Cathepsin K inhibitors based on 2-amino-1,3,4-oxadiazole derivatives

Article abstract of DOI:10.1016/j.bioorg.2021.104662

Two new series of hitherto unknown dipeptides, containing an electrophilic nitrile or a non-electrophilic 2-amino-1,3,4-oxadiazole moiety were synthesized and evaluated in vitro as Cathepsin K (Cat K) inhibitors. From 14 compounds obtained, the oxadiazole derivatives 10a, 10b, 10e, and 10g acted as enzymatic competitive inhibitors with Ki values between 2.13 and 7.33 μM. Molecular docking calculations were carried out and demonstrated that all inhibitors performed hydrogen bonds with residues from the enzyme active site, such as Asn18. The best inhibitors (10a, 10b, 10g) could also perform these bonds with Cys25, and 10a showed the most stabilizing interaction energy (?134.36 kcal mol^?1) with the active cavity. For the first time, derivatives based in 2-amino-1,3,4-oxadiazole scaffolds were evaluated, and the results suggested that this core displays a remarkable potential as a building block for Cat K inhibitors.

Full text of DOI:10.1016/j.bioorg.2021.104662

Bioorganic Chemistry 109 (2021) 104662
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Cathepsin K inhibitors based on 2-amino-1,3,4-oxadiazole derivatives
a
b
b
b
˜
Talita B. Gontijo , Patrícia S. Lima , Marcelo Y. Icimoto , Raquel Leao Neves ,
c
b
d
d
´
Erika C. de Alvarenga , Adriana K. Carmona , Alexandre A. de Castro , Teodorico C. Ramalho ,
a
a,*
ˆ
Eufranio N. da Silva Júnior , Rossimiriam P. de Freitas
a
ˆ
Departamento de Química, ICEx, Universidade Federal de Minas Gerais, Av. Pres. Antonio Carlos 6627 Pampulha, Belo Horizonte, MG 31270-901, Brazil
b
c
˜
˜
Departamento de Biofísica, Universidade Federal de Sao Paulo, Rua Pedro de Toledo, 669, Vila Clementino, Sao Paulo, SP 04039-032, Brazil
ˆ
˜
˜
˜
˜
Departamento de Ciencias Naturais – DCNAT, Universidade Federal de Sao Joao Del-Rei, Bairro Dom Bosco, Sao Joao del-Rei, MG 36301-160, Brazil
d Departamento de Química, Universidade Federal de Lavras, Lavras, MG, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords:
Two new series of hitherto unknown dipeptides, containing an electrophilic nitrile or a non-electrophilic 2-
amino-1,3,4-oxadiazole moiety were synthesized and evaluated in vitro as Cathepsin K (Cat K) inhibitors.
From 14 compounds obtained, the oxadiazole derivatives 10a, 10b, 10e, and 10g acted as enzymatic competitive
inhibitors with Ki values between 2.13 and 7.33 µM. Molecular docking calculations were carried out and
demonstrated that all inhibitors performed hydrogen bonds with residues from the enzyme active site, such as
Asn18. The best inhibitors (10a, 10b, 10g) could also perform these bonds with Cys25, and 10a showed the most
stabilizing interaction energy (ꢀ 134.36 kcal molꢀ 1) with the active cavity. For the first time, derivatives based in
2-amino-1,3,4-oxadiazole scaffolds were evaluated, and the results suggested that this core displays a remarkable
potential as a building block for Cat K inhibitors.
Cathepsin K
Dipeptides
1,3,4-Oxadiazoles
1. Introduction
agents has been extensively explored over the years in medicinal
chemistry.
Cysteine cathepsins are proteases that are involved in various bio-
logical processes such as enzymatic degradation [1a]. These enzymes
comprise a large group of significant macromolecules involved in a
variety of physiological processes, representing viable targets for ther-
apeutic intervention [1]. Among them, cathepsin K (cat K) has been the
most studied one in the last two decades due to its potential relevance in
osteoporosis management [1,2], a chronic disease that affects millions
of people worldwide [3].
In general, Cat K inhibitors are dipeptide derivatives and peptido-
mimetic containing moieties such as ketone, aldehyde, ketoamide,
nitrile and azanitrile groups [7]. Among these electrophilic groups, the
nitrile warhead results in a reversible enzyme inhibition due to the
formation of a thioimidate adduct after linking to the catalytic residue
Cys-25 (present in the S1 subsite) of Cat K (Fig. 1A) [8a]. Besides elec-
tron deficient groups to interact with S1, the molecular scaffold of the
inhibitors also contains hydrophobic residues in P2 and aromatic moi-
eties in P3 which interact with S2 and S3 subsites (according to the
Schechther and Berger nomenclature) [8b], respectively (Fig. 1A).
Moreover, a recent approach using an alkyne moiety as a mild electro-
phile to promote an irreversible inhibition was described as well [9].
Balicatib, odanacatib and ONO-5334 are some of the most advanced
Cat K inhibitors (Fig. 1B). These compounds are under clinical trials or
were studied and discontinued in phase II. Odanacatib, for instance, is a
highly selective and reversible Cat K inhibitor that could be taken
weekly because of its long half-life of 66–93 h [10]. However, due to the
increased side effect risk of stroke in phase III, the clinical trials for
odanacatib were discontinued [11]. Another example of a potent and
Cathepsin K corresponds to the major collagenase expressed in os-
teoclasts, particularly efficient in cleaving type I collagen, with a direct
impact on the response to inflammatory stress on the bone surface [4].
However, its importance surpasses osteoporosis research [2], Cat K is
also able to degrade type II collagen, deteriorating cartilage with
possible implication for rheumatoid arthritis, osteoarthritis, and bone
metastasis [4,5]. Additionally, this enzyme displays an important role in
the nervous and pulmonary tissues, modulating the immunologic and
endocrine systems, especially in pathological conditions, and its elastase
activity could be associated with cardiovascular diseases [1b,6].
Therefore, the development of Cat K inhibitors as potential therapeutical
* Corresponding author at: Departamento de Química, Universidade Federal de Minas Gerais, 31270-901, Brazil.
E-mail address: rossimiriam@qui.ufmg.br (R.P. de Freitas).
https://doi.org/10.1016/j.bioorg.2021.104662
Received 25 October 2020; Received in revised form 6 January 2021; Accepted 12 January 2021
Available online 22 January 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

T.B. Gontijo et al.
Bioorganic Chemistry 109 (2021) 104662
highly selective Cat K inhibitor is MIV-711, which is under phase II
clinical trials for the management of osteoarthritis [10c]. Until the
present time, none of the tested inhibitors have become a commercial
drug for the treatment of the osteoporosis disease. Despite these set-
backs, the understanding of elemental biology of cathepsin K inhibition
and the comprehension of its clinical effects contribute greatly to the
future development of specific drugs for osteoporosis and related
diseases.
market such as raltegravir (antiretroviral), nesapidil (vasodilating
agent) and furamizole (antibacterial agent) (Fig. 1C).
Different approaches using heterocyclic building blocks as scaffolds
for developing Cat K inhibitors have recently been reported [14].
However, to the best of our knowledge, only one report has described
keto-1,3,4-oxadiazole derivatives as Cat K inhibitors so far [15]. In this
context, we report here the synthesis of a series of novel dipeptide ni-
triles and 2-amino-1,3,4-oxadiazole derivatives and their evaluation as
Cat K inhibitors. For the design of the novel molecules, aromatics moi-
eties were used in P3, sec-butyl and isobutyl groups as hydrophobic
residues in the P2 position. Furthermore, the initial molecular strategy
considered the nucleophilic character of the S1 subsite, provided by the
cysteine 25, which allowed the introduction of a nitrile group or
equivalent (Fig. 1D).
1,3,4-Oxadiazole has become an important core for the development
of new drugs due to its broad range of therapeutic effects, including anti-
inflammatory, analgesic, antimicrobial, antifungal, and antitumor ac-
tivities [12]. Its stability in aqueous media and ability for acting as a
hydrogen acceptor are important characteristics for its use in medicinal
chemistry [12f]. Also, the 1,3,4-oxadiazole ring has been employed in
bioisosteric replacement for less stable carbonyl related groups such as
carboxylic acids, esters, amides, carbamates and hydroxyesters [13].
Some drugs containing 1,3,4-oxadiazole moieties are available in the
Fig. 1. Overview.
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T.B. Gontijo et al.
Bioorganic Chemistry 109 (2021) 104662
2. Results and discussion
attempts. For example, the treatment of the hydrazide 9a with the
electrophilic nitrile source cyanogen bromide (BrCN) in presence of
DIPEA as base, employing a mixture of solvents (tetrahydrofuran:
dichloromethane: dioxane 1:1:1) for 30 h [22], furnished a major
product which was characterized as the dipeptide 10a containing a 2-
amino-1,3,4-oxadiazole ring (22%yield, r.t.). Further attempts to
isolate the azadipeptide nitrile N,N-unsubstituted derivatives were made
with the base removal, using the same mixture of solvent and reagents
for 24 h under conventional heating at 50 ◦C. These conditions also
provided compound 10a with 45% of yield (Table 1, entry 1).
A plausible mechanism (Scheme 4) for the formation of the 2-amino-
1,3,4-oxadiazole ring may consider a hydrazide attack on the nitrile
carbon to form intermediate A. After the bromide loss, intermediate B
probably undergoes a cyclization reaction furnishing intermediate C,
which yields the oxadiazole core after aromatization.
2.1. Chemistry
The novel dipeptide nitriles (5a-g) were prepared in three steps
(Scheme 1). Firstly, the amino acid Fmoc-L-isoleucine (1) was treated
with 3-aminopropanenitrile (2), using EDC (1-Ethyl-3-(3-dimethylami-
nopropyl) carbodiimide) as a coupling reagent and DMP (4-Dimethy-
laminopyridine) as catalyst, to provide the intermediate amide 3
[16–17]. The latter was then submitted to a deprotection reaction and
the Fmoc group was removed with piperidine in chloroform [18],
affording the amine 4 with 90% yield (Scheme 1a). Finally, the amine 4
was reacted with different carboxylic acids to form a peptide bond under
classic coupling conditions (EDC/DMP in dichloromethane) [16–17],
yielding the dipeptide nitriles 5a-g (82–96%).
2-amino-1,3,4-oxadiazoles (10a-g) represent the second class of
compounds prepared in this work (Scheme 5). Initially, the protection of
the carboxylic group of L-leucine (6) as methyl ester was accomplished
by the treatment of the amino acid with thionyl chloride in methanol
[7e], affording compound 7 (98% yield). Then, the ester 7 was reacted
with different aromatic carboxylic acids under classic coupling condi-
tions [16–17], furnishing the derivatives 8a-g (49–90% yield) (Scheme
2a). Finally, compounds 8a-g underwent a reaction with hydrazine
Therefore, in order to achieve new dipeptide-2-amino-1,3,4-
oxadiazoles and optimize the yield and time of reaction, microwave or
ultrasonic irradiation were employed since the literature describes them
as excellent and powerful tools for organic synthesis, mainly for the
preparation of heterocycle compounds [23]. Firstly, the synthesis con-
ditions were optimized for 10a, using these techniques, BrCN in DCM/
THF/dioxane, and hydrazide 9a. The dipeptide-oxadiazole 10a was
obtained with 48% (under microwave) and 52% (under ultrasonic
irradiation) yield (Table 1, entries 2 and 3). However, the best condition
(10a, 64% yield) considered isopropanol as solvent under ultrasonic
irradiation and absence of base (Table 1, entry 5).
◦
hydrate in methanol at 70 C [19], yielding the corresponding hydra-
zides 9a-g (90–95% yield).
