Synthesis and biological evaluation of trifluralin analogues as antileishmanial agents

Article abstract of DOI:10.1016/j.bmc.2009.10.059

A series of new analogues of trifluralin (TFL) were synthesized and characterized in view of changing the unfavorable properties that limits its use as antileishmanial agent. Some of the TFL analogues display more activity than a standard drug (miltefosine) against the promastigote forms of Leishmania infantum and Leishmania donovani and the intracellular form (THP-1 infected with L. infantum). All analogues showed a clear advantage over miltefosine, as they are not hemolytic. Some analogues can conjugate these characteristics with reduced cell toxicity and improved intracellular activity.

Full text of DOI:10.1016/j.bmc.2009.10.059

Bioorganic & Medicinal Chemistry 18 (2010) 274–281
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of trifluralin analogues
as antileishmanial agents
M. A. Esteves a, I. Fragiadaki b, R. Lopes c,d, E. Scoulica b, M. E. M. Cruz c,d,
*
a Department of Chemical Industry Technologies, INETI, Estrada do Paço do Lumiar, 1649-038 Lisboa, Portugal
b School of Medicine, Laboratory of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, University of Crete, 71003 Heraklion, Greece
c Unit of New Forms of Bioactive Agents, Faculty of Pharmacy of the University of Lisbon, Estrada do Paço do Lumiar, 1649-038 Lisboa, Portugal
d Research Institute for Medicines and Pharmaceutical Sciences, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new analogues of trifluralin (TFL) were synthesized and characterized in view of changing the
unfavorable properties that limits its use as antileishmanial agent. Some of the TFL analogues display
more activity than a standard drug (miltefosine) against the promastigote forms of Leishmania infantum
and Leishmania donovani and the intracellular form (THP-1 infected with L. infantum). All analogues
showed a clear advantage over miltefosine, as they are not hemolytic. Some analogues can conjugate
these characteristics with reduced cell toxicity and improved intracellular activity.
Received 16 October 2008
Revised 21 October 2009
Accepted 29 October 2009
Available online 31 October 2009
Keywords:
Dinitroanilines
Leishmania
Ó 2009 Elsevier Ltd. All rights reserved.
Trifluralin analogues
Biological activity
1. Introduction
the current therapeutic schemes exhibits high efficiency and low
cytotoxicity together with an affordable cost.2,4,5 For these reasons
Leishmaniasis is a protozoan disease that constitutes a major
public health problem. It affects about 12 million people world-
wide, particularly in tropical and sub-tropical regions. In 2001
leishmaniasis was responsible for almost 60,000 deaths and it rep-
resents an increased risk of infection among the immunosup-
pressed patients.1,2 It attracted a special attention from WHO as
it is considered the second most important protozoan disease.3
Leishmania infantum is the parasite responsible for the visceral
form of the disease in the Mediterranean region. This disease is
reemerging in Europe in association with HIV but also poses a
major veterinary problem in areas where domestic dogs serve as
reservoir host. Inefficient treatment and the absence of vaccine is
the major cause for the high rates of infected dogs that results in
the continuous circulation of the parasite in endemic areas and
in the evolution of resistant phenotypes.
The first line drugs recommended for the treatment of this dis-
ease, such as the pentavalent antimonial agents, have serious side
effects. This and the emergence of resistant strains are responsible
for their progressive abandon. Several other classes of drugs have
been used for the treatment of leishmaniasis, such as pentamidine,
paramomycin, amphotericin B, liposomal amphotericin B, and mil-
tefosine. In spite of the battery of antileishmanial drugs, none of
the development of new effective and less toxic therapies is ur-
gently needed.
Dinitroanilines are tubulin-binding agents than have shown
selective antileishmanial properties in vitro. A member of this class
of compounds, trifluralin (TFL) was effective against the cutaneous
form of the disease, as an ointment.6 However, its use for other
forms of leishmaniasis, namely by parenteral administration, has
been limited by its low water solubility and easy sublimation.7
These two features have created several drawbacks in the develop-
ment of pharmaceutical formulations of dinitroanilines as antipar-
asitic agents.
To overcome the handling problems associated with dinitroani-
lines different strategies can be adopted: one is to introduce chem-
ical modification that will change the water solubility of the
compound; another is to incorporate the compound in liposomes
or both. TFL in its liposomal formulation, showed to be active in
mice and dogs that were experimentally infected with Leishmania
donovani and L. infantum, respectively.8,9 In order to improve the
antiparasitic activity, some dinitroanilines (TFL and oryzalin
analogues) were used in different in vitro models and proved to
be active against C. parvum10 and L. mexicana.6
In this work we have synthesized and characterized a series of
new TFL analogues (Fig. 1) in which the amine group was modified
with different kind of substituents, with increased either water or
lipidic solubility to allow in future better incorporation in
* Corresponding author. Tel.: +35 1210924722; fax: +35 1217163636.
