Green asymmetric synthesis of epoxypeptidomimetics and evaluation as human cathepsin K inhibitors

Article abstract of DOI:10.1016/j.bmc.2020.115597

Cathepsin K (CatK) is a cysteine protease known for its potent collagenolytic activity, being recognized as an important target to the development of therapies for the treatment of bone disorders. Epoxypeptidomimetics have been reported as potent inhibitors of cathepsins, thus in this work we present a green synthesis of new peptidomimetics by using a one-pot asymmetric epoxidation/Ugi multicomponent reaction. The compounds were evaluated against CatK showing selectivity when compared with cathepsin L, with an inhibition profile in the low micromolar IC₅₀ range. Investigation of the mechanism of action carried out for compounds LSPN428 and LSPN694 suggested a mixed inhibition mode and docking studies allowed a better understanding about interactions of inhibitors with the enzyme.

Full text of DOI:10.1016/j.bmc.2020.115597

Journal Pre-proofs
Green Asymmetric Synthesis of Epoxypeptidomimetics and Evaluation as
Human Cathepsin K Inhibitors
Taynara L. Silva, Deborah A. dos Santos, Hugo C.R. de Jesus, Dieter
Brömme, João B. Fernandes, Marcio W. Paixão, Arlene G. Corrêa, Paulo C.
Vieira
PII:
DOI:
Reference:
S0968-0896(20)30427-2
https://doi.org/10.1016/j.bmc.2020.115597
BMC 115597
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
26 March 2020
29 May 2020
8 June 2020
Please cite this article as: Silva, T.L., dos Santos, D.A., de Jesus, H.C.R., Brömme, D., Fernandes, J.B., Paixão,
M.W., Corrêa, A.G., Vieira, P.C., Green Asymmetric Synthesis of Epoxypeptidomimetics and Evaluation as
Human Cathepsin K Inhibitors, Bioorganic & Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.
2020.115597
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Green Asymmetric Synthesis of Epoxypeptidomimetics and Evaluation as
Human Cathepsin K Inhibitors
c
a
b
Taynara L. Silva a,b, Deborah A. dos Santos , Hugo C.R. de Jesus , Dieter Brömme , João B.
Fernandes a, Marcio W. Paixão c, Arlene G. Corrêa c* and Paulo C. Vieira c,d*
aDepartment of Chemistry, Federal University of São Carlos, 13565-905 - São Carlos, SP, Brazil
bFaculty of Dentistry and UBC Center for Blood Research, 2350 Health Sciences Mall, Vancouver,
BC, V6T1Z3 Canada.
cCentre of Excellence for Research on Sustainable Chemistry, Department of Chemistry, Federal
University of São Carlos, 13565-905, São Carlos, SP, Brazil.
dSchool of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, 14040-903, Ribeirão
Preto, SP, Brazil
* Corresponding authors.
E-mail: agcorrea@ufscar.br (A.G. Corrêa) and pcvieira@fcfrp.usp.br (P.C. Vieira).

Abstract
Cathepsin K (CatK) is a cysteine protease known for its potent collagenolytic activity, being
recognized as an important target to the development of therapies for the treatment of bone disorders.
Epoxypeptidomimetics have been reported as potent inhibitors of cathepsins, thus in this work we
present a green synthesis of new peptidomimetics by using a one-pot asymmetric epoxidation/Ugi
multicomponent reaction. The compounds were evaluated against CatK showing selectivity when
compared with cathepsin L, with an inhibition profile in the low micromolar IC50 range. Investigation
of the mechanism of action carried out for compounds LSPN428 and LSPN694 suggested a mixed
inhibition mode and docking studies allowed a better understanding about interactions of inhibitors
with the enzyme.
Keywords: cathepsin K, cysteine proteases, inhibitors, peptidomimetics

1
1. Introduction
2
3
Lysosomal cysteine proteases are members of the papain-like family, comprising
11 members encoded in the human genome (cathepsins B, C, F, H, K, L, O, S, V, W and
X). These enzymes exhibit maximal activity at low pH of the lysosomes and have
variables in their functions.1,2 They are vital to normal physiological functions, however,
numerous pathologies have been attributed to the unregulated activities of these enzymes,
thus being recognized for decades as potent targets for the development of the treatment
of various diseases.2,3
4
5
6
7
8
9
Cathepsin K (CatK) is a cysteine protease highly expressed in osteoclasts,
responsible for the bulk degradation of the collagenous bone matrix playing an important
role in bone resorption. It has the noteworthy ability to cleave type I and II collagens
which are the main components of bone and cartilage. CatK can cleave the triple helix of
collagen in multiple regions, thus it can completely degrade the insoluble collagen of
bone. It is the predominant mammalian bone-degrading protease, and overexpression of
CatK activity increases bone resorption and thus considered one of the major target for
drug development for treatment of diseases such as osteoporosis.4–6
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11
12
13
14
15
16
17
18
19
20
21
22
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24
25
26
Osteoporosis is a skeletal disease that causes low bone mass and microarchitectural
deterioration of bone tissue. This is promoted by deregulation on bone remodeling
process and causes loss bone mass making them more fragile and more susceptible to
fractures.7,8 Osteoporosis affects men and women, however, in women occurs about
fifteen years earlier than in men due to hormonal changes during and after menopause.6
This disease effects about 50% of women aged 50 years or older with medical costs
estimated at 17 to 20 billion dollars annually in the USA.9
CatK inhibition decreases bone resorption and provides a promising approach for
the treatment of bone and cartilage disease, so this enzyme has become an attractive target
in the search for anti-resorptive therapies. Some CatK inhibitors have been evaluated in

