Discovery of Potent, Reversible, and Competitive Cruzain Inhibitors with Trypanocidal Activity: A Structure-Based Drug Design Approach

Article abstract of DOI:10.1021/acs.jcim.9b00802

A virtual screening conducted with nearly 4?000?000 compounds from lead-like and fragment-like subsets enabled the identification of a small-molecule inhibitor (1) of the Trypanosoma cruzi cruzain enzyme, a validated drug target for Chagas disease. Subsequent comprehensive structure-based drug design and structure-activity relationship studies led to the discovery of carbamoyl imidazoles as potent, reversible, and competitive cruzain inhibitors. The most potent carbamoyl imidazole inhibitor (45) exhibited high affinity with a K_i value of 20 nM, presenting both in vitro and in vivo activity against T. cruzi. Furthermore, the most promising compounds reduced parasite burden in vivo and showed no toxicity at a dose of 100 mg/kg. These carbamoyl imidazoles are structurally attractive, nonpeptidic, and easy to prepare and synthetically modify. Finally, these results further advance our understanding of the noncovalent mode of inhibition of this pharmaceutically relevant enzyme, building strong foundations for drug discovery efforts.

Full text of DOI:10.1021/acs.jcim.9b00802

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Pharmaceutical Modeling
Discovery of Potent, Reversible and Competitive Cruzain Inhibitors
with Trypanocidal Activity: A Structure-Based Drug Design Approach
Mariana L Souza, Celso de Oliveira Rezende Júnior, Rafaela Salgado Ferreira, Rocio M
Espinoza Chávez, Leonardo Luiz Gomes Ferreira, Brian W Slafer, Luma Godoy Magalhaes,
Renata Krogh, Glaucius Oliva, Fabio C Cruz, Luiz Dias, and Adriano Defini Andricopulo
J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.9b00802 • Publication Date (Web): 25 Nov 2019
Downloaded from pubs.acs.org on November 26, 2019
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Title
Discovery of Potent, Reversible and Competitive Cruzain Inhibitors with
Trypanocidal Activity: A Structure-Based Drug Design Approach
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Authors
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*
2*
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Mariana L. de Souza , Celso de Oliveira Rezende Junior , Rafaela S. Ferreira ,
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Rocio Marisol Espinoza Chávez , Leonardo L. G. Ferreira , Brian W. Slafer , Luma
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G. Magalhães , Renata Krogh , Glaucius Oliva , Fabio Cardoso Cruz , Luiz Carlos
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**
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Dias , Adriano D. Andricopulo
Affiliations
1Laboratory of Medicinal and Computational Chemistry, Physics Institute of Sao
Carlos, University of Sao Paulo, Sao Carlos – SP, 13563-120, Brazil
2Institute of Chemistry, State University of Campinas, Campinas – SP, 13084-
971, Brazil
3Department of Biochemistry and Immunology, Federal University of Minas Gerais,
Belo Horizonte – MG, 31270-901, Brazil
4Department of Pharmacology, Federal University of Sao Paulo, Sao Paulo – SP,
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4023-062, Brazil
*
These authors contributed equally to this work
**Correspondence: aandrico@ifsc.usp.br (A.D.A); ldias@iqm.unicamp.br (L.C.D)
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ABSTRACT
A virtual screening conducted with nearly 4,000,000 compounds from lead-like and
fragment-like subsets enabled the identification of a small-molecule inhibitor (1) of
the Trypanosoma cruzi cruzain enzyme, a validated drug target for Chagas disease.
Subsequent comprehensive structure-based drug design and structure-activity
relationship studies led to the discovery of carbamoyl imidazoles as potent, reversible
and competitive cruzain inhibitors. The most potent carbamoyl imidazole inhibitor
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(
45) exhibited high affinity with a K value of 20 nM, presenting both in vitro and in
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vivo activity against T. cruzi. Furthermore, the most promising compounds reduced
parasite burden in vivo and showed no toxicity at a dose of 100 mg/kg. These
carbamoyl imidazoles are structurally attractive, nonpeptidic, and easy to prepare and
synthetically modify. Finally, these results further advance our understanding of the
noncovalent mode of inhibition of this pharmaceutically relevant enzyme, building
strong foundations for drug discovery efforts.
