Discovery of benzimidazole-based Leishmania mexicana cysteine protease CPB2.8ΔCTE inhibitors as potential therapeutics for leishmaniasis

Article abstract of DOI:10.1111/cbdd.13326

Chemotherapy is currently the only effective approach to treat all forms of leishmaniasis. However, its effectiveness is severely limited due to high toxicity, long treatment length, drug resistance, or inadequate mode of administration. As a consequence,

Full text of DOI:10.1111/cbdd.13326

PROFESSOR ANTONIO RESCIFINA (Orcid ID : 0000-0001-5039-2151)
DR NICOLA MICALE (Orcid ID : 0000-0002-9294-6033)
Article type : Research Article
Discovery of benzimidazole-based L. mexicana cysteine protease CPB2.8ΔCTE
inhibitors as potential therapeutics for leishmaniasis
a
a
Laura De Luca a, Stefania Ferro , Maria Rosa Buemi a, Anna-Maria Monforte , Rosaria Gitto a, Tanja
Schirmeister b, Louis Maes c, Antonio Rescifina d, Nicola Micale a,*
a Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Viale
F. Stagno D’Alcontres 31, Messina I-98166, Italy
b Institute of Pharmacy and Biochemistry, University of Mainz, Staudinger Weg 5, D-55128 Mainz, Germany
c
Laboratory of Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp, Universiteitsplein 1, B-
2610 Antwerp, Belgium
d Department of Drug Sciences, University of Catania, Viale A. Doria 6, Catania I-95125, Italy
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13326
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*Corresponding author:
Nicola Micale: Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of
Messina, Viale F. Stagno D’Alcontres 31, Messina I-98166, Italy; phone +39 090 6766419, fax +39 090 393897;
E-mail address: nmicale@unime.it
Running title: L. mexicana CPB2.8 inhibitors
Keywords: Benzimidazole derivatives, antileishmanial agents, L. mexicana CPB2.8, docking studies, in
silico profiling
Abstract
Chemotherapy is currently the only effective approach to treat all forms of leishmaniasis. However,
its effectiveness is severely limited due to high toxicity, long treatment length, drug-resistance or
inadequate mode of administration. As a consequence, there is a need to identify new molecular
scaffolds and targets as potential therapeutics for the treatment of this disease.
We report a small series of 1,2-substituted-1H-benzo[d]imidazole derivatives (9a-d) showing affinity
in the submicromolar range (Ki = 0.15-0.69 µM) towards L. mexicana CPB2.8ΔCTE, one of the more
promising targets for antileishmanial drug design. The compounds confirmed activity in vitro against
intracellular amastigotes of L. infantum with the best result being obtained with derivative 9d (IC50 =
6.8 µM), although with some degree of cytotoxicity (CC50 = 8.0 µM on PMM and CC50 = 32.0 µM on
MCR-5). In silico molecular docking studies and ADME-Tox properties prediction were performed to
validate the hypothesis of the interaction with the intended target and to assess the drug-likeness of
these derivatives.
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Introduction
Human leishmaniasis is one of the vector-borne tropical infectious diseases with a high rate of
morbidity and mortality worldwide, predominantly in endemic areas of developing countries.1 The
clinical spectrum ranges from the most common and usually self-resolving cutaneous form to a
disfiguring form affecting mucous membranes predominantly those of nose and mouth, and even to
the most severe visceral form wherein the parasites invade especially liver and spleen. The latter
form is fatal in the absence of a timely chemotherapeutic treatment.2 Despite several drug discovery
efforts that have been made in the recent past to counter the progression of this disease, the
current armamentarium of effective and safe antileishmanial drugs remains quite inadequate.
Pentavalent antimonials (i.e. meglumine antimoniate and sodium stibogluconate) are old and toxic
drugs administered intramuscularly that still constitute, for a large part, the first-line of intervention.