E-mail addresses: eugeniacruz@ff.ul.pt, eugenia.cruz@ineti.pt (M.E.M. Cruz).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.10.059

M. A. Esteves et al. / Bioorg. Med. Chem. 18 (2010) 274–281
275
The method consisted of reacting chloralin, with primary or sec-
ondary amines in the presence of triethylamine using ethanol as
solvent. The substituents R1 and R2 in the amines were aryl and al-
kyl groups. All the new compounds were obtained in 70–80% yield
and are stable and easily-handled crystalline solids, except TFLA4
and TFLA5 which were obtained as oils that solidified to waxy sol-
ids upon storage in a refrigerator. The compounds were fully char-
acterized by FTIR, MS, NMR and elemental analysis and their water
solubility determined by UV spectrophotometry.10 Most of the
analogues showed improved water solubility in comparison with
TFL, but the values are still very low (Table 1). The larger increase
in water solubility was obtained for TFLA1, resulting from the
introduction of a hydroxyethyl group in substitution of one of
the propyl groups in the TFL structure. Moreover, in the series
TFLA1–TFLA5 it can be observed a decrease in water solubility
with the increase of the length of the alkyl substituent, as
expected.
2.2. In vitro biological evaluation
The compounds were evaluated in vitro for their toxicity to hu-
man cells, for their hemolytic activity to human red blood cells
(RBCs) and for their antileishmanial activity. The antileishmanial
activity was evaluated against both L. infantum and L. donovani pro-
mastigote form as well as against the L. infantum intracellular form.
These parameters were systematically evaluated for all TFL ana-
logues and compared with TFL and miltefosine as reference drugs.
2.2.1. Cytotoxicity assays
Figure 1. Trifluralin TFL and analogues TFLA1–TFLA12.
The compounds were evaluated in vitro for their toxicity to hu-
man cells using the monocytic THP-1 cell line also used as a host
cell line of the determination of the antiparasitic activity. The
hemolytic activity of the compounds was also assessed using hu-
man RBCs. The ability of TFL and some TFL analogues to kill
THP-1 is displayed in Figure 2. From these data we calculated the
concentration that was able to kill 50% of the cells (CC50) and the
results are displayed in Table 1.
liposomes. The cytotoxicity and hemolytic activity of the new com-
pounds was assessed against THP1 and whole blood. Several TFL
analogues showed better activity and less toxicity than miltefosine,
a reference compound usually used for the treatment of leishman-
ial infections. The new compounds were also tested in vitro against
the promastigote forms ofL. infantum andL. donovani and the intra-
cellular form (THP1 infected with L. infantum).
From the 12 analogues tested only three TFLA5–TFLA7 revealed
cytotoxicity at concentrations lower than 50
lM, whereas the rest
of the compounds tested showed some cytotoxic potential only at
concentrations above 100 lM.
2. Results and discussion
2.1. Chemistry
2.2.2. Hemolytic activity
The hemolytic activity against RBCs was used as a marker of a
general membrane toxicity effect of the compounds. For concen-
The synthesis of the TFL analogues TFLA1–TFLA12 was accom-
plished by a general procedure10,11 starting from a commercially
available chlorinated precursor, chloralin (Scheme 1).
trations up to 100 lM, TFL and the analogues did not exhibit any
Scheme 1.

276
M. A. Esteves et al. / Bioorg. Med. Chem. 18 (2010) 274–281
Table 1
In vitro biological evaluation of TFL and TFL analogues
Compound Water
Cytotoxicity
CC50 SEM (
Hemolysis
HC50 SEM
% Hemolysis
Potency promastigotes
Potency promastigotes
Intracellular A1 (L.
infantum) IC50 SEM (lM)
name
solubility
l
M)
at 100
lM
IC50 SEM (lM)
IC50 SEM (
lM)
(ppm)
(l
M)
L. infantum
L. donovani
Miltefosine
TFL
—
0.5
157.4
72.3
22.6
6.0
5.7
11.4
9.5
20.0
4.6
18.6
0.1
28.6 2.5
>50
>50
>50
>50
>50
10
38.3 2.8
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
96.1
1.3
0.4
0.2
0.5
0.8
0.7
1.4
1.4
1.2
0.0
0.3
1.1
1.2
23.9 4.2
>100
>100
>100
>100
nd
nd
2.2 1.1
nd
8.7 0.7
>100
>100
>100
>100
nd
nd
0.60
nd
2.7
>50
>50
>50
TFLA1
TFLA2
TFLA3
TFLA4
TFLA5
TFLA6
TFLA7
TFLA8
TFLA9
TFLA10
TFLA11
TFLA12
1.2 0.7
>50
>50
1.8 1.3
2.2 0.5
1.1 0.4
7.3 2.8
0.5 0.1
9.1 0.9
>50
40
35
>50
>50
>50
>50
>50
nd
nd
>100
>100
nd
>100
>100
nd
0.8
nd
nd
nd—not determined.
Figure 3. Hemolytic activity induced by TFL, TFL analogues and miltefosine at
different concentrations. RBCs were exposed to different concentrations of the
compounds and hemolysis was calculated as described in Section 4 by measuring
the absorbance of the cell supernatants at 550 nm with reference filter at 625 nm.
One hundred percentage of hemolysis was induced by incubating RBCs with H2O. –
d–, TFL; –j–, TFLA1; –N–, TFLA2; –w–, TFLA3; –s–, TFLA4; –h–, TFLA5; –4–,
Figure 2. Cytotoxic effect of TFL, TFL analogues and miltefosine against THP-1 cells.
Cells were exposed to increasing concentrations of compounds for 48 h and the
percentage of live/dead cells was measured by flow cytometry after staining with
SYBR14/PI. The control represents non-treated cells. –d–, TFL; –j–, TFLA1; –N–,
TFLA2; –w–, TFLA3; –h–, TFLA5; –4–, TFLA6;
, TFLA7;
, TFLA9;
,
TFLA10;
, miltefosine; –ꢀ–, control.