Keywords: Chagas disease, virtual screening, organic synthesis, structure-activity
relationships, drug design, drug discovery, SBDD, neglected tropical diseases
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INTRODUCTION
Chagas disease is a major cause of death and heart failure in Latin America.1
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This neglected tropical disease (NTD) has also become a relevant public health
problem in nonendemic countries in North America and Europe and in the Western
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Pacific region. According to the World Health Organization (WHO), approximately 8
million people worldwide are infected with the protozoan Trypanosoma cruzi, the
etiological agent of Chagas disease. In addition, Chagas disease causes 10,000 deaths
annually, and more than 25 million people are at risk of infection.3
Chemotherapy for Chagas disease is obsolete; no therapeutic novelty has
entered clinical practice since the early 1970s. This is one of the most remarkable
examples of a lack of pharmaceutical innovation in an area. The only two available
drugs, nifurtimox and benznidazole, are nitroheterocyclic compounds that cause
severe adverse effects in up to 40% of patients and are effective only in the acute
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phase of the disease. For instance, nifurtimox was discontinued in Brazil, Argentina,
Chile and Uruguay because of its remarkable toxicity, which frequently causes
adverse effects such as anorexia, weight loss, psychiatric problems, insomnia, nausea,
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intestinal colic and diarrhea. Consequently, patients in the chronic stage of the
disease, which include those with chagasic cardiomyopathy, are completely deprived
of adequate treatments. Considering these shortcomings, developing safe and effective
drugs for Chagas disease has been headlined as an urgent need by the global scientific
community and international health agencies.5
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The enzyme cruzain (EC 3.4.22.51), the major cysteine protease from T. cruzi,
is expressed during the entire life cycle of the parasite and is essential to several
physiological processes, including nutrition, circumvention of the host immune
response, and invasion of host cells.6,7 Validation of cruzain as a pharmacological
target has relied on multiple studies on the molecular biology of T. cruzi along with
proof-of-concept investigations showing that initial cruzain inhibitors reduce parasite
load in murine models of Chagas disease.8,9 Following these findings, different
inhibitor classes have been discovered, comprising peptidic and nonpeptidic, and
covalent and noncovalent inhibitors. Some examples include vinyl sulfones,
fluoromethyl ketones, triazoles, pyrimidines, thiosemicarbazones, chalcones and
benzimidazoles.6,9-16 The vinyl sulfone K777, which binds irreversibly to the catalytic
thiol group of Cys25 through a Michael addition, is a high-affinity cruzain inhibitor
with remarkable in vivo trypanocidal activity.17,18 Although K777 yielded inspiring
results in preclinical tests, toxicity-related issues were raised and were associated with
the covalent mode of action of the inhibitor, preventing the candidate from
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progressing into advanced clinical development. The results of these investigations
along with the available X-ray structures of the enzyme bound to small-molecule
inhibitors have been fundamental to foster existing studies aimed toward the discovery
of novel inhibitor classes with a different mode of enzyme inhibition.
Structure-based drug design (SBDD) approaches have significantly contributed
to the generation of new hits covering diverse chemical spaces and to the optimization
of lead compounds for a variety of biologically relevant enzymes. In particular,
reversible inhibition is an effective means of modulating the enzymatic activity of
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relevant drug targets. For instance, competitive inhibitors specifically target the free
enzyme (E) to form a binary enzyme-inhibitor (E–I) complex, competing with the
substrate for binding. This phenomenon provides unique opportunities for enzyme
inhibitor drug discovery and the optimization of multiple important properties, such as
potency, affinity and selectivity.
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Considering the current context of Chagas disease drug discovery, the main
goal of this work was the design and optimization of novel, reversible and competitive
cruzain inhibitors with in vitro and in vivo anti-T. cruzi activity. This goal was
pursued by conducting a computational-experimental integrated approach involving
SBDD, organic synthesis, biochemical and biological in vitro assays and in vivo
evaluation. To achieve this general purpose, we settled the following specific goals: (i)
the development of a virtual screening workflow aimed at the selection of virtual hits
for further in vitro profiling; (ii) the use of molecular docking to the design of analogs
of virtual hits that proved to be active; (iii) the synthesis of the designed compounds;
(iv) the in vitro evaluation of the synthesized analogs against cruzain; (v) the
determination of the mechanism of action of the most potent compounds; (vi) the
evaluation of the in vitro trypanocidal activity and cytotoxicity of the most potent
cruzain inhibitors; (vii) and finally the evaluation of the in vivo acute toxicity and
efficacy of the most promising compounds. This work has a distinctive character
among the current literature6,9-16 since it describes the discovery of reversible and
competitive cruzain inhibitors with in vivo efficacy from a virtual screening effort
using a comprehensive combination of computational, synthetic and biological
approaches.
