Fluorine-containing aryloxyethyl thiocyanate derivatives are potent inhibitors of Trypanosoma cruzi and Toxoplasma gondii proliferation

Article abstract of DOI:10.1016/j.bmcl.2007.07.012

As a part of our project aimed at developing new safe chemotherapeutic and chemoprophylactic agents against tropical diseases, fluorine-containing drugs structurally related to 4-phenoxyphenoxyethyl thiocyanate (1) were designed, synthesized, and evaluate

Full text of DOI:10.1016/j.bmcl.2007.07.012

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5068–5071
Fluorine-containing aryloxyethyl thiocyanate derivatives
are potent inhibitors of Trypanosoma cruzi and
Toxoplasma gondii proliferation
a,b
a
a
´
´
Guadalupe Garcıa Linares, Santiago Gismondi, Nicolas Osa Codesido,
˜
Silvia N. J. Moreno,b Roberto Docampob and Juan B. Rodrigueza,*
aDepartamento de Quı´mica Orga´nica and UMYMFOR (CONICET-FCEyN), Facultad de Ciencias Exactas y Naturales,
Universidad de Buenos Aires, Pabello´n 2, Ciudad Universitaria, C1428EHA, Buenos Aires, Argentina
bCenter for Tropical and Emerging Global Diseases and Department of Cellular Biology,
University of Georgia, Athens, GA 30602, USA
Received 17 May 2007; revised 2 July 2007; accepted 6 July 2007
Available online 13 July 2007
Abstract—As a part of our project aimed at developing new safe chemotherapeutic and chemoprophylactic agents against tropical
diseases, fluorine-containing drugs structurally related to 4-phenoxyphenoxyethyl thiocyanate (1) were designed, synthesized, and
evaluated as antiproliferative agents against Trypanosoma cruzi, the parasite responsible of American trypanosomiasis (Chagas’ dis-
ease), and Toxoplasma gondii, the etiological agent of toxoplasmosis. This thiocyanate derivative had previously proven to be an
effective agent against T. cruzi proliferation. Fluorine-containing thiocyanate derivatives 2 and 3 were threefold more potent than
our lead drug 1 against intracellular T. cruzi. The biological evaluation against T. gondii was also very promising. The IC50 values
corresponding to 2 and 3 were at the very low micromolar level against tachyzoites of T. gondii. Both of these drugs are interesting
examples of effective antiparasitic agents that have outstanding potential not only as lead drugs but also to be used for further
in vivo studies.
ꢀ 2007 Elsevier Ltd. All rights reserved.
American trypanosomiasis (Chagas’ disease) and toxo-
plasmosis are among the most prevalent parasitic dis-
eases worldwide.1 It has been estimated that around
18–20 million people are infected and over 40 million
individuals are at risk of infection by the hemoflagellat-
ed protozoan Trypanosoma cruzi, the responsible agent
of this disease.2 As other kinetoplastid parasites, T. cruzi
has a complex life cycle possessing four main morpho-
logical forms. It multiplies within the insect gut as an
epimastigote form and is spread as a non-dividing meta-
cyclic trypomastigote from the insect feces by contami-
nation of intact mucosa or wounds produced by the
blood-sucking activity of the vector. In the mammalian
host, T. cruzi proliferates as the intracellular amastigote
form, which is next released into the bloodstream as a
non-dividing highly infective trypomastigote that can
either invade other tissues or can infect the respective
Chagas’ disease vectors closing the cycle.3–5 Transmis-
sion via the placenta or by blood transfusion is the
responsible mechanism for the occurrence of Chagas’
disease in developed countries where the disease is not
endemic.6,7
Toxoplasmosis is caused by Toxoplasma gondii, a com-
plex eukaryotic parasite that has adopted an essential
intracellular survival strategy.5,8 Most of T. gondii infec-
tions are asymptomatic. There are two asexual forms
that can cause disease in humans. The tachyzoite form,
which can invade all types of cells and divides rapidly
leading to cell death, and the bradyzoite form that di-
vides slowly and forms cysts in muscle and brain.9
The existing chemotherapy either for Chagas’ disease or
for toxoplasmosis remains deficient. The chemotherapy
for Chagas’ disease is based on two drugs empirically
discovered, nifurtimox, now discontinued, and benzni-
dazole. Although both of these compounds are able to
cure at least 50% of recent infections as indicated
by the disappearance of symptoms, and reduction of
Keywords: Chagas’ disease; Trypanosoma cruzi; Toxoplasma gondii;
Aryloxyethyl thiocyanates; Squalene synthase.
