"One-pot" synthesis and antimalarial activity of formamidine derivatives of 4-anilinoquinoline

Article abstract of DOI:10.1248/cpb.49.933

Amodiaquine (AQ) is an antimalarial which is effective against chloroquino-resistant strains of Plasmodium falciparum but whose clinical use is severely restricted because of associated hepatotoxicity and agranulocytosis. "One-pot" synthesis of formamidin

Full text of DOI:10.1248/cpb.49.933

August 2001
Chem. Pharm. Bull. 49(8) 933—937 (2001)
933
“One-Pot” Synthesis and Antimalarial Activity of Formamidine
Derivatives of 4-Anilinoquinoline
Sandrine DELARUE,a Sophie GIRAULT,a Fouad DALI ALI,b Louis MAES,c Philippe GRELLIER,b and
,a
*
Christian SERGHERAERT
UMR CNRS 8525, Institut de Biologie et Institut Pasteur de Lille, Faculté de Pharmacie, Université de Lille II,a 1 rue du
Professeur Calmette, BP 447, 59021 Lille Cedex, France, Laboratoire de Biologie Parasitaire, IFR CNRS 63, Muséum
National d’Histoire Naturelle,b 61 rue Buffon, 75005 Paris, France, and TIBOTEC,c L11 Général de Wittelaan, B-32800
Mechelen, Belgium. Received December 14, 2000; accepted April 13, 2001
Amodiaquine (AQ) is an antimalarial which is effective against chloroquino-resistant strains of Plasmodium
falciparum but whose clinical use is severely restricted because of associated hepatotoxicity and agranulocytosis.
“One-pot” synthesis of formamidines likely to be transformed into AQ derivatives is reported. Compared with
AQ, the new compounds were devoid of in vitro cytotoxicity upon human embryonic lung cells and mouse peri-
toneal macrophages. One showed a potent in vivo activity in mice infected with P. berghei. Transformation of this
compound by reductive amination led to a new type of AQ derivatives that displayed an in vitro activity similar to
that of AQ but did not lead to toxic quinone-imines.
Key words drug design; malaria; Plasmodium falciparum; chloroquine; amodiaquine; formamidine
Today, almost one half of the world’s population is ex- ries of formamidines likely to be transformed into 4Ј-dehy-
posed to the threat of malaria and the disease was responsible droxy AQ derivatives was prepared from the benzylic alcohol
for about 2 million deaths in 1997.1) Quinoline-containing 1 (Fig. 2). Formamidines have been used extensively as phar-
antimalarials, such as chloroquine (CQ, Fig. 1), have long macological agents,10—12) and we have recently described
been used in the struggle against malaria. Since its discovery, their simple preparation in which bromo-tris-pyrrolidino-
CQ has proven to be a highly effective, safe, and well-toler- phosphonium hexafluorophosphate (PyBroP) is used as a
ated drug for the treatment and prophylaxis of malaria. But convenient activator of the formamide group.13) In the new
the emergence of CQ-resistant strains has underlined the series, antimalarial activity could be expected from the pres-
need for a synthetic alternative to CQ. The spread of resis- ence of the amidine side chain capable of interacting with a
tance2) has prompted the re-examination of alternative anti- carboxylate group of heme. The synthesis and antimalarial
malarials, such as amodiaquine (AQ, Fig. 1) which was activity of these compounds (2—6, Fig. 2) are reported as
found to be effective against both CQ-sensitive and CQ-resis- well as those of the AQ analogue (7, Fig. 2) corresponding to
tant strains.3) However, the clinical use of AQ has been se- the most active among them.