The synthesized hydrazides enabled the next step of the synthetic
route targeting azadipeptide nitrile cathepsin K inhibitors (Scheme 3a),
which have been extensively exploited as potential agents for osteopo-
rosis and osteoarthritis therapy in the last decade [7e,20,21]. Azadi-
peptide nitrile N,N-unsubstituted derivatives (B, Scheme 3b), which
represent a group of particular interest due to the electrophilic warhead
present in their structures, were the initial aim of our studies. Never-
theless, the previously published synthesis protocols failed after several
After having optimized the reaction conditions, the new methodol-
ogy was employed for the synthesis of the 2-amino-1,3,4-oxadiazole
derivatives 10a-g (47–76% yield) (Scheme 5). The novel dipeptide ni-
triles and amino-oxadiazole compounds were purificaded by column
chromatographic, as described in the Experimental Section and fully
characterized by spectroscopic techniques (1H NMR, 13C NMR, IR) and
HRMS (see Supplementary Information).
Scheme 1. Synthetic route for the novel dipeptide nitriles 5a-g.
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T.B. Gontijo et al.
Bioorganic Chemistry 109 (2021) 104662
Scheme 2. Synthetic route for the hydrazides 9a-g.
Scheme 3. (a) Azadipeptide nitrile Cat K Inhibitor (b) Synthesis of 2-amino-1,3,4-oxadiazole 10a from hydrazide 9a using BrCN as electrophilic nitrile source in a
basic medium.
Table 1
Optimization of reaction conditions for the synthesis of oxadiazole 10a.
Entry
Radiation source
Solvent
Temperature
Time (h)
Yield (%)
1
2
3
4
5
Conventional heating
MW (100 W)
DCM/THF/Dioxane (1:1:1)
DCM/THF/Dioxane (1:1:1)
DCM/THF/Dioxane (1:1:1)
Isopropanol
50 ◦
45 ◦
40 ◦
50 ◦
40 ◦
C
C
C
C
C
24
3,5
8
45%
48%
52%
52%
64%
Ultrasonic irradiation
MW (100 W)
4
Ultrasonic irradiation
Isopropanol
8
Reaction conditions: 9a (0.25 mmol), BrCN (0.75 mmol), solvents (3 mL). Yield refers to the isolated product
Scheme 4. Proposed mechanism for the formation of the oxadiazole ring from unsubstituted hydrazides using Br-CN.
4

T.B. Gontijo et al.
Bioorganic Chemistry 109 (2021) 104662
Scheme 5. Synthesis route for the novel 2-amino-1,3,4-oxadiazoles 10a-g.
2.2. Inhibition of cathepsin K
contribute to affinity were investigated. Table 3 exhibits the intermo-
lecular interaction energy values, as well as the residues by which the
hydrogen bonds occurred.
The dipeptide nitriles (5a-g) and 2-amino-1,3,4-oxadiazoles (10a-g)
were tested as Cat K inhibitors. Their inhibition constants (Ki) were
calculated by the tight-binding titration data analysis GraFit program
[25] as described in experimental section 4.2.1. and summarized in
Table 2. A representative plot is showed in Fig. 2. Among the evaluated
compounds, it is noteworthy that compound 10a showed the best
inhibitory activity, with a Ki value of 2.1 ± 0.2 µM.
As shown in Table 3, all inhibitors investigated interacted well with
Cathepsin K active cavity, with interaction energy values in the range of
ꢀ 86.57 kcal molꢀ 1 to ꢀ 134.36 kcal molꢀ 1. Based on our findings, all
inhibitors demonstrated, in general, affinity and favorable interactions
within Cathepsin K. We can also observe that the inhibitor (10a) showed
the most stabilizing interaction energy (ꢀ 134.36 kcal molꢀ 1), followed
by the inhibitors (10b) (ꢀ 129.67 kcal molꢀ 1), (10g) (ꢀ 128.62 kcal
molꢀ 1) and also (10e) (ꢀ 126.62 kcal molꢀ 1). Note that the inhibitor
(10a) showed the lowest interaction energy value, that is, this inhibitor
presented the best interaction in the site, which corroborates the
experimental results.
The mechanism of inhibition was determined by enzyme activity
assay using the compound 10a as representative of all studied com-
pounds. As indicated in Fig. 3a, the Lineweaver-Burk plot (double-
reciprocal of Michaelis-Menten equation) indicates similar y-intercept
for 0, 0.9 and 1.9 µM of 10a (same Vmax), as well as increased x-intercept
in the presence of inhibitor (decreased Km). Considering the Dixon plot
(Fig. 3b), using the same data collected in the previous result, the lines
represent the velocity of each substrate, with all lines crossing in the
second quadrant. As observed, both Lineweaver-Burk and Dixon plots
are characteristic for competitive inhibition.
By analysing these outcomes, our calculations protocol employed in
the study revealed that the most reactive compounds (10a, 10b, 10g
and 10e, respectively) showed close interaction energies, with the
largest energy difference of about 7.74 kcal molꢀ 1 between (10a) and
(10e). Another key trend to be highlighted here in the fact of the
interaction energy values increase as the Ki values increase. In this line,
we observe that the interaction with the enzyme is less stabilizing in
increased values of Ki. In order to illustrate this trend, observe that the
inhibitor (5f) had the less stabilizing interaction energy (ꢀ 86.57 kcal
molꢀ 1), which is according to the highest value of Ki (367 ± 22.6 µM).
From our computations, the interaction energy difference between the
least and most active inhibitor (5f and 10a, respectively) was a quite
2.3. Cell viability
The cytotoxic effects of the compounds 10a, 10b, 10e, 10g were
mensurated by the colorimetric MTT assay, using the HUVEC (Human
Umbilical Vein Endothelial Cells) cell line, as described in experimental
section 4.2.3. All compounds tested have not shown cytotoxicity against
the HUVEC cell line at concentrations below 10 µM, as showed in Fig. 4.
These results indicate that compounds were not toxic in the range of
effective inhibition concentration.Fig. 5.
significant value of 47.79 kcal molꢀ 1
.
The hydrogen bonds are essential for stabilizing the ligand in the
active site. Moreover, there are other kinds of interactions equally
important, such as electrostatic and hydrophobic ones, which favor a
stabilizing accommodation of the ligands in the binding site, contrib-
uting to the computed total intermolecular interaction energy. Accord-
ing to Table 3, observe that all inhibitors demonstrated to perform
hydrogen bonds with residues from the active, such as Asn18, which was
common for all compounds. Diverse inhibitors also performed hydrogen
bonds with glycine (such as Gly20, Gly66, Gly64), as well as other amino
acid residues. This computational study is an important step towards
obtaining theoretically the most favorable interaction modes of the in-
hibitors under investigation, in addition to better understanding their
inhibitory activity. It is important to mention that the residue Cys25
performed hydrogen bonds with the best inhibitors (10a, 10b, 10g)
investigated in this computational study.
2.4. Molecular docking
According to the inhibitor structures, the molecular docking calcu-
lations were carried out to adjust these ligands in the Cathepsin K active
cavity, evaluating the affinity among them. For this study, a cavity
prediction algorithm based on a 3D box was employed in order to
generate the binding sites. Thus, the volume of the calculated active
cavity was 311.30 Å3. Due to the big size of the active cavity, no steric
impediment was observed for docking the inhibitors in the active site,
favoring the obtaining of favorable accommodations in the cavity.
In order to comprehend the interaction modes of these inhibitors
within the enzyme active site, in particular, some parameters that
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T.B. Gontijo et al.
Bioorganic Chemistry 109 (2021) 104662
Table 2
Ki values of dipeptide nitriles and 2-amino-1,3,4-oxadiazole compounds against Cathepsin K.
Compound
Ki (µM)
Compound
Ki (µM)
132 ± 11.5
2.1 ± 0.2
110 ± 8.3
95 ± 8.6
64 ± 7.3
4.6 ± 0.3
45 ± 3.5
21 ± 1.5
169 ± 12.7
7.3 ± 0.6
367 ± 22.6
69 ± 5.7
61 ± 5.0
6.7 ± 0.4
Assay conditions: the enzyme was activated with 5 mM DTT for 3 min in 100 mM sodium acetate pH 5.5, at 37 ◦C. The substrate Z-Phe-Arg-AMC was added, and the
residual activity was measured (λex 380 nm and λem 460 nm) in the presence of the inhibitors. The errors were not greater than 5%.
3. Conclusions
cysteine protease cathepsin K for the first time. The 2-amino-1,3,4-oxa-
diazole derivatives 10a, 10b, 10e, and 10g represented the most active
compounds and acted as enzymatic competitive inhibitors with Ki
values between 2.13 and 7.33 µM. Computational studies showed that
the Cat K residue Cys25 performed hydrogen bonds with the best in-
hibitors and the 10a showed the most stabilizing interaction energy
(ꢀ 134.36 kcal molꢀ 1) with the enzyme active cavity. Finally, this work
reported, for the first time, dipeptide 2-amino-1,3,4-oxadiazoles as a
promising class for the development of new Cat K inhibitors and further
In sum, the present work described the synthesis of a series of hith-
erto unknown dipeptides, containing an electrophilic nitrile or a non-
electrophilic 2-amino-1,3,4-oxadiazole moiety. The dipeptide nitriles
were obtained by classic protocols. However, a methodology was
developed to synthesize the dipeptide 2-amino-1,3,4-oxadiazole de-
rivatives from the cyclisation of N,N-unsubstituted hydrazides in the
absence of base. The compounds were evaluated as inhibitors of the
6

T.B. Gontijo et al.
Bioorganic Chemistry 109 (2021) 104662
spectra were recorded using a Bruker maXis and Q-Exactive (Thermo-
Scientific). Infrared spectra were recorded on a Perkin Elmer Spectrum
One FTIR spectrometer in tablets of anhydrous KBr. IR bands are
described by the wavenumber (
ν
‾, cmꢀ 1). Melting points were deter-
mined using the Büchi melting point apparatus and are uncorrected.
4.1.1. Synthesis of dipeptide nitriles 5a-g
4.1.1.1. Synthesis
of
(2S,3S)-2-amino-N-(2-cyanoethyl)-3-methyl-
pentamide (4). Fmoc-L-isoleucine (3 mmol, 1.060 g), 1-Ethyl-3-(3dime-
tylaminopropyl)carbodiimide (EDC) (3.3 mmol, 630 mg) and 4-
dimethylaminopyridine (DMAP) (1.2 mmol, 146 mg) were added to a
round bottom flask containing a stir bar. Dichloromethane (10 mL) was
added, and the reaction mixture was cooled under N2 atmosphere and
stirred for 15 min. After 15 min, the 3-aminopropionitrile (3.3 mmol,
250 µL) was added and then the reaction mixture was stirred at room
temperature for 24 h. After the end of the reaction, the mixture was
diluted with dichloromethane (30 mL) and washed with a aq. solution of
HCl 1 M (2 × 20 mL) and NaHCO3 saturated (1 × 20 mL). The organic
layer was dried over sodium sulfate, filtered, and the filtrate was
evaporated in vacuo to yield a white solid (1.352 g).
Fig. 2. Ki determination for compound 10a. Tight-binding inhibition constant
of cathepsin K was determined by measuring the remaining hydrolytic activity
using 5 µM of Z-Phe-Arg-AMC as substrate and increasing inhibitor concen-
trations. Enzyme was activated by incubation with 5 mM dithiothreitol in 100
mM sodium phosphate buffer, pH 5.5, as described in the material and
methods section.
The white solid (1.352 g), chloroform (30 mL) and piperidine (15
mmol, 1.5 mL) were added to a round bottom flask with a stir bar. The
reaction mixture was stirred at room temperature for 24 h, and the
progress of the reaction was monitored by TLC.
investigations must be performed.
After the end of the reaction, the solvent was evaporated and the
crude was purified by column chromatography on silica gel (n-hexane:
ethyl acetate 1:1) yielded the product corresponding as a yellow oil
(494 mg, 2.7 mmol , 90%).