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MATERIALS AND METHODS
Virtual Screening and Molecular Docking
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The lead-like and fragment-like subsets containing 3,409,091 and 453,539
molecules, respectively, were prepared from SMILES files downloaded from ZINC
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(
University of California at San Francisco – UCSF). The X-ray cruzain structure
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(PDB 3KKU, 1.28 Å) was prepared by removing water molecules and ligands and
adding hydrogen atoms. Enrichment plots were generated using DOCK 3.5.54 and a
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set of 146 competitive cruzain inhibitors that were previously described. The best
curve, in which His162 and Cy25 were kept protonated and negatively charged,
respectively, yielded an enrichment factor of 40 at 1% of the database, corresponding
to an AUC of 0.9313 (see Figure S1 in the Supporting Information). In the SBVS
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using DOCK 3.5.54 (UCSF), the molecular surface was generated, and energy
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potential grids calculated using CHEMGRID and Delphi. Multiple pre-calculated
conformations of ligands were docked, and each pose was scored based on van der
Waals and electrostatic interactions and ligand desolvation. In the SBVS using GOLD
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.2 (Cambridge Crystallographic Data Centre – CCDC, Cambridge, UK) compounds
were docked applying the default parameters and GoldScore function. The binding
site was defined as a sphere with a 10 Å radius centered on the coordinates of the
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Cys25 sulfur atom. In the SBVS using the Surflex module of Sybyl 8.0 (Certara,
Princeton, NJ), the protomol was generated based on the crystallographic ligand. The
Surflex default scoring function was used to score the docking solutions.26
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Examination of the docking output from all used programs was carried out with
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Pymol (Schrödinger, New York, NY) and Chimera (UCSF) (see Figure S2 in the
Supporting Information for the detailed workflow of the SBVS). Virtual screening hits
were purchased from ENAMINE (Kiev, Ukraine).
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Cruzain Expression, Activation and Purification
Cruzain was expressed and purified using a modified version of a previously
published protocol.12 Escherichia Coli (SG13009 strain) harboring the pETM11
plasmid which contains the codifying sequence for cruzain was pre-inoculated in 150
mL of Luria Bertani (LB) medium supplemented with gentamicin (20 µg/mL) and
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kanamicin (50 µg/mL). The cultures were kept overnight at 37 C under agitation (200
rpm). Next, the cultures were added to a medium (1 L) consisting of triptone (10%),
yeast extract (5%), 25 mM NaHPO , 25 mM KH PO 50 mM NH Cl, 5 mM Na SO ,
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glycerol (0.5%), glucose (0.5%), lactose (0.2%), and 2 mM MgSO . The cultures were
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kept at 37 °C under agitation (200 rpm) until reaching an optical density (OD ) of
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.16, which was followed by incubation for 72 hours at 18 °C. Next, the cells were
harvested by centrifugation at 5000 rpm (30 minutes, 4 °C) and the pellet suspended
in 200 mL of lysis buffer (300 mM NaCl, 50 mM Tris, 10 mM imidazole, 1 mM
CaCl , 1 mM MgSO , pH 10). This was followed by sonication (12 cycles of 30
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seconds) and centrifugation at 9000 rpm (30 minutes, 4 C). The supernatant was
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incubated for 3 hours at 4 C with Ni-NTA Superflow resin (5 mL) (Qiagen, Hilden,
Germany) for subsequent Immobilized Metal Affinity Chromatography purification
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(
IMAC). The resin was washed with buffer (300 mM NaCl, 50 mM Tris, 10 mM
imidazole, pH 10). Next, the resin was washed with increasing concentrations of
imidazole (25, 50, 75, 100 and 250 mM). The fractions containing the protein (75 –
250 mM imidazole) were dialyzed in acetate buffer 0.1 M pH 5.5 and concentrated to
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.5 mg/mL in activation buffer (100 mM NaAc, 300 mM NaCl, 5 mM EDTA, 10 mM
DTT, pH 5.5). The N-terminal of cruzain was cleaved by autoproteolysis in the
activation buffer to produce the active enzyme. This process was monitored by the
cleavage of the substrate Z-Phe-Arg-AMC and confirmed by SDS PAGE (12%) gel
electrophoresis. Next, active cruzain was dialyzed in buffer (20 mM NaAc, 150 mM
NaCl, pH 7.2) and mixed with Thiopropyl Sepharose 6B resin (GE Healthcare Life
Sciences, Pittsburgh, PA) previously equilibrated with the same buffer, and stored
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overnight at 4 C under gentle agitation. The elution of the active protein was carried
out by washing the resin with 20 mM DTT buffer. The protein solution was filtrated
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using Amicon filter (Sigma-Aldrich, St. Louis, MO) and stored at − 80 C in buffer
(0.1 M NaAc, 0.000005% Triton X-100, pH 5.5).
Enzyme Kinetics
Cruzain activity was measured by monitoring the cleavage of the fluorogenic
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substrate Z-Phe-Arg-AMC as previously described. Assays were performed in 0.1 M
sodium acetate, 5 mM DTT and 0.01% Triton X-100, pH 5.5. Cruzain concentration
was 1 nM and substrate concentration was 5.0 µM (K = 1.6 µM). For K
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determination, several substrate concentrations were used. Assays were followed for
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00 seconds at 30 C in 96-well flat bottom black plates, and activity was calculated
based on initial rates compared to a DMSO control. A known reversible and
competitive cruzain inhibitor was used as a positive control in all enzyme assays.12
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Wavelengths of 355 nm and 460 nm were used for excitation and emission,
respectively. The IC50 values were determined by using at least six inhibitor
concentrations, each one evaluated in triplicate. To determine the mechanism of
inhibition, eight substrate concentrations (0.31 to 40 µM) were used in 2-fold
increments. K values were determined from Lineweaver-Burk plots generated in
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SigmaPlot 10.0 (Systat Software Inc., Erkrath, Germany). The assays for the
identification of the mechanism of inhibition were performed in triplicates in two
independent experiments. SigmaPlot was used to determine the IC and K values.