verely restricted because of associations with hepatotoxicity
and agranulocytosis.4,5) As paracetamol, due to the presence Chemistry
of the 4Ј-hydroxyl substituent, AQ is known to undergo ex-
The synthesis of compound 1 and that of the formamidine
tensive bioactivation by hepatic cytochrome P-450 enzymes derivatives 2—7 were carried out as shown in Chart 2. Start-
to a chemically reactive quinone–imine (AQQI, Chart 1), fol- ing material 1 was obtained by condensation of 4,7-dichloro-
lowed by nucleophilic addition of glutathione. The formation quinoline with 3,5-diaminobenzyl alcohol according to an
of these reactive species in vivo and their binding to cellular
proteins and lipids could affect cell function either directly or
by immune response.6—8) A 4Ј-dehydroxyfluoro AQ deriva-
tive has been reported9) and proved not to give a quinone-
imine by simple oxidation. This was reflected in its high oxi-
dation potential and no bioactivation. However, compared
with AQ, it was found to be less potent against both CQ-re-
sistant and CQ-sensitive parasites.
With the aim of avoiding bioactivation and toxicity, a se- Fig. 1. Structure of Chloroquine and Amodiaquine
Chart 1. Oxidative Pathways of Amodiaquine and Paracetamol
To whom correspondence should be addressed. e-mail: christian.sergheraert@pasteur-lille.fr
© 2001 Pharmaceutical Society of Japan

934
Vol. 49, No. 8
Fig. 2. Structure of Compounds 1—7
Chart 2. Synthesis of Compounds 1—7
aromatic nucleophilic substitution mechanism.14) Stirring of
the reactive medium for 3 d in a EtOH/CHCl3 55 : 5 mixture,
at room temperature, led to the precipitation of compound 1,
which was collected by filtration. Selective precipitation of
compound 1 was due to its very poor solubility in EtOH and
thus no further purification was necessary. The optimized
procedure leads to yields as high as 88% (Table 1). The ab-
sence of formation of the corresponding bisquinoline can be
explained both by the precipitation of compound 1 and by its
capacity for steric hindrance.
Table 1. Optimization of the Synthesis of Compound 1
Ratio
amine/quinoline
Ratio
EtOH/CHCl3
Assay
Time (h)
Yield (%)
a
b
c
d
1
1
1
1
70 : 10
55 : 5
55 : 5
55 : 5
48
24
48
72
56
50
85
88
Fixation of the amino side chain was carried out in two
steps using a “one-pot” procedure. During the first step, com-
pound 2 was obtained by reaction of the PyBroP-activated
DMF derivative with compound 1.13) During the second step,
the dimethylamino moiety was substituted by various sec-
ondary amines HNRRЈ. Dichloromethane was used as a co-
solvent to permit reduction at reflux temperature and thus
avoid the degradation of the formamidine into formamide.
Amine conversion rates were quantitative, but strong interac-
tions between the final products and silica phase during chro-
matography purification led to moderate yields (27—43%).
Synthesis of compound 7 from compound 1 was carried

August 2001
935
Table 2. In Vitro Antimalarial Activity against P. falciparum FcB1R Strain and in Vitro Cytotoxicity upon MRC-5 Cells and Mouse Peritoneal
Macrophages of Compounds 1—7
Cytotoxicity (%) upon MRC-5 at (mM)
IC50 (nM)
against FcB1R
Cytotoxicity upon MPM
No toxicity at (mM)
Compound
25
12.5
6.3
3.1
CQ
AQ
1
2
3
4
5
6
7
142
20
0
91
20
0
0
0
0
0
90
0
0
0
0
0
0
0
0
5
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
12.5
3.1
300
520
170
190
600
550
20
12.5
12.5
12.5
12.5
12.5
12.5
12.5
out in three steps. The benzylic alcohol group of compound 1 against both CQ-sensitive and -resistant strains.9) In the 4Ј-
was first converted into an aldehyde by oxidation with MnO2, dehydroxyfluoro AQ, the lack of the hydrogen bond, which is
followed by reductive amination with diethylamine using present in AQ between the phenol group and the diethyl-
sodium triacetoxyborohydride as reducing agent (Chart 2).15) amino moiety, was responsible for the decreased activity. In
A low yield was observed because of a side reaction during compound 7, this phenomenon is balanced by an additional
the oxidation step, since the amine moiety of the molecule amino moiety. The increase in activity observed can be ex-
can react with the aldehyde group of another molecule lead- plained by the presence of a bis-cationic site, likely to inter-
ing to a polymerization product. The previous protection of act simultaneously with the two carboxylate groups of heme.
the amine moiety by a Fmoc or by a Boc group was found to Moreover, its synthesis is simple, when compared with the
be unsuccessful. In the final step, the procedure used for five steps, including use of bromine, irradiation, and catalyti-
compounds 3—6 was applied to obtain the desired com- cal hydrogenation, required to prepare 4Ј-dehydroxyfluoro
pound 7 in 45% yield.
