2,4-Diaminopyrimidines as Antimalarials
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1251
prepared from the enol ether 12 (R1 ) R2 ) R3 ) OMe, R4
)
filters was then measured using a microplate scintillation
counter (Topcount, Packard, USA). The concentration of
inhibitor that inhibited 50% of the parasite growth (IC50) was
determined from the sigmoidal curve obtained by plotting the
percentages of [3H]-hypoxanthine incorporation against drug
concentrations. Cytotoxicity tests of some analogues against
vero cells, KB cells, and BC cells were performed according to
the protocol described by Skehan et al. (1990).26
Me) and guanidine according to the procedure given for 9,
followed by recrystallization from MeOH.
Mu ta n ts P r ep a r a tion . The pfDHFR mutants with all
possible amino acids at residue 108 were prepared by cassette
mutagenesis using pUC-pfDHFR, which carries the wild-type
synthetic dhfr gene of P. falciparum as a template and were
those previously described.18 Expression in E. coli BL21(DE3)-
pLysS of the mutant pfDHFRs was achieved by IPTG induc-
tion as previously described18 with slight modification in which
the induction temperature was reduced to 20 °C and the
induction time was increased to 20 h. The cell extract obtained
after disruption of the bacterial cells by a French pressure cell
at 18 000 psi was assayed for DHFR activities. The pfDHFR
mutants with active and/or detectable DHFR activities were
further purified by affinity chromatography using Methotrex-
ate-Sepharose resin.
E n zym e Assa ys a n d In h ib it ion b y An t ifola t es a n d
Der iva tives. The activity of pfDHFR was determined spec-
trophotometrically at 25 °C according to the method previously
described6 using a Hewlette Packard UV-vis spectrophotom-
eter (HP 8453). The reaction (1 mL) contained 1×DHFR buffer
(50 mM TES, pH 7.0, 75 mM â-mercaptoethanol, 1 mg/mL
bovine serum albumin), 100 µM each of the substrate H2folate
and cofactor, reduced nicotinamide adenine dinucleotide phos-
phate (NADPH), and an appropriate amount (0.001-0.005
units in phosphate buffer containing 50 mM KCl) of affinity-
purified enzyme to initiate the reaction. The final concentra-
tion of KCl present in the assay reaction (∼0.5 mM) did not
affect the activity of the enzyme.6 One unit of enzyme is defined
as the amount of enzyme required to produce 1 µmol of
product/min at 25 °C. The inhibition of the enzymes with Pyr
and its substituted derivatives was investigated in a 96 well
plate with 200 µL reaction of the above mixture, in the
presence of antifolate. Verification of the mode of inhibition
of the enzyme by selected analogues was performed by
determining the DHFR activities at different concentrations
of inhibitors in the presence of varying concentration of
substrate H2folate using the standard condition as described
above, and the reciprocal of initial velocity data was plotted
against the reciprocal of substrate concentrations. For the
determination of kinetic parameters, the kinetic reactions at
340 nm were followed using a microplate reader (Labsystems,
Finland). The Ki values of the inhibitors for the wild-type and
mutant enzymes were determined by fitting to the equation
IC50 ) Ki(1 + ([S]/Km)),21 where IC50 is the concentration of
inhibitor that inhibits 50% of the enzyme activity under the
standard assay condition and Km is the Michaelis constant for
the substrate H2folate. This equation assumes competitive
inhibition.
P a r a site Cu ltu r e a n d An tim a la r ia l Testin g In Vitr o.
Two P. falciparum clones, TM4/8.2 (Wild-type DHFR) and
K1CB1 (C59R+S108N-DHFR), were kindly provided by S.
Thaithong, Department of Biology, Faculty of Science, Chu-
lalongkorn University.22 The parasites were maintained con-
tinuously in human erythrocytes at 37 °C under 3% CO2 in
RPMI 1640 culture media supplemented with 25 mM N-(2-
hydroxyethyl)piperazine-N′-ethanesulfonic acid, pH 7.4, 0.2%
NaHCO3, 40 µg/mL gentamicin, and 10% human serum.23 In
vitro antimalarial activity was determined by using the [3H]-
hypoxanthine incorporation method.24
The drugs were initially dissolved in DMSO and diluted
with the culture media. Aliquots (25 µL) of the drug of different
concentrations were dispensed in 96 well plates, and 200 µL
of 1.5% cell suspension of parasitized erythrocytes containing
1-2% parasitemia was added. The final concentration of
DMSO (0.1%) did not affect the parasite growth. The mixtures
were incubated in a 3% CO2 incubator at 37 °C. After 24 h of
incubation, 25 µL (0.25 µCi) of [3H]-hypoxanthine was added
to each well. The parasite cultures were further incubated
under the same conditions for 18-24 h. DNA of parasites was
harvested onto glass filter paper (Unifilter, Packard, USA).
The filters were air-dried, and 20 µL liquid scintillation fluid
(Microscint, Packard) was added. The radioactivity on the
Ack n ow led gm en t. We thank the Bioassay Re-
search Facility of the BIOTEC Center, NSTDA, for
performing cytotoxicity tests. This research was sup-
ported by grants from the World Health Organization
to Y.Y. (TDR and MMV) and W.S. (TDR), from the
European Union (INCO-DC IC18CT970223 and INCO-
DEV) to Y.Y., from the Wellcome Trust to Y.Y., from
Thailand-TDR and Biodiversity Research and Training
(BRT) Programs to S.K., and from NSTDA and the
Thailand Research Fund to Y.T.
Su p p or tin g In for m a tion Ava ila ble: Additional experi-
mental data (1H NMR) of all compounds not listed in the
Experimental Section. This material is available free charge
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