRMS for
(C17H14N2O4 [M+H]+). Calcd: 310.0954. Found: 311.1075.
4.1.2.2.5.
1,4-Dimethoxy-5H-benzo[b]carbazole-6,11-dione
(5e). Orange solid. Yield by method iii (21 mg, 40%). Yield by
method iv (115 mg, 75%), mp 286–288 °C. 1H NMR (400 MHz,
DMSO) d ppm: 3.92 (s, 3H), 4.00 (s, 3H), 6.67 (d, J = 2.0 Hz, 1H),
7.27 (d, J = 2.0 Hz, 1H), 7.84–7.94 (m, 2H), 8.12–8.18 (m, 2H),
13.26 (s, 1H) 13C NMR (CDCl3) d ppm: 55.9, 56.3, 99.7, 118.0,
125.3, 126.0, 126.4, 126.5, 133.4, 133.6, 134.5, 137.0, 148.5,
158.7, 177.2, 180.9. IR (KBr, cmꢂ1): 3254, 1663, 1645, 1621. MS
(ESI): 308.0 (C18H13NO4 [M+H]+). HRMS for (C18H13NO4 [M+H]+).
Calcd: 307.0845. Found: 308.0897.
4.1.2.2.6. 2-Hexyloxy-5H-benzo[b]carbazole-6,11-dione (5f). Orange
solid. Yield by method iii (40 mg, 67%). Yield by method iv (87 mg,
50%), mp 186–187 °C. 1H NMR (400 MHz, CDCl3) d ppm: 0,85 (m, 3H),
1.18 (m, 6H), 1.76 (m, 2H), 3.95 (s, 2H), 6.77 (d, J = 8.0 Hz, 1H), 6.96
(d, J = 8.8 Hz, 1H), 7.40 (d, J = 8.8 Hz, 1H), 7.45 (s, 1H), 7.83 (d,
J = 8.0 Hz, 1H), 8.12 (d, J = 8.8 Hz, 2H). 13C NMR (CDCl3) d ppm: 13.5,
24.1, 25.1, 28.6, 29.0, 72.8, 103.7, 109.0, 114.6, 118.0, 125.2, 125.8,
130.4, 151.5, 152.4, 158.2, 176.6, 180.8. IR (KBr, cmꢂ1): 3264, 2955,
2925, 2852, 1664, 1261. HRMS for (C22H21NO3 [M+H]+). Calcd:
347.1521. Found: 348.1571.
4.1.2.5. 6-((2,5-Dimethoxyphenyl)amino)-2,2-dimethyl-2,3-dih-
ydrobenzofuran-4,7-dione (16).
According to method ii, the
product was prepared in a flask containing 10 mL of ethanol,
100 mg (0.39 mmol) of quinone 15, 119 mg (0.78 mmol) of
2,5-dimethoxyaniline and 75 mg (0.20 mmol) of CeCl3*7H2O. The
mixture was stirred for 2 h to room temperature and then solvent
was removed under reduced pressure. The crude product was puri-
fied by chromatographic column using a mixture of CHCl3/AcOEt
(4/1) as mobile phase. Purple solid, yield (78 mg, 61%), mp:
82–84 °C. 1H NMR (400 MHz, CDCl3) d ppm: 1.57 (s, 6H), 2.89 (s,
2H), 3.77 (s, 3H), 3.85 (s, 3H), 6.06 (s, 1H), 6.65 (dd, J = 2.9,
8.9 Hz, 1H), 6.65 (dd, J = 2.9, 8.9 Hz, 1H), 6.86 (d, J = 8.9 Hz, 1H),
6.95 (d, J = 2.8 Hz, 1H), 8.16 (s, 1H). 13C NMR (101 MHz, CDCl3) d
28.33, 39.39, 55.90, 56.24, 93.25, 97.76, 107.83, 109.80, 111.76,
115.05, 127.39, 144.35, 145.53, 153.77, 161.57, 179.13, 179.52. IR
(KBr, cmꢂ1) 3308, 1655, 1614, 1576, 1208, 1111. HRMS for
(C18H19NO5 [M+H]+). Calcd: 329.1263. Found: 330.1312.
4.1.2.2.7.
2,3-Dimethoxy-5H-benzo[b]carbazole-6,11-dione
(5g). Purple solid. Yield by method iii (22 mg, 42%), mp 275–
276 °C. (lit. 275–277 °C).37 1H NMR (400 MHz, CDCl3) d: 3.92 (s,
3H), 3.93 (s, 3H), 7.04 (s, 1H), 7.62 (s, 1H), 7.83–7.91 (m, 2H),
8.12–8.15 (m, 2H), 12.92 (s, 1H). 13C NMR (CDCl3): d 56.1 (2C),
95.6, 102.3, 117.9, 126.4 (2C), 133.5, 133.6 (2C), 134.2, 134.4,
134.5, 135.8 149.2, 151.4, 176.6, 181.0. IR (KBr, cmꢂ1): 3248,
1643, 1588.MS (ESI): 308.0 (C18H13NO4 [M+H]+).
4.1.2.3. General synthetic procedure for N-methyl-ben-
4.1.2.6. 5,6-Dihydro-1,6,6-trimethyl-1H-furo[3,2-f]indazole-4,8-
zocarbazolediones (6a–b).
To a solution of benzocarbazol-
dione (17).
In a flask containing 15 mL Et2O and 200 mg
edione 5c (140 mg, 0.50 mmol) or 5g (154 mg, 0.50 mmol) in
15 mL of EtOH in a 25 mL flask was added 4.42 mmol of KOH pel-
lets and the mixture was stirred at room temperature until com-
plete dissolution. The EtOH was removed under vacuum and the
residue was treated with acetone (15 mL) and methyl iodide
(3.54 mmol). The product obtained was purified by column chro-
matography on silica gel.