*
Corresponding author. Tel.: +54 11 4576 3346; fax: +54 11 4576
3385; e-mail: jbr@qo.fcen.uba.ar
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.07.012

G. G. Lin˜ares et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5068–5071
5069
parasitemia and serology, they present severe side ef-
fects.10,11 The standard treatment against T. gondii
infections consists in the combination of pyrimeth-
amine, which inhibits the enzymatic activity of dihydro-
folate reductase, and sulfadiazine, whose target is
dihydropteroate synthetase.12 This therapy is frequently
associated with toxic side effects.13
verted into compound 5 in 79% yield. The benzyl group
of the resulting product was removed by catalytic hydro-
genation to give phenol 7 in 90% yield, which after
treatment with 2-bromoethyl tetrahydro-2H-pyran-2-yl
ether in a suspension of potassium hydroxide in methyl
sulfoxide34 afforded 9 in 80% yield. Cleavage of the
tetrahydropyranyl group of 9 by treatment with pyridi-
nium p-toluenesulfonate gave alcohol 11, which on reac-
tion with tosyl chloride gave the corresponding tosylate
13 in 33% yield. The title compound 2 was obtained by
nucleophilic displacement with potassium thiocyanate in
N,N-dimethylformamide at 100 ꢁC in 43% yield.
Compound 3 was prepared following a similar synthetic
approach as illustrated in Scheme 1.
The knowledge of unique aspects of the biochemistry and
physiology of T. cruzi has led to the finding of specific
molecular targets for rational drug design,14–19 among
them, sterol biosynthesis arises as a valid target for Cha-
gas’ disease.20 The parasitic pathway leads to ergosterol,
while cholesterol is the final product in the mammalian
host.21 For example, triazole derivatives,22–24 or
azasterols25,26 had shown to be effective antiparasitic
agents against T. cruzi targeting ergosterol biosynthesis.
In addition, we have demonstrated that aryloxyethyl
thiocyanate derivatives are potent inhibitors of T. cruzi
proliferation.27–29 4-Phenoxyphenoxyethyl thiocyanate
also known as WC-9 (1) is a representative member of
this family of compounds, which proved to be a potent
agent against T. cruzi proliferation whose target is squa-
lene synthase (SQS) (Fig. 1).30
All compounds were routinely characterized by using 1H
and 13C NMR spectroscopy at 500 MHz and 125 MHz,
respectively (Bruker AM-500 apparatus). Elemental
analysis data for all new compounds were satisfactory.38
The title compounds 2 and 3 were potent growth inhib-
itors of the intracellular form of T. cruzi. Certainly,
these drugs exhibited IC50 values of 4.3 and 3.7 lM,
respectively, being fourfold more potent than our lead
drug WC-9, used as a positive control, under the same
assay conditions. The same behavior was observed
against the epimastigote form of T. cruzi where drugs
2 and 3 were more potent than 1 but to a lesser extent
than against amastigotes. The tetrahydropyranyl pre-
cursors of the title drugs, that is, compounds 9 and 10,
were also potent inhibitors of T. cruzi (amastigotes) pro-
liferation showing IC50 values of 19.0 and 14.4 lM,
respectively. This fact was in agreement with our previ-
ous results in other closely related compounds, where
the biological activity of drugs bearing the thiocyanate
group correlated quite well with the activity exhibited
by their natural tetrahydropyranyl ether precursors
when bonded to the same aromatic skeleton.28,29 The
efficacy decreased proportionally in all cases. In addi-
tion, fluorine-containing drugs were also potent inhibi-
tors of T. gondii (tachyzoites) possessing IC50 values at
the low micromolar level. Compounds 1 and 3 proved
to be the more effective growth inhibitors of T. gondii
exhibiting IC50 values of 2.80 and 3.99 lM, respectively.
WC-9, a well-known antiparasitic agent, and atovaqu-
one were used as positive controls for assays on T. cruzi
and T. gondii, respectively. The results are shown in Ta-
ble 1.
