Synthesis and matched molecular pair analysis of covalent reversible inhibitors of the cysteine protease CPB

Article abstract of DOI:10.1016/j.bmcl.2020.127439

Cysteine protease B (CPB) can be targeted by reversible covalent inhibitors that could serve as antileishmanial compounds. Here, sixteen dipeptidyl nitrile derivatives were synthesized, tested against CPB, and analyzed using matched molecular pairs to det

Full text of DOI:10.1016/j.bmcl.2020.127439

Journal Pre-proofs
Synthesis and Matched Molecular Pair Analysis of Covalent Reversible Inhib‐
itors of the Cysteine Protease CPB
Thiago Kelvin Brito Matos, Pedro Henrique Jatai Batista, Fernanda dos Reis
Rocho, Daniela de Vita, Nicholas Pearce, Barrie Kellam, Carlos Alberto
Montanari, Andrei Leitão
PII:
S0960-894X(20)30550-3
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127439
BMCL 127439
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
1 June 2020
20 July 2020
21 July 2020
Please cite this article as: Kelvin Brito Matos, T., Henrique Jatai Batista, P., dos Reis Rocho, F., de Vita, D.,
Pearce, N., Kellam, B., Alberto Montanari, C., Leitão, A., Synthesis and Matched Molecular Pair Analysis of
Covalent Reversible Inhibitors of the Cysteine Protease CPB, Bioorganic & Medicinal Chemistry Letters (2020),
doi: https://doi.org/10.1016/j.bmcl.2020.127439
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version
will undergo additional copyediting, typesetting and review before it is published in its final form, but we are
providing this version to give early visibility of the article. Please note that, during the production process, errors
may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier Ltd.

Synthesis and Matched Molecular Pair Analysis of
Covalent Reversible Inhibitors of the Cysteine Protease CPB
Thiago Kelvin Brito Matosa#, Pedro Henrique Jatai Batistaa#, Fernanda dos Reis Rochoa,
Daniela de Vitaa, Nicholas Pearceb, Barrie Kellamc, Carlos Alberto Montanaria, Andrei
Leitãoa*
a Medicinal & Biological Chemistry Group (NEQUIMED), São Carlos Institute of Chemistry-
University of São Paulo (IQSC-USP) São Carlos -SP – Brazil
b School of Chemistry - University of Nottingham – Nottingham - UK
c School of Pharmacy - University of Nottingham – Nottingham - UK
# Both authors contributed equally to this work
* Corresponding author:
Prof. Dr. Andrei Leitão
Medicinal & Biological Chemistry Group (NEQUIMED), São Carlos Institute of Chemistry-
University of São Paulo (IQSC-USP). Av. Trabalhador São-carlense, 400 São Carlos-SP
Brazil, Zip Code: 13566-590; E-mail: andleitao@iqsc.usp.br
1

Abstract
Cysteine protease B (CPB) can be targeted by reversible covalent inhibitors that could serve
as antileishmanial compounds. Here, sixteen dipeptidyl nitrile derivatives were synthesized,
tested against CPB, and analyzed using matched molecular pairs to determine the effects of
stereochemistry and p-phenyl substitution on enzyme inhibition. The compound (S)-2-(((S)-1-
(4-bromophenyl)-2,2,2-trifluoroethyl)amino)-N-(1-cyanocyclopropyl)-3-phenylpropanamide
(5) was the most potent CPB inhibitor (pKi = 6.82), which was also selective for human
cathepsin B (pKi < 5). The inversion of the stereochemistry from S to R was more detrimental
to potency when placed at the P2 position than at P3. The p-Br derivatives were more potent
than the p-CH3 and p-OCH3 derivatives, probably due to intermolecular interactions with the
S3 subsite.
Keywords: dipeptidyl nitrile derivatives, enzymatic inhibitors, additive effect, SAR,
crystallographic structure.
2