4.1.2.3.1. 3-Methoxy-5-methyl-5H-benzo[b]carbazole-6,11-dione
(6a). Orange solid. Yield (124 mg, 85%), mp 246–249 °C. (lit.
250–253 °C).31 1H NMR (400 MHz, CDCl3) d ppm: 3.91 (s, 3H),
(1.12 mmol) of quinone 15, cooled at 0 °C, it was added an excess
of diazomethane. The reaction was left under magnetic stirring at
0 °C for 15 min. The solvent was the removed under reduced pres-
sure and the reaction mixture was purified by recrystallization in
MeOH. Orange solid, yield (174 mg, 67%), mp 205–206 °C. 1H
NMR (400 MHz, CDCl3) d ppm: 2.17 (s, 6H), 2.91 (s, 2H), 4.20 (s,
3H); 7.83 (s, 1H). 13C NMR (CDCl3) d ppm: 28.3, 30.9, 38.8, 39.3,
93.4 120.4, 120.7, 136.5, 160.5, 173.8 175.3. IR (KBr, cmꢂ1): 3448,
1681, 1645. MS (ESI): 232.0 (C12H12N2O3 [M+H]+). HRMS for
(C12H12N2O3 [M+H]+). Calcd: 232.0848. Found: 233.0904.
Please cite this article in press as: Sieveking, I.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.07.030

I. Sieveking et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
11
4.2. Biology
mixture and treated with the compounds at different concentra-
tions for 72 h. At the end of drug exposure, cells were fixed with
50% trichloroacetic acid at 4 °C (final concentration 10%). After
washing with water, cells were stained with 0.4% sulforhodamine
B (Sigma–Aldrich, St. Louis, MO), dissolved in 1% acetic acid
4.2.1. Trypanocidal assay
4.2.1.1. Cell viability assay.
The effect of drug treatments on
T. cruzi and Vero cells was evaluated using the MTT assay as a via-
bility test.49 Briefly, 10
l
L of 5 mg/mL tetrazolium dye (MTT; 3-
(50
acid to remove unbound stain. The protein-bound stain was solu-
bilized with 100 L of 10 mM unbuffered Tris base, and the cell
density was determined using fluorescence plate reader
lL/well) for 30 min, and subsequently washed with 1% acetic
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) plus
0.22 mg/mL phenazine methosulfate (electron carrier) were added
to each well of a 96-well plate containing Vero cells or epimastig-
l
a
otes in 100
l
L RPMI 1640 without phenol red. Drugs, dissolved in
(wavelength 540 nm). Values shown are the mean SD of three
independent experiments in triplicate.
DMSO, were added to culture media at the concentrations shown
in figures and tables. DMSO final concentration was less than
0.25% v/v. After incubation for 4 h at 37 °C or 28 °C (for Vero cells
or epimastigotes, respectively), the water-insoluble formazan gen-
4.3. Computational
4.3.1. Ligands
erated was dissolved by addition of 100 lL of 10% w/v SDS in
The geometries of all ligands involved in this study were fully
optimized using the Hartree–Fock method with the standard basis
set, 6-31G⁄. Electrostatic potential (ESP) charges were derived from
the calculations. All calculations were performed using the
Gaussian03 suite.54 Druglikeness properties were predicted for
all compounds using from Molsoft LLC and Visual Molecular
Dynamics program (VMD).55
0.01 M HCl. The plates were further incubated overnight at 37 °C,
and optical density (OD) of the wells was determined using a
microplate reader (Asys Hitech, Austria) at 570 nm. Under these
conditions, the OD is directly proportional to the viable cell num-
ber in each well. All experiments were performed at least three
times and data are shown as the means and their standard devia-
tions from triplicate cultures.
Acknowledgments
4.2.1.2. Epimastigote culture growth. Epimastigotes of the
Dm28c strain were grown at 28 °C in modified Diamond’s medium
as described previously.50 The cultures were initiated with a cell
density of 3 ꢃ 106 epimastigotes/mL. Cell density was measured
by direct counting with a hemocytometer. Compounds dissolved
in DMSO were added to the epimastigote suspension, with a final
concentration of DMSO below 1%. After 24 h, viability of the epim-
astigotes was determined by MTT reduction as described above.
Financial support from FONDECYT (Research Grant) N° 1120128
(C.O.S.), N° 1110749 (R.A.T), 1130189 (J.D.M) and 11110182
(R.L.M.) is gratefully acknowledged. P.T is grateful to CONICYT for
a PhD fellowship. J.V. is very grateful to DIUV n° 50/2011 of the
Universidad de Valparaíso for financial support. We also thank
the Xunta de Galicia (CN2011/037) for financial support.
4.2.1.3. T. cruzi trypomastigotes. Vero cells (ATCCÒ CCL-81™)
were infected with bloodstream trypomastigotes from
T. cruzi-infected Balb/c mice. Vero cell cultures infected with
trypomastigotes were incubated at 37 °C in RPMI medium (5%
FBS), in humidified air and 5% CO2 for 5–7 days. After that time,
the culture medium was collected and centrifuged at 3000ꢃg for
5 min, and the trypomastigote-containing pellet was resuspended
in RPMI supplemented with 5% fetal bovine serum and penicil-
lin–streptomycin at a final density of 107 parasites/mL.51 IC50
values were obtained by non-linear regression fitting data to a
four-parameter equation model, using GraphPad Prism 5.0 software.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.07.030.
References and notes
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