Benzophenone-based farnesyltransferase inhibitors with high activity against Trypanosoma cruzi

Article abstract of DOI:10.1021/jm050456x

Less toxic drugs are needed to combat the human parasite Trypanosoma cruzi (Chagas's disease). One novel target for antitrypanosomal drug design is farnesyltransferase. Several farnesyltransferase inhibitors based on the benzophenone scaffold were assayed

Full text of DOI:10.1021/jm050456x

7186
J. Med. Chem. 2005, 48, 7186-7191
Benzophenone-Based Farnesyltransferase Inhibitors with High Activity against
Trypanosoma cruzi
Mo´nica I. Esteva,† Katja Kettler,§ Cristina Maidana,† Laura Fichera,† Andre´s M. Ruiz,† Esteban J. Bontempi,*,†
Bjo¨rn Andersson,| Hans-Martin Dahse, Peter Haebel,X Regina Ortmann,§ Gerhard Klebe,X and
Martin Schlitzer*,§
Center for Genomics and Bioinformatics, Karolinska Institute, Stockholm 171 77, Sweden, Instituto Nacional de Parasitolog´ıa
‘Dr. M. Fatala Chabe´n’, A.N.L.I.S., ‘Dr. Carlos G. Malbra´n’, 1063 Buenos Aires, Argentina, Institut of Pharmaceutical
Chemistry, Philipps-University, D-35032 Marburg, Germany, Leibniz-Institut fu¨r Naturstoff-Forschung e.V., D- Jena,
Germany, and Department of Pharmacy, Center for Pharmaceutical Research, Ludwig-Maximilians-University,
D-81377 Munich, Germany
Received May 12, 2005
Less toxic drugs are needed to combat the human parasite Trypanosoma cruzi (Chagas’s
disease). One novel target for antitrypanosomal drug design is farnesyltransferase. Several
farnesyltransferase inhibitors based on the benzophenone scaffold were assayed in vitro and
in vivo with the parasite. The common structural feature of all inhibitors is an amino function
which can be protonated. Best in vitro activity (LC50 values 1 and 10 nM, respectively) was
recorded for the R-phenylalanine derivative 4a and for the N-propylpiperazinyl derivative 2f.
These inhibitors showed no cytotoxicity to cells. When tested in vivo, the survival rates of
infected animals receiving the inhibitors at 7 mg/kg body weight/day were 80 and 60% at day
115 postinfection, respectively.
Introduction
somatids.8-11 In this context, it is important to note that
the investigations of the antitrypanosomal efficacy of
FTase inhibitors concentrated on T. brucei while only
few inhibitors were tested against the causative agent
of Chagas’s disease, T. cruzi. We therefore focused our
efforts on T. cruzi and joined the active search for better
inhibitors through synthesis and experimental testing
of different series of our previously described12-17 ben-
zophenone-based FTase inhibitors.
Synthesis. The synthesis of most inhibitors employed
in this study has been described.12-16 The preparation
of the heterocyle-substituted derivatives will be outlined
in detail in a different publication.17 The complete sets
of analytical data are provided in these publications.
Briefly, commercially available 2-amino-5-nitroben-
zophenone was acylated by R-chlorophenylacetic acid
chloride. Then, the R-chloro was substituted by different
heterocyclic amines. Reduction of the nitro group and
subsequent acylation with appropriate phenylfurylacryl-
ic acid chlorides yielded the target compounds (Scheme
1). For the preparation of the p-chlorophenylacetic acid-
substituted compound, R-chlorophenylacetic acid was
replaced by R-bromo-p-chlorophenylacetic acid, prepared
from p-chlorophenylacetic acid.
Homology Modeling. The sequences of the alpha
and beta subunits of Trypanosoma cruzi FTase (Q8WR04
and Q8WR02) were retrieved from SWISS-Prot18 and
aligned to rat FTase (Q04631 and Q02293) using
T-COFFEE.19 Based on the rat crystal structure (pdb
code 1qbq), a homology model was computed using
MOBILE20 and MODELLER.21 Significant variation is
observed in the amino acids constituting the far aryl
binding pocket due to a high uncertainty in the sequence
alignment of this region. The procedure for docking of
ligand 2l into the active site of the rat enzyme with
AUTODOCK has been described previously.22,17
The global burden of Chagas’s disease is 649 000
DALYs (disability adjusted life years).1 Trypanosoma
cruzi, the causal agent, is found throughout much of
Mexico and Central and South America. It is transmit-
ted to humans primarily by insects (subfamily Tri-
atominae), which carry trypomastigotes, the infective
stage of the parasite, in their feces. Infected blood is
also a route of Chagas’s disease transmission, especially
since blood control is not implemented in all health
services in affected countries. The probability of getting
an infection can be as high as 2 per 100 transfusions.2
Organs can also function as vehicles, and the parasite
has been transmitted through transplantation.1 For
these reasons novel antitrypanosomal therapeutics are
urgently needed. In this context, farnesyltransferase
inhibitors are of growing importance. Farnesyltrans-
ferase (FTase) is a heterodimeric enzyme which cata-
lyzes the posttranslational modification of several pro-
teins involved in intracellular signal transduction.
Thereby, a farnesyl residue is transferred from farne-
sylpyrophosphate to the thiol of a cysteine residue in
the C-terminal CAAX consensus sequence (C: cysteine;
A: amino acid with aliphatic side chain; X: variable
amino acid) of farnesylated proteins.3,4 FTases of dif-
ferent trypanosomes have been identified and charac-
terized.5-7 Furthermore, it has been shown that differ-
ent FTase inhibitors affect the growth of trypano-
* Corresponding authors. E.J.B.: tel, +54-11-4331-4019; fax, +54-
11-4331-7142; e-mail, ejbon@yahoo.com, M.S.: tel, +49 89 2180 77804;
e-mail, martin.schlitzer@cup.uni-muenchen.de.
| Karolinska Institute.
† Instituto Nacional de Parasitolog´ıa.
X Philipps-University.
Leibniz-Institut fu¨r Naturstoff-Forschung e.V.
§ Ludwig-Maximilians-University.
10.1021/jm050456x CCC: $30.25 © 2005 American Chemical Society
Published on Web 10/20/2005

