N-Sulfonyl dipeptide nitriles as inhibitors of human cathepsin S: In silico design, synthesis and biochemical characterization

Article abstract of DOI:10.1016/j.bmcl.2020.127420

A library of cathepsin S inhibitors of the dipeptide nitrile chemotype, bearing a bioisosteric sulfonamide moiety, was synthesized. Kinetic investigations were performed at four human cysteine proteases, i.e. cathepsins S, B, K and L. Compound 12 with a t

Full text of DOI:10.1016/j.bmcl.2020.127420

Journal Pre-proofs
N-Sulfonyl Dipeptide Nitriles as Inhibitors of Human Cathepsin S: In silico
Design, Synthesis and Biochemical Characterization
Carina Lemke, Lorenzo Cianni, Christian Feldmann, Erik Gilberg, Jiafei Yin,
Fernanda dos Reis Rocho, Daniela de Vita, Ulrike Bartz, Jürgen Bajorath,
Carlos A. Montanari, Michael Gütschow
PII:
DOI:
Reference:
S0960-894X(20)30531-X
https://doi.org/10.1016/j.bmcl.2020.127420
BMCL 127420
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Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
18 May 2020
9 July 2020
14 July 2020
Please cite this article as: Lemke, C., Cianni, L., Feldmann, C., Gilberg, E., Yin, J., dos Reis Rocho, F., de Vita,
D., Bartz, U., Bajorath, J., Montanari, C.A., Gütschow, M., N-Sulfonyl Dipeptide Nitriles as Inhibitors of Human
Cathepsin S: In silico Design, Synthesis and Biochemical Characterization, Bioorganic & Medicinal Chemistry
Letters (2020), doi: https://doi.org/10.1016/j.bmcl.2020.127420
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Graphical Abstract
N-Sulfonyl Dipeptide Nitriles as Inhibitors of
Human Cathepsin S: In silico Design,
Leave this area blank for abstract info.
Synthesis and Biochemical Characterization
Carina Lemke, Lorenzo Cianni, Christian Feldmann, Erik Gilberg, Jiafei Yin, Fernanda dos Reis
Rocho, Daniela de Vita, Ulrike Bartz, Jürgen Bajorath,* Carlos A. Montanari,* and Michael
Gütschow*

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
N-Sulfonyl Dipeptide Nitriles as Inhibitors of Human Cathepsin S:
In silico Design, Synthesis and Biochemical Characterization
a
a,b,c
b
b
a
Carina Lemke, Lorenzo Cianni,
Christian Feldmann, Erik Gilberg, Jiafei Yin, Fernanda dos Reis
c
c
d
b,
c,
Rocho, Daniela de Vita, Ulrike Bartz, Jürgen Bajorath, * Carlos A. Montanari, * and Michael
a,
Gütschow *
a
Pharmaceutical Institute, Pharmaceutical and Medicinal Chemistry, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany
Bonn Aachen International Center for Information Technology BIT, Life Science Informatics, University of Bonn, Endenicher Allee 19c, D-53115 Bonn,
b
Germany
c
Instituto de Química de Sao Carlos, University of Sao Paulo, Avenida Trablhador Sao-carlense 400, BR-13560-970 Sao Carlos, Brazil
Department of Natural Sciences, University of Applied Sciences Bonn-Rhein-Sieg, von-Liebig-Str. 20, 53359 Rheinbach, Germany
d
Article history:
Received
Revised
Accepted
Available online
A library of cathepsin S inhibitors of the dipeptide nitrile chemotype, bearing a
bioisosteric sulfonamide moiety, was synthesized. Kinetic investigations were
performed at four human cysteine proteases, i.e. cathepsins S, B, K and L.
Compound 12 with a terminal 3-biphenyl sulfonamide substituent was the most
potent (K
’-fluoro-4-biphenyl sulfonamide substituent the most selective cathepsin S
inhibitor (K = 35.5 nM; selectivity ratio cathepsin S/K = 57; S/L = 31). In silico
i
= 4.02 nM; selectivity ratio cathepsin S/K = 5.8; S/L = 67) and 24 with a
4
Keywords:
i
cathepsin S
nitriles
reversible inhibition
sulfonamides
design and biochemical evaluation emphasized the impact of the sulfonamide
linkage on selectivity and a possible switch of P2 and P3 substituents with respect
to the occupation of the corresponding binding sites of cathepsin S.
2
020 Elsevier Ltd. All rights reserved.
Peptidomimetic modifications, including the exchange of the
peptide bond by bioisosteric moieties, constitute an important
approach for the development of bioactive compounds. This is
particularly beneficial in the design of peptidomimetic protease
inhibitors, whose amino acid side chains perform affinity-
enhancing interactions with the corresponding specificity pockets
of the protease. For example, the P2-P3 amide bond can be
replaced by the metabolically stable and non-basic
potency of protease inhibitors is multifaceted.4-6 The sulfonamide
moiety has a greater potential to form hydrogen bonds, because
the sulfur may donate the lone pairs of electrons to the oxygens
and enhances their hydrogen bond acceptor capacities. This is a
3
-5,7,8
typical feature of sulfonamide-protein interactions.
Sulfonamide groups in protease inhibitors can be introduced
in place of the scissile bond to mimic the transition state of amide
5
hydrolysis. For example, the sulfonamide oxygens of chymase
1
trifluoroethylamine group, as successfully exemplified by the
inhibitors were expected to accept H-bonds from the serine
protease’s backbone NH hydrogens of Ser195 and Gly193 to
cathepsin K inhibitor odanacatib as well as potent cathepsin S
2
9
inhibitors (Fig. 1).
orientate the sulfonamide moiety towards the oxy-anion hole. In
The sulfonamide group is valued because of its proteolytic
stability. It is more polar and more flexible than the carboxamide
linkage. Hence, the tetrahedral geometry of the sulfonyl group
can lead to a favorable spatial orientation of adjacent substituents,
as it has been detailed for MMP2 inhibitors. However, the
outcome of a carboxamide-sulfonamide replacement for the
this study, it was intended to elaborate the introduction of a
sulfonamide moiety as connecting link between an aryl capping
group and the -carbon of the P2 amino acid. For this approach,
cathepsin S was chosen as target protease and peptide nitriles as
inhibitor chemotype.