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Trypanosoma cruzi Culture Procedures
In vitro assays were performed using T. cruzi Tulahuen strain, provided by
Frederick S. Buckner (University of Washington, Seattle, WA), genetically
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engineered to express the E. coli β-galactosidase gene (lacZ). Epimastigotes were
grown in liver infusion tryptone (LIT) medium supplemented with 10% fetal calf
serum (FCS), penicillin and streptomycin. Metacyclogenesis to trypomastigotes was
induced by seeding epimastigotes in Grace’s Insect Medium (Sigma-Aldrich, St.
Louis, MO) supplemented with 10% FCS. After differentiation, metacyclic
trypomastigotes were harvested and transferred to test plates.
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Intracellular Trypanosoma cruzi Amastigote Assays
Intracellular amastigote assays were performed in 96-well tissue culture plates.
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HFF-1 human fibroblasts were seeded at 2 × 10 cells per well in DMEM medium (80
μL) without phenol red and incubated overnight at 37 °C in a 5% CO humidified
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atmosphere. In the next day, trypomastigotes were added at 1 × 10 cells per well.
After 24 hours, medium was removed to clear extracellular parasites, and 100 μL of
fresh DMEM medium were added to each well. Next, 50 μL of cruzain inhibitors were
added in serial dilutions to yield final concentrations of 0.1 – 100 μM. The compounds
were tested at seven concentrations in 3-fold dilutions, each concentration being tested
in duplicate in three independent experiments. The plates were incubated for 120
hours at 37 °C in a 5% CO humidified atmosphere. All plates included multiple wells
2
with infected fibroblasts without the addition of any inhibitor (negative controls) and
wells with BZ (Sigma-Aldrich, St. Louis, MO) as positive controls. After incubation,
5
0 μL of 1mM chlorophenol red β-D-galactopyranoside (CPRG, Sigma-Aldrich, St.
Louis, MO) and 0.1% Igepal CA-630 (Sigma-Aldrich, St. Louis, MO) were added to
each well and absorbance was measured at 570 nm. Data were transferred to
SigmaPlot to determine the IC values.
50
Cytotoxicity
Cytotoxicity was evaluated using HFF-1 human fibroblasts and the MTS
30
3
Tetrazolium assay (Promega, Madison, WI). Fibroblasts were seeded at 2 × 10 cells
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per well in DMEM medium (80 μL) supplemented with 10% FCS in 96-well plates
and incubated overnight at 37 °C in a 5% CO humidified atmosphere. In the next day,
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0 μL of cruzain inhibitors were added in serial dilutions to yield final concentrations
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of 0.1 –100 μM, and the plates were incubated for 72 hours. Compounds were tested
at seven concentrations in 2-fold dilutions, each dilution being tested in triplicate in
two independent experiments. After 72 hours, 20 µL of MTS were added to the wells.
After 4 hours of incubation, absorbance was measured at 490 nm to assess MTS
reduction by viable cells. All plates included multiple wells with viable fibroblasts
without the addition of any inhibitor (negative controls). Doxorubicin (Sigma-Aldrich,
St. Louis, MO) was used as positive control. Data were transferred to SigmaPlot to
determine the IC values.
5
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In vivo Assays
Pathogen-free female Swiss mice weighing 25 – 30 g, obtained from the animal
breeding facility of the University of Sao Paulo, were used in the in vivo toxicity and
efficacy studies. Groups of five mice were housed in Ventilife mini-isolators (Alesco,
Sao Paulo, Brazil) in rooms maintained at 23 ± 2 ºC. The animals were continuously
maintained on a light cycle (12/12 hours, lights off at 08:00 p.m.) with unrestricted
access to food and water. The experimental protocol was approved by the Ethics
Committee on the Use of Animals of the Sao Paulo Federal University (Protocol
number 5301080816) and was conducted according to the ethics principles of the
National Council for Animal Experimentation Control (CONCEA, Brazil).
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The following solutions were prepared for the in vivo assays: (i) vehicle: 0.9%
NaCl, 10% DMSO in distilled water; (ii) reference drug: BZ (Sigma-Aldrich, St.
Louis, MO), 0.9% NaCl, 10% DMSO in distilled water; (iii) compounds: cruzain
inhibitors 1 and 45, 0.9% NaCl, 10% DMSO in distilled water.