Leishmaniasis is a neglected disease endemic in many countries, including Brazil. It is
considered an emergent and uncontrolled disease, caused by more than 20 species of
Leishmania.1 Leishmaniasis can be classified into three forms, the two most common being
tegumentary and visceral. The most frequent types in Brazil are cutaneous and
mucocutaneous leishmaniasis, caused by Leishmania (Viannia) braziliensis2; however, the
visceral form is the deadliest. According to the World Health Organization (WHO), visceral
leishmaniasis affected 500,000 people in 88 countries worldwide in 2017; 556 million people
live in endemic areas. Six countries, including Brazil, account for 94% of cases.3
The disease is transmitted to humans by the bite of infected female sandflies. These
parasites are present inside the vector gut as extracellular promastigotes; then, they transform
into intracellular amastigotes in mammalian host cells.4 Currently, leishmaniasis treatment
includes pentavalent antimonials, amphotericin B and miltefosine. Pentavalent antimonials are
the first-line treatment; however, these are associated with side effects such as hepatotoxicity
and cardiotoxicity.5 Amphotericin B, another common antileishmanial drug, causes renal
toxicity.6 For these reasons, it is crucial to develop new drugs, and cysteine proteases are
attractive targets; these are associated with physio-pathological processes such as cancer (for
cathepsins),7 Chagas disease that is caused by Trypanosoma cruzi (where cruzipain is
expressed throughout the whole life cycle), and leishmaniasis, caused by Leishmania spp.
(for the enzymes CPA, CPB, and CPC).8 These enzymes irreversibly hydrolyze peptide bonds
by putative mechanism involving nucleophilic attack provided by the thiolate, resulting in
formation of a tetrahedral intermediate that is followed by formation of a thioester
intermediate and its hydrolysis. The active site of the cysteine proteases from the papain
family recognizes the protein sequence to be cleaved (or the biomimetic inhibitor) in which
the substituents in the α-carbon of the amino acids (or the inhibitors) bind to the subsites.9
Many research groups been working with derivatives of dipeptidyl nitrile that bind
reversibly with cysteine proteases.10,11 The covalent bond is formed between the carbon from
the nitrile warhead and the thiolate of the catalytic cysteine forming the thioimidate;
nevertheless, this is unstable and degrades to return the system to the initial state. Therefore,
these compounds are classified as covalent reversible inhibitors.12
There are many reports in the literature of various cysteine proteases inhibitors,
especially reversible and irreversible covalent inhibitors.13,14 Moreover, inhibitors that do not
form a covalent bond with the active site usually show lower potency. By contrast, those that
form irreversible covalent bonds may have toxicity due to off-target effects.15
Here we describe a series of cysteine protease inhibitors based on derivatives of the
3

dipeptidyl nitrile scaffold. These compounds were designed based on a simplification of the
odanacatib structure. Odanacatib is a potent cathepsin K inhibitor that is being tested in phase
3 clinical trials.16 The mode of binding is well-described with substituents interacting at
various subsites of the active site of the cathepsin K (Figure 1a).17 All compounds maintain
the 2,2,2-trifluoroethylamine moiety from odanacatib as an amide isostere, which is often
used to improve the biological stability with respect to the amide itself. However, two
alterations of the chemical structure of odanacatib were made:
(i) The phenylalanine was placed in position P2 (Figure 1b) instead of the unnatural
amino acid 4-fluoroleucine. Our previous study with another parasitic cysteine
protease (cruzain) demonstrated that phenylalanine is also recognized by enzymes
from the papain family,10 in which the homology model of the CPB structure shares
structural features and ligand recognition in the active site.18 The stereochemistry is an
important feature for these compounds, and R and S derivatives were studied for the α-
carbon of the phenylalanine amino acid in position P2 (Figure 1b). The S2 subsite of
these cysteine proteases is deemed as a selective point,15 and it was then exploited in
this study to identify the best stereochemistry.
(ii) A simplification of the chemical structure was made in the position P3, where the
phenyl derivatives substituted in the para position were tested (Figure 1). The
intermolecular interactions in the S3 subsite could improve the potency; hence, two
distinct modifications were devised: (i) the p-phenyl substitution, and (ii) the
stereochemistry of the CF3 group.
(a)
(b)
Figure 1. Representation of odanacatib (a) and the scaffold for the set of compounds
(b) with the numbering of the respective substituents that interact with the subsites of the
cysteine protease. The chiral carbons marked in (b) were studied to identify the stereogenic
recognition by the CPB enzyme. R: H, Br, CH3, or OCH3.
4

