Antimalarial activity of phenazines from lapachol, β-lapachone and its derivatives against Plasmodium falciparum in vitro and Plasmodium berghei in vivo

Article abstract of DOI:10.1016/j.bmcl.2003.12.069

The antimalarial activity of benzo[a]phenazines synthesized from 1,2-naphthoquinone, lapachol, β-lapachone and several derivatives have been tested against Plasmodium falciparum in vitro using isolates of parasites with various susceptibilities to chloroquine and/or mefloquine. Parasite growth in the presence of the test drugs was measured by incorporation of [ ³H]-hipoxanthine in comparison to controls with no drugs, always testing in parallel chloroquine, a standard antimalarial. Among seven benzophenazines tested, four had significant in vitro activities; important, the parasites resistant to chloroquine were more susceptible to the active phenazines in vitro. The doses of phenazines causing 50% inhibition of parasite growth varied from 1.67 to 9.44 μM. The two most active ones were also tested in vivo against Plasmodium berghei in mice, in parallel with lapachol and β-lapachone. The 3-sulfonic acid-β-lapachone-derived phenazine was the most active causing up to 98% inhibition of parasitaemia in long term treatment (7 doses) subcutaneouly, whereas the phenazine from 3-bromo-β-lapachone was inactive. Thus, these simple phenazines, containing polar (-Br,-I) and ionizable (-SO₃H, -OH) groups, easily synthesized from cheap, natural or synthetic precursors (lapachol and β-lapachone), at rather low cost, provide prototypes for development of new antimalarials aiming the chloroquine resistant parasites.

Full text of DOI:10.1016/j.bmcl.2003.12.069

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1145–1149
Antimalarial activity of phenazines from lapachol, ꢀ-lapachone
and its derivatives against Plasmodium falciparum in vitro and
Plasmodium berghei in vivo
a
b
b
Valter F. de Andrade-Neto, Marılia O. F. Goulart, Jorge F. da Silva Filho,
´
c
c
c
Matuzale
´
m J. da Silva, Maria do Carmo F. R. Pinto, Antoˆ nio V. Pinto,
d
a
a,
Mariano G. Zalis, Luzia H. Carvalho and Antoniana U. Krettli *
aCentro de Pesquisas Ren e´ Rachou, Fiocruz and Departamento de Parasitologia-Universidade Federal de Minas Gerais (UFMG),
Belo Horizonte, MG, Brazil
b
Departamento de Quı´mica, Universidade Federal de Alagoas (UFAL), Maceio´, Al, Brazil
cN u´ cleo de Pesquisas de Produtos Naturais, UFRJ, Rio de Janeiro, RJ, Brazil
dInstituto de Biofı´sica Carlos Chagas Filho, UFRJ, RJ, Brazil
Received 27 October 2003; revised 14 December 2003; accepted 18 December 2003
Abstract—The antimalarial activity of benzo[a]phenazines synthesized from 1,2-naphthoquinone, lapachol, b-lapachone and several
derivatives have been tested against Plasmodium falciparum in vitro using isolates of parasites with various susceptibilities to
3
chloroquine and/or mefloquine. Parasite growth in the presence of the test drugs was measured by incorporation of [ H]-hipox-
anthine in comparison to controls with no drugs, always testing in parallel chloroquine, a standard antimalarial. Among seven
benzophenazines tested, four had significant in vitro activities; important, the parasites resistant to chloroquine were more suscep-
tible to the active phenazines in vitro. The doses of phenazines causing 50% inhibition of parasite growth varied from 1.67 to 9.44
mM. The two most active ones were also tested in vivo against Plasmodium berghei in mice, in parallel with lapachol and b-lapa-
chone. The 3-sulfonic acid-b-lapachone-derived phenazine was the most active causing up to 98% inhibition of parasitaemia in long
term treatment (7 doses) subcutaneouly, whereas the phenazine from 3-bromo-b-lapachone was inactive. Thus, these simple phe-
nazines, containing polar (–Br,–I) and ionizable (–SO H, –OH) groups, easily synthesized from cheap, natural or synthetic pre-
3
cursors (lapachol and b-lapachone), at rather low cost, provide prototypes for development of new antimalarials aiming the
chloroquine resistant parasites.
