Mapping the s1 and s1’ subsites of cysteine proteases with new dipeptidyl nitrile inhibitors as trypanocidal agents

Article abstract of DOI:10.1371/journal.pntd.0007755

The cysteine protease cruzipain is considered to be a validated target for therapeutic intervention in the treatment of Chagas disease. A series of 26 new compounds were designed, synthesized, and tested against the recombinant cruzain (Cz) to map its S1/S1′ subsites. The same series was evaluated on a panel of four human cysteine proteases (CatB, CatK, CatL, CatS) and Leishmania mexicana CPB, which is a potential target for the treatment of cutaneous leishmaniasis. The synthesized compounds are dipeptidyl nitriles designed based on the most promising combinations of different moieties in P1 (ten), P2 (six), and P3 (four different building blocks). Eight compounds exhibited a K_i smaller than 20.0 nM for Cz, whereas three compounds met these criteria for LmCPB. Three inhibitors had an EC₅₀ value of ca. 4.0 μM, thus being equipotent to benznidazole according to the antitrypanosomal effects. Our mapping approach and the respective structure-activity relationships provide insights into the specific ligand-target interactions for therapeutically relevant cysteine proteases.

Full text of DOI:10.1371/journal.pntd.0007755

RESEARCH ARTICLE
Mapping the S1 and S1’ subsites of cysteine
proteases with new dipeptidyl nitrile
inhibitors as trypanocidal agents
Lorenzo Cianni1,2,3,4, Carina Lemke2, Erik Gilberg2,3, Christian Feldmann3,
Fabiana Rosini1, Fernanda dos Reis Rocho1, Jean F. R. Ribeiro1, Daiane Y. Tezuka1,5
,
6
Carla D. Lopes , Sergio de Albuquerque 6, Ju¨rgen Bajorath3, Stefan Laufer4,
´
ID
1
ID
Andrei Leitão 1, Michael Gu¨tschow 2*, Carlos A. Montanari
*
ID
ID
1 Medicinal Chemistry Group, Institute of Chemistry of São Carlos, University of São Paulo, São Carlos, São
Paulo, Brazil, 2 Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, Bonn, Germany,
3 Department of Life Science Informatics, B-IT, LIMES Program Unit Chemical Biology and Medicinal
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¨
Chemistry, Rheinische Friedrich-Wilhelms-Universitat, Bonn, Germany, 4 Department of Pharmaceutical/
Medicinal Chemistry, Eberhard Karls University Tu¨bingen, Tu¨bingen, Germany, 5 Ribeirão Preto School of
Pharmaceutical Sciences, University of São Paulo, Ribeirão Preto, São Paulo, Brazil, 6 Department of
Clinical Toxicological and Bromatological Analysis School of Pharmaceutical Sciences of Ribeirão Preto,
University of São Paulo (USP), Ribeirão Preto, São Paulo, Brazil
* guetschow@uni-bonn.de (MG); carlos.montanari@usp.br (CAM)
OPEN ACCESS
Citation: Cianni L, Lemke C, Gilberg E, Feldmann C,
Rosini F, Rocho FdR, et al. (2020) Mapping the S1
and S1’ subsites of cysteine proteases with new
dipeptidyl nitrile inhibitors as trypanocidal agents.
PLoS Negl Trop Dis 14(3): e0007755. https://doi.
org/10.1371/journal.pntd.0007755
Abstract
The cysteine protease cruzipain is considered to be a validated target for therapeutic
intervention in the treatment of Chagas disease. A series of 26 new compounds were
designed, synthesized, and tested against the recombinant cruzain (Cz) to map its S1/S1´
subsites. The same series was evaluated on a panel of four human cysteine proteases
(CatB, CatK, CatL, CatS) and Leishmania mexicana CPB, which is a potential target for
the treatment of cutaneous leishmaniasis. The synthesized compounds are dipeptidyl
nitriles designed based on the most promising combinations of different moieties in P1
(ten), P2 (six), and P3 (four different building blocks). Eight compounds exhibited a Ki
smaller than 20.0 nM for Cz, whereas three compounds met these criteria for LmCPB.
Three inhibitors had an EC50 value of ca. 4.0 μM, thus being equipotent to benznidazole
according to the antitrypanosomal effects. Our mapping approach and the respective
structure-activity relationships provide insights into the specific ligand-target interactions
for therapeutically relevant cysteine proteases.
Editor: Igor C. Almeida, University of Texas at El
Paso, UNITED STATES
Received: August 29, 2019
Accepted: January 30, 2020
Published: March 12, 2020
Copyright: © 2020 Cianni et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Information files.
Author summary
Funding: We are indebted to Fundac¸ão de Amparo
à Pesquisa do Estado de São Paulo – FAPESP
(grant #2013/18009-4, grant #2016/07946-5 and
grant #2017/17386-0) for financing this project.
We also want to acknowledge the National Council
for Scientific and Technological Development
(CNPq, grant # 304030/2018-0) in Brazil for
scholarships. We would also like to thank the
Despite many achievements in identifying novel agents for the treatment of tropical and
neglected diseases, further research continues to be of fundamental importance. Our
research groups have been using the cruzipain cysteine protease in its recombinant form,
cruzain (Cz), to identify new trypanocidal agents. Considering the possible interchange-
ability with other cysteine proteases, the same series of dipeptidyl nitriles was tested in
Leishmania mexicana LmCPB. Other potential targets for such inhibitors are human
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New cysteine protease inhibitors as trypanocidal agents
CAPES Drug Discovery program (Process #139/
2015) that allowed us to evaluate LmCPB
inhibitors. The funders had no role in study design,
data collection and analysis, decision to publish, or
preparation of the manuscript.
cysteine cathepsins, which are involved in different disease states. Thus, the inhibitors
were also tested against cathepsins B, L, K, and S. Our results demonstrate that appropri-
ate structural modifications of dipeptidyl nitriles can lead to inhibition of these cysteine
proteases. It was also possible to identify trypanocidal agents, equipotent to benznidazole,
the current drug of choice used for the treatment of Chagas disease.
Competing interests: The authors have declared
that no competing interests exist.
Introduction
Chagas disease, aka American trypanosomiasis, is a severe health and social problem in Latin
America and in new non-endemic areas such as Japan, East Europe, and the USA. Chagas dis-
ease has an annual incidence of 30,000 new cases and 10,000 deaths per year. An estimated 8
million people are infected with Chagas disease worldwide and more than 25 million people
risk acquiring the disease [1].
The etiological agent, the protozoa parasite Trypanosoma cruzi (T. cruzi), is transmitted by
blood-sucking reduviid bugs of the subfamily Triatominae [2]. The only two existing drugs
in the market, benznidazole and nifurtimox, show strong side effects and inefficiency in the
chronic stage of the disease [3,4]. New safe and efficacious drugs are therefore required to
address with these still unmet medical needs. Initiatives such as the one launched by the
Drugs for Neglected Diseases (DNDi) have led to worldwide collaborative efforts to discover
new therapeutic targets [5]. Cruzain (Cz), a recombinant form of the enzyme cruzipain (EC
3.4.22.51) [6], is the most abundant cysteine protease (CP) present in the parasite and essential
for its development and survival inside and outside the host cell in all forms of its life cycle.
This makes Cz a druggable target for the development of new chemotherapeutic agents against
Chagas disease [7,8].
Cz represents a target for irreversible (or suicide) and reversible inhibitors. K777 was at the
forefront of the first generation of irreversible Cz inhibitors and initially characterized by the
Sandler Center for Research in Tropical Parasitic Disease (University of California, San Fran-
cisco) [9]. Despite its ability to rescue mice of a lethal experimental T. cruzi infection and
reduce parasite growth in dogs, preclinical safety and toxicology studies revealed substantial
side effects of K777 in primates and dogs, even when administered in low doses [10,11]. Cur-
rent research is being focused on reversible Cz inhibitors, as these are assumed to overcome
possible off-target effects [12]. Drug repurposing programs of different pharmaceutical com-
panies have recently put forward reversible inhibitors of Cz as potential drug candidates [13].
Two key compounds, Cz007 and Cz008, exhibited antiparasitic behavior in the low micro-
molar range for the amastigote form of the parasite and they were capable of curing T. cruzi
acute form infection in an in vivo model. Both compounds were orally active, well-tolerated
and worked most effectively at doses of 3 mg/kg [13].
The structure of Cz is closely related to those of mammalian CPs (CatL, CatK and CatS).
Three-dimensional (3D) Cz structures with a variety of ligands have already been resolved
[14]. The target-based molecular design was applied to generate the first series of dipeptidyl
nitrile ligands as reversible Cz inhibitors with trypanocidal activity [14]. In previous work,
some of us reported on low micro-molar inhibitors of Cz as modest trypanocidal agents for
the amastigotes form of the T. cruzi Tulahuen strain (pEC50 around 4). For the most potent
anti-parasite inhibitor with an EC50 value of 28 μM, the activity was not attributed to the gen-
eral cytotoxicity of the compound [14]. More recently, the feasibility of achieving a higher Cz
affinity by benefiting from the S3 structural singularity was underlined [15].
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New cysteine protease inhibitors as trypanocidal agents
In this study, we designed a new, structurally expanded series of 26 Cz-inhibiting dipeptidyl
nitriles, in particular by leveraging the P1-S1/S1‘interactions. We explored the structure-
activity relationships (SARs), mapped the active site of the target enzyme and evaluated the
antichagasic properties of the compounds. Besides that, we have tested them against four
human cysteine cathepsins (CatB, CatL, CatK, CatS) all of which constituting important tar-
gets for human diseases [16], and against the cysteine protease LmCPB, a novel macromolecu-
lar target to fight Leishmania mexicana. As a result of this study, several new low-nanomolar
inhibitors of different CPs were discovered and three new anti-T. cruzi agents, equipotent to
benznidazole, were characterized.
Methods
Modeling
Putative binding modes of novel dipeptidyl nitrile inhibitors compounds were derived from
the crystal structure of N-(2-aminoethyl)-alpha-benzoyl-l-phenylalaninamide (33L) bound to
Cz (PDB ID: 4QH6). This ligand-target-complex served as a template for knowledge-based
modeling and was preprocessed using the “Structure Preparation” and “Protonate3D”-tools of
the modeling software “Molecular Operating Environment” (MOE) [17], version 2018.0101,
with default settings. By modification of moieties, the cocrystallized ligand was structurally
transformed into the compound of interest. Obtained conformations were optimized using
the force field “Amber10:EHT”.
Synthetic chemistry
General considerations. The synthesis was performed as summarized in Figs 1 and 2.
Melting points were determined on a Bu¨chi 510 oil bath apparatus and are uncorrected. Infra-
red spectra were obtained from FT-IR Thermo Scientific Nicolet 380. Reagents, starting materi-
als and solvents were of commercial quality and were used without further purification unless
otherwise stated. All syntheses were started with enantiopure amino acids. TLC analysis was
carried out on Merck 60 F254 silica gel plates and visualized under UV light at 254 nm and 365
nm or by using a ninhydrin staining solution. Preparative column chromatography was carried
out on Grace Davison Davisil LC60A 20–45 micron or Merck Geduran Si60 63–200 micron sil-
ica using the Interchim PuriFlash 430 automated flash chromatography system. The purity of
all tested compounds was determined with one of the three protocols (A-C) noted below.
A). Purity was determined via RP-HPLC on a Hewlett Packard 1090 Series II LC with a
Phenomenex Luna C18 column (150 x 4.6 mm, 5 μm) and detection was performed by
a UV DAD (200–440 nm). Elution was carried out with the following gradient: 0.01 M
KH2PO4, pH 2.30 (solvent A), MeOH (solvent B), 40% B to 85% B in 8 min, 85% B for 5
min, 85% to 40% B in 1 min, 40% B for 2 min, stop time 16 min, flow 1.5 mL/min.
B). Purity was determined using an LC-MS instrument (ABSCIEX API 2000 LC-MS/MS,
HPLC Agilent 1100) with a Phenomenex Luna C18 HPLC column (50 x 2.00 mm, 3 μm)
and detection was performed by a UV DAD (200–440 nm). Elution was carried out with the
following gradient: 0.02 M NH4CH3CO2, pH 7.0 (solvent A), MeOH (solvent B) start with
100%, 10% B in 20 min to 100% B, 10 min 100% B, stop time 20 min, flow 0.25 mL/min.
C). Purity was determined with an LC-MS instrument (AmaZon SL ESI-MS, Shimadzu LC)
with a cellulose-2 Phenomenex column (250 x 4.6 mm, 5 μm) or a Diacel column (IC-
chiralpak, 250 x 4.6 mm, 5 μm). Isocratic elution with MeCN and water was applied as
specified, stop time 60 min, flow 0.5 mL/min.
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New cysteine protease inhibitors as trypanocidal agents
Fig 1. General synthesis of compounds 6–19. Reagents and conditions: a) HATU, DIPEA, 1-amino-
1-cyclopropanecarbonitrile, DMF, rt, 18 h; b) formic acid, rt, 18 h; c) HATU or TBTU, DIPEA, carboxylic acid, DMF,
rt, 18 h; d) DDQ, CH2Cl2, rt, 3–5 days.
https://doi.org/10.1371/journal.pntd.0007755.g001
NMR spectra were recorded on Bruker Avance 200 MHz, Bruker Avance 400 MHz, and
Bruker Avance DRX 500 MHz NMR spectrometers. Chemical shifts are reported in ppm rela-
tive to TMS or the residual proton peak of the re-protonated deuterated solvent, and the spec-
tra were calibrated against the residual proton peak of the used deuterated solvent. The
following symbols indicate spin multiplicities: s (singlet), s br (broad singlet), d (doublet), dd
(doublet of doublet), t (triplet), tt (triplet of triplet), q (quartet), sept (septet), and m (multi-
plet). Standard mass spectra were obtained either as ESI-MS (pos. and/or neg. mode) from a
Advion DCMS interface, (settings as follows: ESI voltage 3.50 kV, capillary voltage 187 V,
source voltage 44 V, capillary temperature 250 ˚C, desolvation gas temperature 250 ˚C, gas
flow 5 L/min) or by an API 2000 mass spectrometer (electron spray ion source, ABSCIEX,
Darmstadt, Germany) coupled to an Agilent 1100 HPLC system.
HRMS spectra were recorded on a Bruker micrOTOF-Q mass spectrometer connected to a
Thermo Scientific Dionex UltiMate 3000 LC via an ESI interface using a Nucleodur C18 Grav-
ity column (50 × 2.0 mm, 3 μm) or were recorded on Thermo Scientific LTQ Velos Orbitrap,
in electrospray ionization (ESI) mode by direct injection.
The synthetic route was developed to optimize the set of substituents to be placed in P1, P2,
and P3 that have been defined after the planning and design studies. Due to the diversity of
building blocks, it was necessary to evaluate different coupling and dehydrating reagents, aim-
ing at the best yield and preventing racemization.
General procedure for amide synthesis. Method A: Isobutyl chloroformate (790 mg, 0.75
mL, 5.5 mmol, 1.1 equiv) was added dropwise to a solution of Boc-(R or S) amino acid (5.0
mmol, 1.0 equiv.), DIPEA (1.6 g, 2.28 mL, 13.0 mmol, 2.6 equiv.) in dry DMF (20 mL), under
argon atmosphere, at -30 ˚C and it was stirred for 0.5 h. Then, an aqueous 2 M NH4Cl solution
(294 mg, 2.75 mL, 5.50 mmol, 1.1 equiv.) was added. The resulting solution was stirred at room
temperature for 20 h. The reaction mixture was dried under reduced pressure. Ethyl acetate
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New cysteine protease inhibitors as trypanocidal agents
Fig 2. General synthesis of compounds 50–60, 65–69. Reagents and conditions: a) Isobutyl chloroformate, NH4Cl 2 M, DIPEA,
DMF, 0 ˚C to rt, 20 h; b) TFA, CH2Cl2, 0 ˚C to rt, 2 h; c) HATU, DIPEA, Boc-AA-OH, DMF, rt, 18 h; d) TFA, CH2Cl2, 0 ˚C to rt, 2
h; e)TBTU, DIPEA, 3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxylic acid, DMF/CH2Cl2, rt, 18 h; f) Cyanuric chloride, DMF, 0 ˚C
to rt, 0.5 h; g) H2 (1 atm), Pd/C, rt, 18 h; h) DDQ, CH2Cl2, rt, 3–5 days; i) TFAA, DIPEA, THF, 0 ˚C to rt, 2 h.
https://doi.org/10.1371/journal.pntd.0007755.g002
(100 mL) was added, and it was washed with a saturated NaHCO3 solution (3 × 50 mL) and
brine (1 × 50 mL). The organic phase was dried over Na2SO4 and evaporated under reduced
pressure to give a crude residue that was purified by flash column chromatography.
