Discovery of synthetic leishmania inhibitors by screening of a 2-arylbenzothiophene library

Article abstract of DOI:10.1111/cbdd.12239

Tamoxifen has been shown to be active in vitro against Leishmania and effective in the treatment for leishmaniasis in murine models. Through the screening of a compound library of estrogen receptor modulator analogs, we identified the major characteristic

Full text of DOI:10.1111/cbdd.12239

Chem Biol Drug Des 2014; 83: 289–296
Research Article
Discovery of Synthetic Leishmania Inhibitors by
Screening of a 2-Arylbenzothiophene Library
Vivian I. Bonano1, Jenicer K. U. Yokoyama-
Yasunaka1, Danilo C. Miguel1,†, Scott A. Jones2,
Jeffrey A. Dodge2 and Silvia R. B. Uliana1,*
Leishmaniasis is a neglected tropical disease caused by
protozoan parasites endemic to 98 countries globally, with
367 million people at risk and about 2 million new cases
yearly (1,2). Over 20 Leishmania species are known to
infect humans. Clinical manifestations range from the dis-
figuring skin lesions of cutaneous leishmaniasis (CL) to the
often fatal visceral leishmaniasis (VL) (3). CL presentation
varies from self-healing cutaneous ulcers, through very
aggressive and disfiguring cases of disseminated or muco-
cutaneous lesions to the rare but life-threatening diffuse
leishmaniasis. VL is characterized by prolonged fever,
enlarged spleen and liver, substantial weight loss and pro-
gressive anemia and invariably leads to death if untreated.
The disease is even more severe in children and in the
course of immuno-depression as is the case for patients
with concomitant HIV infections (4).
1
^
Departamento de Parasitologia, Instituto de Ciencias
ꢀ
~
~
Biomedicas, Universidade de Sao Paulo, Sao Paulo, SP
05508-900, Brazil
2Lilly Research Laboratories, Eli Lilly and Company, Lilly
Corporate Center, Indianapolis, IN 46285, USA
*Corresponding author: Silvia R. B. Uliana,
srbulian@icb.usp.br
†Present address: Departamento de Biologia Animal,
Instituto de Biologia, Universidade Estadual de Campinas,
SP, Brazil
Tamoxifen has been shown to be active in vitro against
Leishmania and effective in the treatment for leishman-
iasis in murine models. Through the screening of a
compound library of estrogen receptor modulator ana-
logs, we identified the major characteristics required
for antileishmanial activity. To overcome the difficulties
presented by tamoxifen’s propensity for E/Z isomeriza-
tion, we used the 2-arylbenzothiophene compound
BTP as a more stable alternative. Directed screening
of a small compound library based on BTP led to
active compounds against Leishmania. Subsequent
structure–activity data for the synthetic 2-arylbenzothi-
ophenes evaluated in this study indicate that optimal
antileishmanial potency is dependent on the presence
of two basic side chains. In addition, the primary struc-
tural features required for estrogen receptor binding,
the phenols, are not required for inhibiting parasitic
growth. Significantly, the most active antileishmanial
benzothiophenes lack the pharmacophore for estrogen
receptor activity and therefore address potential con-
cerns about the undesirable effects of using selective
estrogen receptor modulators in women and children
with leishmaniasis. Three compounds selected from
the screening have shown consistent activity against
all species and stages of Leishmania in vitro although
improvements in selectivity are needed. These com-
pounds represent viable starting points for further opti-
mization as antileishmanial agents.
Human leishmaniasis treatment is based on parenteral
administration of highly toxic drugs, including pentavalent
antimonials, amphotericin B (deoxycholate or as a liposomal
formulation), and pentamidine. Resistance to antimonials is
widespread in India, and oral administration of miltefosine
has emerged as an alternative approach, having been
approved for management of VL. However, the efficacy of
miltefosine outside India is still a matter of investigation. Fur-
thermore, there is reason for concern about the rapid emer-
gence of resistance to miltefosine and its teratogenicity (5).