Cathepsin S belongs to the tissue-specific and not ubiquitously
expressed human cysteine proteases. The enzyme is a key
functioning protein in the major histocompatibility class II (MHC
II) related immune response and, since it cleavages the MHC II-
associated invariant chain (Ii), which itself blocks loading of an
antigen into the peptide-binding groove preventing premature
peptide loading and subsequent presentation on the cell surface.
Hence, cathepsin S supports the antigen presentation by
3
,4
_
_______________
*
Corresponding authors.
J.B.: E-mail: bajorath@bit.uni-bonn.de.
C.A.M.: E-mail: carlos.montanari@usp.br
M.G.: E-mail: guetschow@uni-bonn.de.

H
N
macrophages, B cells and dendritic cells, i.e. components of the
innate immune system that activate specific T-lymphocytes.
Cathepsin S was therefore considered as a promising target for
the treatment of autoimmune and atherosclerotic disorders as
well as the diversification of the antigen repertoire in lymphoma
O
N
R
2
H N
O
3
1
R = CONH2
a
c
2
R = CN
1
0
cells.
b
Nitrile-based peptidomimetic compounds have received
particular attention as cathepsin S inhibitors. Their interaction
with the target enzyme involves the nucleophilic attack of the
active site cysteine at the nitrile carbon leading to the reversible
R1
R1
O
O
O
H
N
H
N
N
N
O
N
O
N
H
O
H
1
1-14
4
5
formation of a thioimidate adduct.
Accordingly, we designed
and synthesized a series of peptide nitriles, equipped their amino
termini with an aromatic sulfonyl group and subjected them to
kinetic evaluation as cathepsin S inhibitors.
d
d
R1
O
R1
O
O
O
O O
H
N
H
N
N
S
N
S
Initially, the possible interaction of cathepsin S with a
prototypic compound of this approach, i.e. 11, was investigated
N
N
H
H
R4
R2
R4
R2
R3
R3
1
5
by molecular docking (Fig. 1). The thioimidate complex was
generated by reaction/transformation method that
6
10
11 25
a
Scheme 1. Synthesis of N-sulfonyl dipeptide nitriles 6-25. Reagents and
conditions. a) TFAA, TEA, THF, 0 °C to rt, 30 min; b) (i) HCO H, rt, 20 h,
ii) HATU, DIPEA, Boc-Leu-OH × H O, DMF, rt, 22 h; c) HATU, DIPEA,
Boc-Leu-OH × H O or Boc-Phe-OH, DMF, rt, 22 h; d) (i) HCO H, rt, 20 h,
(ii) corresponding sulfonyl chloride, DIPEA, CH Cl , 0 °C to rt, 20 h.
computationally forms the single bond between the sulfur of
Cys29 and the carbon of the nitrile inhibitor. With this covalent
constraint, the inhibitor was flexibly docked in the cathepsin S
active site cleft and compared with the binding mode of a
trifluoroethyl dipeptide nitrile, whose binding mode was
2
(
2
2
2
2
2
2
established by X-ray crystal structure analysis. Unexpectedly,
the leucine side chain of 11 occupied the S3 pocket and the
aromatic ring was oriented towards the S2 binding site. Based on
this initial in silico result, an aromatic amino acid in place of
leucine and the expansion of the aromatic sulfonyl capping group
also appeared reasonable and our synthetic efforts were
accordingly oriented.
In general, facile synthetic procedures with good yields were
carried out to obtain the sulfonamide nitriles described in this
work (Scheme 1). The synthetic entry to compounds 6-10 was
achieved by dehydration of the Boc-protected phenylalanine
16
amide
1
to its corresponding nitrile
2
by means of
trifluoroacetic anhydride (TFAA) and triethylamine (TEA) in
THF. After acidic deprotection of 2 using formic acid, a
uronium-salt mediated peptide coupling procedure was
17
performed to obtain the dipeptidic nitrile 4. The hydrochloric
salt of aminocyclopropane nitrile 3, needed for compounds 11-
18,19
2
5, was commercially available and similarly converted to 5.
For the introduction of the amide bioisosteric sulfonamide in
final compounds 6-25, different sulfonyl chlorides were reacted
with the deprotected amines of 4 and 5.
O
2
0-40
O
O
HN
F
S
O O
H
N
F
F
S
N
H
N
N
N
H
N
O
H
O
Table 1. N-Sulfonyl Dipeptide Nitriles Prepared.
F
Cl
Compd
Config.
R1
R2
R3
R4
6
7
8
9
(S)
(S)
(S)
(S)
(S)
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
H
H
H
H
H
H
Cl
H
H
S3
S1
Ph
H
4-F-Ph
H
H
1
0
Ph
1
1
1
1
1
1
1
1
1
2
3
4
5
6
7
8
(S)
(S)
(R)
(S)
(S)
(S)
(S)
(S)
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
H
H
H
H
H
H
H
Cl
Cl
Ph
H
H
Ph
H
Br
H
S2
4-F-Ph
H
H
Ph
4-F-Ph
H
H
Fig. 1. Covalent complex of the N-trifluoroethyl dipeptide nitrile I (top left)2
with human cathepsin S (bottom cyan; PDB-ID: 2FQ9). Proposed covalent
binding mode of the N-sulfonyl dipeptide nitrile 11 (top right, bottom green)
superimposed with the N-trifluoroethyl inhibitor. The protein surface is
depicted in transparent grey. Ligand and residue atoms are shown in stick
representation and specificity pockets are numbered according to the
Schechter-Berger nomenclature.