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29
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31
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35
36
clinical trials for the treatment of bone disease but despite showing efficacy by increasing
bone mineral density and reducing fracture rates, they failed because of various side
effects and thus none have become a commercial drug.10–13
The natural product E-64, isolated from the extract of Aspergillus japonicus, is a
high affinity, nonselective, irreversible cysteine protease inhibitor and has been employed
as a lead compound for the development of new inhibitors.14 In this sense, Asagiri et al.
described a potent orally active CatK inhibitor 1 (IC50 = 34.5 nM), which suppressed
osteoclastic bone resorption both in vivo and in vitro (Fig. 1). Computer-assisted
simulation of the CatK/1 complex indicated that this compound blocked the active-site
cleft where Cys25 and His162 of CatK form the catalytic site.15
O
NH
O
OH
HN
O
O
O
H
O
O
N
N
O
H2N
N
N
O
H
H
H
6
O
O
I
CO2Na
E-64
LSPN423
1
37
38
39
Fig. 1. Epoxypeptidomimetics as inhibitors of cathepsins
40
41
Continuing our efforts in the search for new cathepsin inhibitors,16–19 we have
developed a one-pot synthesis of epoxy--acyloxycarboxamides using an asymmetric
42
43
44
45
46
47
48
49
50
51
organocatalyzed epoxidation of ,-unsaturated aldehydes followed by the Passerini
multicomponent reaction. The carboxamides were evaluated against cathepsin L that
plays important roles in physiological processes as well as in the development of many
pathologies. Among the tested compounds, LSPN423 (Fig. 1) was the most potent
showing a tight binding uncompetitive inhibition profile (IC50 = 5.26 M, Ki = 1.33
μM).20
In order to contribute with the structure activity relationship of the
epoxypeptidomimetics, we report herein the asymmetric synthesis of new derivatives
using the Ugi multicomponent reaction and their inhibitory evaluation against cathepsins
K and L.
2

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2. Results and discussion
2.1. Synthesis
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60
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62
Based on the previously described one-pot asymmetric epoxidation/Passerini
MCR,20 we started this study using the asymmetric organocatalyzed epoxidation of
cinnamaldehyde, with the proline-derived catalyst I, developed in our research group, that
allowed the use of green solvents, in this case an ethanol/water mixture.21,22 The crude
epoxyaldehyde was then challenged to the Ugi reaction conditions without any
chromatographic purification, using benzylamine, benzoic acid and n-butyl isonitrile.
After a screening of solvents usually employed in this MCR, such as EtOH/H2O and TFE,
methanol was selected (Table 1).
63
64
Table 1. Different conditions tested for the interplay between organocatalytic
epoxidation and multicomponent Ugi reaction
nBu
SC6H13
HN
O
N
1) Cat I (10 mol%), H2O2 (3 eq),
EtOH:H2O (3:1), 16 h, r.t.
O
O
(R)
O
SC6H13
Ph
Ph
Ph
H
(S)
N
O
2) BnNH2, PhCO2H, nBuNC
solvent, time, temperature
H
SiMe3
Cat I
Bn
LSPN690
65
Entry
Solvent
EtOH:H2O (3:1)
EtOH:H2O (3:1)
EtOH:H2O (3:1)
EtOH:H2O (3:1)
TFE
Temperature (°C)
Time
24 h
Yield (%)a
1
2
r.t.
60
54
44
-
24 h
3b
4b
5
100
80
40 min
30 min
48 h
42
-
r.t.
r.t.
r.t.
r.t.
80
6
MeOH
4 h
67
43
47
-
7c
8d
9b
MeOH
5 h
MeOH
7 h
MeOH
30 min
66
67
a Isolated yield after purification by column chromatography. b Microwave-assisted
reaction. c First, benzylamine (1 equiv) was added and the mixture stirred for 1 h, then
3

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benzoic acid (1 equiv) and n-butyl isonitrile (1 equiv) were added, and the reaction
occurred in 4 h. d 1.5 eq of benzylamine was used.
70
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Furthermore, the effect of the temperature, including microwave irradiation (entries
3, 4 and 9), has been evaluated, but for our delight at the room temperature the desired
product LSPN690 was obtained in 67% overall yield (Table, entry 6). Using this
optimized condition, a series of new epoxypeptidomimetics was prepared in moderate to
good overall yield and diastereoselectivity (Fig. 2).
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Fig. 2. New epoxy-α-acylaminocarboxamides synthesized via organocatalyzed
epoxidation/Ugi MCR.
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81
2.2. Biochemical evaluation of peptidomimetics
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Continuing our interest in the discovery of potential inhibitors of cathepsins, we
have tested the inhibitory activity of the new epoxy-α-acylaminocarboxamides
(LSPN690-694) and also five epoxy-α-acyloxycarboxamides that were previously
reported (Fig. 3),20 against cathepsins K and L.
4

