2832 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17
Delarue et al.
Ma ter ia ls a n d Meth od s
Refer en ces
(1) WHO Practical Chemotherapy of malaria; Technical Report
Series No. 805; World Health Organization: Geneva, 1990.
(2) Rieckmann, K. Determination of the Drug Sensitivity of Plas-
modium falciparum. J AMA, J . Am. Med. Assoc. 1971, 217, 573-
578.
(3) Watkins, W. M.; Spencer, H. C.; Kariuki, D. M.; Sixsmith, D.
G.; Boriga, D. A.; Kipingor, T.; Koech, D. K. Effectiveness of
Amodiaquine as Treatment for Chloroquine-Resistant Plasmo-
dium falciparum Infections in Kenya. Lancet 1984, 357-359.
(4) Muller, O.; Boele van Hensbroek, M.; J affar, S.; Drakeley, C.;
Okorie, C.; J oof, D.; Pinder, M.; Greenwood, B. A Randomized
Trial of Chloroquine, Amodiaquine and Pyrimethamine-Sulpha-
doxine in Gambian Children with Uncomplicated Malaria. Trop.
Med. Int. Health 1996, 1, 124-132.
(5) Brasseur, P.; Guiguemde, R.; Diallo, S.; Guiyedi, V.; Kombila,
M.; Ringwald, P. Amodiaquine Remains Effective for Uncom-
plicated Infections in West and Central Africa. Trans. R. Soc.
Trop. Med. Hyg. 1999, 93, 645-650.
(6) Hatton, C. S. R.; Bunch, C.; Peto, T. E. A.; Pasvol, G.; Russell,
S. J .; Singer, C. R. J .; Edwards, G.; Winstanley, P. Frequency
of Severe Neutropenia associated with Amodiaquine Prophylaxis
against Malaria. Lancet 1986, 411-414.
(7) Neftel, K. A.; Woodtly, W.; Schmid, M.; Frick, P. G.; Fehr, J .
Amodiaquine Induces Agranulocytosis and Liver Damage. Br.
Med. J . 1986, 292, 721-723.
(8) Warrell, D. A.; Molyneux, M. E.; Beales, P. P. Severe and
Complicated Malaria. Trans. R. Soc. Trop. Med. Hyg. 1990, 84,
1-65.
(9) Ringwald, P.; Keundjian, A.; Ekobo, A.; Basco, L. K. Resistance
of P. falciparum in urban environment at Yaounde, Cameroun.
Part 2: Evaluation of the efficiency of amodiaquine and the
sulfadoxine-pyrimethamine association for the treatment of P.
falciparum malaria at Yaounde, Cameroun. Trop. Med. Int.
Health 2000, 5, 620-627.
(10) Maggs, J . L.; Kitteringham, N. R.; Park, B. K. Drug-Protein
Conjugates. Mechanism of Formation of Protein Arylating
Intermediates from Amodiaquine in Man. Biochem. Pharmacol.
1988, 37, 303-311.
(11) Harrison, A. C.; Kitteringham, N. R.; Clarke, J . B.; Park, B. K.
Mechanism of Bioactivation and Antigene Formation of Amo-
diaquine in the Rat. Biochem. Pharmacol. 1992, 43, 1421-1430.
(12) Clarke, J . B.; Maggs, J . L.; Kitteringham, N. R.; Park, B. K.
Detection of IgG Antibodies in Patients with Adverse Drug
Reactions to Amodiaquine. Int. Arch. Allergy Immunol. 1990,
1335-1342.
(13) Naisbitt, D. J .; Williams, D. P.; O’Neill, P. M.; Maggs, J . L.;
Willock, D. J .; Pirmohamed, M.; Park, B. K. Metabolism-
dependent Neutrophil Cytotoxicity of Amodiaquine: a Compari-
son with Pyronaridine and related Antimalarial Drugs. Chem.
Res. Toxicol. 1998, 11 (12), 1586-1595.
(14) Naisbitt, D. J .; Ruscoe, J . E.; Williams, D. P.; O’Neill, P. M.;
Pirmohamed, M.; Park, B. K. Disposition of Amodiaquine and
related Antimalarial Agents in Human Neutrophils: Implica-
tions for Drug Design. J . Pharmacol. Exp. Ther. 1997, 280 (2),
884-893.
(15) Tingle, M. D.; J ewell, H.; Maggs, J . L.; O’Neill, P. M.; Park, B.
K. The Bioactivation of Amodiaquine by Human Polymorpho-
nuclear Leucocytes in vitro: Chemical Mechanisms and the
Effects of Fluorine Substitution. Biochem. Pharmacol. 1995, 50
(7), 1113-1119.
(16) O’Neill, P. M.; Harrison, A. C.; Storr, R. C.; Hawley, S. R.; Ward,
S. A.; Park, B. K. The Effect of Fluorine Substitution on the
Metabolism and Antimalarial Activity of Amodiaquine. J . Med.
Chem. 1994, 37, 1362-1370.
(17) Blauer, G. Interaction of Ferritoporphyrin IX with the Antima-
larials Amodiaquine and Halofantrine. Biochem. Int. 1988, 17,
729-734.