H
1
2
9
0
(S)
(S)
(R)
(S)
(S)
(S)
(S)
Ph
Ph
Ph
Ph
Ph
Ph
Ph
H
H
H
H
H
H
Cl
Cl
Ph
H
H
21
Ph
H
2
2
2
2
2
3
4
5
4-F-Ph
H
H
Ph
H
4-F-Ph
H
H

In the following, we have evaluated the series of 20 new N-
sulfonyl dipeptide nitriles at the human cathepsins B, K, L, and S
by the use of chromogenic or fluorogenic peptide substrates to
monitor the enzymatic activities (Table 2).41 Linear progress
curves were obtained and IC50 values were determined from
measurements in the presence of different inhibitor
implemented in potent and selective cathepsin S inhibitors.11,12
Moreover, from our initial docking (Fig. 1), a large hydrophobic
residue at the position of the P1 aminonitrile was not expected
to be beneficial and we thus focused on the substructure present
in 11-25. Comparing analog 11 with 7, a slight improvement in
cathepsin S inhibition and selectivity could be seen. Typically,
this P1 variation led to an increase in cathepsin inhibition, much
stronger against cathepsin S than against cathepsin K and L (12
versus 8, 15 versus 9 and 16 versus 10).
concentrations, from which K
of the Cheng-Prusoff equation. Only weak or no inhibition of
cathepsin B, but mostly sub-micromolar K values for the other
i
values were calculated by means
i
three proteases were ascertained. With compound 6, we already
identified a potent inhibitor against cathepsins K, L, and S,
whose potency was somewhat improved when a meta-chloro
substituent (compound 7) was introduced at the
phenylsulfonamide capping group. Exchange of the 3-
chlorophenyl group by different biphenylic moieties did not
result in a better inhibitory activity against cathepsin S (7 versus
To further specify the selectivity and improve the potency
towards cathepsin S, different sulfonamide substructures were
taken into consideration. The exchange of the 3-chloro
substituent by bromine (11 versus 14) or a meta-to-ortho shift (11
versus 18) was not beneficial. The sulfonamide nitrile 12, in
which the 3-chlorophenyl unit was replaced by meta-biphenyl,
was the most potent cathepsin S inhibitor of the whole series with
8
-10). Still, compounds 6-9 were satisfactory dual cathepsin K/S
i
a K value of 4.02 nM, enclosing a strong selectivity towards
inhibitors, but with insufficient selectivity towards cathepsin L.
Unexpectedly, the introduction of a 4’-fluoro-3-biphenylic
capping group instead of the unsubstituted biphenyl appeared to
decrease the affinity for cathepsin L (8 versus 9) and was
considered (and utilized, see below) as an opportunity to improve
the compounds’ selectivity for cathepsin S over L.
We next realized a prominent P1 introduction for cysteine
protease inhibitors and replaced the phenylalanine nitrile by the
cyclopropane nitrile moiety (11-25), which has also been
cathepsin L (67-fold) and a modest one towards cathepsin K (5.8-
fold).
Changing the bent meta-biphenyl of compound 12 to the
linear para-biphenyl of 16 resulted in a drop of activity at
cathepsin S only, yielding a non-selective and moderately potent
cathepsin inhibitor. As already observed for compound 9, a 4’-
fluorine substitution in both the meta- and para-biphenyl systems
increased the selectivity ratio in favor of cathepsin S over L (12
versus 15, 16 versus 17).
.
Table 2
Inhibition of Human Cathepsins by N-Sulfonyl Dipeptide Nitriles 6-25.
a
b
i
K ± SE (µM) or residual activity (%)
Compound
Cathepsin Bc
Cathepsin Kd
Cathepsin Le
Cathepsin Sf
6
7
8
9
84%
0.0672 ± 0.0100
0.0330 ± 0.0051
0.0431 ± 0.0049
0.134 ± 0.013
0.535 ± 0.025
0.128 ± 0.004
0.386 ± 0.014
92%
0.0707 ± 0.0104
0.0426 ± 0.0049
0.0830 ± 0.0130
0.136 ± 0.025
1.20 ± 0.11
26.3 ± 2.5
56.1 ± 9.9
55.6 ± 12.6
92%
1
0
0.641 ± 0.066
0.453 ± 0.009
1
1
1
1
1
1
1
1
1
2
3
4
5
6
7
8
146 ± 16
87%
0.0520 ± 0.0025
0.0234 ± 0.0019
0.0563 ± 0.0077
0.0787 ± 0.0086
0.0454 ± 0.0021
0.0251 ± 0.0029
0.0413 ± 0.0031
0.189 ± 0.012
0.272 ± 0.020
0.269 ± 0.007
0.844 ± 0.052
0.147 ± 0.009
1.37 ± 0.08
0.0297 ± 0.0043
0.00402 ± 0.00078
0.218 ± 0.007
23.8 ± 1.2
83%
0.0738 ± 0.0100
0.0136 ± 0.0014
0.0919 ± 0.0140
0.0657 ± 0.0044
0.177 ± 0.025
91%
86%
0.277 ± 0.002
1.78 ± 0.07
89%
82%
0.238 ± 0.009
1
2
2
2
2
2
2
9
0
1
2
3
4
5
31.1 ± 5.3
86%
0.908 ± 0.067
1.25 ± 0.22
0.111 ± 0.007
0.0963 ± 0.0074
0.343 ± 0.006
0.320 ± 0.029
0.173 ± 0.010
1.09 ± 0.14
0.0260 ± 0.0034
0.0268 ± 0.0039
0.179 ± 0.024
3.52 ± 0.09
30.8 ± 1.7
8.74 ± 0.76
87%
0.635 ± 0.048
0.820 ± 0.056
0.457 ± 0.076
2.03 ± 1.00
0.0295 ± 0.0031
0.0754 ± 0.0109
0.0355 ± 0.0026
0.199 ± 0.0253
20.6 ± 0.5
2.35 ± 0.50
0.156 ± 0.009
a
K
i
values were determined in duplicate measurements with six different inhibitor concentrations when the remaining activity at 10 µM inhibitor concentration
was less than 80%. The equation for non-linear regression used to obtain the IC50 value was v = v /(1+([I]/IC50), where v is the product formation rate at different
inhibitor concentrations, v is the uninhibited product formation rate, [I] is the inhibitor concentration, and IC50 is the half-maximal inhibitory concentration. IC50
values are transformed to K values using the Cheng-Prusoff equation. The standard errors refer to the nonlinear regression.