4. Experimental
4.1. Synthesis of compounds 5a-g and 10a-g
1H NMR (400MHz,CDCl3) δ: 7.87 (s, 1H), 3.53–3.41 (m, 2H), 3.24
(d, J = 3.6 Hz, 1H), 2.58 (t, J = 6.4 Hz, 2H), 1.97–1.87 (m, 1H), 1.69 (bs,
2H), 1.36–1.27 (m, 1H), 1.12–1.01 (m, 1H), 0.92 (d, J = 7.2 Hz, 3H),
0.85 (t, J = 7.2 Hz, 3H). 13C NMR (100MHz, CDCl3) δ: 175.6, 118.3,
59.8, 38.1, 35.3, 23.8, 18.4, 16.2, 11.9. HRMS (ESIꢀ ): 182.12958
[Mꢀ H]ꢀ . Cald. for [C9H16N3O]ꢀ : 182.12934.
Starting materials obtained from commercial suppliers were used as
received unless otherwise stated. Column chromatography (CC) was
performed using silica gel (Aldrich 40–63 µm, 230–400 mesh). Thin
layer chromatography (TLC) was performed using aluminum-backed 60
F254 silica plates. Visualization was achieved by UV fluorescence.
Proton nuclear magnetic resonance (NMR) spectra were recorded using
a Bruker AVANCE DPX 400 spectrometer. 13C NMR spectra were
recorded at 100 MHz as stated. Chemical shifts (δ) are given in parts per
million (ppm). Peaks are described as singlets (s), doublets (d), doublet
of doublets (dd), doublet of triplets (dt), triplets (t), triplet of doublets
(td), quartets (q), quintets and multiplets (m). The 1H and 13C NMR
spectra were referenced to the appropriate residual solvent peak or TMS
peak. Coupling constants (J) were quoted to the nearest 0.5 Hz. Mass
4.1.1.2. General procedure for the synthesis of the compounds 5a-g. The
corresponding benzoic acid (0.5 mmol), 1-Ethyl-3-(3-dimetylamino-
propyl)carbodiimide (EDC) (0.5 mmol, 105 mg) and 4-dimethylamino-
pyridine (DMAP) (0.3 mmol, 37 mg) were added to a round bottom
flask containing a stir bar. Dichloromethane (10 mL) was added, and the
reaction mixture was cooled at 0 ◦C under N2 atmosphere and stirred for
15 min, and then a solution of the compound (4) in dichloromethane (5
Fig. 3. Competitive inhibition profile of compound 10a. (a) Lineweaver-Burk plot; (b) Dixon plot. I = inhibitor 10a concentration (µM). S = substrate Z-Phe-Arg-
AMC concentration (µM). All plots were determined using the GraFit 5.0 software [25]. The error was less than 5% for any obtained values.
7

T.B. Gontijo et al.
Bioorganic Chemistry 109 (2021) 104662
Fig. 4. Effects of compounds 10a, 10b, 10e and 10g on the HUVEC cell viability. The HUVEC cell line was incubated in presence of the compounds 10a, 10b, 10e
and 10g at concentrations 0.25, 0.5, 1, 2.5, 5 and 10 µM for 24 h. After this period, the cellular viability was assessed by the colorimetric MTT assay (final con-
centration 0.5 mg/mL). The experiments were performed in triplicate.
mL) was added. The reaction mixture was stirred, at room temperature
under N2, and the progress of the reaction was monitored by TLC.
After the end of the reaction, the mixture was diluted with
dichloromethane (10 mL) and washed with aq. solution of HCl 1 M (2 ×
10 mL) and NaHCO3 saturated (1 × 10 mL). The organic layer was dried
over sodium sulfate, filtered, and the filtrate was evaporated in vacuo.
Purification was performed by silica gel chromatography (n-hexane/
ethyl acetate), to yield chromatographically and spectroscopically pure
product.
2H), 2.16–2.05 (m, 1H), 1.68–1.55 (m. 1H), 1.30–1.15 (m , 1H), 1.01 (d,
J = 6.8 Hz, 3H), 0.96 (t, J = 7.4 Hz, 3H). 13C NMR (101MHz, CDCl3) δ:
172.3, 165.6, 154.1, 152.1, 121.5, 119.8, 118.0, 115.8, 113.2, 58.6,
56.8, 56.0, 36.8, 35.9, 25.1, 18.3, 16.0, 11.6. HRMS (ESIþ): 348.1918
[M + H]+. Cald. for [C18H26N3O4]+: 348.1923.
4.1.1.4. 3-chloro-N-((2S,3S)-1-((2-cyanoethyl)amino)-3-methyl-1-oxo-
pentan-2-yl)benzamide (5b). The product was obtained after 19 h of
reaction as a white solid (198 mg, 0.48 mmol, 96%); m.p. (◦C)¼
179.7–181.5 ◦C, IV (v, cm¡1): 3293 (
ν NH), 2961 (νass C H), 2250 (ν
–
1
–
–
–
C N), 1659 and 1640 (ν C O). H NMR (400MHz, CD OD) δ: 7.85 (t,
–
–
4.1.1.3. N-((2S,3S)-1-((2-cyanoethyl)amino)-3-methyl-1-oxopentan-2-
yl)-2,5-dimethoxybenzmide (5a). The product was obtained after 24 h of
reaction as a white solid (142 mg, 0.42 mmol, 82%); m.p. (◦C)¼
–
J = 1.6 Hz, 1H), 7.76 (d, J = 8.0 Hz, 1H), 7.55 (d, J = 8.30 Hz, 1H), 7.45
(t, J = 8.0 Hz, 1H), 4.37 (d, J = 8.4 Hz, 1H), 3.60–3.49 (m, 1H), 3.47–3.
36 (m, 1H), 2.76–2.62 (m 2H), 2.03–1.92 (m, 1H), 1.68–1.58 (m, 1H),
149.0–152.3 ◦C, IV (v, cm¡1): 3281 (ν 1NH), 2972–2834 (νass
C H),
2238 (
ν
C
N), 1659 and 1600 (
ν
C
O). H NMR (400MHz, CDCl ) δ:
1.33–1.22 (m, 1H), 1.00 (d, J = 6.8 Hz, 3H), 0.94 (t, J = 7.4 Hz, 3H). 13
C
–
–
–
–
8.50 (d, J = 8.0 Hz, 1H), 7.68 (d, J = 3.2 Hz, 1H), 7.11 (bs, 1H), 7.023(dd,
J = 9.0, 3.2 Hz, 1H), 6.93 (d, J = 9.0 Hz, 1H), 4.55 (dd, J = 8.0, 6.4 Hz,
1H), 3.95 (s, 3H), 3.81 (s, 3H), 3.58–3.45 (m, 2H), 2.61 (t, J = 6.5 Hz,
NMR (100MHz, CD3OD) δ: 173.1, 167.8, 136.1, 134.4, 131.5, 130.0,
127.5, 125.8, 118.3, 58.9, 36.5, 35.4, 25.2, 17.2, 14.8, 10.2. HRMS
(ESIþ): 322.1317 [M + H]+. Cald. for [C16H21ClN3O2]+: 322.1322.
–
8

T.B. Gontijo et al.
Bioorganic Chemistry 109 (2021) 104662
Fig. 5. Representation of the hydrogen bonds performed by the most active inhibitors in the Cathepsin K active site.
306.1618.
Table 3
Values of the parameters obtained by molecular docking calculations for the
inhibitors in the Cathepsin K active site through MVD® software.
4.1.1.6. N-((2S,3S)-1-((2-cyanoethyl)amino)-3-methyl-1-oxopentan-2-
yl)-4 (trifluoromethyl) benzamide (5d). The product was obtained after
5 h of reaction as a white solid (149 mg, 0.42 mmol, 83%); m.p. (◦C)¼
Inhibitor
Ki ± Std Error
(µM)
Interaction Energy (kcal
H-bonds (residues)
molꢀ 1
)
217.5–218.3 C, IV (v, cm¡1): 3291 (
ν
NH), 2964 (νass
C
H), 2241(
ν
◦
–
1
(5a)
132 ± 11.5
ꢀ 106.12
Asn18, Gly20, Gly66,
Cys25
–
–
–
C
N),1634 (
ν
C
O). H NMR (400MHz, CD OD) δ: 8.00 (d, J = 8.4
–
Hz, 2H), 7.78 (d, J = 8.4 Hz, 2H), 4.40 (d, J = 8.34 Hz, 1H), 3.60–3.50 (m,
1H), 3.48–3.37 (m, 1H), 2.76–2.63 (m, 2H), 2.06–1.96 (m, 1H),
1.71–1.58 (m, 1H), 1.37–1.24 (m 1H), 1.02 (d, J = 6.8 Hz, 3H), 0.95 (t, J
= 7.4 Hz, 3H); 13C NMR (100MHz, CD3OD) δ: 174.2, 169.0, 139.2,
–
(5b)
(5c)
(5d)
(5e)
(5f)
110 ± 8.3
95 ± 8.6
ꢀ 108.92
ꢀ 112.06
ꢀ 116.32
ꢀ 96.81
Asn18, Gly66
Asn18, Gly66
64 ± 7.3
Asn18, Gly66, His162
Asn18, Gly64
169 ± 12.7
367 ± 22.6
69 ± 5.7
ꢀ 86.57
Asn18, Gly66, Cys25
Asn18, Gly64
4
134.0, 129.4, 126.5 (q, JCF = 3.5 Hz), 119.4, 60.1, 37.7, 36.6, 26.3,
(5g)
(10a)
ꢀ 114.01
¡134.36
18.4, 16.0, 11.2. HRMS (ESIþ): 356.1580 [M + H]+. Cald. for
2.1 ± 0.2
Asn18, Gly64, Trp26,
Cys25
[C17H21F3N3O2]+: 356.1586;
(10b)
4.6 ± 0.3
¡129.67
Asn18, Gln19, Cys25,
Gly66
4.1.1.7. N-((2S,3S)-1-((2-cyanoethyl)amino)-3-methyl-1-oxopentan-2-
yl)-3-(trifluoromethyl) Benzamide (5e). The product was obtained after
5 h of reaction as a white solid (163 mg, 0.46 mmol, 92%); m.p. (◦C)¼
157.2–159.4 ◦C, IV (v, cm¡1): 3293 (
ν NH), 2972 (νass C H), 2247 (ν
–
(10c)
(10d)
(10e)
(10f)
(10g)
45 ± 3.5
21 ± 1.5
7.3 ± 0.6
61 ± 5.0
6.7 ± 0.4
ꢀ 119.70
ꢀ 122.79
¡126.62
ꢀ 117.23
¡128.62
Asn18, Glu186, Asn187
Asn18, Gly64, His162
Asn18, Tyr67, Gly66
Asn18, Gly64
1
–
Asn18, Ala163, Cys25,
Gly66
–
–
C
N),1663 and 1637 (ν C O). H NMR (400MHz, CD OD) δ: 8.16 (s,
–
1H), 8.10 (d, J = 7.8 Hz, 1H), 7.84 (d, J = 7.8 Hz, 1H),37.67 (t, J = 7.8
Hz, 1H), 4.41 (d, J = 8.5 Hz, 1H), 3.62–3.37 (m,2H), 2.76–2.63 (m, 2H),
2.06–1.93 (m, 1H), 1.69–1.58 (m, 1H), 1.38–1.18 (m, 1H), 1.02 (d, J =
6.8 Hz, 3H), 0.95 (t, J = 7.4 Hz, 3H); 13C NMR (100MHz, CD3OD) δ:
174.3, 168.7, 136.4, 132.2, 132.0–131.7 (m), 130.5, 129.4–129.2 (m),
126.3–124.0 (m), 125.7–125.4 (m), 119.5, 60.1, 37.6, 36.6, 26.4, 18.4,
16.0, 11.2. HRMS (ESIþ): HRMS (ESIþ): 378.13980 [M + Na]+ Cald.
for [C17H21F3N3NaO2]+: 378.14053.