There are few reports of CPB inhibition by reversible covalent dipeptidyl nitrile
derivatives and their effects on leishmania parasites.19,20 Thus, this work demonstrates the
hypothesis-driven asymmetrical synthesis of a series with 16 derivatives of dipeptidyl nitriles
and their evaluation as inhibitors of the leishmanial cysteine protease CPB. As seen in Figure
1b, two stereocenters are found for each compound. All stereoisomers were synthesized and
evaluated in biochemical assays to describe their bioactivity profiles and the influence of their
stereochemistry on CPB inhibition.
The synthesis of the amino acid intermediates used in the peptide coupling is shown
in Scheme 1. D and L-Amino esters were purchased, and the synthetic approach from Chen et
al. was used in this step.21 Amino esters reacted with the opportune 2,2,2-
trifluoroacetophenone in a reflux system in the presence of potassium carbonate and
anhydrous methanol at 75 °C for 18 h, to produce the respective imines (Scheme 1). The basic
reaction condition at elevated temperature promoted the hydrolysis of the methyl ester group,
providing the potassium salts of the 2,2,2-trifluorophenyl imines. Due to the imine instability,
all intermediates were used in the next step without purification.
Scheme 1. Reagents and conditions: (a) 2,2,2-trifluoroacetophenone, K2CO3,
CH3OH, 75 °C, reflux, 18 h; (b) 1 M Zn(BH4)2 in THF, CH3CN/CH3OH (5:1), -40/-45 °C, 4
h; (c) NaBH4, THF, r.t., 6 h; (d) Hexafluorophosphate azabenzotriazole tetramethyl uronium
(HATU), N,N-diisopropylethylamine (DIPEA), opportune amine, r.t. 2 h.
The diastereoselective reduction was described by Hughes et al.,22 where the imines
were reduced with Zn(BH4)2 to obtain the S,S-diastereoisomers (supplementary information,
Figures S1 and S2).23,24,25 The final step of the synthesis to obtain compounds 1–16 involved
the coupling of the amino acids with the respective amine and HATU. All compounds have
the cyano group as a warhead (the reactive group responsible for covalent bond formation).
The overall yields were good (65–70%), with high purity (> 95%), according to the chiral
HPLC system applied to obtain the stereoisomers. All compounds were characterized by
5

NMR spectroscopy, mass spectrometry, IR, and melting point, as described in the supporting
information (characterization data for 1, 2 and 5 were reported elsewhere).10
The stereochemistry was confirmed after the analysis of crystals of compound 12
(C22H22F3N3O, Figure 2). It was crystallized by vapor diffusion of hexane into the
dichloromethane solution over 72 hours. A suitable crystal was selected and mounted in a
fomblin film on micro mount on a SuperNova, Atlas S2 diffractometer.26 Figure 2 shows the
absolute R,R configuration of compound 12. This result confirmed that the experimental
procedure described by Hughes et al.22 indeed produces the syn isomer when the
diastereoselective reduction was performed using Zn(BH4)2. Complementary data are
available in the supplementary information, in which the packing of the compound 12 is
shown (crystallography section), featuring the intermolecular interactions in the crystal. A
network of hydrogen bonds between oxygen and nitrogen atoms of the amide groups and
hydrogen-pi interactions between phenyl groups were observed in the P2 and P3 positions. Of
note, compounds with the anti-configuration could impair the formation of the same set of
interactions and did not provide a crystallographic structure despite the effort made.
Figure 2. Crystalline structure and representation of compound 12 (CCDC
1955373).
All compounds were then tested against the CPB enzyme aiming to identify
submicromolar inhibitors. The determination of the inhibition constant was done using an
indirect assay, in which the inhibitor and substrate were present in solution alongside the
enzyme. The protocol for the competitive inhibition assay was reported elsewhere24 (further
information can be found in the supplementary material).
6