On the other hand, it has been reported that sterol bio-
synthesis inhibitors such as azasterols act as selective
inhibitors of T. gondii proliferation.31,32 In particular,
it was recently reported that two quinuclidine deriva-
tives, SQS inhibitors, showed selective activity against
tachyzoites of T. gondii.33 As compound 1 is a potent
inhibitor of T. cruzi proliferation, and bearing in mind
that previous studies indicated that structural variation at
the B ring had no influence on biological action,27–29,34
it was thought that the introduction of a fluorine atom
at the B ring would be of benefit for biological activity.
The estimated logP values for the title compounds 2 and
3 were 4.71, while the corresponding one for 1 was 4.51.
As the drugs must penetrate two cell membrane to reach
TcSQS, it could be anticipated a better biological action.
Then, fluorine-containing drugs were designed, synthe-
sized, and biologically evaluated against the intracellular
and the epimastigote forms of T. cruzi and the tachyzo-
ite forms of T. gondii.
The introduction of a fluorine atom onto the B ring was
carried out via a coupling reaction that involves an aryl-
boronic acid and an appropriate phenol in the presence
of pyridine and cupric acetate as illustrated in the
Scheme.35–37 The title compounds were synthesized
starting from commercially available 4-benzyloxyphenol
(4), which on reaction with 3-fluorophenylboronic acid
in the presence of pyridine and cupric acetate was con-
Biological assays on epimastigotes were performed as
previously described.34,39 Experiments on the intracellu-
lar form of the parasite were conducted on T. cruzi-in-
fected L6E9 myoblasts (Y strain) as described
before.40–42 Experiments on T. gondii tachyzoites were
carried out as previously published.43,44
2"
It can be concluded that fluorine-containing thiocya-
nate derivatives were potent inhibitors of T. cruzi pro-
liferation exhibiting an efficacy superior than that
presented by WC-9 our lead drug. These compounds
offer excellent prospective as antiparasitic agents be-
cause they also exhibit potent inhibitory action on
T. gondii.
1"
O
2'
2
B
A
1'
SCN
O
1
1
Figure 1. Chemical structure of 4-phenoxyphenylethyl thiocyanate.

5070
G. G. Lin˜ares et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5068–5071
OH
O
O
c
a
OR1
X
OR
X
O
Y
Y
OBn
9, X = H, Y = F, R1 = THP
10, X = F, Y = H, R1 = THP
5, X = H, Y = F, R = Bn
6, X = F, Y = H, R = Bn
4
11, X = H, Y = F, R1 = H
b
7, X = H, Y = F, R = H
8, X = F, Y = H, R = H
d
12, X = F, Y = H, R1 = H
O
O
e
f
OTs
O
SCN
X
X
O
13, X = H, Y = F
14, X = F, Y = H
Y
Y
2, X = H, Y = F
3, X = F, Y = H
Scheme 1. Reagents and conditions: (a) 3-FPhB(OH)2 or 4-FPhB(OH)2, Cu(OAc)2, CH2Cl2, py, rt, 5 days, 79% for 5, 35% for 6; (b) H2/Pd (C), rt,
overnight, 90% for 7, 92% for 8; (c) KOH, DMSO, BrCH2CH2OTHP, rt, 16 h, 80% for 9, 85% for 10; (d) PPTS, MeOH, rt, 14 h, 95% for 11, 78% for
12; (e) ClTs, py, rt, 8 h, 33% for 13, 55% for 14; (f) KSCN, DMF, 100 ꢁC, 5 h, 43% for 2, 48% for 3.
Table 1. Effect of fluorine-containing drugs against Trypanosoma cruzi
and Toxoplasma gondii for compounds 1–3, 9, and 10
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a
a
Compound
IC50 (lg/ml)
IC50 (lM)
IC50 (lM)
T. cruzi
epimastigotes
2.6
T. cruzi
amastigotes
12.0
T. gondii
tachyzoites
2.80
1 (WC-9)
2
1.74
1.95
4.3
3.7
15.8
3
3.85
3.99
9
10
>20.0
>20.0
19.0
14.4
6.16
0.29
Atovaquone
a Values are means of three experiments.
13. Montoya, J. G.; Liesenfeld, O. Lancet 2004, 363, 1965.
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Haemers, A. Curr. Pharm. Des. 2001, 7, 1117.