#
2004 Elsevier Ltd. All rights reserved.
1
. Introduction
mechanism of action, in order to decrease the risk of
1
resistance. We have previously screened the anti-
2
,3
Malaria is one of the most important parasitic infec-
tions of humans due to its high morbidity and mortal-
ity, a threat to over 2 billion people living in areas of
high incidence. Plasmodium falciparum, the causative
agent of the malignant form of malaria, has high
adaptability by mutation and is resistant to various
types of antimalarial drugs, a serious setback to anti-
malarial programs, since it precludes the use of cheap
and previously effective drugs like chloroquine. New
families of active compounds are needed as well as poly
chemotherapy associating molecules with independent
malarial activity of medicinal plants and natural pro-
4
ducts, measuring their blood schizontocidal effects
against Plasmodium berghei and P. falciparum.
Lapachol (1), a prenyl naphthoquinone isolated from
plants of the Bignoniaceae family, is structurally related
to atovaquone, which, together with proguanil, is highly
5
effective in the prevention of P. falciparum malaria.
Curiously, lapachol, considered an antimalarial agent,
6
showed only borderline activity against P. berghei in
7
7,8
mice or against P. falciparum in vitro.
b-lapachone (2), another naturally occurring quinone, is
easily synthesized by sulfuric acid treatment of lapachol
or lomatiol or other methods and has a wide range of
*
Corresponding author. Tel.: +55-31-3295-3566; fax: +55-31-3295-
115; e-mail: akrettli@cpqrr.fiocruz.br
9
10
3
0
960-894X/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.12.069

1146
V. F. de Andrade-Neto et al. / Bioorg. Med. Chem. Lett. 14 (2004)1145–1149
Figure 1. Structures and synthesis of the studied phenazines.
biological activities, including antibacterial, antifungal,
trypanocidal, antipsoriatic, antiinflammatory and anti-
3. Biological activity
1
1
cancer. The synthesis of heterocycles derived from b-
lapachone and their activities against Trypanosoma
All the synthesized phenazines were tested for anti-
2
7
malarial activity in vitro. The compounds were dis-
solved in water+DMSO 0.02% (v/v) and administered
orally, by gavage, to the mice. For the subcutaneous
treatment, instead of water, saline solution was used
and the same amount of DMSO. For the bioassays in
vitro, the amount of DMSO in water was 0.002% (v/v).
The results are in Table 1 and Figure 2, which illustrate
the dose–response curves of one inactive (2a) and one
active (3a) phenazine and their IC . For chloroquine
1
2
cruzi have been reported. One of the possible hetero-
cycles is the phenazinic derivative. In general, natural
and synthetic phenazines have attracted considerable
attention because of their interesting biological activ-
1
3
14
ities, including antimalarial and antihepatitis Cviral
1
5
replication. Benzo[a]phenazines are efficient DNA
intercalating ligands with antitumor activity in leukae-
1
6
mia and solid tumors. The b-lapachone phenazine
3,3 - dimethyl - 2,3 - dihydro - 3H - benzo[c]pyran[3,2 - a]-
5
0
(
phenazine) (2a), known for a considerable time, had
(CQ) the IC50 values were 0.2 to 0.4 mM with the drug-
resistant isolate BHz 26/86 and W2; and 0.02 to 0.08
mM with isolates HB3 and D6, chloroquine sensitive.
The phenazines containing polar [–Br (3a), –I (4a)] or
1
7
been assayed, in vitro, against human tumor cell lines
NCI anticancer screen) and was considered inactive.18
(
That is, to our knowledge, the only report concerning
its biological activity.
