Potent naphthoquinones against antimony-sensitive and -resistant Leishmania parasites: Synthesis of novel α- And nor-α-lapachone-based 1,2,3-triazoles by copper-catalyzed azide-alkyne cycloaddition

Article abstract of DOI:10.1016/j.ejmech.2013.02.038

Continuing our screening program for novel anti-parasite compounds, we synthesized seven 1,4-naphthoquinones coupled to 1,2,3-triazoles, five nor-β-lapachone-based 1,2,3-triazoles and ten α-lapachone-based 1,2,3-triazoles. These and other naphthoquinonoid

Full text of DOI:10.1016/j.ejmech.2013.02.038

European Journal of Medicinal Chemistry 63 (2013) 523e530
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journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Potent naphthoquinones against antimony-sensitive and -resistant
Leishmania parasites: Synthesis of novel - and nor- -lapachone-
a
a
based 1,2,3-triazoles by copper-catalyzed azideealkyne cycloaddition
Tiago T. Guimarães a, Maria do Carmo F.R. Pinto a, Juliane S. Lanza b, Maria N. Melo b,
Rubens L. do Monte-Neto b, Isadora M.M. de Melo c, Emilay B.T. Diogo c, Vitor F. Ferreira d,
,
Celso A. Camara e, Wagner O. Valença e, Ronaldo N. de Oliveira e, Frédéric Frézard b
**
,
,
Eufrânio N. da Silva Júnior c
*
a Núcleo de Pesquisas de Produtos Naturais, UFRJ, 21941-971 Rio de Janeiro, RJ, Brazil
b Instituto de Ciências Biológicas, Departamento de Fisiologia e Biofísica, UFMG, 31270-901 Belo Horizonte, MG, Brazil
c Laboratório de Química Sintética e Heterocíclica, Instituto de Ciências Exatas, Departamento de Química, UFMG, 31270-901 Belo Horizonte, MG, Brazil
d Instituto de Química, Departamento de Química Orgânica, UFF, 24020-150 Niterói, RJ, Brazil
e Departamento de Ciências Moleculares, UFRPE, 52171-900 Recife, PE, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Continuing our screening program for novel anti-parasite compounds, we synthesized seven 1,4-
naphthoquinones coupled to 1,2,3-triazoles, five nor-b-lapachone-based 1,2,3-triazoles and ten a-lapa-
chone-based 1,2,3-triazoles. These and other naphthoquinonoid compounds were evaluated for their
activity against promastigote forms of antimony-sensitive and -resistant strains of Leishmania infantum
(syn. Leishmania chagasi) and Leishmania amazonensis. The toxicity of these compounds to mammalian
cells was also examined. The substances were more potent than an antimonial drug, with IC50 values
Received 8 February 2013
Received in revised form
24 February 2013
Accepted 26 February 2013
Available online 7 March 2013
ranging from 1.0 to 50.7 mM. Nor-a-lapachone derivatives showed the highest antileishmanial activity,
Keywords:
Quinones
Lapachone
Leishmania
Chemotherapy
Click chemistry
with selectivity indices in the range of 10e15. These compounds emerged as important leads for further
investigation as antileishmanial agents. Additionally, one of these compounds exhibited cross-resistance
in Sb-resistant Leishmania and could provide a molecular tool for investigating the multidrug resistance
mechanisms in Leishmania parasites.
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
(America) [5]. Metacyclic promastigotes, the infective stage, are
phagocytized by macrophages and transformed into amastigotes.
Leishmaniasis, a neglected tropical disease [1], is a worldwide
vector borne disease that affects developing countries. This disease is
endemic in 88 countries, with an estimated 12 million cases
worldwide [2]. Approximately 21 Leishmania species are capable of
infecting vertebrate hosts. In humans, the syndrome is mostly
associated with seven species of Leishmania: Leishmania donovani,
Leishmania infantum, Leishmania major, Leishmania tropica, Leish-
mania aethiopica, Leishmania braziliensis and Leishmania mexicana
[3,4]. Infection is caused by the bite of infected female sandflies of
the genera Phelobotomus (Europe, Asia, Africa) and Lutzomyia
These multiply in an intracellular fashion, affecting different tissues.
Leishmaniasis can manifest itself in three different clinical forms,
depending on the Leishmania species: visceral, cutaneous and
mucosal, which arise from parasite replication in the mononuclear
phagocyte system, dermis and naso-oropharyngeal mucosa,
respectively [3,6]. Pentavalent antimonials, miltefosine, paromo-
mycin and amphotericin B are used to clinically treat leishmaniasis;
due to their toxic side effects and the emergence of drug resistance,
however, safer and more effective drugs are still needed [7,8].
Resistance to the first-line antimonial drugs has reached critical
levels in some parts of the world, including Bihar State (India) [8].
The emergence of resistance is attributed to inappropriate drug
exposure, resulting in a build-up of subtherapeutic blood levels of
antimony and increasing the tolerance of the parasites to this
treatment. Although antimonials are still the first-line drugs, they
* Corresponding author. Tel.: þ55 31 34095720; fax: þ55 31 34095700.
** Corresponding author. Tel.: þ55 31 34092940; fax: þ55 31 34092924.
E-mail addresses: frezard@icb.ufmg.br (F. Frézard), eufranio@ufmg.br,
eufranio.junior@yahoo.com.br (E.N. da Silva Júnior).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.02.038

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T.T. Guimarães et al. / European Journal of Medicinal Chemistry 63 (2013) 523e530
exhibit several limitations including severe side effects, the need
for daily parenteral administration and drug resistance [9].
Increased levels of intracellular thiols and/or overexpression of ABC
transporters are usually observed in metal-resistant Leishmania
[10]. Other mechanisms such as diminished biological reduction of
Sb(V) to Sb(III) [11], the loss of a single aquaglyceroporin (or its
down regulation) [12] and hypoxic conditions [13] have been re-
ported to cause increased resistance to pentavalent antimonials.
Thus, to develop new drugs, it is important to ensure that cross-
resistance with conventional drugs does not occur.