AQ.
In conclusion, a series of formamidines without the hy-
droxyl group of AQ, eliciting hepatotoxicity and aganulocy-
Biological Results and Discussion
All compounds were tested for their antimalarial potency tosis by bioactivation, was synthesized using a “one-pot”
against a CQ-resistant FcB1R strain (IC50 CQϭ142 nM) and method from an aminoalcohol, easily obtained by precipita-
for their cytotoxicity upon human MRC-5 cells (human tion. The new compounds are non cytotoxic upon a human
diploid embryonic lung cell line) and mouse primary peri- embryonic lung cell line and upon MPM. The most active
toneal macrophages (MPM) (Table 2).
among them has an in vitro antimalarial activity in the same
Formamidines 2—6 were found to be less active than AQ range as CQ against three strains varying in their resistance
in vitro against the CQ-resistant FcB1R strain but were non to CQ, while also demonstrating an activity against P. berghei
cytotoxic at a concentration of 25 mM upon MRC-5 cells as in mice. These formamidines are intermediates for the facile
well as upon MPM. Two compounds, 3 and 4, displayed an in synthesis of new AQ derivatives, have activity similar to that
vitro antimalarial effect against the FcB1R strain in the same of AQ with less cytotoxicity, and do not lead to toxic
range as that of CQ while the others were less active. There- quinone–imine formation.
fore the presence in the side chain of a cyclic tertiary amine
without an additional hetero atom appears favourable for an- Experimental
Chemistry All reactions were monitored using thin-layer chromatogra-
timalarial activity. The most active, compound 3, was then
tested in vitro against two other strains of P. falciparum, F32a
and PFB, which are respectively more CQ-sensitive (IC50
phy carried out on 0.2 mm E. Merck silica gel plates (60F-254) using UV
1
light as visualizing agent. H-NMR spectra were obtained using a Brücker
300 MHz spectrometer. Mass spectra were recorded on a time-of-flight
CQϭ25 nM) and more CQ-resistant (IC50 CQϭ340 nM) than
the FcB1R strain. IC50 values of compound 3 were, respec-
tively, 80 and 355 nM, thus revealing a correlation with CQ
values and potentially a mode of action similar to both CQ
and AQ, i.e. by inhibiting heme polymerization.16) Com-
pound 3 was also tested at 40 mg/kg in mice infected with
Plasmodium berghei. While the control mice died 7 d follow-
ing infection, animals receiving compound 3 displayed a
60% reduction in parasitemia at 7 d and mean survival time
of about 19 d.
AQ derivative 7, corresponding to the formamidine 3, was
synthesized and displayed an activity similar to that of AQ
against the CQ-resistant FcB1R strain while its cytotoxicity
was slightly inferior to that of AQ. Therefore it proved to be
more active than 4Ј-dehydroxyfluoro AQ which displayed a
2-fold increase in IC50 values when compared with AQ
plasma desorption mass spectrometer (TOF-PDMS) using a Californium
source. Chromatography was carried out using silica gel 60 (230—400 mesh
ASTM) from Macherey-Nagel. Thick-layer chromatography (TLC) was per-
formed using silica gel from Merck and the compounds were extracted from
silica gel using the following solvent system: CH2Cl2/MeOH 80 : 20. The
melting point (mp) of the chlorhydrate form for compounds 1—6 and of the
basic form for compound 7 were determined on a Büchi 535 capillary mp
apparatus and were uncorrected. The purity of final compounds was checked
by high pressure liquid chromatography (PHPLC) with a C18 Vydac column.
