Note
BULLETIN OF THE
ISSN (Print) 0253-2964 | (Online) 1229-5949
KOREAN CHEMICAL SOCIETY
cytotoxicity. Therefore, they were screened for their ability to
inhibit PGE2 production in LPS-induced RAW 264.7 cells at
0.1 μM concentration using NS398 as a positive control. PGE2
concentration in the medium was measured using an enzyme
immunoassay (EIA) kit for PGE2 according to the manufac-
turer's recommendation.18 All experiments were carried out
at least twice, and these new compounds (6c, 6h, and 6k)
exhibited weak or moderate inhibitory activity of PGE2 pro-
duction (15.6%–30.5% inhibition at 0.1 μM concentration)
compared to the previous compounds at the given concentra-
tions (Table 1).
Next, all synthetic compounds were tested in vitro against
T. b. brucei strain 427 using the resazurin assay,19 which
demonstrates cell viability. Pentamidine was included in the
assays as a positive control for T. b. brucei. In this evaluation,
the activity was expressed as EC50 (half maximum effective
concentration on parasite growth). In vitro cytotoxicity was
also evaluated toward both a human embryonic kidney cell
line (HEK293T) and a human liver carcinoma cell line
(HepG2) using chlorpromazine as a positive control. Antipro-
tozoal activity against T. b. brucei and cytotoxicity data of all
tested compounds are given in Table 1.
inactive against two parasites and also not cytotoxic up to
100μM in two host cells when compared with its data against
T. b. brucei. Taken together, these data showed that not all try-
panosomatids are sensitive to s-triazine derivatives, and not all
s-triazine derivatives are equally active on sensitive parasites.
Overall, compound 6e was found to be specifically active
in vitro against T. b. brucei without serious cytotoxicity on
human cell lines and thus can be considered a hit compound
for African trypanosomiasis.
In summary,
a
set of s-triazine derivatives as
potential mPGES-1 inhibitors were evaluated for their
in vitro antiparasite activities against three parasites, namely
T. b. brucei, T. cruzi, andL. donovani. Compound 6eexhibited
an EC50 of 5.51μM against only T. b. brucei and lower toxic
effect on two human cell lines (CC50 = >200 μM). Therefore,
s-triazine ring could be a useful scaffold for the discovery of
novel trypanocidal drug candidates without serious toxic
effects. Accordingly, future work will address the intensive
structure–activity relationship (SAR) of s-triazine derivatives
with a goal to increase the efficacy. Studies on the mechanism
of action of the s-triazine derivatives against T. b. brucei are
under way and will be reported in the future.
In general, all s-triazine compounds showed acceptable
antiprotozoal effects on T. b. brucei strains 427 (EC50
=
Experimental
5.51–13.60 μM) independent of their inhibitory activities of
PGE2 production, but their cytotoxicity toward two human
cell lines exhibited a linear relationship with their PGE2-
related activities, in accordance with the inextricable link
between inflammation and cancer.20 This overall result sug-
gests that the PGE2-related activity was not predictive of
in vitro antiprotozoal activity. Of the compounds, 6e bearing
4-cyclohexylpheyl and 1-naphthyl groups on R1 and R3 posi-
tions, respectively, was found to be the most active compound
(EC50 = 5.51 μM) and also the least toxic one of the series
(CC50 = >200 μM), with T.I. (therapeutic index) value of ~36.
Three compounds showing high cytotoxicity (6c, 6h, and
6k), as well as the active compound 6e, were further screened
on Leishmania donovani-infected THP-1 macrophage
cells (a human acute monocytic leukemia cell line) and
T. cruzi-infected U2OS cells (a human osteosarcoma cell line)
according to the reported procedure by our research team.21
Amphotericin B and benznidazole were used as positive con-
trols, respectively, for activity against each intracellular para-
sites (Table 2). Cytotoxicity was measured by counting host
cell number in an image-based assay. Most of compounds dis-
played weak or no parasite inhibition against L. donovani-
infected THP-1 cells, with less cytotoxicity in host THP-1
macrophage cells compared to amphotericin B. Compound
6h was only compound that showed L. donovani clearance
in host macrophage cells without any cytotoxicity
(Figure 1). In the case of T. cruzi-infected U2OS cells, on
the other hand, three compounds except 6e showed strong par-
asite inhibition with significant cytotoxicity in host U2OS
cells compared to benznidazole, indicating that their antipar-
asite activities could be attributed to nonspecific cytotoxicity
on the host cells. Interestingly, compound 6e was found to be
The Inhibitory Activity Assay of COX-2-catalyzed PGE2.
The RAW 264.7 macrophage cell line was obtained from the
Korea Cell Line Bank (Seoul, Korea). Cells were grown at
37 ꢀC in DMEM supplemented with 10% FBS, penicillin
(100 units/mL),andstreptomycinsulfate(100 μg/mL)inahumi-
dified 5% CO2 atmosphere. Cells were incubated with the
tested samples at increasing concentrations or positive control
chemical (NS398) and then stimulated with LPS 1 μg/mL for
the indicated time. PGE2 concentration in the medium was quan-
tified using EIA kits (R&D Systems, Minneapolis, MN, USA).
T. brucei brucei Activity Assays. T. b. brucei strain
427 bloodstream forms were cultivated at 37 ꢀC with 5%
CO2 in HMI-9 medium supplemented with 10% FBS. Assay
was performed in 384 multiwell plates with 2500 cultivated
parasites in a well with pre-prepared compounds. Compounds
were exposed for 72 h and 120 μM of resazurin sodium salt
(Sigma-Aldrich, St. Louis, MO, USA) and were treated for
additional 5 h. After incubation, the parasites were fixed with
4% paraformaldehyde (Fisher Scientific Co., Fair Lawn, NJ,
USA) and the assay plates were read by a Victor 3 plate reader
(PerkinElmer, Inc., Waltham, MA, USA) at 530 nmEx/590
nmEm. Pentamidine was used as a drug-positive control, and
0.5% of DMSO was used as a drug-negative control.
Cytotoxicity Assays. HEK293T and HepG2 cells lines were
used to evaluate cytotoxicity. Both cell lines were cultured at
37 ꢀC with 5% CO2 in Dulbecco's Modified Eagle Medium
containing 10% FBS. Four thousand HEK239T and 2000
HepG2 cells were seeded individually in 384 multiwell plates
and incubated for 72 h with compounds at twofold dilution
from 100 μM in 10-point concentration. After incubation,
cells were exposed to 40 μM of resazurin sodium salt
Bull. Korean Chem. Soc. 2015, Vol. 36, 2383–2386
© 2015 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Jin, Jae Ho