Method B: The free primary amine (1.0 mmol, 1.0 equiv.) was added to a solution of the
carboxylic acid (1.3 mmol, 1.3 equiv.), HATU (490 mg, 1.3 mmol, 1.3 equiv.) and DIPEA (364
mg, 0.45 mL, 2.60 mmol, 2.6 equiv.) in dry DMF (5 mL) under argon atmosphere. The result-
ing solution was stirred at room temperature for 20 h. The reaction mixture was diluted with
ethyl acetate (10 mL) and washed with a saturated NaHCO3 solution (3 × 20 mL) and brine
(3 × 20 mL). The organic phase was dried over Na2SO4 and evaporated to give a crude residue
that was purified by flash column chromatography.
Method C: The free primary amine (1.0 mmol, 1.0 equiv.) was added to a solution of the
carboxylic acid (1.3 mmol, 1.3 equiv.), TBTU (410 mg, 1.30 mmol, 1.3 equiv.) and DIPEA (364
mg, 0.45 mL, 2.60 mmol) in dry DMF/CH2Cl2 (1:1, 10 mL) under argon atmosphere. The
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New cysteine protease inhibitors as trypanocidal agents
resulting solution was stirred at room temperature for 20 h. The reaction mixture was diluted
with ethyl acetate (10 mL) and washed with a saturated NaHCO3 solution (3 × 20 mL) and
brine (3 × 20 mL). The organic phase was dried over Na2SO4 and evaporated to give a crude
residue that was purified by flash column chromatography.
General procedure for removal of the Boc protecting group. Method A: The Boc-pro-
tected amino compound (0.25 mmol, 1.0 equiv.) was treated with formic acid (2.44 g, 2.0 mL,
47.9 mmol, 47.9 equiv.) at room temperature. The resulting solution was stirred for 18 h. The
reaction mixture was evaporated under reduced pressure to get a yellowish oil. It was treated
with an aqueous solution of 1.0 M NaOH until pH 9 was reached. The product was extracted
with ethyl acetate (4 × 20 mL) and then washed with brine (1 × 20 mL). The organic phase
was evaporated to obtain a colorless oil. The formation of the product was confirmed by TLC
(ethyl acetate). The product was used for the next step without further purification.
Method B: To a solution of Boc-protected amino compound (1.0 mmol, 1.0 equiv.) in dry
CH2Cl2 (3 mL) was added TFA (912 mg, 0.91 mL, 8.00 mmol, 8.0 equiv.) at 0 ˚C. The mixture
was stirred and allowed to reach room temperature within exposed mainly h. The progress of
the reaction was monitored by TLC (ethyl acetate). The reaction mixture was evaporated
under reduced pressure to eliminate the excess of TFA to get a yellowish solid. The product
was used for the next step without further purification.
General procedure for dehydration of primary amides to nitriles. Method A: The pri-
mary amide (1.0 mmol, 1.0 equiv.) was dissolved in dry DMF (5 mL) at 0 ˚C. Then, cyanuric
chloride (73 mg, 0.4 mmol, 1.1 equiv.) was slowly added to the solution under argon atmo-
sphere. The resulted solution was stirred for 0.5 h. Saturated NaHCO3 solution (30 mL) was
added and it was stirred at room temperature for 2 h. The product was extracted with ethyl
acetate (2 x 50 mL), and then the reunited organic phases were washed with an aqueous solu-
tion of 1.0 M KHSO4 (3 × 20 mL), brine (4 × 30 mL) and dried over Na2SO4. The solvent was
removed, and the crude product was purified by flash silica gel chromatography.
Method B: The primary amide (1.0 mmol, 1.0 equiv.) was dissolved in dry THF (5 mL) and
DIPEA (364 mg, 0.45 mL, 2.6 mmol, 2.6 equiv.) was added. Trifluoroacetic anhydride (273
mg, 0.18 mL, 1.30 mmol, 1.3 equiv.) was added over 5 min, at 0 ˚C. The mixture was stirred
and allowed to reach room temperature within 2 h. Then the reaction was quenched with H2O
(20 mL), THF removed in vacuo, and the product was extracted into ethyl acetate (2 × 50 mL).
The organic phase was washed with a solution 1.0 M of KHSO4 (3 × 20 mL) and with a satu-
rated NaHCO3 solution (3 × 20 mL) and brine (3 × 20 mL) and dried over Na2SO4. The sol-
vent was removed, and the crude product was purified by flash silica gel chromatography.
Method C: The primary amide (1.0 mmol, 1.0 equiv.) was dissolved in dry pyridine (5 mL)
at room temperature. Then, p-toluenesulfonyl chloride (572 mg, 3.0 mmol, 3.0 equiv.) was
added to the solution under argon atmosphere. The resulting solution was stirred for 3 days.
Upon the addition of a saturated NaHCO3 solution (30 mL), the reaction mixture was stirred
at room temperature for 2 h. The solution was dried under reduced pressure. The product was
extracted with ethyl acetate (2 × 50 mL), and then the reunited organic phases were washed
with a 1.0 M solution of KHSO4 (2 × 20 mL), brine (4 × 30 mL) and dried over Na2SO4. The
solvent was removed, and the crude product was purified by flash silica gel chromatography.
General procedure for removal of the benzyl protecting group. Method A: The corre-
sponding protected threonine (1.0 mmol, 1.0 equiv.) was dissolved in ethanol absolute (20
mL) in an argon atmosphere. Upon addition of 10% Pd/C, H2 was bubbled in the solution for
0.5 h. The resulting solution was stirred under H2 atmosphere for 12 h. The progress of the
reaction was monitored by TLC (ethyl acetate). The solution was filtered on celite two times
and dried under reduced pressure to afford the desired product as a colorless wax. The product
was used for the next step without further purification.
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New cysteine protease inhibitors as trypanocidal agents
Method B: The corresponding protected threonine (1.0 mmol, 1.0 equiv.) was dissolved in
dry CH2Cl2 (20 mL) under argon atmosphere. Then, DDQ (908 mg, 4.0 mmol, 4.0 equiv.) was
added, and the resulting solution was stirred for 4 days at room temperature. The progress of
the reaction was monitored by TLC (ethyl acetate). The reaction was quenched with an aque-
ous 1.0 M solution of NaHSO3 (20 mL). Then, CH2Cl2 was removed under reduced pressure.
The product was extracted with ethyl acetate (2 × 50 mL), and the reunited organic phases
were washed with an aqueous solution of 1.0 M KHSO4 (2 × 20 mL), brine (4 × 30 mL) and
dried over Na2SO4. The solvent was removed, and the crude product was purified by flash sil-
ica gel chromatography.
Synthesis and characterization of compound 1–5. Compounds 1–5 have been synthe-
sized from the corresponding amino acid and 1-amino-1-cyclopropanecarbonitrile following
the general procedure for amide synthesis (method B) [15].
(S)-tert-Butyl (1-((1-cyanocyclopropyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate
(1). Yield 92%. White solid. Rf = 0.9 (ethyl acetate: n-hexane; 7:3). Mp. 146–147 ˚C. 1H NMR
(500 MHz, CDCl3) δ 7.27–7.04 (m, 5H), 4.58 (m, 1H), 3.19 (dd, J = 13.8, 5.0 Hz, 1H), 2.88 (dd,
J = 9.5, 5.0 Hz, 1H), 1.31 (s, 9H), 1.25 (m, 2H), 1.04 (m, 2H). 13C NMR. (125 MHz, CDCl3) δ
173.06, 155.30, 137.73, 129.35, 128.19, 126.45, 120.80, 78.30, 55.54, 37.36, 28.26, 19.77, 15.80,
15.75. ESI-MS (+) Calc. for [C18H23N3O3] 329.39, found: 352.3 [M+Na]+.
(S)-tert-Butyl (3-(3-chlorophenyl)-1-((1-cyanocyclopropyl)amino)-1-oxopropan-2-yl)
carbamate (2). Yield 83%. White solid. Rf = 0.7 (ethyl acetate: n-hexane; 6:4). Mp. 146–147
˚C. 1H NMR (500 MHz, CDCl3) δ 7.26–7.25 (m, 1H), 7.23–7.21 (m, 1H), 7.08–7.03 (m, 2H),
4.27–4.24 (m, 1H), 3.10 (dd, J = 13.8, 5.0 Hz, 1H), 2.83 (dd, J = 9.5, 5.0 Hz, 1H), 1.52–1.44 (m,
2H), 1.41 (s, 9H), 1.13–1.05 (m, 2H). 13C NMR (125 MHz, CDCl3) 172.02, 155.61, 138.23,
134.32, 129.29, 127.49, 127.25, 119.48, 119.48, 80.55, 55.28, 37.87, 28.18, 20.14, 16.68, 16.56.
ESI-MS (+) Calc. for [C18H22ClN3O3] 363.83, found: 364.3 [M+H]+.
(S)-tert-Butyl (1-((1-cyanocyclopropyl)amino)-1-oxo-3-(pyridin-4-yl)propan-2-yl)car-
bamate (3). Yield 75%. White solid. Rf = 0.5 (ethyl acetate: n-hexane; 4:6). Mp. 134–135 ˚C.
1H NMR (200 MHz, CD3OD) δ 8.45 (d, J = 4.9 Hz, 2H), 7.34 (d, J = 5.8 Hz, 2H), 4.36–4.23 (m,
1H), 2.96 (dd, J = 14.0, 5.0 Hz, 2H), 1.53–1.46 (m, 1H), 1.38 (s, 9H), 1.20–1.14 (m, 2H). 13
C
NMR (50 MHz, CDCl3) δ 174.74, 149.95, 149.15, 126.52, 121.09, 80.79, 56.03, 38.88, 38.31,
28.55, 21.21, 17.05. ESI-MS (+) Calc. for [C17H22N4O3] 330.38, found: 331.2 [M+H]+.
(S)-tert-Butyl (1-((1-cyanocyclopropyl)amino)-4-methyl-1-oxopentan-2-yl)carbamate
(4). Yield 61%. White solid. Rf = 0.7 (ethyl acetate: n-hexane; 4:6). Mp. 162–164 ˚C. 1H NMR
(500 MHz, DMSO-d6) δ 8.76 (s, 1H), 6.86 (d, J = 7.9 Hz, 1H), 3.86 (dt, J = 8.7, 5.5 Hz, 1H),
1.55–1.54 (m, J = 6.6 Hz, 1H), 1.44 (dd, J = 7.9, 5.5 Hz, 2H), 1.37–1.42 (m, 2H), 1.36 (s, 9H),
1.07 (dd, J = 7.7, 5.3 Hz, 2H), 0.84 (2d, J = 6.6 Hz, 6H). 13C NMR (125 MHz, DMSO-d6) δ
174.17, 155.50, 120.92, 78.22, 52.58, 40.49, 28.31, 24.38, 23.01, 21.66, 19.91, 15.87, 15.75.
ESI-MS (+) Calc. for [C15H25N3O3] 295.37, found: 318.3 [M+Na]+.
tert-Butyl ((2S,3R)-3-(benzyloxy)-1-((1-cyanocyclopropyl)amino)-1-oxobutan-2-yl)car-
bamate (5). Yield 89%. White solid. Rf = 0.7 (ethyl acetate: n-hexane; 4:6). Mp. 88–90 ˚C. 1H
NMR (200 MHz, CDCl3) δ 7.36–7.32 (m, 5H), 4.78–4.60 (m, 3H), 4.22–4.12 (m, 1H), 1.57–
1.44 (m, 2H), 1.30–1.27 (m, 9H), 1.17–1.13 (m, 5H). ESI-MS (+) Calc. for [C20H27N3O4]
373.44, found: 396.4 [M+Na]+.
Synthesis of compounds 6–18. Compounds 6–18 have been synthesized in two steps
from compounds 1–5. First, the Boc group was removed (procedure A), and then the free
amine was coupled to the carboxylic acid following the general procedure for amide synthesis
(method B or method C, as indicated).
Synthesis and characterization of compounds 6, 9 and 11 have been already published else-
where [14].
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New cysteine protease inhibitors as trypanocidal agents
(S)-N-(3-(3-Chlorophenyl)-1-((1-cyanocyclopropyl)amino)-1-oxopropan-2-yl)benza-
mide (7). Method B. Yield 86%. White solid. Rf = 0.7 (ethyl acetate: n-hexane; 6:4). Mp. 213–
215 ˚C. 1H NMR (500 MHz, DMSO-d6) δ 9.04 (s, 1H), 8.67 (d, J = 8.1 Hz, 1H), 7.84–7.83 (m,
2H), 7.56–7.54 (m, 1H), 7.53 (t, J = 7.7 Hz, 2H), 7.41 (s, 1H), 7.32–7.25 (m, 3H), 4.65–4.60 (m,
1H), 3.09 (dd, J = 13.6, 5.0 Hz, 1H), 3.02 (dd, J = 15.2, 5.0 Hz, 1H), 1.51–1.49 (m, 2H), 1.12–
1.06 (m, 2H). 13C NMR (125 MHz, DMSO-d6) δ 172.57, 166.48, 140.62, 132.82, 131.52, 130.04,
129.18, 128.31, 128.05, 127.58, 126.50, 120.81, 54.47, 36.60, 19.90, 15.82. HRMS (+) Calc. for
[C20H19ClN3O2]+ 368.11658, found: 368.11615 [M+H]+. HPLC (protocol B): tR (min) = 10.29.
Purity: 99.6%.
(S)-3-(tert-Butyl)-N-(3-(3-chlorophenyl)-1-((1-cyanocyclopropyl)amino)-1-oxopropan-
2-yl)-1-methyl-1H-pyrazole-5-carboxamide (8). Method C. Method B. Yield 72%. Yellow-
ish solid. Rf = 0.7 (ethyl acetate: n-hexane; 5:5). Mp. 152–154 ˚C. 1H NMR (400 MHz, DMSO-
d6) δ 9.03 (s, 1H), 8.59 (d, J = 8.4 Hz, 1H), 7.39 (s, 1H), 7.31–7.24 (m, 3H), 6.79 (s, 1H), 4.57–
4.53 (m, 1H), 3.88 (s, 3H), 3.08 (dd, J = 13.6, 5.1 Hz, 1H), 2.94 (dd, J = 13.6, 10.2 Hz, 1H),
1.50–1.47 (m, 2H), 1.28 (s, 9H), 1.10–1.05 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 172.10,
159.46, 158.70, 140.26, 134.95, 132.64, 129.94, 129.15, 127.85, 126.39, 120.63, 103.82, 55.21,
36.38, 31.58, 30.36, 19.75, 15.67, 15.61. HRMS (+) Calc. for [C22H27ClN5O2]+ 428.18533,
found: 428.1864 [M+H]+. HPLC (protocol B): tR (min) = 11.17. Purity: 98.3%.
(S)-3-(tert-Butyl)-N-(1-((1-cyanocyclopropyl)amino)-1-oxo-3-(pyridin-4-yl)propan-
2-yl)-1-methyl-1H-pyrazole-5-carboxamide (10). Method C. Yield 56%. Yellowish oil. Rf =
0.4 (ethyl acetate: n-hexane; 5:5). 1H NMR (200 MHz, CD3OD) δ 8.48–8.46 (m, 2H), 7.43 (d,
J = 4.9 Hz, 2H), 6.69 (s, 1H), 4.86–4.78 (m, 1H), 3.95 (s, 3H), 3.04–3.02 (m, 1H), 2.89–2.84 (m,
1H), 1.56–1.50 (m, 2H), 1.31 (d, J = 4.3 Hz, 9H), 1.22–1.18 (m, 2H). 13C NMR (50 MHz,
CD3OD) δ 172.80, 160.35, 160.05, 148.62, 147.59, 134.80, 125.00, 119.66, 103.60, 53.18, 37.37,
36.45, 31.42, 29.44, 19.92, 15.60, 15.28. FT-IR (KBr, cm-1) 3297.16, 2966.17, 2242.31, 1670.63,
1601.36, 1531.90, 1425.65, 1352.10, 1278.55, 1241.77, 1049.72, 988.42, 808.63, 755.51, 722.82,
511.19, 506.24, 489.90. HRMS (+) Calc. for [C21H27N6O2]+ 395.21955, found: 395.21973 [M
+H]+. HPLC (protocol B): tR (min) = 3.68. Purity: 94.7%.
N-((2S,3R)-3-(Benzyloxy)-1-((1-cyanocyclopropyl)amino)-1-oxobutan-2-yl)-3-(tert-
butyl)-1-methyl-1H-pyrazole-5-carboxamide (12). Method C. Yield 53%. Yellowish solid.