–
4.1.1.5. N-((2S,3S)-1-((2-cyanoethyl)amino)-3-methyl-1-oxopentan-2-
yl)-4-fluorobenzamide (5c). The product was obtained after 17 h of re-
action as a white solid (126 mg, 0.42 mmol, 83%); m.p. (◦C)¼
189.4–191.◦C, IV (v, cm¡1): 3283 (
ν
–
NH), 2972 (νass C H), 2249 (ν
N), 1663 and 1633 (ν C
O). 1H NMR (400MHz, CD3OD) δ:
–
–
–
–
C
–
7.92–7.88 (m, 2H), 7.19 (t, J = 8.8 Hz, 2H), 4.38 (d, J = 8.4 Hz, 1H),
3.60–3.49 (m, 1H), 3.44–3.37 (m, 1H), 2.79–2.62 (m, 2H), 2.07–1.93
(1H), 1.72–1.58 (m,1H), 1.37–1.20 (m, 1H), 1.01 (d, J = 6.8 Hz, 3H),
0.94 (t, J = 7.4 Hz, 3H); 13C NMR (100MHz, CD3OD) δ: 174.4, 169.1,
166.3 (d, 1JCF = 250.4 Hz), 131.7 (d, 4JCF = 3.2 Hz), 131.2 (d, 3JCF = 10
Hz), 119.4, 116.35 (d, 2JCF = 22.2 Hz), 60.0, 37.8, 36.6, 26.3, 18.3, 16.0,
11.2. HRMS (ESIþ): 306.1612 [M + H]+. Cald. for [C16H21FN3O2]+:
4.1.1.8. N-((2S,3S)-1-((2-cyanoethyl)amino)-3-methyl-1-oxopentan-2-yl)
nicotinamide (5f). The product was obtained from nicotinic acid, after
17 h of reaction, as a white solid (115 mg, 0.40 mmol, 80%); m.p. (◦C)¼
160.7–161.3 ◦C, IV (v, cm¡1): 3281 (
ν NH), 2961 (νass C H), 2247 (ν
–
1
–
–
N),1669 and 1630 (ν C O). H NMR (400MHz, CDCl ) δ: 9.06 (bs,
–
3
–
C
–
9

T.B. Gontijo et al.
Bioorganic Chemistry 109 (2021) 104662
1H), 8.77–8.67 (m, 1H), 8.12 (d, J = 8.0 Hz, 1H), 7.90 (bs, 1H), 7.65 (d,
J = 8.6 Hz, 1H), 7.35 (dd, J = 8.0, 4.9 Hz, 1H), 4.59 (t, J = 8.6 Hz, 1H),
3.67–3.52 (m, 1H), 3.49–3.37 (m, 1H), 2.70–2.51 (m,2H), 2.10–1.96 (m,
1H), 1.71–1.58 (m, 1H), 1.27–1.14 (m, 1H), 0.99 (d, J = 6.7 Hz, 3H),
0.89 (t, J = 7.4 Hz, 3H); 13C NMR (100MHz, CDCl3) δ: 172.5, 166.1,
152.6, 148.6, 135.5, 129.7, 123.6, 118.3, 58.5, 37.1, 35.8, 25.3, 18.4,
6H). 13C NMR (100MHz, CDCl3) δ: δ 173.8, 167.2, 134.2, 131.6, 128.7,
127.2, 52.5, 51.3, 42.1, 25.2, 23.0, 22.2. The product was characterized
by 1H and 13C NMR and the data are consistent with those reported in
the literature [23c].
4.1.2.4. (S)-methyl
2-(2,4-dimethoxybenzamido)-4-methylpentanoate
15.7, 11.1. HRMS (ESIþ): 289.1659 [M
+
H]+. Cald. for
(8b). The product was obtained after 22 h of reaction as a yellow oil
(441 mg, 1.49 mmol, 74% yield). 1H NMR (400MHz, CDCl3) δ: 8.19 (d,
J = 7.3 Hz, 1H), 8.12 (d, J = 8.8 Hz, 1H), 6.56 (dd, J = 8.8, 2.0 Hz, 1H),
6.46 (d, J = 2.0 Hz, 1H), 4.85–4.74 (m, 1H), 3.93 (s, 3H), 3.81 (s, 3H),
3.72 (s, 3H), 1.78–1.60 (m, 3H), 0.95 (d, J = 6.0 Hz, 6H). 13C NMR
(100MHz, CDCl3) δ: 173.9, 164.9, 163.6, 159.2, 134.0, 114.2, 105.4,
98.7, 56.1, 55.6, 51.4, 41.8, 25.3 22.9, 22.2. HRMS (ESIþ): 310.16572
[M + H]+. Cald. for [C16H24NO5]+: 310.16545.
[C15H21N4O2]+: 289.1665.
4.1.1.9. N-((2S,3S)-1-((2-cyanoethyl)amino)-3-methyl-1-oxopentan-2-yl)
thiophene-3-carboxamide (5g). The product was obtained from 2-thio-
phenecarboxylic acid, after 17 h of reaction, as a white solid (129 mg,
0.44 mmol, 88%); m.p. (◦C)¼185.3–187.9 C, IV (v, cm¡1): 3293 (
ν
◦
1
–
–
N),1666 e 1626 (
–
–
ν C O). H NMR
–
–
NH), 2974 (νass
C
H), 2250 (
ν
C
(400MHz, CD3OD) δ: 8.17–8.11 (m, 1H), 7.53 (dd, J = 5.0, 1.1 Hz, 1H),
7.46 (dd, J = 5.0, 3.0 Hz, 1H), 4.37 (d, J = 8.4 Hz, 1H), 3.59–3.48 (m,
1H), 3.46–3.35 (m, 1H), 2.75–2.62 (m, 2H), 2.01–1.92 (m, 1H),
1.67–1.58 (m, 1H), 1.32–1.20 (m, 1H), 1.00 (d, J = 6.8 Hz, 3H), 0.94 (t,
J = 7.4 Hz, 3H); 13C NMR (100MHz, CD3OD) δ: 174.4, 165.5, 137.9,
130.3, 127.7, 127.4, 119.4, 59.6, 37.7, 36.6, 26.3, 18.3, 16.0, 11.2.
4.1.2.5. (S)-methyl
2-(2,5-dimethoxybenzamido)-4-methylpentanoate
(8c). The product was obtained after 44 h of reaction as a colourless oil
(228 mg, 0.73 mmol, 49% yield). 1H NMR (400MHz, CDCl3) δ: 8.46 (d,
J = 7.2 Hz, 1H), 7.74 (d, J = 3.2 Hz, 1H), 7.00 (dd, J = 9.0, 3.2 Hz, 1H),
6.92 (d, J = 9.0 Hz, 1H), 4.86–4.77 (m, 1H), 3.94 (s, 3H), 3.80 (s, 3H),
3.75 (s, 3H), 1.79–1.64 (m , 3H), 0.98 (d, J = 4.7 Hz, 3H). 13C NMR
(10MHz, CDCl3) δ: 173.8, 164.9, 154.1, 152.2, 121.8, 119.9, 115.6,
113.5, 56.9, 56.0, 52.3, 51.5, 41.8, 25.2, 23.0, 22.3. HRMS (ESIþ):
310.16572 [M + H]+. Cald. for [C16H24NO5]+: 310.16545.
HRMS (ESIþ): 294.1271[M
+
H]+. Cald. for [C14H20N3O2S]+:
294.1276.
4.1.2. Synthesis of 2-amino-1,3,4-oxadiazoles 10a-g
4.1.2.1. Synthesis of (S)ꢀ 1-Methoxy-4-methyl-1-oxopentan-2-amine hy-
drochloride (7). L-Leucine (8 mmol, 1.045 g) was added to a round
bottom flask with two necks, containing a stir bar. Dry MeOH (20 mL)
and thionyl chloride (27.2 mmol, 3.2 mL) were added slowly, and the
reaction mixture was stirred under N2 atmosphere at 0 ◦C for 30 min.
After 30 min, the reaction mixture was heated to 70 ◦C for 6 h and then
was stirred at room temperature for 10 h.
4.1.2.6. (S)-methyl 2-(2-methoxybenzamido)-4-methylpentanoate (8d).
The product was obtained after 18 h of reaction as a colourless oil (332
mg, 1.18 mmol, 79% yield). 1H NMR (400MHz, CDCl3) δ: 8.34 (d, J =
6.7 Hz, 1H), 8.17 (dd, J = 7.8, 1.4 Hz, 1H), 7.50–7.39 (m , 1H), 7.05 (t, J
= 7.2 Hz, 1H), 6.97 (d, J = 8.3 Hz, 1H), 4.88–4.76 (m, 1H), 3.97 (s, 3H),
3.74 (s, 3H), 1.83–1.63 (m, 3H), 0.97–0.96 (m, 6H). 13C NMR (100MHz,
CDCl3) δ: 173.8, 165.0, 157.8, 133.1, 132.4, 121.4, 121.1, 111.5, 56.1,
52.3, 51.4, 41.8 25.2, 22.9, 22.2. HRMS (ESIþ): 280.15527 [M + H]+.
Cald. for [C15H22NO4]+: 280.15488.
After 10 h, the reaction mixture was evaporated in vacuo. Diethyl
ether (10 mL) was added to the residue and the so formed suspension
was cooled at 0 ◦C for 2 h. The precipitate was collected by filtration to
give 7, 1.43 g (98%) [7e].
4.1.2.7. (S)-methyl 4-methyl-2-(3-(trifluoromethyl)benzamido)pentanoate
(8e). The product was obtained after 3 h of reaction as a colourless oil
(111 mg, 0.35 mmol, 70% yield). 1H NMR (400MHz, CDCl3) δ: 8.04 (s,
1H), 7.94 (d, J = 7.8 Hz, 1H), 7.74 (d, J = 7.8 Hz, 1H), 7.54 (t, J = 7.8
Hz, 1H), 6.81 (d, J = 8.1 Hz, 1H), 4.94–4.82 (m, 1H), 3.78 (s, 3H),
1.80–1.61 (m, 3H), 0.98 (t, J = 5.9 Hz, 6H). 13C NMR (100MHz, CDCl3)
1H NMR (400MHz, CD3OD) δ: 4.12–3.98 (m, 1H), 3.84 (s, 3H),
1.90–1.65 (m, 3H), (m, 2H), 1.00 (m, 6H), 13C NMR (100MHz, CD3OD)
δ: 171.4, 53.7, 52.5, 40.7, 25.6, 22.5, 22;3.
4.1.2.2. General procedure for the synthesis of the compounds 8a-g. The
corresponding benzoic acid (2 mmol), 1-ethyl-3-(3-dimetylamino-
propyl)carbodiimide (EDC) (2.4 mmol, 458 mg) and 4-dimethylamino-
pyridine (DMAP) (1.2 mmol, 146 mg) were added to a round bottom
flask containing a stir bar. Dichloromethane (10 mL) was added, and the
reaction mixture was cooled under N2 atmosphere and stirred for 15
min. In another round bottom flask, were added (S)-1-methoxy-4-
methyl-1-oxopentan-2-amine hydrochloride (7) (2.4 mmol, 434 mg),
dichorometane (5 mL) and pyridine (2.6 mmol, 210 µL), the mixture was
stirred for 15 min, then was added to round bottom flask containing the
corresponding benzoic acid and others reagents. The progress of reac-
tion was monitored by TLC.