Benzophenone-Based Farnesyltransferase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23 7187
Scheme 1a
a (i) Toluene/dioxane, reflux, 2 h; (ii) (4-oxo)piperidine or morpholine or N-substituted piperazine, acetonitrile, reflux, 24 h; (iii) SnCl2
× 2H2O, ethyl acetate, reflux, 2 h; (iv) 3-[5-(4-nitrophenyl)-2-furyl]acrylic acid chloride, toluene/dioxane, reflux, 2 h.
Table 1. Benzophenone-Based FTase Inhibitors without Basic
Functions in the Side Chain
derivatives 3 and 4 (Tables 3 and 4) and the heterocyle
bearing compounds 2 (Table 2).
In the series of heterocycle substituted FTase inhibi-
tors the methylpiperazine derivative 2a served as a lead
structure. Most variations of the heterocycle led to a
significant reduction in activity with the LC50 values of
most compounds being at 50 µM or higher. However,
also in this series notably more active inhibitors were
found. The 4-oxopiperidyl compound 2d and the cyclo-
hexylpiperazinyl derivative 2i were found to be 10-fold
more active (LC50 ) 1.0 µM) than the methylpiperazinyl
derivative 2a. The propylpiperazinyl derivative 2f with
an LC50 value of 10 nM was 3 orders of magnitude more
active than 2a and again among the most active
antiprotozoal FTase inhibitors described so far. A strik-
ing feature is the inactivity of the structurally closely
related ethylpiperazinyl derivative 2e in comparison to
the propylpiperazinyl derivative 2f and also in compari-
son to the methylpiperazinyl derivative 2a in trypano-
some lysis assay. There is no readily available expla-
nation even on the basis of flexible docking (see infra),
but it should be noted that this compound is also
virtually inactive in the farnesyltransferase inhibition
assay (IC50 > 10 µM), so that the data seem consistent.
To gain insight into the structure-activity relation-
ships of this type of benzophenone-based FTase inhibi-
tors, we previously performed some docking studies with
the inhibitors 2a and 2l17,22 using the crystal structure
of rat FTase.23 In this study, we additionally prepared
a homology model for the T. cruzi enzyme (TcFTase)
(Figure 1). TcFTase shows low sequence homology to
the human or rat enzymes with an identity of 22%.
TcFTase is significantly larger than rat FTase with 628
vs 377 residues in the R-subunit, and 588 vs 437
residues in the â-subunit. Amino acid side chains
contributing to substrate binding, thus forming the
active site, are well-known from crystal structures.23
The active site residues are well conserved with 72%
sequence identity (39 of 54 residues). Amino acid
substitutions are located on opposite sides of the inhibi-
tor binding site affecting the binding of the nitrophe-
nylfurylacryloyl and the 2-(methylpiperazinyl)-2-phe-
nylacetyl moieties, respectively. As stated above, in the
region binding the nitrophenylfurylacryloyl moiety there
Results and Discussion
In the first round of the in vitro lysis assays inhibitors
1a-d (Table 1) and 2a and 2l (Table 2) were employed.
The more potent compounds were inhibitors 2a and 2l
with LC50 values in the low micromolar range. Although
only six inhibitors were employed in the first round, an
important structure activity relationship can be delin-
eated from this sample. Notable activity was only
displayed by the two methylpiperazine substituted
inhibitors (LC50 values: 10 µM). This is most probably
attributable to the higher solubility of this type of
compounds in comparison to those benzophenone-based
inhibitors lacking an amino group which can be proto-
nated. To exemplify this, compounds of type 1 display
a solubility <0.06 mM in water, whereas the solubility
of inhibitor 2a is >3.33 mM.22 Therefore, only com-
pounds possessing protonable amino groups were in-
cluded in the second round. Inhibitors of this second
round can be structurally divided into the amino acid