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In vivo toxicity (MTD) was determined by testing increasing doses of
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compounds on distinct groups of mice (2 animals per group). Non-infected Swiss
female mice were divided into 6 groups and treated via i.p. with four different doses
of each compound (75, 100, 150, and 300 mg/kg). Each mouse received a single dose
of the testing compounds. The animals were observed for seven days according to the
OECD guidelines, which include the following: (i) behavioral functions – alertness,
restlessness, irritability, and fearfulness; (ii) neurological activity – spontaneous
activity, reactivity, touch response, pain response, and gait; and (iii) autonomic
functions – defecation and urination. Animals that received vehicle solution (0.9%
NaCl, 10% DMSO solution) were used as the control group.
Bloodstream T. cruzi trypomastigotes (Y strain) collected from infected Swiss
mice were used to infect the animals used in the in vivo studies. This strain, originally
isolated from an acute-phase chagasic patient in 1950 in Sao Paulo, Brazil, is known
for producing high levels of parasitemia and killing 100% of the animals between
days 12 and 20 after infection.32
Four-week-old female Swiss mice (5 animals per group) were inoculated (i.p.)
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with 5 x 10 trypomastigotes. Parasitemia was monitored daily by counting the
number of motile parasites in 5 µL of blood drawn from the lateral tail vein as
described by Brener.33 At the fifth day after infection, mice showing positive
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parasitemia were randomly selected and treated (i.p.) with seven daily doses of BZ
and compounds 1 and 45 (100 mg/kg of body weight), and vehicle. The mortality rate
was expressed as the cumulative percentage of deaths during a period 15 days after
infection.
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Data were submitted to Repeated Measures ANOVA (Statistics Solutions,
Clearwater, FL). The ANOVA bifactorial module was used to correlate parasitemia
and treatment duration (parasitemia curve). The ANOVA monofactorial module was
used to analyze peak parasitemia. When the ANOVA statistics yielded significant
results (p-value < 0.05), F-test analyses were performed to determine the statistical
significance of intergroup comparisons. The survival rate curves were submitted to
Chi-square analyses.
RESULTS AND DISCUSSION
The present work employed an SBDD approach to identify a new chemically
diverse group of competitive cruzain inhibitors, combining experimental and
computational methods. Structure-based virtual screening (SBVS) studies were
performed with three different molecular docking algorithms and the top-scoring
compounds making the most relevant molecular intermolecular interactions, according
to the available cruzain-inhibitor X-ray structures, were selected (see Figure S2 in the
Supporting Information for the detailed SBVS workflow). Compounds containing
aromatic, hydrophobic or positively charged groups interacting with the S2 pocket in
the enzyme active site were prioritized. Additionally, previously described cruzain
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inhibitors have been shown to form hydrogen bonds with Gln19, Asp161 and Gly66,
which are located at the S1, S1’ and S3 subsites, respectively. Compounds interacting
with these residues were also privileged when selecting candidates for experimental
evaluation. Based on this rationale, the complete SBVS process led to the selection of
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1
4 lead-like and four fragment-like compounds (Figure 1A) for experimental
investigation.
Figure 1. Structure-based virtual screening (SBVS) hits. (A) Structures of the SBVS
hits and the corresponding percent inhibition at 100 µM against the cruzain enzyme.
(B) Structure of compound 1, which was identified in a SBVS as a novel, noncovalent
and nonpeptidic cruzain inhibitor. (C) Lineaweaver-Burk plot showing the
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competitive mechanism of inhibition of compound 1. Curves refer to the following
inhibitor concentrations: control – no inhibitor (); 0.3 µM (); 0.6 µM (); 1.25
µM () and 2.50 µM ().
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The eighteen SBVS hits were evaluated in enzyme kinetics assays for their
activity against cruzain. A fluorescence test relying on the cleavage rate of the
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fluorogenic substrate Z-Phe-Arg-AMC was used. The most potent inhibitor from the
fragment-like library was compound 1 (ZINC3267510, Figure 1B), which had an IC50
value of 1 µM. Compound 1 proved to be a competitive and reversible inhibitor of
cruzain (K = 120 nM), as demonstrated in the Lineaweaver-Burk plot depicted in
i
Figure 1C. The activity of compound 1 on cruzain was neither time dependent nor
3
5
detergent sensitive. Given these results, inhibitor 1 was tested for its in vitro activity
against T. cruzi (Tulahuen LacZ), yielding an IC of 1.1 µM, which is 3-fold better
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than that of the standard drug benznidazole (BZ, IC = 3.3 µM, Table 5). In addition,
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compound 1 features a ligand efficiency (LE)36,37 of 0.53 kcal·mol /non-H atom,
−1
which is a suitable value for fragment-based ligand optimization.