TFLA6;
, TFLA7;
, TFLA8;
, TFLA9;
, TFLA10;
, TFLA11;
,
TFLA12;
, miltefosine.
hemolytic activity (Fig. 3 and Table 1) whereas miltefosine lysed
almost 100% of RBCs.
As it will be discussed in Section 2.3, no clear correspondence
was found between promastigotes and intracellular amastigotes
assays, being the later test identified as the most relevant for the
biological evaluation of the new compounds.
2.2.3. Activity of TFL and TFL analogues against Leishmania
promastigotes
The first screening of antileishmanial activity was performed
against the free-living promastigote form ofL. infantum andL. dono-
vani using selected compounds. We have incubated the promastig-
otes with different concentrations of the compounds and we
measured the cell viability by means of flow cytometry. In Fig. 4a
and b are displayed the kinetics of the parasite killing at different
concentrations and in Table 1 is shown the IC50 for the tested com-
pounds. We observed that only TFLA6 had an antileishmanial
activity significantly higher than that of miltefosine (IC50 2.2 vs
2.2.4. Activity of TFL and TFL analogues against the intracellular
form of Leishmania infantum
The antileishmanial activity of all analogues against the intra-
cellular amastigote form of leishmania was assessed in vitro by
infecting THP-1 cells with a naturally occurring L. infantum strain
(A1), isolated from the bone marrow of a dog with kala-azar. After
treatment with increasing concentrations of the compounds for
48 h, the percentage of the infected cells was measured and plotted
on a semi-logarithmic plot (Fig. 5). The IC50 for each compound was
calculated and displayed in Table 1. The potency of the analogues
23.9
donovani promastigotes), whereas TFL and the other tested ana-
logues are inactive at concentrations up to 100 M.
lM for L. infantum promastigotes and 0.6 vs 8.7 lM for L.
l

M. A. Esteves et al. / Bioorg. Med. Chem. 18 (2010) 274–281
277
Figure 4. Antileishmanial activity of TFL, TFL analogues and miltefosine against promastigotes of L. donovani (LEM703) (a) and of L. infantum (LEM 235) (b). Drug
concentrations ranged from 0.08 V to 50 M were incubated with the cells for 48 h. The percentage of live/death cells was quantified by flow cytometry after staining with
SYBR14/PI. The control represents non-treated promastigotes. –d–, TFL; –j–, TFLA1; –N–, TFLA2; –w–, TFLA3; –h–, TFLA5; –4–, TFLA6; , TFLA7; , TFLA9;
TFLA10;
, miltefosine; –ꢀ–, control.
l
l
,
tefosine). Similarly, most dinitroanilines showed 2-fold lower cyto-
toxic potential than miltefosine.
It was found that the antiparasitic activity of the compounds
was different when tested against promastigotes in culture or
against the intracellular form. Against promastigotes all the tested
dinitroanilines (except TFLA6) display IC50 higher than 100 lM and
higher than miltefosine. Against the intracellular form TFL ana-
logues behave differently, same displaying comparable or even
smaller IC50 values than miltefosine and others being inactive.
Interestingly TFLA6 that displayed lower IC50 against the promas-
tigote form, proved to be also potent against the intracellular form.
The opposite is true with analogues, TFLA3, TFLA9, and TFLA10,
showing antiparasitic activity against the intracellular form but
not against the promastigote form. This different behavior could
not be attributed to the solubility of the analogues, and conse-
quently to the different ability to cross cell membranes, as they
Figure 5. Antiparasitic activity of TFL, TFL analogues and miltefosine against THP-1
cell line infected with L. infantum A1 isolate. –d–, TFL; –j–, TFLA1; –N–, TFLA2; –
w–, TFLA3; –s–, TFLA4; –h–, TFLA5; –4–, TFLA6;
, TFLA7;
, miltefosine.
, TFLA8;
,
TFLA9; , TFLA10; , TFLA11; , TFLA12;
was compared with that of TFL and miltefosine. We observed that
TFLA3 and TFLA6–TFLA11 were active against the intracellular
form of the parasite and in particular the analogues TFLA3, TFLA8,
and TFLA10, significantly reduced the parasite load of the cells
exhibiting IC50 values 2.5–5-fold lower than the IC50 of miltefosine.
However, none of the analogues achieved a complete clearance of
the parasite in this in vitro assay.
2.3. Selection of most potent TFL analogues
Table 1 summarizes the results of the in vitro evaluation of TFL
analogues as compared to TFL and to miltefosine. A common fea-
ture of all dinitroanilines tested was the absence of hemolytic
activity (0–1.4% hemolysis), at concentrations as high as 100
that make them substantially less hemolytic than miltefosine
(HC50 values of analogues are higher than 100 vs 38 M for mil-
lM
Figure 6. Comparative study of TFL and TFL analogues. d, Intracellular activity; h,
cytotoxicity.
, High cytotoxicity and low intracellular activity;
, low cytotox-
l
icity and high intracellular activity regions.

278
M. A. Esteves et al. / Bioorg. Med. Chem. 18 (2010) 274–281
display water solubility in the same range. For this reason, and as
one of the most relevant assays are those against the intracellular
form, the activity against promastigotes was not tested for all the
analogues systematically. Another relevant assay to take into con-
sideration in the selection of the analogues for further tests,
namely in vivo, is the cytotoxicity. The results of these two param-
eters for all analogues were compared in Figure 6.