Efforts have been undertaken to find natural, bioactive com-
pounds that can be used in the treatment of parasitic diseases [14].
Some natural naphthoquinones have emerged as promising subjects
of medicinal chemistry research due to their structural properties.
These compounds can generate reactive oxygen species (ROS), which
lead to oxidative stress and subsequently to parasite death [15a].
Dubey and collaborators have described relevant quinonoid com-
pounds with high leishmanicidal activity. The anticancer drugs,
doxorubicin and mitomycin C, were reported as novel inhibitors of
trypanothione reductase (TryR) in Leishmania and these compounds
also showed significant effect on redox homeostasis of the parasite
[15b]. Goulart and coworkers [16] described the leishmanicidal ac-
tivity of lapachol and some derivatives, showing the potential utility
of these substances against this parasite.
The first group of 1,4-naphthoquinone coupled 1,2,3-triazoles
(3e9) was obtained from 2-bromo-1,4-naphthoquinone (1)
(Scheme 1). From compound 1, 2-azido-1,4-naphthoquinone (2) was
obtained by a simple reaction with sodium azide in dime-
thylformamide, as described previously [25]. Compound 2 was then
submitted to copper-catalyzed azideealkyne cycloaddition (CuAAC)
reaction conditions [26].
The second group of compounds was obtained from lapachol
(10) (Scheme 2) [27]. A four step synthesis afforded 3-azido-nor-
lapachone (11), and nor- -lapachone-based 1,2,3-triazoles 12e16
were prepared from this intermediate in high yields [27].
The third and fourth groups, the - and nor- -lapachone-based
1,2,3-triazoles, were synthesized for the first time as shown in
Schemes 3 and 4. Lapachol (10) was used to prepare -lapachone
(17) by simple cyclization under acidic conditions. Next, the treat-
ment of -lapachone (10) with N-bromosuccinimide in the pres-
ence of benzoyl peroxide in carbon tetrachloride afforded 4-bromo-
-lapachone (18) was performed. Subsequent reaction with sodium
azide gave the novel 4-azido- -lapachone (19), the key interme-
diate used to obtain the triazole derivatives 20e23. -Lapachone-
b-
b
a
a
a
a
a
a
a
based 1,2,3-triazoles 20e23 were then obtained by Cu(I) catalysis
following methodology described by Sharpless and coworkers [26]
(Scheme 3).
The treatment of nor-lapachol (24) with HCl/AcOH provided
In the last few years, our research group has described quino-
noid compounds with anticancer [17] and tuberculostatic activities
[18], as well as compounds that are active against Trypanosoma
cruzi, the etiologic agent of Chagas disease [19]. Guided by the
concept of molecular hybridization and seeking compounds active
against T. cruzi [20], we reported that triazole naphthoquinones are
efficient trypanocidal agents. Treatment with these compounds
causes impaired mitosis and increased ROS production leading to
parasite death by an autophagic mechanism [21].
nor-a-lapachone (25), which was transformed into 3-bromo-nor-a-
lapachone (26). Reaction of 26 with sodium azide in dichloro-
methane gave the corresponding azide compound 27, as described
previously [28]. Azidonaphthoquinone 27 was used to synthesize
the novel triazole derivatives 28e32 in moderate yields (Scheme 4).
3. Results and discussion
We recently reported the synthesis of naphthoquinone coupled
In the present study, triazole naphthoquinones were synthe-
sized, and their leishmanicidal activities were evaluated against
both Sb(III)-sensitive and -resistant L. infantum (syn. Leishmania
chagasi) and Leishmania amazonensis promastigotes. Their toxicity
to murine peritoneal macrophages was also examined.
1,2,3-triazoles 3e9 and nor-b-lapachone-based 1,2,3-triazoles 12e16
and their activity against bloodstream trypomastigotes, the infective
forms of T. cruzi [27]. Their trypanocidal activity, with IC50/24 h
values in the range of 10.9e359.0 mM [27], led us to evaluate them
further against Leishmania: L. infantum (syn. L. chagasi), the etiolog-
ical agent for visceral leishmaniasis [29]. We also studied these
compounds for activity against L. amazonensis, which is related to the
cutaneous form of the disease in the New World [30]. These two
species lead to different clinical manifestations, suggesting different
metabolism and consequently distinct drug sensitivity. This obser-
vation highlights the importance of using different Leishmania spe-
cies in drug screening [31], as well as strains with different
susceptibilities to antimonials.
2. Chemistry
2-Bromo-1,4-naphthoquinone (1) was acquired from Sigmae
Aldrich (St. Louis, MO, USA). Lapachol (10) (2-hydroxy-3-(30-
methyl-20-butenyl)-1,4-naphthoquinone) was extracted from
the heartwood of Tabebuia sp. (Tecoma) and purified by a series of
recrystallizations [22]. From this quinone, nor-lapachol (24)
(2-hydroxy-3-(20-methyl-propenyl)-1,4-naphthoquinone) was ob-
tained as a crystalline orange solid by Hooker oxidation [23,24].
The first series of compounds,1,4-naphthoquinone 1,2,3-triazole
derivatives, was considered to be highly active against all
Scheme 1. Naphthoquinoidal compounds linked 1,2,3-triazoles 3e9.

T.T. Guimarães et al. / European Journal of Medicinal Chemistry 63 (2013) 523e530
525
β
Scheme 2. Nor-b
-lapachone-based 1,2,3-triazoles 12e16. aRef. [27].
Leishmania strains (with the exception of compound 6,
IC50 > 100 M), with IC50 values in the range of 5.8e57.8 M in the
four cell lines studied (Table 1). However, these compounds were
toxic to peritoneal macrophages with CC50 values in a similar range,
The novel
a
- and nor-
a
-lapachone-based 1,2,3-triazoles, com-
m
m
pounds 20e23 and 28e33, respectively, were synthesized and
assessed against all of the Leishmania strains reported here. We
observed that most of the nor-
a-lapachone-based 1,2,3-triazoles
from 5.4 to 50
mM (Table 1), leading to SI (selectivity index) values
were more active than -lapachone coupled 1,2,3-triazoles (with
a
between 0.37 and 1.72 (Table 2). This index is obtained from the
ratio between the CC50 for peritoneal macrophages and the IC50 in
the parasites. Compounds 3e9 were approximately 2e10 times
more active than the standard drug against the wild-type strains of
both L. infantum (syn. L. chagasi) (2e10ꢀ) and L. amazonensis (2.2e
6.6ꢀ).
the exception of compounds 23 and 31), indicating that modifica-
tion of the C-ring (pyran versus furan) is important for the leish-
manicidal activity. On the other hand, nor-
except for compound 29, were less cytotoxic towards macrophages
than were -lapachone compounds.
