Indomethacin amides as a novel molecular scaffold for targeting trypanosoma cruzi sterol 14α-demethylase

Article abstract of DOI:10.1021/jm801643b

Trypanosoma cruzi (TC) causes Chagas disease, which in its chronic stage remains incurable. We have shown recently that specific inhibition of TC sterol 14α-demethylase (TCCYP51) with imidazole derivatives is effective in killing both extracellular and in

Full text of DOI:10.1021/jm801643b

2846
J. Med. Chem. 2009, 52, 2846–2853
Indomethacin Amides as a Novel Molecular Scaffold for Targeting Trypanosoma cruzi Sterol
14r-Demethylase
Mary E. Konkle,† Tatiana Y. Hargrove,‡ Yuliya Y. Kleshchenko,§ Jens P. von Kries,| Whitney Ridenour,‡, Md. Jashim Uddin,‡
Richard M. Caprioli,‡, Lawrence J. Marnett,†,‡,# W. David Nes, Fernando Villalta,§ Michael R. Waterman,‡,# and
Galina I. Lepesheva*,‡
Department of Chemistry and Department of Biochemistry, School of Medicine, Vanderbilt UniVersity, NashVille, Tennessee 37232, Department
of Microbial Pathogenesis and Immune Response, Meharry Medical College, NashVille, Tennessee 37208, Screening Unit, Leibniz Institute for
Molecular Pharmacology (FMP), Berlin 13125, Germany, Mass Spectrometry Research Center, Vanderbilt UniVersity, NashVille,
Tennessee 37232, Vanderbilt Institute of Chemical Biology, NashVille, Tennessee 37232, and Department of Chemistry and Biochemistry,
Texas Tech UniVersity, Lubbock, Texas 79409-1061
ReceiVed December 26, 2008
Trypanosoma cruzi (TC) causes Chagas disease, which in its chronic stage remains incurable. We have
shown recently that specific inhibition of TC sterol 14R-demethylase (TCCYP51) with imidazole derivatives
is effective in killing both extracellular and intracellular human stages of TC. An alternative set of TCCYP51
inhibitors has been identified using optical high throughput screening followed by web-database search for
similar structures. The best TCCYP51 inhibitor from this search was found to have structural similarity to
a class of cyclooxygenase-2-selective inhibitors, the indomethacin-amides. A number of indomethacin-
amides were found to bind to TCCYP51, inhibit its activity in vitro, and produce strong antiparasitic effects
in the cultured TC cells. Analysis of TC sterol composition indicated that the mode of action of the compounds
is by inhibition of sterol biosynthesis in the parasite.
Introduction
ments for Chagas disease.4,5 Inhibition of the TC sterol
biosynthetic pathway is currently among the most promising
approaches.6 Similar to fungi or plants, TC produces 24-
methylated/alkylated (ergosterol-like) sterols, which are neces-
sary for membrane formation and cannot be replaced in the
parasite membranes by the host cholesterol.7
Chagas disease is an insect-borne parasitic disease threatening
millions of lives in Latin America and spreading worldwide as
a result of migration (mammalian hosts and insect vectors), HIV
co-infection, blood transfusion, and organ transplantation (http://
www.cdc.gov/chagas/factsheet.html). For example, a recent
American Red Cross study indicates that approximately one of
4700 blood donors in the United States in 2007 tested positive
for Chagas.1 Victims often do not experience specific symptoms
during the early stages of the disease, which can either pass
unnoticed or be deadly, depending on their immune status to
the protozoan parasite Trypanosoma cruzi (TCa). At the chronic
stage, when TC infects human tissues and exists predominantly
as the intracellular amastigote, the disease is commonly fatal.
Serious cardiac and/or intestinal symptoms develop in 20-40%
of infected individuals 10-20 years later, the probability
increasing up to 70% in immunocompromised patients.2
Only acute TC infection can be cured with the two currently
available drugs, nifurtimox and benznidazole.3 The drugs are
nonspecific, have severe side effects, and induce resistance. The
demand for new drug candidates has given rise to multiple
attempts to use the progress in understanding TC physiology
and biochemistry for the development of more specific treat-
We are focusing our attention on sterol 14R-demethylase
(CYP51). In TC, this cytochrome P450 enzyme catalyzes a
three-step reaction of oxidative removal of the 14R-methyl group
from 24-methylene dihydrolanosterol.8 We have shown recently
that specific inhibition of TCCYP51 with imidazole derivatives
is highly effective in killing the parasite.9 In good cor-
respondence with the fact that TCCYP51 has only about 25%
amino acid identity to the orthologous fungal enzymes, the
structure of the lead compounds we have used differs signifi-
cantly from the structures of the antifungal imidazole and
triazole drugs. However, the compounds still belong to the same
class of CYP51 inhibitors and it is known that azoles currently
used as clinical and agricultural fungicides can cause resis-
tance.10 This may be especially crucial for immunocompromised
(especially HIV-infected) patients with Chagas, as it is very
likely for many of them to also have fungal coinfections and to
be treated with azoles for a long time.