HN
O
O
O
O
O
NH
O
O
HN
O
O
O
O
6
O
6
6
LSPN422
I
LSPN428
I
LSPN423
O
O
NH
O
O
NH
O
O
6
O
6
O
I
LSPN435
LSPN432
87
88
89
Fig. 3. Epoxypeptidomimetics previously prepared by asymmetric epoxidation/Passerini
MCR.20
90
91
As a general procedure, the inhibition bioassay was first carried out at a
concentration of 25 µM. All compounds evaluated had satisfactory inhibition (greater
than 70%) at this concentration, so in order to determine the potency of compounds, IC50
values were obtained by kinetic assays using at least seven different inhibitor
concentrations (Table 2). All compounds exhibited moderate inhibition against CatK with
IC50 values at low micromolar, and selectivity when compared with CatL at 25 µM.20 The
most potent among the evaluated compounds was LSPN432 with IC50 = 2.21 µM.
Comparison with the result obtained for compound LSPN422, where the only difference
is the iodine attached to the aromatic ring, suggests the importance of this atom in the
catalytic inhibition process of this enzyme.
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103
104
105
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Studies with the two new peptidomimetics (LSPN690 and LSPN694) reinforce the
hypothesis of the importance of the aromatic ring-bound iodine atom in inhibiting CatK.
The IC50 of LSPN694 that have an iodine attached to aromatic ring is about one third of
the IC50 value of compound LSPN690. Once again, it was also observed a selectivity of
these two new compounds for CatK inhibition when compared to CatL.
5

107
Table 2: Evaluation of peptidomimetics against cathepsins K and L.
Compound
LSPN422
IC50 (µM) – CatK
IC50 (µM) – CatL
12.47 ± 1.93
86%a
LSPN423
LSPN428
5.72 ± 0.82
3.35 ± 0.28
2.21 ± 0.77
3.81 ± 0.81
7.60 ± 0.18
18.45 ± 1.34
n.d.
5.36 ± 1.62
74%a
65%a
0%a
LSPN432
LSPN434
LSPN435
0%a
LSPN690
0%a
LSPN691
>50
LSPN692
n.d.
>50
LSPN693
n.d.
0%a
LSPN694
6.44 ± 0.54
30%a
108
109
a % inhibition at 25 M.
110
2.3. Mechanism of inhibition
111
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115
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119
120
121
One compound from each class of the evaluated peptidomimetics were selected to
determine the mechanism of action and Ki value: LSPN428 and LSPN694. The
mechanism of action was determined through nonlinear regression, evaluating how the
presence of the inhibitor influences the Vmax and Km values by analyzing of Michaelis
Menten graphs (Fig. 4). Data analysis was performed using the software GraphPad Prisma
5.
Both evaluated compounds showed mixed inhibition mode. Mixed inhibition is
observed when the inhibitor has mechanism of action that may resemble more than one
mode. In this case it is necessary to evaluate the interference caused by presence of the
inhibitor in enzymatic catalysis. This interference can be assessed by the α value, which
determines the degree that inhibitor binding alters the affinity of enzyme for the
6

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substrate.23 Thus, when α = 1 the affinity to bind the free enzyme or the ES complex is
equivalent, which is similar to the noncompetitive inhibition mode. When α > 1 inhibitor
has preference for free enzyme, resembling competitive inhibition mode and when α < 1,
inhibitor has preference for complex ES and resembles uncompetitive inhibition mode.23
There are some divergences about definition of mixed inhibition in the literature.
Some authors consider this mode of inhibition to be noncompetitive. Although the term
noncompetitive is commonly used to inhibitors that have equivalent affinities to bind the
free enzyme or the ES complex (α = 1).
In the case of this study we considered the mixed inhibition and thus evaluated the
α values obtained for the studied inhibitors. For LSPN428 we have α= 0.4 and for
LSPN694 α= 0.3. Thus, inhibitors studied have inhibition that most closely resembles the
uncompetitive mode, that is, they prefer to bind to the complex ES.
135
136
137
Fig. 4. Nonlinear regression graph obtained for mixed inhibition mode for compounds
(A) LSPN428 and (B) LSPN694.
138
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140
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142
143
144
The affinity of the inhibitor by the enzyme was also determined. This affinity is
defined by the Ki value and can be determined by the same kinetic data obtained in
specific experiments to determine the mechanism of action of the inhibitors. The values
obtained for LSPN428 (5.45 µM) and LSPN694 (9.57 µM) confirmed that the
peptidomimetics evaluated are an interesting class of CatK inhibitors and could serve as
models for the planning of new inhibitors for this enzyme.
7

145
2.4. Molecular docking study
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149
150
The predicted CatK-ligand interactions using molecular docking simulations are
consistent with the experimental data obtained from the mechanism of action studies of
compounds LSPN428 and LSPN694, showing that the evaluated inhibitors did not bind
to the enzyme catalytic site and exhibit inhibition mode that resembles the uncompetitive
mode. Both evaluated compounds bound to the same protein site (Fig. 5).
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Fig. 5. Solvent-accessible surface representation of the CatK-LSPN428 (A) and CatK-
LSPN694 (B) predicted complexes.
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162
163
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165
Stabilizing hydrophobic and hydrogen bonding interactions of docked molecules
were observed. As shown in Fig. 6C, LSPN428 iodine atom is oriented between two
lysine residues (Lys41 and Lys214), favoring the formation of hydrogen bonds between
the amino terminal of lysines and the iodine. This interaction allows us to suggest the
importance of this group in the inhibition of CatK, and could explain the IC50 value of
compound LSPN422 obtained in previous studies. It is also possible to observe two
additional hydrogen bond interactions, one involving the LSPN428 ester carbonyl group
and the Arg108 residue and another between the nitrogen of the ligand amide group with
Glu106. Hydrophobic interactions are also favored between LSPN428 and CatK. As
shown in Fig. 6D, the heptyl carbon chain is located in a hydrophobic protein pocket
formed by the residues of Leu38, Leu46, Leu48, Ala104 and Ala105.
8