Most of the naphthoquinoidal compounds were derived from
nor- -lapachone, and compounds 28, 30, 32 and 33 in particular,
a-lapachone derivatives,
a
The second group of compounds, nor-
b
-lapachone-based 1,2,3-
a
triazoles 12e16, contained different electron donating and with-
drawing groups linked to the phenyl group of the triazole ring. All
compounds in this group showed similarly potent activity against
L. infantum (syn. L. chagasi) and L. amazonensis. Activity was
observed in both antimony-sensitive and resistant strains with IC50
exhibit high selectivity index and can be considered as the most
promising drug candidates for use against both antimony-sensitive
and -resistant leishmaniasis.
The need for a safe and highly effective drug against leishmani-
asis is extremely relevant, especially in the cases of drug-resistant
strains. One chemotherapeutic strategy entails the use of drug
combinations to treat visceral leishmaniasis [32]. However, the
long-term efficacy of this approach has been questioned in a recent
study which shows the in vitro development of resistance to drug
associations in L. donovani [33]. Improvements to the chemothera-
peutic arsenal with new antileishmanial agents, starting from drug
screening as described in this study are thus urgently required.
values between 1.00 and 5.57
peritoneal macrophages approximately 4
were also more effective than the naphthoquinoidal precursor
lapachol (10) (IC50 values from 34.72 to 102.05 M) but were less
active than the ortho-quinones, -lapachone and nor- -lapachone
mM and CC50 values for murine
mM. Compounds 12e16
m
b
b
(Table 1). Compounds 12e16 were approximately 20e90 times
more active than potassium antimonyl tartrate against the wild-
type strains of both L. infantum (syn. L. chagasi) (15.5e86ꢀ) and
L. amazonensis (23e98ꢀ).
In comparison with the precursor quinones, lapachol (10),
a-
lapachone (17), nor-lapachol (24) and nor- -lapachone (25) the
a
Scheme 3.
a-Lapachone-based 1,2,3-triazoles 20e23.

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T.T. Guimarães et al. / European Journal of Medicinal Chemistry 63 (2013) 523e530
Scheme 4. Nor-a-lapachone-based 1,2,3-triazoles 28e33.
strategy to insert the triazole ring was efficient since the novel
compounds were, in general, more actives.
compound 32 is highly active against Leishmania and compound 31
presented cross-resistance in Sb-resistant parasites, this modifi-
cation is most likely critical to recognize the drug molecule by the
resistance mechanisms in the Leishmania parasite. These findings
are relevant considering the importance of elucidating drug
resistance mechanisms in Leishmania and the need to identify
agents capable of reversing Leishmania drug resistance. Compound
Of the twenty-seven substances tested herein, only compound
31 showed evidence of cross-resistance with respect to the triva-
lent antimonial potassium antimonyl tartrate. Interestingly, the
only difference between compound 31 and 32 is the presence of an
additional methyl substituent group in the triazole ring. As
Table 1
Cytotoxic effects of naphthoquinones on wild-type (WT) and antimony-resistant (2700R) promastigotes of Leishmania and murine peritoneal macrophages (MPM).
Compounds
L. infantum (syn. L. chagasi) WT L. infantum (syn. L. chagasi) 2700R L. amazonensis WT L. amazonensis 2700R MPM
M)a M)b
IC50 CC50 (m
(
m
3
4
5
6
7
8
9
21.58
15.80
17.25
>100
8.80
39.05
42.13
34.72
w1.00
4.32
5.57
3.25
3.38
13.88
2.26
2.04
5.90
17.73
>100
1.54
1.00
1.12
1.05
1.45
17.16
18.61
14.08
>100
13.55
5.80
8.47
99.88
1.04
4.90
5.39
3.28
3.10
5.85
2.24
2.20
3.43
3.30
>100
1.28
1.01
1.12
1.14
25.36
1.32
1.78
1.60
1.13
>3000
16.68
34.28
57.81
7.94
10.17
16.82
21.42
50.00
15.18
9.51
5.39
59.30
3.42
3.52
4.36
3.35
3.31
3.80
8.59
6.89
14.79
9.63
14.58
0.77
8.01
24.54
50.15
>100
13.79
21.32
40.25
63.28
1.03
4.39
2.49
2.95
3.49
>100
21.25
23.87
50.65
102.05
1.11
4.70
3.87
3.41
3.80
16.08
9.23
6.00
4.37
35.19
Lapachol (10)
12
13
14
15
16
a
20
21
22
-Lapachone (17)
15.15
1.9
1.95
8.04
23
21.55
>100
1.54
1.2
4.23
1.14
27.04
7.31
1.39
1.59
Nor-lapachol (24)
Nor-
28
29
30
31
32
33
Nor-
>100
a
-lapachone (25)
2.72
2.14
11.00
2.19
18.00
14.24
3.50
2.48
2.90
109.7
2.77
15.96
17.92
19.81
18.72
1.55
1.55
1.83
1.14
0.67
b
-lapachone
b
-Lapachone
1.39
>3000
0.79
62.89
Potassium antimonyl tartrate e Sb(III) 86.1
a
IC50: drug concentration that inhibits Leishmania cell growth by 50%.
CC50: drug concentration that decreases the macrophage cell viability by 50%.
b

T.T. Guimarães et al. / European Journal of Medicinal Chemistry 63 (2013) 523e530
527
Table 2
Selectivity index (SI) of the triazole derivatives and the naphthoquinoidal compounds precursor.