The stereochemistry is one of the most critical aspects exploited in this series of
dipeptidyl nitrile derivatives. Therefore, all compounds were tested to quantify the constant of
inhibition (Ki), obtaining their respective pKi values (Table 1). Three of 16 compounds gave
Ki values below 1 µM, and compound 5 was the most potent CPB inhibitor. Compounds that
did not reach the threshold of 10 µM were assigned the percentage inhibition at this cut-off
concentration.
Table 1. The enzymatic inhibition of CPB by the dipeptidyl nitrile derivatives.
CPB
SE pKi
0.05
0.08
0.02
0.06
0.03
0.06
-
0.07
0.08
0.06
-
Cathepsin B
% inh c
74.5
Compound Chirality
R
a
b
Ki (µmol/L)
1.70
pKi
% inh c
1
2
S,S
S,R
R,S
R,R
S,S
S,R
R,S
R,R
S,S
S,R
R,S
R,R
S,S
S,R
R,S
R,R
H
H
H
H
Br
Br
Br
Br
CH3
CH3
CH3
CH3
OCH3
OCH3
OCH3
OCH3
5.77
5.85
5.37
5.11
6.82
6.23
< 5.0
6.10
5.40
5.80
< 5.0
< 5.0
5.50
< 5.0
< 5.0
< 5.0
1.41
4.40
7.65
0.151
0.595
> 10 000
0.795
4.07
1.50
> 10 000
> 10 000
3.23
> 10 000
> 10 000
> 10 000
3
4
5
6
7
8
9
> 95
> 95
92
10
11
12
13
14
15
16
86%
88%
-
0.06
-
-
70%
95%
95%
-
a
b
c
pKi = -log10(Ki/M); Standard Error for pKi; % of inhibition for compounds with a Ki
higher than 10.0 µmol/L.
The set of compounds was analyzed according to the concept of matched molecular
pairs (MMP)27,28 to observe how a single modification of a determined structure would affect
bioactivity. The first subset of compounds proposed in this work (compounds 1–8) was
prepared to determine the influence of the phenylalanine and CF3 configurations, and the
effect of the p-bromine in the P3 position (Figure 3) on enzyme recognition and inhibition
using a kinetic assay. As shown in Table 1 and Figure 3 (1→5 and 4→8), the p-bromine
7

indeed increased the potency of the derivatives by one logarithmic unit. The S,S derivatives
led to more potent inhibitors than the R,R enantiomers. Furthermore, an additive effect was
observed for these compounds (Figure 3).
Another striking result was achieved when the most promising CPB inhibitor (5)
along with some of the derivatives were analyzed against the orthologous human enzyme
cathepsin B (protocol provided in the supplementary information). According to Table 1, all
compounds had pKi < 5, and were therefore considered selective inhibitors for the
Leishmania enzyme CPB.
Figure 3. Matched molecular pair analyses for the first subset (compounds 1–8)
focusing on the enantiomers and the additive effect of the p-bromine addition in P3. The
additive effect was achieved for this subset.
The additivity effect was observed for the comparative analyses between
enantiomers and diastereomers (Figure 3). As expected, the inversion of the stereocenter at
the position P2 (Figure 1) was less tolerated than in position P3. The S2 subsite29,30 (in which
the P2 substituent binds) does not tolerate the inversion of the phenylalanine configuration
(1→3 and 5→7), like the subsite S3, for the CF3 group (1→2 and 5→6). This is observed by
the reduction of the potency in all cases where the S-Phe was changed by R-Phe (Figure 3).
The inversion of the CF3 configuration led to mixed results, depending on the group present in
the p-phenyl position. It did not change the potency for the non-substituted compounds
(1→2), but it led to a reduction in potency for the p-Br derivatives (5→6). The effect is more
pronounced when the enantiomers are compared (1→4 and 5→8).
8