Rf = 0.4 (ethyl acetate: n-hexane; 6:4). Mp. 100–101 ˚C. 1H NMR (200 MHz, CDCl3) δ 7.36–
7.32 (m, 5H), 6.45 (s, 1H), 4.76–4.62 (m, 3H), 4.22–4.12 (m, 1H), 4.09 (s, 3H), 1.57–1.44 (m,
2H), 1.30–1.27 (m, 9H), 1.20–1.13 (m, 5H). 13C NMR (50 MHz, CDCl3) δ 170.36, 160.18,
159.74, 137.15, 134.06, 128.48, 127.98, 127.68, 119.25, 103.18, 74.03, 71.48, 54.81, 38.38, 31.66,
30.16, 28.68, 20.24, 16.91, 15.85. FT-IR (KBr, cm-1) 3288.99, 2953.92, 2917.14, 2214.31,
1646.31, 1589.10, 1495.12, 1049.31, 1294.89, 1200.91, 1033.37, 922.51, 755.51, 681.95. HRMS
(+) Calc. for [C24H32N5O3]+ 437.25051, found: 438.25102 [M+H]+. HPLC (protocol B): tR
(min) = 8.77. Purity: 97.0%.
(S)-7-Chloro-N-(1-((1-cyanocyclopropyl)amino)-1-oxo-3-phenylpropan-2-yl)quino-
line-4-carboxamide (13). Method B. Yield 74%. White solid. Rf = 0.7 (ethyl acetate: n-hex-
ane; 8:2). Mp. 260–262 ˚C. 1H NMR (400 MHz, DMSO-d6) δ 9.15 (d, J = 10.2 Hz, 1H), 9.13 (s,
1H), 8,97 (d, J = 10.2 Hz, 1H), 8.10 (d, J = 2.1 Hz, 1H), 7.72 (d, J = 15 Hz, 1H), 7.56 (dd,
J = 15.1, 10.2 Hz, 1H), 7.44 (d, J = 10.2 Hz, 1H), 7.28–7.24 (m, 5H), 4.75–4.73 (m, 1H), 3.11
(dd, J = 13.8, 5.0 Hz, 1H), 2.87 (dd, J = 9.5, 5.0 Hz, 1H), 1.51–1.49 (m, 2H), 1.09–1.07 (m, 1H).
13C NMR (100 MHz, DMSO-d6) δ 172.34, 166.33, 151.74, 148.36, 141.88, 137.63, 134.53,
129.45, 128.42, 128.00, 127.86, 126.74, 122.89, 120.91, 119.69, 54.56, 37.26, 20.02, 15.89. FT-IR
(KBr, cm-1) 3254.05, 2926.14, 2247.17, 1672.36, 1668.36, 1523.83, 846.79, 831.36. HRMS (+)
Calc. for [C23H20ClN4O2]+ 418.12748, found: 419.12813 [M+H]+. HPLC (protocol C, 50:50
ACN: water): tR (min) = 17.32. Purity: 99.9%.
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New cysteine protease inhibitors as trypanocidal agents
(S)-7-Chloro-N-(1-((1-cyanocyclopropyl)amino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)
quinoline-4-carboxamide (14). Method B. Yield 49%. Yellowish solid. Rf = 0.3 (ethyl ace-
tate). Mp. 196–197 ˚C. 1H NMR (400 MHz, DMSO-d6) δ 10.88 (s, 1H), 9.15 (s, 1H), 9.08 (d,
J = 10.2 Hz, 1H), 8.97 (d, J = 4.2 Hz, 1H), 8.10 (d, J = 2.2 Hz, 1H), 7.68 (t, J = 7.5 Hz, 1H), 7.54–
7.52 (m, 1H), 7.46 (d, J = 4.5 Hz, 1H), 7.38 (d, J = 10.2 Hz 1H), 7.16 (d, J = 2.4 Hz, 1H), 7.09 (t,
J = 5.5 Hz, 1H), 7.00 (t, J = 5.8 Hz, 1H), 4.81–4.79 (m, 1H), 3.20 (dd, J = 13.8, 5.0 Hz, 1H), 3.05
(dd, J = 9.5, 5.0 Hz, 1H), 1.51–1.48 (m, 2H), 1.11–1.08 (m, 2H). 13C NMR (100 MHz, DMSO-
d6) δ 173.02, 166.59, 151.94, 148.61, 142.38, 136.57, 134.72, 128.23, 128.05, 128.03, 127.52,
124.56, 123.19, 121.46, 121.26, 119.97, 119.03, 118.76, 111.81, 110.02, 54.26, 27.79, 20.33, 16.20.
FT-IR (KBr, cm-1) 3254.05, 2926.14, 2247.17, 1672.36, 1665.34, 1522.83, 831.30, 732.98. HRMS
(+) Calc. for [C25H21ClN5O2]+ 458.13838, found: 458.13703 [M+H]+. HPLC (protocol C,
50:50 ACN: water): tR (min) = 18.64. Purity: 99.6%.
(S)-7-Chloro-N-(1-((1-cyanocyclopropyl)amino)-4-methyl-1-oxopentan-2-yl)quino-
line-4-carboxamide (15). Method B. Yield 68%. White solid. Rf = 0.7 (ethyl acetate).
Mp. 169–170 ˚C. 1H NMR (400 MHz, DMSO-d6) δ 9.10 (s, 1H), 9.05 (s, 1H), 9.02 (d, J = 5.5
Hz, 1H) 8.20 (d, J = 11.5 Hz, 1H), 8.15 (d, J = 2.5 Hz, 1H), 7.72 (dd, J = 11.5, 2.5 Hz, 1H), 7.60
(d, J = 5.5 Hz, 1H), 4.48–4.46 (m, 1H), 1.70–1.63 (m, 3H), 1.52–1.48 (m, 2H), 1.19–1.14 (m,
2H), 0.92 (2d, J = 10.5 Hz, 6H). 13C NMR (100 MHz, DMSO-d6) δ 173.63, 166.83, 152.05,
148.70, 142.06, 134.83, 131.26, 128.38, 123.35, 121.21, 120.20, 51.97, 24.87, 23.38, 21.79, 20.34,
16.22, 16.06. FT-IR (KBr, cm-1) 3402.4, 3257.7, 3030.1, 2960.7, 2247.0, 1674.2, 1633.7, 1529.5,
1296.1, 831.31. HRMS (+) Calc. for [C20H22ClN4O2]+ 384.14313, found: 385.14503 [M+H]+.
HPLC (protocol C, 65:35 ACN: water): tR (min) = 11.81. Purity: 96.2%.
(S)-3-((1-((1-Cyanocyclopropyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamoyl)phenyl
benzoate (16). Method B. Yield 89%. White solid. Rf = 0.7 (ethyl acetate: n-hexane; 6:4).
Mp. 185–187 ˚C. 1H NMR (400 MHz, DMSO-d6) δ 9.05 (s, 1H), 8.79 (d, J = 8.0 Hz, 1H), 8.18–
8.16 (m, 2H), 7.81–7.78 (m, 3H), 7.64 (t, J = 8.5 Hz, 2H), 7.57 (t, J = 8.5 Hz, 2H), 7.50–7.48 (m,
1H), 7.31–7.23 (m, 4H), 7.18 (tt, J = 7.0, 1.5 Hz, 1H), 4.64–4.59 (m, 1H), 3.06 (dd, J = 13.6, 5.0
Hz, 1H), 3.00 (dd, J = 13.5, 8.5 Hz, 1H), 1.49–1.45 (m, 2H), 1.06–1.01 (m, 2H). 13C NMR (100
MHz, DMSO-d6) δ 173.04, 165.60, 165.02, 150.88, 138.21, 135.72, 134.66, 130.27, 129.98,
129.57, 129.51, 129.16, 128.57, 126.83, 125.69, 125.53, 121.47, 121.15, 55.10, 37.36, 20.18, 16.15,
16.10, 14.55. FT-IR (KBr, cm-1) 3254.05, 2926.14, 2247.17, 1672.36, 1668.36, 1523.83, 846.79,
831.36. HRMS (+) Calc. for [C27H24N3O4]+ 453.17668, found: 454.17800 [M+H]+. HPLC (pro-
tocol C, 65:35 ACN: water): tR (min) = 19.62. Purity: 99.9%.
(S)-6-Amino-N-(1-((1-cyanocyclopropyl)amino)-4-methyl-1-oxopentan-2-yl)nicotin-
amide (17). Method C. Yield 48%. Yellowish solid. Rf = 0.3 (ethyl acetate: methanol; 8:2).
Mp. 100–101 ˚C. 1H NMR (200 MHz, CD3OD) δ 8.43 (s, 1H), 7.88 (d, J = 8.8, 2.1 Hz, 1H),
6.54 (d, J = 8.9 Hz,1H), 4.53–4.46 (m, 1H), 1.82–1.51 (m, 3H), 1.47–1.43 (m, 2H), 1.24–1.17
(m, 2H), 0.96–0.93 (m, 6H). 13C NMR (50 MHz, CD3OD) δ 176.59, 168.58, 162.86, 149.21,
138.44, 121.42, 119.42, 109.12, 41.60, 26.13 23.54, 21.98, 21.41, 17.15, 16.78. FT-IR (KBr, cm-1)
3288.99, 2953.92, 2917.14, 2214.31,1647.63, 1496.06, 1409.33, 1294.89, 1202.33, 1075.33,
1030.81, 922.54, 763.49, 667.21. ESI-MS (+) Calc. for [C16H22N5O2]+ 316.17735, found:
316.17713 [M+H]+. HPLC (protocol B): tR (min) = 8.77. Purity: 97.0%.
(S)-N-(1-((1-Cyanocyclopropyl)amino)-4-methyl-1-oxopentan-2-yl)-1H-pyrrolo[2,3-b]
pyridine-5-carboxamide (18). Method C. Yield 45%. White solid. Rf = 0.3 (ethyl acetate:
methanol; 8:2). Mp. 79–80 ˚C. 1H NMR (200 MHz, CD3OD) δ 8.71 (d, J = 1.8 Hz, 1H), 8.47 (d,
J = 2.0 Hz, 1H), 7.46 (d, J = 3.5 Hz, 1H), 6.57 (d, J = 3.5 Hz, 1H), 4.59–4.57 (m, 1H), 1.91–1.56
(m, 3H), 1.54–1.49 (m, 2H), 1.29–1.25 (m, 2H), 1.01–0. 89 (m, 6H). 13C NMR (50 MHz,
CD3OD) δ 176.50, 169.61, 163.45, 150.38, 143.32, 129.52, 128.65, 123.21, 120.94, 102.66, 53.50,
38.92, 26.18, 23.41, 22.02, 21.25, 17.17, 16.49. FT-IR (KBr, cm-1) 3286.44, 2933.89, 2924.14,
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New cysteine protease inhibitors as trypanocidal agents
2216.51, 1647.63, 1588.10, 1496.06, 1409.33, 1294.89, 1202.33, 1075.33, 1030.31, 922.54,
763.54, 667.21. ESI-MS (+) Calc. for [C18H22N5O2]+ 340.17753, found: 340.17689 [M+H]+.
HPLC (protocol B): tR (min) = 5.38. Purity: 99.0%.
Synthesis and characterization of compound 19. Compound 19 has been synthesized
from compound 12 by removal of the benzyl protecting group under mild conditions (proto-
col B).
3-(tert-Butyl)-N-((2S,3R)-1-((1-cyanocyclopropyl)amino)-3-hydroxy-1-oxobutan-2-yl)-
1-methyl-1H-pyrazole-5-carboxamide (19). Yield 40%. Yellowish solid. Rf = 0.3 (ethyl ace-
tate). Mp. 201–202 ˚C. 1H NMR (200 MHz, CD3OD) δ 6.81 (s, 1H), 4.40 (d, J = 4.4 Hz, 1H),
4.23–4.18 (m, 1H), 4.04 (s, 3H), 1.52–1.47(m, 2H), 1.32 (s, 9H), 1.29–1.18 (m, 5H). 13C NMR
(50 MHz, CD3OD) δ 174.94, 163.34, 162.74, 137.60, 122.22, 106.16, 69.42, 61.17, 39.91, 34.00,
31.94, 22.41, 21.45, 18.11, 17.83. FT-IR (KBr, cm-1) 3288.89, 2953.92, 2917.14, 2214.31,
1646.31, 1589.10, 1495.12, 1049.31, 1294.89, 1200.91, 1033.97, 922.51, 755.51, 681.95. ESI-MS
(-) Calc. for [C17H25N5O3]+ 348.20356, found: 348.20314 [M+H]+. HPLC (protocol B): tR
(min) = 7.74. Purity: 98.0%.
Synthesis of compounds 20–27. Compounds 20–27 have been synthesized from the
equivalent amino Boc-protected amino acid following the general procedure for amide synthe-
sis (method A).
(S)-tert-Butyl (1-amino-1-oxo-3-phenylpropan-2-yl)carbamate (20). Yield 77%. White
solid. Rf = 0.6 (ethyl acetate). Mp. 146–149 ˚C. 1H NMR (200 MHz, CD3OD) δ 7.26–7.16 (m,
5H), 4.44–4.41 (m, 1H), 3.14 (dd, J = 13.7, 6.0 Hz, 1H), 2.88 (dd, J = 13.7, 8.1 Hz, 1H), 1.23 (s,
9H). 13C NMR (50 MHz, CD3OD) δ 173.98, 155.60, 130.73, 124.93, 122.46, 118.22, 80.20,
54.02, 37.73, 27.94. ESI-MS (+) Calc. for [C14H20N2O3] 264.31, found: 287.3 [M+Na]+.
(R)-tert-butyl (1-amino-1-oxo-3-phenylpropan-2-yl)carbamate (21). Yield 79%. White
solid. Rf = 0.6 (ethyl acetate). Mp. 141–142 ˚C. 1H NMR (200 MHz, CD3OD) δ 7.24–7.17 (m,
5H), 4.42–4.37 (m, 1H), 3.14 (dd, J = 13.7, 6.0 Hz, 1H), 2.92 (dd, J = 13.7, 8.1 Hz, 1H), 1.43 (s,
9H). 13C NMR (50 MHz, CD3OD) δ 173.12, 154.48, 133.36, 122.46, 121.75, 119.38, 83.51,
55.61, 38.38, 28.66. ESI-MS (+) Calc. for [C14H20N2O3] 264.31, found: 287.3 [M+Na]+.
(S)-tert-Butyl (1-amino-4-methyl-1-oxopentan-2-yl)carbamate (22). Yield 74%. White
solid. Rf = 0.4 (ethyl acetate). Mp. 138–141 ˚C. 1H NMR (200 MHz, CDCl3) δ 6.58 (s br, 1H),
6.13 (s br, 1H), 5.25–4.97 (m, 2H), 4.15 (s br, 1H), 1.73–1.43 (m, 3H), 1.41 (s, 9H), 0.92 (d,
J = 3.2 Hz, 6H). 13C NMR (50 MHz, CDCl3) δ 172.34, 155.95, 71.56, 28.57, 28.18, 25.08, 23.21,
19.25. ESI-MS (+) Calc. for [C11H22N2O3] 230.30, found: 253.3 [M+Na]+.
(R)-tert-Butyl (1-amino-4-methyl-1-oxopentan-2-yl)carbamate (23). Yield 71%. White
solid. Rf = 0.4 (ethyl acetate). Mp. 138–141 ˚C. 1H NMR (200 MHz, CDCl3) δ 6.64 (s br, 1H),
6.21 (s br, 1H), 5.21–4.98 (m, 2H), 4.16 (s br, 1H), 1.71–1.45 (m, 3H), 1.42 (s, 9H), 0.93 (d,
J = 3.2 Hz, 6H). 13C NMR (50 MHz, CDCl3) δ 172.11, 155.37, 71.64, 28.61, 28.29, 25.15, 23.34,
19.30. ESI-MS (+) Calc. for [C11H22N2O3] 230.30, found: 253.3 [M+Na]+.
(S)-tert-Butyl (1-amino-3-(3-chlorophenyl)-1-oxopropan-2-yl)carbamate (24). Yield
74%. White solid. Rf = 0.4 (ethyl acetate). Mp. 111–112 ˚C. 1H NMR (200 MHz, CDCl3) δ
7.26–7.13 (m, 4H), 6.16 (s, 1H), 5.75 (s, 1H), 5.21 (d, J = 7.8 Hz, 1H), 4.42–4.36 (m, 1H), 3.19–
2.94 (m, 2H), 1.44 (s, 9H). 13C NMR (50 MHz, CDCl3) δ 173.53, 155.16, 138.90, 134.09, 130.04,
129.44, 127.55, 127.11, 80.31, 37.88, 28.16. ESI-MS (+) Calc. for [C14H19ClN2O3] 298.77,
found: 321.8 [M+Na]+.
(S)-tert-Butyl (1-amino-1-oxo-3-(pyridin-4-yl)propan-2-yl)carbamate (25). Yield 92%.