Compounds
L. infantum (syn. L. chagasi) WT
L. infantum (syn. L. chagasi) 2700R
L. amazonensis WT
L. amazonensis 2700R
3
4
5
6
7
8
9
0.47
1.06
1.24
<0.50
1.72
0.24
0.13
1.71
w3.42
0.81
0.78
1.03
0.98
0.27
3.80
3.38
2.50
0.54
<0.15
0.50
8.01
2.47
15.02
12.36
12.78
10.23
1.36
1.14
0.73
0.59
0.90
1.52
<0.50
1.12
1.64
0.64
0.59
3.29
0.72
0.81
1.02
1.07
0.65
3.83
3.13
4.31
2.92
<0.15
0.60
7.93
2.47
14.0
0.70
15.01
10.51
0.97
0.70
<0.02
0.61
0.49
0.37
<0.50
0.71
0.40
0.11
0.58
3.08
0.75
1.13
0.98
0.87
0.24
0.93
1.15
3.38
0.27
<0.15
0.28
3.74
0.25
7.29
0.99
1.39
5.35
0.62
0.27
0.57
1.28
0.68
0.43
<0.50
1.10
0.45
0.13
0.94
3.32
0.80
1.75
1.35
0.95
0.25
4.52
3.53
1.83
0.45
<0.15
0.50
6.67
0.65
14.0
0.66
2.71
13.47
0.97
0.57
<0.02
Lapachol (10)
12
13
14
15
16
a
20
21
22
-Lapachone (17)
23
Nor-lapachol (24)
Nor-
28
29
30
31
32
33
Nor-
a
-lapachone (25)
b
-lapachone
b
-Lapachone
Potassium antimonyl tartrate e Sb(III)
SI ¼ CC50/IC50; values below 1 mean that the compound is more toxic to mammalian cells than to the parasite.
31 should be investigated further as a molecular tool for the study
and reversal of multidrug resistance mechanisms in Leishmania
parasites.
5. Experimental
5.1. Chemistry
The exposition of sensitive Leishmania to Sb(III) was previously
shown to induce the efflux of thiols and the inhibition of TryR [34a],
promoting redox homeostasis imbalance that leads to parasite
death. Interestingly, naphthoquinones were also proposed to
inhibit TryR and additionally to act as subversive substrates by
converting the enzyme from an anti-oxidant to a pro-oxidant,
resulting in an increase in ROS levels and consequently in cell
death [15b,34b]. Thus, literature reveals some similarities in the
mechanisms of action proposed for Sb(III) and naphthoquinones
against Leishmania parasites. The cross-resistance observed for
compound 31 may be attributed to a similar molecular target
shared with Sb(III), however this feature cannot be generalized
to the other naphthoquinones derivatives. Indeed, the subtle
chemical difference between compounds 31 and 32 leading to
completely different phenotypes was previously observed for other
naphthoquinone-derived compounds in their mode of action and
supports a multi-target mechanism model [34c,d].
Melting points were obtained on a Thomas Hoover melting
point apparatus and are uncorrected. Analytical grade solvents
were used. Column chromatography was performed on silica-gel
(Acros Organics, 0.035e0.070 mm, pore diameter ca. 6 nm).
Infrared spectra were recorded on a Shimadzu IR Prestige-21 FTIR
Spectrometer, 1H and 13C NMR were recorded at room tempera-
ture using
the solvents indicated, with TMS as internal reference. Chemical
shifts ( ) are given in ppm and coupling constants (J) in Hertz.
High resolution mass spectra (electrospray ionization) were ob-
tained using Shimadzu LCMS-IT-TOF. Chemical names for
a VNMRSYS-500, Varian MR-400 instrument, in
d
a
all compounds were generated using CS ChemDraw Ultra
version 10.0.
5.2. General proceduresfor preparationof 4-bromo-a-lapachone (19)
A solution of 4-bromo- -lapachone (18) (320 mg, 1 mmol) in
a
4. Conclusions
dichloromethane (25 mL), was treated with excess sodium azide
(78 mg, 1.2 mmol) at room temperature. The reaction mixture was
stirred for 24 h and filtered. The crude material was purified by
column chromatography over silica-gel, eluting with a gradient
mixture of hexane:ethyl acetate (9:1 to 7:3, v/v) with increasing
polarity. Compound 19 was obtained as an orange solid (141 mg,
0.50 mmol, 50% yield, m.p. 120e125 ꢁC). IR (KBr) 1650 (C]O); 1683
In this study, we described a series of substances with potent
antileishmanialactivity. Weevaluatedtwenty-sevencompounds, and
all naphthoquinones were found to be more active against promas-
tigote forms of antimony-sensitive and resistant strains of Leishmania
than the trivalent antimonial drug potassium antimonyl tartrate.
Compounds 30 and 33, with the largest selectivity indexes in the
range of 10e15, are important drug candidates for further investiga-
tion. The behavior of these compounds was very similar across
different species of Leishmania. Similar effects were also observed in
both the Sb-resistant mutants and their respective parental sensitive
WT strains. The results described here represent an important
contribution to the discovery of novel leishmanicidal drugs.
(C]O); 2107 (N3) cmꢂ1 1H NMR (300 MHz, CDCl3)
; d: 4.88 (dd,
J ¼ 3.1, 5.6 Hz, 1H), 8.17e8.10 (2H, m), 7.76e7.69 (2H, m), 2.15 (dd,
J ¼ 2.9, 14.6 Hz, 1H), 2.02 (dd, J ¼ 5.6, 14.8 Hz, 1H), 1.55 (3H, s),
1.5 (3H, s); 13C NMR (75 MHz, CDCl3)
d: 183.7 (C]O), 179.6 (C]O),
154.7, 133.9, 132.7, 131.6, 130.8, 126.1, 125.9, 116.8, 78.4, 37.6,
25.2, 15.4, 49.5. Compound 27 was prepared as previously
described [28].