Clinical failure rates of CL treatment with antimonials are
also rising in Latin America (6,7), suggesting that the emer-
gence of resistant strains may be occurring (8,9).
The need for the development of new treatment strategies
for leishmaniasis is therefore evident. New drugs or thera-
peutic schemes would ideally be effective against all species
of the parasite, allowing their use in all forms of the disease.
Tamoxifen (Figure 1) is a selective estrogen receptor mod-
ulator (SERM) that has been in clinical use for the treat-
Key words: leishmaniasis, selective estrogen receptor modu-
lator, tamoxifen
Tamoxifen
BTP
Received 3 July 2013, revised 18 September 2013 and
accepted for publication 27 September 2013
Figure 1: Structures of SERMs tamoxifen and BTP.
ª 2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12239
289

Bonano et al.
Table 1: Structures and biological activity of BTP and benzothiophene analogs against promastigotes of L. chagasi
a
Compound
R1
R2
R
X
IC50 (lM Æ SD)
52.2 Æ 18.2
cLogP
5.18
cLogD
4.79
BTPb
OH
OH
C=O
15b
14c
H
H
OH
C=O
C=O
34.6 Æ 4.1
36.5 Æ 5.7
5.55
6.06
5.09
5.25
OMe
12
13
10
H
H
H
OH
OMe
H
OH
OH
OH
C=O
C=O
C=O
88.7 Æ 0.2
79.0 Æ 0.1
65.8 Æ 0.1
5.35
5.49
5.65
5.10
5.35
5.51
19d
20c
21d
22d
23d
H
H
H
H
H
C=O
C=O
C=O
C=O
CH2
3.7 Æ 0.3
28.7 Æ 1.8
4.3 Æ 0.2
5.4 Æ 0.4
3.6 Æ 0.2
6.93
8.10
7.63
8.73
8.26
4.72
6.59
4.37
5.49
4.28
aTamoxifen was used as a positive control in all experiments and the overall mean IC50 for L. chagasi promastigotes was 12.8 Æ 6.0 lM.
bFree base.
cOxalate salt.
dDioxalate salt.
ment and prevention of breast cancer for over 30 years.
However, in an estrogen receptor (ER) independent way,
tamoxifen also demonstrates several other effects in tumor
cells, for example, alkalinization of intracellular organelles
(10), protein kinase C modulation (11), apoptosis induction
(12) and modulation of sphingolipid metabolism (13).
cutaneous and visceral experimental models of leishmania-
sis (15,16). The sensitivity of different species of Leish-
mania, agents of cutaneous (e.g. Leishmania braziliensis,
L. amazonensis, L. major) as well as of visceral disease
(L. donovani and L. chagasi) is uniform with in vitro IC50 in
the low micromolar range. While in the treatment for breast
cancer tamoxifen is usually given continuously for 5 years,
in the leishmaniasis murine models, the drug was effective
when administered for 2–3 weeks. We have previously
Our group has recently described the antileishmanial activ-
ity of tamoxifen in vitro (14,15) as well as in vivo in both
290
Chem Biol Drug Des 2014; 83: 289–296

Discovery of Antileishmanial Benzothiophenes
R
O
Route 1
O
O
b
a
R2
R2
+
R2
Br
S
S
SH
R2
R
9
H
H
Me
H
7 R2 = H
10
11
12
8 R2 = OMe
OMe Me
OH
H
H
13 OMe
OH
OMe
O
N
O
O
S
O
O
c
N
d
OH
OMe
S
S
15
14
13
Route 2
F
O
S
N
N
e
f
B(OH)2
O
O
S
S
Scheme 1: Synthesis of 2-
18
17
16
Arylbenzothiophenes. Reagents: (a)
KOH, PPA; (b) anisoyl chloride,
AlCl3; (c) N-(2-chloroethyl)
pyrrolidine hydrochloride, NaH; (d)
2N NaOH, EtSH; (e) Pd(PPh3)4, 2 N
aqueous Na2CO3, 1-[2-(4-
bromophenoxy)ethyl]pyrrolidine; (f)
p-fluorobenzoyl chloride, TiCl4; (g)
RH, NaH; (h) LiAlH4; (i) Et3SiH, TFA.
g
R
O
R
O
O
S
h, i
N
N
O
O
S
23
19-22
shown that tamoxifen’s antileishmanial activity cannot be
competed out by estradiol, suggesting that the effect
against the parasite is unrelated to ER interaction (14).
