In vivo antimalarial activity of novel 2-hydroxy-3-anilino-1,4- naphthoquinones obtained by epoxide ring-opening reaction

Article abstract of DOI:10.1016/j.bmcl.2013.06.033

1,4-Naphthoquinone derivatives are known to have relevant activities against several parasites. Among the treatment options for malaria, atovaquone, a 1,4-naphthoquinone derivative, is widely applied in the treatment and prophylaxis of such disease. Based on the structure simplification of atovaquone, we designed and synthesized four novel naphthoquinoidal derivatives. The compounds were obtained by the underexplored epoxide-opening reaction of 1,4-naphthoquinone using aniline derivatives as nucleophiles. The antiplasmodial activity of the synthesized compounds was performed in vivo using Peter's 4 days suppression test. Significant parasitemia reduction and increased survival were observed for some of the compounds.

Full text of DOI:10.1016/j.bmcl.2013.06.033

Bioorganic & Medicinal Chemistry Letters 23 (2013) 4583–4586
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
In vivo antimalarial activity of novel 2-hydroxy-3-anilino-1,
4-naphthoquinones obtained by epoxide ring-opening reaction
Lucas Cunha Dias de Rezende a, Fernando Fumagalli a, Marraiana Schiavon Bortolin a,
Marianne Garcia de Oliveira b, Murilo Helder de Paula a, Valter Ferreira de Andrade-Neto b
Flavio da Silva Emery a,
⇑
a Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, 14040-903 Ribeirão Preto-SP, Brazil
b Laboratório de Biologia da Malária e Toxoplasmose, Departamento de Microbiologia e Parasitologia, Centro de Biociências, Universidade Federal do Rio Grande do Norte, 59000-000
Natal, RN, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
1,4-Naphthoquinone derivatives are known to have relevant activities against several parasites. Among
the treatment options for malaria, atovaquone, a 1,4-naphthoquinone derivative, is widely applied in
the treatment and prophylaxis of such disease. Based on the structure simplification of atovaquone,
we designed and synthesized four novel naphthoquinoidal derivatives. The compounds were obtained
by the underexplored epoxide-opening reaction of 1,4-naphthoquinone using aniline derivatives as
nucleophiles. The antiplasmodial activity of the synthesized compounds was performed in vivo using
Peter’s 4 days suppression test. Significant parasitemia reduction and increased survival were observed
for some of the compounds.
Received 26 April 2013
Revised 7 June 2013
Accepted 11 June 2013
Available online 20 June 2013
Keywords:
Naphthoquinones
Epoxides
Ring-opening
Anilines
Ó 2013 Elsevier Ltd. All rights reserved.
Plasmodium
1,4-Naphthoquinones are a class of compounds broadly studied
in organic synthesis, medicinal chemistry and natural products
chemistry.1 Within these studies, the antimalarial activity has been
widely reported for several 1,4-naphthoquinones.2 The antipara-
sitic activity of the quinones is related to several mechanisms such
as the competitive inhibition of the cytochrome bc1 complex, gen-
eration of reactive oxygen species, enzymatic inhibition (e.g., glu-
tathione reductase, dihydroorotate dehydrogenase and glycerol
glyceraldehyde-3-phosphate dehydrogenase) alkylation of biomol-
ecules, depletion of glutathione, among others.3
Lawsone derivatives containing the 2-hydroxy-1,4-naphthoqui-
none scaffold are highly explored for the development of novel
antiparasitics. Atovaquone, parvaquone and buparvaquone are
3-substituted-2-hydroxy-1,4-naphthoquinones clinically applied
as antimalarial (atovaquone) and antipneumocystic (parvaquone
and buparvaquone) agents, which exemplifies the potentiality of
such scaffold in the development of novel drugs. Additionally,
some studies indicate the potential application of natural or syn-
thetic 2-amino-1,4-naphthoquinones and 2-amino-1,2-naphtho-
quinones as antiplasmodial agents.4
Despite the pharmacological relevance of aminated and
hydroxylated naphthoquinoidal derivatives and the wide
structural diversity of 1,4-naphthoquinones, 2-amino-3-hydroxy-
1,4-naphthoquinone is an uncommon structural fragment only
observed in few examples of natural products and bioactive
compounds.5
Recently
2-(4-phenylpiperazinyl)-3-hydroxy-
1,4-naphthoquinone 1,6 which can be seen as a ring bioisostere
of atovaquone, was shown to have inhibitory effects against
Plasmodium falciparum.6
Based on the structures of atovaquone and 1, we propose the
simplification of the common scaffold by modifying the spacer
group (SG) between the naphthoquinoidal moiety and the benzene
ring (Atovaquone, SG = cyclohexane; and 1, SG = piperazine). In our
proposal, the aliphatic rings of these two compounds are substi-
tuted by
a nitrogen atom to produce 3-anilino-substituted
hydroxynaphthoquinones (Fig. 1).