Considering the gathered data, cruzain inhibitor 1 was used as the lead for
molecular optimization and structure–activity relationship (SAR) studies. A series of
analogs were designed considering the predicted binding mode and intermolecular
interactions between 1 and the binding site of cruzain (Figure 2A). The phenyl ring of
compound 1 was observed to interact with the S2 subsite, which is known for
accommodating hydrophobic groups. The urea nitrogen acted as a hydrogen bond
donor to the carbonyl of Asp161, and the urea oxygen formed a hydrogen bond with
the sidechain of Gln19. Another hydrogen bond occurred between the imidazole in 1
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and the indole nitrogen of Trp184. A π stacking interaction was observed between
imidazole rings of the inhibitor and His162. These observations led to the splitting of
1
into three fragments as the spots for structural modification, namely, the phenyl, the
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linker between the rings, and the imidazole (Figure 2B). In the next subsection, we
describe the organic synthesis efforts, which involved the synthesis of virtual
screening hit 1 and its analogs.
Figure 2. Cruzain inhibitor 1 identified in the structure-based virtual screening
(SBVS). (A) Predicted binding mode of inhibitor 1 in the active site of cruzain (PDB
3KKU, 1.28 Å). Cruzain is depicted in cartoon and surface representations. Cruzain
amino acid residues (carbon in white) and 1 (carbon in green) are illustrated as sticks.
Subsites in the cruzain binding site are labeled S1, S1’, S2 and S3. Intermolecular
interactions are indicated as dashed lines. (B) Structure of 1 highlighting the splitting
of the compound into three fragments as a strategy for molecular optimization.
Organic Synthesis
Synthesis of the virtual screening hit 1 and the analogs having different
substituents at the phenyl ring are shown in Scheme 1. Hydrochloride salts 15-27 and
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3-44 were prepared from the corresponding substituted phenols 2-14 and 40-41,
respectively, by an S 2 reaction using alkyl tosylate 42, followed by Boc deprotection
N
under acidic conditions. Subsequent reaction of these salts with carbonyldiimidazole
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(CDI) led to the formation of the target substituted imidazoles 1, 28-39, and 45-46.
Pyridyl analog 50 was prepared by an S Ar reaction between dibromopyridine 47 and
N
alcohol 48, followed by Boc deprotection and acylation using CDI (Scheme 1).
Scheme 1. Synthesis of substituted imidazoles 1, 28-39, 45-46 and 50a
a
Reagents and conditions: (a) 42, K CO , DMF, 60 °C, 6-10 h; (b) HCl (4 M in
2
3
dioxane), DCM, rt, 2-7 h; (c) CDI, DMF, rt, 3-8 h; (d) I) 48, NaH, THF, 0 °C to rt, 20
min; II) 47, rt, 29 h.
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Modifications on the linker between the aromatic rings of compound 1 were
also explored. Replacement of the oxygen by nitrogen (NH) was not possible because
the NH group attacks the activated carbonyl, leading to cyclic urea 53 instead of the
desired product 52 (Scheme 2). To block this undesired cyclization reaction, an N-
methyl (NMe) group, instead of an NH, was used to replace the oxygen in the linker
fragment, leading to the formation of 56 (Scheme 3).
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Scheme 2. Synthesis of compound 53a
a
Reagents and conditions: (a) 42, K CO , DMF, 18 h; (b) HCl (4M in dioxane), DCM,
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rt, 1 h; (c) CDI, DMF, rt, 3 h.
Scheme 3. Synthesis of amine 56a
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aReagents and conditions: (a) p-formaldehyde, MeONa, MeOH, 65 °C, 1 h; (b)
NaBH , 65 °C, 1.5 h; (c) I) NaH, THF, 0 °C to rt, 0.5 h; II) 42, 0 °C to rt, 5 h; (d) HCl
4
(4M in dioxane), DCM, rt, 3 h; (e) CDI, DMF, rt, 3 h.
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Methylaniline 54 was prepared from m-toluidine by reductive amination. This
reaction intermediate was then alkylated using tosylate 42, followed by Boc
deprotection and acylation with CDI to afford 56 (Scheme 3). Ester and amide
derivatives 60 and 61 were prepared from the corresponding phenol and aniline by
esterification and amidation, respectively, using carboxylic acid 57, followed by Boc
deprotection and acylation with CDI (Scheme 4).
Scheme 4. Synthesis of esters 60 and 63 and amide 61a
aReagents and conditions: (a) 57, EDC, DMAP for 60, and HOBt for 61, DCM, rt, 2-4
h; (b) HCl (4M in dioxane), DCM, rt, 3 h; (c) CDI, DMF, rt, 3 h; (d) BnOH, p-TsOH,
toluene, 100 °C, 18 h.
Benzylic ester 63 was prepared from glycine by esterification using benzyl
alcohol and p-TsOH, followed by acylation with CDI (Scheme 4). Esters 68-71 were
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prepared from the carboxylic acids of benzene, pyrazole, furan, and naphthalene,
respectively (Scheme 5). The corresponding carboxylic acids were treated with
alcohol 48, followed by Boc deprotection and acylation using CDI.