Analytical HPLC was performed on a Shimadzu system equipped with a UV
detector set at 254 nm. Compounds were dissolved in MeOH and injected
through a 50 ml loop. The following eluent systems were used: A (H2O/TFA,
100 : 0.05) and B (CH3CN/H2O/TFA, 80 : 20 : 0.05). HPLC retention times
(HPLC tR) were obtained at flow rates of 1 ml/min using a gradient run from
100% eluent A for 5 min and then to 100% eluent B over the next 25 min.
CQ and AQ were obtained from Aldrich and Sigma, respectively. 4,7-
Dichloroquinoline and 3,5-diaminobenzyl alcohol dihydrochloride were ob-
tained from Aldrich.
Procedure for Chlorhydrate Salts Trimethylchlorosilane (1 eq) was

936
Vol. 49, No. 8
added to a solution of formamidine (1 eq) in MeOH. After stirring the mix-
ture for 15 min at room temperature, the solvent was evaporated to yield the
desired chlorhydrate.
Ar-H), 6.88 (1H, s, Ar-H), 6.94 (1H, d, Jϭ5.3 Hz, H quinoline), 7.50 (1H,
dd, Jϭ9.0, 2.2 Hz, H quinoline), 7.76 (1H, s, NϭCH–NRRЈ), 7.85 (1H, d,
Jϭ2.2 Hz, H quinoline), 8.38 (1H, d, Jϭ9.3 Hz, H quinoline), 8.41 (1H, d,
Jϭ5.3 Hz, H quinoline), 8.96 (1H, s, NH). TOF-PDMS m/z: 396.7 (Mϩ).
HPLC: PHPLC 99%, tR 11.09 min.
{3-Amino-5-[(7-chloro-4-quinolyl)amino]phenyl}methanol (1) To a
solution of 4,7-dichloroquinoline (1.88 g, 9.5 mmol, 1 eq), and N-methyl-
morpholine (1.3 ml, 11.8 mmol, 1.2 eq), in EtOH 50 ml was added a solution
of 3,5-diaminobenzyl alcohol dihydrochloride (2 g, 9.5 mmol, 1 eq), and N-
methylmorpholine (1.7 ml, 15.5 mmol, 1.6 eq), in a EtOH/CHCl3 50 : 50
mixture 10 ml, at 0 °C. After stirring for 72 h at room temperature, the mix-
ture was filtered and the residual solid washed with ice cold EtOH and dried
to yield compound 1 as a yellow solid. Yield: 88% (2.5 g). mp: 206 °C. Rf:
0.13 (CH2Cl2/MeOH, 9 : 1). 1H-NMR (DMSO-d6) d: 2.70 (1H, s, OH), 3.60
(2H, br s, NH2), 4.47 (2H, s, CH2), 6.82 (1H, d, Jϭ7.0 Hz, H quinoline),
6.85 (1H, s, Ar-H), 6.92 (2H, s, Ar-H), 7.84 (1H, dd, Jϭ9.1, 2.1 Hz, H
quinoline), 8.10 (1H, d, Jϭ2.0 Hz, H quinoline), 8.52 (1H, d, Jϭ7.0 Hz, H
quinoline), 8.78 (1H, d, Jϭ9.2 Hz, H quinoline), 11.06 (1H, s, NH). TOF-
PDMS m/z: 299.4 (Mϩ). HPLC: PHPLC 98%, tR 11.85 min.