δ: 173.9, 165.8, 134.7, 131.3 (q, 2JCF = 32.9 Hz), 129.3, 128.5 (q, 3JCF
=
3
3.6 Hz), 124.31 (q, JCF = 3.8 Hz), 52.7, 51.4, 41.8, 25.1, 23.0, 22.0.
HRMS (ESIꢀ ): 316.11750 [Mꢀ H]ꢀ . Cald. for [C15H17F3NO3]ꢀ :
316.11605.
4.1.2.8. (S)-methyl 2-(4-fluorobenzamido)-4-methylpentanoate (8f). The
product was obtained after 5 h of reaction as a light yellow solid (102
mg, 0.38 mmol, 76% yield). 1H NMR (400MHz, CDCl3) δ: 7.81 (dd, J =
8.5, JH/F = 5.4 Hz, 2H), 7.11 (t, J = 8.5 Hz, 2H), 6.52 (d, J = 7.6 Hz, 1H),
4.93–4.78 (m, 1H), 3.77 (s, 3H), 1.89–1.57 (m, 3H), 0.98 (t, J = 6.4 Hz,
3H). 13C NMR (100MHz, CDCl3) δ: 173.9, 166.2, 129.6 (d, 3JCF = 8.9
Hz), 115.8 (d, 2JCF = 21.9 Hz), 52.6, 51.3, 42.0, 25.2, 23.0, 22.2. HRMS
(ESIꢀ ): 266.12056 [Mꢀ H]ꢀ . Cald. for [C14H17FNO3]ꢀ : 266.11925.
After the end of the reaction, the mixture was diluted with
dichloromethane (20 mL) and washed with aq. solution of HCl 1 M (2 ×
20 mL) and NaHCO3 saturated (1 × 20 mL). The organic layer was dried
over sodium sulfate, filtered, and the filtrate was evaporated in vacuo.
Purification was performed by silica gel chromatography (n-hexane/
ethyl acetate), to yield chromatographically and spectroscopically pure
product.
4.1.2.9. (S)-methyl 2-(2,4-difluorobenzamido)-4-methylpentanoate (8g).
The product was obtained after 5 h of reaction as a colourless oil (129
1
mg, 0.45 mmol, 90% yield). H NMR (400MHz, CDCl3) δ: 8.09–8.03
(m, 2H), 7.03–6.91 (m, 2H), 6.87–6.83 (m, 1H), 4.91–4.74 (m, 1H), 3.73
(s, 2H), 1.86–1.60 (m, 3H), 0.94 (d, J = 4.5 Hz, 6H). 13C NMR
(100MHz, CDCl3) δ: 173.3, 165.01 (dd, 1JCF = 255.5, 3JCF = 13.1 Hz),
161.17 (dd, 1JCF = 249, 3JCF = 13.0 Hz), 162.2, 133.9 (dd, J = 10.2, 3.7
Hz), 117.2 (dd, J = 11.8, 3.7 Hz), 112.4 (dd, 2J = 21.3, 4J = 3.3 Hz),
104.4 (dd, 2J = 28.7, 25.9 Hz), 52.4, 51.4, 41.6, 25.0, 22.8, 22.0. HRMS
(ESIꢀ ): 284.11124 [Mꢀ H]ꢀ . Cald. for [C14H16F2NO3]ꢀ : 284.10982.
4.1.2.3. (S)-methyl 2-benzamido-4-methylpentanoate (8a). The product
was obtained after 24 h of reaction as a white solid (408 mg, 1.64 mmol,
82% yield. 1H NMR (400MHz, CDCl3) δ: 7.80 (d, J = 7.2 Hz , 2H), 7.51
(t, J = 7.2, 7.2 Hz, 1H), 7.43 (t, J = 7.2, 7.2 Hz, 2H), 6.57 (d, J = 7.2 Hz,
NH), 4.90–4.84 (m, 1H), 3.77 (s, 3H), 1.80–1.65 (m, 3H), 1.00–0.97 (m,
10

T.B. Gontijo et al.
Bioorganic Chemistry 109 (2021) 104662
4.1.2.10. General procedure for the synthesis of the hydrazides (9a-g).
The corresponding compound (8a-g) (1 mmol), methanol (25 mL) and
hydrazine hydrate (25 mmol, 2 mL), were added to a round bottom flask
containing a stir bar. The reaction mixture was heated to 70 ◦C and the
progress of a reaction was monitored by TLC. After the end of the re-
action, the solvent was evaporated in vacuo, and yielded the product
with a small amount of water. To remove the water of the product, 10
mL of toluene was added to the product and the solvent was evaporated
again to yield the corresponding hydrazide.
(m, 2H), 1.05–0.85 (m, 6H). 13C NMR (100MHz, CDCl3) δ: 173.1,
166.7, 129.7 (d, 3JCF = 8.9 Hz), 115.77 (d, 2JCF = 21.9 Hz), 50.9, 41.3,
25.1, 22.9, 22.4. HRMS (ESIþ): 268.14667 [M + H]+. Cald. for
[C13H19FN3O2]+: 268.14613.
4.1.2.17. (S)-2,4-difluoro-N-(1-hydrazinyl-4-methyl-1-oxopentan-2-yl)
benzamide (9g). The product was obtained after 5 h of reaction as a
white solid (90 mg, 0.33 mmol, 91%) 1H NMR (400MHz, DMSO‑d6) δ:
9.25 (s, 1H), 8.34 (d, J = 6.7 Hz, 1H), 7.67 (dd, J = 15.2, 8.5 Hz, 1H),
7.37–7.28 (m, 1H), 7.20–7.12 (m, 1H), 4.52–4.40 (m, 1H), 4.28 (bs, 2H),
1.69–1.54 (m, 2H), 1.50–1.40 (m, 1H), 0.90 (d, J = 6.4 Hz, 3H), 0.87 (d,
J = 6.4 Hz, 3H). 13C NMR (100MHz, DMSO‑d6) δ: 171.0, 162.7, 131.9
(dd, J = 10.2, 4.5 Hz), 120.6 (dd, J = 14.3, 3.7 Hz), 111.6 (dd, J = 21.4,
3.6 Hz), 104.52 (t, 2JCF = 26.4 Hz), 50.4. 41.1, 24.3, 23.0, 21.7. HRMS
(ESIꢀ ): 284.12245 [Mꢀ H]ꢀ . Cald. for [C13H16F2N3O2]ꢀ : 284.12106.
4.1.2.11. (S)-N-(1-hydrazinyl-4-methyl-1-oxopentan-2-yl)benzamide
(9a). The product was obtained after 5 h of reaction as a white solid
(241 mg, 0.9 mmol, 90%) 1H NMR (400MHz, CD3OD) δ: 7.85 (d, , J =
7.2 Hz , 2H), 7.56 (t, J = 7.2, 7.2 Hz, 1H), 7.46 (t, J = 7.2, 7.2 Hz, 2H),
4.64–4.60 (m, 1H), 1.80–1.61 (m, 3H), 0.99 (d, J = 6.4 Hz, 3H), 0.97 (d,
J = 6.4 Hz, 3H). 13C NMR (100MHz, CD3OD) δ: 174.0, 170.2, 135.3,
132.8, 129.5, 128.5, 52.4, 42.0, 26.1, 23.3, 22.1. HRMS (ESIꢀ ):
248.14092 [Mꢀ H]ꢀ . Cald. for [C13H18N3O2]ꢀ : 248.13990.
4.1.2.18. General procedure for the synthesis of the novel 2-amino-1,3,4-
oxadiazoles (10a-g). The corresponding hydrazide (9a-g) (0.28 mmol),
isopropanol (8 mL) and cyanogen bromide (0.33 mmol, 35 mg) were
added to a round bottom flask. The reaction mixture was subjected to
sonication at 40 ◦C. The progress of a reaction was monitored by TLC.
After the end of the reaction, the solvent was evaporated in vacuo and
the residue was purified by column chromatography on silica gel
(Chloroform: MeOH 100:0; 97,5:2,5; 95:5; 90:10; 50:50; 0:100) giving
the corresponding product.
4.1.2.12. (S)-N-(1-hydrazinyl-4-methyl-1-oxopentan-2-yl)-2,4-dimethox-
ybenzamide (9b). The product was obtained after 5 h of reaction as a
yellow oil (370 mg, 0.9 mmol, 90%) 1H NMR (400MHz, CDCl3) δ: 8.34
(bs, 1H), 8.15–8.01(m, 2H), 6.56 (dd, J = 8.8, 2.3 Hz, 1H), 6.45 (d, J =
2.3 Hz, 1H), 4.74–4.58 (m, 1H), 3.92 (s, 3H), 3.82 (s, 3H), 1.85–1.58 (m,
3H), 0.95 (d, J = 6.3 Hz, 3H), 0.92 (d, J = 6.3 Hz, 3H). 13C NMR
(101MHz, CDCl3) δ: 173.0, 165.4, 163.9, 159.1, 134.0, 113.6, 105.6,
98.7, 56.1, 55.6, 50.7, 40.9, 25.0, 23.0, 22.3. The product was charac-
4.1.2.19. (S)-N-(1-(5-amino-1,3,4-oxadiazol-2-yl)-3-methylbutyl)benza-
mide (10a). The product was obtained after 8 h of reaction as a white
solid (50 mg, 0.18 mmol, 64%); m.p. (◦C)¼228–232 ◦C; IV (v, cm¡1):
3317, 2960, 1643. 1H NMR (400MHz, DMSO‑d6) δ: 8.88 (d, J = 8.4 Hz,
1H), 7.88 (d, J = 7.2 Hz, 2H), 7.55 (t, J = 7.2, 7.2 Hz, 1H), 7.47 (t, J =
7.2 Hz, 2H), 6.94 (s, 2H), 5.28–5.18 (m, 1H), 1.92–1.85 (m, 1H),
1.81–1.75 (m, 1H), 1.73–1.58 (m, 1H), 0.93 (d, J = 6.4 Hz, 3H), 0.91 (d,
J = 6.4 Hz, 3H). 13C NMR (100MHz, DMSO‑d6) δ 166.0 163.8, 159.5,
133.7, 131.5, 128.3, 127.4, 43.4, 40.3, 24.2, 22.7, 21.6. HRMS (ESIþ):
275.1496[M + H]+. Cald. for [C14H19N4O2]+: 275.1508.
1
terized by H and 13C NMR and the data are consistent with those re-
ported in the literature [23d]
4.1.2.13. (S)-N-(1-hydrazinyl-4-methyl-1-oxopentan-2-yl)-2,5-dimethox-
ybenzamide (9c). The product was obtained after 6 h of reaction as a
yellow oil (229 mg, 0.27 mmol, 90%) 1H NMR (400MHz, CDCl3) δ: 8
0.34 (d, J = 7.8 Hz, 1H), 7.58 (d, J = 2.8 Hz, 1H), 6.88 (dd, J = 8.9, 2.8
Hz, 1H), 6.79 (d, J = 8.9 Hz, 1H), 4.74–4.60 (m, 1H), 3.82 (s, 3H), 3.69
(s, 3H), 1.80–1.50 (m, 3H), 1.00–0.77 (m 6H). 13C NMR (100MHz,
CDCl3) δ: 172.6, 165.0, 153.6, 151.8, 121.1, 119.4, 115.4, 112.9, 56.4,
55.7, 50.7, 41.2, 24.8, 22.7, 22.1. HRMS (ESIþ): 310.17721 [M + H]+.
Cald. for [C15H24N3O4]+: 310.17668.
