Antitrypanosomal lead discovery: Identification of a ligand-efficient inhibitor of Trypanosoma cruzi CYP51 and parasite growth

Article abstract of DOI:10.1021/jm400012e

Chagas disease is caused by the intracellular protozoan parasite Trypanosomal cruzi, and current drugs are lacking in terms of desired safety and efficacy profiles. Following on a recently reported high-throughput screening campaign, we have explored initial structure-activity relationships around a class of imidazole-based compounds. This profiling has uncovered compounds 4c (NEU321) and 4j (NEU704), which are potent against in vitro cultures of T. cruzi and are greater than 160-fold selective over host cells. We report in vitro drug metabolism and properties profiling of 4c and show that this chemotype inhibits the T. cruzi CYP51 enzyme, an observation confirmed by X-ray crystallographic analysis. We compare the binding orientation of 4c to that of other, previously reported inhibitors. We show that 4c displays a significantly better ligand efficiency and a shorter synthetic route over previously disclosed CYP51 inhibitors, and should therefore be considered a promising lead compound for further optimization.

Full text of DOI:10.1021/jm400012e

Article
pubs.acs.org/jmc
Antitrypanosomal Lead Discovery: Identification of a Ligand-Efficient
Inhibitor of Trypanosoma cruzi CYP51 and Parasite Growth
Grasiella Andriani,§ Emanuele Amata,† Joel Beatty,† Zeke Clements,† Brian J. Coffey,†
Gilles Courtemanche,# William Devine,† Jessey Erath,§ Cristin E. Juda,† Zdzislaw Wawrzak,⊥
JodiAnne T. Wood,‡ Galina I. Lepesheva,*,∥ Ana Rodriguez,*,§ and Michael P. Pollastri*,†,‡
†Department of Chemistry and Chemical Biology, and ‡Center for Drug Discovery, Northeastern University, Boston, Massachusetts
02115, United States
§Division of Parasitology, Department of Microbiology, New York University School of Medicine, New York, New York 10010,
United States
∥Department of Biochemistry School of Medicine, Institute for Global Health, Vanderbilt University School of Medicine, Nashville,
Tennessee 37232, United States
⊥Synchrotron Research Center, Life Science Collaborative Access Team, Northwestern University, Argonne, Illinois 60439, United
States
#Sanofi Research, 195 Route d’Espagne BP13669, 31036 Toulouse Cedex1, France
S
* Supporting Information
ABSTRACT: Chagas disease is caused by the intracellular protozoan parasite Trypanosomal cruzi, and current drugs are lacking
in terms of desired safety and efficacy profiles. Following on a recently reported high-throughput screening campaign, we have
explored initial structure−activity relationships around a class of imidazole-based compounds. This profiling has uncovered
compounds 4c (NEU321) and 4j (NEU704), which are potent against in vitro cultures of T. cruzi and are greater than 160-fold
selective over host cells. We report in vitro drug metabolism and properties profiling of 4c and show that this chemotype inhibits
the T. cruzi CYP51 enzyme, an observation confirmed by X-ray crystallographic analysis. We compare the binding orientation of
4c to that of other, previously reported inhibitors. We show that 4c displays a significantly better ligand efficiency and a shorter
synthetic route over previously disclosed CYP51 inhibitors, and should therefore be considered a promising lead compound for
further optimization.
discovery for a condition such as Chagas disease, which
predominantly affects a very poor population. Nonetheless, due
to immigration into North America and Europe from Latin
America, an increasing burden of disease is becoming
apparent.1
Academic drug discovery has made significant inroads toward
filling the gap for new therapeutics. Repurposed antifungal
agents, such as the approved drug posaconazole,2 the
investigational compound E1224 (a ravuconazole prodrug),
and Tak-187,3 have been assessed in clinical trials for Chagas
INTRODUCTION
■
In Latin America, an estimated 10 million people are infected
with Trypanosoma cruzi, the causative parasite for Chagas
disease. This rate of infection results in over 10 000 deaths per
year, primarily due to the cardiomyopathy end point that is the
most typical pathology resulting from chronic T. cruzi infection.
Current drugs, such as benznidazole and nifurtimox, are fairly
effective when utilized during the acute stage of the disease, yet
success in curing chronic Chagas disease has been limited.
Moreover, these drugs have toxicity limitations. Despite these
limitations and the 25 million people at risk, the pipeline for
new drugs is lacking, primarily due to the lack of financial
incentives for undertaking the expensive process of drug
Received: January 3, 2013
© XXXX American Chemical Society
A
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Figure 1. Anti-T. cruzi compounds previously discovered via HTS approaches, leading to the design principle 4.
a
disease. Other research groups have expanded a class of farnesyl
transferase inhibitors (such as tipifarnib) to improve safety and
efficacy;4,5 the mechanism of action of all of these compounds
is via the sterol biosynthesis pathway, inhibiting cytochrome
P450 sterol 14α-demethylase (T. cruzi CYP51).6,7
Scheme 1. Preparation of Analogs 4a−e
High-throughput screening (HTS) approaches have begun to
reveal new compounds with anti-T. cruzi activity. One such
screening campaign has resulted in compounds that show in
vivo activity.8 We evaluated the screening hits from the HTS
campaign (303 286 compounds) in the context of those
identified in a previous report9 and highlight in Figure 1 a
promising class of imidazole-based inhibitors that effectively
inhibit infection of mammalian host cells by T. cruzi
trypomastigotes. In particular, compounds 1, 2, and related
analogs were identified in the HTS. A third imidazole-bearing
class, typified by 3, showed activity approximately equal to that
of 2. A related set of compounds was recently reported in a
separate HTS that probed T. cruzi CYP51 in a biochemical
assay.10 Taken together, these compounds present a general
structural motif consisting of an imidazole ring that is
connected to a lipophilic tail via a linker of variable length
and flexibility. We therefore looked to optimize this chemotype
with an eye toward improving potency against T. cruzi while
maintaining the low level of host cell toxicity observed in the
HTS. Further, several of the compounds of interest feature long
hydrocarbon linkers, which are implicated in poor bioavail-
ability and metabolic properties.11
a
Reagents and conditions: (a) N-bromosuccinimde, CH2Cl2; (b)
imidazole, DMF, MW 180 °C, 10 min; (c) H2N−Ar, Pd2(dba)3,
DPEPhos, DMF, MW 170 °C, 20 min; (d) CuI, HO−Ar, Cs2CO3,
Me2NCH2CO2H; (e) 4-chlorobenzyl bromide or 1-(2-bromoethyl)-4-
chlorobenzene, K2CO3, DMF, 60 °C, 1 h; (f) LiAlH4, THF; (g) N-
bromosuccinimde, PPh3; and (h) imidazole, DMF, 100 °C, 1 h.
RESULTS
■
on this ring is required; a loss of ∼20-fold in potency was
observed by removal of all substituents (cf., 4i). Readdition of
the chlorine atom improved potency by 2.5-fold (4i versus 4g),
and although addition of a methyl group to 4i provided a 5-fold
improvement in potency (cf., 4f), addition of a second (4e)
showed no effect on T. cruzi cell activity. The regiochemical
requirement for the aryl methyl group was explored, and the
meta-substitution of 4f affords approximately 2× in activity over
the ortho isomer (4h). Extending the linker length by one atom
leads to doubling of potency (4j, NEU704), but further
extension (4k) diminishes activity.
We designed a series of heterocyclic replacements for the
imidazole ring to assess this region of the molecule as well.
Nitrogen-linked analogs 15a−k were accessed by reaction of
the benzyl bromide 14 with various nitrogen heterocycles
(Scheme 2). The bromide 14 was prepared in two steps from 3-
bromobenzaldehyde 12. Carbon-linked analogs 20 and 21 were
prepared from the corresponding bromide 16 via a sequence of
Ulmann coupling, acylhydrazide formation, and dehydrative
cyclization (Scheme 3). Screening data for compounds 15, 20,
and 21 are listed in Table 2, showing that the imidazole moiety
of 4c is by far a more potent growth inhibitor than even
isosteric heterocycles like the pyrazole 15c and triazoles 15a, b,
f, and h.
Medicinal Chemistry. With these goals in mind, we first
sought to replace the alkyl chain linker with a scaffold that
would appropriately present the proposed pharmacophoric
elements. This was pursued by the synthesis of ring-constrained
analogs, typified by structure 4 (Figure 1), which represents an
analog of the potent HTS hit 1c. To that end, benzylic
bromination of 3-bromotoluene followed by nucleophilic
displacement with imidazole provided 7 (Scheme 1). This
was subjected to Buchwald−Hartwig coupling to give diaryl
amines 4a−b or Ullman coupling to give diaryl ethers 4c−i.
Compounds 4j−k were constructed from methyl 3-hydrox-
ybenzoate via a sequence of Ullman coupling and conversion of
the methyl ester to a benzyl bromide, which was substrate for
alkylation of imidazole.
The screening results of this initial SAR exploration are
shown in Table 1. Activity against T. cruzi was determined in a
mammalian host cell infection assay measuring T. cruzi
trypomastigote infection during 96 h, counterscreened against
uninfected host cells (NIH 3T3 cells). Gratifyingly, the ring-
constrained analog 4c (NEU321), directly designed from 1c,
was quite potent (80 nM). Compound 4a was approximately
equipotent to 4c, and all of the analogs tested were at least 60-
fold selective over host cells. Substitution with lipophilic groups
B
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a
Table 1. Potency of Analogs of 4 as T. cruzi Growth Inhibitors
compound
4a
X
R2
R3
R4
R5
T. cruzi EC50 (μM)
NIH 3T3 EC50 (μM)
selectivity
NH
NH
O
CH3
H
H
NO2
H
H
0.072
0.167
0.08
0.282
0.33
0.33
0.65
0.711
1.63
0.042
0.53
1.9
>100
>100
13.5
34
>1000
>500
169
4b
OCH3
CH3
Ph
H
4c
H
Cl
H
CH3
H
4d
O
H
121
4e
O
H
CH3
CH3
H
H
CH3
H
>100
78.7
>100
79.3
>100
76.2
71.1
>50
>300
238
4f
O
H
H
4g
O
H
Cl
H
H
>150
112
4h
O
CH3
H
H
H
4i
O
H
H
H
>60
4j
OCH2
H
H
Cl
Cl
H
1814
134
4k
OCH2CH2
H
H
H
benznidazole
<26
a
EC50 values were determined as the average of three independent experiments performed on different days, and SEM ≤ 30%.
