3930 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 23
J ensen et al.
4,6-Dia m in o-1,2-d ih yd r o-2-n on yl-1-[3′-(2,4,5-t r ich lo-
r oph en oxy)pr opyloxy]-1,3,5-tr iazin e Hydr och lor ide (8ee).
A mixture of 3.54 g (10 mmol) of 3-(2,4,5-trichlorophenoxy)-
propyloxybiguanide17,26 and 1.0 mL of concentrated HCl in 10
mL of EtOH was concentrated to dryness in vacuo and
triturated with 5 mL of ethyl ether and 5 mL of EtOH. The
resultant precipitate was collected on a filter to give 1.7 g (4.3
mmol) of hydrochloride, mp 105-108 °C, which was stirred
for 5 days with 1.0 g (6.4 mM) of decylaldehyde and 15 mL of
DME. The resultant 1.2 g of precipitate (mp 170-173 °C) was
collected on a filter and recrystallized from 8 mL of 90% EtOH
drug solution was added to a well of a 96-well plate, and 2-fold
serial dilutions were made with plain culture medium to cover
the desired range. The parasite-infected erythrocyte suspen-
sion (80 µL) was then added to each well. The final microcul-
ture had hematocrit of 2% and 0.5% concentration of parasit-
ized erythrocytes (>95% rings) in a culture medium containing
10% human serum. The plates were shaken gently and placed
in a humidified, airtight modular incubation chamber in an
atmosphere of 5% O2, 5% CO2, and 90% N2. The parasites were
incubated at 37.5 °C for 24-30 h to allow ring forms in the
control wells to mature to schizonts. After incubation, 80 µL
of supernatant was removed from the culture wells, with
minimal disturbance to the red cell layer at the bottom of the
wells, and 100 µL of phosphate-buffered saline was added to
each well. The microculture plates were then left to stand for
1 h before removing 100 µL of supernatant from each well.
After resuspension of erythrocytes in the remaining fluid, a
thick blood film was made from each well, stained with
Romanowsky stain, and examined microscopically. In the
control wells, ring forms developed into normal schizonts with
well-defined chromatin dots. In the presence of effective drug
concentrations, ring forms did not mature into normal sch-
izonts but appeared as parasites consisting of fragmented
chromatin dots of unequal size scattered over a large area.34
The minimum inhibitory concentration (MIC) for each drug
was determined by assessing the lowest concentration of drug
that prevented more than 90% of parasites from maturing into
normal schizonts.
In Vitr o Activity Aga in st W-2 Str a in P . fa lcipa r u m .
In vitro tests of activity were preformed against the Indochina
W-2 clone (resistant to chloroquine, quinine, sulfadoxine, and
pyrimethamine) strain of P. falciparum using modifications35
of the semiautomated microdilution technique of Desjardins
and others.36 Antimalarial activity in this system was assessed
by inhibition of radiolabeled hypoxanthine incorporation into
parasites by graded concentrations of drugs during incubation
for 66 h in a culture medium containing physiological concen-
trations of folic acid. All drugs studied, as well as control drugs,
were tested in duplicate. In this test system the results are
expressed by estimating the concentrations of drugs that
produced 50% inhibitions of radiolabeled incorporation (IC50).
1
to give 0.8 g of analytically pure product, mp 182-184 °C. H
NMR (DMSO-d6): δ 0.8 (t, 3H), 1.25 (m, 16H), 2.15 (m, 2H),
4.1 (t, 2H), 4.9 (m, 1H), 7.4 (s, 1H), 7.5 (br s, 2H), 7.7 (s, 1H),
8.0 (br s, 1H), 9.0 (br s, 1H), 9.8 (br s, 1H).