Otherwise, the remaining activity at 10 µM inhibitor concentration obtained in duplicate measurements is noted.
i
0
i
0
i
b
c
d
e
3
7 °C, λmax = 405 nm, pH 6.0, substrate: Z-Arg-Arg-pNA (500 µM = 0.45 K
5 °C, λex = 360 nm, λem = 440 nm, pH 5.0, substrate: Z-Leu-Arg-AMC (6 µM = 3.05 K
7 °C, λmax = 405 nm, pH 6.0, substrate: Z-Phe-Arg-pNA (100 µM = 5.88 K ) or 37 °C, λex = 360 nm, λem = 440 nm, pH 6.0, substrate: Z-Phe-Arg-AMC (10.9
).
5 °C, λex = 360 nm, λem = 440 nm, pH 6.0, substrate: Z-Phe-Arg-AMC (40 µM = 0.74 K
m
).
2
3
m
)
m
µM = 4.00 K
m
f
2
m
).

were performed. Plausible binding modes of the most potent (12)
and the selective cathepsin S inhibitor (24) are shown in Figure 3.
The sulfonyl amide moiety of both covalently bound ligands was
found to accommodate the front compartment of the S2
specificity pocket by forming weak hydrogen bonds between the
sulfonyl oxygens and the C-hydrogen donors of residues
Gly133, Asn158 and Gly160, as similarly found for other
8
sulfone/sulfonamide ligands. As a result of this orientation, the
biphenyl substructure bisects the lower S3 specificity pocket and
the S2 cavity by forming edge-to-face and -stacking interactions
with residues Phe67 and Phe205, respectively.
The sulfone/sulfonamide motif is part of several cathepsin S
inhibitors with some of them having a binding mode clarified by
X-ray crystal structure analysis. We superimposed the structures
containing five potent representatives, all equipped with a
2
,12
cyclopropyl unit in P1 (Fig. 4).
These inhibitors and our
compounds share the dipeptide nitrile scaffold, but do not bear a
sulfonyl moiety attached to the N-terminal amino group. In all
2
five cases, the SO motif is located at the very same position,
namely the entrance of the S2 pocket, in accordance to our
modelling study (see Fig. 1).
Fig. 2. Selectivity plot including all inhibitors of Tables 1 and 2. The analog
pairs 12/20 and 17/24 differ in the P2 amino acid, i.e leucine/phenylalanine.
In conclusion, we developed sulfonamide nitriles as selective
and potent cathepsin S inhibitors. Within three subsets, we
altered three points of diversity, introduced several aromatic
To target the selectivity issue of cathepsin S over cathepsin
K, the P2 amino acid leucine was exchanged to phenylalanine in
the third subset of compounds (19-25). It is well known from
substrate mapping studies that cathepsin K prefers leucine,
sulfonamide
residues
and
outlined
structure-activity
relationships. The process was accompanied by computational
studies, leading to the assumption of the sulfonamide moiety
displacing the P2 amino acid residue from the S2 pocket and a
possible deviation from the characteristic backbone alignment of
peptidic protease inhibitors.
4
2
instead of phenylalanine, in P2 position, which is reflected by
several particularly active peptidic cathepsin K inhibitors with
4
3
this feature. Actually, in all leucine-phenylalanine pairs, the
desired effect was attained and affinity for cathepsin K reduced
(11-13, 15-18 versus 19-25). However, due to the P2 preference
of cathepsin L for phenylalanine, the potency of all members of
this subset towards this enzyme was increased (11-13, 15-18
versus 19-25). At this point we could make use of the
aforementioned observation that the introduction of a terminal
fluorine was disadvantageous for cathepsin L (20 versus 22, 23
versus 24). As a result of this interplay of the substituents’
effects, we regained selectivity for cathepsin S over K and L in
the particular case of inhibitor 24. This sulfonamide nitrile
maintained the low-nanomolar inhibitory activity towards
Declaration of Competing Interest
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.
Acknowledgments
The authors thank Marion Schneider, Sabine Terhart-Krabbe,
Rabea Voget and Franka G. Westermann for support. We are also
indebted to FAPESP grant #2013/18009-4 for financing part of
this work.
cathepsin S (K
with respect
i
= 35.5 nM) and possessed a robust selectivity
to the other cathepsins (>30-fold).
Activity/selectivity relationships are summarized in Fig. 2.
In order to verify the in-silico-based approach and the
obtained biochemical data, further molecular docking studies
O
S
O
H
N
O
S
O
N
H
N
N
N
H
N
H
O
O
F
Fig. 3. Putative binding mode of compound 12 (left, carbon atoms are labeled as orange sticks) and compound 24 (right, carbon atoms are labeled as cyan sticks)
in the active site of cathepsin S (PDB-ID: 1MS6).13 Relevant amino acids of the subsites S1, S2 and S3 are labeled as grey sticks, the protein surface in grey.

O
S
O
O
S
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F
R1
R3
O
F
F
H
N
N
R2
H
N
N
N
H
N
O
R1
R2
O
I: R1 = (S)-(5-oxopyrrolidin-2-yl)methyl, R2 = F IV:
II: R1 = 4-morpholino, R = F
R
R
R
1
= H, R2 = CF3, R3 = H
2
1
2
V:
= CO-c-Pr-1-CF3, R = Cl,
= (S)-OCH(CH3)CF3
1
2
3
III:
R
= Bn, R = OH
S3
S1
8
9
.
.
1
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CX3CL1 (fractaline) in vascular smooth muscle cells. Biol Chem.
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Moll S, Popper B, Eberhard JN, Thomasova D, Rufer AC, Gruner S,
Haap W, Hartmann G, Anders HJ. Sci Rep. 2017;7:2775. (d) Thanei S,
Theron M, Silva AP, Reis B, Branco L, Schirmbeck L, Kolb FA, Haap
W, Schindler T, Trendelenburg M. Cathepsin S inhibition suppresses
autoimmune-triggered inflammatory responses in macrophages. Biochem
Pharmacol. 2017;146:151–164. (e) Dheilly E, Battistello E, Katanayeva
N, Sungalee S, Michaux J, Duns G, Wehrle S, Sordet-Dessimoz J, Mina
M, Racle J, Farinha P, Coukos G, Gfeller D, Mottok A, Kridel R, Correia
BE, Steidl C, Bassani-Sternberg M, Ciriello G, Zoete V, Oricchio E.
Cathepsin S regulates antigen processing and T cell activity in Non-
Hodgkin lymphoma. Cancer Cell. 2020:37:1–16.