M2682), Leishmania (Leishmania) amazonensis (MHOM/BR/
1973/M2269) and Leishmania (Viannia) braziliensis (MHOM/
BR/1975/M2903). Culture media and fetal bovine serum
were acquired from Invitrogen. Chemicals used in biological
tests were from Sigma-Aldrich (St. Louis, MO, USA).
While there are several advantages in repurposing a drug
so widely used and with an established safety profile, the
use of tamoxifen in leishmaniasis chemotherapy raises
some safety concerns, including its use in children in
which bone development could be impaired (17) or in
childbearing age women. In addition, treatment for women
with tamoxifen has been associated with an increased risk
of endometrial cancer (18,19). To address these concerns,
we sought to identify alternative chemotypes that have an-
tileishmanial activity yet are devoid of ER activity.
Compound 1 (Tamoxifen [(Z)-1-(p-Dimethylaminoethoxy-
phenyl)-1,2-diphenyl-1-butene, trans-2-[4-(1,2-Diphenyl-1-
butenyl)phenoxy]-N,N-dimethylethylamine]) and compound
2
(2-[4-[(E)-1,2-diphenylbut-1-enyl]phenoxy]-N,N-dimethy-
lethanamine) were purchased from commercial sources
(Sigma-Aldrich).
Compound synthesis
With the aim of identifying hits for further optimization, we
assessed the structural requirements of the tamoxifen scaf-
fold and evaluated two SERM scaffolds, triphenylethylenes
and benzothiophenes, to compare their antiparasitic activity.
We also determined the structural requirements for antileish-
manial activity and whether these determinants are distinct
from those known to be responsible for ER activity.
The syntheses of tamoxifen analogs have been reported,
that is, for analogs 3 and 4 see Shiina et al. (20); for ana-
logs 5 and 6 see (21). The compound library screened for
antileishmanial activity consisted of 147 structurally diver-
gent benzothiophenes from the Lilly compound collection.
These compounds were >95% pure as determined by
LCMS analysis. The synthesis and pharmacology of the
benzothiophene BTP (Figure 1) has been previously
described (22,23). The general synthetic routes for the
preparation of the 2-arylbenzothiophene compounds
shown in Table 1 are depicted in Scheme 1. Two comple-
mentary routes into the benzothiophene scaffold were
developed. In the first route, FriedelÀCrafts acylation (for a
Methods and Materials
Leishmania strains used in this work were as follows: Leish-
mania (Leishmania) infantum chagasi (MHOM/BR/1974/
Chem Biol Drug Des 2014; 83: 289–296
291

Bonano et al.
review of Friedel-Crafts acylation, see (24) with known 2-a-
rylbenzothiophenes 7 and 8 with anisoyl chloride provided
analogs 9 and 11 substituted at the C-3 position of the
benzothiophene. O-Demethylation of 9 and 11 was accom-
plished by complementary methods, that is, pyridine hydro-
chloride was used to make 10 and 12, while selective
removal of the 4′-OMe group was accomplished using
EtSNa (25) to provide 13. Subsequent alkylation with N-(2-
chloroethyl)pyrrolidine hydrochloride followed O-demethyla-
tion with EtSNa gave compound 15. The second general
synthetic approach that was employed utilized benzo[b]
thiophene-2-boronic acid (16). Palladium catalyzed cou-
pling with 1-(2-(4-bromophenoxy)ethyl)pyrrolidine gave
compound 17 which was acylated at the C-3 position with
p-fluorobenzoyl chloride affording the common intermedi-
ate 6. Employing the methods of Schmid et al. (26), a
diverse set of basic side chains was installed at the 4′-posi-
tion through the displacement of the fluoride with a variety
of oxygen nucleophiles to yield derivatives 19–22. Reduc-
tive deoxygenation to the C-3 methylene derivative (23)
was accomplished by a two-step procedure involving initial
hydride reduction to the secondary benzylic alcohols
(LiAlH4), followed by a second reductive deoxygenation
(TFA/Et3SiH) to give the desired methylene product 23.