To investigate other options for the development of alternative
sets of potential antichagastic drugs, optical high throughput
screening of TCCYP51 for binding ligands other than azoles
has been undertaken. Several compounds producing type 1
(substrate-like) or type 2 (azole-like) spectral responses in the
cytochrome P450 Soret band were identified. Following high
throughput screening, a web-database search for similar struc-
tures revealed additional TCCYP51 ligands, some of them of
higher inhibitory potency. The strongest TCCYP51 inhibitor
from this search (more than 2-fold decrease in activity at
equimolar ratio inhibitor/enzyme (I/E2 < 1)9 demonstrated a
clear antiparasitic effect in the TC cells and was found to have
* To whom correspondence should be addressed. Phone: (615)-343-1373.
Fax: (615) 342-4349. E-mail: galina.i.lepesheva@vanderbilt.edu.
†
Department of Chemistry, School of Medicine, Vanderbilt University.
Department of Biochemistry, School of Medicine, Vanderbilt University.
Department of Microbial Pathogenesis and Immune Response, Meharry
‡
§
Medical College.
|
Screening Unit, Leibniz Institute for Molecular Pharmacology (FMP).
Mass Spectrometry Research Center, Vanderbilt University.
Vanderbilt Institute of Chemical Biology.
#
Department of Chemistry and Biochemistry, Texas Tech University.
a Abbreviations: TC, Trypanosoma cruzi; CYP51, sterol 14R-demethy-
lase; COX-2, cyclooxygenase 2; INDO, indomethacin; NSAID, nonsteroidal
anti-inflamatory drug; CPR, cytochrome P450 reductase; MDL, 24-
methylenedihydrolanosterol; I/E2, molar ratio inhibitor/enzyme which causes
a 2-fold decrease in the initial rate of catalysis.
10.1021/jm801643b CCC: $40.75
2009 American Chemical Society
Published on Web 04/08/2009

NoVel CYP51 Inhibitors for Chagas Disease
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2847
significant structural similarity to indomethacin amide deriva-
tives (cyclooxygenase-2 (COX-2) inhibitors).
the pyridyl nitrogen. This has been supported by the results of
the web-database search using compound 5 (N-(4-pyridyl)-
formamide) as a template because similar structures tested
(Figure 1B) produced the same spectral shift in the TCCYP51
absorbance (Soret band maximum 422 nm). On the other hand,
differences in the calculated apparent binding affinities reflect
influence of the configuration of the noncoordinated portion of
the ligand molecule, which (similar to azole inhibitors) might
enhance the inhibitory effect by forming additional interactions
with the amino acid residues in the CYP51 substrate binding
Indomethacin (INDO) is a classic nonsteroidal anti-inflam-
matory drug (NSAID) that has been available commercially to
treat rheumatoid arthritis since 1963. INDO exerts its anti-
inflammatory effect by nonselective inhibition of both cyclooxy-
genase isoforms, COX-1 and COX-2. Despite the high global
sequence homology (>66%) between the COX isoforms, it was
discovered that INDO could be converted to a COX-2-selective
inhibitor by neutralization of the carboxylic acid to an ester or
amide (INDO-amides).11,12 Additionally, it was found that
removing the 2′-methyl group of the INDO scaffold eliminated
potency of INDO against both isoforms of COX.13 INDO is an
attractive scaffold for drug development because it is relatively
inexpensive, readily converted to amide derivatives, and has a
long clinical history. We have synthesized a number of amide
derivatives of INDO and find that several of them are potent
inhibitors of TCCYP51.
cavity. The strongest binding compounds 7 and 9 (Kd 0.75
)
and 0.24 µM, respectively) have bulky structures attached to
the N-(4-pyridyl)-amide moiety. In 9, this portion of the
molecule is larger and more flexible than it is in 7, and this
feature must be preferable because in the reconstituted enzyme
reaction, 9 revealed more than 1 order of magnitude stronger
potency as TCCYP51 inhibitor (I/E2 < 1, Table 1).
Having an inhibitory effect on TCCYP51 comparable with
the effects of antifungal azole drugs fluconazole and ketocona-
zole,9 compound 9 has potential to serve as a lead for the design
of novel nonazole CYP51 inhibitors. Because high flexibility
of the substrate binding cavity is known to be the basis of the
P450 catalysis and ligand binding,16,17 in the absence of
TCCYP51 crystal structure, most preferably in a complex with
9, molecular docking and computer modeling are very unlikely
to produce reliable information on rational 9 structure modifica-
tion, especially because sequence identity of TCCYP51 to the
closest P450 template of known crystal structure is only 28%.
Instead, we continued with investigation of structural similarity
and tested as TCCYP51 inhibitors a number of INDO-amide
derivatives that share structural features with 9. All these
compounds have the same INDO-amide moiety (Figure 1C,
shown in square) but differ in the length of the alkyl linker and
position of the nitrogen in the heterocycle (shown in blue); this
part of the molecule is of special interest because it is expected
to form the sixth coordinate bond with the P450 heme iron.
Results and Discussion
TCCYP51 Ligands Found via High Throughput Screen-
ing. Optical high throughput screening (HTS) is based on the
P450 property of changing the maximum of the Soret band
absorbance upon ligand binding. The changes are connected
with alterations in the surrounding environment of the heme
iron.14 Heterologously expressed CYP51s are usually purified
in the oxidized low-spin ferric form (resting state of the
iron-porphyrin complex with the Soret band maximum at 417
nm), the sixth coordinate position of the iron being occupied
by a water molecule. Binding of a substrate-like ligand in the
active center causes water displacement; the iron becomes penta-
coordinated and pops out of the porphyrin plane. This triggers
a low-to-high-spin transition in the iron-porphyrin complex,
and the Soret band maximum shifts to 390 nm, producing a
so-called type 1 spectral response in the difference spectra.