N-{3-[(7-Chloro-4-quinolyl)amino]-5-[(piperidinomethylidene)-
amino]phenyl}methyl-N,N-diethyl amine (7) To a solution of compound
1 (600 mg, 2 mmol, 1 eq), in a CH2Cl2/DMF 60 : 1 mixture 35 ml were added
DIEA (350 ml, 2 mmol, 2 eq), and MnO2 (2.6 g, 30 mmol, 15 eq). After stir-
ring the mixture for 18 h at room temperature, the mixture was filtrated on
celite and the solid residue washed with CH2Cl2 30 ml. To the filtrate was
then added diethylamine (1 ml, 10 mmol, 5 eq), and, after stirring at room
temperature for 1 h, NaHB(OAc)3 (1.2 g, 6 mmol, 3 eq) was added. Follow-
ing further stirring at room temperature for 18 h, aqueous NaHCO3 1 M was
introduced and the mixture was left to stir for 15 min. The organic layer was
then separated, and the aqueous layer washed with CH2Cl2. The organic lay-
ers were combined, washed with brine, separated and dried over MgSO4.
The solvent was evaporated and the residue purified by TLC (CH2Cl2/
MeOH, 80 : 20) to yield the desired intermediate in 30% yield (210 mg). To
a solution of this intermediate (130 mg, 0.37 mmol, 1 eq) in DMF 10 ml were
added DIEA (65 ml, 0.73 mmol, 2 eq), and PyBroP (180 mg, 0.39 mmol, 1.1
eq). After stirring the mixture for 12 h at room temperature, piperidine was
added (145 ml, 1.47 mmol, 4 eq). Following reflux of the mixture for 5 h, the
solvent was evaporated, the solid residue washed with 1 M NaHCO3 15 ml
and purified by TLC (CH2Cl2/MeOH, 80 : 20), to yield compound 7 as a yel-
low solid. Yield: 45% (75 mg). mp: 79 °C. Rf: 0.30 (CH2Cl2/MeOH, 7 : 3).
1H-NMR (DMSO-d6) d: 1.00 (6H, t, Jϭ4.6 Hz, CH2CH3), 1.52—1.54 (2H,
m, CH2 piperidine), 1.57—1.66 (4H, m, CH2 piperidine), 2.90—3.03 (8H,
m, CH2 piperidine and CH2CH3), 3.55 (2H, s, CH2-Ph), 6.64 (1H, s, Ar-H),
6.72 (1H, s, Ar-H), 6.95 (1H, d, Jϭ5.4 Hz, H quinoline), 7.00 (1H, s, Ar-H),
7.48 (1H, dd, Jϭ9.0, 2.3 Hz, H quinoline), 7.85 (1H, d, Jϭ2.2 Hz, H quino-
line), 8.28 (1H, s, NϭCH–NRRЈ), 8.42 (1H, d, Jϭ9.1 Hz, H quinoline), 8.46
(1H, d, Jϭ5.3 Hz, H quinoline), 9.02 (1H, s, NH). TOF-PDMS m/z: 450.6
(Mϩ). HPLC: PHPLC 100%, tR 12.68 min.
N؅-{3-[(7-Chloro-4-quinolyl)amino]-5-[hydroxymethyl]phenyl}-N,N-
dimethyliminoformamide (2) To a solution of compound 1 (150 mg, 0.5
mmol, 1 eq), in DMF 10 ml were added DIEA (88 ml, 1 mmol, 2 eq), and Py-
BroP (235 mg, 0.5 mmol, 1 eq). After stirring the mixture for 7 h at room
temperature, the solvent was evaporated, the solid residue washed with 1 M
NaHCO3 15 ml and purified by TLC (CH2Cl2/MeOH, 60 : 40), to yield com-
pound 2 as a yellow solid. Yield: 42% (50 mg). mp: 166 °C. Rf: 0.37
1
(CH2Cl2/MeOH, 6 : 4). H-NMR (DMSO-d6) d: 2.87 (3H, br s, CH3), 2.94
(3H, br s, CH3), 4.42 (2H, s, CH2-Ph), 5.11 (1H, br s, OH), 6.63 (1H, s, Ar-
H), 6.71 (1H, s, Ar-H), 6.86 (1H, s, Ar-H), 6.89 (1H, d, Jϭ5.4 Hz, H quino-
line), 7.50 (1H, dd, Jϭ9.0, 2.1 Hz, H quinoline), 7.71 (1H, s, NϭCH–
NRRЈ), 7.83 (1H, d, Jϭ2.2 Hz, H quinoline), 8.38 (1H, d, Jϭ8.8 Hz, H
quinoline), 8.41 (1H, d, Jϭ5.2 Hz, H quinoline), 8.94 (1H, s, NH). TOF-
PDMS m/z: 354.8 (Mϩ). HPLC: PHPLC 94%, tR 11.00 min.