The most common synthetic methodology for obtaining 2-
amino-3-hydroxy-1,4-naphthoquinones and derivatives involves
the nucleophilic substitution of halonaphthoquinones, followed
by hydrolysis depending on the starting material.7 Moreover,
azide,8 nitro or hydrazine reduction,9 amination of aryliodionium
ylides of 2-hydroxy-1,4-naphthoquinones,10 and oxidation of
2-amino-1,4-naphthoquinones are somewhat useful strategies.11
In this work, we describe a two-step method to synthesize
novel 2-hydroxy-3-amino-1,4-naphthoquinone derivatives from
⇑
Corresponding author. Tel.: +55 16 3602 0658; fax: +55 16 3602 4178.
E-mail addresses: flavioemery@fcfrp.usp.br, flavioemery@qhetem.com (Flavio da
Silva Emery).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.06.033

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L. C. D. Rezende et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4583–4586
Figure 1. Synthesis and reaction mechanism of the 2-anilino-3-hydroxy-1,4-naphthoquinone obtained herein.
1,4-naphthoquinone 1, based on aminolysis of epoxide 2 in
aqueous or non-aqueous media. Although epoxide ring-opening
reactions are widely described in the literature, there are only
few examples describing the nucleophilic addition to 2,3-epoxy-
1,4-naphthoquinone 3.12
signals observed for the aniline moiety were similar to the respec-
tive starting anilines, however with slight shielding. In the 13C
NMR, carbonyl carbons were detected as two signal near 181 and
179 ppm, while the others carbons appear as eight signals between
120 and 140 ppm, as expected.
Herein, we report the reactivity of 3 with four anilines to gener-
ate the respective 2-hydroxy-3-anilino-1,4-naphthoquinone deriv-
atives 4a–d as purple solids (Table 1). The epoxide 3 was prepared
by oxidation of 1,4-naphthoquinone 2, in the presence of hydrogen
peroxide, and used after recrystallization (Fig. 1).13 The results
indicate that, following the ring-opening reaction, an oxidation
step regenerates the quinone ring (Fig. 1). In all cases, the typical
procedure involved the addition of the aniline to a stirring solution
of 3, in water or ethanol, at 60 °C, until complete consumption of 3
(observed in TLC). All reactions were reproducible in gram scale. All
compounds were purified by flash chromatography.14
The antiplasmodial activity of compounds 4a–d was assayed
in vivo using a Peter’s20 four days suppression test protocol with
some modifications.21 Groups of six adult Swiss albino mice in-
fected with Plasmodium berghei NK65 were treated for four consec-
utive days with compounds 4a–d (100 mg/kg/day), chloroquine
(10 mg/kg/day positive control group) and vehicle (negative con-
trol group). Parasitemia was determined by blood smear exam on
In order to increase reaction yields, 1 equiv of six Lewis acids
(ZnCl2, CuI, Cu(OAc)2, CuCl2, InCl3, InI), previously described as cat-
alysts for epoxide ring-opening reaction,15 were tested in the aque-
ous reaction of 3 with 2-chloro-aniline. Additionally, 5 mol % of
Sc(OTf)3 in dichloromethane was also tested. In comparison with
noncatalyzed aqueous reaction, the time needed to entirely con-
sume of precursor 3 observed in thin layer chromatography, was
reduced in the presence of catalysts (especially for ZnCl2). Impelled
by these results, ZnCl2 was also tested in aqueous reactions with
the three other anilines. In all cases time for precursor consump-
tion was improved to approximately 2 h, with reduction in the
yield of 4b, expressive increment in the yield of 4c, and no signif-
icant effect in the yields of 4a and 4d (Table 1).