The analysis of the bromine effect in Figure 4 followed the additivity between the
enantiomeric pairs for the S,S (1→5), and R,R (4→8) derivatives, with an increment in the
potency of 10-fold when the bromine atom was in the para position. Intriguingly, other
enantiomeric pairs provided by S,R (2→6), and R,S (3→7) did not follow the same trend
(Figure 4). The S,R derivatives had an increment of almost three times when the compound
bears the bromine in the para position; however, an opposite effect was obtained for the R,S
derivatives. These differences may be associated with the intermolecular interactions, recently
described by us for the T. cruzi cruzipain enzyme as halogen bonding,31 coupled with the
volume of the bromine (and its steric hindrance when misplaced in the S3 subsite). The lack
of a crystallographic structure of the CPB enzyme impairs further studies regarding the
intermolecular interactions between the ligand and the macromolecule. However, we
speculate that the effect of the p-bromine on incremental changes in inhibition potency might
derive from halogen bonding once the p-methyl and p-methoxy derivatives had lower
potencies (Table 1).
Figure 4. Matched molecular pair analyses for the first subset (compounds 1–8),
focusing on the bromine substitution.
The evaluation of the p-methyl and the p-methoxy bearing compounds was also
performed in relation to the non-substituted compounds (Figure 5). However, MMP analyses
were partially impaired by the loss of potency for most of the compounds. This may have
resulted from steric hindrance attributable to the para substituents, with two out of four p-CH3
derivatives considered active (9 and 10) and only one for the bulkier p-OCH3 (13).
9

Our group is investigating a hypothesis regarding the non-additive profile to T. cruzi
cysteine protease cruzain that may also be applicable to CPB. This refers to the
hydrophobicity and the positioning of the P3 substituent.31 The S3 subsite is more exposed to
the solvent for all known cysteine protease three-dimensional structures available in the
Protein Data Bank, where hydrophilic groups are usually placed in this region to interact with
the amino acids. This may be the reason why the lipophilic derivatives were less potent
(compounds 9–16, Figure 5).
Interestingly, all derivatives with the R-Phe were weaker inhibitors than the S-Phe
ones, reinforcing the notion of selectivity of the S2 subsite for S-Phe (Figure 5). The
lipophilic p-CH3 (9) had almost the same potency of the hydrogen bond donor p-OCH3
compound (13). This suggests that the presence of a hydrogen bond acceptor did not improve
the potency, because the methoxy group could not interact with any hydrogen bond donor
from the amino acids of the CPB enzyme. It is now necessary to increase the number of
derivatives to further advance the insight described here regarding the putative formation of a
halogen bonding, instead of hydrogen bonding.
Figure 5. Schematic analyses of compounds 1–4, 9–12, and 13–16. The upper
threshold for activity was 10 µmol/L.
This work described the synthesis and characterization of sixteen compounds against
the cysteine protease CPB. SAR analysis for CPB inhibition using MMP showed that the p-
bromine compound with (S,S) configuration was the most potent (5, pKi = 6.82). This result is
probably due to a combination of (i) S-Phe fitting into the S2 subsite, with the change of the
stereochemistry (S to R) in P2 leading to a loss of inhibition capacity (Figure 1 and Table 1);
and (ii) a putative halogen bonding that could form between the p-Br derivative and an amino
10