Scheme 2. Synthesis of Various N-Linked Imidazole
Replacements 15
96 h of the assay, while benznidazole required 48−72 h (Figure
2).
a
We then evaluated the phenotypic effect of 4c on T. cruzi
infection of host cells. We observed that 4c had a pronounced
inhibitory effect on T. cruzi replication comparable to
benznidazole, because individual parasites that were able to
invade, but not replicate, are observed within host cells (Figure
3).
In Vitro Profiling of Absorption, Distribution, Metab-
olism, and Excretion Properties. We evaluated 4c in in vitro
assessments of metabolic and physicochemical properties, to
ascertain parameters that required further optimization (Table
3). This compound shows good microsomal stability in human,
rat, and mouse microsomes, and the percentage is only slightly
reduced in the presence of quinidine (a CYP2D6 inhibitor) and
ketoconazole (CYP3A4 inhibitor), indicating that metabolism
of 4c is mediated in part, but not entirely, by CYP2D6 and
CYP3A4. Compound 4c is also fully stable in human and
mouse plasma, and is highly permeable through Caco-2 cell
monolayers. However, perhaps not surprisingly, 4c is highly
plasma protein bound (>99.8%), which suggests low
bioavailability in vivo. Further, this compound displays strong
inhibition of the hERG ion channel (94% at 10 uM); because
this target is implicated in cardiotoxicity, there are significant
concerns regarding this compound’s safety in chronic chagasic
patients that are likely to have existing cardiovascular issues due
to the infection.
a
Reagents and conditions: (a) 4-chloro-3,5-dimethylphenol, Cs2CO3,
CuI, 2-(dimethylamino)acetic acid, 1,4-dioxane, 180 °C, 12 h; (b)
TMSCl, 1,1,3,3-tetramethyldisiloxane, LiBr, CH3CN, 80 °C, over-
night; (c) heterocycle, amine or indole, K2CO3, DMF, 50 °C, 3 h.
Scheme 3. Synthesis of Various C-Linked Imidazole
Replacements 20 and 21
a
Mechanism of Action Hypothesis. Confident that the
imidazole is key to binding, we noted that this moiety is indeed
reminiscent of other inhibitors described for T. cruzi activity,
such as tipifarnib and its analogs,12 posaconazole,13 and
VNF13,14 (Figure 4), all of which inhibit parasite growth via
blockade of T. cruzi sterol 14α-demethylase (CYP51). This has
been shown to be a central enzyme for parasitic sterol
biosynthesis, the pathway essential in all life stages of T. cruzi
that leads to formation of ergosterol and related structures from
lanosterol (through eburicol as the T. cruzi CYP51 substrate).13
We hypothesized that the class of compounds typified by 4 may
be inhibiting parasite growth via the same mechanism.
a
Reagents and conditions: (a) 4-chloro-3,5-dimethylphenol, Cs2CO3,
CuI, 2-(dimethylamino)acetic acid, 1,4-dioxane, 180 °C, 9 h; (b) THF,
LiOH, H2O, room temperature, 2 h; (c) H2NNHAc, HOBT, EDC,
DMF, room temperature, 5 h; (d) POCl3, 110 °C, 1 h; or Lawesson’s
reagent, 1,4-dioxane, 100 °C, 2 h.
To investigate whether the effect of 4c on T. cruzi is
reversible, the compound was washed off the cultures at
different times after infection. In two distantly related strains of
T. cruzi (Tulahuen and Y), we observed that incubation for 24
h with 4c is sufficient to inhibit T. cruzi replication during the
C
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a
the LE values calculated for those compounds for which the T.
cruzi CYP51 binding data were obtained, in comparison with
other established inhibitors. Compounds 4b, 4c, and 4h do
indeed represent an attractive starting point for further
optimization.
Table 2. Potency of Analogs 15, 20, and 21
X-ray Crystallographic Analysis. X-ray crystallography
has revealed the binding orientations of posaconazole and VNF
complexed to T. cruzi CYP51, and it was noted that the
inhibitors bound in the pocket in opposite orientations to each
other.12 Briefly, while the long arm of posaconazole lies inside
the CYP51 substrate access channel, protruding through the
channel entrance above the protein surface, the two-ring arm of
VNF is oriented deeper into the active site cavity, intercalating
between helices B′, C, and the N-terminal portion of helix I.
We wished to compare the structure of 4c to ascertain which
conformation this scaffold would adopt. We therefore
performed cocrystallization of T. cruzi CYP51 in complex
with compound 4c, and observed that 4c binds to the heme, as
VNF and posaconazole do, and the vector of 4c follows that of
VNF (Figure 6). Table 5 summarizes the diffraction and
refinement statistics; electron density maps for the inhibitor
area are presented in the Supporting Information (Figure S3).
DISCUSSION AND CONCLUSIONS
■
Scaffold Refinement. We began our exploration of the
series of imidazole-containing HTS hits by simply installing a
ring that restricted compound flexibility. This first round of
modification led immediately to 4c, a potent inhibitor of T.
cruzi growth that has 169-fold selectivity over host cells.
Exploration of the structural substitution of the diaryl ether and
replacements of the imidazole heterocycle confirmed that 4c
was already of optimal properties for this scaffold for potency.
Although analog 4a was of equal potency to 4c, we
deprioritized this structure due to the aryl nitro group, which
is often cited as a toxicophoric motif that generates reactive
oxygen species in vivo.16 Compound 4j also showed modestly
improved activity, selectivity, and ligand efficiency over 4c,
although we had concerns about the metabolic stability of the
benzyl ether motif. We therefore focused on 4c as a tool to
characterize the mechanism of action of this class of growth
inhibitors and to better understand the binding modality.
Further study of the in vitro ADME properties of 4c
uncovered further shortcomings of this scaffold that will be
directive of future optimization efforts. Importantly, although
we are confident in the imidazole functionality’s importance to
T. cruzi CYP51 binding, much work remains for optimization of
the central phenyl scaffold combined with other replacements
of the 4-chloro-3,5-dimethylphenyl tail group. We anticipate
that these further modifications can be aptly informed by the
rich structural biology information we have uncovered.
Inhibitor Affinity of T. cruzi CYP51. All four tested
compounds produce the characteristic type 2 spectral responses
in T. cruzi CYP51,17 the Soret band maximum of the
hemoprotein shifting from 417 to 424 nm, which is indicative
of the formation of the coordination bond between the heme
iron and the N3 atom of an imidazole ring. The apparent Kd
values shown in Table 4 suggested 4c as the strongest ligand
among those tested, the saturation being achieved at an
approximately equimolar ratio to enzyme. The apparent
binding affinity of 4c correlates well with its inhibitory effect
on the T. cruzi CYP51 activity. We observed only a 7.5%
substrate conversion at a 2-fold molar excess of the inhibitor
over the enzyme (1 h of reaction), indicating a very high
a
EC50 values were determined as the average of three independent
experiments performed on different days, and SEM ≤ 30%.
To experimentally confirm this, we tested binding of 4b, 4c,
and 4h to recombinant T. cruzi CYP51, measuring a dose-
responsive type 2 spectroscopic shift that occurs upon
coordination of a Lewis-basic atom of the inhibitor to the
heme iron of the enzyme (Figure 5, Table 4).14 These
experiments uncovered Kd values that correlated well with the
EC50 values observed in parasite cultures.
As a measure of binding energy per heavy atom of a drug
compound interacting with a target, ligand efficiency (LE) is a
useful metric for comparing compounds.15 LE is typically
calculated by the dividing the pKd or pEC50 by the number of
heavy atoms in the molecule. From this standpoint, a
compound that is intended to serve as a starting point for
further optimization should have an LE ≥ 0.3.15 Table 4 lists
D
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Figure 2. Reversibility of compound 4c. T. cruzi growth inhibition assays were performed by adding the compounds 4c or benznidazole at the time
of infection and washing them off at the indicated times. EC50 was calculated for each condition after 96 h of incubation. Left panel shows T. cruzi
Tulahuen, and right panel shows T. cruzi Y strains.
Figure 3. NIH-3T3 fibroblasts were incubated with T. cruzi trypomastigotes for 2 h before washing of extracellular T. cruzi and addition of drugs.
Cells were incubated for 3 days, and stained with an anti-T. cruzi antibody and DAPI to visualize DNA. Control infection (A) or infection in the
presence of benznidazole (B) and 4c (C) at 9.5 and 0.4 μM, respectively (5 times the EC50 concentration). These are representative images from a
total of 50 fields observed in each condition.
Table 3. In Vitro ADME Parameters for 4c
a
microsomal stability (%)
mouse
29
36
39
rat
human
plasma protein binding (%)
mouse
>99.8
>99.8
human
b
plasma stability (%)
mouse
human
4
−9.7
hERG affinity (%)
19 (1 μM)
94 (10 μM)
209.7
Caco-2 permeability (nm s−1
)
a
b
Percent metabolized at 20 min. Mean percent of difference between
Co and C4h.