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T.T. Guimarães et al. / European Journal of Medicinal Chemistry 63 (2013) 523e530
5.3. General procedures for preparation of
based 1,2,3-triazoles
a
- and nor-
a
-lapachone-
and sodium ascorbate (27 mg, 0.11 mmol) in CH2Cl2:H2O (12 mL,1:1,
v/v). Compound 23 was obtained as a yellow solid (304 mg,
0.8 mmol, 80% yield, mp 205e208 ꢁC); IR (KBr) 1611 (C]O), 1650
The azidoquinones were reacted with CuSO4$5H2O (9.3 mg,
0.04 mmol) and sodium ascorbate (22 mg, 0.11 mmol) and the
desired alkyne in a mixture of CH2Cl2:H2O (12 mL, 1:1, v/v). The
mixture was stirred at room temperature until product formation
was complete as determined by thin layer chromatography. The
aqueous phase was extracted with dichloromethane, dried over
anhydrous Na2SO4 and concentrated under reduced pressure. The
residue was purified by column chromatography on silica gel
eluting with a gradient mixture of hexane:ethyl acetate with
increasing polarity to 100% ethyl acetate.
(C]O) cmꢂ1; 1H NMR (500 MHz, CDCl3)
d: 8.17e8.14 (m, 1H), 8.04e
8.00 (m, 1H), 7.77e7.70 (m, 2H), 7.45 (d, J ¼ 6.87 Hz, 1H), 5.80 (dt,
J ¼ 6.25, 3.33 Hz, 1H), 2.84 (dt, J ¼ 14.41, 6.21 Hz, 1H), 2.41e2.33 (m,
2H), 1.95e1.82 (m, 2H), 1.57 (d, J ¼ 6.89 Hz, 3H), 1.54 (d, J ¼ 1.81 Hz,
3H), 1.29 (d, J ¼ 1.81 Hz, 3H), 0.83 (q, J ¼ 7.45 Hz, 3H); 13C NMR
(125 MHz, CDCl3) d: 182.7 (C]O), 179.4 (C]O), 156.1, 154.1, 134.5,
133.5,131.7,131.0,126.6,126.4,120.0,115.0, 79.2, 71.3, 49.4, 39.1, 35.9,
28.0, 26.7, 26.3, 8.2; EI/HRMS (m/z) [M þ H]þ 382.1770. Calcd for
[C21H23N3O4H]þ: 382.4329.
5.3.5. 2,2-Dimethyl-3-(4-phenyl-1H-1,2,3-triazol-1-yl)-2,3-dihydr
5.3.1. 4-(4-Cyclohexyl-1H-1,2,3-triazol-1-yl)-2,2-dimethyl-3,4-dihyro-
onaphtho[2,3-b]furan-4,9-dione 28
2H-benzo[g]chromene-5,10-dione 20
Thesubstitutedalkyne (1 mmol)wasreacted with3-azido-nor-a-
The substituted alkyne (1 mmol) was reacted with 4-azido-
a
-
lapachone (27), (269mg,1 mmol), CuSO4$5H2O (11.3 mg, 0.04 mmol)
and sodium ascorbate (27 mg, 0.11 mmol) in CH2Cl2:H2O (12 mL,1:1,
v/v). Compound28wasobtainedasayellowsolid(204mg, 0.5mmol,
lapachone(19), (283mg,1 mmol), CuSO4$5H2O(11.3 mg, 0.04 mmol)
and sodium ascorbate (27 mg, 0.11 mmol) in CH2Cl2:H2O (12 mL,1:1,
v/v). Compound 20 was obtained as a yellow solid (195 mg,
0.5 mmol, 50% yield, mp 130e135 ꢁC); IR (KBr) 1682 (C]O), 1651
55% yield, mp 227e230 ꢁC); IR (KBr) 1649 (C]O),1628 (C]O) cmꢂ1
;
1H NMR (400 MHz, CDCl3)
d: 8.22e8.17 (m, 1H), 8.11e8.06 (m, 1H),
(C]O) cmꢂ1; 1H NMR (400 MHz, CDCl3)
d: 8.16e8.12 (m, 1H), 8.04e
7.83e7.74 (m, 4H), 7.72e7.69 (m, 1H), 7.43e7.37 (m, 2H), 7.36e7.30
7.99 (m, 1H), 7.77e7.68 (m, 2H), 7.42 (s, 1H), 5.78 (t, J ¼ 6.36 Hz, 1H),
2.80 (dd, J ¼ 14.60, 6.41 Hz, 1H), 2.40e2.31 (m, 3H), 2.21e2.14 (m,
2H), 1.78e1.70 (m, 3H), 1.68e1.60 (m, 4H), 1.53 (s, 3H), 1.33 (s, 3H);
(m, 1H), 6.08 (s, 1H), 1.76 (s, 3H), 1.24 (s, 3H); 13C NMR (125 MHz,
CDCl3)
d: 180.7 (C]O), 177.9 (C]O), 161.3, 147.8, 134.9, 133.5,
132.7, 131.6, 130.0, 128.8, 128.4, 126.8, 126.5, 125.7, 119.1, 118.4, 94.6,
67.5, 27.5, 21.0; EI/HRMS (m/z) [M þ H]þ 372.1213. Calcd for
[C22H17N3O3H]þ: 372.13483.
13C NMR (100 MHz, CDCl3)
d: 182.7 (C]O),179.4 (C]O),156.1,149.0,
134.5, 133.4, 131.7, 131.0, 127.1, 126.6, 126.4, 125.1, 118.3, 115.2, 79.3,
49.5, 39.1, 26.6, 26.3, 25.2, 22.4, 22.1.