acid residue in the S3 subsite, which is corroborated by the other less potent p-CH3 and p-
OCH3 compounds. Furthermore, an additive effect was observed for non-substituted
compounds concerning the p-bromine derivatives (Figure 3).
Finally, we reported the determination of the absolute stereochemical configuration
of compound 12 using X-ray crystallography (Figure 2). The structure corroborated the
mechanism of the asymmetric synthetic route used to produce the compound with the desired
stereochemistry.
Competing interest
The authors declare no competing interests.
Acknowledgment
The authors thank the financial support from the São Paulo Research Foundation
(FAPESP – grants 2013/18009-4, 2016/07946-5, 2018/15904-6), the Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES – Finance code 001)
139/2015 and 1632465, and the Conselho Nacional de Desenvolvimento Científico, CNPq
(process number 14242212016-9). The valuable contributions of the reviewers are also
acknowledged.
References
1 Lainson R. The Neotropical Leishmania species: a brief historical review of their discovery,
ecology and taxonomy. Rev Pan-Amaz Saude 2010;1:13-32. https://doi.org/10.5123/S2176-
62232010000200002
2
Marzochi MCA, Marzochi KBF. Tegumentary and visceral leishmaniases in Brazil -
emerging anthropozoonosis and possibilities for their control. Cad Saúde Públ.
1994;10:suppl.2,359-375. http://doi.org/10.1590/S0102-311X1994000800014
3
World Health Organization
–
Leishmaniasis
–
Epidemiological situation.
https://www.who.int/leishmaniasis/burden/en/; Accessed 27 May 2020.
11

4 Torres-Guerrero E, Quintanilla-Cedillo MR, Ruiz-Esmenjaud J, Arenasa R. Leishmaniasis: a
review. F1000Res. 2017;6:750. https://doi.org/10.12688/f1000research.11120.1
5 Andrade-Neto VV, Cunha-Junior EF, Dos Santos Faioes V, Pereira TM, Silva RL, Leon LL,
Torres-Santos EC. Leishmaniasis treatment: update of possibilities for drug repurposing.
Front Biosci. 2018;23:967-996. https://doi.org/10.2741/4629
6
Fanos V, Cataldi L. Amphotericin B-induced nephrotoxicity: A Review. J Chemother.
2000;12:463-70. https://doi.org/10.1179/joc.2000.12.6.463
7
Siklos M, Benaissa M, Thatcher GR. Cysteine proteases as therapeutic targets: does
selectivity matter? A systematic review of calpain and cathepsin inhibitors. Acta Pharm Sin B.
2015;5:506-519. https://doi.org/10.1016/j.apsb.2015.08.001
8
Rebello KM, Britto C, Pereira BAS, Pita-Pereira D, Moraes MO, Ferreira ABR, Cysne-
Finkelstein L, Otto TD, Cortês LMC, da-Silva GG, Alves CR. Leishmania (Viannia)
braziliensis: Influence of successive in vitro cultivation on the expression of promastigote
proteinases. Exp Parasitol. 2010;126:570-576. https://doi.org/10.1016/j.exppara.2010.06.009
9
Otto H-H, Schirmeister T. Cysteine proteases and their inhibitors. Chem Rev. 1997;97:133-
172. https://doi.org/10.1021/cr950025u
10 Gomes JC, Cianni L, Ribeiro JFR, Rocho FR, Silva SCM, Batista PHJ, Moraes CB, Franco
CH, Junior LF, Kenny P, Leitão A, Burtoloso A, De Vita D, Montanari C. Synthesis and
structure-activity relationship of nitrile-based cruzain inhibitors incorporating a
trifluoroethylamine-based P2 amide replacement. Bioorg Med Chem. 2019;27:115083.
https://doi.org/10.1016/j.bmc.2019.115083
11 Giroud M, Kuhn B, Saint-Auret S, Kuratli C, Martin RE, Schuler F, Diederich F, Kaiser M,
Brun R, Schirmeister T, Haap W. Triazole-Based Dipeptidyl Nitriles: Potent, Selective, and
Trypanocidal Rhodesain Inhibitors by Structure-Based Design. J. Med. Chem. 2018;61:3370-
3388. https://doi.org/10.1021/acs.jmedchem.7b01870
12
Dos Santos AM, Cianni L, De Vita D, Rosini F, Leitao A, Laughton CA, Lameira J,
Montanari CA. Experimental study and computational modelling of cruzain cysteine protease
inhibition by dipeptidyl nitriles. Phys Chem Chem Phys. 2018;20:24317-24328.
http://doi.org/10.1039/c8cp03320j
12