1
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Scheme 5. Synthesis of esters 68-71a
aReagents and conditions: (a) 48, EDC, DMAP, DCM, rt, 3-4 h; (b) HCl (4M in
dioxane), DCM, rt, 3-5 h; (c) CDI, DMF, rt, 5-6 h.
To probe the relevance of the imidazole group present in virtual screening hit 1
for activity toward cruzain, structurally related heterocyclic derivatives were prepared
(Scheme 6). Treatment of anilines 72 and 73 with bromoacetyl bromide under
Schotten‒Baumann conditions followed by displacement of the bromide with sodium
azide afforded azides 74 and 75. Reduction of the azide group using Pd/C and H2,
followed by coupling with 5-pyrimidinecarboxylic acid, nicotinic acid, and 1H-
imidazole-5-carboxylic acid led to amides 76-79. Pyrimidine, pyridine, and imidazole
derivatives 80-82 were prepared from ammonium hydrochloride salt 15 by amidation
with the corresponding carboxylic acid (Scheme 6). Amide 84 was prepared from an
2
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SN2 reaction between m-cresol and ethyl 2-bromoacetate followed by basic hydrolysis
of the ethyl ester and acylation with CDI. Ureas 85-92 were prepared from 1 by a
carbonyl substitution reaction using different amines (Scheme 7). See the Supporting
Information for additional data on the synthesis of the compounds.
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8
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1
2
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4
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6
7
8
9
0
Scheme 6. Synthesis of heterocyclic derivatives 76-82a
a
Reagents and conditions: (a) bromoacetyl bromide, EtOAc, NaHCO , 0 °C to rt, 1-3
3
h; (b) sodium azide, DMF, rt, 18-48 h; (c) H , 5% Pd/C, MeOH, rt, 1-2 h; (d)
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carboxylic acid, EDC, DMF, rt, 18 h; (e) carboxylic acid, EDC, HOBt, Et N, DMF, rt,
3
15-18 h.
1
1
1
1
1
1
1
1
1
1
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2
2
2
2
2
2
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2
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0
1
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5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 7. Synthesis of amide 84 and ureas 85-92a
a
Reagents and conditions: (a) ethyl 2-bromoacetate, K CO , DMF, rt, 3 h; (b) I) NaOH
2
3
(
6M), MeOH, rt, 10 min; II) HCl (6M), 0°C, 10 min; (c) CDI, CH CN, rt, 8 h; (d)
3
Amine, NaH for 85 and 86, DMF, rt or 100°C, 6-14 h.
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1
1
1
1
1
1
1
1
1
1
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5
6
Structure-Activity Relationships and the Discovery of Cruzain Inhibitors
with in vitro Trypanocidal Activity
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1
2
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0
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9
0
Modification of the phenyl ring of inhibitor 1 was explored by adding diverse
substituents to it and by expanding it to a naphthyl, leading to compounds 28-39, 45,
46 and 50. Some of these analogs showed increased potency toward cruzain (1.5- to
8
.3-fold) compared to 1 (Table 1). Otherwise, inhibitors 28, 32, 35-37, 39 and 50
proved to be less active, with the potency loss ranging from 1.7- to 3.1-fold.
The data in Table 1 indicate that modifying the phenyl ring, which interacts
with the S2 subsite in the cruzain binding cavity, is a worthwhile strategy to increase
the potency against the enzyme. Replacing the m-methyl group on the phenyl ring
with a chlorine or bromine improved potency as observed for compounds 29 and 30
(
IC = 0.57 and 0.41 µM, respectively). Compound 31 (IC = 0.36 µM), which has
5
0
50
an iodine replacing the methyl, was approximately 3-fold more potent than 1. Among
compounds having a halogen at the meta position, m-fluorophenyl derivative 28
exhibited a moderate decrease in potency, showing an IC value of 1.7 µM.
50
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Table 1. Structure and cruzain inhibitory activity of analogs 28-39, 45, 46 and 50
having phenyl group modifications (compound 1 as reference).
