Synthesis and antimalarial activity of quinones and structurally-related oxirane derivatives

Article abstract of DOI:10.1016/j.ejmech.2015.11.020

A series of eighteen quinones and structurally-related oxiranes were synthesized and evaluated for in vitro inhibitory activity against the chloroquine-sensitive 3D7 clone of the human malaria parasite Plasmodium falciparum. 2-amino and 2-allyloxynaphthoquinones exhibited important antiplasmodial activity (median inhibitory concentrations (IC₅₀) < 10 μM). Oxiranes 6 and 25, prepared respectively by reaction of α-lapachone and tetrachloro-p-quinone with diazomethane in a mixture of ether and ethanol, exhibited the highest antiplasmodial activity and low cytotoxicity against human fibroblasts (MCR-5 cell line). The active compounds could represent a good prototype for an antimalarial lead molecule.

Full text of DOI:10.1016/j.ejmech.2015.11.020

European Journal of Medicinal Chemistry 108 (2016) 134e140
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and antimalarial activity of quinones and
structurally-related oxirane derivatives
Paula F. Carneiro a, Maria C.R.F. Pinto a, Roberta K.F. Marra b, Fernando de C. da Silva b,
Jackson A.L.C. Resende c, Luiz F. Rocha e Silva d, Hilkem G. Alves e, Gleyce S. Barbosa e,
,
*
Marne C. de Vasconcellos e, Emerson S. Lima e, Adrian M. Pohlit d, Vitor F. Ferreira b
a Universidade Federal do Rio de Janeiro, Instituto de Pesquisas de Produtos Naturais, 21944-970 Rio de Janeiro, RJ, Brazil
b
^
ꢀ
Universidade Federal Fluminense, Instituto de Química, Departamento de Química Organica, 24020-150 Niteroi, RJ, Brazil
c
^
ꢀ
Universidade Federal Fluminense, Instituto de Química, Departamento de Química Inorganica, 24020-150 Niteroi, RJ, Brazil
d
e
^
ꢀ
^
ꢀ
Instituto Nacional de Pesquisas da Amazonia, Laboratorio de Princípios Ativos da Amazonia, Av. Andre Araújo, 2936 Manaus, Brazil
^
^
Universidade Federal do Amazonas, Faculdade de Ciencias Farmaceuticas, 69010-300 Manaus, AM, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of eighteen quinones and structurally-related oxiranes were synthesized and evaluated for
in vitro inhibitory activity against the chloroquine-sensitive 3D7 clone of the human malaria parasite
Plasmodium falciparum. 2-amino and 2-allyloxynaphthoquinones exhibited important antiplasmodial
Received 3 October 2015
Received in revised form
10 November 2015
Accepted 13 November 2015
Available online xxx
activity (median inhibitory concentrations (IC50) < 10
mM). Oxiranes 6 and 25, prepared respectively by
reaction of -lapachone and tetrachloro-p-quinone with diazomethane in a mixture of ether and ethanol,
a
exhibited the highest antiplasmodial activity and low cytotoxicity against human fibroblasts (MCR-5 cell
line). The active compounds could represent a good prototype for an antimalarial lead molecule.
© 2015 Published by Elsevier Masson SAS.
Keywords:
Naphthoquinones
Lapachone
Epoxides
Malaria
Infectious disease
1. Introduction
cyclodextrin-encapsulated b-lapachone (2) was named ARQ501
(ArQule Inc.) and investigated in a phase II clinical [11] in combi-
nation with Taxol® or gemcitabine [12,13]. An important variation,
the water-soluble prodrug of 2, ARQ 761 (Code C99146, structure
not reported) [14], is currently in clinical trial [15] and requires less
solvent in the formulation for intravenous administration and
consequently less hemolytic anemia is associated with adminis-
tration of ARQ 761.
The antitumor mechanism of action of quinones is based on
redox cycling that represents a cyclic process of reduction of a
compound, followed by (auto)-oxidation of the reaction product
and generation of reactive oxygen species (ROS) [16]. Conversely,
the reduction reaction of the quinone moiety via acceptance of one
or two electrons followed by oxidation by oxygen cause the for-
mation of ROS. Also, it is known that reactive oxygen species
formed in excess in the intracellular environment are able to acti-
vate the intrinsic pathway of apoptosis by the permeabilization of
mitochondria and activation of caspase 9. This may be the mech-
anism responsible for the cytotoxic action of these substances, both
on micro-organisms, as well as on tumor cells [17].