Liposomal amphotericin B, pentamidine salts, paromomycin sulphate and oral miltefosine are the
available second-line drugs in case of antimonial treatment failure.3 As for antimonials, these
second-line drugs also have several drawbacks including toxicity, high costs, long-term treatments
and repeated doses, emergence of resistance, and need of constant medical care (which is difficult
to afford in areas strongly related to poverty and lack of health services). In this scenario and in the
absence of an effective vaccine, the identification of new targets and the development of new
antileishmanial agents remain of primary importance.4 In this regard, the Leishmania parasite
expresses high levels of several classes of cysteine proteases (CPs) belonging to the papain family
whose activity has been recognized as essential for parasite survival and infectivity to the
mammalian host.5 Among these CPs, cysteine proteases group B (CPBs) of L. mexicana have been
identified as virulence factors and putative druggable targets.6 In particular, the isoform CPB2.8,
which is a typical cathepsin L-like endo-peptidase endowed also with carboxydipeptidase activity,7
remains relatively unexplored. Since only few reports describe specific inhibitors of L. mexicana
CPB2.8ΔCTE (the recombinant form of the isoform CPB2.8 expressed without the C-terminal
extension). These include peptide-based inhibitors bearing an electrophilic warhead that covalently
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traps the catalytic thiolate such as dipeptidyl vinyl sulfone 1, dipeptidyl α-ketoheterocycles 2 and
aziridinyl peptides (e.g. 3),8-12 semicarbazone 4, thiosemicarbazones 5, triazine nitrile 6,13 natural
compounds (e.g. morelloflavones 7),14,15 and a decorated fused benzo[b]thiophene inhibitor 8 acting
through a time-dependent bimodal mechanism of action (Fig. 1).16
Fig. 1: Chemical structures of known L. mexicana CPB2.8ΔCTE inhibitors.
In our ongoing search for novel antileishmanial agents that might specifically target L. mexicana
CPB2.8ΔCTE,16-18 we have made a screening campaign on our in-house database CHIME 1.6
containing compounds synthesized and stocked in laboratory. Therefore, we have initially selected
small compounds possessing the benzimidazole or imidazole ring as common chemical feature. The
idea of testing these derivatives arose from literature data indicating that compounds containing an
indole/benzimidazole scaffold showed potent anti-Leishmania properties with no specific insights
about the possible targets. This extensive list of indole-based compounds contains
indolylquinolines,19 azoles,20 alkaloids,21 indole-2-hydrazones,22 indole-2-carboxamides,23 C3-mono-
functionalized oxindoles,24 and thiophene-indole hybrids.25 Some of selected small compounds have
been previously studied as potential HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs)26
as well as negative allosteric modulators of GluN2B-containing NMDA receptors in mammalian
central nervous system.27-32
As a result of our preliminary screening of a set of twelve compounds, four 1,2-disubstituted-1H-
benzo[d]imidazole derivatives (i.e. 9a–d; Fig. 2) turned out to be active against the listed intended
target. The remaining eight indole derivatives I-VIII proved to be low inhibitors of L. mexicana
CPB2.8ΔCTE at 20µM concentration (see Supplementary Data).
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Fig. 2. Chemical structures of the 1,2-disubstituted-1H-benzo[d]imidazole derivatives tested against L.
mexicana CPB2.8ΔCTE.
1. Results and discussion
1.1. Chemistry
The synthesis of the selected panel of 1,2-disubstituted-1H-benzo[d]imidazole derivatives 9a–d was
33-35,26
accomplished following a straightforward procedure developed in our laboratories
and is
depicted in Scheme 1. After obtaining the two main fragments N1-aryl-1,3-dihydro-2H-
benzimidazole-2-thiones (16–18) and 2-chloro- N-phenylacetamides (19–21) (Schemes 1A,B), both
were condensed by reaction in dimethylformamide and in presence of K2CO3 (Scheme 1C) to yield
the target compounds 9a–d.
Scheme 1. Reagents and conditions: (a) DMF, NaH, rt, 2–6 h (b) Zn/HCl, EtOH, 80 °C, 1 h; (c) TCDI, pyridine, rt, 1 h;
(d) chloroacetyl chloride, DIPEA, CH2Cl2, rt, 1 h; (e) K2CO3, DMF, rt, 1.5 h.
The final products 9a–d were purified by recrystallization from ethanol or by column flash-
chromatography on silica gel to afford pure samples for biological assays. Analytical and spectral
data (1H NMR) of all synthesized compounds are in full agreement with the proposed structures.