5.3.6. 3-(4-Hexyl-1H-1,2,3-triazol-1-yl)-2,2-dimethyl-2,3-dihydr
5.3.2. 2,2-Dimethyl-4-(4-pentyl-1H-1,2,3-triazol-1-yl)-3,4-dihydro-
onaphtho[2,3-b]furan-4,9-dione 29
2H-benzo[g]chromene-5,10-dione 21
Thesubstitutedalkyne (1 mmol)wasreacted with3-azido-nor-a-
The substituted alkyne (1 mmol) was reacted with 4-azido-
a
-
lapachone (27), (269mg,1 mmol), CuSO4$5H2O (11.3 mg, 0.04 mmol)
and sodium ascorbate (27 mg, 0.11 mmol) in CH2Cl2:H2O (12 mL,1:1,
v/v). Compound 29was obtained as ayellowsolid (155mg, 0.4mmol,
lapachone (19), (283 mg,1 mmol), CuSO4$5H2O (11.3 mg, 0.04 mmol)
and sodium ascorbate (27 mg, 0.11 mmol) in CH2Cl2:H2O (12 mL,1:1,
v/v). Compound 21 was obtained as ayellow solid (227 mg, 0.6 mmol,
41% yield, mp 173e175 ꢁC); IR (KBr) 1659 (C]O),1637 (C]O) cmꢂ1
;
60% yield, mp 176e178 ꢁC); IR (KBr) 1653 (C]O), 1611 (C]O) cmꢂ1
;
1H NMR (400 MHz, CDCl3)
d
: 8.18 (dd, J ¼ 7.53, 1.39 Hz,1H), 8.08 (dd,
1H NMR (500 MHz, CDCl3)
d: 8.16e8.12 (m, 1H), 8.04e8.01 (m, 1H),
J ¼ 6.99,1.91 Hz,1H), 7.81e7.73 (m, 2H), 7.20 (s,1H), 6.00 (s,1H), 2.68
(dt, J¼ 7.39, 2.83Hz, 2H),1.72 (s, 3H),1.67e1.59(m, 2H),1.34e1.24 (m,
6H), 1.16 (s, 3H), 0.85 (t, J ¼ 6.53 Hz, 3H); 13C NMR (100 MHz, DMSO-
7.76e7.69 (m, 2H), 7.29 (s, 1H), 5.78e5.75 (m, 1H), 2.85e2.80 (dd,
J ¼ 14.67, 6.35 Hz, 1H), 2.70e2.66 (m, 2H), 2.37e2.32 (dd, J ¼ 14.67,
6.35 Hz,1H),1.67e1.60 (m, 4H),1.59 (s, 3H),1.33e1.29 (m, 2H),1.30 (s,
d6) d: 181.1 (C]O),178.1 (C]O),161.8,147.6,135.0,134.0,133.0,132.3,
3H) 0.87 (t, J ¼ 7.00 Hz, 3H); 13C NMR (125 MHz, CDCl3)
d
: 182.7 (C]
126.4, 126.0, 122.4, 118.7, 94.3, 66.6, 31.3, 29.1, 28.5, 27.0, 25.4, 22.4,
O), 179.4 (C]O), 156.1, 148.0, 134.5, 133.4, 131.7, 131.0, 126.6, 126.4,
120.6, 115.3, 79.3, 49.4, 39.1, 31.4, 29.0, 26.7, 26.5, 25.6, 22.3, 14.0; EI/
HRMS (m/z) [M þ H]þ 380.1891. Calcd for [C22H25N3O3H]þ: 380.1974.
20.7, 14.3; EI/HRMS (m/z) [M
þ
H]þ 380.2038. Calcd for
[C22H25N3O3H]þ: 380.46014.
5.3.7. 2,2-Dimethyl-3-(4-pentyl-1H-1,2,3-triazol-1-yl)-2,3-dihydr
onaphtho[2,3-b]furan-4,9-dione 30
5.3.3. 2,2-Dimethyl-4-(4-propyl-1H-1,2,3-triazol-1-yl)-3,4-dihydro-
2H-benzo[g]chromene-5,10-dione 22
The substituted alkyne (1 mmol) was reacted with 3-azido-nor-a-
The substituted alkyne (1 mmol) was reacted with 4-azido-
a
-
lapachone (27), (269 mg,1 mmol), CuSO4$5H2O (11.3 mg, 0.04 mmol)
and sodium ascorbate (27 mg, 0.11 mmol) in CH2Cl2:H2O (12 mL, 1:1,
v/v). Compound 30 was obtained as a yellow solid (109 mg, 0.3 mmol,
lapachone(19), (283mg,1 mmol), CuSO4$5H2O(11.3 mg, 0.04 mmol)
and sodium ascorbate (27 mg, 0.11 mmol) in CH2Cl2:H2O (12 mL,1:1,
v/v). Compound 22 was obtained as a yellow solid (175 mg,
0.5 mmol, 50% yield, mp 214.2e215.2 ꢁC); IR (KBr) 1609 (C]O),1651
30% yield, mp 105e110 ꢁC); IR (KBr) 1649 (C]O), 1632 (C]O) cmꢂ1
;
1H NMR (400 MHz, CDCl3)
d
: 8.17 (dd, J ¼ 7.28, 1.53 Hz, 1H), 8.08 (dd,
(C]O) cmꢂ1; 1H NMR (500 MHz, CDCl3)
d: 8.15e8.12 (m, 1H), 8.02e
J ¼ 7.03,1.89 Hz, 1H), 7.83e7.72 (m, 2H), 7.20 (s,1H), 5.99 (s, 1H), 2.68
(dt, J ¼ 7.42, 3.06 Hz, 2H),1.72 (s, 3H),1.67e1.59 (m, 3H),1.34e1.27 (m,
5H),1.16(s,3H),0.87(t,J¼ 6.90Hz,3H);13CNMR(100MHz, DMSO-d6)
7.99 (m, 1H), 7.75e7.69 (m, 2H), 7.32 (s, 1H), 5.77 (t, J ¼ 6.37 Hz, 1H),
2.83e2.76 (m,1H), 2.66 (dt, J ¼ 7.24,1.44 Hz, 2H), 2.35 (dd, J ¼ 14.59,
6.32 Hz, 1H), 1.71e1.62 (m, 2H), 1.53 (s, 3H), 1.31 (s, 3H), 0.93 (t,
d
: 181.1 (C]O), 178.1 (C]O), 161.8, 147.6, 135.0, 134.0, 133.0, 132.3,
J ¼ 7.37 Hz, 3H); 13C NMR (125 MHz, CDCl3)
d
: 182.7 (C]O), 179.4
126.4, 126.0, 122.4, 118.7, 94.4, 66.5, 31.2, 28.9, 27.0, 25.4, 22.2, 20.7,
14.3; EI/HRMS (m/z) [M þ H]þ 366.1880. Calcd for [C21H23N3O3H]þ:
366.1817.