General Procedure for Synthesis of Compounds 3—6 To a solution
of compound 1 (150 mg, 0.5 mmol, 1 eq), in DMF 10 ml were added DIEA
(88 ml, 1 mmol, 2 eq), and PyBroP (235 mg, 0.5 mmol, 1 eq). After stirring
the mixture for 12 h at room temperature, the appropriate amine (4 mmol, 8
eq) was added. Following reflux of the mixture for 5 h, the solvent was evap-
orated, the solid residue washed with 1 M NaHCO3 15 ml and purified by
TLC (CH2Cl2/MeOH, 70 : 30), to yield the desired compound.
{3-[(7-Chloro-4-quinolyl)amino]-5-[(piperidinomethylidene)amino]-
phenyl}methanol (3) Yellow solid. Yield: 40% (80 mg). mp: 231 °C. Rf:
0.66 (CH2Cl2/MeOH, 7 : 3). 1H-NMR (DMSO-d6) d: 1.55—1.57 (2H, m,
CH2 piperidine), 1.66—1.70 (4H, m, CH2 piperidine), 2.93—2.99 (4H, m,
CH2 piperidine), 4.42 (2H, s, CH2-Ph), 5.10 (1H, br s, OH), 6.63 (1H, s, Ar-
H), 6.70 (1H, s, Ar-H), 6.86 (1H, s, Ar-H), 6.89 (1H, d, Jϭ5.4 Hz, H quino-
line), 7.49 (1H, dd, Jϭ9.0, 2.3 Hz, H quinoline), 7.69 (1H, s, NϭCH–
NRRЈ), 7.83 (1H, d, Jϭ2.2 Hz, H quinoline), 8.38 (1H, d, Jϭ9.1 Hz, H
quinoline), 8.40 (1H, d, Jϭ5.3 Hz, H quinoline), 8.95 (1H, s, NH). TOF-
PDMS m/z: 394.8 (Mϩ). HPLC: PHPLC 95%, tR 12.12 min.
{3-[(7-Chloro-4-quinolyl)amino]-5-[({4-methylpiperidino}methyli-
dene)amino]phenyl}methanol (4) Yellow solid. Yield: 37% (75 mg). mp:
188 °C. Rf: 0.70 (CH2Cl2/MeOH, 8 : 2). 1H-NMR (DMSO-d6) d: 0.99 (3H, d,
Jϭ6.4 Hz, CH3), 1.12 (2H, m, CH2 piperidine), 1.29 (1H, s, CH piperidine),
1.65—1.80 (4H, m, CH2 piperidine), 3.06 (2H, m, CH2 piperidine), 4.52
(2H, d, Jϭ5.8 Hz, CH2-Ph), 5.19 (1H, t, Jϭ5.8 Hz, OH), 6.73 (1H, s, Ar-H),
6.80 (1H, s, Ar-H), 6.97 (1H, s, Ar-H), 6.99 (1H, d, Jϭ5.4 Hz, H quinoline),
7.60 (1H, dd, Jϭ9.0, 2.2 Hz, H quinoline), 7.80 (1H, s, NϭCH–NRRЈ), 7.93
(1H, d, Jϭ2.2 Hz, H quinoline), 8.48 (1H, d, Jϭ9.1 Hz, H quinoline), 8.51
(1H, d, Jϭ5.3 Hz, H quinoline), 9.04 (1H, s, NH). TOF-PDMS m/z: 408.4
(Mϩ). HPLC: PHPLC 95%, tR 13.19 min.