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Fig. 6. Predicting interaction between compound LSPN428 and CatK. (C) Hydrogen
bonds interactions involving polar groups of the ligand and CatK amino acid side-chains;
(D) Hydrophobic interactions in the pocket formed by Leu38, Leu46, Leu48, Ala104 and
Ala105.
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Consistent with the aforementioned LSPN428-CatK, the predicted docking model
of CatK with LSPN694 (Fig. 7E) shows stabilizing hydrogen interactions involving the
ligand iodine atom with three lysine residues (Lys40, Lys41 and Lys214). Similarly,
hydrogen bonds between the inhibitor epoxide group with the Arg79 and Arg108 residues
provide additional stabilization to the LSPN694-CatK complex. Unlike LSPN428, which
has a long nonpolar heptyl chain facing the hydrophobic pocket of the enzyme, LSPN694
present the nitrogen-bound butyl group of the amide occupying the CatK hydrophobic
site formed by Leu38, Leu46, Leu48, Ala104 and Ala105 (Fig. 7F).
9

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183
184
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Fig. 7. Predicting interaction between compound LSPN694 and CatK. (E) Hydrogen
bonds interactions involving polar groups of the ligand and CatK amino acid side-chains;
(F) Hydrophobic interactions in the pocket formed by Leu38, Leu46, Leu48, Ala104 and
Ala105.
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191
No effective interaction between LSPN694 nitrogen-bound isopropyl group (-
C3H7) and the protein side chain residues was observed. We hypothesize that the variation
in the inhibitory activity of the compounds LSPN690 and LSPN694 is associated with
the presence of the iodine atom in LSPN694. It corroborates with the crucial importance
of the iodine atom for the inhibitory activity of the evaluated peptidomimetics.
192
3. Conclusion
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201
New epoxypeptidomimetics were efficiently synthesized through a green one-pot
methodology employing asymmetric organocatalyzed epoxidation followed by Ugi
multicomponent reaction. Then, a series of epoxypeptidomimetics were evaluated against
cathepsins K and L and some of them presented selectivity and potency with IC50 values
at low micromolar range. A preliminary structure-activity relationship study allowed us
to infer the importance of iodine groups for the inhibitory activity of these compounds.
The inhibition mechanisms were determined for the compounds LSPN428 and LSPN694
showing the mixed inhibition mode for both, being more similar to the uncompetitive
mode (α < 1). Molecular docking studies carried out corroborate the experimental data
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revealing favorable interactions between the evaluated inhibitors and the enzyme. It was
also possible to observe that the evaluated compounds do not bind to the CatK catalytic
site, being in agreement with the experimental data.
205
4. Experimental section
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4.1. Synthesis
4.1.1.General methods
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All commercially available reagents were purchased from Sigma-Aldrich and,
when necessary, purified according to the literature procedures. Non-commercial
reagents and catalyst I were synthesized as reported.21
The synthesized products were purified through chromatographic column using
silica gel 60, 230-400 mesh. TLC were performed in silica gel 60 F254 supported in
aluminum sheets.
¹H and ¹³C NMR spectra were recorded on a Bruker ARX 400 MHz spectrometer.
Chemical shifts (δ) were presented in ppm units and the coupling constants (J) in Hertz
(Hz). Signals multiplicity were expressed by the following abbreviations: s (singlet), d
(doublet), t (triplet), q (quadruplet), m (multiplet).
Optical rotation was measured using a Schmidt Haensch polarimeter, model
Polatronic H100. The analysis wavelength was 589 nm. Solutions were prepared in ethyl
acetate and analyzed at the temperature of 24 ºC.
The exact mass measurement was carried out using a Shimadzu Nexera X2 UHPLC
system coupled to a Bruker Impact HD QqTOF mass spectrometer (FAPESP PROEM
2014/50244-6). A Betasil® C18 (20 x 2.1 mm, 5µm) pre-column was used for injection.
224
4.1.2.General procedure for the synthesis of epoxy-α-acyloxycarboxamides
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230
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234
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238
Compounds LSPN428, LSPN432, LSPN434 and LSPN435 were synthesized
according to the methodology described previously.20 In a 5 mL vial, organocatalyst I
(0.03 mmol, 16.7 mg) was dissolved in ethanol/H2O 3:1 (0.6 mL), then the trans-2-
decenal (0.3 mmol, 0.055 mL) and hydrogen peroxide (0.9 mmol, 0.075 mL, 35%
aqueous solution) were added. The mixture was stirred for 16 h at room temperature, after
that, in the same reaction flask the carboxylic acid (0.3 mmol) and the isocyanide (0.5
mmol) were added. The reaction was stirred for 24 h at room temperature, followed by
the addition of distilled water (10 mL), then the mixture was extracted with ethyl acetate
(3 x 5 mL) and the organic phase was dried over Na2SO4 and concentrated under vaccum.
The desired products were purified by column chromatography using silica gel and
hexane/ethyl acetate mixtures as eluent. The synthesized compounds were fully
characterized, and the NMR spectra correspond to a mixture of three diastereoisomers
(d1-Major; d2-Minor; d3-Minor).
239
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241
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243
244
245
246
247
248
249
2-(Butylamino)-1-((2S,3R)-3-heptyloxyran-2-yl)-2-oxo-ethyl-4-iodobenzoate
(LSPN428): 1H NMR (400 MHz, CDCl3) δ (ppm): 7.88 – 7.81 (m, 6H), 7.81 – 7.75 (m,
6H), 6.07 (bs, 3H), 5.35 (d1) (d, J = 4.7 Hz, 1H), 5.33 (d2) (d, J = 5.0 Hz, 1H), 499 (d3)
(d, J = 8.8 Hz, 1H), 3.43 (d3) (dd, J = 8.9, 3.9 Hz, 1H), 3.34 – 3.26 (m, 7H), 3.21 (d1)
(dd, J = 4.6, 2.1 Hz, 1H), 3.12 (d3) (m, 1H) 3.07 (d1) (td, J = 5.7, 2.1 Hz, 1H), 3.02 (d2)
(td, J = 5.9, 2.1 Hz, 1H), 1.61 – 1.55 (m, 6H), 1.53 – 1.40 (m, 12H), 1.37 – 1.21 (m, 30H),
0.97 – 082 (m, 18H). Major diastereoisomer – d1: 13C NMR (100 MHz, CDCl3) δ (ppm):
166.23, 16461, 138.11, 138.02 (2), 131.23 (2), 128.44, 101.74, 73.76, 73.23, 57.44, 5623,
39.21, 31.71, 31.49, 31.33, 29.23, 25.76, 22.62, 19.94, 14.07, 1368. [α]D24= +1003 (c =
0.005 g/mL, in EtOAc). HRMS: calculated to C22H32INNaO4 [M + Na]+ m/z = 524.1268,
found m/z = 524.1271.
250
251
2-(tert-Butylamino)-1-((2S,3R)-3-heptyloxyran-2-yl)-2-oxoethyl-4-iodobenzoate
12