The compounds can be assembled in different groups with re-
spect to their antileishmanial activity and cytotoxicity. The upper
shadowed level comprises those analogues with low activity and
high toxicity, whereas in the lower shadowed level are grouped
analogues displaying high activity and low toxicity. According to
these results the analogues TFLA3, TFLA8 to TFLA10 are the most
promising for further investigations.
toxicity problem of some compounds with promising intracellular
activity (TFLA6 and TFLA7) and to further increase cellular acces-
sibility, these compounds could benefit from the association with
macrophage targeted drug delivery systems such as liposomes.
This strategy already proved to be beneficial for TFL by improving
the therapeutic index of this compound in vivo.8,9 In this respect, of
special concern was the incomplete clearance of the intracellular
parasite even by the most active compounds, (TFLA3, TFLA8, and
TFLA10) and the low activity of others (TFLA9 and TFLA11) that
may be due to the reduced accessibility of the parasitophorous vac-
uole or to efficient removal of the drug from the host cell.
We believe that the association to liposomes will be of great
interest to further improve the pharmacological characteristics of
this class of compounds. Therefore, the most promising analogues
will be tested in appropriated animal models, both in the free form
and in the respective liposomal formulations.
3. Conclusion
4. Experimental
Due to the absence of effective alternatives for the treatment of
leishmania infections it is of high priority to develop derivatives
from new classes of chemical compounds that exhibit a potent
antiparasitic activity combined with reduced cytotoxic effects.
Dinitroanilines were identified as promising antileishmanial
drugs, based on their antiparasitic activity, their mechanism of ac-
tion and their reduced toxicity to human cells. In this context, we
recently reported the successful use of a dinitroaniline, TFL, as an
antileishmanial agent in animal models.8,9 In order to improve
the physicochemical properties of this compound such as solubil-
ity, and to enhance the antiparasitic activity we designed and syn-
thesized TFL chemical derivatives.
Twelve TFL analogues were synthesized and tested for their
antileishmanial activity and their cytotoxicity to human cells as
compared with the parent compound, TFL and one standard drug,
miltefosine that is currently used as antileishmanial agent.
Most of the new compounds were substantially less cytotoxic to
human cells when compared to miltefosine with exception of
TFLA5–TFLA7. Seven of them (TFLA3, TFLA6–TFLA11) exhibited
antileishmanial activity against the intracellular form of the para-
site. In particular, three compounds (TFLA3, TFLA8, and TFLA10)
showed 2.5–5-fold higher potency than miltefosine.
The design of the derivatives allowed us to elucidate some
structural features with important contribution to the antileishma-
nial activity of the compounds. For the analogues with a linear al-
kyl substituent in the amino group (TFLA1–TFLA5), the length of
the alkyl chain seems to be critical for the potency against the
intracellular parasite. An optimal length of the alkyl chain, specif-
ically four carbons (TFLA3), was identified. The analogue with six
carbons (TFLA5) was the most toxic and less active compound.
The three isomers (TFLA6–TFLA8) showed similar intracellular
activity which suggests that the position of the hydroxy group in
the aryl ring in this case is not relevant. On the contrary, the posi-
tion of that substituent seems to have some influence in the cyto-
toxicity since only the ortho-isomer (TFLA8) is not cytotoxic.
It was also observed, by comparing TFLA10 and TFLA12, a
remarkable improvement in the intracellular activity due to the
introduction of an oxygen atom in the heterocyclic ring (TFLA10).
All the tested analogues were inactive against the promastigote
form of Leishmania, except for TFLA6. This analogue is the only one
that shows activity in both forms of the parasite. This could prob-
ably be due to the differences in membrane transport between the
two parasite forms and differences in solubility in cell medium.
Surprisingly, TFLA6 also shows cytotoxicity.
Reagents and solvents were of the purest grade available, and
generally were used without further treatment. The starting mate-
rial, chloralin was purchased from Aldrich. 2-Pentylaminoethanol
and 2-hexylaminoethanol were prepared by a previously described
procedure.12
Melting points were determined in a Reichert Thermovar appa-
ratus and are uncorrected. FTIR spectra were recorded on a Perkin–
Elmer Spectrum BX v5.3.1 spectrometer. UV–vis spectra were re-
corded on a Hitachi U-2800A spectrophotometer. Fourier trans-
form (FT) NMR spectra were run on
a Brucker QE-300
spectrometer with resonance frequency of 300.65 MHz for 1H
using an appropriate solvent. The chemical shifts are reported in
d (ppm, TMS) and coupling constants in Hz. Electronic impact
(EI) mass spectra (MS) and EI gas-chromatography–mass spectra
(GC–MS) were determined on a Kratos MS 25RF instrument at
70 eV and electron spray ionization (ESI) mass spectra were per-
formed on a Brucker Daltonics Apex-Qe instrument at 300.0 V at
CACTI of Vigo University. Microanalyses were performed on a Fi-
sons EA-1108 microanalyzer at CACTI of Vigo University. Thin-
layer chromatography (TLC) was performed on Silica Gel 60 F254
plates with 0.2 mm layer thickness from Macherey-Naguel, and
the compounds visualized by illumination under UV light at
254 nm. Column chromatography (CC) was carried out with
Macherey-Naguel Si Gel 60 (230–400 mesh).