Figure 4. Other azole-based inhibitors of T. cruzi CYP51.
potency that is approaching the potencies of VNF and
posaconazole (Table 4 and Figure S4 in the Supporting
Information).18
plane, the B′C loop (residues F110, A115, and Y116 in Figure
6b), the C-helix (Q120, M123, L127, and L130), and the N-
terminal portion of helix I (M284, A287, A288, A291, and
T295). Thus, of the 13 amino acid residues forming van der
Waals contacts with 4c, 12 are provided by these three
secondary structural elements. Only one inhibitor-contacting
residue, L356, derives from the substrate access channel area
(K/β1−4 loop, CYP51 SRS5).20
Interestingly, the location of 4c in the cocrystal structure is
highly similar to that of VNF (Figure 6c), the CYP51 inhibitor
that so far has been the only example of a ligand molecule
whose orientation inside the CYP51 binding site cavity is
opposite to that of posaconazole (Figure 6d),18 the VNF analog
X-ray Cocrystal Structure of 4c Complexed with T.
cruzi CYP51. The goal of this structural work was to uncover
the molecular basis for the observed high inhibitory potency of
4c as well as to gain further information on possible directions
for the future scaffold enhancement. Figure 6a and b depicts an
overall view of the enzyme−inhibitor complex and an enlarged
view of the inhibitor binding area, respectively. As anticipated,
in the T. cruzi CYP51 active site, the imidazole ring on 4c
coordinates to the heme iron, the distance between Fe and N3
being only 2.05 Å, while the nonligated 4-chloro-3,5-
dimethyphenyl region of the molecule occupies the deepest
hydrophobic pocket19 formed by the distal surface of the heme
E
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visible detector, using acetonitrile/H2O gradients with 0.1% formic
acid. Fractions were collected on the basis of triggering using UV and
mass detection. Yields reported for products obtained by preparative
HPLC represent the amount of pure material isolated; impure
fractions were not repurified.
N-(3-((1H-Imidazol-1-yl)methyl)phenyl)-2-methyl-4-nitroani-
line (4a). Compound 7 (0.020 g, 0.084 mmol) dissolved in DMF (0.5
mL) was added to 2-methyl-4-nitroaniline (19.25 mg, 0.127 mmol),
Pd2(dba)3 (15.45 mg, 0.017 mmol), 2,2′-oxybis(2,1-phenylene)bis-
(diphenylphosphine) (18.17 mg, 0.034 mmol), and potassium tert-
butoxide (0.169 mL, 0.169 mmol). The reaction was run for 20 min in
the microwave at 170 °C. The crude mixture was diluted with H2O,
and the aqueous layer was extracted with EtOAc (3×). The combined
organic layers were washed with brine, dried under anhydrous sodium
sulfate, and concentrated. The crude product was purified via
preparative HPLC to give 4a (yield: 40%). 1H NMR (500 MHz,
CDCl3): δ 8.08 (d, J = 2.44 Hz, 1H), 7.98 (dd, J = 2.44, 8.79 Hz, 1H),
7.59 (s, 1H), 7.37 (t, J = 7.75 Hz, 1H), 7.17 (d, J = 7.81 Hz, 1H), 7.13
(s, 1H), 7.07 (d, J = 9.28 Hz, 1H), 6.93−6.98 (m, 2H), 6.89 (s, 1H),
5.88 (br s, 1H), 5.14 (s, 2H), 2.33 (s, 3H). LCMS found 309.01, [M +
H]+.
Figure 5. Spectroscopic assay data for 4c binding to T. cruzi CYP51.
Table 4. Binding of Compounds to Recombinant T. cruzi
3-((1H-Imidazol-1-yl)methyl)-N-(3-methoxyphenyl)aniline
(4b). Compound 7 (0.0437 g, 0.184 mmol) dissolved in DMF (1 mL)
was added to 3-methoxyaniline (0.031 mL, 0.276 mmol), Pd2(dba)3
(33.8 mg, 0.037 mmol), 2,2′-oxybis(2,1-phenylene)bis-
(diphenylphosphine) (39.7 mg, 0.074 mmol), and potassium tert-
butoxide (0.369 mL, 0.369 mmol). The reaction was heated for 20 min
in the microwave at 170 °C. The crude mixture was diluted with H2O,
and the aqueous layer was extracted with EtOAc (3×). The combined
organic layers were washed with brine, dried under anhydrous sodium
sulfate, and concentrated. The crude product was purified via
preparative HPLC to give 4b (yield: 54%). 1H NMR (400 MHz,
CDCl3): δ 7.60 (s, 1H), 7.24 (t, J = 8.06 Hz, 1H), 7.18 (t, J = 8.06 Hz,
1H), 7.11 (s, 1H), 7.02−7.07 (m, 1H), 6.94 (s, 1H), 6.82 (s, 1H), 6.71
(d, J = 7.33 Hz, 1H), 6.60−6.66 (m, 2H), 6.52 (dd, J = 1.83, 8.43 Hz,
1H), 5.81 (br s, 1H), 5.07 (s, 2H), 3.78 (s, 3H). LCMS found 280.01,
[M + H]+.
1-(3-(4-Chloro-3,5-dimethylphenoxy)benzyl)-1H-imidazole
(4c). Compound 7 (0.6403 g, 2.70 mmol) dissolved in DMF (8 mL)
was added to copper(I) iodide (257 mg, 1.350 mmol), 4-chloro-3,5-
dimethylphenol (634 mg, 4.05 mmol), cesium carbonate (1760 mg,
5.40 mmol), and 2-(dimethylamino)acetic acid (418 mg, 4.05 mmol).
The reaction was heated for 20 min in the microwave at 180 °C. The
crude mixture was diluted with H2O, and the aqueous layer was
extracted with EtOAc (6×). The combined organic layers were washed
with brine, dried under anhydrous sodium sulfate, and concentrated.
The crude product was purified via preparative HPLC to give 4c
(yield: 40%).1H NMR (500 MHz, CDCl3): δ 7.54 (s, 1H), 7.29 (t, J =
7.81 Hz, 1H), 7.10 (s, 1H), 6.89−6.94 (m, 2H), 6.86 (d, J = 7.81 Hz,
1H), 6.80 (s, 1H), 6.73 (s, 2H), 5.08 (s, 2H), 2.34 (s, 6H). LCMS
found 313.01, [M + H]+.
CYP51
number
Kd
LE
4c
4h
4b
0.067
0.18
0.21
0.07
0.06
0.33
0.34
0.32
0.25
0.14
VNF
posaconazole
VNI,21 or the tipifarnib derivative JK35.22 Moreover, binding of
both VNF and 4c requires shifting of the side chain of Y116
away from the heme, thus causing loss of the hydrogen bond
between the Y116 hydroxyl and the porphyrin ring B
propionate.23 This is an interaction conserved in the CYP51
family that is known to play a role in maintaining a catalytically
favorable heme environment. Disruption of this heme support
from the protein moiety may be one of the reasons for the
elevated inhibitory potencies of these relatively smal molecules.
In summary, hit-to-lead chemistry follow-up efforts were
launched following a phenotypic high-throughput screen,
leading to identification of a new, ligand-efficient series of T.
cruzi growth inhibitors that mediate their effect via inhibition of
T. cruzi CYP51. Early structure−activity relationships have
been outlined, as well as structural biology information that will
drive further optimization efforts, which will be reported in due
course.
EXPERIMENTAL SECTION
1-(3-(Biphenyl-3-yloxy)benzyl)-1H-imidazole (4d). Com-
pound 7 (0.020 g, 0.084 mmol) dissolved in DMF (5 mL) was
added to copper(I) iodide (8.03 mg, 0.042 mmol), 3-phenylphenol
(21.54 mg, 0.127 mmol), cesium carbonate (55.0 mg, 0.169 mmol),
and 2-(dimethylamino)acetic acid (13.05 mg, 0.127 mmol). The
reaction was heated for 20 min in the microwave at 180 °C. The crude
mixture was diluted with H2O, and the aqueous layer was extracted
with EtOAc (3×). The combined organic layers were washed with
brine, dried under anhydrous sodium sulfate, and concentrated. The
crude product was purified via preparative HPLC to give 4d (yield:
■
Chemical Synthesis. Unless otherwise noted, reagents were
obtained from Sigma-Aldrich, Inc. (St. Louis, MO), or Frontier
Scientific Services, Inc. (Newark, DE), and were used as received.
Boronic acids and aniline reagents were purchased, unless the
synthesis is specifically described below. Reaction solvents were
purified by passage through alumina columns on a purification system
manufactured by Innovative Technology (Newburyport, MA). Micro-
wave reactions were performed using a Biotage Initiatior-8 instrument.
NMR spectra were obtained with Varian NMR systems, operating at
1
1
5%). H NMR (500 MHz, CDCl3): δ 8.58 (br s, 1H), 7.52−7.63 (m,
400 or 500 MHz for H acquisitions as noted. LCMS analysis was
2H), 7.32−7.49 (m, 7H), 7.24−7.26 (m, 1H), 7.04−7.13 (m, 2H),
7.00 (d, J = 6.84 Hz, 1H), 6.93 (br s, 2H), 5.22 (br s, 2H). LCMS
found 327.01, [M + H]+.
performed using a Waters Alliance reverse-phase HPLC, with single-
wavelength UV−visible detector and LCT Premier time-of-flight mass
spectrometer (electrospray ionization). All newly synthesized com-
pounds that were submitted for biological testing were deemed >95%
pure by LCMS analysis (UV and ESI−MS detection) prior to
submission for biological testing. Preparative LCMS was performed on
a Waters FractionLynx system with a Waters MicroMass ZQ mass
spectrometer (electrospray ionization) and a single-wavelength UV−
1-(3-(3,5-Dimethylphenoxy)benzyl)-1H-imidazole (4e). Com-
pound 7 (30 mg, 0.127 mmol) dissolved in DMF (0.8 mL) was added
to copper(I) iodide (12.05 mg, 0.063 mmol), 3,5-dimethylphenol
(23.19 mg, 0.190 mmol), cesium carbonate (82 mg, 0.253 mmol), and
2-(dimethylamino)acetic acid (19.57 mg, 0.190 mmol). The reaction
F
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Figure 6. X-ray structure of T. cruzi CYP51 complexed to 4c (PDB code 4H6O). Electron density maps and stereoview of the active site are available
in the Supporting Information. (a) Overall view from the distal side of the CYP51 molecule. The polypeptide chain is depicted as a ribbon diagram,
and the secondary structural elements forming the substrate access channel entrance are colored in purple. The C atoms of the heme and 4c are
displayed as orange and blue stick models, respectively. The semitransparent surface of 4c (probe radius 1.4 Å) is colored by interpolated charge. (b)
Enlarged view of 4c in the T. cruzi CYP51 active site. The 13 residues located within 4.5 Å from the inhibitor are depicted by lines and labeled. (c)
Superimposed cocrystal structures of T. cruzi CYP51 with 4c and VNF (PDB code 3KSW). VNF and the heme from the 3KSW structure are colored
in salmon. The distance between the heme ring propionate and OH of Y116 (3.7 Å, dashed line) is 1 Å longer than in the ligand-free or
posaconazole-bound CYP51 structures (2.7 Å). (d) Superimposed cocrystal structures of T. cruzi CYP51 with 4c and posaconazole (PDB code
3K1O). Posaconazole, heme, and Y116 from the 3K1O structure are colored in green. VNF and compound 4c both adopt the orientation in the
CYP51 active site, which is opposite to the orientation of posaconazole.