Fig. 4. Superposition of five selected crystallized cathepsin S inhibitors with
a sulfone/sulfonamide moiety (I, cyan, PDB-ID: 2FQ9; II, yellow, 2FT2, III,
dark green, 2FRA; IV, magenta, 4BS5; V, blue, 4BPV).2,12
Appendix A. Supplementary Data
Representative NMR spectra. Supplementary data to this
article can be found online at
1
1. (a) Gauthier JY, Black WC, Courchesne I, Cromlish W, Desmarais S,
Houle R, Lamontagne S, Li CS, Massé F, McKay DJ, Ouellet M,
Robichaud J, Truchon JF, Truong VL, Wang Q, Percival MD. The
identification of potent, selective, and bioavailable cathepsin S inhibitors.
Bioorg Med Chem Lett. 2007;17:4929–4933. (b) Moss N, Xiong Z,
Burke M, Cogan D, Gao DA, Haverty K, Heim-Riether A, Hickey ER,
Nagaraja R, Netherton M, O’Shea K, Ramsden P, Schwartz R, Shih DT,
Ward Y, Young E, Zhang Q. Exploration of cathepsin S inhibitors
characterized by a triazole P1-P2 amide replacement. Bioorg Med Chem
Lett. 2012;22:7189–7193.
2. Hilpert H, Mauser H, Humm R, Anselm L, Kuehne H, Hartmann G,
Gruener S, Banner DW, Benz J, Gsell B, Kuglstatter A, Stihle M, Thoma
R, Sanchez RA, Iding H, Wirz B, Haap W. Identification of potent and
selective cathepsin S inhibitors containing different central cyclic
scaffolds. J Med Chem. 2013;56:9789–9801.
3. Ward YD, Thomson DS, Frye LL, Cywin CL, Morwick T, Emmanuel
MJ, Zindell R, McNeil D, Bekkali Y, Girardot M, Hrapchak M, DeTuri
M, Crane K, White D, Pav S, Wang Y, Hao MH, Grygon CA, Labadia
ME, Freeman DM, Davidson W, Hopkins JL, Brown ML, Spero DM.
Design and synthesis of dipeptide nitriles as reversible and potent
cathepsin S inhibitors. J Med Chem. 2002;45:5471–5482.
4. Frizler M, Lohr F, Lülsdorff M, Gütschow M. Facing the gem-dialkyl
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References and Notes
1
.
(a) Volonterio A, Bellosta S, Bravin F, Bellucci MC, Bruché L, Colombo
G, Malpezzo L, Mazzini S, Meille SV, Meli M, Ramírez De Arellano C,
Zanda M. Synthesis, structure and conformation of partially-modified
retro- and retro-inverso ψ[NHCH(CF
003;9:4510–4522. (b) Black WC, Bayly CI, Davis DE, Desmarais S,
Falgueyret JP, Léger S, Li CS, Massé F, McKay DJ, Palmer JT, Percival
MD, Robichaud J, Tsou N, Zamboni R. Trifluoroethylamines as amide
isosteres in inhibitors of cathepsin K. Bioorg Med Chem Lett.
3
)]Gly peptides. Chem Eur J.
2
1
1
2
005;15:4741–4744. (c) Elie BT, Gocheva V, Shree T, Dalrymple SA,
Holsinger LJ, Joyce JA. Identification and pre-clinical testing of a
reversible cathepsin protease inhibitor reveals anti-tumor efficacy in a
pancreatic cancer model. Biochimie. 2010;92:1618–1624.
2
3
.
.
Link JO, Mossman CJ, Woo SH, Zipfel SM. Preparation of
(
hetero)aryltrifluoroethyl containing amino acid cyanoalkylamides as
cysteine protease (cathepsin S) inhibitors. Patent US 2003-60504680.
004.
Adhikari N, Halder AK, Mallick S, Saha A, Saha KD, Jha T. Robust
design of some selective matrix metalloproteinase-2 inhibitors over
matrix metalloproteinase-9 through in silico/fragment-based lead
identification and de novo lead modification: syntheses and biological
assays. Bioorg Med Chem. 2016;24:4291–4309.
2
1
1
5. Computational Methods. Superposition of co-crystallized cathepsin S
inhibitors. X-ray structures of human and mouse cathepsin S bound to
sulfone-type inhibitors were obtained from the RCSB Protein Data
4
5
6
.
.
.
Adhikari N, Mukherjee A, Saha A, Jha T. Arylsulfonamides and
selectivity of matrix metalloproteinase-2: an overview. Eur J Med Chem.
4
4
Bank. The following crystallographic complexes were used: 2FT2,
2
were performed with the Molecular Operating Environment (MOE
2
cathepsin S (PDB ID: 2FQ9 and PDB-ID: 1MS6) were extracted from
the RCSB Protein Data Bank. Covalent Docking was performed as
described elsewhere.
2
017;31:72–109.
2
12
FRA, 2FQ9, and 4BS5, 4BPV. Protein alignment and superposition
Obreza A, Gobec S. Recent advances in design, synthesis and biological
activity of aminoalkylsulfonates and sulfonamidopeptides. Curr Med
Chem. 2004;11:3263–3278.
4
5
018.0101). Molecular docking. The X-ray structures of human
(a) Supuran CT, Casini A, Scozzafava A. Protease inhibitors of the
sulfonamide type: anticancer, antiinflammatory, and antiviral agents.
Med Res Rev. 2003;23:535–558. (b) Poliakov A, Johansson A, Akerblom
E, Oscarsson K, Samuelsson B, Hallberg A, Danielson UH. Structure-
activity relationships for the selectivity of hepatitis C virus NS3 protease
inhibitors. Biochim Biophys Acta. 2004;1672:51–59. (c) Ramesh VV,
Kale SS, Kotmale AS, Gawade RL, Puranik VG, Rajamohanan PR,
Sanjayan GJ. Carboxamide versus sulfonamide in peptide folding: a case
study with a hetero foldamer. Org Lett. 2013;15:1504–1507.
4
6
1
1
6. Thompson SA, Andrews PR, Hanzlik RP. Carboxyl-modified amino
acids and peptides as protease inhibitors. J Med Chem. 1986;29:104–111.