4.1. General procedure for trifluralin analogues synthesis
To a solution of chloralin (1 mmol) in ethanol (5 mL) under an
nitrogen atmosphere was added simultaneously triethylamine
(1.1 mmol) and the amine (1.1 mmol) via a syringe. The mixture
was refluxed until complete consumption of amine (0.5–1 h), as
monitored by TLC, cooled to room temperature, poured into ice-
water and extracted with ethyl acetate. The organic extracts were
dried over magnesium sulfate, filtered and concentrated in vacuum
to obtain a solid or oily residue. The residues were purified by silica
column chromatography and/or recrystallization to afford the
products as crystalline solids.
4.1.1. 2-((2,6-Dinitro-4-trifluoromethyl-phenyl)-ethylamino)-
ethanol (TFLA1)
The general method was used with chloralin (0.273 g, 1 mmol),
triethylamine (0.15 mL, 1.1 mmol), and 2-ethylamino-ethanol
(0.10 mL, 1.1 mmol) to afford the product as white crystals (diethyl
Taking into consideration that the targets of the disease are in-
fected macrophages, the results on the intracellular form of the
parasite are more critical for the assessment of the compounds that
should be studied further, namely in vivo. In order to overcome the
ether/hexane,
1411, 1317 cmꢁ1
H5), 3.73 (2H, m, CH2OH), 3.19 (4H, m, CH2N), 2.62 (1H, t,
g
= 62%). Mp 59–60 °C. FTIR (KBr):
m 2926, 1458,
.
1H NMR (CDCl3): d (ppm) 8.12 (2H, s, H3 and

M. A. Esteves et al. / Bioorg. Med. Chem. 18 (2010) 274–281
279
J = 6 Hz, OH), 1.22 (3H, t, J = 7 Hz, CH3CH2). MS (EI) m/z 323 [M+]
(3), 292 (100), 264 (23), 145 (16), 43 (25). Anal. Calcd for
C11H12F3N3O5: C, 40.87; H, 3.74; N, 13.00. Found: C, 40.92; H,
3.92; N, 13.01.
ether/hexane,
g
= 79%). Mp 180–183 °C. FTIR (KBr):
.
m
3483, 3311,
1305, 730 cmꢁ1
1H NMR (CDCl3): d (ppm) 9.90 (1H, s, OH), 8.42
(2H, s, H3 and H5), 6.95 (2H, d, J = 9 Hz, CHCOH), 6.80 (2H, d,
J = 9 Hz, CHCN), 5.02 (1H, s, NH). MS (EI) m/z 343 [M+] (100), 296
(18), 263 (22), 251 (36), 43 (50). Anal. Calcd for C13H8F3N3O5: C,
45.49; H, 2.35; N, 12.24. Found: C, 45.44; H, 2.12; N, 12.24.
4.1.2. 2-((2,6-Dinitro-4-trifluoromethyl-phenyl)-propylamino)-
ethanol (TFLA2)
The general method was used with chloralin (0.273 g, 1 mmol),
2-propylaminoethanol (0.13 mL, 1.1 mmol), and triethylamine
(0.15 mL, 1.1 mmol) to afford the product as orange crystals
4.1.7. 3-(2,6-Dinitro-4-trifluoromethyl-phenylamino)-phenol
(TFLA7)
The general method was used with chloralin (0.273 g, 1 mmol),
3-aminophenol (0.12 g, 1.1 mmol), and triethylamine (0.15 mL,
1.1 mmol) to afford the product as dark red crystals (dichlorometh-
(diethyl ether/hexane,
g
= 76%). Mp 50–52 °C. FTIR (KBr):
m 3560,
2971, 1445, 1411, 1320 cmꢁ1
.
1H NMR (CDCl3): d (ppm) 8.12 (2H,
s, H3 and H5), 3.74 (2H, m, CH2OH), 3.21 (2H, t, J = 5 Hz,
NCH2CH2OH), 3.02 (2H, m, NCH2CH2CH3), 2.67 (1H, t, J = 7 Hz,
OH), 1.64 (2H, m, CH2CH3), 0.89 (3H, t, J = 7 Hz, CH3). GC-MS (EI)
m/z 338 [M++1] (2), 306 (65), 264 (100), 206 (30), 160 (19). Anal.
Calcd for C12H14F3N3O5: C, 42.74; H, 4.18; N, 12.46. Found: C,
42.72; H, 4.44; N, 12.46.
ane/hexane,
g
= 90%). Mp 156–157 °C. FTIR (KBr):
m
3461, 3340,
.
3088, 1638, 1602, 1541, 1311, 1276, 1131, 773 cmꢁ1
1H NMR
(CDCl3): d (ppm) 9.88 (1H, s, OH), 8.48 (2H, s, H3 and H5), 7.20
(1H, t, J = 8.1 Hz, CHCHCH), 6.59 (1H, d, J = 8.1 Hz, C(NH)CHCH),
6.69 (1H, dd, J = 2.1 Hz, CHCHC(OH)), 6.54 (1H, d, J = 2.1 Hz,
C(NH)CHC(OH), 5.31 (1H, s, NH). MS (ESI) m/z 343 [M+] (100),
269 (12), 245 (11), 231 (55). Anal. Calcd for C13H8F3N3O5, C,
45.49; H, 2.35; N, 12.24. Found: C, 45.44; H, 2.27; N, 12.17.