White solid. Rf = 0.2 (ethyl acetate). Mp. 131–133 ˚C. 1H NMR (200 MHz, CDCl3) δ 8.42 (d,
J = 5.7 Hz, 2H), 7.22 (d, J = 5.9 Hz, 2H), 4.35–4.31 (m, 1H), 3.18 (dd, J = 13.7, 6.0 Hz, 1H), 2.81
(dd, J = 13.7, 8.1 Hz, 1H), 1.49–1.33 (s br, 9H). 13C NMR (50 MHz, CDCl3) δ 173.69, 155.86,
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New cysteine protease inhibitors as trypanocidal agents
148.48, 147.61, 118.42, 80.22, 54.51, 37.77, 27.89. ESI-MS (+) Calc. for [C13H19N3O3] 265.31,
found: 266.2 [M+H]+.
tert-Butyl ((2S,3R)-1-amino-3-(benzyloxy)-1-oxobutan-2-yl)carbamate (26). Yield
65%. White solid. Rf = 0.5 (ethyl acetate). Mp. 145–147 ˚C. 1H NMR (500 MHz, CDCl3) δ
7.37–7.28 (m, 5H), 6.50 (s, 1H), 5.70–5.51 (m, 2H), 4.62 (q, J = 4.6 Hz, 2H), 4.34–4.31 (m, 1H),
4.18–4.11 (m, 1H), 1.45 (s, 9H), 1.19 (d, J = 6.3 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ
172.09, 155.66, 137.78, 128.41, 127.82, 127.73, 80.03, 74.53, 71.61, 57.13, 28.23, 27.84, 18.92.
ESI-MS (+) Calc. for [C16H24N2O4] 308.37, found: 331.4 [M+Na]+.
tert-Butyl ((2R,3S)-1-amino-3-(benzyloxy)-1-oxobutan-2-yl)carbamate (27). Yield
68%. White solid. Rf = 0.5 (ethyl acetate). Mp. 143–145 ˚C. 1H NMR (500 MHz, CDCl3) δ
7.37–7.28 (m, 5H), 6.50 (s, 1H), 5.70–5.51 (m, 2H), 4.62 (q, J = 4.6 Hz, 2H), 4.33 (s br, 1H),
4.27–4.19 (m, 1H), 1.45 (s, 9H), 1.19 (d, J = 6.3 Hz, 3H) .13C NMR (125 MHz, CDCl3) δ
172.74, 156.31, 138.44, 129.06, 128.47, 128.38, 80.68, 77.16, 75.18, 72.26, 57.78, 28.89, 28.49,
19.57. ESI-MS (+) Calc. for [C16H24N2O4] 308.37, found: 331.4 [M+Na]+.
Synthesis of compounds 28–38. Compounds 28–38 have been synthesized in two steps
from their precursors 20–27. After removal of the Boc-protecting group (method B), the
resulting free amine was coupled to the carboxylic acid following the general procedure for
amide synthesis (method B).
tert-Butyl ((S)-1-(((S)-1-amino-1-oxo-3-phenylpropan-2-yl)amino)-1-oxo-3-phenylpro-
pan-2-yl)carbamate (28). Yield 92%. White solid. Rf = 0.8 (ethyl acetate). Mp. 186–188 ˚C.
1H NMR (200 MHz, CD3OD) δ 7.37–7.22 (m, 6H), 7.16–7.09 (m, 4H), 4.76–4.63 (m, 1H),
4.34–4.20 (m, 1H), 3.15–2.95 (m, 2H), 2.91–2.62 (m, 2H), 1.39 (s, 9H). 13C NMR (50 MHz,
CD3OD) δ 174.36, 172.35, 156.09, 136.44, 129.04, 128.35, 128.27, 126.78, 126.64, 79.89, 56.00,
53.78, 37.73, 37.24, 27.84. ESI-MS (+) Calc. for [C23H29N3O4] 411.49, found: 434.3 [M+Na]+.
tert-Butyl ((S)-1-(((R)-1-amino-1-oxo-3-phenylpropan-2-yl)amino)-1-oxo-3-phenyl-
propan-2-yl)carbamate (29). Yield 92%. White solid. Rf = 0.8 (ethyl acetate). Mp. 185–187
˚C. 1H NMR (200 MHz, CD3OD) δ 7.75–7.60 (m, 6H), 7.56–7.49 (m, 4H), 5.14–5.01 (m, 1H),
4.72–4.57 (m, 1H), 3.53–3.33 (m, 2H), 3.29–3.00 (m, 2H), 1.77 (s, 9H). 13C NMR (50 MHz,
CD3OD) δ 174.85, 172.83, 156.58, 136.93, 129.53, 128.83, 128.76, 127.26, 127.13, 80.38, 56.49,
54.26, 38.22, 37.73, 28.33. ESI-MS (+) Calc. for [C23H29N3O4] 411.49, found: 434.3 [M+Na]+.
tert-Butyl ((S)-1-(((S)-1-amino-4-methyl-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpro-
pan-2-yl)carbamate (30). Yield 83%. White solid. Rf = 0.8 (ethyl acetate). Mp. 164–165 ˚C.
1H NMR (200 MHz, CD3OD) δ 7.24–7.20 (m, 5H), 4.33–4.28 (m, 1H), 4.11–4.05 (m, 1H),
3.03–2.93 (m, 2H), 1.56–1.38 (m, 2H), 1.36 (s, 9H), 1.26–1.24 (m, 1H), 0.89 (d, J = 2.6 Hz, 6H).
13C NMR (50 MHz, CD3OD) δ 175.39, 154.59, 143.42, 136.55, 128.99, 128.29, 126.65, 84.91,
72.86, 52.36, 51.47, 27.61, 24.41, 22.20, 21.01. ESI-MS (+) Calc. for [C20H31N3O4] 377.48,
found: 400.5 [M+Na]+.
tert-Butyl ((S)-1-(((R)-1-amino-4-methyl-1-oxopentan-2-yl)amino)-1-oxo-3-phenyl-
propan-2-yl)carbamate (31). Yield 88%. White solid. Rf = 0.8 (ethyl acetate). Mp. 168–170
˚C. 1H NMR (200 MHz, CD3OD) δ 7.24–7.21 (m, 5H), 4.31–4.21 (m, 1H), 4.11–4.09 (m, 1H),
3.04–2.94 (m, 2H), 1.57–1.40 (m, 2H), 1.39 (s, 9H), 1.27–1.24 (m, 1H), 0.90 (d, J = 2.6 Hz, 6H).
13C NMR (50 MHz, CD3OD) δ 175.80, 155.00, 143.83, 136.96, 129.40, 128.70, 127.06, 85.32,
77.16, 73.27, 52.78, 51.89, 28.02, 24.82, 22.61, 21.42. ESI-MS (+) Calc. for [C20H31N3O4]
377.48, found: 400.5 [M+Na]+.
tert-Butyl ((S)-1-(((S)-1-amino-3-(3-chlorophenyl)-1-oxopropan-2-yl)amino)-1-oxo-
3-phenylpropan-2-yl)carbamate (32). Yield 78%. White solid. Rf = 0.6 (ethyl acetate).
Mp. 148–150 ˚C. 1H NMR (200 MHz, CDCl3) δ 7.38–7.26 (m, 7H), 7.10–7.06 (m, 2H), 6.63 (s
br, 1H), 6.39 (s br, 1H), 4.81–4.77 (m, 1H), 4.38–4.35 (m, 1H), 3.05–2.97 (m, 4H), 1.41 (s, 9H).
13C NMR (50 MHz, CDCl3) δ 172.83, 161.82, 138.54, 136.14, 134.46, 133.26, 130.05, 129.51,
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New cysteine protease inhibitors as trypanocidal agents
129.36, 129.02, 127.69, 127.36, 126.78, 80.95, 62.51, 53.21, 52.76, 38.74, 28.23. ESI-MS (+) Calc.
for [C23H28ClN3O4] 445.94, found: 468.8 [M+Na]+.
tert-Butyl ((S)-1-(((S)-1-amino-1-oxo-3-(pyridin-4-yl)propan-2-yl)amino)-1-oxo-
3-phenylpropan-2-yl)carbamate (33). Yield 71%. Yellowish solid. Rf = 0.4 (ethyl acetate).
Mp. 155–157 ˚C. 1H NMR (200 MHz, CD3OD) δ 8.63 (d, J = 5.7 Hz, 2H), 7.58–7.30 (m, 7H),
4.94–4.89 (1H, m), 4.55–4.40 (m, 1H), 3.44 (dd, J = 13.8, 5.5 Hz, 2H), 3.28–2.93 (m, 2H), 1.60
(s, 9H). 13C NMR (50 MHz, CDCl3) δ 173.27, 172.38, 148.58, 140.95, 136.44, 135.10, 128.97,
128.32, 126.68, 124.97, 99.74, 55.40, 27.73, 23.92, 13.06, 10.47.ESI-MS (+) Calc. for
[C23H28ClN3O4] 445.94, found: 468.8 [M+Na]+.
tert-Butyl ((S)-1-(((2S,3R)-1-amino-3-(benzyloxy)-1-oxobutan-2-yl)amino)-1-oxo-
3-phenylpropan-2-yl)carbamate (34). Yield 88%. Yellowish solid. Rf = 0.8 (ethyl acetate).
Mp. 161–163 ˚C. 1H NMR (200 MHz, CDCl3) δ 7.38–7.26 (m, 7H), 7.10–7.06 (m, 2H), 6.63 (s
br, 1H), 6.39 (s br, 1H), 5.65 (s br, 1H), 5.08 (s br, 1H), 4.81–4.77 (m, 1H), 4.38–4.35 (m, 1H),
3.15–2.97 (m, 5H), 1.74–1.47 (m, 3H), 1.41 (s, 9H). 13C NMR (50 MHz, CDCl3) δ 172.98,
171.90, 156.38, 137.70, 136.53, 128.99, 128.42, 128.13, 127.64, 127.58, 126.75, 80.30, 74.03,
71.36, 60.44, 38.05, 27.67, 20.33, 15.75. ESI-MS (+) Calc. for [C25H33N3O5] 455.55, found:
478.5 [M+Na]+.
tert-Butyl ((S)-1-(((2R,3S)-1-amino-3-(benzyloxy)-1-oxobutan-2-yl)amino)-1-oxo-
3-phenylpropan-2-yl)carbamate (35). Yield 78%. Yellowish solid. Rf = 0.8 (ethyl acetate).
Mp. 138–139 ˚C. 1H NMR (200 MHz, CDCl3) δ 7.40–7.37 (m, 7H), 7.11–7.08 (m, 2H), 6.58 (s
br, 1H), 6.43 (s br, 1H), 5.61 (s br, 1H), 5.23 (s br, 1H), 4.83–4.78 (m, 1H), 4.34–4.31 (m, 1H),
3.32–2.85 (m, 5H), 1.39 (s, 9H), 1.19–1.09 m, 3H). 13C NMR (50 MHz, CDCl3) δ 173.34,
172.27, 156.74, 138.07, 136.90, 129.35, 128.79, 128.49, 128.01, 127.95, 127.12, 80.66, 74.40,
71.73, 60.81, 38.42, 28.04, 20.70, 16.12.ESI-MS (+) Calc. for [C25H33N3O5] 455.55, found:
478.5 [M+Na]+.
tert-Butyl ((S)-1-(((2S,3R)-1-amino-3-(benzyloxy)-1-oxobutan-2-yl)amino)-4-methyl-
1-oxopentan-2-yl)carbamate (36). Yield 76%. Yellowish solid. Rf = 0.8 (ethyl acetate).
Mp. 88–91 ˚C. 1H NMR (200 MHz, CDCl3) δ 7.30–7.22 (m, 5H), 7.19–7.15 (m, 1H), 6.24 (s br,
1H), 6.04 (s br, 1H), 5.36 (s br, 2H), 4.57 (s, 2H), 4.56–4.52 (m, 1H), 4.36–4.21 (m, 1H), 4.17–
4.11 (m, 1H), 1.77–1.48 (m, 3H), 1.38 (s, 9H), 1.13 (d, J = 6.4 Hz, 3H), 0.94–0.90 (m, 6H). 13
C
NMR (50 MHz, CDCl3) δ 172.84, 171.86, 155.98, 137.88, 128.29, 127.66, 127.63, 80.38, 73.89,
71.58, 56.40, 54.01, 40.85, 28.12, 24.74, 22.92, 21.62. ESI-MS (+) Calc. for [C22H35N3O5]
421.53, found: 444.7 [M+Na]+.
tert-Butyl ((S)-1-(((2R,3S)-1-amino-3-(benzyloxy)-1-oxobutan-2-yl)amino)-4-methyl-
1-oxopentan-2-yl)carbamate (37). Yield 73%. Yellowish solid. Rf = 0.8 (ethyl acetate).
Mp. 86–88 ˚C. 1H NMR (200 MHz, CDCl3) δ 7.31–7.22 (m, 5H), 7.20–7.11 (m, 1H), 6.26 (s br,
1H), 6.02 (s br, 1H), 5.36 (s br, 2H), 4.54 (s, 2H), 4.56–4.53 (m, 1H), 4.33–4.28 (m, 1H), 4.16–
4.11 (m, 1H), 1.76–1.48 (m, 3H), 1.48 (s, 9H), 1.11 (d, J = 6.4 Hz, 3H), 0.96–0.90 (m, 6H). 13
C
NMR (50 MHz, CDCl3) δ 172.84, 171.86, 155.98, 137.88, 128.29, 127.66, 127.63, 80.38, 73.89,
71.58, 56.40, 54.01, 40.85, 28.12, 24.74, 22.92, 21.62. ESI-MS (+) Calc. for [C22H35N3O5]
421.53, found: 444.5 [M+Na]+.
tert-Butyl ((S)-1-(((2S,3R)-1-amino-3-(benzyloxy)-1-oxobutan-2-yl)amino)-3-
(3-chlorophenyl)-1-oxopropan-2-yl)carbamate (38). Yield 77%. Yellowish solid. Rf = 0.6
(ethyl acetate). Mp. 164–165 ˚C. 1H NMR (200 MHz, CD3OD) δ 7.47–7.22 (m, 9H), 4.55–
4.37 (m, 1H), 4.29–4.17 (m, 2H), 3.26 (dd, J = 13.9, 5.1 Hz, 1H), 3.07–2.96 (m, 1H), 2.92 (s,
2H), 1.44 (s, 9H), 1.27 (d, J = 6.3 Hz, 3H). 13C NMR (50 MHz, CD3OD) δ 172.84, 171.76,
156.24, 138.90, 137.56, 136.39,130.04, 128.85, 128.28, 127.99, 127.50, 127.44, 126.61, 80.16,
73.89, 71.22, 60.30, 49.00, 37.92, 27.53, 20.19. ESI-MS (+) Calc. for [C25H32ClN3O5] 489.99,
found: 513.1 [M+Na]+.
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New cysteine protease inhibitors as trypanocidal agents
Synthesis of compounds 39–49. Compounds 39–49 have been synthesized in two steps
from their precursors 28–38. After removal of the Boc-protecting group (method B), the free
amine was coupled to the carboxylic acid following the general procedure for amide synthesis
(method C).
N-((S)-1-(((S)-1-Amino-1-oxo-3-phenylpropan-2-yl)amino)-1-oxo-3-phenylpropan-
2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (39). Yield 79%. Yellowish
solid. Rf = 0.3 (ethyl acetate). Mp. 109–110 ˚C. 1H NMR (200 MHz, CDCl3) δ7.32–6.96 (m,
10H), .6.65 (s br, 1H), 6.26 (s, 1H), 6.09 (s br, 1H), 4.68–4.58 (m, 2H), 3.81 (s, 3H), 2.87–2.74
(m, 4H), 1.11 (s, 9H). 13C NMR (50 MHz, CDCl3) δ 173.37, 165.56, 162.46, 160.20, 159.99,
136.34, 136.16, 134.40, 129.13, 128.39, 126.87, 126.81, 124.46, 103.30, 54.93, 53.91, 38.44, 36.34,
31.69, 31.28, 30.27. ESI-MS (+) Calc. for [C27H33N5O3] 475.58, found: 498.7 [M+Na]+.
N-((S)-1-(((R)-1-Amino-1-oxo-3-phenylpropan-2-yl)amino)-1-oxo-3-phenylpropan-
2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (40). Yield 72%. Yellowish
solid. Rf = 0.4 (ethyl acetate). Mp. 121–122 ˚C. 1H NMR (200 MHz, CDCl3) δ 7.59–7.13 (m,
10H), 6.88 (s, 1H), 6.50 (s, 1H), 6.32 (s br, 1H), 4.92–4.82 (m, 2H), 4.04 (s, 3H), 3.10–2.91 (m,
4H), 1.34 (s, 9H). 13C NMR (50 MHz, CDCl3) δ 172.94, 165.13, 162.04, 159.77, 159.56, 135.92,
135.74, 133.97, 128.70, 127.97, 126.45, 126.38, 124.04, 102.88, 54.50, 53.48, 38.01, 35.91, 31.26,
30.86, 29.84. ESI-MS (+) Calc. for [C27H33N5O3] 475.58, found: 498.7 [M+Na]+.