(18) O’Neill, P. M.; Willock, D. J .; Hawley, S. R.; Bray, P. G.; Storr,
R. C.; Ward, S. A.; Park, B. K. Synthesis, Antimalarial Activity,
and Molecular Modeling of Tebuquine Analogues. J . Med. Chem.
1997, 40, 437-448.
Biologica l Eva lu a tion . Compounds 1-21 were evaluated
in their chlorohydrate form.
In Vitr o P . fa lcipa r u m Cu ltu r e a n d Dr u g Assa ys. P.
falciparum strains were maintained continuously in culture
on human erythrocytes as described by Trager and J ensen.30
In vitro antiplasmodial activity was determined using a
modification of the semiautomated microdilution technique of
Desjardins et al.31 P. falciparum CQ-sensitive (THAI/Thailand)
and CQ-resistant (FcB1R/Colombia and K1/Thailand) strains
were used in sensitivity testing. Stock solutions of chloroquine
diphosphate and test compounds were prepared in sterile
distilled water and DMSO, respectively. Drug solutions were
serially diluted with culture medium and introduced to
asynchronous parasite cultures (0.5% parasitemia and 1% final
hematocrite) on 96-well plates for 24 h at 37 °C prior to the
addition of 0.5 µCi of [3H]hypoxanthine (1-5 Ci/mmol; Amer-
sham, Les Ulis, France) per well for 24 h. The growth
inhibition for each drug concentration was determined by
comparison of the radioactivity incorporated into the treated
culture with that in the control culture (without drug) main-
tained on the same plate. The concentration causing 50%
inhibition (IC50) was obtained from the drug concentration-
response curve, and the results were expressed as the mean
( standard deviation determined from several independent
experiments. The DMSO concentration never exceeded 0.1%
and did not inhibit the parasite growth.
Cytotoxicity Test u p on MRC-5 Cells a n d Mou se P er i-
ton ea l Ma cr op h a ges. A human diploid embryonic lung cell
line (MRC-5, Bio-Whittaker 72211D) and mouse primary
peritoneal macrophages were used to assess the cytotoxic
effects toward host cells. The peritoneal macrophages were
collected from the peritoneal cavity 48 h after stimulation with
potato starch, and they were seeded in 96-well microplates at
30000 cells per well. MRC-5 cells were seeded at 5000 cells
per well. After 24 h, the cells were washed, and 2-fold dilutions
of the drug were added in 200 µL of standard culture medium
(RPMI + 5% FCS). The final DMSO concentration in the
culture remained below 0.5%. The cultures were incubated
with several concentrations of compounds (between 32 and 1.6
µM) at 37 °C in 5% CO2-95% air for 7 days. Untreated cultures
were included as controls. For MRC-5 cells, the cytotoxicity
was determined using the colorimetric MTT assay31 and scored
as a percent reduction of absorption at 540 nm of treated
cultures versus untreated control cultures. For macrophages,
scoring was performed microscopically.
In Vivo Dr u g Assa ys u p on P . ber gh ei. The antimalarial
activities were determined in mice infected with P. berghei
(ANKA 65 strain). Four week old female Swiss mice (CD-1,
20-25 g) were intraperitoneally infected with about 107
parasitized erythrocytes, collected from the blood of an acutely
infected donor animal. At the same time, the animals (three
animals per group) were orally treated with the test compound
at 40 mg/kg (drug formulation in 100% DMSO). The treatment
was continued during the following 4 days by the intraperi-
toneal route. Untreated control animals generally die between
7 and 10 days following infection. Drug activity was evaluated
by the reduction of parasitaemia at day 4 and by the prolonga-
tion of the mean survival time compared to that of untreated
controls. Three infected, DMSO-dosed mice were used as
controls.
(19) Chou, A. C.; Fitch C. D. Control of Heme Polymerase by
Chloroquine and other Quinoline Derivatives. Biochem. Biophys.
Res. Commun. 1993, 195, 422-427.
(20) Slater, A. F. G. Chloroquine: Mechanism of Drug Action and
Resistance in Plasmodium falciparum. Pharmacol. Ther. 1993,
57, 203-235.
(21) Koh, H. L.; Go, M. L.; Ngiam, T. L.; Mak, J . W. Conformational
and Structural Features Determining in vitro Antimalarial
Activity in some Indolo Quinolines, Anilinoquinolines and Tet-
rahydroindolo-benzazepines. Eur. J . Med. Chem. 1994, 29, 107-
113.
(22) Fitch, C. D. Chloroquine Resistant Plasmodium falciparum:
Difference in the Handling of 14C-Amodiaquine and 14C-
Chloroquine. Antimicrob. Agents Chemother. 1973, 3, 545-548.
Ack n ow led gm en t. We express our thanks to Dr.
Elisabeth Davioud-Charvet for her help, Ge´rard Mon-
tagne for NMR experiments, and Dr. Steve Brooks for
proofreading. This work was supported by the CNRS
(GDR 1077, IFR CNRS 63, UMR CNRS 8525) and
Universite´ de Lille II. S.D. is a recipient of fellowships
from the Re´gion Nord-Pas de Calais.
Su p p or tin g In for m a tion Ava ila ble: Details of chemical
procedures and spectral data. This material is available free