Alternatively, a type 2 spectral response (red-shift in the Soret
band maximum to 422-424 nm) reflects direct coordination of
a ligand other than water to the low-spin heme iron.
All “substrate-like” ligands identified by HTS produced rather
weak spectral responses in TCCYP51. The three best com-
pounds (Figure 1A) revealed apparent dissociation constants in
the concentration range of 50-100 µM, which suggests binding
affinities at least 50-fold lower than the affinity of the enzyme/
substrate complex formation (Kd around 1 µM with the preferred
substrate 24-methylenedihydrolanosterol).9 Correspondingly, the
inhibitory potencies of these compounds in the reconstituted
enzyme reaction were very low (I/E2 > 50 (not shown)). We
believe this might be connected with the narrowness of the
CYP51 substrate specificity:15 to preserve their catalytic function
through hundreds of million of years of evolution, the CYP51
family enzymes developed very strict requirements toward their
substrates so that minor alterations in the spatial structure would
differentiate a foreign compound and prevent it from being
metabolized or interfere with the substrate in the CYP51 active
center. The finding supports the notion that the most likely way
to design potent substrate-analogous CYP51 inhibitors would
be through the subtle modifications of the basic sterol structure
in the regions adjusted to the reactive 14R-methyl group.8
The HTS hits that induced type 2 spectral response in
TCCYP51 demonstrated higher binding affinities, the most
potent inhibitor (5) exhibiting a Kd of 0.44 µM and I/E2 of about
10 (Table 1). Contrary to compounds 4 and 6, which must
interact with the TCCYP51 heme iron through their five-
membered imidazole ring, 5 is most likely to coordinate through
INDO-Amide Derivatives as TCCYP51 Ligands. While
spectral responses of TCCYP51 to the compounds 7-11 were
very similar to the responses caused by imidazole and triazole
derivatives, INDO-amides induced a variety of spectral changes
in the hemoprotein (Figure 2, Table 1). It could be connected
with the variations in the location of the nitrogen in the
heterocyclic ring. Thus, when the nitrogen is in the para position
(as in compounds 7-11), the Soret band maximum shows a
normal shift to 422 nm (14 and 15). The same response is
produced if the nitrogen is in the meta position (12) and the
ring does not contain any additional substituents. If it does (18
and 19), the maximum is observed at 420 nm. Taking into
account that the same spectral changes are also caused by two
compounds that do not carry any nitrogen atoms in the ring
(21 and 22), it is not excluded that this modified type 2 response
may result from accommodation of the inhibitor in the TC-
CYP51 active center, which would strengthen the water ligation
to the heme iron. This can also be true for 13 (nitrogen in the
ortho position), although in this case the iron coordination is
weaker and the absorbance maximum shifts only to 418 nm.
Having a shorter arm between the ortho-nitrogen ring and the
amide, 16 definitely does not participate in iron coordination
as the compound causes very weak substrate-like type 1 spectral
response in TCCYP51. Finally, 17 (pyrimidine ring with both
nitrogens ortho to the arm) did not induce any spectral changes
in TCCYP51.
Although they differ in the location of the Soret band
maximum, the majority of the spectral responses upon titration

2848 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Konkle et al.
Figure 1. Search for TCCYP51 inhibitors. (A) Ligands (1-6) found via HTS using FMP library of small organic molecules (20000 compounds
selected from ChemDiv, San Diego, CA). (B) Inhibitors (7-11) found using web-database search for structural similarity to 5 (http://
chemdiv.emolecules.com). (C) 9-Like INDO-amide derivatives. Calculated from spectral responses. Apparent dissociation constants (µM) for the
compounds not included in Table 1 are shown in brackets.
of TCCYP51 with the increasing concentrations of the ligands
produced typical hyperbolic curves. The only exception was
observed in the case of 20 (Figure 2C). The sigmoid shape of
the titration curve was reproducible upon variations in the buffer
composition, salt gradient, increase in the concentration of
TCCYP51, or use for the titration of more soluble 20-
hydrochloride. The Hill coefficient of 2.3 suggested a possibility
of simultaneous binding of two inhibitor molecules to the
enzyme. This has been tested and confirmed using ESI-mass
spectrometry (Supporting Information, Figure S1). Allosteric
effects (non-Michaelis-Menten kinetics) have been reported for
several microsomal drug-metabolizing CYPs (e.g., 1A2, 2A6,
2C9, 3A418), but this is the first example in a CYP51 orthologue
and binding of a second molecule may have no inhibitory effect.
Regardless of the quite pronounced differences in the apparent
binding affinities calculated from the spectral responses, most
of the INDO-amides demonstrated comparably strong inhibitory
effects on TCCYP51 (I/E2 around 5-10, reversible) except for
21 and 22 (I/E2 < 1, functionally irreversible) (Table 1).
Altogether this supports our previous observation that, being
indicative of binding of a ligand near the heme iron, spectral
responses do not necessarily reflect real inhibitory potency9 and
the interactions in the regions of the CYP51 substrate binding
cavity, remote from the heme iron, might also provide a strong
inhibitory effect on the enzyme catalysis and should be con-
sidered in the design of new inhibitors.