Attempts to affect both the reduction and deoxygenation
steps in a single transformation using NaBH4/TFA were
unsuccessful. Experimental procedures and characteriza-
tion for all compounds in Table 1 are included in the Sup-
porting Information. Further information on the preparation
of these and other analogs has been described (27,28).
of each compound with ORIGIN PRO 7.5 analysis software
(OriginLab Corporation, Northampton, MA, USA).
Cytotoxicity against mammalian cells in vitro was evaluated
by testing each compound against Vero cells. Cells were
seeded in 24-well plates at a density of 4–5 9 105 cells/
well and allowed to adhere overnight at 37 °C, in a 5%
CO2 atmosphere. Cells were then treated for 48 h with
various concentrations (0.3–81 lM) of the compounds of
interest, replacing the media with drug after the initial
24 h. Cell viability after treatment was assayed by MTT as
described above.
In vitro activity testing against Leishmania intracellular am-
astigotes was performed using BALB/c bone marrow-derived
macrophages (BMDM) which were seeded at a density of
4 9 105 cells/well in 24-well plates containing round cover-
slips. Cells were allowed to adhere overnight at 37 °C with
5% CO2 and then infected for 3 h with L. chagasi promastig-
otes at
a ratio of 10 parasites/macrophage. Infected
macrophages were then treated with various concentrations
of each compound (0.3–8 lM) for 48 h, replacing the media
with drug at 24 h. Cells were then washed, fixed, and stained
using Instant ProV (Newprov, Pinhais, PR, Brazil). Coverslips
containing stained cells were fixed onto slides using Entellan
(Merck KGaA, Darmstadt, Germany), and cells were evalu-
ated by light microscopy (Nikon Eclipse E200; Nikon Cor-
poration, Tokyo, Japan) to determine the number of infected
cells and the number of amastigotes/cell. Percent survival
was calculated for each treatment performed intriplicate.
Biological evaluation
Results and Discussion
Promastigotes were cultured at 25 °C in M199 supplemented
with 40 mM HEPES pH 7.4, 0.1 mM adenine, 0.005% hemine,
10% fetal bovine serum, and 100 lg/mL penicillin/streptomy-
cin. Additionally, cultures of L. braziliensis and L. chagasi
were supplemented with 2% human male sterile urine.
The E and Z isomers of tamoxifen analogs are known to
have distinct estrogen-dependent activities (30). While the
(Z)-isomer of tamoxifen (1) is a SERM, the cis isomer (E) (2)
is a pure estrogen agonist with no estrogen-antagonistic
actions (31). We wanted to test whether these isomers dem-
onstrated different antileishmanial activity. (Z)-Tamoxifen (1)
kills L. chagasi promastigotes in a concentration dependent
way with a calculated 50% inhibitory concentration (IC50) of
17.7 Æ 0.8 lM (14). When (E)-tamoxifen (2) (Table 2) was
used, a calculated IC50 of 12.6 Æ 1.1 lM was obtained indi-
cating that both isomers are active against Leishmania. We
also tested other (Z) and (E) analogs of tamoxifen, as shown
in the comparison between compounds 3–4 and 5–6,
respectively, in Table 2. In each case, the isomeric pairs
were similar in potency. Because interconversion between
(Z) and (E) isomers is known to occur among tamoxifen ana-
logs (32), we could not rule out that the similarities in
potency seen between each pair are due to E/Z conversion
under the assay conditions. Rather than explore the propen-
sity for isomerization in promastigotes, we elected to look
for other scaffolds with fewer structural liabilities.