4.1.3. 2-((2,6-Dinitro-4-trifluoromethyl-phenyl)-butylamino)-
ethanol (TFLA3)
The general method was used with chloralin (0.273 g, 1 mmol),
2-butylaminoethanol (0.14 mL, 1.1 mmol), and triethylamine
(0.15 mL, 1.1 mmol) to afford the product as orange crystals
4.1.8. 2-(2,6-Dinitro-4-trifluoromethyl-phenylamino)-phenol
(TFLA8)
The general method was used with chloralin (0.273 g, 1 mmol),
2-aminophenol (0.12 g, 1.1 mmol), and triethylamine (0.15 mL,
1.1 mmol) to afford the product as dark red crystals (dichlorometh-
(diethyl ether/hexane,
g
= 62%). Mp 56–58 °C. FTIR (KBr):
m 3398,
2962, 1447, 1412, 1321 cmꢁ1
.
1H NMR (CDCl3): d (ppm) 8.12 (2H,
s, H3 and H5), 3.73 (2H, m, CH2OH), 3.20 (2H, t, J = 5 Hz,
NCH2CH2OH), 3.06 (2H, t, J = 7 Hz, NCH2CH2CH2), 2.66 (1H, t,
J = 7 Hz, OH), 1.57 (2H, m, NCH2CH2CH2), 1.28 (2H, m, CH2CH3),
0.90 (3H, t, J = 7 Hz, CH3). MS (EI) m/z 351 [M+] (3), 320 (57), 264
(38), 251 (36), 57 (100). Anal. Calcd for C13H16F3N3O5: C, 44.45;
H, 4.59; N, 11.96. Found C, 44.40; H, 4.58; N, 11.96.
ane/hexane,
g
= 52%). Mp 157–159 °C. FTIR (KBr):
m 3448, 3351,
3090, 1638, 1533, 1517, 1142, 756 cmꢁ1
.
1H NMR (CDCl3): d
(ppm) 9.77 (1H, s, OH), 8.47 (2H, s, H3 and H5), 7.17 (1H, dt,
J = 2.4 Hz, C(NH)CHCH), 6.87-6.97 (3H, m, C(NH)CH + C(OH)CH + -
C(OH)CHCH), 5.36 (1H, s, NH). MS (ESI) m/z 343 [M+] (100), 269
(20), 245 (17). Anal. Calcd for C13H8F3N3O5: C, 45.49; H, 2.35; N,
12.24. Found: C, 45.28; H, 2.20; N, 12.02.
4.1.4. 2-((2,6-Dinitro-4-trifluoromethyl-phenyl)-pentylamino)-
ethanol (TFLA4)
The general method was used with chloralin (0.273 g, 1 mmol),
2-pentylaminoethanol (0.14 g, 1.1 mmol), and triethylamine
(0.15 mL, 1.1 mmol) to afford the product as a orange oil which
solidified to a waxy orange solid upon storage in a refrigerator
4.1.9. N-(2,6-Dinitro-4-trifluoromethyl-phenyl)-
isopropylamine (TFLA9)
The general method was used with chloralin (0.273 g, 1 mmol),
triethylamine (0.15 mL, 1.1 mmol), and isopropylamine (0.10 mL,
1.1 mmol) to afford the product as yellow crystals (petroleum
(g
= 83%). FTIR (KBr):
m
3324, 2963, 2938, 2876, 1630, 1549,
.
ether/hexane,
g
= 78%). Mp 68–72 °C. FTIR (KBr):
m 3304, 2999,
1309, 1140, 909, 711 cmꢁ1
1H NMR (CDCl3): d (ppm) 8.12 (2H, s,
1468, 1414, 1296, 729 cmꢁ1
.
1H NMR (CDCl3): d (ppm) 8.52 (1H,
H3 and H5), 3.74 (2H, d, J = 3.5 Hz, CH2OH), 3.21 (2H, t, J = 4.7 Hz,
NCH2CH2OH), 3.05 (2H, t, J = 7.6 Hz, NCH2CH2CH2), 2.66 (1H, s,
OH), 1.56–1.66 (2H, m, NCH2CH2CH2), 1.22–1.31 (4H, m,
CH2CH2CH3), 0.88 (3H, t, J = 6.5 Hz, CH3). HRMS (ESI-TOF) m/z calcd
for [C14H18F3N3O5+H]+ 366.1278, found 366.1271.
s, NH), 8.37 (2H, s, H3 and H5), 3.56 (1H, hept, J = 9 Hz, CH3CHCH3),
1.25 (6H, d, J = 9 Hz, CH3CH). GC–MS (EI) m/z 294 [M++1] (16), 275
(81), 232 (76), 202 (100), 186 (70). Anal. Calcd for C10H10F3N3O4: C,
40.96; H, 3.44; N, 14.33. Found: C, 40.71; H, 3.64; N, 14.18.
4.1.10. 4-(2,6-Dinitro-4-trifluoromethylphenyl)-morpholine
(TFLA10)
4.1.5. 2-((2,6-Dinitro-4-trifluoromethyl-phenyl)-hexylamino)-
ethanol (TFLA5)
The general method was used with chloralin (0.273 g, 1 mmol),
2-hexylaminoethanol (0.16 g, 1.1 mmol), and triethylamine
(0.15 mL, 1.1 mmol) to afford the product as a orange oil which
solidified to a waxy orange solid upon storage in a refrigerator
The general method was used with chloralin (0.273 g, 1 mmol),
morpholine (0.10 mL, 1.1 mmol), and triethylamine (0.15 mL,
1.1 mmol) to afford the product as yellow crystals (diethyl ether/
hexane,
g m
= 82%). Mp 141–142 °C (139–140 °C).1 FTIR (KBr):
2993, 1458, 1312, 1243 cmꢁ1
.