4,6-Diam in o-1,2-dih ydr o-2,2-dim eth yl-1-[3′-(1-n aph th yl-
oxy)p r op yloxy]-1,3,5-tr ia zin e Hyd r obr om id e (8gg). A 0.93
g (5.0 mmol) portion of 4,6-diamino-1,2-dihydro-2,2-dimethyl-
1-hydroxy-1,3,5-triazine26 was suspended in 10 mL of DMF
and stirred with 1.33 g (5.0 mmol) of 3-(1-naphthyloxy)propyl
bromide (2gg), which was prepared in the same manner as
2c and used crude. After 16 h of stirring, 0.10 g (0.35 mmol)
of 2gg was added, and the mixture was stirred for an
additional 16 h. The solvent was removed on a rotavap, and
the residue was dissolved in 20 mL of hot water. When the
mixture was cooled, an oil separated, so the aqueous mixture
was rewarmed to give a solution that was washed with 5 mL
of EtOAc. When the mixture was cooled, crystals separated
that were collected and recrystallized from acetonitrile to give
1.04 g (49%) of product as the hydrobromide salt, mp 200-
201 °C. 1H NMR (DMSO-d6 + D2O): δ 1.40 (s, 6H), 2.30 (br
m, 2), 4.30 (br m, 4), 7.10 (m,1), 7.6 (br m, 4), 7.95 (m, 1), 8.10
(m, 1).
P h a r m a cology. In vivo31 antimalarial activity was tested
in male or female Charles River CD-1 mice that were 4-5
weeks old and weighed 20-25 g. They were housed in groups
of three or four in standard plastic cages with wire tops, Bed-
o-cob bedding, and 12 h/day of light and maintained at 75 °F.
They were fed a standard Ralston Purina mouse chow, and
the cages and water bottles were changed twice a week. Test
compounds were ground in a mortar and pestle and diluted
with enough vehicle to give a volume of 10 mL/kg of mouse
weight. The oral doses were prepared in 0.5% hydroxyethyl-
cellulose/0.1% Tween-80. The amount of drug was calculated
on the free base weight. The mice were infected intraperito-
neally on day 0 with 5 × 104 erythrocytes parasitized with
Plasmodium berghei (KBG-173 strain) from a donor mouse
having a parasitemia between 5% and 10%. On days 3, 4, and
5 the test compounds were administered bid, spaced 6 h apart,
to the mice. Smears were made from tail blood on day +6 and
twice a week thereafter. The smears were stained with Geimsa
and examined microscopically. Parasitemias are reported as
the percentage of the red blood cells that are infected. On day
6 the (suppression of) parasitemias of treated animals may
be compared to the parasitemias of infected nontreated
controls, but these infected nontreated controls die on days
7-12. Activity was also measured by survival. Full activity is
defined as all animals living on day 31. Partial activity is
defined as days of increased survival vs the infected nontreated
controls.
Refer en ces
(1) Report of the Ad Hoc Committee on Health Research Relating
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(2) World Health Report 1999, World Health Organization: Geneva,
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(3) Wirth, D. F. Malaria: A Third World Disease in Need of First
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(4) Snow, R. W.; Craig, M. H.; Deichman, U.; LeSueur, D. A
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In Vitr o Activity Aga in st K-1 Str a in P . fa lcipa r u m .
Malaria parasites were maintained in continuous culture32
using 10% O-positive human serum, erythrocytes, and RPMI-
1640 LPLF medium containing 0.01 mg of folic acid per liter
and 0.0005 mg/L p-aminobenzoic acid (GIBCO, Grand Island,
NY) and supplemented with 2 g/L glucose, 50 mg/L hypoxan-
thine, 5.97 g/L N-(2-hydroxyethylpiperazine)-N′-2-ethane-
sulfonic acid, and 2.1 g/L sodium carbonate. Parasites were
synchronized repeatedly with 5% sorbitol to produce experi-
mental cultures consisting of parasites 6-12 h into the cycle.33
Cultures were grown in flat-bottomed wells of a 96-well
microculture plate. The drugs were dissolved in methanol or
in DMSO and subsequently diluted with plain culture medium
to obtain the desired concentration. A total of 20 µL of final
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Jensen