7188 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
Esteva et al.
Table 3. FTase Inhibitors with Phenylglycine Substructure
Table 2. FTase Inhibitors with an R-Heterocycle-Substituted
Residue
is a high uncertainty in the sequence alignment. How-
ever, the replacements of Lys 356â by Cys, Gln 48â by
Met, Glu 47â by Pro and Thr 44â by Asp are likely not
to have any significant effect on inhibitor 2l binding
because they are too distant from the inhibitor. The
majority of amino acid substitutions occurs in the pocket
where the 2-(methylpiperazinyl)-2-phenylacetyl moiety
is bound. While most substitutions probably do not
affect inhibitor binding significantly, the replacement
of Ala 129R by Gly and Gln 162R by Asn creates more
space in the vicinity of the N-4 of the piperazinyl
residue. While this could be a possible explanation of
the high activity of the N-propylpiperazinyl-substituted
inhibitor 2f, the inactivity of inhibitor 2e remains
unexplained.
With the phenylglycine derivatives 3, notable activity
was only recorded for the p-trifluoromethyl derivative
3e with an LC50 value of 10 µM while other phenylgy-
cine derivatives were at least 5-fold less active. For
comparison, the boc-protected derivative of 3e (3d) was
also tested. Not surprisingly, it displayed a significantly
lower activity than its parent compound, again, most
probably attributable to its lower solubility. When the
homologue of the phenylglycine derivative 3b, the
R-phenylalanine derivative 4a, was assayed, a striking
enhancement in antitrypanosomal activity was ob-
served. The phenylalanine derivative 4a displayed the
highest activity of the second test series with an LC50
value of 1 nM. Furthermore, this compound is among
the FTase inhibitors with the highest in vitro activity
against trypanosome parasites recorded. Because of the
high activity of the phenylalanine derivative 4a, several
more amino acid derivatives were prepared (Table 4).
The most important compound of these additional amino
acid derivatives is the enantiomer of 4a, the S-phenyl-