Naphthoquinones and derivatives are important due to their
demonstrated activity against several pathogenic microorganisms
such as Trypanosoma cruzi (protozoan that causes American leish-
maniasis or Chagas' Disease) [1e3], Plasmodium falciparum and
Plasmodium berghei (protozoans that cause severe human and ro-
dent malaria, respectively) [4,5], and human immunodeficiency
virus (HIV) [6] and insects such as the mosquito species Aedes
aegypti [7]. The naphthoquinones lapachol (1) and b-lapachone (2)
are isolated from South American trees of the genus Tabebuia that
have been used traditionally by indigenous people to treat many
parasitic infections, including malaria [8].
Compound 1 has been used in the past as a co-adjuvant in the
treatment of malignant solid tumors but its use was discontinued
due to concerns about its toxicity [9,10]. Later, hydroxypropyl-b-
* Corresponding author.
E-mail address: cegvito@vm.uff.br (V.F. Ferreira).
http://dx.doi.org/10.1016/j.ejmech.2015.11.020
0223-5234/© 2015 Published by Elsevier Masson SAS.

P.F. Carneiro et al. / European Journal of Medicinal Chemistry 108 (2016) 134e140
135
The general structures of compounds 1 and 2 have been widely
exploited in medicinal chemistry as prototypes for new candidates
against cancer and parasitic infections. Atovaquone (3) is synthe-
sized starting from 1 and is used as a therapeutic drug in the
treatment or prevention of mild cases of infection by Plasmodium
vivax (in combination with proguanil) [18e20], although there have
been cases of resistance reported [21]. It also can be used for
pneumocystosis, toxoplasmosis and babesiosis (usually in combi-
nation with azithromycin). Two similar compounds, buparvaquone
(4) and parvaquone (5), are pharmaceuticals for veterinary use
(Fig. 1).
Fig. 2. General rationale used in this study.
Substitution of a carbonyl of naphthoquinone by another group
has generated new compounds with important biological activities.
Also, epoxynaphthoquinones distributed in nature and the natural
against T. cruzi and Leishmania spp. we decided to synthesize a
series of oxiranes and to investigate their activity against the
chloroquine-sensitive 3D7 clone of the human malaria parasite P.
falciparum. It was assumed that these oxiranes would have anti-
malarial activity. The purpose of this study was to prepare oxirane
derivatives of naphthoquinones and test them for antiplasmodial
activity and cytotoxicity as a means to search for new antimalarial
compounds. Herein we report our findings on the antiplasmodial
and cytotoxic activity of these naphthoquinone-derived oxiranes.
6
a-acetoxygedunin (8) exhibit interesting biological activities [22].
Substance 6 was synthesized from -lapachone and was the only
a
oxirane that showed high trypanocidal activity with excellent
minimal cytotoxicity in VERO cells. Comparatively, oxirane 7
exhibited similar trypanocidal activity to
b-lapachone (2). The
major mechanism of trypanocidal action of naphthoquinones is by
inducing intracellular damage caused by oxidative stress due to the
quinonoid moiety. Our group synthesized compounds 6 [23] and 7
[24] and found that they inhibited the serine proteinase of T. cruzi
leading to interference in the establishment of infections [25e27].
Recently we have shown that oxirane 6 has leishmanicidal effects
on Leishmania (Viannia) braziliensis and L. amazonensis [28]. This
compound was able to cause death of promastigote and amastigote
forms of Leishmania spp. after 3 h of exposure [29].
Recently we reported naphthoquinones with activity against
P. falciparum [30,31]. These compounds act via ROS production.
However, oxiranes act by inhibiting proteinase enzymes (Fig. 2).
Protozoans comprise a very diverse group of unicellular eukaryotic
organisms [32], which include Plasmodium, Trypanosoma and
Leishmania parasites, among others. Based on the good biological
activity obtained with oxiranes derived from naphthoquinones
2. Results and discussion
The substances used in the screening against the chloroquine-
sensitive 3D7 clone of P. falciparum were prepared in one step
from the appropriate quinone by adding a freshly prepared solution
of diazomethane in ether (Scheme 1). The reaction proceeds by
nucleophilic attack of diazomethane on the more electrophilic
carbonyl of quinones 9e17 and fluorenone (18) yielding oxiranes 6,
7, 19e23, 25 and the unexpected non-oxirane compounds 24, 26
and 27. In general, reaction occurred at carbonyl C-1 due to the
higher electrophilicity provided by the adjacent heteroatom
substituent.
