2590
DELMAS ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
high-performance liquid chromatography and nuclear magnetic resonance spec-
troscopy. Amphotericin B (Sigma) and metronidazole were used as standard
drugs for positive controls. All compounds were dissolved in sterile DMSO
(analytical grade; Sigma) and stored frozen at Ϫ70°C until used. Antileishmanial
activities were assessed on the referenced strain L. infantum (MHOM/FR/78/
LEM75). Antitrichomonal activities were assessed on the referenced strain T.
vaginalis (TVR87).
Predictive values of biological properties. Physicochemical values such as
lipophilicity (LogP, representing the n-octanol–water partition coefficient) and
solubility in water (LogS) were estimated by predictive mathematical methods
using ALOGPS version 2.0 software according to the methodology described by
Tetko et al. (18). Predictive values of antileishmanial and antitrichomonal activ-
ities, together with toxicity, were also investigated using the chemistry software
model and the database developed by Poroikov et al. (16) and Lagunin et al. (13),
respectively.
Activity against Trichomonas vaginalis. Parasites were maintained in continu-
ous culture in the Trichomonas medium TM 161 (Oxoid) supplemented with 8%
heat-inactivated horse serum (Eurobio, Paris, France). Parasites in late log phase
were incubated at an average of 104 cells/ml, and a range of benzothiazole
concentrations were aseptically incorporated into duplicate cultures (final
DMSO concentration, less than 5%). Negative controls treated by solvent
(DMSO) and positive controls containing a range of metronidazole (Sigma)
concentrations were added to each set of experiments. After a 48-h incubation
period at 37°C, viable parasites were identified and counted microscopically on
trations of compound C11 were aseptically incorporated into duplicate promas-
tigote and extracellular amastigote cultures and incubated at 25 and 37°C,
respectively. Following a 48-h incubation period, parasite viability was estimated
by flow cytometry after staining with 1 M propidium iodide.
Effect of compound C11 on nitric oxide production. Maturation of human
monocytes into adherent macrophages was induced by treating exponentially
growing monocytes (105 cells/ml) with 1 M phorbol myristate acetate (Sigma).
After a 48-h incubation period at 37°C (5% CO2) in chamber slides (Fisher), cells
were rinsed with fresh medium and suspended in RPMI medium containing
various concentrations of compound C11, in the presence or absence of 10 U of
human recombinant gamma interferon (IFN␥)/ml. After 48 h at 37°C, NO
production was measured by assessing the nitrite content of culture supernatants
by the method described by Ding et al. (6). Fresh Griess reagent (100 l) was
added to equal volumes of culture supernatants, and the optical density at 540
nm was measured after 15 min of incubation at room temperature. Nitrite
concentrations were determined using NaNO2 diluted in Dulbecco’s modified
Eagle’s medium as the standard.
Effect of compound C11 on phagocytic capacities of human macrophages.
Assays were performed on human monocyte-derived macrophages. Maturation
of monocytes into adherent macrophages was performed by treating exponen-
tially growing monocytes (105 cells/ml) with 1 M phorbol myristate acetate
(Sigma). After a 48-h incubation period at 37°C (5% CO2) in chamber slides
(Fisher), cells were rinsed with fresh medium and various concentrations of
compound C11 were incorporated into duplicate cultures. After a 48-h incuba-
tion period at 37°C, cells were rinsed with fresh medium and infected with RPMI
medium containing stationary-phase promastigotes (cell/promastigote ratio,
1/10). After a 4-h incubation period at 37°C (5% CO2), promastigotes were
removed by four successive washes with fresh medium, fixed with methanol, and
stained with 10% Giemsa stain. The percentage of macrophages containing
adherent or intracellular parasites was analyzed microscopically at a magnifica-
tion of ϫ1,000.
the basis of their aspect and motility and 50% inhibitory concentration (IC50
values were determined.
)
Antileishmanial activity against promastigotes. Leishmania infantum promas-
tigotes in late log phase were incubated in RPMI medium supplemented with
12% fetal calf serum at an average of 105 cells/ml, and a range of benzothiazole
concentrations were aseptically incorporated into duplicate cultures (final
DMSO concentration, less than 5%). Following a 48-h incubation period at 25°C,
promastigote growth was estimated by counting parasites with a hemacytometer
and IC50 values were determined.
RESULTS
Antileishmanial activity against intracellular amastigotes. Intracellular amas-
tigote culturing was performed in human monocyte-derived macrophages ac-
cording to the methodology previously described by Ogunkolade et al. (15).