was heated for 20 min in the microwave at 180 °C. The crude mixture
was diluted with H2O, and the aqueous layer was extracted with
EtOAc (6×). The combined organic layers were washed with brine,
dried under anhydrous sodium sulfate, and concentrated. The crude
product was purified via preparative HPLC, followed by further
purification via silica gel chromatography (100% hexanes, followed by
(t, J = 7.69 Hz, 1H), 7.12 (br s, 1H), 6.89−6.97 (m, 3H), 6.76−6.88
(m, 4H), 5.09 (s, 2H), 2.33 (s, 3H). LCMS found 265.16, [M + H]+.
1-(3-(4-Chlorophenoxy)benzyl-1H-imidazole (4g). Compound
7 (30 mg, 0.127 mmol) dissolved in DMF (0.8 mL) was added to
copper(I) iodide (12.05 mg, 0.063 mmol), 4-chlorophenol (24.40 mg,
0.190 mmol), cesium carbonate (82 mg, 0.253 mmol), and 2-
(dimethylamino)acetic acid (19.57 mg, 0.190 mmol). The reaction
was heated for 20 min in the microwave at 180 °C. The crude mixture
was diluted with H2O, and the aqueous layer was extracted with
EtOAc (6×). The combined organic layers were washed with brine,
dried under anhydrous sodium sulfate, and concentrated. The crude
product was purified via preparative HPLC, followed by further
purification via silica gel chromatography (100% hexanes, followed by
1
10% MeOH in DCM) to give 4e (yield: 2%). H NMR (500 MHz,
CDCl3): δ 7.54 (br s, 1H), 7.28 (t, J = 8.00 Hz, 1H), 7.09 (br s, 1H),
6.90−6.92 (m, 2H), 6.83 (d, J = 8.00 Hz, 1H), 6.81 (s, 1H), 6.76 (s,
1H), 6.61 (s, 2H), 5.09 (s, 2H), 2.29 (s, 6H). LCMS found 279.01, [M
+ H]+.
1-(3-(m-Tolyloxy)benzyl)-1H-imidazole (4f). Compound 7
(65.8 mg, 0.277 mmol) dissolved in DMF (1.2 mL) was added to
copper(I) iodide (26.4 mg, 0.139 mmol), m-cresol (0.044 mL, 0.416
mmol), cesium carbonate (181 mg, 0.555 mmol), and 2-
(dimethylamino)acetic acid (42.9 mg, 0.416 mmol). The reaction
was heated for 20 min in the microwave at 180 °C. The crude mixture
was diluted with H2O, and the aqueous layer was extracted with DCM
(5×). The combined organic layers were washed with brine, dried
under anhydrous sodium sulfate, and concentrated. The crude product
1
10% MeOH in DCM) to give 4g (yield: 6%). H NMR (500 MHz,
CDCl3): δ 7.54 (s, 1H), 7.28−7.34 (m, 3H), 7.10 (s, 1H), 6.90−6.95
(m, 4H), 6.88 (d, J = 7.32 Hz, 1H), 6.81 (s, 1H), 5.09 (s, 2H). LCMS
found 285.01, [M + H]+.
1-(3-(o-Tolyloxy)benzyl)-1H-imidazole (4h). Compound 7
(0.040 g, 0.169 mmol) dissolved in DMF (1 mL) was added to
copper(I) iodide (16.07 mg, 0.084 mmol), o-cresol (27.4 mg, 0.253
mmol), cesium carbonate (110 mg, 0.337 mmol), and 2-
(dimethylamino)acetic acid (26.1 mg, 0.253 mmol). The reaction
1
was purified via preparative HPLC to give 4f (yield: 1%). H NMR
(400 MHz, CDCl3): δ 7.64 (br s, 1H), 7.29 (t, J = 7.69 Hz, 1H), 7.22
G
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mixture was cooled to room temperature, diluted with H2O (25 mL),
and the aqueous layer was extracted with EtOAc (5 × 20 mL). The
combined organic layers were washed with H2O (3 × 15 mL), washed
with brine (15 mL), dried under anhydrous sodium sulfate, and
concentrated. The crude product was then purified by flash column
chromatography using a gradient of 0−1% MeOH in EtOAc to give 4j
Table 5. Data Collection and Refinement Statistics for T.
cruzi CYP51, in Complex with 4a (PDB Code 4H6O)
Data Collection
wavelength, Å
0.9786
space group
P3(1)21
1
as a white solid (yield: 68%). H NMR (500 MHz, CDCl3): δ ppm
cell dimensions
7.55 (s, 1H), 7.31−7.38 (m, 4H), 7.28 (t, J = 7.8 Hz, 1H), 7.10 (s,
1H), 6.88−6.92 (m, 2H), 6.77 (d, J = 7.8 Hz, 1H), 6.70 (t, J = 2.0 Hz,
1H), 5.09 (s, 2H), 4.99 (s, 2H). LCMS found 299.24, [M + H]+.
1-(3-(4-Chlorophenethoxy)benzyl)-1H-imidazole (4k). To
11b (107 mg, 0.33 mmol) dissolved in DMF (3 mL) were added
potassium carbonate (188 mg, 1.36 mmol) and imidazole (92 mg, 1.35
mmol). The mixture was stirred and heated to 100 °C for 1 h. The
mixture was cooled to room temperature, diluted with H2O (25 mL),
and the aqueous layer was extracted with EtOAc (5 × 20 mL). The
combined organic layers were washed with H2O (3 × 15 mL), washed
with brine (15 mL), dried under anhydrous sodium sulfate, and
concentrated. The crude product was then purified by flash column
chromatography using a gradient of 0−1% MeOH in EtOAc to give 4k
a, b, c, Å
63.290, 63.290, 223.995
90.00, 90.00, 120.00
α, β, γ, deg
molecules per asymmetric unit
solvent content, %
resolution (last shell), Å
Rmerge (last shell)
I/σ (last shell)
52.5
30−2.55 (2.64−2.55)
0.047 (0.610)
39.3 (2.9)
completeness (last shell), %
redundancy (last shell)
99 (100)
8.8 (6.0)
Refinement
resolution, Å
27.5−2.8
0.263
0.275
12 800
5.3
1
R-factor
as a clear oil (yield: 53%). H NMR (500 MHz, CDCl3) δ ppm 7.54
R-free
(s, 1H), 7.29 (d, J = 8.3 Hz, 2H), 7.25 (d, J = 7.8 Hz, 1H), 7.20 (dt, J =
8.3, 2.4 Hz, 2H), 7.10 (s, 1H), 6.90 (s, 1H), 6.83 (dd, J = 8.3, 2.4 Hz,
1H), 6.74 (d, J = 7.8 Hz, 1H), 6.64 (t, J = 2.0 Hz, 1H), 5.07 (s, 2H),
4.11 (t, J = 6.6 Hz, 2H), 3.04 (t, J = 6.8 Hz, 2H). LCMS found 313.25,
[M + H]+.
reflections used
test set size, %
rms deviations from ideal geometry
bond lengths, Å
bond angles, deg
Ramachandran plot
residues in favorable regions (%)
residues in allowed regions (%)
outliers (%)
0.007
1.87
1-(3-Bromobenzyl)-1H-imidazole (7). To 6 (1.50 g, 6.00 mmol)
were added imidazole (899 mg, 13.20 mmol) and DMF (15 mL). The
reaction was heated for 10 min in the microwave at 180 °C. The crude
mixture was then diluted with H2O, and the aqueous layer was
extracted with DCM (5×). The combined organic layers were washed
with brine, dried under anhydrous sodium sulfate, and concentrated.
The crude product was purified via silica gel chromatography, eluting
with 0−5% MeOH in DCM to give 7 as a yellow oil (yield: 55%). 1H
NMR (500 MHz, CDCl3): δ 7.55 (s, 1H), 7.46 (d, J = 7.81 Hz, 1H),
7.30 (s, 1H), 7.23 (t, J = 8.06 Hz, 1H), 7.12 (s, 1H), 7.07 (d, J = 7.32
Hz, 1H), 6.90 (s, 1H), 5.10 (s, 2H). LCMS found 237.01, [M + H]+.