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Work aimed at optimizing the chemical structure of
compounds such as 2 and 3 as well as to establish a more
complete structure–activity relationship is currently
being pursued in our laboratory.
17. Docampo, R. Curr. Pharm. Des. 2001, 7, 1157.
18. Docampo, R.; Moreno, S. N. J. Curr. Drug Targets Infect.
Disord. 2001, 1, 51.
Acknowledgments
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We thank Cuing Jiang for technical help with the T. gondii
assays. This work was supported by grants from the
National Research Council of Argentina (PIP 5508),
ANPCyT (PICT2004 #21897), and the Universidad de
Buenos Aires (X-252) to J.B.R., and the U.S. National
Institutes of Health to R.D. (AI-68647) and S.N.J.M.
(AI-68467). G.G.L. thanks the Ellison Medical Founda-
tion for a Fellowship.
23. Gonzalez-Cappa, S. M.; Stoppani, A. O. M. Biochem.
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inhibition, the following formula was used: percent
inhibition = 100 ꢀ (DAd · 100)/DAc, where DAc and DAd
are the differences in the absorbance of control cultures
and drug-treated cultures, respectively, at the beginning
and at day 5.
´
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42. Gamma-irradiated L6E9 myoblasts (1 · 107 cells/plate) in
DMEM containing 20% FCS were plated in 12-well tissue
culture plates and incubated at 37 ꢁC in a 5% CO2
atmosphere for 24 h. After 24 h, wells were washed once
and fresh media were added containing 4.17 · 106 try-
pomastigotes or amastigotes/well in DMEM. One well was
left without parasites for control. After 2 h of incubation
at 37 ꢁC in a 7% CO2 atmosphere, cultures were washed
twice with Hanks’ solution, and culture medium was
replaced to remove extracellular parasites. At this time,
0.5–1 lCi of [5,6-3H]uracil/well (specific activity, 40–50 Ci/
mmol) and drug solutions in water were added. Two wells
were left for infection control. Cultures were incubated for
72 h. Incorporation of the [5,6-3H]uracil into trichloro-
acetic acid (TCA)-precipitable material was measured at
day 3. The supernatants from the monolayers were
transferred to glass tubes, the cells were dissolved with
1.3 ml of 1% sodium dodecyl sulfate containing 100 lg of
cold uracil per ml, and the suspension was transferred to
the glass tubes. The wells were rinsed with 3 ml of 5%
TCA (ice-cold) which was combined with the previous
suspension. The samples were maintained in ice for 15 min
and collected on glass fiber filters (Whatman GF/B) by
using a sampling manifold (Millipore, Bedford, MA).
After filtering, the tubes were rinsed twice with 4 ml of 5%
TCA and the filters were rinsed twice with TCA and once
with 95% ethanol. After drying the filters, they were
placed in scintillation vials containing 4–5 ml of scintilla-
tion cocktail Ecolume. Vials were vortexed for 20 s
and counted. The percent inhibition was calculated by
employing the following formula: percent inhibi-
tion = 100 ꢀ (DCd · 100)/DCc, where DCc and DCd are
the differences in the count per minute of control cultures
and drug-treated cultures, respectively.
38. Selected data for title compounds 2 and 3.
4-(3-Fluorophenoxy)phenoxyethyl thiocyanate (2): yellow
pale oil; Rf 0.5 (hexane/EtOAc, 7:3); IR (film, cmꢀ1) 3075,
2927, 2872, 2158, 1607, 1509, 1271, 1122, 1080, 968, 772;
1H NMR (CDCl3) d 3.34 (t, J = 5.8 Hz, 2H, H-1), 4.31 (t,
J = 5.8 Hz, 2H, H-2), 6.64 (dt, J = 10.4, 2.3 Hz, 1H, H-20),
6.74 (m, 2H, H-500, H-600), 6.93 (d, J = 9.0 Hz, 2H, H-20),
7.05 (d, J = 8.9 Hz, 2H, H-30), 7.24 (dt, J = 8.2, 6.8 Hz,
1H, H-400); 13C NMR (CDCl3) d 33.26 (C-1), 66.34 (C-2),
105.03 (d, J = 24.6 Hz, C-200), 109.31 (d, J = 21.2 Hz, C-
400), 111.70 (SCN), 112.98 (d, J = 3.4 Hz, C-600), 115.93 (C-
20), 121.29 (C-30), 130.39 (d, J = 10.2 Hz, C-500), 150.17 (C-
40), 154.45 (C-10), 159.69 (d, J = 11.0 Hz, C-100), 163.47 (d,
J = 246.7 Hz, C-300); MS (m/z, relative intensity) 289 (M+,
96), 203 (100), 147 (27), 95 (63), 86 (67); Anal. Calcd for
C15H12FNO2S: C, 62.28; H, 4.15; N, 5.19; S, 11.07.