Antiparasitic Effect of CYP51 Inhibitors in Extracellu-
lar and Intracellular Trypanosomes. TC is an obligate parasite
with a complex life cycle that includes four morphologically
and metabolically different stages. Proliferative epimastigotes
and infective metacyclic trypomastigotes are present in the insect
vectors (kissing bugs). Upon infection of mammalian hosts, TC
transforms into bloodstream trypomastigotes and proliferative
intracellular amastigotes (amastigotes dominate in the chronic
stage of Chagas). Pretreatment of TC trypomastigotes with
compounds 9, 13, 18, and 20 produced a clear dose-dependent
antiparasitic effect in both human forms of TC (Figure 3). At a
20 µM compound concentration, the parasite was practically

NoVel CYP51 Inhibitors for Chagas Disease
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2849
Table 1. Binding Parameters, Inhibitory Potencies, and Antiparasitic Effects of Selected TCCYP51 Ligands
d
spectral response in TCCYP51
effect on enzymatic activity (I/E2 )
compd
type
Soret band maximum, nm
Kd, µM
TCCYP51
mouse COX-2
5
7
9
2
2
2
422
422
422
422
418
422
422
416
417
420
420
422
419
419
0.44 ( 0.02
0.75 ( 0.03
0.24 ( 0.01
0.29 ( 0.01
7.6 ( 0.9
0.54 ( 0.02
0.37 ( 0.05
3.3 ( 0.48
10
14
<1
9.1
13
10
8.5
11
12
13
14
15
16
17
19
18
20
21
22
2
2.1
>10
10
1.7
2.1
3.6
<1
<1
24
2b
2
2
1a
none
2b
2b
2c
2b
2b
15
0.56 ( 0.08
0.26 ( 0.04
2.31 ( 0.09
0.36 ( 0.02
0.15 ( 0.01
7.7
4.2
6.3
<1
<1
<1
>40
a Weak type 1 response with only ∼7% of P450 experiencing low to high spin transition. b Modified type 2 response with the smaller shift in the Soret
band maximum. c Sigmoid curve, Hill coefficient 2.3 ( 0.2 (see Figure 2). d Inhibitory potencies of the compounds toward TCCYP51 (minimal final
concentration required in the reconstituted reaction 1 µM9) and mouse COX-2 (final concentration 0.1 µM) are compared as molar ratios inhibitor/enzyme
that cause a 2-fold decrease in the initial rate of catalysis.
eliminated from cardiomyocytes. The strongest growth inhibition
(ED50 < 1 µM in TC amastigotes) was observed with 20, which
may be at least in part due to its better cellular permeability or
the longest lifetime/highest metabolic stability.19 For the com-
parison, published ED50 values in TC cells for benznidazole,
one of the two drugs currently used for clinical treatment of
TC infections, were reported to be within 25-50 µM.20-22
Sterol Composition of TC Cells upon Treatment with
20. We have shown recently that treatment of TC cells with a
potent TCCYP51 inhibitor profoundly alters their sterol com-
position.9 While untreated parasites, where sterol biosynthesis
is highly efficient, essentially contain only the pathway products
(ergosterol and its 24-ethylated analogue) and exogenous
cholesterol from the medium (Figure 4A), in the inhibitor-treated
TC (N-(1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-
phenyl-1,3,4-oxadiazol-2-yl)benzamide (SDZ-284629), 1 µM),
the pathway practically stops at the stage of the C14-methylated
sterol precursors, predominantly lanosterol, the first cyclized
compound of the pathway, and 24-methylenedihydrolanosterol,
the preferred TCCYP51 substrate. Treatment of TC with 50 µM
20 produces a very similar effect on cellular sterol composition
(Figure 4B), indicating that the mechanism of action of the
compound is connected with blocking sterol biosynthesis in the
parasite via inhibition of TCCYP51 activity.
attractive for the acute stage because drugs traditionally aimed
to ease nonspecific symptoms of inflammation and fever can
actually provide treatment to the disease. Finally, HTS of larger
libraries of small bioactive molecules in combination with the
database search for structural similarity may help to discover
other novel CYP51 inhibitors.
Experimental Section
The TCCYP51 gene subcloned into the pCW vector (Nde I/Hind
III cloning sites) was expressed in E. coli and purified as previously
described.8 The protein was electrophoretically pure, had a spec-
trophotometric index of OD417/OD278 of 1.55, and a specific heme
content of 17 nmol/mg. P450 concentration was determined from
the difference spectra of the reduced carbon monoxide complexes
using the extinction coefficient of 91 mM-1 cm-1 (450-490 nm)24
or from the absolute absorbance spectra of the ferric water-coor-
dinated TCCYP51 in the Soret band maximum (417 nm) using the
8
extinction coefficient of 117 mM-1 cm-1
.