{3-[(7-Chloro-4-quinolyl)amino]-5-[({4-methylpiperazino}methyli-
dene)amino]phenyl}methanol (5) Yellow solid. Yield: 27% (55 mg). mp:
195 °C. Rf: 0.28 (CH2Cl2/MeOH, 7 : 3). 1H-NMR (DMSO-d6) d: 2.16 (3H, s,
CH3), 2.27 (4H, t, Jϭ5.1 Hz, CH2 piperazine), 3.40—3.50 (4H, m, CH2
piperazine), 4.42 (2H, s, CH2-Ph), 5.10 (1H, br s, OH), 6.64 (1H, s, Ar-H),
6.72 (1H, s, Ar-H), 6.88 (1H, s, Ar-H), 6.89 (1H, d, Jϭ5.4 Hz, H quinoline),
7.50 (1H, dd, Jϭ9.0, 2.2 Hz, H quinoline), 7.73 (1H, s, NϭCH–NRRЈ), 7.83
(1H, d, Jϭ2.2 Hz, H quinoline), 8.38 (1H, d, Jϭ8.9 Hz, H quinoline), 8.41
(1H, d, Jϭ5.3 Hz, H quinoline), 8.95 (1H, s, NH). TOF-PDMS m/z: 409.9
(Mϩ). HPLC: PHPLC 95%, tR 12.09 min.
Biological Evaluation Compounds 1—6 were evaluated in their chlorhy-
drate form and compound 7 in its basic form.
In Vitro P. falciparum Culture and Drug Assays P. falciparum strains
were maintained continuously in culture on human erythrocytes as described
by Trager and Jensen.17) In vitro antiplasmodial activity was determined
using a modification of the semi-automated microdilution technique of Des-
jardins et al.18) P. falciparum CQ-sensitive (F32a/Tanzania) and CQ-resistant
(FcB1R/Colombia, PFB/Brazil) strains were used in sensitivity testing.
FcB1R, F32a, and PFB strains were obtained by limit dilution. Stock solu-
tions of CQ diphosphate and test compounds were prepared in sterile, dis-
tilled water and DMSO, respectively. Drug solutions were serially diluted
with culture medium and added to asynchronous parasite cultures (0.5% par-
asitemia and 1% final hematocrit) in 96-well plates for 24 h, at 37 °C, prior
to the addition of [3H]hypoxanthine 0.5 mCi (1 to 5 Ci/mmol; Amersham,
Les Ulis, France) per well for 24 h. The growth inhibition for each drug con-
centration was determined by comparison of the radioactivity incorporated
in 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 from two independent experiments. The DMSO
concentration never exceeded 0.1% and did not inhibit parasite growth.
Cytotoxicity Evaluation upon Human MRC-5 Cells and MPM
A
human diploid embryonic lung cell line (MRC-5, Bio-Whittaker 72211D)
and MPM were used to assess cytotoxicity in host cells. The MPM were col-
lected from the peritoneal cavity 48 h after stimulation with potato starch
and 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 standard culture medium (RPMIϩ5%
fetal calf serum) 200 ml. The final DMSO concentration in the culture re-
mained below 0.5%. The cultures were incubated with four concentrations
of compounds (25, 12.5, 6.3 and 3.1 mM) at 37 °C in 5% CO2–95% air for 7
d. Untreated cultures were included as controls. For MRC-5 cells, the cyto-
toxicity was determined using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl-tetrazolium bromide (thiazolyl blue) assay.19) The concentra-
tion causing 50% reduction of viable cells (CC50) was obtained from the
drug concentration–response curve. For macrophages, scoring was per-
formed microscopically.