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1.2. Biological activity
1.2.1. Enzyme assays
Compounds 9a–d were preliminarily screened against L. mexicana CPB2.8ΔCTE at a fixed 20 µM
concentration to assess their ability to target the enzyme. Cross-reactivity assays against highly
similar human cysteine proteases cathepsin-B (Cat-B) and cathepsin-L (Cat-L) were performed in
parallel under the same experimental conditions. An equivalent volume of DMSO was used as
negative control and Cbz-Phe-Arg-AMC was employed as the fluorogenic substrate. All compounds
strongly inhibited the intended parasitic target (>90%) without significantly affecting (from “no
inhibition” for Cat-B to a maximum of 20% of inhibition for Cat-L) the human counterparts (Table 1)
pointing out a remarkable selectivity of the new ‘lead’ structures. Hence, compounds 9a–d were
further evaluated against the parasite target by progress curve analysis (Fig. 3: inhibition of
CPB2.8ΔCTE by compound 9c) using a continuous readout.36 As can be seen from the data in Table 1,
the three compounds having the sulfone group as connection unit between the benzimidazole
scaffold and the aryl group at its N1 (i.e. 9a–c) showed IC50 values in the submicromolar range,
whereas the compound having the methylene group as connection unit at the same position (i.e. 9d)
turned out to be roughly one order of magnitude less potent. These results suggest that the
electron-rich sulfone group may take part considerably to the binding network involved within the
catalytic site of the enzyme, presumably as H-bond acceptor. In addition, the introduction of
electron-withdrawing substituents to the phenyl ring of the 2-ylthioacetamide side chain of the
benzimidazole core (e.g. 9a vs 9b) positively affects the binding affinity towards the target, as well as
the introduction of additional substituents with the same characteristics to the C6 position of the
base scaffold (e.g. 9b vs 9c).
Table 1
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Fig. 3. Progress curves of substrate hydrolysis in the presence of inhibitors 9a-c (0, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0,
10.0 µM from top to bottom; A, B and C, respectively), and 9d (0, 0.25, 0.5, 1.0, 2.5, 5.0, 10.0, 20.0 µM; D)
continuously determined over a period of 10 min. Substrate: Cbz-Phe-Arg-AMC. Experiments were performed
in duplicate. A representative graph is shown.
1.2.2. In vitro antileishmanial activity and cytotoxicity
Compounds 9a–d were evaluated in vitro against intracellular amastigotes of L. infantum to establish
whether any correlation exists between the inhibition of the target enzyme and the activity against
the whole organism. Cytotoxicity assays were also performed on primary peritoneal mouse
macrophages (PMM) and human fetal lung fibroblasts (MCR-5) to assess the selectivity. The in vitro
profiling was carried out as previously described.37 The results point out that there is not a clear
correlation between the activity against the selected L. mexicana CPB2.8ΔCTE target and the activity
against the parasite (Table 2). The most potent compound in the cell-based assay turned out to be
9d, with an IC50 value in the micromolar range (6.82 µM) comparable to that of the reference
compound miltefosine (4.50 µM). The other three derivatives 9a–c, which inhibit the molecular
target more efficaciously than 9d, showed fair activity (range 20–50 µM) against the whole
organism. Since the most relevant difference in terms of chemical structure between 9a–c and 9d is
the sulfone group vs methylene group as connection unit at the N1 of the benzimidazole scaffold, we
presume that one reason of this discrepancy in terms of activity could be the presence of the sulfone
group that unfavorably (compared to the methylene group) may affect the diffusion through the
biological barriers. A similar profile has been detected in the cytotoxicity assays against PMM and
MRC-5 cells (Table 2). Indeed, the most active compound 9d proved to be a more cytotoxic agent
against PMM and MCR-5 when compared with inhibitors 9a–c.
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Table 2
1.3. Docking studies
To better rationalize the ligand-enzyme interactions, more accurate docking experiments were
carried out utilizing the homology model of mature L. mexicana CPB2.8ΔCTE that had been
previously generated and validated.16
Inactivation of a protease by an active-site directed irreversible inhibitor usually proceeds by the
rapid formation of a non-covalent reversible enzyme-inhibitor complex (E•I), and successively in a
slower chemical step, a covalent bond is formed with the enzyme to generate the enzyme-inhibitor
adduct (E-I).38 Since the test compounds do not contain an electrophilic group which could
covalently react with the target enzyme, we conducted the study utilizing a sequence inherent to
only the first stadium of enzyme recognition: i) non-covalent docking of ligand upon mature L.
mexicana CPB2.8ΔCTE enzyme; ii) 25 ns of MD simulation of the best pose obtained for ligand-
CPB2.8ΔCTE complex, to accommodate the ligand; iii) non-covalent re-docking of the complex
obtained from the last 3 ns of MD simulation averaged frames. The above sequence has been
appropriately adapted from that previously used,16 which was shown to be effective in performing a
suitable level of docking accuracy.
The calculated values of Ki obtained by the non-covalent re-docking for compounds 9a–d are
reported in Table 3 and, in all cases, they are in parallel with those obtained experimentally.