In the present work, we performed chemical reactions
with the quinones lapachol (1), represented by a tauto-
1
9
20
meric mixture (Fig. 1), b-lapachone (2) and its deri-
vatives (3–6)
2
0À22
and 1,2-naphthoquinone (7) with
2
3
phenylenediamine (Fig. 1), following previously descri-
7
1
bed procedures, and tested their antimalarial activities
27
in vitro and in vivo.31
2. Synthesis
Phenazines were synthesized23 from lapachol, easily
obtained and commercially available (PVP, Piauı, Bra-
´
zil) at low cost, giving 1a, from b-lapachone, giving 2a,
from 3-bromo-b-lapachone to 3a, from 3-iodo-b-lapa-
chone to 4a, from 3-allyl-b-lapachone to 5a and from 3-
sulfonic acid-b-lapachone to 6a (Fig. 1), using a simpler
phenazine (7a), synthesized from 1,2-naphthoquinone
3
Figure 2. Dose-response curve from [ H]-hipoxanthine incorporation
by the P. falciparum BHz 26/86 parasites (chloroquine partially resis-
tant) in the presence of various concentrations of two phenazines tes-
ted, one active, 3a (&) in which the value of the IC50 is illustrated and
one inactive 2a (&) up to the dose of 20 mM.
(7), as a pattern. The phenazines 4a, 5a, and 6a are new
compounds. Their analytical data are in full agreement
2
4À26
with the proposed structure.

V. F. de Andrade-Neto et al. / Bioorg. Med. Chem. Lett. 14 (2004)1145–1149
1147
Table 1. Susceptibility of four different Plasmodium falciparum strains to benzo[ꢀ]phenazines and to chloroquine (CQ), an antimalarial reference
drug, in vitro, measured by the inhibitory concentration (IC50) dose of the parasite growth in relation to control cultures with no drugs
Parasite strains
IC50 values (mM)
3
a
6a
4a
1a
2a
5a
7
a
CQ
a
BHz26/86
b
1.90Æ0.05
8.75Æ0.14
4.07Æ0.03
3.04Æ0.1
1.75Æ0.02
6.14Æ0.18
2.43Æ0.02
1.67Æ0.05
2.30Æ0.13
6.65Æ0.24
4.26Æ0.15
3.22Æ0.09
3.14Æ0.04
9.44Æ0.16
3.72Æ0.025
3.54Æ0.03
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
0.2Æ0.03
0.02Æ0.01
0.08Æ0.05
0.4Æ0.04
HB3
c
D6
W2
d
a
CQ partially resistant.
CQ and mefloquine sensitive.
b
c
CQ sensitive and mefloquine resistant.
CQ resistant and mefloquine sensitive. Values are meanÆstandard deviation in one representative experiment in triplicate.
d
Table 2. Antimalarial activity of lapachol (1), b-lapachone (2) and
two benzo[ꢀ]phenazines (3a, 6a) against Plasmodium berghei in mice
infected with blood parasites, treated by oral or subcutaneous routes,
during four consecutive days
considered inactive. In conclusion, three degrees of sus-
ceptibility to the phenazines in vitro were observed,
namely the highly susceptible group (BHz 26/86 and W2
parasites), the partly susceptible group (D6 parasites),
and the less susceptible group (HB3 parasites) with IC50
Compd
Route
Dose
mg/kg)
% Inhibition of parasite growtha
(
values 3 up to 4.6-times higher than the values detected
Exp 1
Exp 2
Exp3b
for BHz 26/86 and W2 parasites (Table 1). Important,
the chloroquine-resistant P. falciparum strains (W2 and
BHz) were more sensitive to the phenazines than the
chloroquine-sensitive strains (HB3 and D6) and these
differences were statistically significant (values of
p<0.01 for HB3 and <0.05 for the D6 isolate) within
each tested molecule (Mann–Whitney U test, NCSS2000/
PASS 2000 Statistical package, Kaysville, UT, USA).