Fig. 1. Natural and synthetic substances with activity against parasitic infections.

136
P.F. Carneiro et al. / European Journal of Medicinal Chemistry 108 (2016) 134e140
Scheme 1. General reactions used for preparing the oxiranes (and other products).
Although substance 23 has been described previously, the NMR
The reaction of diazomethane with fluorenone (18) did not
provide the desired oxirane product. Instead, after initial nucleo-
philic attack by diazomethane at the carbonyl group, ring expan-
sion occurred to form 9-phenanthrol which in turn was methylated
by diazomethane to form methyl 9-phenanthryl ether (27). In the
1H and 13C NMR (APT type) spectra signals related to a CH2 group
were not observed. Instead a singlet was observed at dH 4.0 that is
characteristic of a methoxy group. In the high resolution mass
spectrum a hydrogen ion adduct [MþHþ] m/z 209.0954 was
observed that is consistent with the molecular formula C15H12O of
compound 27. Similar to the result with fluorenone (18), quinone 17
did not produce the desired oxirane in the reaction with diazo-
methane and instead afforded dimethylated derivative 26 whose
reaction mechanism has not been elucidated.
A series of quinones 9e17 were used for preparing the oxiranes
19e25. The inhibitory concentrations of these compounds against
P. falciparum 3D7 strain and median toxicities against MRC-5 cells
(IC50) were determined and the results are presented in Table 1.
Structurally diverse oxiranes 6, 19 and 25 were among the most
active antiplasmodial compounds and were more active than the
naphthoquinono-3,4-dihydro-2H-pyran 15, dichloronaphthoquin
data presented in the literature for this compound was not
consistent with this structure [33]. The hydrogen chemical shifts
less than dH 3.00 ppm for CH2 of the published compound clearly
show that reaction (cyclopropanation) occurred at the double bond
to form compound 24. It is noteworthy that the synthesis of 24 and
its spectral characterization have been published previously [24].
Therefore, we established that treatment of 14 or 15 with diazo-
methane in ethyl ether can provide 23 (dH 3.70 and 3.44 ppm for
the oxirane H atoms) or 24 (dH 1.95 and 1.73 ppm for the cyclo-
propane H atoms) [24], respectively. The correct structures are re-
ported herein. These structures were confirmed by X-ray
crystallography and ORTEP projections are presented in Fig. 3.
Reaction of 17 with diazomethane yielded product 26. In the 1H
NMR spectrum of 26 the two characteristic doublets of an epoxy
group were not observed. Only a singlet was observed at dH
4.15 ppm due to the CH3 groups. In the 13C NMR spectrum (APT
type) at dC 62.7 ppm and the high resolution mass spectrum
exhibited a sodium ion adduct [MþNaþ] at m/z 278.9700 consistent
with the molecular formula C10H6Cl2N2O2 of compound 26 whose
structure was confirmed by X-ray diffraction (Fig. 3).

P.F. Carneiro et al. / European Journal of Medicinal Chemistry 108 (2016) 134e140
137
Fig. 3. ORTEP representation (50% probability displacement ellipsoid) of oxirane 23 (A) and cyclopropane 24 (B) derivatives and compound 26 (C). Hydrogen, carbon, nitrogen,
chlorine and oxygen atoms are represented in white, gray, blue, green and red, respectively. (For interpretation of the references to color in this figure legend, the reader is referred
to the web version of this article.)
Table 1
Median inhibitory concentrations (IC50) against Plasmodium falciparum 3D7 strain, toxicities to MRC-5 cells and selectivity indices (SI) of quinones and oxiranes.