N
H
N
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
1
O
% Inhibitiona
b
Compound
Structure
IC (µM)
5
0
N
H
N
O
N
2
8
100
1.7 ± 0.2
F
Cl
Br
I
O
N
H
N
O
N
N
29
100
100
100
100
100
100
100
0.57 ± 0.03
0.41 ± 0.02
0.36 ± 0.07
2.5 ± 0.5
O
N
H
N
O
3
3
3
3
0
1
2
3
O
N
H
N
O
N
O
N
N
N
N
H
N
N
N
N
N
O
O
O
O
O
O
O
O
O
H
N
0.5 ± 0.1
O N
2
H
N
34
0.50 ± 0.08
1.80 ± 0.01
F3C
NC
H
N
3
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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N
H
N
N
36
37
100
100
2.8 ± 0.6
3.1 ± 0.9
O
Br
O
Br
N
0
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3
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9
0
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0
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2
3
4
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9
0
H
N
N
O
O
O
F3C
N
H
N
38
N
100
0.60 ± 0.06
O
F
F
N
H
39
45
N
N
100
100
100
1.8 ± 0.3
0.12 ± 0.02
0.66 ± 0.01
O
O
N
H
N
N
O
Br
O
N
H
N
N
4
5
6
0
O
O
N
H
N
N
100
100
3.0 ± 0.2
Br
N
O
O
H
H
N
N
3c
0.21 ± 0.01
O
9
Br
O
N
aThe percent inhibition values are the average of three measurements (inhibitor
b
concentration = 100 µM). The IC values were determined by obtaining rate
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c
measurements in triplicate for at least six inhibitor concentrations. Positive control.
The values represent the mean of three individual experiments.
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These results revealed that the greater the atomic radius of the halogen
substituent at the meta position, the higher the activity. This correlation indicates that
bulkier groups at this position promote a better interaction between the phenyl ring
and the S2 subsite, as observed in the molecular modeling studies. Adding a bromine
at the ortho or para position resulted in a potency loss of approximately 3-fold, as
observed for compounds 36 and 37 (IC = 2.8 and 3.1 µM, respectively). Likewise,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
3
3
4
4
4
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4
4
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5
5
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5
5
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0
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0
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0
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9
0
5
0
introducing a meta- and para-fluorine led to a moderate potency loss, as noted for
inhibitor 39 (IC = 1.8 µM). Similarly, attaching a nitrile group led to a moderate
5
0
potency reduction (35, IC = 1.80 µM), and adding a methoxy decreased activity by
5
0
2
.5-fold (32, IC = 2.5 µM). Otherwise, adding electron-withdrawing groups such as
50
a nitro group at the meta position (33, IC = 0.5 µM) and trifluoromethyl at the meta
5
0
(
34, IC = 0.5 µM) and para (38, IC = 0.6 µM) positions led to more potent
50
50
inhibitors. Replacing the phenyl with a 2-bromopyridyl, a less hydrophobic fragment
(
50, IC = 3.0 µM) led to a 3-fold less active ligand.
50
In general, introducing bulkier substituents at the phenyl moiety resulted in
more potent inhibitors. Naphthyl derivative 46, yielding an IC value of 0.66 µM,
50
was nearly 1.5-fold more potent than 1. The best compound in this initial series,
bromonaphthyl 45 (IC = 0.12 µM), showed an activity increment of approximately
50
8
-fold over 1. As shown in the molecular docking studies, the bromonaphthyl
occupies the same binding site region as the m-tolyl of 1 (Figure 3); this phenomenon
reinforces the argument that bulkier groups capable of interacting more extensively
with the S2 subsite are able to improve the activity of cruzain inhibitors. These
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findings are consistent with the results reported in our previous publication on
cruzain.12
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Figure 3. Binding modes of inhibitors 1 and 45. Cruzain is depicted in cartoon and
surface representations (PDB 3KKU, 1.28 Å). Cruzain amino acid residues (carbon in
blue) and inhibitors 1 (carbon in green) and 45 (carbon in yellow) are illustrated as
sticks. Subsites in the cruzain binding site are labeled S1, S1’, S2 and S3.
Intermolecular interactions are indicated as dashed lines.
Further molecular modifications focused on the linker between the phenyl and
imidazole rings (Table 2). Thus, seven compounds were synthesized bearing
modifications to this fragment, particularly by introducing amine, amide, ester, methyl
and piperazine groups. Tertiary amine 56 showed a potency increase of approximately
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2
-fold (IC = 0.52 µM) over compound 1. Inhibitor 60 (IC = 2.0 µM), having an
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0
50
ester group on the linker, showed a 2-fold potency decrease. Amide 61 displayed a
more pronounced activity decline, having an IC value of 6.2 µM. Compound 84
5
0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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proved to be inactive, which can be related to the shortening of the linker and the
resulting loss of the key interactions, such as the hydrogen bond from the urea
fragment of 1, supporting the activity of the inhibitors. The benzyl derivative 63 (IC50
=
0.6 µM), having a methylene and a carbonyl added to the linker and the methyl
removed from the phenyl, showed an improved inhibition profile over 1. This
enhanced activity could be related to the introduction of the methylene, which
projected the phenyl group more deeply into the S2 subsite, compensating for the loss
of the m-methyl. The introduction of a rotatable bond by adding a methylene in the
linker of 63, which consequently placed the phenyl deeper into S2, reinforced the
rationale that optimizing the steric complementarity with S2 is favorable for activity.
In addition, the added carbonyl was observed to act as a hydrogen bond acceptor for
the main chain nitrogen of Gly66, enhancing the interaction with the S3 subsite
(Figure 4).
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