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To avoid an over-scoring of the calculated energy of the database of the compounds, the “ligand
efficiency” (LE) of all the molecular structures was calculated,39,40 taking into account the slight
different molecular weight between the molecules and the co-crystallized ligand. In fact, an increase
in the molecular weight may influence the amount of van der Waals interactions, representing an
important factor for the calculations with docking software. The concept of ligand efficiency has
recently emerged as a measure for lead compound selection.41,42 This parameter is useful and
efficient for the prediction of the activity in the process of drug discovery. The ligand efficiency
depends on the dimension of the ligand, as smaller ligands have a higher efficiency than the larger
ligands. One of the causes behind this principle is the reduction in the area accessible to the ligand
on increasing the size of the ligand. These considerations play an important role for the screening of
compound databases. Ligand efficiency is calculated using the equation LE = ∆GB/N, where N is the
number of non-hydrogen atoms.
On this basis, we calculated the LE of our compounds and for the standard ligands (Table 3) from
which it emerged that compounds 9a and 9d are a little more efficient than 9b and 9c.
Table 3
The best-docked pose of compounds 9a-d together with amino acid interactions in the binding
pocket of the CPB2.8ΔCTE are represented in Figure 4 as a 2D arrangement. Almost all interactions
are due to hydrogen bond, although one π-H interaction for each of 9b and 9c, and three π-π
interactions for 9d are also proposed. The energies of these interactions have been reported in Table
4. Notably, all compounds explore a different interacting space on the enzymatic surface depending
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on substituents diversification; this is clearly evidenced in the 3D superposition of the best-docked
poses reported in Figure 5.
Fig. 4. 2D Schematic view of hydrophobic, hydrogen bond, π-H, and π-π interactions for compounds 9a–d.
From top left to bottom right.
Fig. 5. 3D superposition of the best-docked pose for compounds 9a–d (a in red, b in green, c in blue, and d in
magenta).
Table 4
1.4. In silico profiling
ADME-Tox properties of compounds 9a-d were investigated with the same criteria previously
adopted by us, and the results of these preliminary in silico studies are reported in Tables 5-6. The
“drug-likeness” of 9a-d was searched for the Lipinski’s rule and for the standard ADME prediction,
that is the Human Intestinal Absorption (HIA), Caco-2 cell permeability, Plasma Protein Binding
(PPB), and Blood-Brain Barrier (BBB) penetration. Ames mutagenic and carcinogenic potentials were
calculated for the toxicity profiling.
The overview of the physicochemical properties reported in Table 5 indicate that compounds 9a-d
do not satisfy the whole Lipinski’s rule of five and that they cannot be considered at first sight “drug-
like” according to Oprea’s descriptor-based scoring scheme.43 Nevertheless, compound 9a shows
only a single violation, viz. log P.
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The in silico ADME results instead (Table 6), clearly indicate that compounds 9a-d possess a
promising oral availability (e.g. optimal HIA >95%; suboptimal Caco-2 cell permeability) and a strong
plasma protein binding (PPB = 100%). Interestingly, compounds 9a–c are supposed to moderately
permeate the BBB (BBB penetration <1) unlike 9d (BBB penetration = 0.08); since the penetration
through BBB is not required for the treatment of leishmaniasis, compound 9d turns out to be less
likely to cause neurotoxicity. Finally, with the exception of compounds 9c, all these benzimidazole
derivatives resulted in a non-mutagen profiling and 9a,d as non-carcinogenic in rat and mouse.
Table 5
Table 6
2. Conclusion
To sum up, we discovered a new class of 1,2-substituted-1H-benzo[d]imidazole derivatives (i.e. 9a-d)
acting as non-covalent and selective inhibitors of the L. mexicana cysteine protease CPB2.8ΔCTE, one
of the most promising target within anti-Leishmania drug design. Overall, the most interesting
compound turned out to be 9d which showed affinity towards the enzyme in the submicromolar
range (Ki = 0.69 µM) and activity in vitro against the intracellular form of the parasite (amastigotes of
L. infantum) in the micromolar range (IC50 = 6.8 µM). Moreover, preliminary in silico ADME-Tox
studies indicated that 9d exhibits a good oral availability and results in a non-mutagen and non-
carcinogenic profiling. Taking together, these outcomes make 9d a new lead structure for further
drug design of anti-Leishmania agents.