N-((S)-1-(((S)-1-Amino-4-methyl-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-
2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (41). Yield 85%. Yellowish
solid. Rf = 0.3 (ethyl acetate). Mp. 152–153 ˚C. 1H-NMR (200 MHz, CDCl3) δ. 7.42–7.25 (m,
5H), 6.81 (s, 1H), 4.77–4.65 (m, 1H), 4.36–4.08 (m, 1H), 4.08 (s, 3H), 3.22–3.18 (m, 2H), 1.89–
1.46 (m, 3H), 1.40 (s, 9H), 0.88–0.80 (m, 6H). 13C NMR (50 MHz, CDCl3) δ 173.57, 168.11,
162.00, 161.25, 137.23, 135.94, 130.02, 129.31, 127.64, 104.97, 57.07, 49.00, 38.87, 32.59, 31.75,
30.78, 24.80, 23.57, 21.42. ESI-MS (+) Calc. for [C24H35N5O3] 441.57, found: 464.5 [M+Na]+.
N-((S)-1-(((S)-1-Amino-3-(3-chlorophenyl)-1-oxopropan-2-yl)amino)-1-oxo-3-phenyl-
propan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (42). Yield 72%.
Yellowish solid. Rf = 0.3 (ethyl acetate). Mp. 131–133 ˚C. 1H NMR (200 MHz, CDCl3) δ 7.45–
7.28 (s, 5H), 6.83 (s, 1H), 4.87–4.68 (m, 1H), 4.39–4.11 (m, 1H), 4.11 (s, 3H), 3.25–3.21 (m,
2H), 1.76–1.49 (m, 3H), 1.42 (s, 9H), 0.88–0.83 (m, 6H). 13C NMR (50 MHz, CDCl3) δ 176.49,
171.02, 164.92, 164.17, 140.15, 138.86, 132.94, 132.23, 130.56, 107.89, 76.35, 59.99, 41.79, 35.51,
34.67, 33.70, 27.72, 26.49, 24.34. ESI-MS (+) Calc. for [C24H35N5O3] 441.57, found: 464.5 [M
+Na]+.
tert-Butyl ((S)-1-(((S)-1-amino-3-(3-chlorophenyl)-1-oxopropan-2-yl)amino)-1-oxo-
3-phenylpropan-2-yl)carbamate (43). Yield 80%. Yellowish solid. Rf = 0.2 (ethyl acetate).
Mp. 101–103 ˚C. 1H NMR (200 MHz, CDCl3:CD3OD 10:1) δ. 7.58–6.99 (m, 9H), 6.47 (s, 1H),
4.77–4.62 (m, 2H), 4.04 (s, 3H), 3.39–2.91 (m, 4H), 1.34 (s, 9H). 13C NMR (50 MHz, CDCl3:
CD3OD 10:1) δ 172.23, 172.21, 161.25, 161.03, 137.40, 136.96, 135.83, 135.15, 130.68, 129.87,
129.23, 128.42, 128.30, 127.69, 117.62, 104.55, 55.12, 49.00, 42.41, 38.76, 38.62, 32.51, 30.78.
ESI-MS (+) Calc. for [C27H32ClN5O3] 510.03, found: 532.9 [M+Na]+.
N-((S)-1-(((S)-1-Amino-1-oxo-3-(pyridin-4-yl)propan-2-yl)amino)-1-oxo-3-phenylpro-
pan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (44). Yield 66%. Yellowish
oil. Rf = 0.2 (ethyl acetate). 1H NMR (200 MHz, CDCl3) δ 8.63 (d, J = 5.7 Hz, 2H), 7.58–7.30
(m, 7H), 7.36–7.13 (m, 7H), 6.59 (s, 1H), 4.84–4.68 (m, 2H), 3.89 (s, 3H), 3.35–3.07 (m, 2H),
1.27 (s, 9H) .13C NMR (50 MHz, CDCl3) δ 174.42, 173.04, 161.51, 161.12, 149.40, 148.81,
138.04, 136.11, 129.86, 129.09, 127.45, 126.03, 104.55, 55.73, 54.08, 38.35, 37.93, 37.75, 32.45,
30.45. ESI-MS (+) Calc. for [C26H32N6O3] 476.57, found:477.3 [M+H]+.
N-((S)-1-(((2S,3R)-1-Amino-3-(benzyloxy)-1-oxobutan-2-yl)amino)-1-oxo-3-phenyl-
propan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (45). Yield 65%.
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New cysteine protease inhibitors as trypanocidal agents
Yellowish solid. Rf = 0.3 (ethyl acetate). Mp 109–110 ˚C. 1H NMR (200 MHz, CDCl3) δ 7.30–
7.08 (m, 9H), 6.40 (d, J = 14.4 Hz, 1H), 6.26 (s, 1H), 5.00–4.76 (m, 1H), 4.52 (s, 2H), 4.17–4.06
(m, 2H), 3.98 (s, 3H), 3.20–3.09 (m, 2H), 1.27 (s, 9H), 1.11 (d, J = 6.2 Hz, 3H). 13C NMR (50
MHz, CDCl3) δ 171.79, 171.19, 160.58, 160.20, 137.80, 136.25, 134.62, 129.26, 128.78, 128.44,
127.84, 127.78, 127.21, 103.32, 74.00, 71.58, 60.39, 56.64, 38.86, 38.61, 31.91, 30.49, 21.02.
ESI-MS (+) Calc. for [C29H37N5O4] 519.64, found:549.4 [M+Na]+.
N-((S)-1-(((2R,3S)-1-Amino-3-(benzyloxy)-1-oxobutan-2-yl)amino)-1-oxo-3-phenyl-
propan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (46). Yield 61%.
Yellowish solid. Rf = 0.3 (ethyl acetate). Mp 119–120 ˚C. 1H NMR (200 MHz, CDCl3) δ.7.27–
7.04 (m, 9H), 6.37 (d, J = 14.4 Hz, 1H), 6.22 (s, 1H), 4.97–4.73 (m, 1H), 4.48 (s, 2H), 4.14–4.03
(m, 2H), 3.95 (s, 3H), 3.17–3.06 (dd, J = 11.3, 5.1 Hz, 2H), 1.23 (s, 9H), 1.08 (d, J = 6.2 Hz, 3H).
13C NMR (50 MHz, CDCl3) δ 171.79, 171.19, 160.58, 160.20, 137.80, 136.25, 134.62, 129.26,
128.78, 128.44, 127.84, 127.78, 127.21, 103.32, 74.00, 71.58, 60.39, 56.64, 38.86, 38.61, 31.91,
30.49, 21.02. ESI-MS (+) Calc. for [C29H37N5O4] 519.64, found: 549.4 [M+Na]+.
N-((S)-1-(((2S,3R)-1-Amino-3-(benzyloxy)-1-oxobutan-2-yl)amino)-4-methyl-1-oxo-
pentan-2-yl)-3-(tert-butyl)-1-methyl-1H3-pyrazole-5-carboxamide (47). Yield 73%.
Yellowish solid. Rf = 0.3 (ethyl acetate). Mp 177–178 ˚C. 1H NMR (200 MHz, CDCl3) δ. 7.27–
7.20 (m, 4H), 7.14 (d, J = 8.5 Hz, 1H), 6.42 (s, 1H), 4.85–4.82 (m, 1H), 4.62–4.52 (s, 2H), 3.96
(s, 3H), 3.87–3.84 (m, 1H), 1.69–1.59 (m, 3H), 1.22 (s, 9H). 1.18 (d, J = 6.3 Hz, 3H), 0.90 (d,
J = 6.0 Hz, 3H), 0.97 (d, J = 6.0 Hz, 3H). 13C NMR (50 MHz, CDCl3) δ 172.35, 171.66, 160.37,
160.31, 137.82, 134.69, 128.57, 128.00, 127.74, 103.21, 74.07, 71.71, 56.24, 52.12, 39.03, 38.69,
32.02, 30.59, 25.00, 23.01, 22.0. ESI-MS (+) Calc. for [C26H39N5O4] 485.62, found: 508.5 [M
+Na]+.
N-((S)-1-(((2R,3S)-1-Amino-3-(benzyloxy)-1-oxobutan-2-yl)amino)-4-methyl-1-oxo-
pentan-2-yl)-3-(tert-butyl)-1-methyl-1H3-pyrazole-5-carboxamide (48). Yield 70%.
Yellowish solid. Rf = 0.3 (ethyl acetate). Mp 170–171 ˚C. 1H NMR (200 MHz, CDCl3) δ 7.33–
7.21 (m, 4H), 7.15 (d, J = 8.5 Hz, 1H), 6.34 (s, 1H), 4.85–4.82 (m, 1H), 4.64–4.53 (s, 2H), 3.98
(s, 3H), 3.88–3.86 (m, 1H), 1.71–1.60 (m, 3H), 1.24 (s, 9H), 1.21 (d, J = 6.3 Hz, 3H), 0.91 (d,
J = 6.0 Hz, 3H), 0.89 (d, J = 6.0 Hz, 3H). 13C NMR (50 MHz, CDCl3) δ 171.72, 171.02, 159.73,
159.68, 137.18, 134.05, 127.94, 127.36, 127.10, 102.58, 73.43, 71.08, 55.60, 51.49, 38.39, 38.06,
31.39, 29.95, 24.37, 22.38, 21.44. ESI-MS (+) Calc. for [C26H39N5O4] 485.62, found: 508.5 [M
+Na]+.
N-((S)-1-(((2S,3R)-1-Amino-3-(benzyloxy)-1-oxobutan-2-yl)amino)-3-(3-chlorophe-
nyl)-1-oxopropan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (49). Yield
65%. Yellowish solid. Rf = 0.2 (ethyl acetate). Mp 102–104 ˚C. 1H NMR (200 MHz, CD3OD) δ
8.59 (d, J = 7.8 Hz, 1H), 8.03 (d, J = 8.4 Hz, 1H), 7.26 (dd, J = 5.6, 2.2 Hz, 9H), 6.59 (s, 1H),
4.64–4.45 (m, 3H), 4.16–4.11 (m, 1H), 3.86 (s, 3H), 3.27–3.25 (m, 1H), 3.03 (dd, J = 14.7, 10.8
Hz, 1H), 1.28 (s, 9H), 1.22 (d, J = 6.3 Hz, 3H). 13C NMR (50 MHz, CD3OD) δ 174.44, 173.31,
162.05, 161.35, 140.90, 139.31, 136.28, 134.96, 130.78, 130.33, 129.08, 128.62, 128.52, 128.44,
127.71, 104.84, 75.78, 72.24, 58.45, 55.77, 38.46, 37.34, 32.66, 30.66, 16.58. ESI-MS (+) Calc. for
[C29H36ClN5O4] 554.08, found: 577.1 [M+Na]+.
Synthesis of compounds 50–60. Compounds 50–60 have been synthesized by dehydra-
tion of the corresponding primary amide precursor 39–49 with cyanuric chloride (method A).
3-(tert-Butyl)-N-((S)-1-(((S)-1-cyano-2-phenylethyl)amino)-1-oxo-3-phenylpropan-
2-yl)-1-methyl-1H-pyrazole-5-carboxamide (50). Yield 89%. Yellowish solid. Rf = 0.6 (ethyl
acetate: n-hexane; 6:4). Mp. 89–90 ˚C. 1H NMR (400 MHz, CDCl3) δ 7.32–7.21 (m, 8H), 7.16
(dd, J = 6.4, 2.8 Hz, 2H), 6.90 (d, J = 8.2 Hz, 1H), 6.82 (d, J = 8.2 Hz, 1H), 6.35 (s, 1H), 5.04–
5.02 (m, 1H), 4.82–4.80 (m, 1H), 4.02 (s, 3H), 3.21–3.07 (m, 2H), 3.00–2.98 (m, 2H), 1.33–1.30
(m, 9H). 13C NMR (100 MHz, CDCl3) δ 170.30, 160.25, 160.30, 135.55, 134.04, 133.37, 129.08,
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New cysteine protease inhibitors as trypanocidal agents
129.04, 128.75, 128.72, 127.71, 127.26, 117.25, 103.06, 54.17, 41.48, 38.77, 38.35, 37.95, 31.75,
30.26. FT-IR (KBr, cm-1) 3300.81, 2951.21, 2909.60, 2243.70, 1677.69, 1648.55, 1544.51,
1278.15, 1232.37, 986.82, 751.92, 670.52, 524.68, 424.97. HRMS (+) Calc. for [C27H32N5O2]+
458.25560, found: 458.2586 [M+H]+. HPLC (protocol A): tR (min) = 9.07. Purity 99.0%.
3-(tert-Butyl)-N-((S)-1-(((R)-1-cyano-2-phenylethyl)amino)-1-oxo-3-phenylpropan-
2-yl)-1-methyl-1H-pyrazole-5-carboxamide (51). Yield 92%. Yellowish solid. Rf = 0.6 (ethyl
acetate: n-hexane; 6:4). Mp. 98–99 ˚C. 1H NMR (200 MHz, CDCl3) δ 7.34–7.12 (m, 9H), 6.91
(d, J = 7.9 Hz, 1H), 6.34 (s, 1H), 5.09–5.05 (m, 1H), 4.94–4.91 (m, 1H), 4.00 (s, 3H), 3.12 (dd,
J = 13.8, 6.4 Hz, 2H), 2.98 (dd, J = 13.8, 6.4 Hz, 2H), 1.30 (s, 9H). 13C NMR (50 MHz, CDCl3) δ
170.75, 160.43, 160.33, 135.98, 134.32, 133.78, 129.42, 129.33, 129.01, 128.86, 127.99, 127.42,
118.87, 103.48, 54.38, 41.82, 38.96, 38.48, 38.26, 31.98, 30.51. FT-IR (KBr, cm-1) 3350.28,
3256.30, 2953.92, 2165.27, 1638.14, 1511.6, 1307.15, 1160.05, 1086.49, 906.70, 820.89, 694.21,
669.69, 543.02. HRMS (+) Calc. for [C27H32N5O2]+ 458.25560, found: 458.2586 [M+H]+.
HPLC (protocol A): tR (min) = 17.32. Purity 99.3%.
3-(tert-Butyl)-N-((S)-1-(((S)-1-cyano-3-methylbutyl)amino)-1-oxo-3-phenylpropan-2-yl)-
1-methyl-1H-pyrazole-5-carboxamide (52). Yield 84%. Yellowish solid. Rf = 0.7 (ethyl acetate:
n-hexane; 6:4). Mp 81–82 ˚C. 1H NMR (200 MHz, CD3OD) δ 7.29–7.20 (m, 5H), 6.64 (s, 1H),
4.80–4.69 (m, 2H), 3.90 (s, 3H), 3.23–2.94 (m, 2H), 1.78–1.62 (m, 3H), 1.27 (s, 9H), 0.95–0.91 (m,
6H). 13C NMR (50 MHz, CD3OD) δ 173.33, 161.87, 161.51, 138.07, 136.59, 130.36, 129.56,
127.93, 119.68, 105.02, 56.04, 49.00, 42.00, 40.04, 38.69, 32.86, 30.88, 25.80, 22.35, 22.19. FT-IR
(KBr, cm-1) 3403.41, 3309.42, 3203.18, 2953.92, 2937.57, 1695.35, 1634.05, 1523.72, 1368.44,
1274.46, 1245.86, 1168.22, 835.58, 702.38, 669.69, 567.54. HRMS (+) Calc. for [C24H34N5O2]+
423.27125, found: 424.27572 [M+H]+. HPLC (protocol A): tR (min) = 9.16. Purity 97.4%.
3-(tert-Butyl)-N-((S)-1-(((S)-1-cyano-3-methylbutyl)amino)-1-oxo-3-phenylpropan-
2-yl)-1-methyl-1H-pyrazole-5-carboxamide (53). Yield 79%. White solid. Rf = 0.7 (ethyl
Acetate: n-hexane; 6:4). Mp. 91–94 ˚C. 1H NMR (200 MHz, CDCl3) δ 7.32–7.21 (m, 3H), 6.98
(d, J = 8.2 Hz, 1H), 6.88 (d, J = 8.2 Hz, 1H), 6.34 (s, 1H), 4.92–4.86 (m, 1H), 4.77–4.71 (m, 1H),
3.99 (s, 3H), 3.29–3.19 (m, 2H), 1.63–1.46 (m, 3H), 1.27 (s, 9H), 0.88 (2d, J = 5.9 Hz, 6H). 13
NMR (50 MHz, CDCl3) δ 171.28, 161.15, 161.04, 136.68, 135.04, 129.97, 129.56, 128.05,
C
119.17, 104.16, 55.16, 41.95, 39.62, 39.12, 32.67, 31.19, 25.31, 22.79, 22.53. FT-IR (KBr, cm-1)
3350.28, 3256.30, 2953.92, 2165.27, 1638.14, 1511.46, 1307.15, 1160.05, 1086.49, 906.70,
820.89, 694.21, 669.69, 543.02. HRMS (+) Calc. for [C24H34N5O2]+ 423.27125, found:
424.27572 [M+H]+. HPLC (protocol A): tR (min) = 9.42. Purity 99.1%.