(C]O),156.1,147.7,134.5,133.5,131.7,131.0,126.6,126.4,120.8,115.2,
79.3, 49.4, 39.1, 27.5, 26.7, 26.5, 22.5, 13.7; EI/HRMS (m/z) [M þ H]þ
352.1636. Calcd for [C20H21N3O3]þ: 352.1661.
5.3.8. 3-(4-(2-Hydroxybutan-2-yl)-1H-1,2,3-triazol-1-yl)-2,2-
5.3.4. 4-(4-(2-Hydroxybutan-2-yl)-1H-1,2,3-triazol-1-yl)-2,2-
dimethyl-2,3-dihydronaphtho[2,3-b]furan-4,9-dione 31
dimethyl-3,4-dihydro-2H-benzo[g]chromene-5,10-dione 23
The substituted alkyne (1 mmol) was reacted with 3-azido-nor-a-
The substituted alkyne (1 mmol) was reacted with 4-azido-
a
-
lapachone (27), (269 mg,1 mmol), CuSO4$5H2O (11.3 mg, 0.04 mmol)
lapachone(19), (283mg,1 mmol), CuSO4$5H2O(11.3 mg, 0.04 mmol)
and sodium ascorbate (27 mg, 0.11 mmol) in CH2Cl2:H2O (12 mL,1:1,

T.T. Guimarães et al. / European Journal of Medicinal Chemistry 63 (2013) 523e530
529
v/v). Compound 31 was obtained as a yellow solid (201 mg, 0.5 mmol,
55% yield, mp 154e156 ꢁC); IR (KBr) 3312 (OH), 1657 (C]O), 1634
(syn. L. chagasi) promastigotes (1 ꢀ106 parasites/mL) were seeded in
24-well cell culture plates with 1.5 mL of a-MEM. The cells were
(C]O) cmꢂ1; 1H NMR (400 MHz, CDCl3)
d
: 8.18 (dd, J ¼ 7.29, 1.39 Hz,
incubated under shaking at 25 ꢁC for 72 h in the presence of seven
different concentrations of the compound to be tested. Non-treated
parasites were established for growth comparison. Stock solutions
1H), 8.07 (dd, J ¼ 7.44, 1.22 Hz, 1H), 7.81e7.73 (m, 2H), 7.37 (d,
J ¼ 1.31 Hz, 1H), 6.01 (d, J ¼ 11.57 Hz, 1H), 1.95e1.80 (m, 2H), 1.72 (s,
3H),1.56 (d, J ¼ 10.10 Hz, 3H),1.15 (s, 3H), 0.80 (q, J ¼ 7.37 Hz, 3H); 13
C
of the drugs were prepared in DMSO and diluted in a-MEM cell
NMR (100 MHz, DMSO-d6)
d
: 181.1 (C]O),178.0 (C]O), 161.8,155.2,
culture medium to obtain the range of tested concentrations. The
final DMSO concentration did not exceed 0.2%, which is known to be
nontoxic to Leishmania parasites [38,39]. For drug susceptibility
assays, Leishmania growth curves were constructed by measuring
135.0, 133.9, 133.0, 132.3, 126.4, 126.0, 122.2, 118.8, 94.4, 70.1, 66.6,
35.9, 28.6, 27.1, 20.7, 8.7; EI/HRMS (m/z) [M þ H]þ 368.1667. Calcd for
[C20H21N3O4H]þ: 368.1610.
absorbance at 600 nm. Antileishmanial activity is expressed as IC50
/
5.3.9. 3-(4-(2-Hydroxypropan-2-yl)-1H-1,2,3-triazol-1-yl)-2,2-
dimethyl-2,3-dihydronaphtho[2,3-b]furan-4,9-dione 32
72 h, which is the compound concentration that reduces cell growth
by 50% compared to untreated control (relative growth). All ex-
periments were performed in triplicate.
The substituted alkyne (1 mmol) was reacted with 3-azido-nor-a-
lapachone (27), (269 mg,1 mmol), CuSO4$5H2O (11.3 mg, 0.04 mmol)
and sodium ascorbate (27 mg, 0.11 mmol) in CH2Cl2:H2O (12 mL, 1:1,
v/v). Compound 32 was obtained as a yellow solid (141 mg, 0.4 mmol,
40% yield, mp 202e205 ꢁC); IR (KBr) 3428 (OH), 1693 (C]O), 1645
6.3. Cytotoxicity assay against peritoneal macrophages
The cytotoxicity of the compounds towards murine peritoneal
macrophages was evaluated using the classical 3-(4,5-dimethyl
thiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) method
[40]. Briefly, macrophages were obtained by lavage of the perito-
neal cavities of Swiss mice with 10 mL cold RPMI-1640 without FBS.
After washing, the cell suspension (4.0 ꢀ 106/mL) was seeded
(0.1 mL) in 96-well flat bottom plates. Macrophages were allowed
to adhere for 1 h and non-adherent cells were removed by washing
with RPMI. The compounds to be studied were then added to the
(C]O) cmꢂ1; 1H NMR (400 MHz, CDCl3)
d
: 8.18 (dd, J ¼ 7.05,1.65,1H),
8.07 (dd, J ¼ 7.05,1.56,1H), 7.82e7.73 (m, 2H), 7.39 (s,1H), 6.00 (s,1H),
1.73 (s, 3H), 1.61 (s, 3H), 1.60 (s, 3H), 1.15 (s, 3H); 13C NMR (100 MHz,
DMSO-d6)d:181.2 (C]O),178.1 (C]O),161.9,156.4,135.0,133.9,133.0,
132.4,126.4,126.0,121.3,118.6, 94.5, 67.4, 66.6, 31.1, 31.0, 27.0, 20.7; EI/
HRMS (m/z) [M]þ 354.1501. Calcd for [C19H19N3O4]þ: 354.1454.