252
253
254
255
256
257
258
259
260
261
(LSPN432): 1H NMR (400 MHz, CDCl3) δ (ppm): 7.85 (dd, J = 8.3, 5.3 Hz, 6H), 7.79
(t, J = 9.3 Hz, 6H), 5.89 (s, 3H), 5.23 (d2) (d, J = 4.9 Hz, 3H), 5.18 (d1) (d, J = 51 Hz,
1H), 4.92 (d3) (d, J = 9.1 Hz, 1H), 3.40 (d3) (dd, J = 8.7, 3.9 Hz, 1H), 3.26 (d2) (dd, J =
4.7, 2.0 Hz, 1H), 3.18 (d1) (dd, J = 5.0, 1.9 Hz, 1H), 3.14 – 3.09 (d3) (m, 1H), 3.06 (d1)
(td, J = 5.6, 1.8 Hz, 1H), 3.00 (d2) (td, J = 5.6, 1.8 Hz, 1H), 1.64 – 1.52 (m, 6H), 1.47 –
1.40 (m, 6H), 1.37 (d, J = 2.6 Hz, 27H), 1.31 – 1.22 (m, 24H), 087 (t, J = 6.6 Hz, 9H).
Major diastereoisomer – d1: 13C NMR (100 MHz, Acetone-d6) δ (ppm): 166,51, 165,23,
138,90 (2), 132,11 (2), 13019, 101.61, 76.95, 7381, 57.23, 56.75, 51.89, 32.49, 32.26,
24
32.16, 2995, 28.86, 26.61, 23.30, 14.35. [α]D = +2.32 (c = 0.007 g/mL, in EtOAc).
HRMS: calculated to C20H40INNaO5 [M + Na]+ m/z = 524.1843, found m/z = 524.1840.
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269
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272
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274
2-(tert-Butylamino)-1-((2S,3R)-3-heptyloxyran-2-yl)-2-oxoethyl-6-bromohexanoate
1
(LSPN434): H NMR (400 MHz, CDCl3) δ (ppm): 5.83 (bs, 3H), 5.02 (d1) (d, J = 4.8
Hz, 1H), 5.01 (d2) (d, J = 5.0 Hz, 1H), 4.71 (d3) (d, J = 8.8 Hz, 1H), 3.41 (t, J = 6.7 Hz,
6H), 3.25 (d3) (dd, J = 8.9, 4.0 Hz, 1H), 3.12 (d2) (dd, J = 5.0, 2.2 Hz, 1H), 3.06 (d1) (dd,
J = 4.8, 2.2 Hz, 1H), 2.97 (d1) (td, J = 5.7, 2.1 Hz, 1H), 2.93 (d2) (ddd, J = 6.0, 5.3, 2.2
Hz, 1H), 2.45 (td, J = 7.2, 1.0 Hz, 6H), 1.93 – 1.84 (m, 6H), 1.70 (dt, J = 15.3, 7.6 Hz,
6H), 1.62 (s, 3H), 1.59 – 1.47 (m, 12H), 1.44 -1.40 (m, 6H), 1.36 (s, 27H), 1.28 (dd, J =
6.1, 4.4 Hz, 27H), 0.90 – 0.85 (m, 9H). Major diastereoisomer – d1: 13C NMR (101 MHz,
CDCl3) δ (ppm): 171.66, 165.65, 72.37, 57.23, 56.29, 51.62, 33.80, 33.38, 32.32, 31.71,
31.33, 29.23, 29.14, 28.67 (3), 27.54, 25.74, 23.95, 22.62, 14.08. [α]D24 = +1.45 (c= 0.006
g/mL, in EtOAc). HRMS: calculated to C21H38BrNNaO4 [M + Na]+ m/z = 470.1876,
found m/z = 470.1877.
275
276
277
2-(tert-Butylamino)-1-((2S,3R)-3-heptyloxyran-2-yl)-2-oxo=-ethylcyclo-
hexanocarboxylate (LSPN435): 1H NMR (400 MHz, CDCl3) δ (ppm): 5.83 (bs, 3H), 5.08
13

