Synthesis and biological characterization of new aryloxyindole-4,9-diones as potent trypanosomicidal agents

Article abstract of DOI:10.1016/j.bmcl.2014.06.044

A new indole-4,9-dione and their phenoxy derivatives were synthesized and evaluated in vitro against the epimastigote form of Trypanosoma cruzi, Y strain. All of these novel compounds were found to be extremely potent and selective that the standard drug nifurtimox. Interestingly, phenoxyindole-4,9-dione 9d displayed excellent nanomolar inhibitory activity, IC₅₀ = 20 nM, and high selectivity index, SI = 625. In silico studies using MOE program were performed to generate a preliminary pharmacophore model.

Full text of DOI:10.1016/j.bmcl.2014.06.044

Accepted Manuscript
Synthesis and biological characterization of new aryloxyindole-4,9-diones as
potent trypanosomicidal agents
Ricardo A. Tapia, Cristian O. Salas, Karina Vázquez, Christian Espinosa-
Bustos, Jorge Soto-Delgado, Javier Varela, Estefanía Birriel, Hugo Cerecetto,
Mercedes González, Margot Paulino
PII:
S0960-894X(14)00672-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.06.044
BMCL 21763
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
29 April 2014
12 June 2014
16 June 2014
Please cite this article as: Tapia, R.A., Salas, C.O., Vázquez, K., Espinosa-Bustos, C., Soto-Delgado, J., Varela, J.,
Birriel, E., Cerecetto, H., González, M., Paulino, M., Synthesis and biological characterization of new
aryloxyindole-4,9-diones as potent trypanosomicidal agents, Bioorganic & Medicinal Chemistry Letters (2014),
doi: http://dx.doi.org/10.1016/j.bmcl.2014.06.044
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Synthesis and biological characterization of new aryloxyindole-4,9-
diones as potent trypanosomicidal agents
a,
Ricardo A. Tapia *, Cristian O. Salas , Karina Vázquez , Christian Espinosa-Bustos ,
a
a
a
a
b
b
Jorge Soto-Delgado , Javier Varela , Estefanía Birriel , Hugo Cerecetto b,c, Mercedes
González b, Margot Paulino d
a Departamento de Química Orgánica, Facultad de Química, Pontificia Universidad Católica de Chile, Santiago 6094411, Chile
b
Grupo de Química Medicinal, Instituto de Química Biológica, Facultad de Ciencias, Universidad de la República, Iguá 4225,
Montevideo, Uruguay.
c
Área de Radiofarmacia, Centro de Investigaciones Nucleares, Facultad de Ciencias, Universidad de la República, Mataojo 2055,
Montevideo, Uruguay.
d Centro de Bioinformática Estructural-DETEMA, Facultad de Química, Universidad de la República, C.C. 1157, Montevideo, Uruguay.
Abstract
A new indole-4,9-dione and their phenoxy derivatives were synthesized and evaluated in
vitro against the epimastigote form of Trypanosoma cruzi, Y strain. All of these novel
compounds were found to be extremely potent and selective that the standard drug
nifurtimox. Interestingly, phenoxyindole-4,9-dione 9d displayed excellent nanomolar
inhibitory activity, IC50=20 nM, and high selectivity index, SI = 625. In silico studies using
MOE program were performed to generate a preliminary pharmacophore model.
Keywords: Indolequinones, anti-T. cruzi, cytotoxicity, pharmacophore model.
*Corresponding authors. E-mail address
rtapia@uc.cl (R.A. Tapia)
:
Chagas disease, or American tripanosomiasis, is a neglected tropical disease caused by
the protozoan Trypanosoma cruzi, which affects 16-18 million people in many rural areas
of Latin America.1 There are two main drugs used for the treatment of this disease,
nifurtimox (Nfx) and benznidazole, but both are toxic and present severe side effects.2,3
Therefore, there is a need for more effective drugs and many efforts have been done in the
search for new compounds with potential clinical utility.4 Taking into account the anti-
trypanosomal activity of natural naphthoquinones, the preparation of many compounds