4.1.2.20. (S)-N-(1-(5-amino-1,3,4-oxadiazol-2-yl)-3-methylbutyl)-2,4-
dimethoxybenzamide (10b). The product was obtained after 8 h of re-
action as a yellow light solid (63 mg, 0.18 mmol, 67%); m.p. (◦C)¼
136.9–138.3 ◦C; IV (v, cm¡1): 3373, 3091, 2962, 1654. 1H NMR
(400MHz, CDCl3) δ: 8.16–8.07 (m, 2H), 6.57 (dd, J = 8.8, 2.4 Hz, 1H),
6.46 (d, J = 2.4 Hz, 1H), 5.68 (bs, 1H), 5.49–5.30 (m, 1H), 3.92 (s, 3H),
3.82 (s, 3H), 1.96–1.69 (m, 3H), 0.97 (d, J = 4.4 Hz, 3H), 0.95 (d, J =
4.4 Hz, 3H). 13C NMR (100MHz, CDCl3) δ: 164.9, 163.9, 163.5, 161.3,
159.2, 134.2, 113.8, 105.6, 98.8, 56.2, 55.6, 43.9, 42.2, 24.9, 22.8, 22.3.
HRMS (ESIþ): 335.1714 [M + H]+, Cald. for [C16H23N4O4]+: 335.1719.
4.1.2.14. (S)-N-(1-hydrazinyl-4-methyl-1-oxopentan-2-yl)-2-methox-
ybenzamide (9d). The product was obtained after 3.5 h of reaction as a
white solid (286 mg, 0.9 mmol, 95%) 1H NMR (400MHz, CDCl3) δ: 8.19
(d, J = 7.6 Hz, 1H), 8.13 (dd, J = 7.6, 1.7 Hz, 1H), 7.40–7.48 (m, 1H),
7.05 (t, J = 7.6 Hz, 1H), 6.96 (d, J = 8.3 Hz, 1H), 4.72–4.63 (m, 1H),
3.96 (s, 3H), 3.20 (bs, 3H), 1.83, 1.61 (m, 3H), 0.96 (d, J = 6.2 Hz, 3H),
0.96 (d, J = 6.3 Hz, 3H). 13C NMR (100MHz, CDCl3) δ: 172.0, 164.5,
157.0, 132.4, 131.2, 120.5, 110.9, 55.4, 50.2, 24.3, 22.4, 21.6. HRMS
(ESIꢀ ): 278.15169 [Mꢀ H]ꢀ . Cald. for [C14H20N3O3]ꢀ : 278.15047.
4.1.2.21. (S)-N-(1-(5-amino-1,3,4-oxadiazol-2-yl)-3-methylbutyl)-2,5-
dimethoxybenzamide (10c). The product was obtained after 8 h of re-
action as a yellow light solid (45 mg, 0.14 mmol, 52%); m.p. (◦C)¼
144.5–146.8 ◦C; IV (v, cm¡1): 3371, 3303, 2963, 1637. 1H NMR
(400MHz, CDCl3) δ: 8.38 (d, J = 8.4 Hz, 1H), 7.73 (d, J = 3.2 Hz, 1H),
7.00 (dd, J = 9.0, 3.6 Hz, 1H), 6.91 (d, J = 9.0 Hz, 1H), 5.54 (s, 2H),
5.49–5.42 (m, 1H), 3.93 (s, 3H), 3.80 (s, 3H), 1.97–1.70 (m, 3H), 0.99
(d, J = 4.8 Hz, 3H), 0.97 (d, J = 4.8 Hz, 3H). 13C NMR (100MHz,
CDCl3) δ: 164.9, 163.4, 161.3, 154.1, 152.1, 121.3, 120.1, 115.7, 113.3,
56.8, 56.0, 44.0, 42.2, 24.9, 22.9, 22.3. HRMS (ESIþ): 335.1714 [M +
H]+, Cald. for [C16H23N4O4]+: 335.1719.
4.1.2.15. (S)-N-(1-hydrazinyl-4-methyl-1-oxopentan-2-yl)-3(tri-
fluoromethyl)benzamide (9e). The product was obtained after 5 h of re-
1
action as a white solid (241 mg, 0.9 mmol, 90%) H NMR (400MHz,
DMSO‑d6) δ: 9.31 (s, 1H), 8.78 (d, J = 8.1 Hz, 1H), 8.26 (s, 1H), 8.19 (d,
J = 7.8 Hz, 1H), 7.90 (d, J = 7.8 Hz, 1H), 7.71 (t, J = 7.8 Hz, 1H),
4.57–4.44 (m, 1H), 4.24 (bs, 2H), 1.76–1.44 (m, 3H), 0.90 (d, J = 6.4 Hz,
3H), 0.90 (d, J = 6.4 Hz, 3H). 13C NMR (100MHz, DMSO‑d6) δ: 171.2,
164.8, 130.0, 131.8, 129.5, 127.9, 127.8, 124.3–124.2, 50.7, 40.6, 24.4,
23.0, 21.5. HRMS (ESIþ): 318.14340 [M
+
H]+. Cald. for
[C14H19F3N3O2]+: 318.14294.
4.1.2.22. (S)-N-(1-(5-amino-1,3,4-oxadiazol-2-yl)-3-methylbutyl)-2-
methoxybenzamide (10d). The product was obtained after 8 h of reac-
tion as a yellow light solid (53 mg, 0.17 mmol, 62%); m.p. (◦C)¼
162.5–166.3 ◦C; IV (v, cm¡1): 3376, 3142, 2969, 1649. 1H NMR
(400MHz, DMSO‑d6) δ: 8.51 (d, J = 8.2 Hz, 1H), 7.65 (d, J = 7.8 Hz,
4.1.2.16. (S)-4-fluoro-N-(1-hydrazinyl-4-methyl-1-oxopentan-2-yl)benza-
mide (9f). The product was obtained after 5 h of reaction as a white
solid (90 mg, 0.33 mmol, 91%) 1H NMR (400MHz, CDCl3) δ: 7.81–7.73
(m, 2H), 7.17–6.98 (m, 3H), 4.80–4.66 (m, 1H), 3.30 (bs, 2H), 1.84–1.61
11

T.B. Gontijo et al.
Bioorganic Chemistry 109 (2021) 104662
1H), 7.48 (t, J = 7.8 Hz, 1H), 7.14 (d, J = 7.8 Hz, 1H), 7.06–6.96 (m,
3H), 5.24–5.11 (m, 1H), 3.86 (s, 3H), 1.90–1.60 (m, 3H), 0.98–0.83 (m,
6H). 13C NMR (100MHz, DMSO‑d6) δ: 165.0, 163.7, 159.4, 156.9,
132.4, 130.3, 123.0, 120.5, 112.1, 56.0, 43.4, 40.8, 24.3, 22.7, 21.7.
HRMS (ESIþ): 305.1608, Cald. for [C15H21N4O3]+: 305.1614.
Kiapp
Ki =
[S]
1 + K
m
The Ki values were calculated by the tight-binding titration data
analysis GraFit program [25]
4.1.2.23. (S)-N-(1-(5-amino-1,3,4-oxadiazol-2-yl)-3-methylbutyl)-3-(tri-
fluoromethyl) benzamide (10e). The product was obtained after 8 h of
reaction as a yellow light solid (65 mg, 0.19 mmol, 76%); m.p. (◦C)¼
211.4–213.7 ◦C; IV (v, cm¡1): 3321, 2966, 1656. 1H NMR (400MHz,
DMSO‑d6) δ: 9.19 (d, J = 8.2 Hz, 1H), 8.24 (s, 1H), 8.19 (d, J = 7.6 Hz,
1H), 7.94 (d, J = 7.6 Hz, 1H), 7.74 (t, J = 7.6 Hz, 1H), 6.99 (s, 2H),
5.30–5.18 (m, 1H), 1.94–1.58 (m, 3H), 0.93 (d, J = 6.7 Hz, 3H,), 0.91 (d,
J = 6.7 Hz, 3H). 13C NMR (100MHz, DMSO‑d6) δ: 164.5, 163.9, 159.2,
134.5, 131.7, 129.5–128.8 (m), 128.3–128.2 (m, 1C), 125.4–122.7 (m,
2C), 43.6, 40.3, 24.3, 22.7, 21.6. HRMS (ESIþ): 343.13739 [M + H]+,
Cald. for [C15H18F3N4O2]+: 343.13819.
4.2.2. Mechanism of inhibition determination
The type of mechanism was determined for the compound 10a as
representative of this class of compounds. Cathepsin K was incubated for
5 min at 37 ◦C in 100 mM sodium acetate buffer containing 5 mM DTT,
pH 5.5, and two inhibitor specified concentrations (0.9 μM and 1.8 μM).
Subsequently, the substrate Z-Phe-Arg-AMC was added (concentrations
ranging between 1.25 and 20 M) and the enzyme activity was contin-
μ
uously monitored in a quartz cuvette that was maintained in a ther-
mostated compartment (37 ◦C).). The kinetic constants were determined
using the GraFit software (version 5.0, Erithacus Software, UK) [25] and
the errors were less than 5% for all the values obtained. The mechanism
of action was determined using the Lineweaver-Burk double-reciprocal
and the Dixon plot methods. All plots were determined using the GraFit
software (version 5.0, Erithacus Software, UK) [25].
4.1.2.24. (S)-N-(1-(5-amino-1,3,4-oxadiazol-2-yl)-3-methylbutyl)-4-fluo-
robenzamide (10f). The product was obtained after 8 h of reaction as a
yellow light solid (60 mg, 0.20 mmol, 70%); m.p. (◦C)¼139.2–142.1 ◦C;
IV (v, cm¡1): 3141, 2923, 1647. 1H NMR (400MHz, DMSO‑d6) δ: 8.93
(d, J = 8.3 Hz, 1H), 7.96 (dd, J = 8.8, 5.5 Hz, 2H) 7.31 (t, J = 8.8 Hz,
2H), 6.97 (s, 2H), 5.27–5.16 (m, 1H), 1.93–1.58 (m, 3H), 0.93 (d, J =
6.6 Hz, 3H), 0.90 (d, J = 6.6 Hz, H). 13C NMR (100MHz, DMSO‑d6) δ:
4.2.3. Cell viability assay
The cytotoxic effects (in vitro) of the compounds 10a, 10b, 10e and
10g were evaluated using the HUVEC (Human Umbilical Vein Endo-
thelial Cells) cell line (ATCC® CRL-1730™). Initially, the cells were
cultivated in a mixture (1:1) of DMEM/F12 (Dulbecco’s Modified Eagle
Medium/Nutrient mixture F12) with 10U/mL of penicillin and 10 µg/
mL of streptomycin, supplemented with 10% FBS (fetal bovine serum)
(Gibco/Thermo Fisher Scientific Inc. – Waltham, Massachusetts, USA).
The cell viability was assessed using the MTT (Thiazolyl Blue Tetrazo-
lium Bromide) colorimetric assay (Sigma Aldrich – Chemie, Steinheim,
GER). The HUVEC cells were plated (5 × 103 cells/well) 24 h before the
experiments in a 96 well flat-bottom plate and maintained in a humid-
ified incubator at 37 ◦C containing 5% CO2. After this period, the cells
were incubated with increasing concentrations of the compounds 10a,
10b, 10e and 10g (0.25, 0.5, 1, 2.5, 5 and 10 µM) for 24 h. Subsequently,
the supernatant was removed, and the cells were washed 3 times with
PBS (phosphate-buffered saline), followed by the incubation with MTT
reagent (final concentration 0.5 mg/mL). After 4 h, the supernatant was
removed and DMSO (dimethyl sulfoxide) was added to dissolve the
formazan products. The absorbance was measured at 570 nm. The ex-
periments were performed in triplicate.
164.1 (d, 1JCF = 249.0 Hz), 164.0, 163.8, 162.8, 159.4, 130.2 (d, 3JCF
=
9.0 Hz), 115.32 (d, 2JCF = 21.8 Hz), 43.5, 40.3, 24.3, 22.7, 21.6. HRMS
(ESIþ): 293.14053 [M + H]+, Cald. For [C14H18FN4O2]+: 293.14138.