Compound
IC50
(
m
M) P. falciparum 3D7
IC50
(m
M) MRC-5 cells
Selectivity index, SIa
Naphthoquinones
9
10
11
12
13
14
15
13.9 (12.6e16.5)
6.30 (4.41e7.42)b
5.14 (3.18e8.66)b
5.18 (4.29e6.77)b
>50
98.2 (90.7e107)
>100
7.2
>15
63
322 (153e681)
64.8 (60.2e64.4)
5.2 (4.5e5.9)
56.1 (51.5e60.9)
>100
12
<0.10
<1.1
ND
>50
>50
Quinones
16
17
22.4 (19.5e29.0)
>50
>100
>100
>4.4
ND
Oxiranes
19
6.22 (5.43e8.21)b
92.5 (26.1e331)
135 (112e164)
>100
15
<2.7
ND
20
21
>50
>50
22
7
23
6
25
26
>50
>50
>50
26.4 (23.7e29.5)
116 (90.0e148)
55.7 (45.8e68.1)
>100
368 (302e449)
>100
<0.53
<2.3
<1.1
>27
93
3.71 (3.12e4.60)b
3.95 (3.58e5.13)b
>50
ND
Chloroquine (diphosphate)
0.18 (0.14e0.22)
ND
ND
a
SI ¼ IC50(cytotoxicity) ÷ IC50(antiplasmodial)
.
b
Compounds active against P. falciparum (1 < IC50 < 10
m
M) e ND ¼ not determined.
one 9 and tetrachloro-p-benzoquinone (16) from which these
oxiranes were respectively synthesized. Thus, specific improve-
ment of antiplasmodial action may be attributable to the presence
of the oxirane moiety in this small group of structurally diverse
compounds. However, the oxirane moiety is not in general
responsible for enhanced antiplasmodial activity. Thus, the 2-
aminonaphthoquinones 10 and 11 and 2-allyloxynaphthoquinone
(12) were among the most active antimalarial compounds yet
their corresponding oxirane derivatives 20e22 exhibited no anti-
plasmodial activity. Thus, among quinones, naphthoquinones
having a 2-amino, 2-aminoaryl or 2-oxyalkyl group may be closer
to an optimal antiplasmodial structure than other quinones studied
herein. Also, it is not clear what the structural requirements are for
an oxirane to exhibit enhanced antiplasmodial activity compared to
its quinone precursor. None of the compounds tested was more
active than chloroquine diphosphate used as drug standard (posi-
tive controls). While screening for new antimalarial substances it is
normally assumed that active compounds will have IC50 values
presented high leishmanicidal activity against Leishmania (Vian-
nia) braziliensis and Leishmania (Leishmania) amazonensis [28]
wherein it inhibited serine proteinases of these parasites [29].
The mechanism of action of oxirane 6 in P. falciparum may also be
inhibition of the serine proteinase of the parasite. The second most
active compound was oxirane 25 that was derived from tetra-
chlorobenzoquinone 16. The latter compound has more electro-
philic centers that are able to react with an amino group. Both
compounds exhibited good selectivity indices, which is an
important indication that both are low toxicity antiplasmodial
agents.
Aminonaphthoquinones 9e11 were quite active with low cyto-
toxicity leading to high selectivity indices. Oxirane 19 exhibited
more antiplasmodial activity and had a higher selectivity index
than its precursor 9.
The quinone 17 did not produce the desired oxirane in the re-
action with diazomethane and in its place afforded the dimethy-
lated derivative 26 whose reaction mechanism has not been
elucidated.
<10
The most active compound in this series was oxirane 6 (IC50
3.71 M), i.e. the adduct derived from -lapachone. Importantly,
mM [34e36].
m
a
3. Conclusion
this compound was also active against T. cruzi [23] where it spe-
cifically inhibited serine proteinases [25e27]. Also, this compound
In summary, a series of oxiranes was synthesized and analysis

138
P.F. Carneiro et al. / European Journal of Medicinal Chemistry 108 (2016) 134e140
of the antiplasmodial properties of these oxiranes and their
precursors was performed. Optimization of naphthoquinone
antiplasmodial activity should be possible in future work by
varying the substituents at the 2 and 3 positions. Similarly, the
3:1 mixture of ethyl ether and ethanol. After dissolution of the
quinone was complete, diazomethane in ethyl ether (10 mL) was
added. The reaction was left at room temperature for 3e5 days. The
solvent was evaporated under reduced pressure and the crude
product was purified on a column of silica gel by eluting with a
gradient of increasing polarity of ethyl acetate/hexane [24].