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3. Experimental methods
3.1. Chemistry
3.1.1 General information
Characterization of each new intermediate and final compound (i.e. melting points, elemental
analyses and NMR spectra) were determined by means of equipments previously reported by our
group, as well as materials and purification methods.27
3.1.2 General procedures for the synthesis of N-(2-nitrophenyl)-benzenesulfonamides
(10,11) and N-substituted-2-nitroaniline (12)
Derivatives 10–12 were prepared following the synthetic procedures previously reported by
us.26-28 A mixture of anhydrous sodium hydride (5 mmol) and 2-nitroaniline (138 mg, 1
mmol) or 5-chloro-2-nitroaniline (173 mg, 1 mmol) in DMF (5 mL) was stirred for 10 min at
0
°C and then 3,5-dimethylbenzyl bromide (597 mg,
3
mmol) or 3,5-
dimethylbenzensulphonyl chloride (614 mg, 3 mmol) was added. When the reaction was
completed (2–6 h), a saturated NaHCO3 aqueous solution was added. The mixture was
extracted with dichloromethane (3 × 10 mL) and dried over Na2SO4. After removal of the
solvent under reduced pressure, the residue was triturated by treatment with diethyl ether and
crystallized from ethanol.
Spectral data of compounds 10,26 11,28 and 1227 are in accordance with the literature.
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3.1.3 General procedures for the synthesis of N-(2-aminophenyl)-benzene sulfonamides (13,14) and
N1-(substituted-benzyl)-2-aminoaniline (15)
Derivatives 13–15 were prepared following the synthetic procedures previously reported by us.26-28
The mixture of N-substituted-2-nitroanilines (1 mmol) or N-(2-nitrophenyl)-benzenesulfonamides (1
mmol) in HCl conc. (5 mL) and EtOH (7 mL) was stirred vigorously, then zinc dust (2.18 g, 33 mmol)
was added in several portions at room temperature. When the addition was completed, the reaction
was refluxed (80 °C) for 1 h. Then, the resulting mixture was cooled, made alkaline with NaOH 2N
aqueous solution and extracted with ethyl acetate (3 × 10 mL). The organic phases were collected,
washed with water, dried over Na2SO4 and evaporated. The residue was crystallized from ethanol or
purified by column flash chromatography using cyclohexane/AcOEt as eluent.
Spectral data of compounds 13,26, 14,28 and 1527 are in accordance with the literature.
3.1.4 General procedures for the synthesis of 1-(3,5-dimethylbenzyl)-1,3-dihydro-2H-
benzimidazol-2-one (16) and 1-(3,5-dimethylphenylsulfonyl)-1,3-dihydro-2H-benzimidazol-
2-ones (17,18)
Derivatives 16–18 were prepared following the synthetic procedures previously reported by us.26-28
To a solution of N1-(substituted-benzyl)-2-amino-aniline (1 mmol) or N-(2-aminophenyl)-
benzenesulfonamides (1 mmol) in pyridine (10 mL) 1,1’-thiocarbonyldiimidazole (250 mg, 1.4 mmol)
was added at room temperature and the resulting mixture was maintained under stirring for 1 h.
After this time, distilled water was added to quench the reaction and the precipitate was filtered off
to give the desired products after cooling.
Spectral data of compounds 16,26 17,28 and 1827 are in accordance with the literature.
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3.1.5 General procedures for the synthesis of 2-chloro-N-phenylacetamides (19–21).
Derivatives 19–21 were prepared following the synthetic procedures previously reported by us.26,35
N,N-Diisopropylethylamine (175 µL, 1 mmol) and then chloroacetyl chloride (78 µL, 1 mmol) were
added dropwise to a solution of suitable substituted anilines (1 mmol) in dichloromethane (5 mL).
The mixture was stirred for 1 h at room temperature. Successively, the reaction was quenched with
a saturated NaHCO3 aqueous solution. The reaction mixture was extracted with ethyl acetate (3 × 10
mL), dried over Na2SO4 and evaporated under reduced pressure. The residue was crystallized from
ethanol.
2-Chloro-N-phenylacetamide (19)
1
Yield 65%; m.p.: 115–117 °C. H NMR (DMSO-d6): 4.23 (s, 2H, CH2); 7.07 (m, 1H, ArH); 7.31 (m, 2H,
ArH); 7.56 (m, 2H, ArH); 10.29 (bs, 1H, NH). Anal. Calcd for C8H8ClNO: C, 56.65; H, 4.75; N, 8.26.
Found: C, 56.72; H, 4.79; N, 8.30.
Spectral data of compounds 20 and 21 are in accordance with the literature.26,35
3.1.6 General procedure for the synthesis of 1,2-substituted-1H-benzo[d]imidazole derivatives 9a–
d.