1H NMR (CDCl3): d (ppm) 8.07 (2H,
(
g
= 68%). FTIR (KBr):
m
3461, 2933, 2860, 1630, 1542, 1314,
s, H3 and H5), 3.79 (4H, t, J = 4 Hz, CH2O), 3.14 (4H, t, J = 4 Hz,
CH2N). GC–MS (EI) m/z 321 [M++1] (14), 200 (90),186 (100), 159
(69), 145 (58). Anal. Calcd for C11H10F3N3O5: C, 41.13; H, 3.14; N,
13.08. Found: C, 41.08; H, 3.02; N, 13.03.
1137, 909, 713 cmꢁ1
.
1H NMR (CDCl3): d (ppm) 8.12 (2H, s, H3
and H5), 3.74 (2H, d, J = 3.8 Hz, CH2OH), 3.21 (2H, t, J = 4.7 Hz,
NCH2CH2OH), 3.05 (2H, t, J = 7.5 Hz, NCH2CH2CH2), 2.66 (1H, t,
J = 6.6 Hz, OH), 1.57–1.60 (2H, m, NCH2CH2CH2), 1.18–1.31 (6H,
m, CH2CH2CH2CH3), 0.86 (3H, t, J = 6.7 Hz, CH3). HRMS (ESI-TOF)
m/z calcd for [C15H20F3N3O5+H]+ 380.1434, found 380.1428.
4.1.11. N-(2,6-Dinitro-4-trifluoromethylphenyl)
cyclohexylamine (TFLA11)
The general method was used with chloralin (0.273 g, 1 mmol),
cyclohexylamine (0.13 mL, 1.1 mmol), and triethylamine (0.15 mL,
1.1 mmol) to afford the product as yellow crystals (dichlorometh-
4.1.6. 4-(2,6-Dinitro-4-trifluoromethyl-phenylamino)-phenol
(TFLA6)
The general method was used with chloralin (0.273 g, 1 mmol),
4-aminophenol (0.12 g, 1.1 mmol), and triethylamine (0.15 mL,
1.1 mmol) to afford the product as dark red crystals (diethyl
ane/hexane, g = 73%). Mp 83–84 °C. FTIR (KBr): m 3313, 2942, 2924,
2857, 1646, 1526, 1509, 1310, 1255, 1128, 915, 764 cmꢁ1. 1H NMR
(CDCl3): d (ppm) 8.62 (1H, d, J = 7.2 Hz, NH), 8.36 (2H, s, H3 and

280
M. A. Esteves et al. / Bioorg. Med. Chem. 18 (2010) 274–281
H5), 3.20-3.25 (1H, m, NCH), 1.28–1.98 (10H, m, 5 ꢀ CH2). MS (ESI)
m/z 334 [M++1] (100), 202 (7). Anal. Calcd for C13H14F3N3O4: C,
46.85; H, 4.23; N, 12.61. Found: C, 46.76; H, 4.18; N, 12.50.
in the following section. The IC50 r values were calculated using
sigmoidal regression analysis13,14 from the data of three indepen-
dent experiments.
4.1.12. 1-(2,6-Dinitro-4-trifluoromethyl-phenyl)-piperidine
(TFLA12)
4.3.3. Determination of in vitro antileishmanial activity against
the intracellular L. infantum
The general method was used with chloralin (0.273 g, 1 mmol),
piperidine (0.11 mL, 1.1 mmol), and triethylamine (0.15 mL,
1.1 mmol) to afford the product as yellow crystals (diethyl ether/
THP-1 cells were differentiated with 1 lM retinoic acid (Sigma)
for 72 h at 37 °C/5% CO2. Cells were then washed twice in PBS and
once in plain RPMI medium, and incubated overnight with prom-
astigotes at 8:1 parasite/cell ratio at 37 °C/5% CO2. Following incu-
bation, cells were harvested, collected in RPMI medium and
carefully layered on 4 mL of Histopaque 1077 (Sigma). Free prom-
astigotes were removed by centrifugation at 1000g for 20 min. The
opaque cell layer containing the mononuclear cells was collected,
washed thrice in PBS and once in plain RPMI, and resuspended at
a concentration of 4 ꢀ 105cells/mL. Cells were then incubated with
the appropriate compound concentrations in 24-well tissue culture
plates (Cellstar, Greiner) for 48 h at 37 °C/5% CO2. The percentage
of infected cells was assessed microscopically after Giemsa stain-
hexane,
g
= 82%). Mp 109–110 °C. FTIR (KBr):
m
.
3080, 2954, 2865,
1634, 1526, 1361, 1160, 1123, 910, 712 cmꢁ1
1H NMR (CDCl3): d
(ppm) 8.01 (2H, s, H3 and H5), 3.05 (4H, d, J = 4.8 Hz, 2 ꢀ NCH2),
1.66 (6H, s, 3 ꢀ CH2). MS (ESI) m/z 320 [M++1] (100), 252 (4), 209
(5), 187 (7). Anal. Calcd for C12H12F3N3O4: C, 45.15; H, 3.79; N,
13.16. Found: C, 45.21; H, 3.69; N, 13.09.
4.2. Determination of water solubility
The water solubility values were obtained from the UV spectra
recorded for each compound. The samples were prepared accord-
ing to the following general procedure: a suspension of each com-
pound (5 mg) in water (10 mL) was stirred at room temperature for
1 h, then it was filtered and the UV spectra of the filtrate (3 mL)
was recorded and the value of the absorbance at the maximum
wavelength (kmax) was obtained. In order to determine the value
of the absorptivity at kmax, a standard solution of known concentra-
tion was prepared by dissolving the compound (4 mg) in methanol
(10 mL) and then diluting this solution in water (5, 12.5, and 25
times) followed by recording the UV spectra for the three dilutions.