7. Gomes JC, Cianni L, Ribeiro J, Dos Reis Rocho F, da Costa Martins
Silva S, Batista PHJ, Moraes CB, Franco CH, Freitas-Junior LHG,
Kenny PW, Leitão A, Burtoloso ACB, de Vita D, Montanari CA.
Synthesis and structure-activity relationship of nitrile-based cruzain
inhibitors incorporating
replacement. Bioorg Med Chem. 2019;27:115083.
a
trifluoroethylamine-based P2 amide
7
.
(a) Johansson A, Poliakov A, Akerblom E, Wiklund K, Lindeberg G,
Winiwarter S, Danielson UH, Samuelsson B, Hallberg A. Acyl

1
8. Giroud M, Kuhn B, Saint-Auret S, Kuratli C, Martin RE, Schuler F,
Diederich F, Kaiser M, Brun R, Schirmeister T, Haap, W. 2H-1,2,3-
Triazole based dipeptidyl nitriles: potent, selective, and trypanocidal
128.26, 128.41, 128.96, 129.08, 129.39, 135.34, 138.68, 139.92, 143.79,
170.93. LC/MS (ESI): >99% purity, m/z [M+Na] 498.1.
+
26. 3-Chlorophenylsulfonyl-(S)-leucyl-aminocyclopropane-nitrile (11): Yield
1
3
rhodesain inhibitors by structure-based design.
018;61:3370–3388.
J
Med Chem.
81%. Mp 166-168 °C. H NMR (400 MHz, DMSO-d
6
) δ 0.86 (d, J = 6.8
3
2
Hz, 3H), 0.88 (d, J = 6.8 Hz, 3H), 1.07-1.11 (m, 2H), 1.32-1.36 (m, 1H),
1
2
9. Cianni L, Sartori G, Rosini F, De Vita D, Pires G, Lopes BR, Leitão A,
Burtoloso ACB, Montanari CA. Leveraging the cruzain S3 subsite to
increase affinity for reversible covalent inhibitors. Bioorg Chem.
1.41-1.45 (m, 1H), 1.46-1.49 (m, 2H), 1.54-1.61 (m, 1H), 3.86-3.94 (m,
3
3
4
1H), 7.67 (t, J = 8.0 Hz, 1H), 7.87 (dt, J = 8.0 Hz, J = 1.5 Hz, 1H),
4
3
8.01 (t, J = 2.0 Hz, 1H), 8.06-8.21 (m, 1H), 8.82 (d, J = 8.1 Hz, 1H).
1
3
2
018;79:285–292.
6
One NH signal is missing. C NMR (100 MHz, DMSO-d ) δ 14.54,
0. General procedures for preparation of the final compounds 6-25: A
solution of the corresponding Boc-protected dipeptide nitrile (0.2 mmol)
was dissolved in formic acid (2 mL) and stirred at room temperature for
15.21, 18.85, 20.84, 22.21, 23.38, 40.97, 53.73, 119.83, 124.21, 124.90,
127.80, 130.08, 138.24, 171.76. One carbon signal is missing due to peak
+
overlay. LC/MS (ESI): 97% purity, m/z [M+Na] 392.1.
2
2 h. The solvent was evaporated and the remaining crude intermediate
27. (Biphenyl-3-yl)sulfonyl-(S)-leucyl-aminocyclopropane-nitrile (12): Yield
1
was dissolved in H O (5 mL). By using 1 M NaOH, the pH was adjusted
2
51%. Mp 163-165 °C. H NMR (400 MHz, DMSO-d
1H), 0.72-0.82 (m, 4H), 0.84 (d, J = 6.8 Hz, 3H), 1.20-1.40 (m, 4H)
6
) δ 0.41-0.49 (m,
3
to 12-13 and it was extracted with ethyl acetate (3 × 20 mL). The
combined organic layers were dried and the solvent was evaporated to
obtain the corresponding free amine, which was used without further
purification. It was dissolved in CH
mg, 0.4 mmol) was added. Under argon atmosphere, this mixture was
injected into a solution of the corresponding sulfonyl chloride (0.3-0.4
mmol) in CH
After evaporation of the solvent, the remaining crude material was
partitioned between ethyl acetate (30 mL) and sat. NaHCO solution (30
mL) and extracted. The organic layer was further washed with sat.
NaHCO solution (2 × 30 mL) and brine (3 × 30 mL) and dried over
Na SO or MgSO . Evaporation and purification by preparative column
1.51-1.60 (m, 1H), 3.60-3.68 (m, 1H), 7.41-7.48 (m, 1H), 7.50-7.57 (m,
3
2H), 7.64 (t, J = 7.7 Hz, 1H), 7.68-7.74 (m, 3H), 7.89-7.93 (m, 1H), 7.98
4
3
13
2
Cl
2
(10 mL) and DIPEA (68 µL, 52
(t, J = 1.7 Hz, 1H), 8.17 (d, J = 8.4 Hz, 1H), 8.88 (s, 1H); C NMR
(100 MHz, DMSO-d ) δ 14.75, 15.42, 19.06, 21.05, 22.42, 23.59, 41.18,
6
53.94, 120.04, 124.42, 125.11, 126.62, 128.01, 128.91, 129.47, 130.29,
+
2
Cl
2
(10 mL) at 0 °C. The mixture was stirred for 20 h.
138.45, 140.60, 141.47, 171.97. LC/MS (ESI): 96% purity, m/z [M+H]
412.1.
3
28. (Biphenyl-3-yl)sulfonyl-(R)-leucyl-aminocyclopropane-nitrile (13): Yield
1
78%. Mp 160-162 °C. H NMR (500 MHz, DMSO-d
6
) δ 0.44-0.50 (m,
3
3
3
1H), 0.72 (d, J = 6.6 Hz, 3H), 0.72-0.80 (m, 1H), 0.81 (d, J = 6.5 Hz,
2
4
4
3H), 1.21-1.32 (m, 3H), 1.33-1.43 (m, 1H), 1.50-1.59 (m, 1H), 3.67 (app.