Derivatives 9a–d were prepared following the synthetic procedures previously reported by us.26
A
solution of 1-(3,5-dimethylbenzyl or 3,5-dimethylphenylsulfonyl)-1H-benzo[d]imidazole-2(3H)-thione
derivative (1 mmol) (16–18) in DMF (3 mL), anhydrous potassium carbonate (138 mg, 1 mmol) and
the appropriate 2-chloro-N-phenylacetamide (1 mmol) (19–21) was stirred at room temperature for
1.5 h. The reaction was quenched by the addition of saturated NaHCO3 aqueous solution and the
mixture was extracted with ethyl acetate (3 × 10 mL). After removal of the solvent under reduced
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pressure, the residue was crystallized by treatment with ethanol or purified by column flash
chromatography using cyclohexane/AcOEt as eluent.
2-(1-(3,5-Dimethylphenylsulfonyl)-1H-benzo[d]imidazol-2-ylthio)-N-phenylacetamide (9a)
Yield 61%; m.p.: 159–161 °C. 1H NMR (DMSO-d6): 2.32 (s, 6H, (CH3)2); 4.35 (s, 2H, CH2); 7.01–7.06 (m,
1H, ArH); 7.26–7.31 (m, 4H, ArH); 7.40 (s, 1H, ArH); 7.55–7.57 (m, 3H, ArH); 7.80–7.88 (m, 3H, ArH);
10.38 (bs, 1H, NH). Anal. Calcd for C23H21N3O3S2: C, 61.18; H, 4.69; N, 9.31. Found: C, 61.32; H, 4.79;
N, 9.21.
Spectral
data
of
methyl-4-(2-(1-(3,5-dimethylphenylsulfonyl)-1H-benzo[d]imidazol-2-
ylthio)acetamido) benzoate (9b), methyl 3-chloro-4-(2-(6-chloro-1-(3,5-dimethylphenylsulfonyl)-1H-
benzo[d]imidazol-2-ylthio)acetamido)benzoate (9c) and methyl 3-chloro-4-(2-(6-chloro-1-(3,5-
dimethylbenzyl)-1H-benzo[d]imidazol-2-ylthio)acetamido)benzoate (9d) are in accordance with the
literature.26,35 Data of elemental analysis for the last three resynthesized compounds are the
following: Anal. Calcd for C25H23N3O5S2 (9b): C, 58.92; H, 4.55; N, 8.25. Found: C, 58.98; H, 4.59; N,
8.20. Anal. Calcd for C25H21Cl2N3O5S2 (9c): C, 51.91; H, 3.66; N, 7.26. Found: C, 52.00; H, 3.69; N, 7.20.
Anal. Calcd for C26H23Cl2N3O3S (9d): C, 59.09; H, 4.39; N, 7.95. Found: C, 59.17; H, 4.41; N, 7.92.
3.2.Biological activity
3.2.1. Enzyme assays
Recombinant L. mexicana cysteine protease CPB2.8ΔCTE was expressed and purified as previously
described,44,45 whereas human cathepsins B and L were purchased from Calbiochem. The initial
screening of benzimidazole and indole derivatives against the parasite enzyme was performed at 20
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μM concentration according to well-established methods previously reported.16 Compounds
showing at least 50% inhibition (i.e. 9a-d) were subjected to detailed follow-up assays against the
target and cross-reactivity screening against the above-mentioned cathepsins.16,46
3.2.2. In vitro amastigote assay
The in vitro anti-Leishmania assay of 9a-d was performed as previously described by using L.
infantum MHOM/MA (BE)/67 intracellular amastigotes collected from the spleen of an infected
donor hamster and used to infect primary peritoneal mouse macrophages.47 Miltefosine was
included as reference drug.
3.2.3. Cytotoxicity assays
Cytotoxicity assays were performed both on MRC5SV2 and PMM cells according to procedures
previously reported.47 Tamoxifen was included as the reference drug.