In vitro assays against Leishmania promastigotes were
performed by incubating 5 9 106 parasites in media con-
taining increasing concentrations (0.3–250 lM) of the com-
pounds dissolved in DMSO at 25 °C for 24 h, in 96-well
plates. The maximal concentration of DMSO used to add
the compounds was 3%, which was used as a negative
control, while tamoxifen and amphotericin B were used as
positive controls. The effective antileishmanial concentra-
tion was assessed by colorimetry using MTT (3-(4,5-dim-
ethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) as
described previously (29). MTT reduction was quantified
by measuring the absorbance at 595 nm using 690 nm as
a reference, in a Multiscan EX spectrophotometer (Lab
Systems, Inc., Vantaa, Finland). Assays were performed in
triplicate, and results are expressed as the mean percent-
age reduction in parasite numbers compared with
untreated control wells calculated for at least two indepen-
dent experiments. Percent survival was used to determine
The 2-arylbenzothiophene scaffold has been shown to
the 50% and 90% inhibitory concentration (IC50 and IC90
)
mimic the stilbene core of tamoxifen (1) based on molecu-
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Discovery of Antileishmanial Benzothiophenes
Table 2: Structures and biological activity of (Z) and (E) geometrical isomers of tamoxifen and diarylethylene analogs against promastig-
otes of Leishmania chagasi
Isomer (Z) R1
R2
IC50 (lM Æ SD) IC90 (lM) Isomer (E) R1
R2
IC50 (lM Æ SD) IC90 (lM) cLogP cLogD
1
3
5
H
3-OH
H
H
17.7 Æ 0.8
13.4 Æ 4.5
7.0 Æ 0.8
46.6
27.6
15.2
2
4
6
H
3-OH
H
H
12.6 Æ 1.1
9.2 Æ 0.5
6.6 Æ 0.9
26.1
22.6
20.8
6.35
5.68
6.19
4.97
4.66
4.81
4-OMe 4-OMe
4-OMe 4-OMe
lar modeling overlays and crystal structures (33,34). In
addition, the thiophene ring prevents potential isomeriza-
tion issues by locking the three aryl groups in a topological
display similar to 1 but not 2. Because of our experience
with SERMS, we selected the 2-arylbenzothiophene BTP
(Figure 1) to test for antileishmanial activity. Like tamoxifen,
BTP is a SERM and possesses pharmacological properties
similar to second generation SERMs in rodents (22,23).
The synthesis and pharmacology of the benzothiophene
BTP has been previously described (22,23).
phenols (R1 and R2, Table 1) and the basic side chain (R,
Table 1). We were particularly interested in determining
whether the structural features responsible for antiparasitic
activity were related to those responsible for ER activity. A
phenol at R1 is known to be important for binding to ER
for BTP (27).
To determine whether the phenol is important for antileish-
manial activity, we analyzed benzothiophene 15 (Table 1)
in which the phenol at the 6-position of the benzothioph-
ene is not present. This compound inhibits parasitic
growth with a half-maximal effective concentration of
34.6 Æ 4.1 lM, marginally more potent than BTP
(52.2 lM), indicating that this phenol is not a requirement
for antileishmanial activity. As the 4′-phenol is also known
to play a role in ER binding, we tested benzothiophene 14
in which the OH has been masked as a methyl ether, a
group which is known to diminish ER binding (33). These
two compounds, 14 and 15, are equipotent, that is,
36.5 Æ 5.7 lM versus 34.6 Æ 4.1 lM, respectively. Taken
together, this indicates that neither phenol is required for
antileishmanial activity, validating our previous findings that
indicated that tamoxifen’s antileishmanial activity and ER
binding are not related (14).