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280
281
282
283
284
285
286
287
(d1) (d, J = 4.4 Hz, 1H), 5.04 (d2) (d, J = 4.7 Hz, 1H), 4.71 (d3) (d, J = 8.8 Hz, 1H), 3.26
(dd, J = 8.8, 4.0 Hz, 3H), 3.14 (d2) (dd, J = 4.7, 2.2 Hz, 1H), 3.06 (d1) (dd, J = 4.5, 2.1
Hz, 1H), 2.97 (d1) (td, J = 5.7, 2.1 Hz, 1H), 2.92 (d2) (ddd, J = 6.2, 5.2, 2.1 Hz, 1H), 246
– 2.35 (m, 3H), 1.99 – 1.88 (m, 6H), 1.81 – 1.74 (m, 6H), 1.70 – 1.63 (s, 3H), 157 – 1.42
(m, 12H), 1.35 (s, 27H), 1.31 – 1.24 (m, 36H), 1.17 (td, J = 7.0, 44 Hz, 3H), 0.88 (t, J =
6.5 Hz, 9H). Major diastereoisomer – d1: 13C NMR (100 MHz, CDCl3) δ (ppm): 174.13,
165.83, 71.85, 5685, 56.38, 51.51, 42.89, 31.70, 31.37, 2922, 29.15, 29.06, 29.00, 28.87,
24
28.66 (3), 2574, 25.62, 25.33, 25.25, 22.62, 14.07. [α]D = +6.06 (c= 0.004 g/mL, in
EtOAc). HRMS: calculated to C22H39NNaO4 [M + Na]+ m/z = 404.277129, found m/z =
404.277815.
288
4.1.3.General procedure for the synthesis of epoxy-α-acylaminocarboxamides
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290
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292
293
294
295
296
297
298
299
300
In a 5 mL vial, the organocatalyst I (0.022 mmol, 0.012 g) was dissolved in
ethanol:H2O 3:1 mixture (0.4 mL) followed by the addition of the ,-unsaturated
aldehyde (0.22 mmol) and hydrogen peroxide (0.066 mmol, 0.055 mL; 35% aqueous
solution). The solution was stirred at room temperature for 16 h. Then, the reaction media
was concentrated under reduced pressure and the mixture was redissolved in methanol
(0.4 mL) followed by the addition of the amine (0.2 mmol), carboxylic acid (0.2 mmol)
and n-butyl isocyanide (0.2 mmol, 0.021 mL). The mixture was stirred at room
temperature up to completion (monitored by TLC analysis). The work up of the reaction
was performed by adding water (10 mL) to the reaction flask and extracted with ethyl
acetate (3 x 5 mL). The organic phase was dried with anhydrous Na2SO4 and concentrated
under vacuum. The desired products were purified by column chromatography with silica
gel and hexane:EtOAc mixtures as eluent.
301
302
303
From the crude mixture two diastereoisomers were identified. Once we were able
to isolate both from the chromatographic column in silica, only the majors
diastereoisomers were used in the enzymatic assays.
14

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306
307
308
309
310
311
312
313
314
N-benzyl-N-(2-(butylamino)-2-oxo-1-((2R,3R)-3-phenyl-oxyran-2-yl)ethyl)benzamide
(LSPN690), major diastereoisomer: 1H NMR (400 MHz, CDCl3) δ (ppm): 8.01 (dd, J =
8.2, 1.1 Hz, 2H), 7.58 – 7.51 (m, 2H), 7.47 – 7.38 (m, 2H), 7.38 – 7.32 (m, 6H), 7.31 –
7.26 (m, 3H), 6.09 (s, 1H), 5.41 (dd, J = 4.1, 2.8 Hz, 1H), 5.21 (d, J = 4.1 Hz, 1H), 3.83
(d, J = 12.6 Hz, 1H), 3.59 (d, J = 12.6 Hz, 1H), 3.40 (d, J = 2.8 Hz, 1H), 3.23 – 3.09 (m,
1H), 1.40 – 1.29 (m, 2H), 1.29 – 1.17 (m, 2H), 0.80 (t, J = 7.3 Hz, 3H). 13C NMR (101
MHz, CDCl3) δ (ppm): 170.01, 165.73, 140.99, 138.50, 133.30, 130.06, 129.87 (2),
128.89 (2), 128.75 (2), 128.55 (2), 128.43 (2), 127.66 (2), 126.10 (2), 76.58, 75.49, 60.31,
27
52.66, 39.37, 31.49, 19.95, 13.60. [α]D = +2.45 (c= 0.005 g/mL, in EtOAc). HRMS:
+
calculated to C28H33N2O4 [M + H3O]+ m/z = 461.2435, found m/z = 461.2379.
315
316
317
318
319
320
321
322
323
324
325
326
327
N-benzyl-N-(1-((2R,3R)-3-(2-bromophenyl)oxyran-2-yl)-2-(butylamino)-2-
oxoethy)benzamide (LSPN691), Major diastereoisomer: 1H NMR (400 MHz, CDCl3) δ
(ppm): 8.06 (dd, J = 8.4, 1.3 Hz, 2H), 7.67 (dd, J = 7.8, 1.6 Hz, 2H), 7.60 – 7.54 (m, 2H),
7.46 – 7.41 (m, 2H), 7.34 – 7.29 (m, 1H), 7.24 – 7.19 (m, 3H), 7.16 (dd, J = 7.6, 1.7 Hz,
1H), 6.95 (dd, J = 6.6, 2.9 Hz, 1H), 5.81 (dd, J = 4.5, 1.7 Hz, 1H), 5.42 (d, J = 4.5 Hz,
1H), 3.67 (d, J = 13.1 Hz, 1H), 3.36 (d, J = 1.5 Hz, 1H), 3.32 (d, J = 13.1 Hz, 1H), 3.28
(dt, J = 13.6, 6.7 Hz, 1H), 3.13 (dt, J = 13.1, 6.4 Hz, 1H), 1.38 – 1.29 (m, 2H), 1.24 – 1.13
(m, 2H), 0.77 (t, J = 7.3 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ (ppm): 171.37, 166.72,
140.31, 138.11, 133.53, 132.84, 130.05 (2), 129.35, 129.20, 128.47 (4), 128.32 (2),
128.25, 127.99, 127.41, 122.03, 77.08, 73.20, 60.28, 51.85, 39.15, 31.43, 19.92, 13.58.
27
+
[α]D = -1.23 (c= 0.006 g/mL, in EtOAc). HRMS: calculated to C28H32BrN2O4 [M +
H3O]+ m/z = 539,1540, found m/z = 539.1420.
328
329
N-benzyl-N-(2-(butylamino)-1-((2R,3R)-3-(2-nitro-phenyl)oxyran-2-yl)-2-
15