containing the naphthoquinone pharmacophore and their activity on Trypanosoma cruzi
were described.5 In addition some heterocyclic quinones such as naphthopyranoquinones,
naphthofuranoquinones showed interesting trypanosomicidal activities.6-8 Furthermore, the
potent trypanosomicidal activity of 2-phenoxy-1,4-naphthoquinone (1, Fig. 1) against the
amastigote forms of T. cruzi with an IC50 of 1.70 µM but low selectivity index (SI < 5) was
described.9 Some studies suggest that the anti-trypanosomal activity of quinones is related
to oxygen radical formation and consequently redox cycling.10 Moreover, the presence of
heteroatoms in the aromatic moiety contribute to an enhancement of the anti-trypanosomal
activity.8 On the other hand, the potent cytotoxic activity of some indolequinones, such as
compound 2 (Fig. 1), has been described by Moody et al.11,12 The proposed mechanism of
action of this indolequinone implicates the induction of caspase-dependent apoptosis but
does not involve redox cycling or oxidative stress in all cancer cells assay. Based on these
precedents, we became interested in the synthesis of new aryloxy indolequinones (using as
template compound 3, Fig. 1) as compounds with enhanced trypanosomicidal activity and
decreased cell cytotoxicity.
Figure 1. Chemical structures of compounds with biological properties and designed
template for new trypanosomicidal agents.
Knowing that the usual approach for the preparation of aryloxyquinones is done by
nucleophilic substitution reaction of haloquinones with phenols,13-15 we decided to
synthesize the unknown bromoindol-4,7-dione 8 (Scheme 2). Moreover, considering the
versatility of the Hemetsberger-Knittel reaction16-18 for the preparation of indoles, we take
advantage of this methodology to obtain bromoindol 7 (Scheme 1). Thus, reaction of 4-
bromo-2,5-dimethoxybenzaldehyde (4)19 with ethyl azidoacetate and subsequent thermal
cyclization of 2-azidocinnamate 5 resulted in the formation of indole 6 in 52% yield (two

steps). Next, treatment of compound 6 with bromoethane in the presence of NaH in DMF
afforded the N-alkylated indole 7 (Scheme 1).
Scheme 1. Reagents and conditions: i) Ethyl azidoacetate, Na, ethanol, -10 °C, 3 h, 57%;
ii) toluene, reflux, 12 h, 92%; iii) NaH, bromoethane, DMF, 1 h, 98%.
Oxidative demethylation of indol 7 with excess silver (II) oxide and HNO3 in
tetrahydrofuran20 gave bromoquinone 8. Finally, aryloxyindole-4,9-diones 9a-e were
obtained in 36-72% yield by nucleophilic substitution reaction of 8 with phenols and
sodium hydride in tetrahydrofuran (Scheme 2). Compound 1 was prepared as described,9 by
reaction of 2-bromo-1,4-naphthoquinone with phenol and potassium carbonate in dimethyl
formamide (DMF).
Scheme 2. Reagents and conditions: i) AgO, HNO3 6N, THF, rt, 5 min, 68%; ii) NaH,
ArOH, THF, rt, 10 min, 36-72%.
The in vitro trypanosomicidal activity of compounds 6-8 and phenoxyquinones 1, 9a-e
was initially tested against the epimastigote form of T. cruzi, Y strain (Tc II).21 For each
derivative a dose-response assay, between 0.01 and 50 µM, was evaluated to calculate the
IC50 concentration (50% inhibitory concentration). Nfx (Bayer) was used as the reference
trypanosomicidal drug.11 All phenoxyquinones showed potent trypanosomicidal activity

and they are over tenfold more active that the reference drug Nfx, which has an IC50 of 7.0
µM (Table 1). Among them, compounds 9b and 9d displayed the most potent inhibitory
activity (IC50 = 0.02 µM for epimastigotes). While it is not possible to establish a structure–
relationship with these compounds, there are some preliminary analyses that we can do
with these results. First, the presence of donor groups at C-4 in the phenyl ring of the
phenoxyindolequinone derivatives have a remarkable effect on the trypanosomicidal
activity and the most active compounds were 9b (4-Me) and 9d (4-OMe) with an IC50 of
0.02 µM. On the other hand, compounds without a phenoxy group bound to the
indolequinone system, had the lowest activity (compound 8). This fact indicates the
importance of the phenoxy moiety on the eventual pharmacophore structure. Finally, a
potential antichagasic drug must show low toxicity in mammalian host cells and for this
reason, the cytotoxic effects in J774 murine macrophage-like cells (ATCC, USA) of
compounds with the strongest trypanosomicidal effects vs epimastigotes were determined.23
It is well known that naphthoquinones generate ROS (Reactive oxygen species) and, as
expected several of the tested compounds were quite cytotoxic. According to the strategies
for the development of novel drugs for tropical disease the selective index should be higher
than 50.22,23 Table 1 shows for some compounds with higher epimastigote toxicity with
IC50 values for J774 murine cell proliferation and their selectivity indexes as the ratios of
the IC50 in epimastigotes. These results indicate all of these compounds tested are more
selective than Nfx (SI = 40). The most remarkable results came from compound 9d, which
exhibits greater selectivity in regard of its toxicity toward murine cells (SI = 625). This
result is very promising for the developing of new selective and more potent
trypanosomicidal agents.