Methyl 3-((4-Chlorobenzyl)oxy)benzoate (9a). To methyl 3-
hydroxybenzoate (620 mg, 4.07 mmol) dissolved in DMF (4 mL)
were added 4-chlorobenzyl bromide (763 mg, 3.71 mmol) and
potassium carbonate (560 mg, 4.05 mmol). The mixture was stirred
and heated to 60 °C for 1 h. The crude mixture was diluted with H2O
(25 mL), and the aqueous layer was extracted with a 1:1 mixture of
EtOAc and hexanes (3 × 25 mL). The combined organic layers were
washed with H2O (30 mL), washed with brine (20 mL), dried under
anhydrous sodium sulfate, and concentrated. The crude product was
then purified by flash column chromatography using a gradient of 1−
97.2
100
0
Model
total number of atoms (average B-factor, Å)
3592 (75.3)
residues
protein
heme
435 (75.1)
1 (34.9)
4c (NEE)
water
1 (45.5)
55 (86.9)
was heated for 20 min in the microwave at 180 °C. The crude mixture
was diluted with H2O, and the aqueous layer was extracted with
EtOAc (6×). The combined organic layers were washed with brine,
dried under anhydrous sodium sulfate, and concentrated. The crude
product was purified via silica gel chromatography to give 4h (yield:
33%). 1H NMR (400 MHz, CDCl3): δ 7.56 (br s, 1H), 7.24−7.28 (m,
2H), 7.15−7.20 (m, 1H), 7.05−7.12 (m, 2H), 6.86−6.94 (m, 2H),
6.80 (d, J = 7.50 Hz, 2H), 6.74 (s, 1H), 5.07 (s, 2H), 2.20 (s, 3H).
LCMS found 265.01, [M + H]+.
1-(3-Phenoxybenzyl)-1H-imidazole (4i). Compound 7 (30 mg,
0.127 mmol) dissolved in DMF (0.8 mL) was added to copper(I)
iodide (12.05 mg, 0.063 mmol), phenol (17.86 mg, 0.190 mmol),
cesium carbonate (82 mg, 0.253 mmol), and 2-(dimethylamino)acetic
acid (19.57 mg, 0.190 mmol). The reaction was heated for 20 min in
the microwave at 180 °C. The crude mixture was diluted with H2O,
and the aqueous layer was extracted with EtOAc (6×). The combined
organic layers were washed with brine, dried under anhydrous sodium
sulfate, and concentrated. The crude product was purified via
preparative HPLC, followed by further purification via silica gel
chromatography (100% hexanes, followed by 10% MeOH in DCM) to
give 4i (yield: 5%). 1H NMR (500 MHz, CDCl3): δ 7.54 (s, 1H), 7.35
(t, J = 8.06 Hz, 2H), 7.30 (t, J = 8.06 Hz, 1H), 7.13 (t, J = 7.57 Hz,
1H), 7.09 (s, 1H), 6.99 (d, J = 7.81 Hz, 2H), 6.93 (dd, J = 1.95, 8.30
Hz, 1H), 6.91 (s, 1H), 6.86 (d, J = 7.81 Hz, 1H), 6.83 (s, 1H), 5.08 (s,
2H). LCMS found 251.01, [M + H]+.
1
20% EtOAc in hexanes to give 9 as a white solid (yield: 84%). H
NMR (500 MHz, CDCl3): δ ppm 7.67 (dt, J = 7.8, 1.2 Hz, 1H), 7.64
(dd, J = 2.4, 1.5 Hz, 1H), 7.34−7.41 (m, 5H), 7.16 (ddd, J = 8.3, 2.9,
1.0 Hz, 1H), 5.08 (s, 2H), 3.92 (s, 3H). LCMS found 277.25, [M +
H]+.
Methyl 3-(4-Chlorophenethoxy)benzoate (9b). To methyl 3-
hydroxybenzoate (630 mg, 4.14 mmol) dissolved in DMF (4 mL)
were added 1-(2-bromoethyl)-4-chlorobenzene (3.87 g, 17.63 mmol)
and potassium carbonate (2.90 g, 20.98 mmol). The mixture was
stirred and heated to 60 °C for 4 h. The crude mixture was diluted
with H2O (40 mL), and the aqueous layer was extracted with a 1:1
mixture of EtOAc and hexanes (3 × 40 mL). The combined organic
layers were washed with H2O (30 mL), washed with brine (20 mL),
dried under anhydrous sodium sulfate, and concentrated. The crude
product was then purified by flash column chromatography using a
gradient of 1−20% EtOAc in hexanes to give 9b as a white solid (yield:
59%). 1H NMR (500 MHz, CDCl3): δ ppm 7.64 (d, J = 7.8 Hz, 1H),
7.55 (dd, J = 2.4, 1.5 Hz, 1H), 7.34 (t, J = 8.1 Hz, 1H), 7.30 (dt, J =
8.3, 2.0 Hz, 2H), 7.23 (dt, J = 8.3, 2.0 Hz, 2H), 7.09 (ddd, J = 8.3, 2.9,
1.0 Hz, 1H), 4.20 (t, J = 6.6 Hz, 2H), 3.92 (s, 3H), 3.08 (t, J = 6.8 Hz,
2H). LCMS found 291.28, [M + H]+.
1-(3-((4-Chlorobenzyl)oxy)benzyl)-1H-imidazole (4j). To 11a
(106 mg, 0.34 mmol) dissolved in DMF (3 mL) were added
potassium carbonate (195 mg, 1.41 mmol) and imidazole (97 mg, 1.43
mmol). The mixture was stirred and heated to 100 °C for 1 h. The
(3-((4-Chlorobenzyl)oxy)phenyl)methanol (10a). 9a (794 mg,
2.87 mmol) was dissolved in anhydrous THF (6 mL) and cooled to 0
H
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°C. Lithium aluminum hydride (218 mg, 5.74 mmol) was then added
slowly, and the mixture was stirred for 15 min. The mixture was
warmed to room temperature and was stirred for 45 min. The mixture
was cooled to 0 °C and was quenched by the dropwise addition with
H2O (0.3 mL), 15% aqueous KOH (0.3 mL), and H2O (0.6 mL). The
mixture was warmed to room temperature and stirred for 15 min.
Magnesium sulfate was added, and the mixture was stirred a further 15
min. The solids were vacuum filtered, and to the filtrate were added
H2O (20 mL), EtOAc (10 mL), and hexanes (10 mL). The organic
layer was removed, and the aqueous layer was extracted with EtOAc (3
× 15 mL). The combined organic layers were washed with brine (10
mL), dried under anhydrous sodium sulfate, and concentrated onto
silica. The crude product was then purified by flash column
chromatography using a gradient of 10−30% EtOAc in hexanes to
give 10a as a white solid (yield: 71%). 1H NMR (500 MHz, CDCl3): δ
ppm 7.26−7.40 (m, 5H), 7.01 (s, 1H), 6.97 (d, J = 7.3 Hz, 1H), 6.89
(dd, J = 8.1, 2.7 Hz, 1H), 5.05 (s, 2H), 4.67 (s, 2H), 1.81 (br s, 1H).
LCMS found 231.22, [M + H − H2O]+.
3-(4-Chloro-3,5-dimethylphenoxy)benzaldehyde (13). 3-Bro-
mobenzaldehyde (1.0 g, 0.630 mL, 5.40 mmol) was dissolved in 1,4-
dioxane (40.0 mL), and then 4-chloro-3,5-dimethylphenol (1.270 g,
8.11 mmol), 2-(dimethylamino)acetic acid (0.836 g, 8.11 mmol),
cesium carbonate (5.28 g, 16.21 mmol), copper(I) iodide (0.515 g,
2.70 mmol), and other 20 mL of 1,4-dioxane were added to the
solution. The reaction was heated to 180 °C. After 4 h, another 0.5
equiv of all of the reagents except the aldehyde were added, and
heating continued. After 12 h, the crude was evaporated to dryness and
then dissolved in DCM (100 mL). The solution was filtered two times
on filter paper. The organic phase was washed with NaOH 10 N
solution (2 × 30 mL), and then aqueous layers were washed with
DCM (3 × 30 mL). The combined organic layers were washed with
brine, dried over anhydrous sodium sulfate, and concentrated. The
crude product was purified via silica gel chromatography (9:1 hexane/
EtOAc) to give 13 as a yellow oil (1.320 g, 94% yield). 1H NMR (500
MHz, CDCl3): δ 9.96 (s, 1H), 7.60 (d, J = 7.80 Hz, 1H), 7.50 (t, J =
7.80 Hz, 1H), 7.41−7.43 (m, 1H), 7.25−7.28 (m, 1H), 6.78 (s, 2H),
2.36 (s, 6H). LCMS found 261.03, [M + H]+.
(3-(4-Chlorophenethoxy)phenyl)methanol (10b). Compound
9b (681 mg, 2.34 mmol) was dissolved in anhydrous THF (5 mL) and
cooled to 0 °C. Lithium aluminum hydride (190 mg, 5.01 mmol) was
then added slowly, and the mixture was stirred for 15 min. The
mixture was then warmed to room temperature and was stirred for 45
min. The mixture was cooled to 0 °C and was quenched by the
dropwise addition with H2O (0.3 mL), 15% aqueous KOH (0.3 mL),
and H2O (0.6 mL). The mixture was warmed to room temperature
and stirred for 15 min. Magnesium sulfate was added, and the mixture
was stirred a further 15 min. The solids were vacuum filtered, and to
the filtrate were added H2O (20 mL), EtOAc (10 mL), and hexanes
(10 mL). The organic layer was removed, and aqueous layer was
extracted with EtOAc (3 × 20 mL). The combined organic layers were
washed with brine (15 mL), dried under anhydrous sodium sulfate,
and adsorbed onto silica. The crude product was then purified by flash
column chromatography using a gradient of 10−30% EtOAc in
5-(3-(Bromomethyl)phenoxy)-2-chloro-1,3-dimethylben-
zene (14). To a solution of 3-(4-chloro-3,5-dimethylphenoxy)-
benzaldehyde (807 mg, 3.10 mmol) in CH3CN (5 mL) was added
lithium bromide (512 mg, 5.90 mmol) followed by the dropwise
addition of chlorotrimethylsilane (0.786 mL, 6.19 mmol). After 15
min, the reaction mixture was cooled to 0 °C, 1,1,3,3-tetramethyldi-
siloxane (1.368 mL, 7.74 mmol) was added dropwise, and the resulting
mixture was heated to 80 °C overnight. The mixture was separated
into two layers. The lower layer was removed, was diluted with DCM,
and was filtered through filter paper. The filtrate was concentrated
under reduced pressure, was dissolved in DCM, and was refiltered.