Maturation of monocytes into adherent macrophages was induced by treating
exponentially growing monocytes (105 cells/ml) with 1 M phorbol myristate
acetate (Sigma). After a 48-h incubation period at 37°C (5% CO2) in chamber
slides (Fisher, Paris, France), cells were rinsed with fresh medium and suspended
in RPMI medium containing stationary-phase promastigotes (cell/promastigote
ratio, 1/10). After a 24-h incubation period at 37°C (5% CO2), promastigotes
were removed by four successive washes with fresh medium. Adapted dilutions
of chemical compounds were added in duplicate chambers, and cultures were
incubated for 96 h at 37°C (5% CO2). Negative controls treated by solvent
(DMSO) and positive controls containing a range of amphotericin B (Sigma)
concentrations were added to each set of experiments. At the end of the incu-
bation period, cells were harvested with analytical-grade methanol (Sigma) and
stained with 10% Giemsa stain (Eurobio). The percentage of infected macro-
phages in each assay was determined microscopically at magnification of ϫ1,000,
and IC50 values for the infected macrophages were determined.
Toxicity against human monocytes. In vitro toxicity of benzothiazoles was
assessed for human monocytes maintained in RPMI medium (Eurobio) supple-
mented with 10% fetal calf serum (Eurobio) at 37°C in 5% CO2 and replicated
every 7 days. A range of benzothiazole concentrations were incorporated in late-
log-phase monocytes (105 cells/ml), and cultures were incubated at 37°C with 5%
CO2. After a 72-h incubation period, cell growth and viability were measured by
flow cytometry after staining monocytes with propidium iodide (1 M final
concentration in culture medium). IC50 values and 50% lethal concentration
(LC50) values were determined for cell growth and viability, respectively. An in
vitro selective index (SI) value, corresponding to the ratio between antiparasitic
and cytotoxic activities, was calculated for each parasite according to the follow-
ing formula: SI ϭ LC50 against human monocytes/IC50 against intracellular
amastigotes or SI ϭ LC50 against human monocytes/IC50 against T. vaginalis.
Toxicity of compound C11 against promastigotes and extracellular amasti-
gotes. Promastigotes were incubated in RPMI medium supplemented with 12%
fetal calf serum and incubated at 25°C. Amastigotes were obtained from human
macrophages previously infected with promastigotes according to the protocol
described by Ogunkolade et al. (15). They were transferred into RPMI medium
supplemented with 20% fetal calf serum, titers were determined at pH 5.5, and
the mixture was incubated at 37°C (5% CO2). Under these conditions, extracel-
lular amastigotes could be maintained for more than 1 week. Various concen-
Physical properties of and predictive values for 6-nitro-ben-
zothiazoles, 6-amino-benzothiazoles, and the corresponding
anthranilic acids are summarized in Table 1. Lipophilicity was
estimated by prediction of n-octanol–water partition coeffi-
cient LogP values (defined as the ratio of concentration in an
immiscible solvent such as n-octanol to concentration in the
aqueous phase). In practice, a LogP value of 1 signifies that the
corresponding molecule could be partitioned according to the
ratio 10/1 (organic solvent/aqueous phase), a LogP value of 0
demonstrates that the corresponding molecule could be parti-
tioned according to the ratio 1/1, and a LogP value of Ϫ1
indicates that the corresponding molecule could be partitioned
according to the ratio 1/10. Data showed that almost all par-
tition coefficient values were Ͼ1 (ranging from 0.99 for com-
pound B3 to 5.32 for compound C10), suggesting that the
chemical compounds exhibited high lipophilic properties. An-
thranilic acids (series C) presented the highest affinity for
organic solvents, while 6-nitro- and 6-amino-benzothiazoles
demonstrated lower lipophilicity. Results observed for benzo-
thiazoles showed that replacement of 6-amino groups by 6-ni-
tro groups led to lipophilicity decreases. As expected, data also
demonstrated that all chemical compounds tested exhibited
weak solubility in water. Predictive values concerning biologi-
cal activities were obtained by comparing the chemical struc-
tures of the compounds with structures or substructures of
more than 30,000 well-known biologically active drugs. Results
of prediction are presented as estimates of the probability Pa
that the compounds are active. For Pa values Ͼ0.7, the corre-
sponding compound is very likely to reveal this activity in
experiments, but in that case, the chance of the compound
being the analogue of a known pharmaceutical agent is also
Delmas, Florence