Despite the fact that the more active compounds in vitro
are at least 5 to 10-times less active than chloroquine, their
IC50 values are in the micromolar concentration range
1
2
3
6
Oral
200
200
200
200
25
100
200
100
200
15
0
28
11
33
99
0
0
34
10
36
98
0
—
—
—
—
—
0
a
a
CQ
3
3
6
6
a
a
a
a
Subcutaneous
0
0
0
34
50
99
31
40
99
—
98
99
CQ
a
Calculated in relation to the control non-treated mice, at day 5 after
parasite inoculation (n=3–5 animals per group).
The same, except by the 7-day treatment schedule.
8
,34
(Table 1), comparable to recently reported results.
b
Some available molecules were also tested in P. berghei-
1
3
infected mice (Table 2) against the NK65 strain, as
3
3
ionizable groups [–SO H (6a), –OH (1a)] had the high-
3
described. Our data show that lapachol (1) was inac-
tive and b-lapachone (2) had a borderline activity (up to
200 mg/kg) given orally, by gavage, in 4 doses. The two
most active phenazines in vitro were either inactive (3a)
or had a borderline activity (6a) in vivo at the doses of
200 mg/kg by oral route. Given by subcutaneous route
3a was inactive; 6a caused up to 50% inhibition of
est antimalarial activities. The IC50 value for each drug
was determined using a non-linear curve fitting pro-
gram. The IC50 were confined to the following ranges
for each drug: 3a, 1.90–8.75 mM; 6a, 1.67–6.14 mM; 4a,
2
.3 to 6.65 mM and, 1a, 3.14–9.44 mM (Table 1). For 2a,
5
a and 7a, the IC50 values were higher than 20 mM, thus
Figure 3. Course of parasitaemia by P. berghei in mice at different days after infection and treatment with: saline (control) [&], 6a ~], and chloroquine
~], by subcutaneous route for 7 consecutive days. The values in parenthesis show parasite inhibition growth by 6a in relation to control groups.
Cumulative mortality rates are shown for controls (black bars), 6a (white bars) and chloroquine (gray bars) treated mice.
[

1148
V. F. de Andrade-Neto et al. / Bioorg. Med. Chem. Lett. 14 (2004)1145–1149
parasite growth (100 and 200 mg/kg), in four doses
Table 2). Neither of them increased mice survival in
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ability of the two drugs in rodents, we next performed
two additional experiments, treating the mice for 7 days
with compounds 3a and 6a (200 mg/kg, subcutaneous).
Whereas 3a remained inactive (data not shown), 6a
showed up to 98% inhibition of parasite growth (Fig. 3)
and a significant reduction in mortality (pꢀ0.05) in
relation to non-treated control groups by day 20.
Chloroquine used in parallel (15–25 mg/kg) caused
intense reduction of parasitaemia and of mortality in all
experiments (Table 2, Fig. 3).
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The in vivo low antimalarial activity of such phenazines,
by oral route, may be a result of slow uptake or rapid
elimination of the active metabolites to an intracellular
compartmentalization, or inactivation of the compound
in vivo as shown for other molecules.35 Indeed, this
seems to be the case of 6a if used in longer term treat-
ment (7 doses) instead of the 4-day suppressive routine
test.
1
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6. Rewcastle, G. W.; Denny, W. A.; Baguley, B. C. J. Med.
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Assayed as antimalarial for the first time, these phena-
zines, some of them new and rather active, should pro-
vide leads for development of antiparasitic drugs since
they are easily synthesized at low cost, and have shown
activities in the micromolar ranges, especially the sulfo-
2
1. Pereira, M. A. A structural analysis of lapachol and ꢁ-
˜
lapachones, PhD thesis, Sa˜o Paulo University; Sao Carlos/
nide derivative (R=SO H), active in vivo and in vitro
3
against malaria.
SP: Brazil, 1989.
22. Carvalho, C. E. M.; Brinn, I. M.; Pinto, A. V.; Pinto,
M. C. F. R. J. Photochem. Photobiol. A: Chem. 2000, 136,
2
5.