High Throughput Screening (HTS) and Web Search for
Similar Structures. HTS of the FMP library of small organic
molecules (20000 compounds selected from ChemDiv, San Diego,
CA) was performed using a pipetting robot (Sciclone 3000; Caliper
Lifesciences), a plate reader for absorbance scanning (Safire; Tecan),
and various robots for washing and dispensing. Compounds were
dissolved in DMSO at 10 mM stock concentrations and distributed
in 0.4 µL aliquots into 384-well microtiter plates. Buffer (40 µL
of 50 mM potassium-phosphate buffer, pH 7.4, containing 100 mM
NaCl) was added to each well to achieve a 100 µM concentration
of the tested compounds. Plates were incubated for 15 min at 37
°C and then sonicated for 5 min to allow solubilization of sus-
pensions of the most hydrophobic compounds. Compound-specific
changes in absorption spectra (350-450 nm) were recorded
automatically after adding 10 µL of 5 µM TCCYP51 in 50 mM
potassium-phosphate buffer, pH 7.4, containing 100 mM NaCl and
0.1 mM EDTA, optical path length 7 mm, and then validated
manually by spectral titration. Search for similar structures was
done using the ChemDiv database (http://chemdiv.emolecules.com).
The examples of TCCYP51 ligands (shown in Figure 1) identified
by HTS are: 1-(3-amino-4-chlorophenyl)(thien-2-yl)methanone (1),
3-(4-aminophenyl)-2H-chromen-2-one (2), [5-(4-fluoro-benzylidene)-
4-oxo-2-thioxo-thiazolidin-3-yl]-acetic acid (3), 3-thien-2-yl-5,6-
dihydroimidazo[2,1-b][1,3]thiazole (4), 3-(2-furyl)-5,6-dihydroim-
idazo[2,1-b][1,3]thiazole (5), 2-phenyl-N-pyridin-4-ylbutanamide
(6). Compound 5-like ligands found via web search, ChemDiv
numbers: 5556-0480 (7), 6903-0018 (8), C155-0123 (9),
3124-0167 (10), 000 L-5707 (11).
Summary
Inhibition of sterol biosynthesis in TC is becoming one of
the very promising directions in the development of anti-
Chagasic drugs. Several antifungal azoles have recently entered
clinical trials for antitrypanosomal chemotherapy.4 However,
azole derivatives (at least those currently used as fungicides)
have one serious disadvantage: upon long-term treatment they
often cause resistance. Experimental development of resistance
to fluconazole in the cultured TC cells also has been described.23
Though strong inhibition of TCCYP51 with specific azoles
might shorten the treatment time and decrease the probability
of resistance, an alternative set of TCCYP51 inhibitors would
be highly desirable. The strong antiparasitic effect of indo-
methacin amide derivatives in both extracellular and intracellular
forms of human stages of TC indicates that this group of
compounds might find a new application as potential anti-
trypanosomal agents. While additional directed modifications,
targeted to decrease COX-2 inhibitory potency, might be
desirable for use as a lead structure 20 in the chronic (especially
cardiac) forms of Chagas, this class of compounds is particularly
Synthesis and Characterization of INDO-Amide Derivatives.
Purity of the compounds was determined using NMR and ESI-MS
as described19 and was g95%. Materials and instrumentation were

2850 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Konkle et al.
Figure 3. Cellular effects of TCCYP51 inhibitors in TC trypomas-
tigotes and amastigotes. (A) Dose-dependent inhibition of T. cruzi
intracellular multiplication and infection in cardiomyocytes by TC-
CYP51 inhibitors. Trypomastigotes were pretreated with several
concentrations of TCCYP51 inhibitors or mock-treated, exposed to
cardiomyocyte monolayers, and T. cruzi multiplication was evaluated
at 72 h by determining the number of T. cruzi/cell (left panel) and the
percent of infection (right panel). The data represent the mean (
standard deviation (SD) of results from triplicate samples. SD did not
exceed 10% of the mean. Differences in T. cruzi per cell and % infection
between trypomastigotes-mock treated (DMSO) and trypomastigotes
treated with each TCCYP51 inhibitor (5-50 µM) are p < 0.05,
determined by the Student t test. The two most potent TCCYP51
inhibitors (21 and 22) show antiparasitic activities at e10 µM
concentrations) but release cardiomyocyte monolayers at higher
concentrations. (B) Microscopic observation of the inhibition of T. cruzi
multiplication by 20 µM TCCYP51 inhibitors within cardiomyocytes
at 72 h. T. cruzi pretreated with control DMSO showed high levels of
parasite multiplication, whereas cells exposed to trypanosomes prein-
cubated with the inhibitors showed a dramatic decrease in the number
of intracellular parasites.
crude product was purified by trituration with ethanol to give a
precipitate. The solid was collected in a fritted funnel. The HPLC
analysis was done using a C18 column with UV detection at 235
nm using a gradient of 90:10 A:B to 100% B over 30 min with
solvent A ) water with 0.05% TFA and B ) acetonitrile with
0.05% TFA.
Compound 12 was obtained as an off-white solid (320 mg, 69%
isolated yield). Melting point ) 183 °C, 1H NMR (300 MHz,
CDCl3, ppm) δ 8.40 (d, J ) 1.4 Hz, 1H) 8.32 (dd, J1 ) 4.7 Hz, J2
) 1.4 Hz, 1H), 8.06 (m, 1H,), 7.65 (d, J ) 8.6 Hz, 2H), 7.50 (m,
3H), 7.25 (m, 1H), 6.94 (d, J ) 2.4 Hz, 1H), 6.86 (d, J ) 9.0 Hz,
1H), 6.71 (dd, J1 ) 9.0 Hz, J2 ) 2.4 Hz, 1H), 3.84 (s, 2H), 3.81 (s,
3H), 2.46 (s, 3H). ESI-MS m/z (positive) 434 [C24H20ClN3O3 +
H]+. HPLC: retention time ) 18.1 min.