Benzophenone-Based Farnesyltransferase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23 7189
Table 4. FTase Inhibitors with Different R-Aminoacyl
Residues
Figure 1. Homology model of farnesyl transferase from
Trypanosoma cruzi based on the crystal structure of the rat
enzyme (pdb entry 1qbq). The docked inhibitor 2l (yellow) lies
on top of the farnesylpyrophosphate analogue FHP (white) and
the Zn2+ ion (magenta). The majority of amino acid substitu-
tions are located in the specificity pocket.
reliable docking solution could be obtained possibly
helping in the interpretation of the SAR.
Since in this second round two compounds were active
in the nanomolar range these two inhibitors (4a and
2f) were selected for further in vivo evaluation. No
toxicity in terms of cell morphology or confluent growth
was detected on Vero cells treated with drug diluent
(DMSO 1/1000) or with compounds 4a and 2f at
maximal concentrations of 100 µM. Confirming these
results, an independent assessment established the
CC50 values against HeLa cell of selected inhibitors of
the first two test series being in the range of 37 to >78
µM with the best inhibitors being at 68 µM (2f) and >
78 µM (4a).
When performing the in vivo assays, no alteration in
behavior or aspect was recorded in the uninfected/
treated toxicity control group, receiving 39 doses. Para-
sitemia levels in blood were assessed twice a week by
microscopic examination. Infected groups (five animals
per group) reached the parasitemia peaks (0.8-1.2 ×
106 parasites/mL) at around 24-27 days pi. Groups
treated daily with inhibitors at ≈3.5 mg/kg body weight/
day presented higher parasitemia than the control
group, while those receiving at ≈7 mg/kg/day behaved
like the control (not shown). On day 50 pi parasites were
no more detectable in surviving mice with our method
of optical microscopic examination (detection limit g104
parasites/mL).24
Animal mortality stopped at day 34 pi (Figure 2).
Animals treated at ≈3.5 mg/kg/day performed as the
controls. Unexpectedly, group 4a at 3.2 mg/kg/day
displayed 0% survival. Although it is in accordance to
the higher parasitemia registered, we have no explana-
tion for this result. The survival in the control group
(infected, no drug) was 16% (1/6). Survival rates of 60%
(3/5) and 80% (4/5) were observed in mice treated with
inhibitors at 7 and 6.4 mg/kg/day (2f and 4a, respec-
tively). On day 115 pi the animals were still alive. These
results, and the fact that toxicity seems to be low or
absent, single out compounds 2f and 4a as the most
promising inhibitors.
alanine derivative 4b. For these two compounds a
striking stereodifferentiation is visible. While the R-
enantiomer 4a is the most active compound of the whole
study (LC50 ) 1 nM) the S-enantiomer 4b is more than
4 orders of magnitude less active with an LC50 value >
10 µM. Apart from the methionine derivatives 4j and
4k which both display only weak activity, stereodiffer-
entiation is also observed for other amino acid deriva-
tives although this effect is not as pronounced as in the
case of the phenylalanine enantiomers. No general
preference for the R- or S-enantiomer is visible in this
series. The activity of most inhibitors in this series was
in the micromolar range with LC50 values between 1
and 10 µM. However, the R-leucine derivative 4h and
the S-isoleucine derivative 4i displayed submicromolar
activity with LC50 values of 100 nM. As in the series of
the heterocylic substituted inhibitors 2, also in this
series there is no obvious explanation for the structure-
activity relationships. Because of the large number of
rotatable bonds in the amino acid derivatives, no