{3-[(7-Chloro-4-quinolyl)amino]-5-[(morpholinomethylidene)amino]-
phenyl}methanol (6) Yellow solid. Yield: 43% (85 mg). mpϾ250 °C. Rf:
0.83 (CH2Cl2/MeOH, 7 : 3). 1H-NMR (DMSO-d6) d: 3.37—3.49 (4H, m,
CH2 morpholine), 3.55—3.58 (4H, m, CH2 morpholine), 4.43 (2H, d, Jϭ5.5
Hz, CH2-Ph), 5.13 (1H, t, Jϭ5.5 Hz, OH), 6.65 (1H, s, Ar-H), 6.73 (1H, s,
In Vivo Drug Assays in P. berghei-Infected Mice The in vivo anti-
malarial activities were determined in mice infected with P. berghei (ANKA
65 strain). Four-week-old female Swiss mice (CD-1, 20—25 g) were in-

August 2001
937
traperitoneally infected with about 107 parasitized erythrocytes, collected
from the blood of an acutely infected donor animal. At the same time, the
animals (3 animals per group) were orally administrated the test compound
at a dose of 40 mg/kg (drug formulation in 100% DMSO). The administra-
tion continued on the 4 subsequent days by the intraperitoneal route. Un-
treated control animals generally die between 7—10 d following infection.
Drug activity was evaluated as the prolongation of the mean survival time
observed in untreated controls and/or by the reduction in parasitemia at 7 d
after infection. The suppression of parasitemia was calculated by the for-
mula: (average % parasitemia in controlsϪaverage % parasitemia in treated
mice)/average % parasitemia in controls. Three infected, DMSO-dosed mice
were used as controls.
4) Neftel K. A., Woodtly W., Schmid M., Br. Med. J., 292, 721—723
(1986).
5) Lind D. E., Leir J. A., Vincent P. C., Br. Med. J., 1, 458—460 (1973).
6) Maggs J. L., Tingle M. D., Kitteringham N. R., Parks B. K., Biochem.
Pharmacol., 37, 303—311 (1988).
7) Christie G., Brechenridge A. M., Park B. K., Biochem. Pharmacol.,
38, 1451—1458 (1989).
8) Harrison A. C., Kitteringham N. R., Clarke J. B., Park B. K., Biochem.
Pharmacol., 43, 1421—1430 (1992).
9) O’Neill P. M., Harrison A. C., Storr R. C., Hawley S. R., Ward S. A.,
Park B. K., J. Med. Chem., 37, 1362—1370 (1994).
10) Aziz S. A., Knowles C. O., Nature (London), 242, 417—418 (1973).
11) Beeman R. W., Matsumura F., Nature (London), 242, 273—274
(1973).
12) Meyers A. I., Elworthy T. R., J. Org. Chem., 57, 4732—4740 (1992).
13) Delarue S., Sergheraert C., Tetrahedron Lett., 40, 5847—5890 (1999).
14) Su T. S., Chou T. C., Kim J. Y., Huang J. T., Ciszewska G., Rens W. Y.,
Otter G. M., Sirotnak F. M., Watanabe K. A., J. Med. Chem., 38,
3226—3235 (1995).
Acknowledgments We express our thanks to Gérard Montagne for
NMR experiments and Dr. Steve Brooks for proof reading. This work was
supported by CNRS (EP CNRS 1790, UMR CNRS 8525) and Université de
Lille II. S. D. is a recipient of fellowships from the Région Nord-Pas de
Calais.
15) Abdel Magid A. F., Carson K. G., Harris B. D., Maryanoff C. A., Shah
R. D., J. Org. Chem., 61, 3849—3862 (1996).
16) Ridley R. G., J. Pharm. Pharmacol., 49, 43—48 (1997).
17) Trager W., Jensen J. B., Science, 193, 673—677 (1976).
18) Desjardins R. E., Canfield C. J., Haynes J. D., Chulay J. D., Antimicrob.
Agents Chemother., 16, 710—718 (1979).
References
1) World Health Organisation, “The World Health Report, Conquering,
Suffering, Enriching Humanity,” World Health Organisation Publish-
ers, Geneva, 1997.
2) World Health Organisation, Trans. R. Trop. Med. Hyg., 80, 1—50
(1986).
19) Carmichael J., DeGraff W. G., Gazdar A. F., Minna I. D., Mitchell J.
B., Cancer Res., 47, 936—942 (1987).
3) Watkins W. M., Sixsmith D. G., Spencer H. G., Boriga D. A., Karjuki
D. M., Kipingor T., Koech D. K., Lancet, 1984-i, 357—359.