When tested against L. chagasi, BTP inhibits parasitic
growth with an IC50 of 52.2 Æ 18.2 lM (see Table 1). Apart
from being active against a viscerotropic species, BTP was
also shown to be effective against the cutaneous species
L. amazonensis with an IC50 of 50.6 Æ 21.3 lM. While BTP
is approximately fivefold less potent than tamoxifen, it is
configurationally stable, and thus provided a seed for further
hit expansion. Thus, holding the core 2-arylbenzothiophene
constant, we conducted a similarity search of the Lilly com-
pound collection that resulted in the identification of 147
divergent 2-arylbenzothiophenes which were collected for
screening purposes and subsequently tested against
L. chagasi. An initial screen was performed against
L. chagasi promastigotes in a dose-response test with 0.5,
5, 50, and 250 lM of each compound. Compounds with
IC50 lower than 10 lM were subsequently subjected to
more detailed dose-response tests.
To determine the significance of the basic side chain R,
the inhibition potencies for compounds 15 and 14 were
compared to analogs that lack the side chain, compounds
10, 12, and 13 (Table 1). An approximate twofold drop-off
in potency is observed for these compounds indicating
that the presence of the side chain is important for antile-
ishmanial potency. However, one of the more potent com-
pounds in the original screening cassette was compound
19 (Table 1), which contains a pyrrolidine and a piperidine
base and is 10-fold more potent than 15 which contains
only a single basic pyrrolidine, that is, compare 3.7 lM for
19 to 34.6 lM for 15. Thus, we expanded the SAR in
these regions. Replacement of either of the basic nitro-
gen’s with a non-basic carbon atom results in a loss of
activity, that is, cyclopentyl analog 20 has an IC50 of
In general, most molecules tested exhibited activity (123
compounds) with an IC50 that ranged from 3.6 Æ 0.2
to 121.9 Æ 0.2 lM. Among these, 101 compounds exhib-
ited an IC50 ≤ BTP (IC50 52.6 Æ 18.4 lM) and 59 had an
IC50 ≤ tamoxifen (IC50 17.7 Æ 0.8 lM), respectively.
Twenty-four compounds with an IC50 over 250 lM were
deemed unsuitable for our purposes, and their exact activ-
ity was not determined.
Structure–activity relationships in the benzothiophene scaf-
fold initially examined two of its structural subunits, the
Chem Biol Drug Des 2014; 83: 289–296
293

Bonano et al.
28.7 lM, respectively, compared with 3.7 lM for 19. These
data further demonstrate the significance of the nitrogen
for biological activity.
Cytotoxicity against mammalian cells in vitro was evaluated
in Vero cell cultures. Selectivity indexes were around 3.0
(Table 3). We also evaluated 19 for cell-based activity
against the ER and found it to be inactive (>25 lM) in a
GAL4 luciferase cotransfected with ERa or ERb (data not
shown).
Variation in the base is well tolerated. For example, the
potencies of N,N-di-isopropylamine in compound 21
(4.3 lM) and N,N-di-n-butylamine in 22 (5.4 lM) are similar to
pyrrolidine in 19 (3.7 lM). Changing the carbonyl group to
the corresponding methylene (compare 22–23 in Table 1)
does not alter the potency. In summary, the structure–activity
data for the synthetic 2-arylbenzothiophenes evaluated in
this study indicate that optimal antileishmanial potency is
dependent on the presence of two basic side chains. In addi-
tion, the primary structural features required for ER binding,
the phenols, are not required for inhibiting parasitic growth.
Concentrations in the range of the IC50 against promastig-
otes (4 lM) were used to test the activity against intracellu-
lar amastigotes in L. chagasi infected macrophages. At
these concentrations, the treatment with compounds 19,
23 and 21 reduced the number of infected cells by
70.1 Æ 8.1, 64.8 Æ 11.8, and 34.4 Æ 6.3%, respectively,
demonstrating the compounds’ activity against intracellular
amastigotes. While it is desirable that selectivity indexes in
vitro are as high as possible, for some drugs, in vitro data
do not correlate well with in vivo drug safety. For example,
an in vitro selective index of only 1.3 was found for tamoxi-
fen (14), while in vivo safety data allow very wide safety
margins for this drug.