ꢁ
oxirane moiety provided greater antiplasmodial activity vis-a-vis
that of a few structurally diverse quinone precursors. Two oxir-
anes exhibited good antiplasmodial activity and high selectivity
and can be considered for the development of new antimalarial
agents with the initial step being the optimization of oxirane
structure-activity relationships. The antiplasmodial mechanism of
action of these oxiranes is unknown but may be related to the
inhibition of P. falciparum serine proteinase based on the obser-
vation that oxiranes act on this target of Trypanosoma cruzi and
Leishmania (Viannia) braziliensis and Leishmania (Leishmania)
amazonensis.
2,3-Dichloro-4H-spiro[naphthalene-1,20-oxirane]-4-one
(19)
was obtained as a white solid in 52% yield. m.p. 149e150 ꢀC; FT-IR
nmax (cmꢁ1, KBr): 3103, 3074, 3045, 2924, 1664, 1595, 1568, 1456,
1325, 1282, 1238, 1165, 1141, 904, 866, 840, 810, 756; 1H NMR
(400 MHz, CDCl3)
d
(ppm): 8.23 (d, J ¼ 7.7 Hz, 1H), 7.66 (t, J ¼ 8.1 Hz,
1H), 7.55 (t, J ¼ 7.7 Hz, 1H), 7.31 (d, J ¼ 8.1 Hz, 1H), 3.77 (d, J ¼ 6.1 Hz,
1H), 3.37 (d, J ¼ 6.1 Hz, 1H); 13C NMR (100 MHz, CDCl3)
d (ppm):
176.1 (C), 148.4 (C), 137.7 (C), 135.2 (C), 133.8 (CH), 131.0 (C), 129.4
(CH),127.9 (CH),123.4 (CH), 58.2 (CH2), 55.2 (C); MS (70 eV) m/z (%):
239 (16, Mþ), 212 (41), 205 (12), 184 (33), 182 (52), 177 (83), 147
(100),135 (7), 113 (34), 99 (15), 97 (19), 74 (22), 63 (24), 50 (13); HR-
ESI-MS m/z calcd. for C11H7Cl2O2 [MþH]þ: 240.9818. Found: m/z
4. Experimental section
4.1. Chemistry
240.9817.
D
¼ 0.41 ppm.
2-(Phenylamino)-4H-spiro[naphthlene-1,20-oxirane]-4-one
(20) was obtained as a red solid in 69% yield. m.p. 154e155 ꢀC; FT-IR
nmax (cmꢁ1, KBr): 3205, 3178, 3116, 3049, 3035, 2964, 1623, 1593,
1568, 1524, 1497, 1448, 1141, 1131; 1H NMR (400 MHz, CDCl3)
Melting points were obtained on a Thomas Hoover apparatus
(Philadelphia, USA) 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) and the
reactions was monitored by analytical thin-layer chromatography
was performed with silica gel plates (Merck, TLC silica gel 60
F254), and the plots were visualized using UV light. Infrared
spectra were recorded on a Shimadzu IR Prestige-21 FTIR spec-
trometer (Kyoto, Japan). 1H and 13C NMR were recorded at room
temperature using a VNMRSYS-500 or a Varian MR 400 instru-
ment, in the solvents indicated, with TMS as internal standard.
d
(ppm): 8.18 (d, J ¼ 8.8 Hz,1H), 7.52 (m, 2H), 7.43e7.36 (m, 2H), 7.29
(d, J ¼ 7.5 Hz, 1H), 7.22 (d, J ¼ 8.8 Hz, 2H), 6.58 (s, 1H), 6.23 (s, 1H),
3.51 (d, J ¼ 6.1 Hz, 1H), 3.28 (d, J ¼ 6.1 Hz, 1H); 13C NMR (125 MHz,
CDCl3)
d (ppm): 183.2 (C), 155.2 (C), 137.4 (C), 136.2 (C), 133.6 (C),
131.7 (CH),129.6 (CH),128.6 (CH),126.3 (CH),125.6 (CH),123.2 (CH),
121.5 (CH), 102.0 (CH), 60.8 (CH2), 53.8 (C); MS (70 eV) m/z (%): 264
(18), 263 (100), 262 (50), 246 (4), 235 (10), 234 (46), 218 (8), 217
(10), 206 (14), 204 (15), 185 (15), 179 (7),157 (6), 142 (8), 130 (9),130
(9), 116 (9), 109 (11), 102 (21), 93 (9), 84 (10), 77 (15), 63 (5); HR-ESI-
MS m/z calcd. for C17H13NO2Naþ: 286.0844. Found: m/z 286.0838.