The structure of 4a,16 4b,17 4c18 and 4d19 were confirmed based
on 1H NMR, 13C NMR and HRMS-ESI data. A common pattern of sig-
nals could be identified in 1H NMR spectra, where the four hydro-
gen atoms of the naphthoquinoidal ring (H-5 to H-8) originates
two signals (near 8.0 ppm and 7.6 ppm). Additionally, the aromatic
Figure 2. Evolution of strategies to design novel antimalarial derivatives.
Table 1
Reaction of 2,3-epoxy-1,4-naphthoquinone 3 with nucleophiles on ethanol and water
yielding the respective 2-anilino-3-hydroxy-1,4-naphthoquinones
Table 2
Effects of compounds 4a, 4b and 4d over parasitemia
Nucleophile
Prod.
Yielda (%)
B
Data
Compd
Parasitemia (%)
Day 5 Day 7
Parasitemia inhibition (%)
A
C
Day 5
Day 7
Aniline
4a
4b
4c
4d
88.0
50.0
61.6
48.5
94.7
23.7
92.2
55.0
65.2
9.5
93.0
30.5
Ref.14
Ref.15
Ref.16
Ref.17
2-chloro-aniline
4-methoxy-aniline
2,6-dimethyl-aniline
4a
4c
4d
Cont.
0.7
0.5
1.5
1.5
2.2
2.0
2.7
2.7
53.3
66.6
0
18.5
25.9
0
0
0
a
A—water; B—water + Zinc chloride (1 equiv), C—ethanol.

L. C. D. Rezende et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4583–4586
4585
be responsible for some antiparasitic activity of this class of com-
pounds. Further studies will furnish molecular evidences of the
mechanism of action of the antiplasmodial compounds showed
herein.
Acknowledgements
The authors acknowledge São Paulo Research Foundation (FA-
PESP – grants #2009/14184-0 and 2011/01945-3), CNPq and IN-
CT_if for the grants and scholarships. We also would like to
acknowledge the Analytical Centre of NPPNS and the Analytical
Chemistry Laboratory (FCFRP) for the Mass Spectrometry and IR
spectroscopy analysis, respectively.
Figure 3. Mean survival of animals treated with 4a, 4b and 4d and the negative
control.
References and notes
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growth inhibition of each drug was calculated.22 The compounds
were considered partially active if parasitemia decreased by 30%
or more.20 Mortality was monitored in all the groups for four
weeks after inoculation.
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As shown in Table 2, significant parasitemia reduction was ob-
served for compounds 4a and 4c, mainly at day five after parasite
inoculation when parasite growth inhibition was 53% and 66%
respectively. No relevant activity over parasitemia was observed
in the group of animals treated with compound 4d. Since the activ-
ities were tested in vivo, this result may be related to poor pharma-
cokinetic profile and bioavailability of this compound. The
compound 4b could not be tested due to solubility problems. The
positive control group, treated with chloroquine, showed a parasite
growth inhibition of 98%, showing the necessity of chemical mod-
ifications to obtain antiplasmodial activity consistent for clinical
tests. It is worth to note that the drugs were administered via ga-
vage, consequently, first pass metabolism may have significant
influence over their activities. Future tests with subcutaneous
administration and higher attack doses will enable us to outline
in detail the potentiality of these compounds.
The mean survival of the animals under different therapeutic
regimens is shown in Figure 2. While the animals treated with
chloroquine survived for more than 60 days after parasite inocula-
tion, the group treated with vehicle showed a mean survival of
20 2.7 days. Our results (Fig. 3) indicate a slight higher survival
for the animals treated with 4c and 4d (26.0 4.0 and
26.7 2.5 days, respectively), however with no statistical signifi-
cance (T-test p > 0.05 when compared to control group). Com-
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pound 4a, however, showed
a
statistically significant
enhancement of mean survival (32.5 6.6, T-test p < 0.01 when
compared to control group).