Found: C, 62.71; H, 4.41; N, 4.77; S, 10.78.
4-(4-Fluorophenoxy)phenoxyethyl thiocyanate (3): yellow
pale oil: Rf 0.48 (hexane/EtOAc, 7:3); IR (film, cmꢀ1
)
2930, 2169, 1515, 1219, 1041, 842; 1H NMR (CDCl3)d
3.33 (t, J = 5.8 Hz, 2H, H-1), 4.30 (t, J = 5.8 Hz, 2H, H-2),
6.90 (d, J = 9.2 Hz, 2H, H-30), 6.92 (m, 2H, H-300), 6.95 (d,
J = 9.2 Hz, 2H, H-20), 7.00 (m, 2H, H-200); 13C NMR
(CDCl3)d 33.28 (C-1), 66.40 (C-2), 111.71 (SCN), 115.99
(d, J = 18.7 Hz, C-300), 116.24 (C-20), 119.41 (d, J = 8.5 Hz,
C-200), 120.14 (C-30), 151.69 (C-40), 153.86 (C-10), 158.45
(d, J = 241.6 Hz, C-400); MS (m/z, relative intensity) 289
(M+, 100), 203 (99), 147 (24), 95 (41), 86 (42); Anal. Calcd
for C15H12FNO2S: C, 62.28; H, 4.15; N, 5.19; S, 11.07.
Found: C, 62.42; H, 4.30; N, 4.81; S, 10.93.
43. Toxoplasma gondii tachyzoites of the m2m3 clone express-
ing yellow fluorescence protein44 were routinely maintained
in vitro in human foreskin fibroblast monolayers (HFF) in
Dulbecco’s modified Eagle’s medium (DMEM) supple-
mented with 10% fetal bovine serum, 2 mM glutamine,
1 mM pyruvate, at 37 ꢁC in a humid 5% CO2 atmosphere.
Confluent HFF monolayers grown in 96-well black plates
with optical bottoms (Falcon/Becton–Dickinson, Franklin
Lakes, NJ) were used and drugs dissolved in the same
medium and serially diluted in the plates. Freshly isolated
tachyzoites were filtered through a 3 lm filter and passed
through a 22 gauge needle, before use. The cultures were
inoculated with 104 tachyzoites/ml in the same media. The
plates were incubated at 37 ꢁC and read daily in a Molecular
Devices fluorescent plate reader. To preserve sterility the
plates were read with covered lids, and both excitation
(510 nm) and emission (540 nm) were read from the
bottom.44 For the calculation of the IC50, the percent of
growth inhibition was plotted as a function of drug
concentration by fitting the values to the function: I = Imax
C/(IC50 + C), where I is the percent inhibition, Imax = 100%
inhibition, C is the concentration of the inhibitor, and IC50
is the concentration for 50% growth inhibition.
39. Trypanosoma cruzi epimastigotes (Y strain) were grown in
a LIT medium containing 5% NCS and 1% P/S. Five-day-
old culture was centrifuged and resuspended in fresh
medium to get a 2–3 · 106 cell/ml suspension. Parasites
were then placed in sterile screw-cap tubes (2 ml/tube).
Two tubes were filled with medium for blank. Each drug
was tested at four different concentrations (1, 2.5, 5, 10,
and 20 lg/ml), each one in triplicate. Drugs stock solu-
tions were prepared in absolute ethanol and then were
diluted in a LIT medium. A control without drug was
done for each group tested. The concentrations of cells
were determined by measuring the absorbance of the
culture medium containing parasites at 600 nm against a
blank with culture medium alone. To calculate percent
44. Gubbels, M.-J.; Striepen, B. Antimicrob. Agents Chemo-
ther. 2003, 47, 309.