4.1.2.25. (S)-N-(1-(5-amino-1,3,4-oxadiazol-2-yl)-3-methylbutyl)-2,4-
difluorobenzamide (10g). The product was obtained after 8 h of reaction
as a yellow light solid (40 mg, 0.13 mmol, 47%); m.p. (◦C)¼
191.1–192.5 ◦C; IV (v, cm¡1): 3315, 2966, 1647. 1H NMR (400MHz,
DMSO‑d6) δ: 8.89 (d, J = 8.2 Hz, 1H), 7.63 (dd, J = 15.1, 8.3 Hz, 1H),
7.40–7.28 (m, 1H), 7.22–7.11 (m, 1H), 7.00 (s, 2H), 5.21–5.10 (m, 1H),
1.87–1.60 (m, 3H), 0.92 (t, J = 6.8 Hz, 6H). 13C NMR (100MHz,
1
3
DMSO‑d6) δ: 164.2, 163.9 (dd, JCF = 250.5, JCF = 12.2 Hz), 163.6,
160.0 (dd, 1JCF = 252.6, 3JCF = 12.8 Hz), 159.6, 132.03 (dd, 3JCF = 10.4,
3JCF = 4.3 Hz), 120.70 (dd, 2JCF = 14.8, 4JCF = 3.6 Hz), 112.23 (dd, 2JCF
= 21.5, 4JCF = 3.6 Hz), 104.99 (t, 2J CF = 26.2 Hz), 44.0, 40.7, 24.6, 22.9,
21.9. HRMS (ESIþ): 311.13147 [M + H]+, Cald. For [C14H17F2N4O2]+
:
311.13196.
4.2. Enzyme inhibition assays
4.3. Molecular docking
4.2.1. Ki values determination
The chemical structures of the inhibitors were prepared for this study
as shown in Table 1. All structures were optimized (as the starting point
for molecular docking studies) at the PM6 theoretical level, from using
the Gaussian 09 package [26]. The partial charges of the atoms were also
elucidated.
The recombinant human cathepsin K (EC 3.4.22.38) was purchased
from Sigma-Aldrich (Mo, USA) and the molar concentration was deter-
mined by titration of the active site using the irreversible E-64 inhibitor
[24]. For the determination of the Ki values the enzyme was incubated
for 3 min at 37 ◦C in 100 mM sodium acetate buffer containing 5 mM
DTT, pH 5.5. Subsequently, the fluorogenic substrate Z-Phe-Arg-AMC
In the molecular docking studies, the affinity of the inhibitors within
the Cathepsin K active site was investigated. The inhibitors were then
docked inside the crystallographic structure of Cathepsin K enzyme
(PDB code 1TU6; resolution = 1.75 Å) [27], using the Molegro Virtual
Docker program (MVD®) [3], taking into account the same procedures
employed previously [29–31]. According to our calculation protocol, it
was considered a radius of about 10 Å, where the residues were kept as
flexible. Due to the nature of the docking methods, the calculations were
carried out, generating 50 poses (conformation and orientation) for each
ligand investigated.
(Z-Phe-Arg-4-methyl-coumaryl-7-amide, Sigma-Aldrich) (5
μM) was
added, and the residual activity was measured (λex 380 nm and λem 460
nm) in the presence of different concentrations of the inhibitors. The
apparent inhibition constants (Kiapp) values were obtained under the
same assay conditions and calculated using the following equation:
v0
v1
[I]
= 1 +
Kiapp
where v0 and v1 are the velocity of substrate hydrolysis in the absence
and presence of different inhibitor concentrations [I], respectively. The
Ki parameters were obtained from the following equation:
In the MVD program, the MolDock score algorithm method used as a
scoring function is based on the piecewise linear potential, which
fundamentally is a simplified potential whose parameters are in turn
fitted to protein–ligand structures, binding data scoring functions and
further extended in Generic Evolutionary Method for molecular dock-
12

T.B. Gontijo et al.
Bioorganic Chemistry 109 (2021) 104662
opportunities, EMBO J. 20 (2001) 4629–4633, https://doi.org/10.1093/emboj/
20.17.4629;
ing, including a new hydrogen bonding term as well as new charge
schemes [28]. Along this line, the docking scoring function values,
Escore, are usually defined by Eq. (1):
(c) R. Dai, Z. Wu, H.Y. Chu, J. Lu, A. Lyu, J. Liu, G. Zhang, Cathepsin K: The action
in and beyond bone, Front. Cell Dev. Biol. 8 (2020) 1–13, https://doi.org/10.3389/
fcell.2020.00433.
Escore = Einter + Eintra
(1)
[2] T.D. Rachner, S. Khosla, L.C. Hofbauer, Osteoporosis: now and the future, Lancet
377 (2011) 1276–1287, https://doi.org/10.1016/S0140- 6736(10)62349-5.
[3] T.-M. Ozra, P. Salari, P. Khashayar, B. Larijani, New horizons in treatment of
osteoporosis, J. Pharm. Sci. 25 (2017) 2–16, https://doi.org/10.1186/s40199-017-
0167-z.
Wherein:
[
]
∑
∑
ꢀ
)
qiqj
Einter
=
EPLP rij + 332.0
(2)
[4] L. Troeberg, H. Nagase, Proteases involved in cartilage matrix degradation in
osteoarthritis, Biochim. Biophys. Acta 2012 (1824) 133–145, https://doi.org/
10.1016/j.bbapap.2011.06.020.
4ri2j
i
ε
ligandj
ε
protein
Note that the EPLP represents ‘‘piecewise linear potential’’, which
consists of the use of two different parameter sets, as described forward:
one for the approximation of the steric term (i.e., Van der Waals) among
atoms, as well as the other potential for the hydrogen bonding. As can be
seen, the second term is related to the electrostatic interactions among
overloaded atoms. Typically, it is a Coulomb potential with a dielectric
constant dependent on the distance (which can be approximately
described as D(r) = 4r). Hence, for this, the numerical value of 332.0 is
responsible for the electrostatic energy unit to be given in kilocalories
per molecule [28].
[5] A.A. Rose, P.M. Siegel, Emerging therapeutic targets in breast cancer bone
metastasis, Future Oncol. 6 (2010) 55–74.
[6] Y. Hua, T.J. Robinson, Y. Cao, Shi Guo-Ping, J. Ren, S. Nair, Cathepsin K knockout
alleviates aging-induced cardiac dysfunction, Aging Cell 14 (2015) 345–351,
https://doi.org/10.1111/acel.12276.
[7] (a) M.J. Boyd, et al., Investigation of ketone warheads as alternatives to the nitrile
for preparation of potent and selective cathepsin K inhibitors, Bioorg. Med. Chem.
Lett. 19 (2009) 675–679, https://doi.org/10.1016/j.bmcl.2008.12.053;
(b) J.G. Catalano, et al., Exploration of the P1 SAR of aldehyde cathepsin K
inhibitors, Bioorg. Med. Chem. Lett. 14 (2004) 275–278, https://doi.org/10.1016/
j.bmcl.2003.09.088;
(c) D.G. Barrett, et al., A structural screening approach to ketoamide-based
inhibitors of cathepsin K, Bioorg. Med. Chem. Lett. 15 (2005) 2209–2213;
(d) M. Frizler, M. Stirnberg, M.T. Sisay, M. Gütschow, Development of nitrile-
based peptidic inhibitors of cysteine cathepsins, Curr. Top. Med. Chem. 10 (2010)
294–322, https://doi.org/10.2174/156802610790725452;
E
intra is defined as the internal energy of each ligand. That is:
∑
∑
∑
Eintra
=
EPLP(rij) +
A[1 ꢀ cos(m.θ ꢀ θ0)] + Eclash
(3)
(e) Ren Xing-Feng Li, Xuexun F. Hong-Wei, W. Yuqing, W. Lincong, Z. Shuxue,
Highly selective azadipeptide nitrile inhibitors for cathepsin K: design, synthesis
and activity assays, Org. Biomol. Chem. 11 (2013) 1143–1148, https://doi.org/
10.1039/C2OB26624E.
i
εligandj
εligand
flexiblebonds
Note that the first part of the equation (double summation) is among
all pairs of atoms in the ligand, taking off those connected by two bonds.
Thus, in this equation, the second term denotes the torsional energy,
where θ is the torsional angle of the bond. Hence, if several torsions
could be determined, then, each torsional energy is considered as an
average among them. The last term, Eclash, assigns as a penalty of about
1.000 if the distance between two heavy atoms (e.g., more than two
bonds apart) is smaller than 2.0 Å, but not taking into account infeasible
ligand conformations [28]. Thus, the docking search algorithm that is
applied in the MVD program considers an evolutionary algorithm, that
is, based on the interactive optimization techniques (inspired by
Darwinian evolution theory), which implies a new hybrid search algo-
rithm conveniently so-called guided differential evolution. As such, this
hybrid combines the differential evolution optimization technique with
a cavity prediction algorithm during the search process, allowing that
way a fast and accurate identification of potential binding modes (poses)
[28,32,33].
[8] (a) J. Lu, M. Wang, Z. Wang, Z. Fu, A. Lu, G. Zhang, Advances in the discovery of
cathepsin K inhibitors on bone resorption, J. Enzym. Inhib. Med. Chem. 33 (2018)
890–904, https://doi.org/10.1080/14756366.2018.1465417;
(b) I. Schechter, A. Berge, On the size of the active site in proteases I Papain,
Biochem. Biophys. Res. Commun. 27 (1967) 157–162, https://doi.org/10.1016/
s0006-291x(67)80055-x.
[9] E. Mons, I.D.C. Jansen, J. Loboda, B.R. van Doodewaerd, J. Hermans, M. Verdoes,
C.A.A. van Boeckel, P.A. van Veelen, B. Turk, D. Turk, H. Ovaa, The alkyne moiety
as a latent electrophile in irreversible covalent small molecule inhibitors of
cathepsin K, J. Am. Chem. Soc. 141 (2019) 3507–3514, https://doi.org/10.1021/
jacs.8b11027.
[10] (a) K. Mukherjee, N. Chattopadhyay, Pharmacological inhibition of cathepsin K: A
promising novel approach for postmenopausal osteoporosis therapy, Biochem.
Pharmacol. 117 (2016) 10–19, https://doi.org/10.1016/j.bcp.2016.04.010;
(b) M.R. McClung, M.L. O’Donoghue, S.E. Papapoulos, et al., Odanacatib for the
treatment of postmenopausal osteoporosis: results of the LOFT multicentre,
randomised, double-blind, placebo-controlled trial and LOFT Extension study,
Lancet Diabetes Endocrinol. 7 (2019) 899–911, https://doi.org/10.1016/S2213-
8587(19)30346-8;
(c) P.G. Conaghan, M.A. Bowes, S.R. Kingsbury, A. Brett, G. Guillard, B. Rizoska,
¨
¨
N. Sjogren, P. Graham, Å. Jansson, C. Wadell, R. Bethell, J. Ohd, Disease-modifying
effects of a novel cathepsin K inhibitor in osteoarthritis, Ann. Intern.. Med. 172
(2020) 147–148, https://doi.org/10.7326/M19-3809.
Declaration of Competing Interest
[11] Merck, Merck provides update on odanacatib development program. Sept 2, 2016.
Merck Newsroom Home. https://www.mrknewsroom.com/news-release/research-
and-development-news/merck-provides-updateodanacatib-development-program
(accessed September, 29, 2020).