The antiplasmodial activity observed for 4a and 4c gives sup-
ports to the potential use of 2-amino-3-hydroxy-1,4-naphthoqui-
none derivatives as antiplasmodial agents, which justifies further
studies for the development of a library of 2-hydroxy-3-amino-
1,4-naphthoquinone derivatives applying a diversity-oriented syn-
thesis approach. In this study, we were able to obtain compound 3
in moderate yields, in multi-gram, scale and compounds 4a–d
were obtained in moderate to good yields with a simple work-
up. Three of the four novel compounds were active in vivo against
P. berghei NK65.
Due to their structural similarity with atovaquone these novel
compounds may probably act through similar mechanism. Ato-
vaquone is believed to inhibit electron flow in aerobic respiration,
by binding to cytochrome b in place of ubiquinone in the parasite
mitochondria. Such inhibition hampers the activity of dihydrooro-
tate dehydrogenase, an enzyme linked to the mitochondrial elec-
tron transport system that is required in the de novo synthesis of
pyrimidines.23 Additionally, the redox potential of naphthoqui-
nones is suggested to cause an oxidative stress which may also
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14. (a) General method for reaction of 3 with anilines in water: The aniline
(3 equiv) was added to
a suspension of 3 in water, at 60 °C. After the
consumption of 3, the precipitate was filtered through reduced pressure, dried
in a dessicator purified by flash chromatography. (b) General method for
reaction of 3 with anilines in ethanol: The aniline (3 equiv) was added to a
solution of 3 in ethanol, at 60 °C. After the consumption of 3, the solvent was
distilled and the product was purified by flash chromatography.
15. (a) Durán Pachón, L.; Gamez, P.; van Brussel, J. J. M.; Reedijk, J. Tetrahedron Lett.
2003, 44(32), 6025; (b) Placzek, A. T.; Donelson, J. L.; Trivedi, R.; Gibbs, R. A.;
De, S. K. Tetrahedron Lett. 2005, 46, 9029; (c) Rajender Reddy, L.; Arjun Reddy,
M.; Bhanumathi, N.; Rama Rao, K. New J. Chem. 2001, 25, 221; (d) Kokubo, M.;
Naito, T.; Kobayashi, S. Tetrahedron 2010, 66(5), 1111.
16. 2-Hydroxy-3-(phenylamino)-1,4-naphthoquinone, 4a, dark purple solid
purified by flash chromatography (hexane/ethyl acetate 4:1). Yield: 95.1%
(water) 65.2% (ethanol) 1H NMR (500 MHz, CDCl3) dH: 8.0 (m, 2H), 7.6 (m, 2H),
7.3 (t, 2H), 7.08 (t, 1H), 7.0 (d, 3H); 13C NMR (125 MHz, CDCl3) dC: 182.3, 179,5.
138.9, 136.3, 134.0, 133.6, 133.0, 130.8, 130.3, 128.2, 126.6, 126.0, 124.7, 123.4,

4586
L. C. D. Rezende et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4583–4586
121.4, 122.7; HRMS-ESI: [M+H]+ calcd for C16H12NO3, 266.0817; found,
266.0812; IV (ATR)
(cmÀ1): 3357, 3290, 1647, 1612, 1570, 1512, 1500,
1445, 1380, 1332, 1264, 711, 694. Melting point:>215 °C
2.17 (s, 6H); 13C NMR (125 MHz, CDCl3) dC: 181.0, 178,0. 135.8, 134.9, 133.6,
132.9, 132.0, 129.6, 127.0, 126.5, 125.7, 125.3, 124.7, 17.1; HRMS-ESI: [M+H]+
calcd for C18H16NO3, 294,1125 found: 294,1124; IV (ATR)
(cmÀ1): 3739, 3422,
m
m
17. 2-[(2-Chlorophenyl)amino]-3-hydroxy-1,4-napthoquinone, 4b, dark purple
solid purified by flash chromatography (hexane/ethyl acetate 9:1). Yield:
60.5% (water) 40.3% (ethanol) 1H NMR (500 MHz, CDCl3) dH: 8.04–7.99 (m, 2H),
7.65–7.60 (m, 2H), 7.31–7.29 (dd, J = 7.5 Hz, 2.0 Hz, 1H), 7.15–7.12 (dt,
J = 7.5 Hz, 7.5 Hz, 2.0 Hz, 1H), 6.93–6.90 (dt, J = 7.5 Hz, 7.5 Hz, 2.0 Hz, 1H),
6.83–6.81 (dd, J = 7.5 Hz, 2.0 Hz, 1H); 13C NMR (125 MHz, CDCl3) dC: 181.9,
179,7. 137.1, 135.9, 134.0, 133.8, 130.9, 130.1, 129.0, 126.7, 126.4, 126.1, 125.2,
123.8, 123.4, 122.2; HRMS-ESI: [M+Na]+ calcd for C16H10ClNNaO3, 322.0247;
3376, 3289, 3065, 2956, 2922, 2847, 2644, 1974, 1779, 1710, 1649, 1619, 1591,
1572, 1478, 1382, 1317, 1296, 1266, 1223, 1075, 1024, 975, 916, 879, 789, 775,
719, 569. Melting point: 151 °C.