Figure 2. Spectral responses of TCCYP51 (2 µM) to three INDO-
amide derivatives (A) 18, (B) 16, (C) 20. Upper spectra: absolute
absorbance in the Soret band region (black line, no ligand; red line,
plus ligand). Lower: difference spectra upon titration with the ligands
(0.5 µM steps). Insets: titration curves.
as previously described.11 A reaction mixture containing in-
domethacin (300 mg, 1.0 equiv, 0.84 mmol) in 6 mL of anhydrous
CH2CL2 was treated with dicyclohexylcarbodiimide (192 mg, 1.1
equiv, 0.92 mmol), dimethylaminopyridine (10 mg, 0.1 equiv, 0.084
mmol), and the appropriate amine (1.1 equiv, 0.92 mmol). After
stirring at room temperature for 5 h, the reaction mixture was filtered
and the filtrate was concentrated in vacuo. The residue was diluted
with water (2 × 15 mL) and extracted with CH2CL2 (3 × 15 mL).
The combined organic solution was washed with brine (2 × 15
mL), dried over MgSO4, filtered, and concentrated in vacuo. The
Compound 13 was obtained as a yellow solid (120 mg, 32%
isolated yield). Melting point ) 181 °C. 1H NMR (CDCl3) δ 8.39
(d, J ) 4.3 Hz, 1H), 7.68 (d, J ) 13.1 Hz, 2H), 7.62 (dt, J ) 1.8,
7.7 Hz, 1H), 7.47 (d, J ) 13.1 Hz, 2H), 7.15 (m, 2H), 6.92 (d, J

NoVel CYP51 Inhibitors for Chagas Disease
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2851
Figure 4. Sterols in TC. (A) Structural formulas of neutral lipids formed upon sterol biosynthesis (squalene (1), squalene epoxide (2), lanosterol
(3), 24-methylenedihydrolanosterol (4), obtusifoliol (5), ergosterol (6), 24-methylergosterol (7)) or up taken from the media (cholesterol (8)) 14R-
methyl group and formed by CYP51. ∆14-15 double bonds are shown in red. (B) TLC analysis of sterols extracted from TC epimastigotes. St,
sterol standards; C, sterols in untreated cells; T, sterols in TC incubated for 24 h with 50 µM 20.
) 2.4 Hz, 2H), 6.88 (s, 1H), 6.69 (dd, J ) 2.4, 9.0 Hz, 1H), 4.52
(d, J ) 5.1 Hz, 2H), 3.79 (s, 3H), 3.72 (s, 2H), 3.39 (s, 3H). ESI-
MS m/z 469 [C25H22ClN3O3 + Na]+. HPLC: retention time ) 18.6
min.
Compound 14 was obtained as an off-white solid (204 mg, 55%
isolated yield). Melting point ) 178 °C. 1H NMR (CDCl3) δ 8.47
(dd, J ) 1.8, 4.3 Hz, 2H), 7.63 (d, J ) 13.1 Hz, 2H), 7.47 (d, J )
13.1 Hz, 2H), 7.05 (dd, J ) 1.8, 4.3 Hz, 2H), 6.89 (d, J ) 2.4 Hz,
1H), 6.84 (d, J ) 9.0 Hz, 1H), 6.70 (dd, J ) 2.4, 9.0 Hz, 1H), 4.40
(d, J ) 6.3 Hz, 2H), 3.80 (s, 3H), 3.74 (s, 2H), 2.41 (s, 3H). ESI-
MS m/z 469 [C25H22ClN3O3 + Na]+. HPLC: retention time ) 17.2
min.
Compound 15 was obtained as an off-white solid (53 mg, 13%
isolated yield). Melting point ) 181 °C. 1H NMR (CDCl3) δ 8.48
(dd, J ) 1.6, 4.4 Hz, 2H), 7.65 (d, J ) 13.1 Hz, 2H), 7.49 (d, J )
13.1 Hz, 2H), 7.04 (dd, J ) 1.6, 4.4 Hz, 2H), 6.85 (m, 1H), 6.72
(dd, J ) 2.5, 9.0 Hz, 1H), 5.83 (d, J ) 7.8 Hz, 1H), 5.10 (quint,
J ) 7.4 Hz, 1H), 3.78 (s, 3H), 3.68 (s, 2H), 2.40 (s, 3H), 1.35 (d,
J ) 7.4 Hz, 3H). ESI-MS m/z 483 [C26H24ClN3O3 + Na]+. HPLC:
retention time ) 16.2 min.
Compound 16 was obtained as a yellow solid (310 mg, 85%
isolated yield). Melting point ) 163 °C. 1H NMR (CDCl3) δ 8.24
(d, J ) 8.4 Hz, 1H), 8.10 (d, J ) 5.9 Hz, 1H), 7.95 (s, 1H), 7.51
(d, J ) 13.1 Hz, 2H), 7.08 (m, 1H), 6.94 (d, J ) 2.4 Hz, 1H), 6.87
(d, J ) 9.0 Hz, 1H), 3.87 (s, 2H), 3.82 (s, 3H), 2.45 (s, 3H). ESI-
MS m/z 455 [C24H20ClN3O3 + Na]+. HPLC: retention time ) 17.1
min.