3.3. Docking
3.3.1. Preparation of ligands
The 3D structures of ligands were built using Gabedit (2.4.8) software48 and all geometries were fully
optimized, in the same software, with the semi-empirical PM6 Hamiltonian49 implemented in
MOPAC2016 (17.130W).50
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3.3.2. Molecular dynamics simulations
The molecular dynamics simulations of the mature L. mexicana CPB2.8ΔCTE/ligand complexes
(based on the PDBs prepared as described above) were performed with the YASARA Structure
package (17.4.17).51 A periodic simulation cell with boundaries extending 10 Å from the surface of
the complex was employed. The box was filled with water, with a maximum sum of all bumps per
water of 1.0 Å, and a density of 0.997 g/mL with explicit solvent. YASARA’s pKa utility was used to
assign pKa values at pH 7.2,52 and the cell was neutralized with NaCl (0.9% by mass); in these
conditions ligands 9 result protonated at pyrrolidinic N-Me. Waters were deleted to readjust the
solvent density to 0.997 g/mL. The YASARA2 force field was used with long-range electrostatic
potentials calculated with the Particle Mesh Ewald (PME) method, with a cutoff of 8.0 Å.53-55 The
ligand force field parameters were generated with the AutoSMILES utility,56 which employs
semiempirical AM1 geometry optimization and assignment of charges, followed by the assignment
of the AM1BCC atom and bond types with refinement using the RESP charges, and finally the
assignments of general AMBER force field atom types. Optimization of the hydrogen bond network
of the various enzyme-ligand complexes was obtained using the method established by Hooft et
al.,57 in order to address ambiguities arising from multiple side chain conformations and protonation
states that are not well resolved in the electron density.58 A short MD was run on the solvent only.
The entire system was then energy minimized using first a steepest descent minimization to remove
conformational stress, followed by a simulated annealing minimization until convergence (<0.01
kcal/mol Å). The MD simulation was then initiated, using the NVT ensemble at 298 K, and integration
time steps for intramolecular and intermolecular forces every 1.25 fs and 2.5 fs, respectively. The
MD simulation was stopped after 40 ns and, on the averaged structure of the last 3 ns frames, a
second cycle of energy minimization, identical to the first, was applied.
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3.3.3. Docking protocol
Macromolecules and ligands, as obtained after MD simulation and energy minimization, were
prepared with Vega ZZ (3.1.1)59 assigning Gasteiger charges to the protein and AM1BCC ones to the
ligand. Fine docking was performed by applying the Lamarckian genetic algorithm (LGA)
implemented in AutoDock 4.2.6.60 The ligand-centered maps were generated by the program
AutoGrid (4.2.6) with a spacing of 0.375 Å and dimensions that encompass all atoms extending 5 Å
from the surface of the ligand. All of the parameters were inserted at their default settings. In the
docking tab, the macromolecule and ligand are selected, and GA parameters are set as ga_runs =
100, ga_pop_size = 150, ga_num_evals = 20000000, ga_num_generations = 27000, ga_elitism = 1,
ga_mutation_rate = 0.02, ga_crossover_rate = 0.8, ga_crossover_mode = two points,
ga_cauchy_alpha = 0.0, ga_cauchy_beta = 1.0, number of generations for picking worst individual =
10.
Because no water molecule is directly involved in complex stabilization they were not considered in
the docking process. All protein amino acidic residues were kept rigid whereas all single bonds of
ligands were treated as full flexible.
Acknowledgments
Free academic license from ChemAxon for its suite of programs is gratefully acknowledged.
Conflict of Interest
The authors declare no competing financial interest.
This article is protected by copyright. All rights reserved.

Figure legends
Fig. 1: Chemical structures of known L. mexicana CPB2.8ΔCTE inhibitors.
Fig. 2. Chemical structures of the 1,2-disubstituted-1H-benzo[d]imidazole derivatives tested against L.
mexicana CPB2.8ΔCTE.
Fig. 3. Progress curves of substrate hydrolysis in the presence of inhibitor 9c (0, 0.25, 0.5, 1.0, 2.5, 5.0, 10.0 µM
from top to bottom), continuously determined over a period of 10 min. Substrate: Cbz-Phe-Arg-AMC.
Experiments were performed in duplicate. A representative graph is shown.
Fig. 4. 2D Schematic view of hydrophobic, hydrogen bond, π-H, and π-π interactions for compounds 9a–d.
From top left to bottom right.
Fig. 5. 3D superposition of the best-docked pose for compounds 9a–d (a in red, b in green, c in blue, and d in
magenta).
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Table 1. Screening at 20µM of 9a–d against L. mexicana CPB2.8ΔCTE. Human Cat-B and Cat-L were used in the
counter assay to test the selectivity profile. IC50 values include standard deviation from two independent
experiments, each performed in duplicate. Ki ±SD values have been calculated using the Cheng-Prusoff
equation.