2
3. Analytical grade reagents were used throughout the
experiments. All the phenazines (1a–7a) were prepared
according to the classical procedure described by Hoo-
Acknowledgements
To CNPq, CAPES, FAPEAL, FAPEMIG and FUJB/
UFRJ for financial support; to Luciana A. Oliveira and
Brian Njaine, for help with the antimalarial assays. The
authors wish to thank PVP (PVP - Produtos Vegetais do
1
7
ker, from the reaction of the appropriated quinones (1–
) with o-phenylenediamine (Scheme 1). General proce-
7
17
dure : a mixture of the quinone (20.6 mmol), 158.5 mmol
of crystalline sodium acetate, 44.5 mmol of phenylene-
diamine hydrochloride and 90 mL of glacial acetic acid
were heated on steam-bath for a short time and mon-
itored by silica gel TLC. After reaction, the mixture was
diluted with water and allowed to stay overnight. The
resulting oil was dissolved in diethyl ether and the organic
solution washed with distilled water, dried over anhy-
drous sodium sulphate, followed by concentration under
vacuum to an amorphous residue from where the phena-
zines were easily isolated in crystalline forms after silica
gel chromatography, using a mixture of hexane–ethyl
acetate of increasing polarity. The chemical structures
Piauı
Parnaı
kind gift of lapachol.
´
˜ ˜
, Rua Dr. Joao Emılio Falcao Costa, 148, Centro,
´
´
ba, Piauı, Brazil, Fax: +55-86-3158006) for a
´
References and notes
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. Kano, S. Prog. Med. 2001, 21, 319.
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. Branda
C. G. C.; Marinuzzi, H. C. J. Ethnopharmacol. 1997, 57,
31.
˜
1
13
1
were checked by H, CNMR, elemental analysis (C, H,
N), IR and UV–vis spectroscopy and are in full accor-
dance with the assigned structures. The new compounds
are 4a, 5a and 6a. The main data for the compounds are
3
4
˜
o, M. G. L.;
1
ꢁ
˜
o, M. G. L.; Krettli, A. U.; Soares, L. S. R.; Nery,
presented: (1a) purple needles, 161.5–162.5 C[161.5–
ꢁ
24
ꢁ
162.5 C, from ETOH ]; (2a) yellow needles, 130 C[lit.
ꢁ
24,25
26
ꢁ
1
130.5–133.5 C
]; (3a) dark yellow needles, 189 C
1
5
6
. McKeage, K.; Scott, L. J. Drugs 2003, 63, 597.
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with decomposition, H NMR: d H [200 MHz, CDCl ; J
3
(Hz)] 9.33–9.27 (m, 1H, H-8), 8.32–8.16 (m, 3H, H-10, H-
13, H-5), 7.84–7.70 (m, 4H, H-6, H-7, H-11, H-12), 4.50
(dd, J=9 and 6 Hz, H-2); 4.00 (dd, J=18 and 6 Hz, H-1),
3.70 (dd, J=18 and 6 Hz, H-1), 1.73 (s, 3H, –CH
3
), 1.62
): d C
-1), 52.5 (CH-2), 78.6
(C-3), 108.6 (C-14b), 122.2, 125.1, 128.1 (C), 128.5 (C),
1
3
7
. Carvalho, L. H.; Rocha, E. M. M.; Raslan, D. S.; Oli-
veira, A. B.; Krettli, A. U. Braz. J. Med. Biol. Res. 1988,
(s, 3H, –CH
3
) ppm. C NMR (50.32 MHz, CDCl
), 30.2 (CH
3
22.3 (CH ), 26.7 (CH
3
3
2
2
1, 485.