2.4 Hz, 1H), 6.87(d, J ) 9.0 Hz, 1H), 6.69 (dd, J ) 2.4, 9.0 Hz,
1H), 4.03 (s, 2H), 3.80 (s, 3H), 2.44 (s, 3H). ESI-MS m/z 456
[C23H19ClN4O3 + Na]+. HPLC: retention time ) 18.9 min.
Compound 18 was obtained as an off-white solid (250 mg, 59%
isolated yield). Melting point ) 177 °C. 1H NMR (300 MHz,
CDCl3, ppm) δ 8.15 (d, 1H, J ) 2.1 Hz), 7.50 (m, 5H), 7.422 (d,
1H, J ) 8.4 Hz), 6.83 (m, 2H), 6.69 (dd, J1 ) 9 Hz, J2 ) 2.4 Hz
1H), 6.19 (br t, J ) 6 Hz, 1H), 4.37 (d, J ) 6 Hz, 2H), 3.79 (s,
3H), 3.71 (s, 2H), 2.36 (s, 3H). ESI-MS m/z (positive) 482
[C25H21Cl2N3O3]+. HPLC: retention time ) 19.1 min.
Compound 19 was obtained as an off-white solid (264 mg, 69%
isolated yield). Melting point ) 183 °C. 1H NMR (300 MHz,
CDCl3, ppm) δ 8.54 (s, 1H), 7.60 (m, 4H), 7.49 (m, 2H), 6.85 (m,
2H), 6.70 (d, J ) 8.7 Hz, 1H), 6.12 (br s, 1H), 4.47 (s, 2H), 3.79
(s, 3H), 3.72 (s, 2H), 2.39 (s, 3H). ESI-MS m/z (positive) 516
[C26H21ClF3N3O3]+. HPLC: retention time ) 20.0 min.
Compound 20 was obtained as a yellow solid (280 mg, 59%
isolated yield), Melting point ) 188 °C. 1H NMR (300 MHz,
CDCl3, ppm) δ 8.40 (d, J ) 1.4 Hz, 1H), 8.32 (dd, J1 ) 4.7 Hz, J2
) 1.4 Hz, 1H), 8.06 (m, 1H), 7.65 (d, J ) 8.6, 2H), 7.48 (d, J )
8.6, 2H), 7.24 (m, 1H), 6.94 (d, J ) 2.5 Hz, 1H), 6.86 (d, J ) 9.0
Hz, 1H), 6.72 (dd, J1 ) 9.0, J2 ) 2.5, 1H), 5.61 (br s, 1H), 3.81 (s,
3H), 3.59 (s, 2H), 3.45 (m, 2H), 2.70 (t, J ) 6.7 Hz, 2H), 2.04 (s,
3H). ESI-MS m/z (positive) 462 [C26H24ClN3O3 + H]+. HPLC:
retention time ) 15.1 min.
Compound 21 was obtained as an off-white solid (360 mg, 72%
isolated yield). Melting point ) 191 °C. 1H NMR (300 MHz,
CDCl3, ppm) δ 7.65 (d, J ) 8.6, 2H), 7.53 (br s, 1H), 7.48 (d, J )
8.6, 2H), 7.30 (m, 2H), 7.18 (dd, J1 ) 9.0, J2 ) 2.5, 1H), 6.94 (d,
J ) 2.5 Hz, 1H), 6.86 (d, J ) 9.0 Hz, 1H), 6.72 (dd, J1 ) 9.0, J2
) 2.5, 1H), 5.61 (br s, 1H), 3.81 (s, 3H), 3.59 (s, 2H), 3.45 (m,
Compound 17 was obtained as a yellow solid (60 mg, 16%
isolated yield). Melting point ) 167 °C. 1H NMR (CDCl3) δ 8.61
(d, J ) 4.9 Hz, 2H), 8.41 (br s, 1H), 7.69 (d, J ) 13.1 Hz, 2H),
7.47 (d, J ) 13.1 Hz, 2H), 7.02 (t, J ) 3.2 Hz, 1H), 6.96 (d, J )

2852 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Konkle et al.
2H), 2.70 (t, J ) 6.7 Hz, 2H), 2.04 (s, 3H). ESI-MS m/z (positive)
495 [C27H24Cl2N2O3 + H]+. HPLC: retention time ) 16.1 min.
Compound 22 was obtained as an off-white solid (301 mg, 62%
isolated yield). Melting point ) 180 °C. 1H NMR (300 MHz,
CDCl3, ppm) δ 7.59 (d, J ) 8.4 Hz, 2H), 7.47 (d, J ) 8.4 Hz,,
2H), 7.13 (m, 3H), 6.90 (m, 4H), 6.71 (dd, J1 ) 8.9, J2 ) 2.4, 1H),
5.61 (br s, 1H), 3.81 (s, 3H), 3.59 (s, 2H), 3.45 (m, 2H), 2.70 (m,
1H), 2.04 (s, 3H), 1.28 (d, J ) 6.8 Hz, 3H). ESI-MS m/z (positive)
475 [C28H27ClN2O3 + H]+. HPLC: retention time ) 17.3 min.
Spectral Titration of TCCYP51. The apparent affinities of
ligand binding were evaluated by the spectral changes they induced
in the TCCYP51.9 The titration experiments were carried out at
24 °C in 2 mL tandem cuvettes, containing 2 µM TCCYP51 in
buffer A in the wavelength range 350-500 nm using a Shimadzu
UV-2401PC spectrophotometer. The tested compounds (1 or 10
mM stock solutions in DMSO, depending on the affinity of the
interaction) were added in 1 µL aliquots to the test cuvette until
the maximum in the TCCYP51 spectral response was reached.