The solvent was removed in vacuo to provide 14 as a light yellow oil
1
(0.88 g, 87% yield). H NMR (500 MHz, CDCl3): δ 7.31 (t, J = 7.80
Hz, 1H), 7.12 (d, J = 7.80 Hz, 1H), 7.00 (t, J = 2.40 Hz, 1H), 6.92 (dd,
J = 2.40, 7.80 Hz, 1H), 6.75 (s, 2H), 4.54 (s, 2H), 2.35 (s, 6H). LCMS
found 325.39, [M + H]+.
1
hexanes to give 10b as a clear oil (yield: 96%). H NMR (500 MHz,
CDCL3): δ ppm 7.29 (dt, J = 8.3, 2.0 Hz, 2H), 7.26 (d, J = 7.8 Hz,
1H), 7.23 (dd, J = 8.8, 2.0 Hz, 2H), 6.90−6.96 (m, 2H), 6.82 (dd, J =
7.8, 2.4 Hz, 1H), 4.67 (s, 2H), 4.17 (t, J = 6.8 Hz, 2H), 3.07 (t, J = 6.8
Hz, 2H), 1.70 (br. s., 1H). LCMS found 245.25, [M + H − H2O]+.
1-(Bromomethyl)-3-((4-chlorobenzyl)oxy)benzene (11a).
Compound 10a (396 mg, 1.59 mmol) was dissolved in anhydrous
THF (8 mL) under a stream of nitrogen. Triphenylphosphine (463
mg, 1.77 mmol) was added, and the solution was stirred for 5 min
before the addition of N-bromosuccinimide (312 mg, 1.75 mmol). The
mixture was stirred at room temperature for 2 h. The reaction was
quenched with H2O (15 mL), and the aqueous mixture was extracted
with a 1:1 mixture of hexanes and EtOAc (3 × 15 mL). The combined
organic layers were washed with brine (10 mL), dried under
anhydrous sodium sulfate, and concentrated. The crude product was
then purified by flash column chromatography using a gradient of 0−
General Alkylation Procedure for Compounds 15a−k. To a
solution of 14 (20 mg, 0.061 mmol) in 0.5 mL of DMF was added the
appropriate heterocycle in 1 mL of DMF (0.092 mmol), then K2CO3
(16.98 mg, 0.123 mmol), and the mixtures were stirred for 3 h at 50
°C. After 3 h, reactions were cooled and filtered through 17 mm
cellulose syringe filters (0.45 μm) and purified via preparative HPLC
to give the desired products.
1-(3-(4-Chloro-3,5-dimethylphenoxy)benzyl)-1H-1,2,3-tria-
zole (15a). Yield: 25%. 1H NMR (500 MHz, CDCl3): δ 7.72 (s, 1H),
7.50 (s, 1H), 7.31 (t, J = 8.0 Hz, 1H), 6.91−6.97 (m, 2H), 6.88 (br s,
1H), 6.72 (s, 2H), 5.53 (s, 2H), 2.34 (s, 6H). LCMS found 314.03, [M
+ H]+.
1-(3-(4-Chloro-3,5-dimethylphenoxy)benzyl)-1H-1,2,4-tria-
1
zole (15b). Yield: 3.1%. H NMR (500 MHz, CDCl3): δ 8.07 (br s,
1H), 7.97 (s, 1H), 7.31 (t, J = 7.80 Hz, 1H), 6.95 (d, J = 7.80 Hz, 1H),
6.93 (dd, J = 2.40, 7.80 Hz, 1H), 6.87−6.89 (m, 1H), 6.73 (s, 2H),
5.31 (s, 2H), 2.34 (s, 6H). LCMS found 314.00, [M + H]+.
1-(3-(4-Chloro-3,5-dimethylphenoxy)benzyl)-1H-pyrazole
1
5% EtOAc in hexanes to give 11a as a white solid (yield: 86%). H
NMR (500 MHz, CDCl3): δ ppm 7.38 (s, 4H), 7.27 (t, J = 8.1 Hz,
1H), 6.99−7.04 (m, 2H), 6.90 (dd, J = 8.3, 2.4 Hz, 1H), 5.04 (s, 2H),
4.47 (s, 2H). GCMS found 309.9, [M+·]
1
(15c). Yield: 20%. H NMR (500 MHz, CDCl3): δ 7.55 (d, J = 1.95
1-(Bromomethyl)-3-(4-chlorophenethoxy)benzene (11b).
Compound 10b (549 mg, 2.09 mmol) was dissolved in anhydrous
THF (5 mL) under a stream of nitrogen. Triphenylphosphine (626
mg, 2.39 mmol) was added, and the solution was stirred for 5 min
before the addition of N-bromosuccinimide (437 mg, 2.46 mmol). The
mixture was stirred at room temperature for 2 h. The reaction was
quenched with H2O (20 mL), and the aqueous mixture was extracted
with a 1:1 mixture of hexanes and EtOAc (3 × 15 mL). The combined
organic layers were washed with brine (15 mL), dried under
anhydrous sodium sulfate, and concentrated. The crude product was
then purified by flash column chromatography using a gradient of 0−
Hz, 1H), 7.40 (d, J = 1.95 Hz, 1H), 7.29 (t, J = 7.80 Hz, 1H), 6.87−
6.91 (m, 2H), 6.82 (s, 1H), 6.72 (s, 2H), 6.29 (t, J = 1.95 Hz, 1H),
5.30 (s, 2H), 2.33 (s, 6H). LCMS found 312.99, [M + H]+.
4-(3-(4-Chloro-3,5-dimethylphenoxy)benzyl)morpholine
1
Formate (15d). Yield: 7%. H NMR (500 MHz, CDCl3): δ 8.13 (s,
1H), 7.30 (t, J = 7.80 Hz, 1H), 7.09 (d, J = 7.80 Hz, 1H), 6.99 (br. s,
1H), 6.91 (dd, J = 2.40, 7.80 Hz, 1H), 6.74 (s, 2H), 3.79 (t, J = 4.64
Hz, 4H), 3.69 (s, 2H), 2.65 (br s, 4H), 2.34 (s, 6H). LCMS found
332.02, [M + H]+.
1-(3-(4-Chloro-3,5-dimethylphenoxy)benzyl)-1H-benzo[d]-
imidazole (15e). Yield: 17%. 1H NMR (500 MHz, CDCl3): δ 8.01 (s,
1H), 7.82−7.85 (m, 1H), 7.26−7.32 (m, 4H), 6.88−6.92 (m, 2H),
6.83 (t, J = 2.00 Hz, 1H), 6.69 (s, 2H), 5.33 (s, 2H), 2.32 (s, 6H).
LCMS found 362.97, [M + H]+.
1
5% EtOAc in hexanes to give 11b white solid (yield: 88%). H NMR
(500 MHz, CDCl3): δ ppm 7.31 (dt, J = 8.3, 2.0 Hz, 2H), 7.22−7.26
(m, 3H), 6.99 (d, J = 7.3 Hz, 1H), 6.93 (t, J = 2.0 Hz, 1H), 6.84 (dd, J
= 8.3, 2.4 Hz, 1H), 4.46 (s, 2H), 4.17 (t, J = 6.8 Hz, 2H), 3.08 (t, J =
6.8 Hz, 2H). GCMS found 323.9, [M+·]
2-(3-(4-Chloro-3,5-dimethylphenoxy)benzyl)-2H-1,2,3-tria-
1
zole (15f). Yield: 8%. H NMR (500 MHz, CDCl3): δ 7.63 (s, 2H),
I
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7.29 (dt, J = 2.44, 7.80 Hz, 1H), 6.99 (d, J = 7.80 Hz, 1H), 6.88−6.91
(m, 2H), 6.72 (s, 2H), 5.58 (s, 2H), 2.33 (s, 6H). LCMS found
314.00, [M + H]+.
1-(3-(4-Chloro-3-methylphenoxy)benzyl)-4-methylpipera-
zine (15g). Yield: 7%. 1H NMR (500 MHz, CDCl3): δ 7.23−7.28 (m,
1H), 7.06 (d, J = 7.30 Hz, 1H), 6.99 (br s, 1H), 6.85 (dt, J = 2.40, 7.30
Hz, 1H), 6.73 (s, 2H), 3.49 (s, 2H), 2.46 (br s, 8H), 2.34 (s, 6H), 2.28
(s, 3H). LCMS found 345.04, [M + H]+.
4-(3-(4-Chloro-3,5-dimethylphenoxy)benzyl)-4H-1,2,4-tria-
zole (15h). Yield: 3.1%. 1H NMR (500 MHz, CDCl3): δ 8.17 (s, 2H),
7.33 (t, J = 7.30 Hz, 1H), 6.95 (dd, J = 2.40, 7.30 Hz, 1H), 6.88 (d, J =
7.30 Hz, 1H), 6.81−6.83 (m, 1H), 6.73 (s, 2H), 5.14 (s, 2H), 2.35 (s,
6H). LCMS found 314.03, [M + H]+.
2-(3-(4-Chloro-3,5-dimethylphenoxy)phenyl)acetic Acid
1
(18b). Yield: 96%. H NMR (500 MHz, CDCl3): δ 7.29 (t, J = 7.80
Hz, 1H), 7.03 (d, J = 7.80 Hz, 1H), 6.93 (t, J = 1.00 Hz, 1H), 6.89 (dd,
J = 2.40, 8.30 Hz, 1H), 6.75 (s, 2H), 3.64 (s, 2H), 2.34 (s, 6H). LCMS
found 290.58, [M + H]+.
General Procedure for Compounds 19. The appropriate acid
18 (1.19 mmol) was dissolved in DMF (7 mL), and then
acetohydrazide (2.39 mmol), HOBT (1.789 mmol), and EDC (1.79
mmol) were added to the solution. The reaction was stirred at room
temperature for 5 h. The reaction was evaporated to dryness and
solubilized in DCM (20 mL) and H2O (20 mL). The organic layer was
removed, and the aqueous was extracted with DCM (2 × 20 mL). The
combined organic layers were washed with H2O (20 mL), washed with
brine (5 mL), and dried under anhydrous Na2SO4. The solvent was
removed in vacuo, and the residue was purified via silica gel
chromatography (2% MeOH in DCM) to provide the desired product.