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
[12] (a) Bansal S., Bala M., Suthar S K, Choudhary S., Bhattacharya S., Bhardwaj V.,
Singla S., Joseph A. Design and synthesis of novel 2-phenyl-5-(1,3-diphenyl-1H-
pyrazol-4-yl)-1,3,4-oxadiazoles as selective COX-2 inhibitors with potent anti-
inflammatory activity. Eur. J. Med. Chem. 80 (2014) 167-174 Doi:10.1016/j.
ejmech.2014.04.045 (b) Husain, A., Ajmal M., Synthesis of novel 1,3,4-oxadiazole
derivativesand their biological properties Acta Pharm. 59 (2009) 223–233 doi:
10.2478/v10007-009-0011-1 (c) Altıntop M. D., Sever B., Turan-Zitouni G.,
Acknowledgments
˜
`
The authors wish to thank Fundaçao do Amparo a Pesquisa do Estado
de Minas Gerais (FAPEMIG), Conselho Nacional de Desenvolvimento
Científico (CNPq) for financial support and grants, and Centro Regional
¨
Kaplancıklı Z. A., Ozdemir A. Design, synthesis, in vitro and in silico evaluation of a
new series of oxadiazole-based anticancer agents as potential Akt and FAK
inhibitors Eur. J. Med. Chem. 155 (2018) 905-924 doi:10.1016/j.
´
˜
para o Desenvolvimento Tecnologico e Inovaçao (CRTI) for HRMS
ejmech.2018.06.049 (d). Hannouna M. H, Hagrasa M., Kotba A., El-Attarb Abdul-
Aziz M. M., Abulkhair H. S. Synthesis and antibacterial evaluation of a novel
library of 2-(thiazol-5-yl)- 1,3,4-oxadiazole derivatives against methicillin-resistant
Staphylococcus aureus (MRSA). Bioorg. Chem. 94 (2020) 103364 doi: 10.1016/j.
bioorg.2019.103364 (e) Abd-Ellah H. S., Abdel-Aziz M., Shoman Mai E., Beshr E.
A. M., Kaoud T. S., Ahmed Al-Shaimaa F.F. Novel 1,3,4-oxadiazole/oxime hybrids:
Synthesis, docking studies and investigation of anti-inflammatory, ulcerogenic
liability and analgesic activities. Bioorg, Chem. 69 (2016) 48–63 doi:10.1016/j.
bioorg.2016.09.005 (f) Pitasse-Santos, Paulo, Sueth-Santiago, Vitor, & Lima, Marco
E. F.. 1,2,4- and 1,3,4-Oxadiazoles as Scaffolds in the Development of Antiparasitic
Agents J. Braz. Chem. Soc. 29 (2018) 435-456.
analysis. There are no conflicts of interest to declare.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104662.
References
[13] S. Borg, G. Estenne-Bouhtou, K. Luthman, I. Csoregh, W. Hesselink, U. Hacksell,
Synthesis of 1,2,4-oxadiazole-, 1,3,4-oxadiazole-, and 1,2,4-triazole-derived
dipeptidomimetics, J. Org. Chem. 60 (1995) 3112–3120, https://doi.org/10.1021/
jo00115a029.
[1] (a) S. Verma, R. Dixit, K.C. Pandey, Cysteine proteases modes of activation and
future prospects as pharmacological targets, Front. Pharmacol. 7 (2016) 1–12,
https://doi.org/10.3389/fphar.2016.00107;
(b) V. Turk, B. Turk, D. Turk, Lysosomal cysteine proteases: facts and
13

T.B. Gontijo et al.
Bioorganic Chemistry 109 (2021) 104662
[14] (a) F.X. Tavares, D.N. Deaton, A.B. Miller, L.R. Miller, L.L. Wright,
Ketoheterocycle-based inhibitors of cathepsin K: A novel entry into the synthesis of
peptidic ketoheterocycles, Bioorg. Med. Chem. Lett. 15 (2005) 3891–3895, https://
doi.org/10.1016/j.bmcl.2005.05.091;
10406638.2019.1635173;
´
(c) N. Klai, M. Berredjem, N. Khettache, M.Y. Belghit, Z. Regaïnia, A. Nour-Eddine,
Simple and efficient cleavage reaction of the boc group in heterocyclic compounds,
J. Heterocycl. Chem. 41 (2004) 57–60, https://doi.org/10.1002/jhet.5570410109;
(d) E.I. Al-afaleq, S.A. Abubshait, L-amino acid esters studies: Part II: Synthesis of
N-(dimethoxy/3,5-diacetoxybenzoyl)-L-amino acid hydrazides and their reactions
with aldehydes and ketones, Synth. Commun. 29 (1999) 1317–1331, https://doi.
org/10.1080/00397919908086107.
(b) T.L. Silva, A.A. Bartolomeu, H.C.R. Jesus, K.T. Oliveira, J.B. Fernandes,
¨
D. Bromme, P.C. Vieira, New synthetic quinolines as cathepsin K inhibitors, J. Braz.
Chem. Soc. 31 (2020) 1605–1613, https://doi.org/10.21577/0103-
5053.20200046;
ˇ
(c) N. Petek, B. Stefane, M. Novinec, J. Svete, Synthesis and biological evaluation
[24] A.J. Barrett, A.A. Kembhavi, M.A. Brown, H. Kirschke, C.G. Knight, M. Tamai,
K. Hanada, L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its
analogues as inhibitors of cysteine proteinases including cathepsins B, H and L,
Biochem. J. 201 (1982) 189–198, https://doi.org/10.1042/bj2010189.
[25] R.J. Leatherbarrow, GraFit Version 5, Erithacus Software Ltd, Horley, UK, 2001.
[26] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M. Robb, J. Cheeseman, G.
Scalmani, V. Barone, B. Mennucci, G.A.H. Petersson, et al., Gaussian 09 (Revision
A02), 2009.
of 7-(aminoalkyl)pyrazolo[1,5-a]pyrimidine derivatives as cathepsin K inhibitors,
Bioorg. Chem. 84 (2019) 226–238, https://doi.org/10.1016/j.bioorg.2018.11.029.
[15] J.T. Palmer, B.L. Hirschbein, H. Cheung, J. McCarter, J.W. Janc, Z. Walter Yu,
G. Wesolowski, Keto-1,3,4-oxadiazoles as cathepsin K inhibitors, Bioorg. Med.
Chem. Lett. 16 (2006) 2909–2914, https://doi.org/10.1016/j.bmcl.2006.03.001.
[16] X. Chen, J. Lawrence, S. Parelkar, T. Emrick, Novel zwitterionic copolymers with
dihydrolipoic acid: synthesis and preparation of nonfouling nanorods,
Macromolecules 46 (2013) 119–127, https://doi.org/10.1021/ma301288m.
[17] T.A. Davis, A.E. Vilgem, A. Richmond, J.N. Johnston, Preparation of (ꢀ )-Nutlin–3
using enantioselective organocatalysis at decagram scale, J. Org. Chem. 78 (2013)
10605–10616, https://doi.org/10.1021/jo401321a.
[27] D.G. Barrett, J.G. Catalano, D.N. Deaton, A.M. Hassell, S.T. Long, A.B. Miller, L.
R. Miller, L.M. Shewchuk, K.J. Wells-Knecht, D.H.M. Willard, et al., Potent and
selective P2–P3 ketoamide inhibitors of cathepsin K with good pharmacokinetic
properties via favorable P1′, P1, and/or P3 substitutions, Bioorg. Med. Chem. Lett.
14 (2004) 4897–4902, https://doi.org/10.1016/j.bmcl.2004.07.031.
[28] R. Thomsen, M.H. Christensen, MolDock: A new technique for high-accuracy
molecular docking, J. Med. Chem. 49 (2006) 3315–3321, https://doi.org/
10.1021/jm051197e.
[18] E. Jahnke, J. Weiss, S. Neuhaus, T.N. Hoheisel, H. Frauenrath, Synthesis of
diacetylene-containing peptide building blocks and amphiphiles, their self-
assembly and topochemical polymerization in organic solvents, Chem. Eur. J. 15
(2009) 388–404, https://doi.org/10.1002/chem.200801668.
[19] G. Tu, Y. Yan, X. Chen, Q. Lv, J. Wang, S. Li, Synthesis and antiproliferative assay
of 1,3,4-oxadiazole and 1,2,4-triazole derivatives in cancer cells, Drug Discov.
Ther. 7 (2013) 58–65, https://doi.org/10.5582/ddt.2013.v7.2.58.
[29] T.C. Silva, M. dos S. Pires, A.A. de Castro, E.F.F. da Cunha, M.S. Caetano, T.
C. Ramalho, Molecular insight into the inhibition mechanism of plant and rat 4-
hydroxyphenylpyruvate dioxygenase by molecular docking and DFT calculations,
Med. Chem. Res. 24 (2015) 3958–3971, https://doi.org/10.1007/s00044-015-
1436-3.
[20] Z.-Y. Ren, I. Machuca-Gayet, C. Domenget, R. Buchet, Y. Wu, P. Jurdic,
S. Merabeck, Azanitrile cathepsin K inhibitors: effects on cell toxicity, osteoblast-
induced mineralization and osteoclast – Mediated bone resorption, PLoS ONE 10
(2015) 1–14, https://doi.org/10.1371/journal.pone.0132513.
[30] A.P. Guimaraes, A.A. Oliveira, E.F.F. da Cunha, T.C. Ramalho, T.C.C. Franco,
Analysis of Bacillus anthracis nucleoside hydrolase via in silico docking with
inhibitors and molecular dynamics simulation, J. Mol. Model. 17 (2011)
2939–2951, https://doi.org/10.1007/s00894-011-0968-9.
[21] X.-Y. Yuan, Z. Ren, Y. Wu, C. Bougault, L. Brizuela, D. Magne, R. Buchet,
S. Mebarek, Design, synthesis and biological evaluation of inhibitors of cathepsin K
on dedifferentiated chondrocytes, Bioorg. Med. Chem. 27 (2019) 1034–1042,
https://doi.org/10.1016/j.bmc.2019.02.003.
[31] K.S. Matos, D.T. Mancini, E.F.F. da Cunha, K. Kuca, T.C.C. Franca, T.C. Ramalho,
Molecular aspects of the reactivation process of acetylcholinesterase inhibited by
cyclosarin, J. Braz. Chem. Soc. 22 (2011) 1999–2004, https://doi.org/10.1590/
S0103-50532011001000023.
[22] (a)Buehlmayer P. Substituted benzenesulfonamide derivatives and their
pharmaceutical use. WO1999032466A1, 1994.
(b)Kadiyala V. S. N. M., Desai D. M., et al. Preparation of novel hydrazide
containing heterocyclic compounds as Bruton tyrosine kinase (BTK) inhibitors
WO/2018/00295, 2018.
[32] E.F.F. da Cunha, E.F. Barbosa, A.A. Oliveira, T.C. Ramalho, Molecular modeling of
mycobacterium tuberculosis DNA gyrase and its molecular docking study with
gatifloxacin inhibitors, J. Biomol. Struct. Dyn. 27 (2010) 619–625, https://doi.org/
10.1080/07391102.2010.10508576.
[23] (a) C. Madhu, G. Prabhu, R. Pal, T.N. Guru Row, V.V. Sureshbabu, Synthesis of
optically active 2-amino-1,3,4-oxadiazoles and their hybrid peptides, J. Heterocycl.
Chem. 52 (2015) 1438–1446, https://doi.org/10.1002/jhet.2166;
[33] T.C.S. Souza, D. Josa, T.C. Ramalho, M.S. Caetano, E.F.F. da Cunha, Molecular
modelling of Mycobacterium tuberculosis acetolactate synthase catalytic subunit
and its molecular docking study with inhibitors, Mol. Simul. 34 (2008) 707–713,
https://doi.org/10.1080/08927020802129974.
(b) Bharat K. Dhotre, M.A. Patharia, R.V. Santosh, M.A. Pathan, An efficient,
microwave-assisted, one-pot synthesis of fluorinated 2,5-disubstituted 1,3,4-
oxdiazole derivative, Polycycl. Aromatic Compd. (2019), https://doi.org/10.1080/
14