Chemical shifts (d) are given in ppm and coupling constants (J) in
Hertz (Hz). Low resolution mass spectra were obtained using
Shimadzu GCMSQP2010 Plus and GCMS-QP5000 (70 eV) gas
chromatograph-mass spectrometer (Tokyo, Japan) systems with a
DB-5MS column. High resolution mass spectra (electrospray
ionization) were obtained using a Waters QTOF Micro (Man-
chester, UK) mass spectrometer. Ions were described in mass-to-
charge units (m/z) and relative abundance was expressed as a
percentage of the base peak intensity. Diazomethane was pre-
pared by reaction of Diazald® (N-methyl-N-nitroso-p-toluene-
sulfonyl nitrosamide, Steinheim, Germany) with potassium
hydroxide.
The quinones 9, 16, 17 and compound 18 are commercially
available (SigmaeAldrich, Brazil). The naphtoquinones 10, 11, 12, 13,
14 and 15 were prepared by standard procedures as described in
the literature [37e42]. Oxiranes 6, 7, 19, 22, 26 and 27 were pre-
viously synthesized [23,24,43,44], but proper comparison of NMR
spectra was not possible due to lack of spectral data for compounds
20 and 25 in the literature.
The structure of compounds 23, 24 and 26 were confirmed using
single crystal X-ray diffraction experiments. The analysis for 23 and
24 were performed on an Oxford Gemini A-Ultra diffractometer at
LabCri/Federal University of Minas Gerais (UFMG), Brazil and for 26
was performed on a Kappa CCD diffractometer at LARE-DRX (Uni-
versidade Federal Fluminense), Brazil. The structures were solved
by direct methods and refined by full-matrix least-squares on F2
using the SHELX-2013 package (1). All non-hydrogen atoms were
refined with anisotropic displacement parameters. Hydrogen
atoms bonded to C atoms were placed at their idealized positions
using standard geometric criteria [45].
D
¼ 2.1 ppm.
2-Amino-4H-spiro[naphthlene-1,20-oxirane]-4-one (21) was
obtained as a red solid in 55% yield. m.p. 281e283 ꢀC; FT-IR nmax
(cmꢁ1, KBr): 3336, 3154, 3080, 3062, 2992, 1662, 1612, 1595, 1538,
1470, 1443, 1273, 1141; 1H NMR (400 MHz, CD3OD)
d (ppm): 7.94 (d,
J ¼ 7.7 Hz, 1H), 7.45 (t, J ¼ 7.5 Hz, 1H), 7.37 (t, J ¼ 7.5 Hz, 1H), 7.16 (d,
J ¼ 7.7 Hz, 1H), 5.67 (s, 1H), 3.36 (d, J ¼ 6.4 Hz,1H), 3.18 (d, J ¼ 6.4 Hz,
1H); 13C NMR (125 MHz, CDCl3)
d (ppm): 187.2 (C), 168.6 (C), 140.7
(C), 137.3 (C), 135.1 (CH), 131.8 (CH), 129.1 (CH), 125.5 (CH), 103.2
(CH), 65.0 (CH2), 56.3 (C); MS (70 eV) m/z (%): 187 (56), 172 (4), 171
(9), 160 (9), 159 (60), 143 (11), 131 (19), 130 (63), 115 (14), 103 (25),
102 (100), 89 (12), 76 (21), 63 (12), 51 (15); HR-ESI-MS m/z calcd. for
C
11H8NOꢁ2 [MꢁH]ꢁ: 186.0557. Found: m/z 186.0560.
D
¼ 1.6 ppm.