Table 1. Effect of phenoxyindolequinones upon culture growth of T. cruzi and selectivity
index vs. murine cells.
epimastigote IC50
(µM)b
0.05 0.02
Compound
%GIa,b
87.2 2.1
26.9 + 7.2
2.9 + 1.3
97.1 + 2.2
84.7 + 2.4
92.1 + 3.2
100.0
J774
IC50 (µM)
12.5
SIc
250
-
1
6
-
-
-
-
-
7
0.53 + 0.12
0.22 + 0.08
0.02 + 0.01
0.14 + 0.05
0.02 + 0.01
0.13 + 0.05
7.00
n.d.
22
-
8
100
-
9a
9b
9c
9d
9e
Nfxd
n.d.
12.5
12.5
n.d.
316
89.3
625
-
90.5 + 4.7
94.6 + 2.3
49.9
40
a %GI = Percentage of growth inhibition on T.cruzi culture Y at 10 µM.
b
The results are means of three independent experiments.
c Selectivity Index: expressed as the ratio of IC50 in J774 cells to IC50 in epimastigotes.
d 5µM was used for this assay.
n.d.: not determined.
Then, MOE program24 was used to generate a preliminary pharmacophore model25,26
related with in vitro trypanosomicidal activity (IC50) of compounds 1, 7-9a-e. First, to
determinate the essential chemical features of those molecules with more relevant (lower)
IC50 values, compounds 9b and 9d were chosen to create the pharmacophore model
(“Activity Pharmacophore”, Figure 2). With fully optimized structures of compounds 9b
and 9d, it was possible identify regions of pharmacophoric importance such as; aromatic
ring (Aro), hydrogen bond acceptor (Acc) and hydrophobic rings (Hyd) (Figure 2b-c).
Furthermore, the distance between the pharmacophoric features was measured and shown
in the Figure 2d.

a)
b)
c)
d)
Figure 2. Graphical display of “Activity Pharmacophore”. a) 2D picture of the modelled
9d molecule. b) 3D drawing of 9d molecule overlapped with the pharmacophore picture. c)
3D drawing of isolated pharmacophore. The different features are depicted in yellow
(aromatic), red (H-bond acceptor) and blue (hydrophobic). d) Distances between the centre
of pharmacophoric features for 9a and 9d molecules are shown in green numbers.
In summary, the pharmacophoric features found as relevant for both 9b and 9d
molecules were: i) Aromatic (Aro); ii) Hydrogen bond Acceptor (Acc) and iii)
Hydrophobic moiety (Hyd). These three essential characteristics of 9b and 9d, were also
identified in molecules 9a, 9c and 9e. It is noticeable that all these molecules having the
above pharmacophoric features showed lowers IC50 values, when compared with the IC50
value of Nfx (7µM), suggesting that the generated pharmacophore model is an identifier of
the essential characteristics that are responsible for biological activity.
Finally, once the pharmacophoric features that make a molecule reactive against T.
cruzi in vitro were elucidated, a “Selectivity Pharmacophore” was designed, assessing the
same three essential characteristics centred in the O-phenyl scaffold of the molecules.

Modifying and assigning these features in a spatial arrangement as it is presented in the
Figure 3a, and applying them in the molecules database of molecules previously resulted
selected by the “Activity Pharmacophore” it was possible to identify just the compound 9d
that is the one with the best IC50 and IS (Figure 3b).
a)
b)
Figure 3. a) “Selectivity Pharmacophore” showing the spatial arrangement of three
essential features Aro (green), Acc (blue) and Hyd (red). b) “Selectivity pharmacophore”
showed as big green, blue and red spheres over the 3D image of 9d best active and selective
compound. Small spheres are features related with the main tripanosomicidal activity
according to the “Activity pharmacophore” previously designed.
In conclusion, the present study provides useful information concerning the optimal
structural requirements necessary for designing new, more potent and selective
trypanosomicidal agents.
Acknowledgements: We are grateful to Fondecyt (Grant 1110749), KV thanks to
PROMEP-México (Grant 103.5 -10-5345), CSIC-UdelaR (Proyecto Grupos Nº 661) of
Uruguay, MP is grateful to the PEDECIBA-UDelaR Master in Bioinformatics. JV, and EB
thank ANII (Uruguay) for their scholarships.
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Synthesis and biological characterization of new
aryloxyindole-4,9-diones as potent trypanosomicidal
agents
Leave this area blank for abstract info.
Ricardo A. Tapia,*, Cristian O. Salas, Karina Vázquez, Christian Espinosa-Bustos, Jorge Soto-Delgado, Javier
Varela, Estefania Birriel, Hugo Cerecetto, Mercedes González, Margot Paulino.
O
O
Me
OMe
EtOOC
EtOOC
N
N
O
O
Et
Et
O
O
IC50 = 20 nM (On epimastigote culture)
20 nM