Equal volumes of DMSO were added to the reference cuvette. The
apparent Kd’s were determined from the equilibrium titration curves
by plotting absorbance changes against the concentration of free
ligand and fitting the data to a rectangular hyperbola using
SigmaPlot Statistics.25
ESI-MS Analysis of Noncovalent Interactions in TCCYP51. The
experiments were conducted with an ESI-oaTOF mass spectrometer
(micrOTOF, Bruker Daltonics, Inc., Billirica, MA), which has been
modified for enhanced collisional cooling in the source for the
analysis of noncovalent protein complexes. This was completed
by the addition of a valve in turbo pump line restricting vacuum.
The standard instrument parameters were used with the exception
of the following: capillary voltage 3.5 kV, capillary exit 250 V,
fore pressure 4.99 mbar, and TOF pressure 6.29 × 10-7 mbar.
TCCYP51 was buffer exchanged into 50 mM ammonium acetate,
pH 7.0; 20-HCl was dissolved in ethanol and added to the protein
solution to give the final concentration of 100 µM. The ESI flow
rate was 180 µL h-1. Spectra were acquired in positive polarity
mode, externally calibrated, and processed using DataAnalysis
software (Bruker Daltonics, Inc.) by smoothing and baseline
subtracting.
Reconstitution of Enzymatic Activity and Inhibition Assay.
Sterol 14R-demethylase activity of TCCYP51 in vitro was recon-
stituted with cytochrome P450 reductase (CPR) from Trypanosoma
brucei as an electron donor partner26 and 24-methylenedihydrol-
anosterol (MDL) as a substrate.8 The concentrated proteins were
preincubated for 10 min at room temperature with dilauroyl-R-
phosphatidylcholine (DLPC) at molar ratio TCCYP51:CPR:DLPC
) 1:2:50. The final reaction mixture contained 1 µM TCCYP51
and 50 µM substrate (unlabeled and [3H]-MDL were mixed to give
∼2000 cpm/nmol and added from 1 mM stock solution in 45%,
2-hydroxypropyl-ꢀ-cyclodextrin) in 20 mM MOPS (pH 7.4), 50
mM KCl, 5 mM MgCl2, 10% glycerol, 0.4 mg/mL isocitrate
dehydrogenase, and 25 mM sodium isocitrate. For the inhibition
assay, the reaction was performed in the presence of the increasing
concentrations of the compounds tested as TCCYP51 inhibitors
(concentration range 1-100 µM). After 5 min of preincubation with
the inhibitors at 37 °C, the reaction was initiated with the addition
of NADPH (5 µM). Sterols were extracted with ethyl acetate and
analyzed by reverse phase HPLC in the linear gradient of methanol:
acetonitrile:H2O ) 9:9:2 (solution A) and methanol (solution B)
(0-100%) using a Waters C18 column and a ꢀ-RAM radioactivity
detector. The inhibitory potency was estimated as molar ratio
inhibitor/enzyme at which the activity decreased 2-fold (I/E2).9
Inhibitory effect of the compounds on COX-2 enzyme was
determined at 100 nM final concentration of mouse cyclooxygenase
2 as previously described,19 and the potency was expressed as I/E2
to correspond to the way it is expressed for TCCYP51.
affect their motility. The parasites were then exposed in triplicate
to rat cardiomyocyte monolayers at the ratio 10 parasites/cell in
Laboratory Tech chambers for 2 h as described.27 To verify that
the inhibitory effect was on trypanosomaes and not on cardiomyo-
cytes, in additional experiments, excess compound was removed
from the trypanosomes prior to infection, producing similar results.
After removing the unbound parasites, monolayers were incubated
with DMEM supplemented with 10% FBS for 72 h to allow parasite
intracellular multiplication and the number of T. cruzi per 200 cells,
and the percent of infection were microscopically determined in
Giemsa stained monolayers.27
TC Cellular Sterol Analysis. Epimastigotes (plating density 107
cells/mL) were cultured in brain heart infusion supplemented with
hemin and 10% calf serum for 120 h without an inhibitor and in
the presence of 20. The inhibitor was added daily to maintain 50
µM concentration. The cell pellet was washed with Hanks’ balanced
salt solution without phenol red to remove excess cholesterol from
the serum and saponified using 10% KOH in 98% aqueous methanol
at reflux temperature for 1 h. The TC sterols were extracted with
hexane and analyzed by silica gel TLC in hexane:ethyl acetate
(8:2) as described previously.9
Acknowledgment. This work was supported by grants from
the American Heart Association (0535121N to G.I.L.), the
National Institutes of Health (GM067871 to M.R.W. and G.I.L.,
ES00267 to M.R.W. and L.J.M., AI080580, AI007281, and
HL007737 to F.V. and CA89450 to L.J.M.), Welch Foundation
(D-1276 to W.D.N.), and from the Department of Defense
(W81XWH-05-01-0179 to R.M.C,).
Supporting Information Available: ESI-MS of TCCYP51,
ligand free and compound 20 bound. This material is available free
of charge via the Internet at http://pubs.acs.org.
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