2-(Allyloxy)-4H-spiro[naphthalene-1,20-oxirane]-4-one
(22)
was obtained as a white solid in 35% yield. m.p. 92e94 ꢀC; FT-IR
nmax (cmꢁ1, KBr): 3082, 2924, 1656, 1610, 1570, 1460, 1408, 1365,
1325, 1269, 1236, 1203, 1145, 1103, 1060, 1028, 966, 906, 873, 831,
781, 750; 1H NMR (400 MHz, CDCl3)
d
(ppm): 8.17 (d, J ¼ 7.8 Hz, 1H),
7.57 (t, J ¼ 7.8 Hz, 2H), 7.24 (d, J ¼ 7.8 Hz, 1H), 6.05e5.94 (m, 1H),
6.00 (s, 1H), 5.39 (m, Hz, 2H), 4.55 (m, 2H), 3.72 (d, J ¼ 6.8 Hz, 1H),
3.31 (d, J ¼ 6.8 Hz, 1H); 13C NMR (125 MHz, CDCl3)
d (ppm): 184.6
(C), 168.0 (C), 137.1 (C), 132.6 (CH), 132.5 (C), 130.7 (CH), 128.5 (CH),
126.4 (CH), 122.8 (CH), 119.4 (CH2), 106.9 (CH), 69.7 (CH2), 57.9
(CH2); MS (70 eV) m/z (%): 228 (100, Mþ), 213 (61), 199 (12), 185
(15), 181 (9), 152 (16), 128 (24), 115 (29), 102 (6), 89 (12), 77 (14), 63
(12), 51 (10). HR-ESI-MS m/z calcd. for
C
14H13Oþ3 : 229.0859
([MþH]þ). Found m/z 229.0863.
D
¼ 1.7 ppm.
2,2-Dimethyl-2,3-dihydro-4H-spiro[naphtho[2,3-b]furan-9,20-
oxiran]-4-one (23) was obtained as a white solid in 60% yield. m.p.
123e126 ꢀC; FT-IR nmax (cmꢁ1, KBr): 3066, 2966, 2926, 2858, 1660,
1600, 1568, 1460, 1417, 1375, 1271, 1211, 1165, 1122, 1060, 891, 866,
4.2. General method for the preparation of oxiranes 19e27
The appropriate quinone (1 mmol) was dissolved in 20 mL of a
754; 1H NMR (400 MHz, CDCl3)
d
(ppm): 8.18 (d, J ¼ 7.6 Hz,1H), 7.52

P.F. Carneiro et al. / European Journal of Medicinal Chemistry 108 (2016) 134e140
139
(m, 2H), 7.20 (d, J ¼ 7.6 Hz, 1H), 3.70 (d, J ¼ 6.5 Hz, 1H), 3.44 (d,
J ¼ 6.5 Hz, 1H), 2.93 (s, 2H), 1.52 (s, 3H), 1.51 (s, 3H); 13C NMR
was determined by comparing the sample with untreated controls,
according to the formula below:
ðparasitemia of untreated controls ꢁ parasitemia of sampleÞ
% inhibition ¼
x 100
parasitemia of untreated controls
(125 MHz, CDCl3)
d
(ppm): 181.5 (C), 168.1 (C), 136.2 (C), 134.2 (C),
4.4. Cytotoxicity assay
131.8 (CH), 128.6 (CH), 126.4 (CH), 122.6 (CH), 117.4 (C), 92.2 (C), 57.3
(CH2), 50.8 (C), 40.2 (CH2), 28.3 (CH3), 28.2 (CH3); MS (70 eV) m/z
(%): 242 (100, Mþ), 227 (20), 211 (5), 200 (19), 183 (9), 171 (12), 156
(11), 141 (16), 128 (24),115 (35), 105 (13), 89 (12), 77 (19), 63(12), 43
(45); HR-ESI-MS m/z calcd. for C12H11Oþ3 : 243.1028 [MþH]þ. Found:
The MRC-5 cell line of human fibroblasts was grown in Dul-
becco's Modified Eagle's Medium (DMEM) supplemented with 10%
fetal bovine serum, 2 mM glutamine 100 mg/mL streptomycin and
100 U/mL penicillin, and in-cubated at 37 ꢀC with a 5% atmosphere
of CO2. For assays, the cells were plated in 96-well plates
(2.5 ꢂ 104 cells/well) and the Alamar Blue™ assay was performed
using a previously described procedure [48,49]. After incubation for
24 h, the compounds were individually dissolved in DMSO and the
resulting solutions were diluted in culture medium. The resulting
dilute solutions of each sample were added to wells at final (well)
m/z 243.1016.
D
¼ 4.9 ppm.