14
Li Y-Y, Fang J, Ao G-Z. Cathepsin B and L inhibitors: a patent review (2010 - present).
Expert Opin Ther Pat. 2017;27:6, 643-656. https://doi.org/10.1080/13543776.2017.1272572
15
Cianni L, Feldmann CW, Gilberg E, Gütschow M, Juliano L, Leitão A, Bajorath J,
Montanari CA. Can Cysteine Protease Cross-Class Inhibitors Achieve Selectivity? J Med
Chem. 2019;62:10497-10525. https://doi.org/10.1021/acs.jmedchem.9b00683
16 Stone JA, McCrea JB, Witter R, Zajic S, Stoch SA. clinical and translational pharmacology
of the cathepsin K inhibitor odanacatib studied for osteoporosis. Br J Clin Pharmacol.
2019;85:1072-1083. https://doi.org/10.1111/bcp.13869
17
Law S, Andrault PM, Aguda AH, Nguyen NT, Kruglyak N, Brayer GD, Bromme D.
Identification of mouse cathepsin K structural elements that regulate the potency of
odanacatib. Biochem J. 2017;474:851-864. http://dx.doi.org/10.1042/BCJ20160985
18
Schröder J, Noack S, Marhöfer RJ, Mottram JC, Coombs GH, Selzer M. Identification of
semicarbazones, thiosemicarbazones and triazine nitriles as inhibitors of leishmania mexicana
cysteine protease CPB. PLoS One 2013;8:e77460. doi:10.1371/journal.pone.0077460
19
Machado PA, Carneiro MPD, Sousa-Batista AJ, Lopes FJP, Lima APCA, Chaves SP,
Sodero ACR, de Matos Guedes HL. Leishmanicidal therapy targeted to parasite proteases.
Life Sci. 2019;219:163-181. https://doi.org/10.1016/j.lfs.2019.01.015
20
Quilles JC Jr, Tezuka DY, Lopes CD, Ribeiro FL, Laughton CA, de Albuquerque S,
Montanari CA, Leitão A. Dipeptidyl nitrile derivatives have cytostatic effects against
Leishmania
spp.
promastigotes.
Exp
Parasitol.
2019;200:84-91.
https://doi.org/10.1016/j.exppara.2019.04.001
21 Chen C, Devine P, Foster B, Hughes G, O'Shea, P. US Patent US200600307317.
22
Hughes G, Devine PN, Naber JR, O'shea PD, Foster BS, McKay DJ, Volante RP.
Diastereoselective reductive amination of aryl trifluoromethyl ketones and α-amino esters.
Angew Chem Int Ed. 2007;46:1839-1842. https://doi.org/10.1002/anie.200603745
13
13 Siqueira-Neto JL, Debnath A, McCall L-I, Bernatchez JA, Ndao M, Reed SL, Rosenthal PJ.
Cysteine proteases in protozoan parasites. PLOS Negl Trop Dis. 2018;12:e0006512.