330
331
332
333
334
335
336
337
338
339
340
oxoethyl)benzamide (LSPN692), major diastereoisomer: 1H NMR (400 MHz, CDCl3) δ
(ppm): 7.98 (dd, J = 8.4, 1.3 Hz, 2H), 7.89 (dt, J = 7.7, 1.5 Hz, 2H), 7.63 – 7.51 (m, 3H),
7.48 – 7.37 (m, 5H), 7.25 – 7.20 (m, 2H), 7.05 (dd, J = 7.1, 2.4 Hz, 1H), 5.73 (d, J = 5.5
Hz, 1H), 5.68 (dd, J = 5.5, 2.8 Hz, 1H), 3.69 (d, J = 13.3 Hz, 1H), 3.55 (d, J = 2.7 Hz,
1H), 3.52 (d, J = 13.3 Hz, 1H), 3.28 (dt, J = 13,6, 6,8 Hz, 1H), 3.14 (dt, J = 13.0, 6.9 Hz,
1H), 1.37 – 1.28 (m, 2H), 1.24 – 1.15 (m, 2H), 0.78 (t, J = 7.3 Hz, 3H). 13C NMR (101
MHz, CDCl3) δ (ppm): 171.16, 166.18, 148.19, 138.11, 136.41, 133.58, 133.53, 133.39,
130.12, 129.92 (2), 129.62, 128.96, 128.64, 128.59 (2), 128.47, 128.17, 127.52, 124.58,
77.66, 70.05, 61.41, 52.23, 39.25, 31.42, 19.94, 1357. [α]D27 = -11.45 (c= 0.008 g/mL, in
+
EtOAc). HRMS: calculated to C28H29N3NaO5 [M + Na]+ m/z = 510.1999, found m/z =
510.1697.
341
342
343
344
345
346
347
348
349
350
351
352
N-benzyl-N-(2-(butylamino)-1-((2R,3R)-3-ethyloxiran-2-yl)-2-oxoethyl)benzamide
(LSPN693), Major diastereoisomer: 1H NMR (400 MHz, CDCl3) δ (ppm): 8.09 (dd, J =
8.3, 1.3 Hz, 1H), 8.00 (dd, J = 8.4, 1.3 Hz, 2H), 7.59 (tt, J = 1.1, 7.4, 1H), 7.48 – 7.42 (m,
4H), 7.34 – 7.29 (m, 2H), 7.26 – 7.23 (m, 1H), 5.29 (dd, J = 7.8, 3.3 Hz, 1H), 3.83 – 3.79
(m, 1H), 3.79 – 3.74 (m, 3H), 3.33 (dtd, J = 6.4, 7.0, 13.6, 1H), 3.20 (dtd, J = 5.6, 6.9,
13.6, 1H), 1.59 – 1.50 (m, 1H), 1.49 – 1.37 (m, 2H), 1.35 – 1.22 (m, 2H), 0.98 (t, J = 7.4
13
Hz, 3H), 0.94 – 0.89 (m, 1H), 0.87 (t, J = 7.3 Hz, 3H). C NMR (101 MHz, CDCl3) δ
(ppm): 170.58, 166.72, 138.15, 133.56, 129.82 (2), 129.37, 128.74 (2), 128.53 (2), 128.26
27
(2), 127.68, 77.53, 72.11, 62.35, 52.62, 39.05, 31.65, 26.89, 20.04, 13.68, 9.87. [α]D
=
+
-4.96 (c= 0.005 g/mL, in EtOAc). HRMS: calculated to C24H33N2O4 [M + H3O]+ m/z =
413.2435, found m/z = 413.2393.
353
354
355
N-(tert-butyl)-N-(2-(butylamino)-2-oxo-1-((2R,3R)-3-phenyloxyran-2-yl)ethyl)-4-
1
iodobenzamide (LSPN694), major diastereoisomer: H NMR (400 MHz, CDCl3) δ
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360
361
362
363
(ppm): 7.78 (d, J = 8.6 Hz, 2H), 762 (d, J = 8.6 Hz, 2H), 7.49 (d, J = 7.6 Hz, 2H), 7.32 (t,
J = 7.5 Hz, 3H), 6.53 (t, J = 5.9 Hz, 1H), 5.31 (dd, J = 5.5, 4.5 Hz, 1H), 5.13 (d, J = 5.6
Hz, 1H), 3.61 (d, J = 45 Hz, 1H), 3.22 – 3.06 (m, 2H), 1.38 – 1.32 (m, 2H), 1.29 – 1.20
13
(m, 2H), 1.08 (s, 9H), 0.85 (t, J = 7.3 Hz, 3H). C MNR (101 MHz, CDCl3) δ (ppm):
172.06, 165.36, 140.90, 137.82 (2), 137.68, 131.10 (2), 128.62, 128.32 (2), 127.74,
27
126.56 (2), 101.25, 74.95, 56.75, 39.27, 31.38, 29.17 (3), 28.40, 20.01, 13.63. [α]D
=
+
+18.27 (c= 0.004 g/mL, in EtOAc). HRMS: calculated to C25H34IN2O4 [M + H3O]+ m/z
= 553.1558, found m/z = 553.1532.
364
4.2. Ezymatic assay
365
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369
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374
375
376
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379
380
381
Cathepsin K used in this study is human recombinant enzyme. It was expressed in
the Pichia pastoris system and purified according to protocols described in24,25. After
purification, the molar concentration of enzyme were determined by active site titration
using E-64 following the conditions previously described.26
The enzymatic activity of CatK was monitored by measuring the rate of release of
the fluorogenic group MCA (amino-methyl coumarin). All assays were performed at
room temperature in 0.1 M sodium acetate, pH 5.5, containing 2.5 mM DTT and 2.5 mM
EDTA. The cleavage of the fluorogenic substrates, Z-Phe-Arg-MCA
(benzyloxycarbonyl-phenylalanyl-arginine-4-methyl-7-coumarylamide), was recorded
with a Perkin Elmer LS 50B Fluorimeter (Perkin Elmer, λex = 380 nm, λem = 460 nm).
The protease inhibitory activity was performed in cuvette and the final volume of
reaction was 1000 µL. Each inhibitor was incubated with enzyme in buffer for 5 min.
After the incubation the fluorogenic substrate was added and the fluorescence reading
was performed for 5 minutes. The used concentration of enzyme was 2 nM and substrate
were 10 μM. Experiments were performed in triplicate and a positive control (irreversible
inhibitor, E-64) and a negative control (without inhibitor) were used. The percentage of
inhibition was calculated according to the equation:
17