3-(tert-Butyl)-N-((S)-1-(((S)-2-(3-chlorophenyl)-1-cyanoethyl)amino)-1-oxo-3-phenyl-
propan-2-yl)-1-methyl-1H-pyrazole-5-carboxamide (54). Yield 50%. Yellowish solid. Rf =
0.6 (ethyl acetate: n-hexane; 6:4). Mp 132–133 ˚C. 1H NMR (200 MHz, CD3OD/ CDCl3) δ
7.54–7.35 (m, 9H), 6.75 (s, 1H), 5.19–5.11 (m, 1H), 4.51–4.48 (m, 1H), 4.17 (s, 3H), 3.33–3.21
(m, 4H), 1.49 (s, 9H). 13C NMR (50 MHz, CD3OD) δ 172.23, 161.25, 161.03, 137.40, 136.96,
135.83, 135.15, 130.68, 130.07, 129.87, 129.23, 128.42, 128.30, 127.69, 118.18, 104.55, 55.12,
42.41, 38.76, 38.62, 32.51, 30.78. FT-IR (cm-1) 3288.99, 2925.31, 2855.85, 2161.18, 1666.74,
1634.05, 1544.15, 1507.38, 1450.17, 1266.29, 1221.34, 1074.23, 861.75, 747.33, 706.47, 694.21.
HRMS (+) Calc. for [C27H31ClN5O2]+ 491.21663, found: 492.21034 [M+H]+. HPLC (protocol
A): tR (min) = 9.61. Purity > 99.9%.
3-(tert-Butyl)-N-((S)-1-(((S)-1-cyano-2-(pyridin-4-yl)ethyl)amino)-1-oxo-3-phenylpro-
pan-2-yl)-1-methyl-1H-pyrazole-5-carboxamide (55). Yield 35%. Yellowish wax. R = 0.4
(ethyl acetate: n-hexane; 6:4). 1H NMR (400 MHz, CD3OD) δ 8.44 (s br, 2H), 7.38 (d, J = 5.3
Hz, 2H), 7.30–7.22 (m, 5H), 6.63 (s, 1H), 5.14 (t, J = 7.5 Hz, 1H), 4.66 (dd, J = 8.6, 6.8 Hz, 1H),
3.94 (s, 3H), 3.26–3.13 (m, 4H), 1.30 (s, 9H). 13C NMR (100 MHz, DMSO-d6) δ 171.17, 170.90,
159.29, 158.46, 137.89, 136.81, 135.23, 134.81, 129.27, 128.86, 128.13, 127.92, 126.93, 126.34,
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New cysteine protease inhibitors as trypanocidal agents
118.94, 60.46, 53.87, 53.24, 41.15, 31.39, 30.18, 13.74. FT-IR (cm-1) 3264.47, 2953.92, 2913.05,
2851.76, 2161.43, 1662.66, 1605.45, 1548.24, 1466.62, 1204.99, 1115.10, 996.59, 800.45, 735.07,
681.95. HRMS (+) Calc. for [C26H31N6O2]+ 459.25085, found: 459.25627 [M+H]+. HPLC (pro-
tocol A): tR (min) = 6.84. Purity 98.6%.
N-((S)-1-(((1R,2R)-2-(Benzyloxy)-1-cyanopropyl)amino)-1-oxo-3-phenylpropan-2-yl)-
3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (56). Yield 80%. White solid. Rf = 0.5
(ethyl acetate: n-hexane; 6:4). Mp. 115–116 ˚C. 1H-NMR (200 MHz, CDCl3) δ 7.35–7.26 (m,
10H), 6.95 (d, J = 8.7 Hz, 1H), 6.61 (d, J = 7.0 Hz, 1H), 6.30 (s, 1H), 4.88–4.85 (m, 2H), 4.62–
4.58 (m, 1H), 4.03 (s, 3H), 3.82–3. 78 (m, 1H), 3.29–3.24 (m, 2H), 1.31 (s, 9H), 1.03 (d, J = 6.0
Hz, 3H). 13C NMR (50 MHz, CDCl3) δ 170.30, 159.83, 159.62, 136.42, 135.27, 133.75, 128.67,
128.41, 128.00, 127.60, 127.30, 126.87, 116.63, 102.65, 72.60, 70.91, 53.85, 44.51, 38.33, 37.60,
31.40, 29.94, 15.29. FT-IR (cm-1) 3354.37, 3252.21, 2181.61, 1654.61, 1654.48, 1540.07,
1486.95, 1290.81, 1151.87, 1086.48, 894.44, 808.63, 710.56, 661.32, 543.02. HRMS (+) Calc. for
[C29H36N5O3]+ 502.28182, found: 502.28095 [M+H]+. HPLC (protocol A): tR (min) = 9.54.
Purity 99.4%.
N-((S)-1-(((1S,2S)-2-(Benzyloxy)-1-cyanopropyl)amino)-1-oxo-3-phenylpropan-2-yl)-
3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (57). Yield 82%. White solid. Rf = 0.5
(ethyl acetate: n-hexane; 6:4). Mp. 123–124 ˚C. 1H NMR (200 MHz, CDCl3) δ 7.36–7.25 (m,
10H), 6.98 (d, J = 8.7 Hz, 1H), 6.65 (d, J = 7.0 Hz, 1H), 6.32 (s, 1H), 4.91–4.83 (m, 2H), 4.63–
4.60 (m, 1H), 4.05 (s, 3H), 3.80 (s br, 1H), 3.24–3.20 (m, 2H), 1.33 (s, 9H), 1.05 (d, J = 6.0 Hz,
3H). 13C NMR (50 MHz, CDCl3) δ 171.01, 160.54, 160.34, 137.14, 135.98, 134.46, 129.39,
129.12, 128.71, 128.32, 128.02, 127.59, 117.34, 103.37, 72.85, 71.15, 54.56, 45.22, 39.04, 38.32,
32.12, 30.65, 16.00. FT-IR (cm-1) 2376.73, 2958.00, 2169.35, 1642.22, 15400.07, 1499.21,
1442.00, 1290.81, 1225.43, 1111.01, 1033.37, 988.42, 743.25, 690.12. HRMS (+) Calc. for
[C29H36N5O3]+ 502.28182, found: 502.28095 [M+H]+. HPLC (protocol A): tR (min) = 9.43.
Purity 98.3%.
N-((S)-1-(((1R,2R)-2-(Benzyloxy)-1-cyanopropyl)amino)-4-methyl-1-oxopentan-2-yl)-
3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (58). Yield 78%. White solid. Rf = 0.5
(ethyl acetate: n-hexane; 6:4). Mp. 96–97 ˚C. 1H NMR (400 MHz, DMSO-d6) δ 8.93 (d, J = 8.1
Hz, 1H), 8.43 (d, J = 7.6 Hz, 1H), 7.38–7.29 (m, 5H), 6.88 (s, 1H), 5.07–5.04 (m, 1H), 4.64–4.61
(m, 2H), 4.59–4.56 (m, 1H), 3.97–3.94 (m, 3H), 3.86–3.84 (m, 1H), 1.72–1.64 (m, 2H), 1.50–
1.46 (m, 2H), 1.26–1.20 (m, 12H), 0.93 (d, J = 6.3 Hz, 3H), 0.88 (d, J = 6.5 Hz, 3H). 13C NMR
(100 MHz, DMSO-d6) δ 172.36, 159.56, 158.66, 137.92, 134.92, 128.10, 127.53, 127.45, 117.82,
103.73, 73.39, 70.40, 51.05, 44.75, 38.44, 31.53, 30.31, 24.25, 22.91, 21.19, 15.69. FT-IR (cm-1)
3378.89, 3350.28, 3186.83, 2958.00, 2116.43, 1674.91, 1650.40, 1531.90, 1368.44, 1262.20,
1160.05, 1057.89, 1033.37, 780.02, 735.07, 649.26, 604.31. HRMS (+) Calc. for [C26H38N5O3]
468.29746, found: 468.29785 [M+H]+. HPLC (protocol A): tR (min) = 9.61. Purity 98.3%.
N-((S)-1-(((1S,2S)-2-(Benzyloxy)-1-cyanopropyl)amino)-4-methyl-1-oxopentan-2-yl)-
3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (59). Yield 70%. White solid. Rf = 0.5
(ethyl acetate: n-hexane; 6:4). Mp. 110–111 ˚C. 1H NMR (400 MHz, CDCl3) δ 7.27–7.20 (m,
5H), 7.14 (d, J = 8.5 Hz, 1H), 6.42 (d, J = 7.8 Hz, 1H), 6.33 (s, 1H), 4.85–4.82 (m, 1H), 4.62–
4.52 (m, 3H), 3.96 (s, 3H), 3.87–3.84 (m, 1H), 1.69–1.59 (m, 3H), 1.22 (s, 9H). 1.18 (d, J = 6.3
Hz, 3H) 1.24–1.19 (m, 12H), 0.92–0.88 (m, 6H). 13C NMR (100 MHz, CDCl3) δ 172.10,
160.65, 160.51, 137.21, 134.43, 128.66, 128.23, 127.92, 117.45, 103.16, 73.69, 71.80, 51.75, 45.28,
40.81, 38.06, 32.10, 30.62, 25.04, 22.97, 22.13, 16.36. FT-IR (cm-1) 3333.94, 3284.90, 2962.09,
2868.10, 2255.16, 1647.44, 1540.07, 1507.38, 1442.00, 1274.46, 1245.86, 988.42, 739.16, 661.52.
HRMS (+) Calc. for [C26H38N5O3] 468.29746, found: 468.29785 [M+H]+. HPLC (protocol A):
tR (min) = 9.56. Purity 96.9%.
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N-((S)-1-(((1R,2R)-2-(Benzyloxy)-1-cyanopropyl)amino)-3-(3-chlorophenyl)-1-oxopro-
pan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (60). Yield 50%. Yellowish
wax. Rf = 0.4 (ethyl acetate: n-hexane; 6:4). 1H NMR (400 MHz, CDCl3) δ 7.30–7.13 (m, 6H),
7.04 (dt, J = 7.0, 1.7 Hz, 1H), 6.62 (dd, J = 17.8, 8.2 Hz, 2H), 6.25 (s, 1H), 4.77–4.71 (m, 2H),
4.48 (dd, J = 36.1, 11.7 Hz, 2H), 3.95 (s, 3H), 3.82–3.74 (m, 1H), 3.11–3.00 (m, 2H), 1.20 (s,
9H), 1.08 (d, J = 6.3 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 170.73, 160.54, 160.12, 137.80,
136.91, 134.84, 134.30, 130.44, 129.51, 128.71, 128.34, 128.03, 127.84, 127.62, 117.06, 103.35,
73.35, 71.72, 54.28, 45.07, 39.06, 38.13, 32.07, 30.58, 16.21. FT-IR (cm-1) 3357.24, 3264.21,
2193.15, 1654.33, 1538.29, 1487.88, 1209.18, 1141.78, 1036.43, 863.33, 807.36, 701.44, 658.23,
534.57. ESI-MS (+) Calc. for [C29H345ClN5O3] 536.24284, found: 536.24235 [M+H]+.HPLC
(protocol B): tR (min) = 12.06. Purity 96.5%.
Synthesis of compounds 61–64. Compounds 61–64 have been synthesized from com-
pounds 34–37 by removal of the benzyl group through hydrogenation on Pd/C (method A).
N-((S)-1-(((2S,3R)-1-Amino-3-hydroxy-1-oxobutan-2-yl)amino)-1-oxo-3-phenylpro-
pan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide.(61). Yield 92%. Colorless
wax. Rf = 0.2 (ethyl acetate). 1H NMR (200 MHz, CDCl3) δ 8.03 (s, 1H), 7.36–6.94 (m, 5H),
6.54 (s, 1H), 4.44–4.39 (m, 2H), 4.03–4.01 (m, 1H), 3.96 (s, 3H), 3.18–3.14 (m, 1H), 2.87 (s,
1H), 1.30 (s, 9H), 1.03–1.01 (m, 3H). 13C NMR (50 MHz, CDCl3) δ 173.54, 172.15, 161.25,
160.35, 136.25, 135.98, 134.58, 129.36, 128.76, 127.25, 66.60, 58.51, 55.55, 54.30, 38.70, 31.98,
30.51, 18.92. ESI-MS (+) Calc. for [C22H31N5O4] 429.51, found:452.4 [M+Na]+.
N-((S)-1-(((2R,3S)-1-Amino-3-hydroxy-1-oxobutan-2-yl)amino)-1-oxo-3-phenylpro-
pan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide.(62). Yield 95%. Colorless
wax. Rf = 0.2 (ethyl acetate). 1H NMR (200 MHz, CDCl3) δ 8.01 (s, 1H), 7.36–6.98 (m, 5H),
6.59 (s, 1H), 4.42–4.36 (m, 2H), 4.04–4.01 (m, 1H), 3.96 (s, 3H), 3.12–3.09 (m, 1H), 2.89 (s,
1H), 1.33 (s, 9H), 1.03–0.99 (m, 3H). 13C NMR (50 MHz, CDCl3) δ 173.54, 172.15, 161.25,
160.35, 136.25, 135.98, 134.58, 129.36, 128.76, 127.25, 66.60, 58.51, 55.55, 54.30, 38.70, 31.98,
30.51, 18.92. ESI-MS (+) Calc. for [C22H31N5O4] 429.51, found: 452.4 [M+Na]+.
N-((S)-1-(((2S,3R)-1-Amino-3-hydroxy-1-oxobutan-2-yl)amino)-4-methyl-1-oxopen-
tan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide(63). Yield 90%. Colorless
wax. Rf = 0.2 (ethyl acetate). 1H NMR (200 MHz, CDCl3) δ 7.37 (d, J = 3.9 Hz, 1H), 6.74–6.71
(m, 2H), 6.42 (s, 1H), 4.71–4.68 (m, 1H), 4.42–4.38 (m, 2H), 4.06 (s, 3H), 1.71–1.68 (m, 3H),
1.27 (s, 9H), 1.14–1.11 (m, 3H), 0.98–0.92 (m, 6H). ESI-MS (+) Calc. for [C19H33N5O4] 395.51,
found: 418.5 [M+Na]+.
N-((S)-1-(((2R,3S)-1-Amino-3-hydroxy-1-oxobutan-2-yl)amino)-4-methyl-1-oxopen-
tan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (64). Yield 93%. Colorless
wax. Rf = 0.2 (ethyl acetate). 1H NMR (200 MHz, CDCl3) δ 7.34 (d, J = 3.9 Hz, 1H), 6.79–6.75
(m, 2H), 6.46 (s, 1H), 4.73–4.70(m, 1H), 4.45–4.41 (m, 2H), 4.11 (s, 3H), 1.77–1.74 (m, 3H),
1.28 (s, 9H), 1.19–1.17 (m, 3H), 0.99–0.91 (m, 6H). ESI-MS (+) Calc. for [C19H33N5O4] 395.51,
found: 418.5 [M+Na]+.
Synthesis of compounds 65–68. Compounds 65–68 have been synthesized by dehydra-
tion of the corresponding primary amide precursor 61–64 with trifluoroacetic anhydride
(method B).
3-(tert-Butyl)-N-((S)-1-(((1R,2R)-1-cyano-2-hydroxypropyl)amino)-1-oxo-3-phenyl-
propan-2-yl)-1-methyl-1H-pyrazole-5-carboxamide (65). Yield 45%. White solid. Rf = 0.7
(ethyl acetate). Mp. 159–160 ˚C. 1H NMR (500 MHz, CDCl3) δ 7.32–7.26 (m, 4H), 7.26–7.21
(m, 1H), 6.65 (s, 1H), 4.84–4.79 (m, 2H), 3.96 (s, 3H), 3.88–3.84 (m, 1H), 3.20 (dd, J = 13.6, 7.1
Hz, 1H), 3.10 (dd, J = 13.6, 7.1 Hz, 1H), 1.31 (s, 9H), 1.07 (d, J = 6.3 Hz, 3H). 13C NMR (125
MHz, CDCl3) δ 173.35, 162.06, 161.60, 138.15, 136.69, 130.42, 129.57, 127.98, 118.24, 105.04,
67.67, 56.05, 38.86, 38.68, 32.90, 30.87, 18.89, 18.55. FT-IR (KBr, cm-1) 3293.08, 2953.92,
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2913.05, 2868.10, 1646.10, 1540.07, 1446.08, 1286.72, 1237.68, 1098.75, 922.51, 739.16, 689.30,
469.47. HRMS (+) Calc. for [C22H30N5O3]+ 412.23486, found: 412.23811 [M+H]+. HPLC (pro-
tocol A): tR (min) = 7.65. Purity 97.8%.