3
3
chromatography yielded the desired products 6-25.
q, J = 7.0 Hz, 1H), 7.40-7.47 (m, 1H), 7.48-7.56 (m, 2H), 7.63 (t, J =
3
4
2
1. Phenylsulfonyl-(S)-leucyl-phenylalanine-nitrile (6): Yield 77%. Mp 130-
7.8 Hz, 1H), 7.65-7.74 (m, 3H), 7.90 (dt, J = 7.7, J = 1.4 Hz, 1H), 7.98
1
3
4
3
13
1
31 °C. H NMR (400 MHz, DMSO-d
6
) δ 0.63 (d, J = 6.0 Hz, 3H), 0.78
(t, J = 1.8 Hz, 1H), 8.13 (d, J = 7.0 Hz, 1H), 8.86 (s, 1H); C NMR
(125 MHz, DMSO-d ) δ 15.14, 15.80, 19.45, 21.43, 22.79, 23.98, 41.57,
3
(d, J = 6.0 Hz, 3H), 1.15-1.22 (m, 2H), 1.39-1.45 (m, 1H), 2.87-3.00 (m,
6
2
H), 3.60-3.73 (m, 1H), 4.62-4.75 (m, 1H), 7.26-7.38 (m, 5H), 7.54-7.60
54.35, 120.42, 124.81, 125.50, 127.00, 128.39, 129.29, 129.85, 130.67,
138.84, 141.00, 141.87, 172.37. LC/MS (ESI): 96% purity, m/z [M+H]
412.1.
3
4
+
(m, 2H), 7.53 (tt, J = 7.4 Hz, J = 1.2 Hz, 1H), 7.75-7.79 (m, 2H), 8.08
3
3
13
(
d, J = 8.0 Hz, 1H), 8.87 (d, J = 7.5 Hz, 1H); C NMR (100 MHz,
) δ 21.64, 23.06, 24.14, 37.52, 41.73, 42.00, 54.83, 119.05,
26.97, 127.63, 128.88, 129.36, 129.79, 132.78, 135.73, 141.28, 171.58.
DMSO-d
1
6
29. 3-Bromophenylsulfonyl-(S)-leucyl-aminocyclopropane nitrile (14): Yield
1
30%. Mp 175-177 °C. H NMR (400 MHz, DMSO-d
6
) δ 0.56-0.61 (m,
+
3
3
LC/MS (ESI): 99% purity, m/z [M+Na] 422.2.
1H), 0.75 (d, J = 6.4 Hz, 3H), 0.84 (d, J = 6.4 Hz, 3H), 0.91-0.95 (m,
1H), 1.24-1.43 (m, 4H), 1.49-1.60 (m, 1H), 3.67-3.71 (m, 1H), 7.52 (t, J
= 8.0 Hz, 1H), 7.72 (ddd, J = 8.0 Hz, J = 2.0 Hz, J = 1.2 Hz, 1H), 7.83
(ddd, J = 7.9 Hz, J = 2.0 Hz, J = 0.8 Hz, 1H), 7.87 (t, J = 1.6 Hz, 1H),
3
2
2. 3-Chlorophenylsulfonyl-(S)-leucyl-phenylalanine-nitrile (7): Yield 65%.
1
3
3
4
4
Mp 139-141 °C. H NMR (400 MHz, DMSO-d
6
) δ 0.68 (d, J = 6.1 Hz,
3
3
4
4
4
3
1
1
7
1
4
1
H), 0.80 (d, J = 6.1 Hz, 3H), 1.18-1.23 (m, 1H), 1.28-1.34 (m, 1H),
3
13
.45-1.50 (m, 1H), 2.85-2.96 (m, 2H), 3.70-3.75 (m, 1H), 4.64-4.69 (m,
6
8.31 (d, J = 8.8 Hz, 1H), 8.90 (s, 1H); C NMR (100 MHz, DMSO-d ) δ
3
H), 7.23-7.36 (m, 5H), 7.58 (t, J = 8.0 Hz, 1H), 7.67-7.72 (m, 2H),
15.11, 15.66, 19.33, 21.20, 22.63, 23.80, 41.29, 54.11, 120.21, 121.81,
125.42, 128.92, 131.29, 135.17, 143.07, 172.02. LC/MS (ESI): 96%
4
3
3
.78 (t, J = 2.0 Hz, 1H), 8.32 (d, J = 8.5 Hz, 1H), 8.95 (d, J = 7.5 Hz,
1
3
+
H); C NMR (100 MHz, DMSO-d
6
) δ 21.60, 23.10, 24.20, 37.53,
purity, m/z [M+Na] 437.9.
1.73, 42.09, 54.77, 118.92, 125.61, 126.72, 127.69, 128.92, 129.77,
30. (4'-Fluorobiphenyl-3-yl)sulfonyl-(S)-leucyl-aminocyclopropane-nitrile
1
2
31.52, 132.76, 133.97, 135.63, 143.25, 171.35. LC/MS (ESI): 97%
6
(15): Yield 75%. H NMR (500 MHz, DMSO-d ) δ 0.47 (ddd, J = 12.8
+
3
3
3
purity, m/z [M+Na] 456.1.
Hz, J = 7.1 Hz, J = 4.6 Hz, 1H), 0.72 (d, J = 6.6 Hz, 3H), 0.74-0.77 (m,
1H), 0.81 (d, J = 6.7 Hz, 3H), 1.26-1.29 (m, 2H), 1.33-1.45 (m, 2H),
1.50-1.59 (m, 1H), 3.66 (dt, J = 9.0 Hz, J = 5.4, 1H), 7.33-7.38 (m, 2H),
7.62 (t, J = 7.8 Hz, 1H), 7.69-7.77 (m, 3H), 7.88 (dt, J = 7.7 Hz, J =
1.4 Hz, 1H), 7.95 (t, J = 1.8 Hz, 1H), 8.14 (d, J = 8.7 Hz, 1H), 8.85 (s,
6
1H); C NMR (125 MHz, DMSO-d ) δ 15.17, 15.77, 19.45, 21.42,
22.79, 23.97, 41.56, 54.36, 116.14 (d, J = 21.4 Hz), 120.39, 124.75,
125.46, 129.10 (d, J = 8.3 Hz), 129.87, 130.64, 135.24 (d, J = 3.0 Hz),
3
2
3. (Biphenyl-3-yl)sulfonyl-(S)-leucyl-phenylalanine-nitrile (8): Yield 58%.