V. F. de Andrade-Neto et al. / Bioorg. Med. Chem. Lett. 14 (2004)1145–1149
1149
1
1
28.8 (C), 129.5, 129.6, 129.7, 130.7, 140.0, 140.4, 142.5,
ꢁ
previously diluted in medium without hypoxanthine in 96-
well culture plates. After 24 h of incubation parasite-
drugs, 25 mL/well of medium containing [ H]-hypox-
43.5, 150.5 ppm; (4a), dark yellow needles, 185.5 C . IR
À1
3
(
KBr, cm ): 3057, 2973, 2932, 1625, 1597, 1530, 1494,
1
1
414, 1383, 1348, 1333, 1318, 1236, 1158, 1111, 1100,
053, 1031, 958, 768, 755, 654, 604, 557. UV (lmax., nm,
anthine (0.5 mCi/well) were added followed by 18 h at
ꢁ
30
37 C. The plate was frozen (6–18 h) and thawed, the
cells were harvested [Tomtec 96-Harvester (Tomtec Inc.,
Handen, CT, USA)] in glass fiber filters (Wallac Oy,
Turku, Finland), which were placed in sample bags (Wal-
lac) and immersed in scintillation fluid (Optiphase super
mix, Wallac), followed by radioactive emissions counted
in a 1205 Betaplate reader (Wallac). The reduction in
hypoxanthine uptake, a marker of inhibition of parasite
growth, was calculated in relation to control samples with
no drugs, then plot as function of drug concentrations.
The 50% inhibitory concentration (IC50) values were
determined by linear regression analyses of the linear
segments of the curves.
ꢁ
log ): 424 (4.13), 407 (4.13), 359 (3.88), 301 (4.71), 291
1
(
4.71), 263 (4.65), 275 (4.66), 237 (4.72), 224 (4.68). H
NMR (200 MHz, CDCl , d): 9.27 (m, 1H, H-8); 8.34–8.10
m, 3H); 7.78–7.68 (m, 4H); 4.58 (dd, J=9 and 6 Hz, H-
3
(
1
3
2); 4.1–3.66 (m, 2H); 1.78 (s, 3H); 1.63 (s, 3H); CNMR
(
50.32 MHz, CDCl
3
): d 150.5 (C), 142.4 (C), 139.9 (C),
1
1
39.7 (s); 130.3(C), 129.5 (CH), 129.4 (CH), 129.3 (CH),
29.1 (C), 128.4 (CH), 128.3 (CH), 127.7 (CH), 125.0
(
(
1
1
1
2
CH), 121.8 (CH), 109.3 (C), 78.4 (C), 32.6 (CH ), 31.5
CH
3
ꢁ
), 27.5 (CH
50 C. IR (KBr, cm ): 3055, 2980, 2935, 2840, 1624,
3
), 23.6 (CH); (5a), yellow needles, 149–
À1
597, 1528, 1494, 1413, 1386, 1347, 1334, 1294, 1159,
126, 1035, 987, 913, 859, 755, 659, 605, 560. UV (lmax.
,
28. Trager, W.; Jensen, J. B. Science 1976, 193, 673.
29. Lambros, C.; Vanderberg, J. P. J. Parasitol. 1979, 65, 418.
30. Zalis, M. G.; Pang, L.; Silveira, M. S.; Milhous, W. K.;
Wirth, D. F. Am. J. Trop. Med. Hyg. 1998, 58, 630.