N′-Acetyl-3-(4-chloro-3,5-dimethylphenoxy)benzohydrazide
1-(3-(4-Chloro-3,5-dimethylphenoxy)benzyl)-1H-indole
1
(15i). Yield: 3.6%. H NMR (500 MHz, CDCl3): δ 7.64 (d, J = 7.80
Hz, 1H), 7.25−7.28 (m, 1H), 7.23 (t, J = 7.80 Hz, 1H), 7.17 (t, J =
7.80 Hz, 1H), 7.13 (d, J = 3.40 Hz, 1H), 7.11 (d, J = 7.80 Hz, 1H),
6.80−6.85 (m, 2H), 6.76−6.78 (m, 1H), 6.68 (s, 2H), 6.54 (d, J = 3.40
Hz, 1H), 5.29 (s, 2H), 2.31 (s, 6H). LCMS found 362.07, [M + H]+.
2-(3-(4-Chloro-3,5-dimethylphenoxy)benzyl)-2H-indazole
1
(19a). Yield: 78%. H NMR (500 MHz, CDCl3): δ 9.78 (br s, 1H),
9.58 (br s, 1H), 7.51 (d, J = 8.05 Hz, 1H), 7.43 (t, J = 2.20 Hz, 1H),
7.32 (t, J = 8.05 Hz, 1H), 7.07−7.11 (m, 1H), 6.71 (s, 2H), 2.32 (s,
6H), 2.02−2.08 (m, 3H). LCMS found 333.03, [M + H]+.
1
(15j). Yield: 6%. H NMR (500 MHz, CDCl3): δ 7.92 (s, 1H), 7.72
N′-Acetyl-2-(3-(4-chloro-3,5-dimethylphenoxy)phenyl)-
(d, J = 8.80 Hz, 1H), 7.64 (d, J = 8.80 Hz, 1H), 7.29 (t, J = 7.80 Hz,
2H), 7.09 (s, 1H), 6.98 (d, J = 7.80 Hz, 1H), 6.89−6.92 (m, 2H), 6.71
(s, 2H), 5.57 (s, 2H), 2.31 (s, 6H). Regiochemistry confirmed by NOE
experiments (Supporting Information). LCMS found 362.99, [M +
H]+.
1
acetohydrazide (19b). Yield: 67.1%. H NMR (500 MHz, CDCl3):
δ 8.45−8.55 (br s, 1H), 8.01 (br s, 1H), 7.28 (t, J = 7.80 Hz, 1H), 7.02
(d, J = 7.80 Hz, 1H), 6.91 (t, J = 2.00 Hz, 1H), 6.88 (dd, J = 2.40, 8.80
Hz, 1H), 6.74 (s, 2H), 5.30 (s, 2H), 2.94−2.97 (m, 6H), 2.88 (s, 3H).
LCMS found 346.5, [M + H]+.
General Procedure for Compounds 20. The appropriate
hydrazide (0.120 mmol) was added to POCl3 (2.164 mmol), and
the reaction mixture was heated at reflux (110 °C) for 1 h. The
reaction mixture was evaporated to dryness, and the residue was
purified via preparative HPLC.
1-(3-(4-Chloro-3,5-dimethylphenoxy)benzyl)-1H-indazole
1
(15k). Yield: 3%. H NMR (500 MHz, CDCl3): δ 8.03 (s, 1H), 7.75
(d, J = 8.30 Hz, 1H), 7.33−7.35 (m, 2H), 7.23 (d, J = 7.81 Hz, 1H),
7.13−7.17 (m, 1H), 6.90 (d, J = 7.81 Hz, 1H), 6.84 (dd, J = 2.44, 7.80
Hz, 1H), 6.79−6.81 (m, 1H), 6.67 (s, 2H), 5.57 (s, 2H), 2.30 (s, 6H).
LCMS found 362.99, [M + H]+.
2-(3-(4-Chloro-3,5-dimethylbenzyl)phenyl)-5-methyl-1,3,4-
oxadiazole (20a). Yield: 5.3%. 1H NMR (500 MHz, CDCl3): δ 7.76
(d, J = 7.80 Hz, 1H), 7.60 (t, J = 1.00 Hz, 1H), 7.45 (t, J = 7.80 Hz,
1H), 7.14 (dd, J = 2.45, 8.30 Hz, 1H), 6.78 (s, 2H), 2.61 (s, 3H), 2.36
(s, 6H). LCMS found 313.01, [M + H]+.
General Procedure for Compounds 17. The appropriate ester
16 (2.325 mmol) was dissolved in 1,4-dioxane (20.0 mL), and then 4-
chloro-3,5-dimethylphenol (3.49 mmol), 2-(dimethylamino)acetic acid
(3.49 mmol), cesium carbonate (4.65 mmol), and copper(I) iodide
(1.16 mmol) were added to the solution. The reaction was refluxed in
a preheated oil bath at 180 °C. After 9 h, the crude was evaporated to
dryness and then dissolved in DCM (70 mL). The solution was
filtered on filter paper. The organic phase was washed with Na2CO3
5% solution (2 × 50 mL), and then aqueous layers were washed with
DCM (3 × 30 mL). The combined organic layers were washed with
brine, dried under anhydrous Na2SO4, and concentrated. The crude
product was purified via silica gel chromatography (9:1 hexane/
EtOAc) to give the desired product.
2-(3-(4-Chloro-3,5-dimethylphenoxy)benzyl)-5-methyl-
1
1,3,4-oxadiazole (20b). Yield: 2.2%. H NMR (400 MHz, CDCl3):
δ 7.28 (t, J = 8.05 Hz, 1H), 7.02 (d, J = 7.33 Hz, 1H), 6.92 (t, J = 2.20
Hz, 1H), 6.88 (dd, J = 2.20, 8.05 Hz, 1H), 6.74 (s, 2H), 4.12 (s, 2H),
2.48 (s, 3H), 2.34 (s, 6H). LCMS found 328.49, [M + H]+.
General Procedure for Compounds 21. To a solution of the
appropriate hydrazide (0.096 mmol) in 1,4-dioxane (2 mL) was added
Lawesson’s Reagent (0.288 mmol) portion-wise, and the reaction
mixture was heated at reflux (100 °C) for 2 h. The reaction was
evaporated to dryness and solubilized in DCM (20 mL). The solution
was washed with a saturated solution of NaHCO3 (2 × 20 mL) and
brine (1 × 10 mL). The organic layer was dried under anhydrous
Na2SO4. The solvent was removed in vacuo. Reaction mixture was
evaporated to dryness, and the residue was purified via preparative
HPLC.
Methyl 3-(4-Chloro-3,5-dimethylphenoxy)benzoate (17a).
1
Yield: 72%. H NMR (500 MHz, CDCl3): δ 7.78 (d, J = 7.80 Hz,
1H), 7.61−7.63 (m, 1H), 7.40 (t, J = 7.80 Hz, 1H), 7.18 (dd, J = 2.45,
8.30 Hz, 1H), 6.74 (s, 2H), 3.90 (s, 3H), 2.35 (s, 6H). LCMS found
291.0, [M + H]+.
Methyl 2-(3-(4-Chloro-3,5-dimethylphenoxy)phenyl)-
acetate. (17b). Yield: 31.5%. 1H NMR (500 MHz, CDCl3): δ
7.27−7.29 (m, 1H), 7.01 (d, J = 7.80 Hz, 1H), 6.91 (t, J = 1.95 Hz,
1H), 6.87 (dd, J = 1.95, 8.30 Hz, 1H), 6.75 (s, 2H), 3.70 (s, 2H),
3.59−3.62 (m, 3H), 2.34 (s, 6H). LCMS found 305.10, [M + H]+.
General Procedure for Compounds 18. To a solution of
appropriate ester (1.469 mmol) in THF (2 mL) was added lithium
hydroxide (2.94 mmol) in H2O (2 mL). The reaction was stirred at
room temperature for 2 h. The reaction was evaporated to dryness and
dissolved in H2O. The mixture was washed with EtOAc (30 mL), and
the aqueous was acidified to pH 2−3 with 3 M HCl (10 mL). Product
was extracted using EtOAc (4 × 20 mL), and the solution was dried
under anhydrous Na2SO4. The solvent was removed in vacuo to
provide the desired product, which was used without further
purification
2-(3-(4-Chloro-3,5-dimethylphenoxy)phenyl)-5-methyl-
1
1,3,4-thiadiazole (21a). Yield: 2.5%. H NMR (500 MHz, CDCl3):
δ 7.62 (dd, J = 0.98, 7.81 Hz, 1H), 7.57 (t, J = 2.40 Hz, 1H), 7.41 (t, J
= 8.30 Hz, 1H), 7.08 (dd, J = 2.40, 8.30 Hz, 1H), 6.78 (s, 2H), 2.81 (s,
3H), 2.36 (s, 6H). LCMS found 330.95, [M + H]+.
2-(3-(4-Chloro-3,5-dimethylphenoxy)benzyl)-5-methyl-
1
1,3,4-thiadiazole (21b). Yield: 6.0%. H NMR (500 MHz, CDCl3):
δ 7.28 (t, J = 8.05 Hz, 1H), 7.02 (d, J = 7.32 Hz, 1H), 6.91 (t, J = 2.20
Hz, 1H), 6.88 (dd, J = 2.20, 8.05 Hz, 1H), 6.73 (s, 2H), 4.35 (s, 2H),
2.71 (s, 3H), 2.34 (s, 6H). LCMS found 344.49, [M + H]+.