% inhibition at 20µM
Compound
IC50 (µM)
Ki (µM)b
CPB2.8ΔCTE
95.6 ±0.5
97.2 ±0.4
97.4 ±0.6
92.0 ±0.9
Cat-B
n.i.a
Cat-L
9a
9b
9c
9d
13.7 ±2.0 0.68 ±0.06 0.23 ±0.02
20.3 ±3.5 0.54 ±0.13 0.18 ±0.04
12.5 ±2.1 0.45 ±0.03 0.15 ±0.01
16.5 ±2.2 2.06 ±0.92 0.69 ±0.31
n.i.
n.i.
12.8 ±1.8
a n.i. = no inhibition. b assuming compounds are competitive inhibitors
Table 2. In vitro antileishmanial activity and cytotoxicity (IC50 µM and CC50 µM, respectively) of
selected benzimidazole-based derivatives 9a–d.
L. infantuma
50.8
PMMb
>64.0
32.0
32.0
8.0
MCR-5c
>64.0
>64.0
>64.0
32.0
Compound
9a
20.3
9b
32.5
9c
6.8
9d
Tamoxifen
Miltefosine
—
—
10.9
4.5
—
—
aL. infantum MHOM/MA/67/ITMAP263. bPrimary peritoneal mouse macrophages. cHuman fetal lung fibroblast
(MRC-5) cell line toxicity.
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Table 3. Calculated Ki and LE values for compounds 9a–d.
Compound
Experimental Ki (nM)
Calculated Ki (nM)
N
Ligand efficiency
9a
9b
9c
9d
230
180
150
690
383
194
136
750
31
35
37
35
0.28
0.26
0.25
0.28
Table 4. Calculated interaction energies for hydrogen bond interactions of compounds 9a–d with CPB2.8ΔCTE.
Compound Ligand
Receptor
SG CYS25
O GLY23
Interaction
H-donor
Distance (Å)
4.02
E (kcal/mol)a
9a
N3
–1.7
C4
H-donor
2.83
–0.8
O1
N GLY66
H-acceptor
H-acceptor
H-acceptor
pi-H
3.00
–3.1
O17
O17
6-ring
SG CYS25
ND1 HIS163
CB ASN162
3.11
–1.0
2.99
–5.9
3.66
–0.6
9b
N13
O1
SG CYS25
N CYS25
CA GLY65
N GLY66
H-donor
3.37
3.14
2.67
3.04
–2.1
–3.1
–0.7
–2.7
H-acceptor
H-acceptor
H-acceptor
O30
O31
9c
O11
NE2 GLN19
NE1 TRP185
CA ASN162
H-acceptor
H-acceptor
pi-H
2.82
2.89
3.72
–1.2
–3.8
–1.4
O11
5-ring
9d
O1
N CYS25
H-acceptor
H-acceptor
pi-pi
2.78
2.86
3.79
3.47
3.94
–5.7
–2.0
–0.0
–0.0
–0.0
O34
N GLY66
5-ring
6-ring
6-ring
5-ring TRP185
5-ring TRP185
6-ring TRP185
pi-pi
pi-pi
aCalculated by the Analyze Interactions subroutine present in YASARA software (v. 17.4.17,
http://www.yasara.org/).
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Table 5. In silico Lipinski’s rule of five parameters and drug-likeness of compounds 9a–da.
Compound MW
Number
of H-
Number
of H-
log P
log D5.0 log D7.4 TPSA
Number
of
Drug-
likeness
bond
acceptors donors
bond
Lipinski’s
violations
9a
9b
9c
9d
451.56
4
5
5
3
1
1
1
1
5.13
5.13
6.34
7.36
5.13
5.13
6.34
7.34
5.13
5.13
6.34
7.36
81.06
1
2
2
2
False
False
False
False
509.60
578.48
528.45
107.36
107.36
73.22
aJChem for Excel (version 17.4.300.1589) was used for structure-property prediction and calculation,
ChemAxon (http://www.chemaxon.com).
Table 6. Selected in silico ADME-Tox profiling of compounds 9a–da.
Compd.
Absorption
Distribution
Toxicity
Carcinoge- Carcinoge
HIA In vitro Caco-
In vitro
In vivo BBB
Ames test
(%)
2 cell
PPB (%)
penetration
(Cbrain/Cblood)
nicity in
rat
-nicity in
mouse
permeabi-lity
(nm s–1)
9a
9b
96.97
98.03
21.48
21.15
100
100
0.51
non-
mutagen
negative
negative
negative
positive
0.57
non-
mutagen
9c
97.82
97.63
11.92
48.11
100
100
0.93
0.08
mutagen
negative
negative
positive
negative
9d
non-
mutagen
aThe properties related to ADME were predicted using PreADMET web-based application
(http://preadmet.bmdrc.kr).
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