In terms of the physical properties of these compounds,
the calculated logP’s, which are a measure of drug-like-
ness, are shown in Tables 1 and 2. Historically, launched
drugs have cLogP’s of approximately 2.5, while tamoxifen
and raloxifene, the most closely structurally related
launched drugs to the chemical entities in our study, have
clogP’s of 6.35 and 6.34, respectively. The most active of
the benzothiophene analogs against Leishmania, 19, 21,
22 and 23, has clogP’s in the 6.93–8.26 range. Likewise,
the molecular masses are somewhat high relative to mar-
keted drugs. As these physicochemical parameters are
somewhat higher than both the average drug and even
structurally related ones, optimization of these properties
may be required in future SAR.
The antileishmanial mechanism of action of tamoxifen is
presently under study. Dibasic benzothiophenes such as
19 have been shown to inhibit the serine protease throm-
bin, but there appears to be little correlation between the
reported thrombin activity and the observed antileishmanial
activity (28). For example, the thrombin inhibitory activity of
the compound 19 is 30 times more potent than of com-
pound 23 (28), while they are equipotent in their leishmani-
cidal effect.
Based on these findings, we selected the compounds with
the most potent activity to be tested further. Tamoxifen
was previously shown to be active against all Leishmania
species tested. Furthermore, it was demonstrated to be
equally active against the promastigote stage as well as
against the intracellular amastigote stage, present in the
mammalian host (14,15). The activity of compounds 19,
21 and 23 was also homogeneous across the different
Leishmania species tested, with values of IC50 in the sub-
10 lM range (see Table 3), a desirable characteristic for a
future drug development.
Some SERMs (clomiphene, toremifene, tamoxifen, and
raloxifene) were recently shown to possess in vitro and in
vivo potent inhibitory activity against Ebola virus infection
(35). This activity was also unrelated to ER expression and
response. Mechanistic studies suggested that this inhibi-
tory activity (for toremifene and clomiphene) is related to a
late step in viral entry, possibly through interference in
endosome membrane composition (35).
Studies in progress in our laboratories indicate that distur-
bances of membrane fluidity and interference with sphin-
Table 3: IC50 of selected compounds against promastigotes of different Leishmania speciesa and toxicity to mammalian cells
Leishmania
amazonensis
Leishmania chagasi
Leishmania braziliensis
VERO
Compound
IC50 (lM Æ SD)
IC90 (lM)
IC50
IC90
IC50
IC90
IC50
IC90
SIb
19
21
3.7 Æ 0.3
4.3 Æ 0.2
8.2
7.9
9.2
ND
9.9 Æ 0.5
8.0 Æ 1.2
20.4
13
11.4
ND
5.2 Æ 0.8
10.0
6.3
5.4
11.7 Æ 3.6
13.6 Æ 1.6
13.0 Æ 2.9
ND
15.2
16.7
16
3.16
3.16
3.6
3.5 Æ 0.9
4.2 Æ 0.1
23
Amphotericin B
3.6 Æ 0.2
7.3 Æ 1.8
0.111 Æ 0.013
0.091 Æ 0.006
0.070 Æ 0.013
ND
ND
ND
ND, not determined.
aLeishmania strains used in this work were as follows: L. (L.) infantum chagasi (MHOM/BR/1974/M2682), L. (L.) amazonensis (MHOM/BR/
1973/M2269), and L. (V.) braziliensis (MHOM/BR/1975/M2903).
bSelectivity index calculated for L. chagasi: IC50 mammalian cell/IC50 L. chagasi.