T. cruzi and Mammalian Cells Cultures. LLC-MK2 were used to
cultivate T. cruzi and NIH/3T3 for infection assay to test compounds
activity. Both cell lines were grown in DMEM supplemented with 10%
FBS, 100 U/ml penicillin, 0.1 mg/mL streptomycin, and 0.292 mg/mL
glutamine (Pen-Strep-Glut) at 37 °C and 5% CO2.
3-(4-Chloro-3,5-dimethylphenoxy)benzoic Acid (18a). Yield:
T. cruzi Tulahuen strain expressing the β-gal gene (clone C4)24 and
T. cruzi Y strain expressing the firefly Luciferase gene8 were grown on
LLC-MK2 cells in DMEM with 2% FBS and 1% Pen-Strep-Glut at 37
1
87%. H NMR (500 MHz, DMSO-d6): δ 7.69 (d, J = 7.8 Hz, 1H),
7.47 (t, J = 7.8 Hz, 1H), 7.42 (m, J = 1.5 Hz, 1H), 7.23 (dd, J = 7.6, 2.2
Hz, 1H), 6.87 (s, 2H), 2.30 (s, 6H). LCMS found 277.03, [M + H]+.
J
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Journal of Medicinal Chemistry
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°C and 5% CO2 and harvested every 5−7 days. Trypomastigotes in the
harvested medium were centrifuged for 7 min at 2500 rpm. To
separate from amastigotes and cellular debris, the trypomastigotes
were allowed to swim out of the pellet for at least 3 h at 37 °C.
T. cruzi in Vitro Inhibition Assay. Harvested trypomastigotes
were centrifuged and washed with DMEM without phenol red
supplemented with 2% FBS and Pen-Strep-Glut. Phenol red interferes
with absorbance readings at 590 nM and needs to be avoided. NIH/
3T3 host cells (50 000 per well) and purified T. cruzi trypomastigotes
(50 000 per well) were seeded in 96-well plates. Test compounds were
added at a maximum concentration of 50 μM. DMSO was always
lower than 0.5%, which does not affect the viability of the parasites.
Each determination was performed in duplicated wells. Amphotericin
B (Sigma-Aldrich) was used as control to obtain maximal inhibition at
a final concentration of 4 μM. Six wells of each negative and positive
controls were carried in every plate. The plates were incubated for 96 h
before processing. When the assay was performed with T. cruzi
expressing β-galactosidase,24 50 μL of PBS with 0.5% NP40 and 100
μM chlorophenol Red-β-D-galactoside (CPRG) (Sigma) were added
per well, plates were incubated at 37 °C for 4 h, and absorbance was
read at 590 nm. When the assay was performed with T. cruzi
expressing the firefly Luciferase gene,8 D-Luciferin potassium salt
dissolved in PBS was added at a final concentration of 150 μg/mL, and
luminescence was measured for 1 s right after addition. Both assays
were read using a Victor X3 Perkin-Elmer plate reader.
The absorbance or luminescence obtained was proportional to the
viability of the parasite. To determine EC50 values, the absorbance or
luminescence was converted to the activity anti-T. cruzi of the
compounds according to the formula: % activity = 100 − 100*[(CPD
− CPA)/(CP − CPA)], where CPD = cells + parasites + drug; CPA =
cells + parasites + amphotericin B; and CP = cells + parasites. The log
of the molar tested concentrations were plotted against the % activity
in the Graph Prism Software, and the value of EC50 was determined.
EC50 values are relative to the maximal inhibition values, which are
obtained when cultures are incubated with amphotericin B (4 μM).
These CPA values are significantly lower than the CP controls
(typically 7-fold lower), but are higher than the values of cells alone
(approximately 2-fold), because there is a background of extracellular
trypomastigotes that do not invade host cells and do not die over the 4
days of the assay.
Immunofluorescence Assay. NIH-3T3 cells plated on coverslips
were infected with either T. cruzi Tulahuen expressing β-galactosidase
or T. cruzi Y expressing luciferase and incubated with culture medium
alone or containing compound 4c or benznidazole at 5 times the value
of the EC50 obtained in the in vitro assay. After 3 days, they were fixed
with 4% of paraformaldehyde, rinsed with PBS, permeabilized for 15
min in PBS with 0.1% Triton X-100 (Sigma-Aldrich), and blocked for
20 min in PBS with 10% goat serum, 1% bovine serum albumin, 100
mM glycine, and 0.05% sodium azide. The cells were incubated for 1 h
at room temperature with a polyclonal rabbit anti-T. cruzi (kindly
provided by Dr. Barbara Burleigh, Harvard University, Boston) at
1:2000 dilution. After rinsing, they were incubated for 1 h at a 1:800
dilution with an Alexa Fluor 488 goat antirabbit IgG secondary
antibody (Molecular Probes, Invitrogen), followed by DAPI to stain
the nuclei. Coverslips were mounted on Mowiol and analyzed using an
inverted Olympus IX70 microscope with a 60× oil-immersion
objective.
T. cruzi in Vitro Reversibility Assay. This assay was performed
with T. cruzi Tulahuen expressing β-galactosidase24 and T. cruzi Y
strain expressing the firefly Luciferase gene.8 The same procedures for
the T. cruzi in vitro inhibition assay were followed, but compound 4c
or benznidazole was washed out from the well and replaced by fresh
culture medium at different time points (24, 48, and 72h) after
addition of the compounds. After 96 h of incubation, the specific
substrates for each strain of parasite were added to the wells, and
absorbance or luminescence was read using a Victor X3 Perkin-Elmer
plate reader. EC50 values were determined as previously described.
Testing T. cruzi CYP51 as a Potential Target for Inhibitor
Compounds. Compounds 4b, 4c, and 4h were studied as CYP51
ligand/inhibitors in vitro using purified full-length T. cruzi CYP51
protein sample. Binding affinities of the compounds were estimated
using spectral titration.17 The experiments were performed on a dual-
beam Shimadzu UV-240IPC spectrophotometer in the wavelength
range 350−450 nm at 25 °C in 2 mL tandem cuvettes at 2 μM P450
and ligand concentrations ranging from 0.5 to 10 μM. The apparent
dissociation constants were calculated by plotting the absorbance
changes in the difference spectra (ΔA425−390) upon titration against
free ligand concentration and fitting the data to a rectangular
hyperbola in Sigma Plot Statistics. Inhibitory potencies of the
compounds were compared in the reconstituted CYP51 reaction,25
5
min and 1 h, at 50/2/1 molar ratio substrate/inhibitor/enzyme.
Posaconazole served as positive control;18 P450 concentration in the
reaction mixture was 1 μM. The reaction products were analyzed on a
reverse-phase HPLC system (Waters) equipped with a C18Nova Pak
column and a β-RAM detector (INUS Systems, Inc.).20
X-ray Crystallography. Crystallographic studies were carried out
using the Δ31 N-terminus truncated expression construct of T. cruzi
CYP51 (MAKKT-P32).18 A 20 mM stock solution of 4c in DMSO was
added to the 300 μM solution of the protein in 20 mM K-phosphate
buffer, pH 7.2, containing 200 mM NaCl, 0.1 mM EDTA, 10%
glycerol, and 8 mM Cymal 4, to produce the final concentration of 600
μM. Co-crystals were obtained by hanging-drop vapor diffusion at 22
°C against a well solution containing 50 mM K-phosphate buffer, pH
7.6, 0.2 M magnesium chloride hexahydrate, and 25% (w/v) PEG
4000. Data were collected at LS-CAT, Advanced Photon Source,
Argonne National Laboratory, beamline 21ID-F, and processed with
HKL2000 software package. The structure was determined in CCP4
Program Suite 6.2.0. Solvent content was estimated with Matthews
probability calculator. A single solution with one protein monomer in
the asymmetric was found with PhaserMR using posaconazole-bound
T. cruzi CYP51 structure (PDB code 3K1O)18 as a search model, an
initial Rfactor of 0.44, and log-likelihood gain of 640. Model building and
refinement were performed with COOT and REFMAC5, respectively.
Table 5 summarizes the diffraction and refinement statistics; electron
density maps for the inhibitor area are presented in the Supporting
Information (Figure S3). The coordinates and structure factors of T.
cruzi CYP51 in complex with compound 4c (PDB ID NEE) have been
deposited at the RCSB Protein Data Bank, PDB code 4H6O.
ASSOCIATED CONTENT
* Supporting Information
A tabulation of all inhibitors and screening data is publically
■
S
a v a i l a b l e a s
a
s h a r e d d a t a s e t a t w w w .
collaborativedrugdiscovery.com. Synthetic details and charac-
terization of the compounds described, cross-correlation of the
manuscript compound numbers and NEU registration
numbers, and the amino acid sequence alignment of T. cruzi
CYP51 with selected fungal CYP51 orthologs. This material is
available free of charge via the Internet at http://pubs.acs.org.
Accession Codes
The coordinates and structure factors of T. cruzi CYP51 in
complex with compound 4c (PDB ID NEE) have been
deposited at the RCSB Protein Data Bank, PDB code 4H6O.
AUTHOR INFORMATION
Corresponding Author
■
*Phone: (617) 373-2703 (M.P.P.); (212) 263-6757 (A.R.);
(615) 343-1373 (G.I.L.). E-mail: m.pollastri@neu.edu
(M.P.P.); ana.rodriguez@nyumc.org (A.R.); galina.i.
lepesheva@vanderbilt.edu (G.I.L.).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
Funding from Northeastern University and the National
Institutes of Health for the medicinal chemistry
■
K
dx.doi.org/10.1021/jm400012e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
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(R01AI082577 to M.P.P.), T. cruzi CYP51, and X-ray
crystallography (NIH GM067871 to G.I.L.) is gratefully
acknowledged. We appreciate a free academic license to the
OpenEye suite of software.
ABBREVIATIONS USED
■
HTS, high-throughput screening; CYP51, cytochrome P450
sterol 14α-demethylase; SAR, structure−activity relationship;
T. cruzi, Trypanosoma cruzi; VNF, (4-(4- chlorophenyl)-n-[2-
(1h-imidazol-1-yl)-1-phenylethyl]benzamide
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