20. (a) Peters, W. Exp. Parasitol. 1965, 17, 80; (b) Carvalho, L. H.; Brandão, M. G. L.;
Santos-Filho, D.; Lopes, J. L. C.; Krettli, A. U. Braz. J. Med. Biol. Res. 1991, 24, 1113.
21. Adult Swiss albino mice weighing 20 2 g were infected intraperitoneally with
infected blood containing 1 Â 105 P. berghei NK65. The mice were randomly
allocated to groups of three to five animals per cage. The compounds were
diluted in a Tween 20 solution with distilled water (final concentration of 2%
found, 322.0241 ; IV (ATR) m
(cmÀ1): 3739, 3313, 2922, 2852, 2347, 1660, 1625,
1592, 1573, 1516, 1467, 1376, 1327, 1295, 1264, 1226, 1179, 1136, 1075, 1013,
979, 793, 750, 720, 601, 554; Melting point: 155 °C
Tween 20), 200 lL of the concentration of test solution was administrated
orally, using the gavage technique, to each animal in dose of 100 mg/kg/day.
The chloroquine was dissolved in distilled water and was administered orally
as an antimalarial control reference at a dose of 10 mg/kg/day. A negative
control group consisted of a 2% Tween 20 solution in distilled water that was
orally administered to each animal.
18. 2-Hydroxy-3-[(4-methoxyphenyl)amino]-1,4-napthoquinone, 4c, dark purple
solid purified by flash chromatography (hexane/ethyl acetate 4:1). Yield: 61,6%
(water) 17.3% (ethanol) 1H NMR(500 MHz, CDCl3) dH: 8.03–8.06 (t, J = 8.5 Hz,
2H), 7.60–7.57 (m, 2H), 7.02–6.98 (d, J = 8.3 Hz, 2H), 6.88–6.84 (d, J = 8.3 Hz,
2H), 3.81 (s, 3H); 13C NMR (125 MHz, CDCl3) dC: 182.5, 179.3. 156.4, 135.5,
134.1, 133.4, 132.0, 130.8, 130.6, 126.7, 126.0, 126.8, 123.8, 113.6, 55.6, HRMS-
22. Blood smears from all the mice were prepared, fixed with methanol, stained
with Giemsa and examined microscopically (1000Â magnification). The
parasitemia was determined by counting the number of parasitized
erythrocytes out of 1000–3000 in random fields of the microscope. Parasite
growth inhibition in the drug-treated groups was defined as the parasitemia in
the nontreated control group minus the parasitemia in the treated control
group, divided by the parasitemia in the nontreated control group, expressed
in percentages.
ESI: [M+Na]+ calcd for C17H13NNaO4, 318,0742; found, 318,0736; IV (ATR)
m
(cmÀ1): 3743, 3422, 3315, 2924, 2854, 1642, 1619, 1573, 1509, 1462, 1380,
1328, 1265, 1118, 1079, 1016, 981, 822, 795, 716, 589. Melting point: 141 °C.
19. 2-[(2,6-Dimethylphenyl)amino]-3-hydroxy-1,4-napthoquinone,
4d,
dark
purple solid purified by flash chromatography (hexane/ethyl acetate 9:1).
Yield: 62.2% (water) 30.5% (ethanol) 1H NMR (500 MHz, CDCl3) dH: 7.99–7.97
(d, 7.2 Hz, 1H), 7.92–7.91 (d, 7.2 Hz, 1H), 7.61–7.52 (m, 2H), 7.06–7.0 (m, 3H),
23. Baggish, A. L.; Hill, D. R. Antimicrob. Agents Chemother. 2002, 46, 1163.