294
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Discovery of Antileishmanial Benzothiophenes
golipid biosynthesis seem to be responsible for tamoxifen’s
leishmanicidal activity (unpublished data). Future experi-
ments using the compounds identified in this work will be
directed at investigating whether they induce the same
biochemical modifications as tamoxifen.
(2007) Inactivation of the miltefosine transporter, LdMT,
causes miltefosine resistance that is conferred to the
amastigote stage of Leishmania donovani and persists
in vivo. Int J Antimicrob Agents;30:229–235.
6. Machado P.R., Ampuero J., Guimaraes L.H., Villasboas
L., Rocha A.T., Schriefer A., Sousa R.S., Talhari A.,
Penna G., Carvalho E.M. (2010) Miltefosine in the treat-
ment of cutaneous leishmaniasis caused by Leishmania
braziliensis in Brazil: a randomized and controlled trial.
PLoS Negl Trop Dis;4:e912.
7. Neves L.O., Talhari A.C., Gadelha E.P., Silva Junior
R.M., Guerra J.A., Ferreira L.C., Talhari S. (2011) A
randomized clinical trial comparing meglumine antimo-
niate, pentamidine and amphotericin B for the treat-
ment of cutaneous leishmaniasis by Leishmania
guyanensis. An Bras Dermatol;86:1092–1101.
8. Inocencio da Luz R., Romero G.A., Dorval M.E., Cruz
I., Canavate C., Dujardin J.C., Van Assche T., Cos P.,
Maes L. (2011) Drug susceptibility of Leishmania infan-
tum (syn. Leishmania chagasi) isolates from Brazilian
HIV-positive and HIV-negative patients. J Antimicrob
Chemother;66:677–679.
Conclusions and Future Directions
The directed screening of a 2-arylbenzothiophene library
based on the SERM BTP resulted in the discovery of a
novel class of potent antileishmanial agents. Structure–
activity studies indicate that the critical determinant for an-
tileishmanial potency is the presence of two basic side
chains. Representative compounds within this structural
class have shown consistent activity against all species
and stages of Leishmania in vitro although improvements
in selectivity are needed. As such, these compounds rep-
resent viable starting points for further optimization as an-
tileishmanial agents. Significantly, the most active
antileishmanial benzothiophenes lack the pharmacophore
for ER activity, and therefore address potential concerns
about the undesirable effects of using SERMs in women
and children with leishmaniasis.
9. Rojas R., Valderrama L., Valderrama M., Varona M.X.,
Ouellette M., Saravia N.G. (2006) Resistance to anti-
mony and treatment failure in human Leishmania (Vian-
nia) infection. J Infect Dis;193:1375–1383.
Acknowledgments
10. Altan N., Chen Y., Schindler M., Simon S.M. (1999)
Tamoxifen inhibits acidification in cells independent of
the estrogen receptor. Proc Natl Acad Sci
USA;96:4432–4437.
We thank Andrew S. Bell for the critical reading of the
manuscript. This work was supported by Grants #2011/
~
20484-7, Sao Paulo Research Foundation (FAPESP) and
11. O’Brian C.A., Liskamp R.M., Solomon D.H., Weinstein
I.B. (1985) Inhibition of protein kinase C by tamoxifen.
Cancer Res;45:2462–2465.
12. Mandlekar S., Kong A.N. (2001) Mechanisms of
tamoxifen-induced apoptosis. Apoptosis;6:469–477.
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human cancer cells. FEBS Lett;394:129–131.
473343/2012-6, Conselho Nacional de Desenvolvimento
Cientıfico e Tecnologico (CNPq). V.I.B. was supported by
a PNPD/CAPES fellowship (2289/2009).
ꢀ
ꢀ
Conflict of Interest
None.
14. Miguel D.C., Yokoyama-Yasunaka J.K., Andreoli W.K.,
Mortara R.A., Uliana S.R. (2007) Tamoxifen is effective
against Leishmania and induces a rapid alkalinization
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(2009) Tamoxifen as a potential antileishmanial agent:
efficacy in the treatment of Leishmania braziliensis and
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
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