7190 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
Esteva et al.
each well was then microscopically examined under a 18 mm-
diameter coverslip at 400× magnification.24 Each dilution
condition was done in triplicate. Each complete experiment
(samples and control) was repeated 3-4 times for confirmation.
The percentage of lysis was calculated considering the control
as 100%. The LC50 (lythic concentration 50) was defined as
the minimal concentration of compound required to lyse 50%
of the trypomastigotes.
In Vivo Assays. For the in vivo assays, 60 days old BALB/c
males were used. The uninfected/treated toxicity control group
received the same drug concentration as the treated groups,
and the general aspect of the animals was recorded. The
infected/treated groups consisted of five animals, while the
infected/non treated control group had six. All of them were
injected by intraperitoneal route with 500 T. cruzi-bloodstream
trypomastigotes. From 30 min after infection, doses of the most
promising drugs (4a and 2f) or physiological solution were
administered orally (25 µL) five times a week. The total
number of doses was 39, except group 4a 3.2 mg/kg body
weight/day, that received 21 doses (see survival in Figure 2).
The final drug doses were ≈3.5 or ≈7 mg/kg/day. Parasitemia
levels were estimated by microscopic examination of blood
samples taken from the tail twice a week. Parasitemia and
mortality were recorded.
Cytotoxicity Assays. The test substances were dissolved
in DMSO (10 mM) before being diluted in the cell culture
medium (1:200). HeLa (DSM ACC 57) cells were grown in
RPMI 1640 culture medium (GIBCO BRL 21875-034) supple-
mented with 25 µg/mL gentamicin sulfate (BioWhittaker 17-
528Z) and 10% heat inactivated fetal bovine serum (GIBCO
BRL 10500-064) at 37 °C in high density polyethylene flasks
(NUNC 156340). The adherent HeLa cells were harvested at
the logarithmic growth phase after soft trypsinization, using
0.25% trypsin in PBS-containing 0.02% EDTA (Biochrom KG
L2163). For each experiment approximately 10 000 cells were
seeded with 0.1 mL of RPMI 1640 containing 25 µg/mL
gentamicin sulfate (BioWhittaker 17-528Z), but without
HEPES, per well of the 96-well microplates (NUNC 167008).
For the cytotoxic assay, HeLa cells were preincubated for 48
h without the test substances. The dilutions of the test
substances were carried out carefully on the monolayers of
HeLa cells after the preincubation time. The HeLa cells were
further incubated for 72 h at 37 °C in a humidified atmosphere
and 5% CO2. The adherent HeLa cells were fixed with 25%
glutaraldehyde and stained with a 0.05% solution of methylene
blue for 15 min. After gently washing, the stain was eluted
with 0.2 mL of 0.33 N HCl per well. The optical densities were
measured at 660 nm in SUNRISE microplate reader (TECAN).
For data analysis, the Magellan software (TECAN) was used.
Vero cells were grown in RPMI 1640 culture medium
(DIFCO) and 5% heat inactivated fetal bovine serum (BIOSER,
Buenos Aires). The toxicity control was performed incubating
cell cultures (104 cells/well, in triplicate for each drug concen-
tration) with different concentrations of drugs at 37 °C in 5%
CO2. DMSO was used as drug diluent, reaching a final
concentration of 1/1000 in the assays. Drugs (10 mM stock
solutions) were used at final concentrations of 1 nM, 10 nM,
100 nM, and 1 µM. Cell morphology and confluent growth were
checked at 24, 48, and 72 h.
Figure 2. Survival of infected and treated animals. Mice
groups received the following inhibitors: 2f 3.5 mg/kg/day (2),
2f 7 mg/kg/day (1), 4a 3.2 mg/kg/day (0), 4a 6.4 mg/kg/day
(9). The control group (O) received no drug.
We compared these results with those obtained previ-
ously with other molecules. We reported that some
Green tea (Camelia sinensis) catechins were very active
in vitro against T. cruzi trypomastigotes (MBC50 at the
picomol level),25 but we cannot compare their in vivo
effect since is currently under study. We also com-
municated that the in vitro effect of methoprene (a
juvenile hormone analogue) on T. cruzi trypomastigotes
was excellent, but only 60% of the treated infected mice
survived.26 Our present survival result obtained admin-
istering 4a is similar to the obtained by Garzoni et al.27
using Risedronate, an inhibitor of the farnesyl pyro-
phosphate synthase. Both results are not comparable
due to differences in the treatment protocols. However,
it is remarkable that our administration route (oral vs
iv) is more demanding, speaking in favor of the stability
and efficacy of our compounds. Interestingly, both
chemotherapeutic targets contribute to the same meta-
bolic products.
Summary and Conclusion
From several benzophenone-based FTase inhibitors
carrying a protonable nitrogen in the side chain two
inhibitors (4a and 2f) displayed promising in vitro
activity against T. cruzi with LC50 values of 1 nM and
10 nM, respectively. When further evaluated in vivo,
the phenylalanine derivative 4a, which was more active
in vitro, proved to be slightly superiour to the propy-
lpiperazinyl-substituted inhibitor 2f. However, further
structural variation of the amino acid portion of 4a did
not yield better inhibitors. Therefore, the propylpiper-
azinyl derivative 2f seems to be the most promising lead
structure for further development of antitrypanosomal
FTase inhibitors.
Experimental Section
Acknowledgment. L.F. and A.M.R. are members
of the Research Career of the Consejo Nacional de
Investigaciones Cient´ıficas y Te´cnicas (CONICET), Ar-
gentina. This research was supported by the Instituto
Nacional de Parasitolog´ıa “Dr. Mario Fatala Chaben”,
A.N.L.I.S. “Dr. Carlos G. Malbra´n”, the NASA/Cha-
gaSpace network, CONICET (Argentina), the Network
for Research and Training in Parasitic Diseases at the
Southern Cone of Latin America SIDA/SAREC, and
Wallenberg Consortium North.
Farnesyltransferase Inhibition. Yeast farnesyltrans-
ferase inhibition of all compounds employed in this study has
been published.12-17 The assay is described and referenced
there.
Parasites. Mice blood containing T. cruzi bloodstream
trypomastigotes (Tulahuen strain, Tul 2 stock) was obtained
by cardiac puncture from infected BALB/c mice.
In Vitro Assays. Test substances were dissolved in DMSO
(10 mM) before being diluted in the culture medium BHI (brain
heart infussion 0.37%, DIFCO). Five microliters of each
compound was added to 95 µL of blood containing 5 × 104
trypomastigotes. Controls received just DMSO (final dilution
1/1000). Incubations were performed in polystyrene 96-well
U-plates at 4 °C during 24 h. Five microliters of blood from
Supporting Information Available: Sequence alignment
for T. cruzi and rat farnesyltransferase. This material is
available free of charge via the Internet at http://pubs.acs.org.

Benzophenone-Based Farnesyltransferase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23 7191
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