23
The preference of the syn isomers over the anti comes from the formation of a complex
between the Zn2+ cation and the nitrogen and oxygen atoms at the 1,3-positions of the imine.
The chelated complex only allows the attack to the opposed face, once the si face is blocked.
Considering that the more stable conformation of these salts puts the methine proton in the
same plane of the imine face, the re face is more sterically hindered than the si face, due to the
presence of bulky groups such as the benzyl group in P2 (Figure S1). To obtain the R,S-
diastereoisomers (anti isomers), the reducing agent used was NaBH4 for the L-amino acid
derivatives. The same effect is observed for the D-amino acid, but now the final products after
the reduction with Zn(BH4)2 and NaBH4 are R,R-diastereoisomer (syn isomer) and the S,R-
diastereoisomer (anti isomer), respectively.
24
Avelar LAA, Camilo CD, de Albuquerque S, Fernandes WB, Gonçalez C, Kenny PW,
Leitão A, Mckerrow JH, Montanari CA, Orozco EVM, Ribeiro JFR, Rocha JR, Rosini F,
Saidel ME. Molecular design, synthesis and trypanocidal activity of dipeptidyl nitriles as
cruzain
inhibitors.
PLoS
Negl
Trop
Dis.
2015;9:e0003916.
http://dx.doi.org/10.1371/journal.pntd.0003916
25 Burtoloso ACB, de Albuquerque S, Furber M, Gomes JC, Gonçalez C, Kenny, PW, Leitão
A, Montanari CA, Júnior JCQ, Ribeiro, JFR, Rocha, JR. Anti-trypanosomal activity of non-
peptidic nitrile-based cysteine protease inhibitors. PLOS Negl Trop Dis. 2017;11:e0005343.
https://doi.org/10.1371/journal.pntd.0005343
26
Crystal Data for C22H22F3N3O: (M = 401.42 g/mol): orthorhombic, space group P212121
(no. 19), a = 9.65500 (10) Å, b = 13.59830(10) Å, c = 15.78070(10) Å, V = 2071.87(3) Å3, Z
= 4, T = 120(2) K, μ(CuKα) = 0.832 mm-1, Dcalc = 1.287 g/cm3, 19803 reflections measured
(8.584° ≤ 2Θ ≤ 149.212°), 4179 unique (Rint = 0.0221, Rsigma = 0.0145) which were used in
all calculations. The final R1 was 0.0256 (I > 2σ(I)) and wR2 was 0.0689 (all data).
Refinement model description: Number of restraints - 0, number of constraints - unknown.
27
Griffen E, Leach AG, Robb GR, Warner DJ. Matched molecular pairs as a medicinal
chemistry tool. J. Med. Chem. 2011:54;7739-7750. https://doi.org/10.1021/jm200452d
28
Chang G, Huard K, Kauffman GW, Stepan AF, Keefer CE. A multi-endpoint matched
molecular pair (MMP) analysis of 6-membered heterocycles. Bioorg Med Chem.
2017;25:381-388. http://doi.org/10.1016/j.bmc.2016.11.004
14

29
Lecaille F, Chowdhury S, Purisima E, Brömme D, Lalmanach G. The S2 subsites of
cathepsins K and L and their contribution to collagen degradation. Protein Sci. 2007;16:662-
670. http://doi.org/10.1110/ps.062666607
30
Alves LC, Melo RL, Cezari MH, Sanderson SJ, Mottram JC, Coombs GH, Juliano L,
Juliano MA. Analysis of the S(2) subsite specificities of the recombinant cysteine proteinases
CPB of Leishmania mexicana, and cruzain of Trypanosoma cruzi, using fluorescent substrates
containing non-natural basic amino acids. Mol Biochem Parasitol. 2001;117:137-143.
https://doi.org/10.1016/s0166-6851(01)00340-1
31
Cianni L, Sartori GR, Rosini F, De Vita D, Pires G, Rebelo B, Leitão A, Burtoloso ACB,
Montanari CA . Leveraging the cruzain S3 subsite to increase affinity for reversible covalent
inhibitors. Bioorg Chem. 2018;79:285-292. http://dx.doi.org/10.1016/j.bioorg.2018.04.006
15