5.3.10. 3-(4-Cyclohexyl-1H-1,2,3-triazol-1-yl)-2,2-dimethyl-2,3-
dihydronaphtho[2,3-b]furan-4,9-dione 33
wells at concentrations ranging from 1 to 20
further cultured in RPMI supplemented with 10% FBS for 24 h at
38 ꢁC in a humidified 5% CO2 atmosphere. At that point, 10
L of
MTT solution (5 mg/mL) was added to each well, and the plates
were incubated for an additional 4 h. Supernatants were aspirated,
mM and the cells were
The substituted alkyne (1 mmol) was reacted with 3-azido-nor-a-
lapachone (27), (269 mg,1 mmol), CuSO4$5H2O (11.3 mg, 0.04 mmol)
and sodium ascorbate (27 mg, 0.11 mmol) in CH2Cl2:H2O (12 mL,1:1,
v/v). Compound 33 was obtained as a yellow solid (150 mg, 0.4 mmol,
m
40% yield, mp 208e210 ꢁC); IR (KBr) 1657 (C]O),1630 (C]O) cmꢂ1
;
and the formazan crystals formed were dissolved in 100 mL of
1H NMR (500 MHz, CDCl3)
d
: 8.18 (dd, J ¼ 7.45, 1.39 Hz,1H), 8.08 (dd,
DMSO. After 15 min of incubation at room temperature, the
absorbance of solubilized MTT formazan product was measured
spectrophotometrically at 570 nm. Cell viability was calculated
from the ratio of the absorbance of the well treated with the drug to
that of the non-treated well. The concentration of drug that
decreased cell viability by 50% (CC50) was determined.
J ¼ 7.44,1.35 Hz,1H), 7.77 (m, 2H), 7.29 (s,1H), 6.00 (s,1H), 2.36e2.31
(m, 2H), 2.20e2.15 (m, 2H), 1.72 (s, 3H), 1.67e1.62 (m, 2H), 1.62e1.59
(m, 5H),1.19 (s, 3H); 13C NMR (100 MHz, CDCl3)
d: 180.6 (C]O),178.0
(C]O),161.1,149.5,134.9,133.5,132.7,131.6,126.8,126.7,126.5,125.7,
118.6, 117.6, 94.6, 67.3, 27.4, 26.2, 25.2, 22.3, 22.1, 20.8.
6. Antileishmanial activity
6.4. Statistical analysis
6.1. Parasite
IC50 and CC50 values were calculated by nonlinear regression
using the software GraphPad Prism 5.0. Significance was set to
p < 0.05.
Leishmania (Leishmania) amazonensis (strains MHOM/BR/1989/
BA199, sensitive and resistant to Sb(III) at concentration up to
2700
(strains MCAN/BR/2002/BH400, sensitive and resistant to Sb(III) at
concentration up to 2700 M) promastigotes were maintained in
minimum essential culture medium ( -MEM) (Gibco, Invitrogen
m
M) and Leishmania (Leishmania) infantum (syn. L. chagasi)
6.5. Selectivity index
m
The selectivity index (SI) of the compound was calculated as the
ratio between the 50% cytotoxic concentration for murine perito-
neal macrophages (CC50) and the 50% inhibitory growth concen-
tration in Leishmania promastigote (IC50); SI ¼ CC50/IC50. An SI value
less than 1 means that a compound is more toxic to mammalian
cells than it is effective against the parasite.
a
NY, USA) supplemented with 10% (v/v) heat inactivated fetal calf
serum (FBS, Cultilab, Campinas, SP, Brazil), 100 mg/mL kanamycin,
50 mg/mL ampicillin, 2 mmol Lꢂ1
L-glutamine, 5 mg/mL hemin,
5 mmol Lꢂ1 biopterin, (SigmaeAldrich, St Louis, USA), at pH 7.0 and
incubated at 25 ꢁC. L. amazonensis and L. infantum (syn. L. chagasi)
were selected for Sb(III) resistance as described previously [35,36].
The Sb(III)-resistant mutants L. amazonensis BA199 Sb(III) 2700.2
and L. infantum (syn. L. chagasi) BH400 Sb(III) 2700.2 were selected
Conflict of interest
Authors declare no conflict of interest.
Acknowledgments
in 25 cm2 flasks containing 5 mL of
a-MEM in the presence of
increasing Sb(III) concentrations up to 2700 m .
mol Lꢂ1
6.2. Antileishmanial activity assay
This research was supported by grants from the Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Proj-
ect Universal e MCTI/CNPq nꢁ 14/2012 (480719/2012-8), FAPEMIG
(APQ-04166-10, REDEe40/11, PRONEX/2009, and PPMe00382-11),
PRONEX-FAPERJ (E-26/110.574/2010), PRONEM-FACEPE (1232.1.06/
Compounds were evaluated in vitro for their activity against both
Sb(III)-sensitive and -resistant Leishmania parasites, as described
previously [37]. Briefly, log-phase L. amazonensis and L. infantum

530
T.T. Guimarães et al. / European Journal of Medicinal Chemistry 63 (2013) 523e530
[20] (a) E.N. da Silva Júnior, R.F.S. Menna-Barreto, M.C.F.R. Pinto, R.S.F. Silva,
D.V. Teixeira, M.C.B.V. de Souza, C.A. de Simone, S.L. de Castro, V.F. Ferreira,
A.V. Pinto, Eur. J. Med. Chem. 43 (2008) 1774;
10) and CAPES. F Frézard and M.N. Melo are recipients of the CNPq
research fellowship. Dr. E.N. da Silva Júnior also thanks the Programa
Institucional de Auxílio à Pesquisa de Doutores Recém-Contratados
(Edital 12/2011) and Universidade Federal de Minas Gerais.
(b) S.B. Ferreira, K. Salomão, F.C. da Silva, A.V. Pinto, C.R. Kaiser, A.C. Pinto,
V.F. Ferreira, S.L. de Castro, Eur. J. Med. Chem. 46 (2011) 3071;
(c) P.F. Carneiro, S.B. do Nascimento, A.V. Pinto, M.C.F.R. Pinto, G.C. Lechuga,
D.O. Santos, H.M. dos Santos Júnior, J.A.L.C. Resende, S.C. Bourguignon,
V.F. Ferreira, Bioorg. Med. Chem. 15 (2012) 4995.
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