3-(tert-Butyl)-N-((S)-1-(((1S,2S)-1-cyano-2-hydroxypropyl)amino)-1-oxo-3-phenylpro-
pan-2-yl)-1-methyl-1H-pyrazole-5-carboxamide (66). Yield 42%. White solid. Rf = 0.7
(ethyl acetate). Mp. 145–147 ˚C. 1H NMR (400 MHz, CDCl3) δ 7.31–7.23 (m, 5H), 7.07 (d,
J = 8.0 Hz, 1H), 6.67 (d, J = 7.5 Hz, 1H), 6.31 (s, 1H), 4.85–4.84 (m, 1H), 4.72–4.68 (m, 1H),
4.15–4.10 (m, 1H), 4.02 (s, 3H), 3.21–3.15 (m, 2H), 1.29–1.27 (m, 9H), 1.08 (d, J = 6.2 Hz, 3H).
13C NMR (100 MHz, CDCl3) δ 172.41, 171.26, 160.58, 135.89, 134.27, 129.38, 129.14, 127.65,
117.25, 103.52, 67.39, 54.76, 47.02, 39.11, 38.38, 32.09, 30.59, 19.02. FT-IR (KBr, cm-1) 3289.11,
2955.43, 2918.55, 2860.11, 1649.54, 1543.16, 1444.02, 1277.62, 1233.66, 1091.87, 944.77,
745.55, 690.34, 477.11. HRMS (+) Calc. for [C22H30N5O3]+ 412.23486, found: 412.23811 [M
+H]+.HPLC (protocol A): tR (min) = 7.71. Purity 95.7%.
3-(tert-Butyl)-N-((S)-1-(((1R,2R)-1-cyano-2-hydroxypropyl)amino)-4-methyl-1-oxo-
pentan-2-yl)-1-methyl-1H-pyrazole-5-carboxamide (67). Yield 33%. White solid. Rf = 0.5
(ethyl acetate). Mp. 118–119 ˚C. 1H NMR (400 MHz, CDCl3) δ 7.56–7.51 (m, 1H), 6.80–6.75
(m, 1H), 6.43 (s, 1H), 4.87–4.83 (m, 1H), 4.65–4.63 (m, 1H), 4.15–4.11 (m, 1H), 4.06 (s, 3H),
1.31–1.20 (m, 15H), 0.97 (2d, J = 6.2 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 174.32, 161.62,
162.14, 135.77, 117.22, 104.56, 67.34, 52.68, 41.12, 38.28, 31.46, 32.15, 26.11, 23.56, 21.65, 19.03.
FT-IR (KBr, cm-1) 3297.16, 2953.92, 2913.05, 2868.10, 1646.31, 1535.89, 1503.29, 1462.52,
1364.36, 1282.63, 1172.38, 1131.44, 996.59, 730.99, 592.05. HRMS (+) Calc. for [C19H32N5O3]+
378.25071, found: 378.25231 [M+H]+.HPLC (protocol A): tR (min) = 8.08. Purity: 96.7%.
3-(tert-Butyl)-N-((S)-1-(((1S,2S)-1-cyano-2-hydroxypropyl)amino)-4-methyl-1-oxopen-
tan-2-yl)-1-methyl-1H-pyrazole-5-carboxamide (68). Yield 35%. White solid. Rf = 0.5 (ethyl
acetate). Mp. 111–113 ˚C. 1H NMR (400 MHz, CD3OD) δ 6.78 (s, 1H), 4.88–4.84 (m, 1H), 4.65–
4.60 (m, 1H), 4.07–4.05 (m, 1H), 4.05 (s, 3H), 1.81–1.61 (m, 4H), 1.32 (s, 9H), 1.26 (d, J = 6.3
Hz, 3H), 1.00 (2d, J = 6.3 Hz, 6H). 13C NMR (100 MHz,CD3OD) δ 175.01, 162.46, 161.76,
136.76, 118.59, 105.26, 68.00, 53.28, 41.76, 39.00, 33.07, 31.04, 26.27, 23.54, 21.94, 19.44. FT-IR
(KBr, cm-1) 3293.08, 2962.09, 2917.14, 2847.67, 1646.31, 1540.07, 1458.34, 1278.55, 1131.44,
1078.32, 1000.68, 853.58, 812.71, 780.02, 730.99, 559.36. HRMS (+) Calc. for [C19H32N5O3]+
378.25071, found: 378.25231 [M+H]+. HPLC (protocol A): tR (min) = 7.94. Purity: 98.6%.
Synthesis of compound 69. Compound 69 has been synthesized by removal of the benzyl
group from compound 60 with DDQ (method B).
3-(tert-butyl)-N-((S)-3-(3-chlorophenyl)-1-(((1R,2R)-1-cyano-2-hydroxypropyl)
amino)-1-oxopropan-2-yl)-1-methyl-1H-pyrazole-5-carboxamide (69). Yield 32%. White
solid. Rf = 0.3 (ethyl acetate). Mp. 102–103 ˚C. 1H NMR (400 MHz, CD3OD) δ 7.35–7.28 (m,
4H), 6.69 (s, 1H), 4.87–4.83 (m, 2H), 3.99 (s, 3H), 3.90–3.84 (m, 1H), 3.24 (dd, J = 13.6, 7.1 Hz,
1H), 3.08 (dd, J = 13.6, 6.5 Hz, 1H), 1.34 (s, 9H), 1.11 (d, J = 6.3 Hz, 3H). 13C NMR (100 MHz,
CD3OD) δ 175.23, 163.23, 151.94, 151.48, 128.04, 126.57, 120.30, 120.10, 119.52, 119.45,
117.86, 108.12, 94.92, 57.55, 45.93, 28.74, 28.56, 22.78, 20.75, 18.78. FT-IR (KBr, cm-1) 3284.90,
3056.07, 2958.00, 2925.31, 2868.10, 1638.14, 1556.41, 1499.21, 1462.43, 1364.36, 1270.37,
1229.51, 1098.75, 878.09, 730.99, 702.38, 453.12. ESI-MS (+) Calc. for [C22H29ClN5O3]+
446.19589, found: 446.19745 [M+H]+.HPLC (protocol A): tR (min) = 8.55. Purity 99.7%.
Enzyme inhibition studies
Enzyme inhibition studies for Cz and LmCPB were performed as previously described for Cz
[15]. The experimental procedures are described in the Supporting Information (see chapter
S3). Cathepsins B, L, S were assayed as reported [18,19].
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New cysteine protease inhibitors as trypanocidal agents
All experiments were performed in triplicate and for the most potent compounds (pKi >
7.5) two other independent experiments were performed.
Cathepsin K assay. Human recombinant cathepsin K was assayed on a FLUOSTAR
Optima plate reader at 25 ˚C with an excitation wavelength of 360 nm and an emission wave-
length of 440 nm on a 96 well plate. The enzyme solution (23 μg/mL in 50 mM sodium acetate
pH 5.5, 50 mM NaCl, 0.5 mM EDTA, 5 mM DTT) was diluted 1:100 with assay buffer (100
mM sodium citrate buffer pH 5.0, 100 mM NaCl, 1 mM EDTA, 0.01% CHAPS) containing 5
mM DTT and was then incubated at 37 ˚C for 30 min for activation. A 1.5 mM stock solution
of the substrate Z-Leu-Arg-AMC was prepared in DMSO. The final substrate concentration
was 6 μM (= 3.05 × Km). The assay was performed with a final concentration of cathepsin K of
1.73 ng/mL. Stock solutions of inhibitors were prepared in DMSO. The final DMSO concen-
tration was 2% (4 μL). Into a well containing 194.5 μL assay buffer, 0.8 μL of the fluorogenic
substrate, DMSO and inhibitor solution (3.2 μL) were added. Upon addition of cathepsin K
(1.5 μL), the measurement was started and followed for 20 min.
Mammalian cytotoxicity assay
LLCMK2 cells were cultured in 96-well plates at a concentration of 5 x 104 cells/ml. After 48 h,
the plates were washed twice with PBS and 200 μL RPMI medium was added with serial dilu-
tions of the compounds and benznidazole (1.95 μM to 250 μM) in triplicate. After 72 h at 37
˚C, the cytotoxic activity of the compounds was determined by the classical MTT [3-
(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide] method. Briefly, 50 μL MTT dis-
solved in PBS (2.0 mg mL-1) was added to each well and the plates were incubated for 4 h at 37
˚C. The formed formazan crystals were dissolved with DMSO (50 μL/well) and the absorbance
of the samples was measured by spectrophotometer at 570 nm after 30 min. The cytotoxicity
results (CC50) were calculated as a percentage by the formula [(ABScontrol-ABSample / ABS
control) � 100], where ABScontrol represents the mean absorbance of the untreated control
(viable cells) and ABSample, the absorbance of each cellular treatment.
All experiments were performed in triplicate and for the most potent compounds (EC50
100) two other independent experiments were performed.
<
In vitro trypanocidal activity evaluation on intracellular amastigote forms
(Tulahuen strain)
Cells were evaluated in 96-well plates. Cells from the LLCMK2 lineage were plated at a concen-
tration of 5x104 cells mL-1. Trypomastigote forms of the Tulahuen LacZ strain were added at a
concentration of 5×105 parasites mL-1 and placed in the incubator at 37˚C with 5% CO2 for 24
h. After the incubation period, the trypomastigote forms present were removed by successive
washes with PBS, remaining only as intracellular amastigote forms. Compounds were added at
different concentrations (1.95 μM to 250 μM serial dilutions) and incubated for 72 h. At the
end of this period, the substrate CPRG (chlorophenol red β-D-galactopyranoside, 400 μM in
0.3% Triton X-100, pH 7.4) was added. After 4 h of incubation at 37 ˚C, the plates were ana-
lyzed in a spectrophotometer at 570 nm to obtain the effective concentration (EC50) to reduce
the parasitemia inside the host cell. Benznidazole was used as a positive control in the same
concentrations as the substances, and DMSO as a negative control. Compounds were solubi-
lized in DMSO. The selectivity index (SI) was calculated using the formula: SI = EC50/CC50.
All statistical analyses were done with the program GraphPad Prism v.5.
All experiments were performed in triplicate and for the most potent compounds (CC50
100) two other independent experiments were performed.
<
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New cysteine protease inhibitors as trypanocidal agents
Results
Structure-based design, modeling studies, and compound synthesis
Cz, the recombinant form of cruzipain, is a monomeric enzyme, composed of two folded and
equally sized domains. These domains are divided by the enzyme’s active site, which is V-
shaped and primarily exposed to solvent. A catalytic triad cysteine-histidine-asparagine forms
the active site [14]. The main polar interactions between the protein and inhibitor are well con-
served involving the residues Gln19, Gly66, Asp161, His162, and Trp184 (residue numbers
correspond to the cruzipain sequence) of the enzyme. Cz is a cathepsin L-like cysteine protease
and is closely related to the mammalian CPs such as CatB, CatK, CatL, and CatS.
A variety of studies have been conducted on optimization strategies for the interactions of
different classes of inhibitors with the S1, S2 and S3 binding sites of cruzain and related cyste-
ine proteases [12,14,15,20]. Nonetheless, far less is known about the attainable interactions at
S10 for dipeptidyl nitrile inhibitors [21]. The high-resolution crystal structure of cruzain shows
that there is a large open surface characterized by Trp177 in the primed binding site region
(Fig 3) [22]. The design of compounds to exploit this cavity would provide enhanced enzyme-
inhibitor interactions. This concept has already been applied for a class of different dipeptidic
vinyl sulfone inhibitors [23]. As Fig 3 (left) exemplarily illustrates, the substituents of vinyl sul-
fone inhibitors predominantly sit on top of the shelf formed by residues Ser139, Met142 and
Asp158 rather than adopting an orientation for an intense aromatic–aromatic interactions
with Trp177. The nitrile inhibitor 33L does not bear an appropriate substituent that would
allow for interaction with the primed binding region of cruzain (Fig 3, right).
The nitrile warhead has been applied successfully for a variety of series of cathepsin inhibi-
tors. Peptidic nitriles are known to interact with the active site cysteine by forming a covalent,
but reversible thioimidate adduct [24]. The nitrile warhead was also repurposed for Cz inhibi-
tion as trypanocidal agents, displaying low toxicity, probably due to the reversible character of
interaction [25]. Therefore, starting from our recent study on dipeptidyl nitriles as trypanoci-
dal agents, we expanded our previous inhibitor series to map the S1/S1´ subsites of Cz [14]. By
applying a knowledge-based design approach, we have explored different amino acids as possi-
ble building blocks for the P1 moiety. Based on a template crystal structure of the dipeptidyl
nitrile inhibitor 33L bound to Cz (PDB ID: 4QH6), structural modifications have been exe-
cuted that might increase the affinity towards the S1’ specificity pocket. Fig 4 shows dipeptidyl
nitriles 50, 52, 56, and 58 with different lipophilic substitution patterns at the P1 position,
which were assumed to accommodate the S1´ pocket through hydrophobic interactions with-
out interfering in the general mode of binding.
Fig 3. Crystal structures of vinyl sulfone derivative K777 and dipeptidyl nitrile 33L covalently bound to cruzain.
Left. K777 in the active site of cruzain (PDB-ID: 1F2B). Right 33L in the active site of cruzain (PDB-ID 4QH6).
https://doi.org/10.1371/journal.pntd.0007755.g003
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New cysteine protease inhibitors as trypanocidal agents
Fig 4. The putative orientation of P1 moieties in compounds 50, 52, 56, and 58.
https://doi.org/10.1371/journal.pntd.0007755.g004
Possible interactions of inhibitors with residues forming the S1 and S1’ pockets of cruzain
(PDB ID: 4QH6). Residue numbers are referring to the cruzipain sequence.
Compound 9 (Fig 5) was adopted as an archetype, with the cyclopropyl group at P1 position
and phenylalanine in P2 as well as a pyrazole moiety in P3 for advantageous interactions with
the non-primed binding region of the target protease. We mainly used different natural and
unnatural amino acids for the P1 moiety and maintained the nitrile warhead. Leucine (Leu)
and phenylalanine (Phe) were incorporated (compounds 50–54, Fig 6) as molecular sensors in
P1 for aliphatic and aromatic interactions. 4-Pyridylalanine was implemented to leverage the
affinity by polar interaction with Asp161 (S1´). Thr-O-Bzl, an unusual building block for
peptide inhibitors, was used as a chimera in P1 for aliphatic and aromatic interactions in the
S1/S1´ area. After removal of the benzyl protecting group from Thr-O-Bzl, the so produced
alcoholic moiety should allow the evaluation of whether a hydrogen bond donor is tolerated in
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New cysteine protease inhibitors as trypanocidal agents
Fig 5. Structure representation of compounds 6–19.
https://doi.org/10.1371/journal.pntd.0007755.g005
the S1/S1´ area. Moreover, it was intended to investigate how the stereochemistry in this
region will influence the affinity with Cz.
3-(tert-Butyl)-1-methyl-1H-pyrazole-5-carboxylic acid is a privileged building block
applied for the inhibition of Cz and CatL [26]. Thus, we have explored some possible bioisos-
teres in order to increase the affinity and the selectivity towards Cz, such as 7-chloroquinoline
carboxylic acid, 1H-indole-5-carboxylic acid, or 6-aminonicotinic acid.
Accordingly, we synthesized a new series of dipeptidyl nitriles (Figs 1 and 2). For com-
pounds 6–12 and 14–19 bearing a cyclopropyl moiety in P1, the synthesis was carried out as
known from the literature [15]. The peptide coupling reaction was performed twice, first to
connect the enantiomerically pure, Boc-protected P2 amino acid with the aminonitrile moiety,
and secondly, after removing the Boc group, to introduce the corresponding aroyl acids (Fig
1). Compound 19 was synthesized from compound 12 by removing the benzyl group under
mild oxidative conditions (Fig 1) [27].
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New cysteine protease inhibitors as trypanocidal agents
Fig 6. Structure of compounds 50–60 and 65–69.
https://doi.org/10.1371/journal.pntd.0007755.g006
For the synthesis of compounds 50–60 (Fig 6), we have adopted a different synthetic strat-
egy. In general, the desired dipeptidyl primary amide was synthesized, followed by the dehy-
dration reaction to form the dipeptidyl nitrile. Due to the diversity of building blocks, it was
necessary to evaluate different dehydrating reagents, aiming at the best yield and prevention of
racemization. For compounds 65–68 the cleavage of the benzyl group was performed by
hydrogenolysis before the conversion of the primary amide to the nitrile, while for compound
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New cysteine protease inhibitors as trypanocidal agents
69, considering the lability of the chlorine atom under hydrogenolysis, we first transformed
the primary amide to the nitrile and then removed the benzyl group under mild oxidative con-
ditions [27]. The absolute geometry of the P1 group did not change, but, owing to CIP priority
rules, the configuration at the α−carbon for the Thr-O-Bzl building block changed two times:
(i) in the dehydration step to form the nitrile group and, (ii) when the catalytic cysteine attacks
the carbon atom of the nitrile warhead to form a covalent bond (Fig 7).