4,5,7,8-Tetrachloro-1-oxaspiro[2.5]octa-4,7-dien-6-one
(25)
was obtained as a white solid in 74% yield. m.p. 158e160 ꢀC; FT-IR
nmax (cmꢁ1, KBr): 3334, 1674, 1613, 1583, 1119, 857; 1H NMR
(500 MHz, CDCl3)
(ppm): 146.2 (C), 134.0 (C), 55.1 (CH2); MS (70 eV) m/z (%): 260
d
(ppm): 3.70 (s, 2H); 13C NMR (125 MHz, CDCl3)
d
(57), 258 (45), 262 (24), 244 (2), 230 (13), 225 (16), 223 (16), 211
(11), 209 (11), 204 (15), 202 (31), 198 (25), 196 (44), 194 (46), 169
(45), 168 (46); HR-ESI-MS m/z calcd. for C7HCl4Oꢁ2 : 258.8707
(100.0%) and 256.8736 (75%) [MꢁH]ꢁ. Found: m/z 258.8727 (100%)
concentrations of 1.56e100 mg/mL. Control groups had final well
concentrations of 0.1% DMSO. The plates were further incubated for
48 h. Three hours before the end of the incubation period, Alamar
Blue™ (10 mL) was added to each well. The fluorescent signal was
monitored with a multiplate reader using 530e560 nm excitation
and m/z 256.8745 (75%).
D
¼ 7.7 and 3.5 ppm, respectively.
4,5-Dichloro-3,6-dimethoxyphthalonitrile (26) was obtained as
a white solid in 50% yield. m.p. 185e186 ꢀC; FT-IR nmax (cmꢁ1, KBr):
2947, 2925, 2238, 1545, 1463, 1386, 1020, 844; 1H NMR (400 MHz,
and 590 nm emission wavelengths.
Acknowledgments
CDCl3)
d d (ppm):
(ppm): 4.15 (s, 6H); 13C NMR (75 MHz, CDCl3)
156.0 (C), 135.2 (C), 111.8 (C), 108.5 (C), 62.7 (CH3). MS (70 eV) m/z
(%): 257 (9), 258 (43), 256 (67), 243 (62), 241 (100), 215 (8), 213
The authors would like to acknowledge the agencies that fund
our research: CNPq (National Council of Research of Brazil), CAPES
(scholarship), FAPERJ, in particular, through project financing of
PRONEX FAPERJ (E-26/110.574/2010) coordinated by Prof. Vitor F.
Ferreira (UFF), CNPq (304716/2014-6) coordinated by Fernando de
C. da Silva and PRONEX FAPEAM NOSSAPLAM (Edital 023/2009)
Project coordinated by Adrian M. Pohlit. This work is part of the
activities of ResNet NPND.
(11); HR-ESI-MS m/z calcd. for
C
10H6Cl2N2O2Naþ: 278.9698
[MþNa]þ. Found: m/z 278.9706.
D
¼ 2.9 ppm.
Methyl 9-phenanthryl ether (27) was obtained as a white solid
in 45% yield. m.p. 64e66 ꢀC; FT-IR nmax (cmꢁ1, KBr): 3445, 3424,
3057, 3009, 2964, 2873, 2836, 1623, 1596, 1459, 1426, 1394, 1309,
1234, 1117, 1095, 829, 766, 743, 722; 1H NMR (500 MHz, CDCl3)
d
(ppm): 8.62 (d, J ¼ 7.9 Hz, 1H), 8.55 (d, J ¼ 8.0 Hz, 1H), 8.35 (dd,
J ¼ 8.0, 1.0 Hz, 1H), 7.75 (d, J ¼ 7.9 Hz, 1H), 7.68e7.63 (m, 1H),
Appendix A. Supplementary data
7.63e7.57 (m, 1H), 7.54e7.45 (m, 2H), 6.95 (s, 1H), 4.05 (s, 3H); 13C
NMR (125 MHz, CDCl3) d (ppm): 153.5 (C), 132.9 (C), 131.2 (C), 127.3
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.11.020.
(CH), 127.2 (CH), 127.1 (CH), 126.8 (CH), 126.5 (C), 126.4 (C), 126.3
(CH), 124.2 (CH), 124.1 (CH), 122.5 (CH), 101.9 (CH), 55.4 (CH3). MS
(70 eV) m/z (%): 208 (78), 193 (7), 165 (100), 139 (7), 115 (5), 104 (8);
HR-ESI-MS m/z calcd. for C15H12OHþ: 209.0961 [MþH]þ. Found: m/
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