The value of absorptivity obtained is the average of the absorptiv-
ity values calculated for each dilution by applying the Beer law to
the UV data. With this value of absorptivity it was then calculated
the concentration of each compound in the water sample (water
solubility).
ing. The IC50
4.3.2.
r values were calculated as described in Section
4.3.4. Assessment of hemolytic potential
EDTA-preserved peripheral blood from healthy volunteers was
centrifuged in order to remove serum and red blood cells were
washed twice in PBS. After the final wash, cells were distributed
in 96-well microplates (100 lL/well) and an equal volume of the
appropriate compound concentration was added. The tested com-
pounds were diluted in PBS in concentrations ranging from 100 to
6.25 lM. Incubation proceeded at 37 °C for 1 h, and the micro-
plates were centrifuged at 800g for 10 min. Absorbance of the
supernatants were measured at 550 nm with reference filter at
620 nm. The percentage of hemolytic activity of each drug at differ-
ent concentrations was estimated as the (A ꢁ A0/Amax ꢁ A0) ꢀ 100
where A0 is the background hemolysis obtained by incubation with
PBS and Amax is the 100% hemolysis achieved after incubation in
water. Each compound was assayed in triplicate.
4.3. Evaluation of in vitro antiparasitic activity
4.3.1. Cell culture and parasite strains
The human monocytic leukemia THP-1 cell line was used as a
host for leishmania parasites. Infection rate of this cell line ranged
between 35% and 45% and was significantly lower than the infec-
tion rate achieved with mouse peritoneal macrophages (85–95%).
However, the use of THP-1 as the parasite host cell for the evalua-
tion of the compounds’ antileishmanial activity against the intra-
cellular parasite, proved to be highly reproducible and
convenient for the high number of compounds to be tested in three
independent experiments. THP-1 cells were maintained in RPMI
4.3.5. Assessment of cytotoxicity in THP-1 monocytic cells
As a quantitative measurement of the cell damage after incuba-
tion with different concentrations of drugs, dual staining with
SYBR-14 and PI was carried out, using the LIVE/DEAD Sperm Viabil-
ity kit (Molecular Probes). THP-1 cells were incubated at a final
concentration of 1 ꢀ 106 cells/mL with different concentrations of
the compounds ranging from 50 to 1.56
lM. After an incubation
period of 72 h, cells were stained with 12
lM PI and 100 nM
SYBR-14, as described for promastigotes (Section 4.3.2). The ratio
live/dead cells was assessed by flow cytometry as described below.
Each compound was assayed in triplicate.
1640 supplemented with 10% FCS, L-glutamine and antibiotics at
37 °C, in 5% CO2 atmosphere. Promastigotes of L. infantum
MHOM/TN/80/IPT1/LEM 235 and L. donovani MHOM/IN/80/DD8/
LEM 703, were grown in RPMI 1640 supplemented with 10% FCS,
4.3.6. Flow cytometric analysis
L-glutamine and antibiotics, at 26 °C.
Cell samples were analyzed on an Epics Elite model flow cytom-
eter (Coulter, Miami, FL), equipped with a 488 nm argon laser. Dif-
ferential monitoring of the dyes was achieved by detecting green
fluorescence of SYBR-14 at 545 nm and red fluorescence of PI at
645 nm. At least 10,000 cells were analyzed per sample, and data
analysis was performed on fluorescence intensities that excluded
cell autofluorescence and cell debris.
4.3.2. Determination of in vitro antileishmanial activity in
promastigote cultures
All compounds were dissolved in DMSO/ethanol 50:50 v/v to a
final concentration of 65 mM and linear 2-fold dilutions ranging
from 100 to 0.8 lM were done in the culture medium. Promastig-
otes of L. infantum and L. donovani at a final concentration of
1 ꢀ 106 cells/mL, were incubated with the appropriate compound
concentration in 24-well tissue culture plates (Cellstar, Greiner)
at 26 °C. After 72 h, cells were harvested, resuspended in Hepes-
buffered saline solution, containing 10% BSA, and stained with
12 lM PI and 100 nM SYBR-14, using the LIVE/DEAD Sperm Viabil-
ity kit (Molecular Probes) according to the manufacturer recom-
mendations. Cells were analyzed by flow cytometry as described
Acknowledgements
Funding for this work was provided by the Project Research
Grant POCTI/CVT/56995/2004 by Fundação para a Ciência e Tecno-
logia and POCI 2010, Portugal, partially supported by the European
Union FEDER. The authors thank Prof. J.A.G. Morais from the Fac-

M. A. Esteves et al. / Bioorg. Med. Chem. 18 (2010) 274–281
281
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ulty of Pharmacy of Lisbon University for the fruitful discussions
during the project conception.
8. Carvalheiro, M.; Jorge, J.; Eleutério, C.; Pinhal, A. F.; Sousa, A. C.; Morais, J. G.;
Cruz, M. E. M. Eur. J. Pharm. Biopharm. 2009, 71, 292.
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