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395
% inhibition = 100 x (1 - Vi/V0)
Where Vi and V0 are initial velocities (enzyme activities) determined in the
presence and in the absence of inhibitor, respectively. Values of IC50 were determined by
making rate measurements for at least seven inhibitor concentrations.
The inhibitory activity of the synthetic compounds against cathepsin L was carried
out as described in the literature.20
To determine the mechanism of inhibition and Ki value an inhibitor was selected
from each class of compounds. The experimental method of this study is to measure the
reaction rate in the absence and at different substrate concentrations, as well as in the
absence and at different inhibitor concentrations. Substrate concentrations were chosen
based on the experimental Km value obtained and inhibitor concentrations were chosen
based on the IC50 value of the compound to be tested.
396
4.3. Molecular modelling
397
398
399
400
401
402
403
404
405
406
407
The RosettaScripts application,27 available in the Rosetta software version 3.8,28
was used to dock the ligands into the protein structure. After preparing the protein
structure (CatK; Protein Data Bank ID: 1ATK), through energy minimization using the
Rosetta Fast Relax protocol with a knowledge-based all-atom energy function,29,30
a
three-dimensional conformer library for each ligand was generated using the BCL::Conf,
31 by providing ligand in the SDF format. The initial geometric center of the ligand in the
CatK-LSPN428 and CatK-LSPN694 complex was predicted using PatchDock server.32
Beginning from the initial ligand pose, 10000 cycles of sampling were performed in the
Monte Carlo simulation and the best scoring ligand pose was kept. A total of 1000 models
was generated using the routines described by Combs et al. 2013.33 Best 5% of models
were selected by Rosetta energy function, a proxy for the free energy which consists of a
18

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411
combination of physics-based and statistics-based potentials.33 Finally, the subset of
models was grouped based on the mean square root deviation (RMSD) between the ligand
heavy atoms for all pairs of models, and the one with the lowest average RMSD to all
other structures was chosen as a representative model.
412
Acknowledgment
413
414
415
416
417
The authors gratefully acknowledge FAPESP (grants 2013/07600-3, 2014/50249-8
and 2017/15455-4), GlaxoSmithKline, CAPES (Finance Code 001), CNPq (grant
429748/2018-3) and the Canadian Institutes of Health Research (MOP-89974; DB) for
funding and fellowships. TLS acknowledges a sandwich scholarship from CAPES (grant
PDSE - 88881.134061/2016-01).
418
419
Conflict of interest
The authors declare no conflicts of interest.
420
421
Appendix A. Supplementary data
Supplementary data are available in the online version, at http://.
422
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