Structure-activity relationships for inhibition of cysteine proteases by
dipeptidyl nitriles
The pKi values were determined for parasite cysteine proteases (Cz, LmCPB) and also for
human cysteine cathepsins (CatB, CatK, CatL, CatS) and are reported in Table 1. Compounds
6, 8, 9 and 11 have already been described as competitive inhibitors that bind reversibly to Cz
[14]. Several new compounds are Cz nanomolar inhibitors and exhibit good affinity for
LmCPB, CatL, CatS and CatK (pKi over 7.5). The application of such inhibitors extends to can-
didates for antiprotozoal action and as inhibitors of cysteine cathepsins.
One crucial question is the cross-reactivity of CP inhibitors, for which an extrathermody-
namic relationship can be formulated [28,29]. Ligand-target affinity involves enthalpy and
entropy, which, in most cases, results in enthalpy-entropy compensation (EEC) [30]. The com-
pensation effect should lead to an observable linear relationship between enthalpy and entropy
when ligands differ by a single molecular modification. EEC is a fundamental extrathermody-
namic phenomenon in medicinal chemistry and one of its grand challenges [31]. A thermody-
namic analysis is commonly applied to a series of compounds that act on the same target.
Here, the ligand is kept invariant while the targets are changed. Therefore, the nature of the
ligand-target interaction governed by the thermodynamic parameter of the free energy change
(via the estimation of the dissociation constant) results in extrathermodynamic relationships
for the set of derivatives.
Hence, we investigated the degree of linear correlation between Cz and the other CPs by
plotting the pKi data against each other. The results (see S1 Fig) indicated an extrathermody-
namic relationship between Cz and LmCPB, while this was not observed for all the other CPs.
This finding highlights that the mode of inhibition for this series of compounds is similar for
Cz and LmCPB, corroborating the fact that all the structural transformations of prototype
compounds 9 and 11 affected the affinity towards the two protozoa CPs with the same magni-
tude (Figs 8 and 9). Therefore, known Cz inhibitors can be repurposed as LmCPB inhibitors,
and the knowledge acquired in the design of Cz inhibitors can be translated into future
LmCPB inhibition studies.
Fig 7. Change in stereochemistry for compounds bearing Thr or Thr-O-Bzl group in P1.
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New cysteine protease inhibitors as trypanocidal agents
Table 1. Number identification, pKi values for CatB, CatK, CatL, CatS, Cz and LmCPB.
pKi valuesa or remaining activity (%) at 10 μMb
Cmpd.
CatB
73%b
5.5
CatK
CatL
7.4
7.3
8.6
8.2
7.6
7.6
5.9
7.2
5.7
6.6
6.3
6.5
7.1
5.2
8.5
6.9
8.3
7.0
8.3
8.3
7.0
85%b
7.2
90%b
8.1
5.3
5.6
7.0
88%b
8.2
CatS
7.1
7.7
6.7
6.8
5.6
7.4
5.9
6.7
6.0
6.9
7.3
7.6
7.3
5.0
7.3
5.9
6.9
6.0
7.1
7.2
6.0
n.i.c
7.3
85%b
6.5
5.3
4.7
5.0
83%b
6.6
Cz
6.6
LmCPB
6.6
6
6.4
7
6.2
6.9
6.2
8
5.4
6.4
7.4
6.7
9
4.8
93%b
6.5
7.3
7.1
10
11
12
13
14
15
16
17
18
19
50
51
52
53
54
55
56
57
58
59
60
65
66
67
68
69
5.0
6.6
6.4
4.5
8.3
7.8
7.3
4.4
96%b
81%b
n.i.c
6.9
5.1
5.5
4.9
6.7
6.9
4.7
89%b
6.2
5.7
7.8
7.3
5.2
6.2
6.9
7.0
n.i.c
4.5
8.0
7.1
6.8
8.7
7.7
51%b
7.2
67%b
4.6
5.5
4.5
94%b
6.3
7.7
7.8
5.6
6.3
6.1
5.1
6.7
7.5
7.4
90%b
4.9
6.0
6.5
6.4
6.3
7.5
7.5
4.8
6.5
7.5
7.2
5.2
n.i.c
5.7
97%b
6.3
75%b
6.5
72%b
5.1
7.8
7.9
7.7
n.i.c
6.3
n.i.c
6.0
86%b
81%b
7.8
60%b
7.2
68%b
75%b
6.8
69%b
92%b
89%b
92%b
93%b
5.2
5.4
4.8
7.1
6.7
54%b
5.1
5.4
6.1
6.9
6.0
a The standard deviation was lower than 15% for all reported pKi values.
b Percentage of remaining activity at 10 μM, number of replicates = 2.
c n.i. = no inhibition observed at 10 μM.
https://doi.org/10.1371/journal.pntd.0007755.t001
One common approach to SAR analysis is to examine ΔpKi values associated with particu-
lar structural transformations, and these can be specified concisely using the square bracket
notation previously described [32]. For example, the structural transformation of the phenylal-
anine in P2 of compound 6 to the corresponding 3-chloro-phenylalanine (8) can be noted as
[6!8]. As already described for Cz [14], the exchange of benzoyl by 1-methyl-3-tert-butyl-
pyrazolyl-carbonyl [6!9] led to a potency increase of 0.5 log units. Following this path, we
inserted a meta-benzoic ester in the P3 position in the attempt to design a prodrug analog
[15]. The transformation [6!16] unfortunately displayed a slight increment as compared to
the transformation [6!13]. Hence, compound 9 (pKi of 7.4 for Cz) has been used as a proto-
type for mapping S1-P1 interaction on different targets (Fig 8).
The effects on affinity resulting from stereochemical modifications in P1 of the structural
prototype 9 are shown in Fig 8. In general, the stereochemistry of P1 moiety strongly
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New cysteine protease inhibitors as trypanocidal agents
Fig 8. SAR summary for P1-S1/S1´ interactions. Values are reported as differences in pKi and are color-coded as red (negative), green (positive), grey
(no significant difference, ΔpKi < 0.2).
https://doi.org/10.1371/journal.pntd.0007755.g008
influences the affinity towards all the CPs. The (S) ! (R) conversion in [50!51] and
[52!53] decrease the pKi values for Cz and LmCP both by about one log unit. Likewise, the
double stereochemical modification from (R,R) benzyl-protected threonine to the (S,S) diaste-
reomer [56!57] led to a complete all-target affinity loss. Instead, the structural transforma-
tion from the cyclopropyl unit to CH attached with a benzyl group [9!50] resulted in a
significant affinity increment for Cz (pKi of 7.7) and LmCPB (pKi of 7.8) of 0.4 and 0.7 log
units, respectively. Replacement of the P1 cyclopropane linker with CH attached to isopropyl
[9!52], 3-chlorobenzyl [9!54], or even 4-pyridyl [9!55] led to a small increase or no signif-
icant difference in affinity against those two protozoa enzymes. Moreover, the insertion of the
benzyl protected threonine in P1 [9!56] and [9!57] decreased the affinity for Cz and
LmCPB. Remarkably, replacement by the hydroxybutyl residues led to an almost one hun-
dredfold affinity loss for both enzymes. In general, those results are corroborated by the dock-
ing studies which display a reasonable accommodation of the aromatic and hydrophobic
moieties in the S1´ site without forming any specific interaction with the residues present in
this area. While, for smaller moieties as cyclopropane or Thr-OH the interaction is manly in
the interface between the S1 and the S1´. Primarily, the same trend in affinity was observed for
the four mammalian CPs, when the structural modifications in P1 were realized starting from
the prototype compound 9, as illustrated in Fig 8. Singularly, the introduction of a benzyl-pro-
tected threonine [9!50] resulted in a pKi decrease of 0.3 log units towards CatB.
As recently described [14], the effects on affinity when replacing the P2 phenylalanine (9)
with leucine (11) appears to depend on the substructural context, and this relates to non-addi-
tivity in the SAR. Accordingly, we used compound 11 as a starting prototype for another SAR
considering P1, P2, and P3 for structural modifications, as summarized in Fig 9.
Substitution of the in P3 positioned 1-methyl-3-tert-butyl-pyrazole ring (11) with 7-chloro-
quinoline (15), or 1H-indole (19) preserved the high affinity towards Cz and LmCPB, and,
strikingly, this substitution led to a decrease of 1.0 and 0.5 in the pKi value for CatL (Fig 9).
Insertion of a basic moiety, i.e., 2-amino pyridine [11!18], produced a significant reduction
of potency for Cz (-0.7) and LmCPB (-0.5). Compound 19 showed a high affinity for CatK
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New cysteine protease inhibitors as trypanocidal agents
Fig 9. SAR summary starting from compound 11. Values are reported as differences in pKi and are color-coded as red (negative), green (positive),
grey (no significant difference, ΔpKi < 0.2).
https://doi.org/10.1371/journal.pntd.0007755.g009
(pKi of 8.7) with a significant selectivity over the other mammalian CPs (Table 1). At position
P2, the transformation of the leucine moiety into tryptophan, phenylalanine, or derivatives
thereof resulted in an affinity loss of up to one log unit for Cz. A similar replacement led to a
gain in affinity towards CatL and CatB, and it is consistent with previously reported data [33].
For compound 11, as for compound 9, the stereochemistry of the substituent in P1 was vitally
for the bimolecular recognition process. Moreover, the transformation [11!58] kept the pKi
in the same range for Cz while increasing it by 0.4 log units for LmCPB.
Non-additivity in SAR is of considerable interest [34], and this is illustrated for the six cys-
teine proteases in Fig 10. Non-additivity can be quantified by comparing the ΔpKi value result-
ing from a pair of substructural transformations with the sum of ΔpKi values that result from
the individual transformations. The Cz ΔpKi values for [9!11] (0.5) and [9!56] (-1.0) shown
in Fig 10A sum up to -0.5. Nevertheless, [9!58], which corresponds to the simultaneous
application of the pair transformations, is associated with a ΔpKi value increase of +0.6, thus
indicating that the effects of this pair of transformations on Cz affinity are superadditive. The
same was true for the effects of these two transformations on the other five CPs. Analogous
analysis of the results in Fig 10B displays the effects of two transformations to be superadditive
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New cysteine protease inhibitors as trypanocidal agents
Fig 10. Non-additivity of SAR. Values are reported as differences in pKi and are color-coded as red (negative), green (positive), grey
(no significant difference, ΔpKi< 0.2).
https://doi.org/10.1371/journal.pntd.0007755.g010
for the entire CP targets investigated herein. These results entail the P1-S1 and P1’-S1’ interac-
tions to be driven by the molecular recognition in P2.
Pairwise plots for the selectivity towards Cz in relation to other human cathepsins are pro-
vided in Fig 11. It is not trivial to achieve a significant selectivity for Cz inhibitors (ΔpKi > 1.0)
over mammalian CPs due to their high structural similarity of the active site. Undeniably,
CatB has a different mode of binding due to the larger S2 and S3 pockets [33]. Compounds 15
(pKi of 7.8 for Cz) and 67 (pKi of 7.1) displayed a significant selectivity toward CatL (pKi of
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New cysteine protease inhibitors as trypanocidal agents
Fig 11. Selectivity pairwise plots. Values are given in pKi. The X-axis represents the difference in pKi for the same inhibitor for a
pair of CPs. Y-axis represents the mean value of pKi for the same inhibitor for a pair of CPs. The black dashed line highlights no
selectivity. The magenta dashed line highlights a significant selectivity. Positive differences correspond to Cz pKi values that are
greater than those for CatB, CatK, CatL or CatS.
https://doi.org/10.1371/journal.pntd.0007755.g011
6.6) and CatS (pKi of 5.0), respectively. The Cz selectivity in the case of compound 15 is driven
by the S3-P3, while that of 67 is driven by S1-P1 interaction. Additionally, the hydrophobic
interaction in S1 and S1´ with P1 of compounds 50 (pKi of 7.7 for Cz), 54 (pKi of 7.5 for Cz)
and 60 (pKi of 7.2 for Cz) resulted in a good selectivity over CatK (pKi of 6.3, 6.3 and 6.0,
respectively, for 50, 54 and 60). On the other hand, our study highlights two low nanomolar
inhibitors of mammalian CPs: compounds 8 (pKi of 8.6 for CatL) and 19 (pKi of 8.7 for CatK)
which showed an affinity difference of more than one log unit compared to the other mamma-
lian cathepsins.
Biological evaluation
All compounds synthesized were evaluated for their trypanocidal activity against the amasti-
gote form of the Tulahuen T. cruzi strain, and the best results are presented in Table 2. A num-
ber of 15 compounds had pEC50 values above 4 against T. cruzi amastigotes and three (52, 57
and 60) were equipotent to the gold standard benznidazole as trypanocidal agents. In particu-
lar, compounds 52 and 60 are both low nanomolar Cz inhibitors and one-digit nanomolar
inhibitors for CatL. Compound 57 had no affinity for any of the six CPs reported herein,
which excluded the possibility that its mechanism of action is similar to compound 52 and 60.
Physiochemical properties (logP, MM, TPSA, LogS) play an important role in drug design. As
well, for potential trypanocidal agents, which had been designed as protozoan cysteine prote-
ases inhibitors, physicochemical properties can influence their outcome. Therefore, we have
included TPSA, calculated logP (ilogP), and LogS (Ali_LogS) in this discussion (Figs 12, 13
and 14) [35].
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New cysteine protease inhibitors as trypanocidal agents
Table 2. Biological data for trypanocidal activity (EC50), cytotoxicity (CC50), and selective index (SI) for the series of
dipeptidyl nitriles.
Compounds
EC50 (μM)
4.4 0.47
72 5.7
CC50 (μM)
> 100
SI
Benznidazole
> 23
> 1.4
> 1.5
> 3.1
1.14
9
> 100
10
12
15
16
50
51
52
53
54
57
60
65
66
67
68
69
67.6 8.22
32.7 3.86
64.7 4.32
47.3 3.26
63.3 5.74
8.6 0.53
4.1 0.47
26 2.6
> 100
> 100
73.6 8.74
> 100
> 2.1
1.31
83.1 6.85
42.3 3.94
97.9 6.52
> 100
4.9
24
> 3.8
2.83
15.8 1.43
4.3 0.32
4.9 0.45
70.8 8.79
30.0 2.71
24.4 2.36
16 4.0
44.7 5.24
> 100
> 23
> 20
> 1.4
> 3.3
> 4.1
> 6.3
> 3.3
> 100
> 100
> 100
> 100
> 100
30.6 3.11
> 100
Benznidazole was used as a positive control. DMSO was used to dissolve compounds and as a negative control. All
data were obtained using at least two independent experiments. Compounds with EC50 > 100 μM were considered to
be not active, and they are not reported in this Table. Green areas highlight the most potent compounds.
https://doi.org/10.1371/journal.pntd.0007755.t002
In general, the substitution of the P3 or P2 moieties from the prototype compound (9) did
not result in any increment of potency, except in the case of compound 12 (Fig 12). Modifica-
tion in P1 strongly modulated the trypanocidal effect. Substitution of Phe for (S,R)-Thr-O-Bn
led to a modest trypanocidal activity. If we consider the physicochemical properties of the
whole set of compounds, there is no consistent correlation with their trypanocidal activities.
However, when considering discontinuity in small sets of compounds related to their matched
molecular pairs (MMP) [36], we observed, in some specific cases, that modulation in com-
pound lipophilicity yields to high trypanocidal potency. For instance, the existence of the
molecular pair between compounds 52 (EC50 = 4.1 μM; ilogP = 2.3) and 9 (EC50 = 71.8 μM;
ilogP = 3.2) suggests that the 17.5-fold increase in potency in favor of 52 may be due to its
reduced lipophilicity. In addition, single modification in P2 or P1 of compound 9 did not
always produce a beneficial effect for the trypanocidal activity. However, considering both
modifications [960], compound 60 (EC50 = 4.9 μM) exhibited a 14.5 times higher trypanocidal
potency than compound 9 (EC50 = 71.8 μM). Compound 60 displayed activity against the par-
asite similar to that observed for benznidazole–EC50 of 4.4 (Fig 13). Interestingly, the ilogP of
4.1 for 60 was the highest among the entire series and more than one log unit larger than that
of compound 52. Too, compound 57, which also has ilogP of 4.1, is equipotent to compound
52 whose ilogP is only 2.3! It is noteworthy that compound 57, exhibiting the same values for
TPSA and ilogP as 60 (a high affinity cruzain inhibitor), had no affinity for any of the CPs
studied herein. Assumably, compound 57 has a different mode of action than 60. Further-
more, inhibitor 57 (CC50 ’ 124.7 μM) showed the highest selectivity and lowest cytotoxicity in
our assays. This guarantees an SI (selective index) ratio of over 20, as well as for compounds 60
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