ꢁ
nm, log ): 429 (3.98), 410 (3.99), 359 (3.72), 342 (3.66),
1
3
02 (4.60), 283 (1.86), 276 (4.50), 237 (4.59), 224 (4.54). H
NMR (200 MHz, CDCl ) d H: 9.16 (m, 1H, H-8), 8.14 (m,
H), 7.78 (m, 4H), 6.00 (m, 1H), 5.10 (m, 2H), 3.6 (dd,
3
3
2
3
1
3
31. Antimalarial tests in vivo: the suppressive Peters test as
2
H), 2.82 (dd, 1H), 2.58 (m, 1H), 2.05 (m, 2H), 1.62 (s,
ꢁ
modified was used. Briefly, adult Swiss outbread mice,
20Æ2 g weight, inoculated by intraperitoneal route with
H), 1.18 (s, 3H); (6a) orange needles, 245 Cwith
À1
5
decomposition. IR (KBr, cm ): 3539, 3477, 3337, 1688,
P. berghei-infected red blood cells (1Â10 ), were daily
1
1
5
629, 1596, 1567, 1488, 1452, 1408, 1337, 1240, 1217,
175, 1115, 1094, 1055, 1042, 994, 936, 857, 782, 729, 628,
35, 452. H NMR (200 MHz, CDCl ) d H: 9.2 (m, 1H,
treated by oral or subcutaneous route, during 4 days. The
samples were dissolved in an aqueous solution of DMSO
0.02% (v:v) and this solution was, later, diluted to the
final volume with saline and given in a 200 mL volume per
animal. Two control groups (n5) were used each time, one
treated with non-curative doses of chloroquine (ꢀ25 mg/
kg), one untreated or treated with saline, as specified in
the results. On day 5 after parasite inoculation blood
smears were prepared, methanol fixed, stained with
Giemsa, and microscopically examined by counting para-
sitaemia in 1000 up to 6000 erythrocytes. Overall mortal-
ity was daily monitored until all controls died. The
inhibition of parasite growth in the drug-treated groups
1
3
H-8); 8.32–8.18 (m, 1H); 7.96–7.76 (m, 2H); 7.04–6.92 (m,
2
1
1
H); 6.90–6.80 (m, 2H); 3.28 (d, 1H); 3.18–3.02 (m, 1H);
.87 (s, 3H); 1.47 (s, 3H) and (7a), yellow needles,
28 C, H NMR (200 MHz, CDCl ) d H 9.42–9.39 (m,
ꢁ
25 1
3
ArH); 8.38–8.26 (m, 2 ArH); 8.03–7.75 (m, 7 ArH).
4. Hooker, S. C. J. Am. Chem. Soc. 1936, 58, 1190.
5. Benedetti-Doctorovich, V.; Escola, N.; Burton, G. Magn.
Res. Chem. 1998, 36, 529.
2
2
2
2
6. Kehrmann, F.; Mermod, C. Helv. Chim. Acta 1927, 10,
65.
ꢁ
33
7. The phenazines (1a–7a, Fig. 1) were maintained at À20 C
as 10 mg/mL stock solutions in dimethylsulfoxide (Sigma,
St. Louis, MO, USA) and used as a solution with final
concentration of DMSO as 0.002%. P. falciparum culture
and antimalarial tests in vitro: four P. falciparum isolates
with different susceptibility to chloroquine and mefloquine
were used: clone W2 (chloroquine-resistant, mefloquine-
sensitive); clone D6 (chloroquine-sensitive, mefloquine-
resistant); clone HB3 (chloroquine and mefloquine-sensi-
tive); and isolate BHz 26/86 (chloroquine partially resistant),
previously isolated from an imported case of malaria from
was calculated in relation to non-treated controls. All
phenazines were tested in 3 independent experiments.
For 6a, an additional experiment was performed, using a
7-day schedule, by subcutaneous route, with parasitaemia
monitored at several days after inoculation.
32. Peters, W. Exp. Parasitol. 1965, 17, 80.
˜
33. Andrade-Neto, V. F.; Brandao, M. G. L.; Stehmann,
J. R.; Oliveira, L. A.; Krettli, A. U. J. Ethnopharmacol.
2003, 87, 253.
34. Ren, H.; Grady, S.; Gamenara, D.; Heinzen, H.; Moyna,
P.; Croft, S. L.; Kendrick, H.; Yardley, V.; Moyna, G.
Bioorg. Med. Chem. Lett. 2001, 11, 1851.
35. Zani, C. L.; Chiari, E.; Krettli, A. U.; Murta, S. M. F.;
Cunningham, M. L.; Fairlamb, A. H.; Romanha, A. J.
Bioorg. Med. Chem. 1997, 5, 2185.
7
the Amazon region. All parasites were maintained in
2
8
continuous culture on human erythrocytes as described.
2
9
Briefly, trophozoites synchronized in sorbitol were cul-
tured at 1–2% parasitaemia, incubated with phenazines