1
3
3
3
Mp 187-188 °C. H NMR (400 MHz, DMSO-d
3
1
6
) δ 0.58 (d, J = 6.4 Hz,
3
3
3
4
H), 0.67 (d, J = 6.4 Hz, 3H), 0.94-1.02 (m, 1H), 1.03-1.12 (m, 1H),
2
3
2
4
3
.18-1.28 (m, 1H), 2.85 (dd, J = 13.6 Hz, J = 9.2 Hz, 1 H), 3.04 (dd, J
3
13
=
13.2 Hz, J = 6.4 Hz, 1H), 3.61-3.69 (m, 1H), 4.66-4.74 (m, 1H), 7.14-
.25 (m, 5H), 7.38-7.45 (m, 1H), 7.48-7.55 (m, 2H), 7.64 (t, J = 8.0 Hz,
H), 7.68-7.71 (m, 2H), 7.74-7.80 (m, 1H), 7.85-7.90 (m, 1H), 8.02 (t, J
1.6 Hz, 1H), 8.08 (d, J = 8.0 Hz, 1H), 8.84 (d, J = 8.0 Hz, 1H);
) δ 21.32, 22.49, 23.54, 37.39, 40.90, 41.32,
4.34, 118.72, 124.59, 125.12, 126,82, 126.99, 128.14 128.22, 129.08,
29.32, 129.62, 130.54, 135.29, 138.79, 140.88, 141.70, 170.91. LC/MS
3
2
7
1
=
4
3
4
3
3
13
1
C
139.96, 141.89, 162.45 (d, J = 243.9 Hz), 172.35. LC/MS (ESI): >99%
+
NMR (100 MHz, DMSO-d
5
1
6
purity, m/z [M+H] 430.0
31. (Biphenyl-4-yl)sulfonyl-(S)-leucyl-aminocyclopropane-nitrile (16): Yield
1
2
3
54%. Mp 183-185 °C. H NMR (400 MHz, CDCl ) δ 0.51-0.58 (ddd, J =
+
3
3
3
(ESI): 95 % purity, m/z [M+H] = 476.2.
12.7 Hz, J = 7.0 Hz, J = 4.5 Hz, 1H), 0.74 (d, J = 6.5 Hz, 3H), 0.80-
3
2
4. (4'-Fluorobiphenyl-3-yl)sulfonyl-(S)-leucyl-phenylalanine-nitrile
(9):
) δ 0.66 (d, 3J
7.0 Hz, 3H), 0.77 (d, J = 7.0 Hz, 3H), 1.15-1.24 (m, 1H), 1.26-1.36
0.90 (m, 4H), 1.21-1.43 (m, 4H), 1.54-1.60 (m, 1H), 3.66 (dt, J = 9.3 Hz,
J = 5.5 Hz, 1H), 7.40-7.45 (m, 1H), 7.47-7.53 (m, 2H), 7.68-7.72 (m,
2H), 7.76-7.85 (m, 4H), 8.12 (s, 1H), 8.90 (s, 1H); C NMR (100 MHz,
1
3
Yield 53%. Mp 193-194 °C. H NMR (400 MHz, DMSO-d
=
6
3
13
2
3
(m, 1H), 1.40-1.54 (m, 1H), 2.72 (dd, J = 13.5 Hz, J = 9.0 Hz, 1H),
3
CDCl ) δ 14.90, 15.31, 19.11, 21.06, 22.46, 23.61, 41.19, 53.98, 120.12,
2
3
2
1
7
.83 (dd, J = 13.5 Hz, J = 6.5 Hz, 1H), 3.71-3.75 (m, 1H), 4.60-4.67 (m,
126.81, 126.91, 126.95, 128.21, 128.89, 138.40, 139.59, 143.70, 172.05.
3
+
H), 7.10-7.16 (m, 2H), 7.22-7.46 (m, 5H), 7.63 (t, J = 7.5 Hz, 1H),
.71-7.81 (m, 3H), 7.87-7.91 (m, 1H), 8.02 (t, J = 2.0 Hz, 1H), 8.17 (br
LC/MS (ESI): 97% purity, m/z [M+Na] 434.1.
4
32. (4'-Fluorobiphenyl-4-yl)sulfonyl-(S)-leucyl-aminocyclopropane-nitrile
3
3
13
1
d, J = 7.5 Hz, 1H), 8.89 (d, J = 8.0 Hz, 1H); C NMR (100 MHz,
DMSO-d ) δ 20.96, 22.40, 23.51, 36.87, 41.15, 41.29, 54.16, 115.78 (d,
(17): Yield 80%. Mp 186-188 °C. H NMR (500 MHz, DMSO-d
6
) δ 0.57
2
3
3
3
6
(ddd, J = 10.8 Hz, J = 7.4 Hz, J = 4.8 Hz, 1H), 0.75 (d, J = 6.5 Hz,
3H), 0.83 (d, J = 6.6 Hz, 3H), 0.84-0.88 (m, 1H), 1.22-1.35 (m, 4H),
1.57 (m, 1H), 3.66 (dt, J = 9.1 Hz, 5.5 Hz, 1H), 7.29-7.35 (m, 2H), 7.72-
7.83 (m, 6H), 8.09 (d, J = 8.8 Hz, 1H), 8.87 (s, 1H); C NMR (125
MHz, DMSO-d ) δ 15.31, 15.67, 19.49, 21.42, 22.81, 23.98, 41.54,
54.37, 116.10 (d, J = 21.4 Hz), 120.46, 127.23, 127.33, 129.30 (d, J =
8.3 Hz), 135.24 (d, J = 3.0 Hz), 139.95, 143.01, 162.58 (d, J = 244.3
Hz), 172.39. LC/MS (ESI): >99% purity, m/z [M+H] 430.0.
2
3
3
J = 21.5 Hz), 118.23, 124.58, 125.11, 126.94, 128.16, 128.65 (d, J = 8.0
4
3
Hz), 128.99, 129.47, 130.50, 134.88 (d, J = 2.0 Hz), 139.52, 141.35,
1
peak overlay. LC/MS (ESI): 98% purity, m/z [M+Na] 516.1.
1
3
13
62.06 (d, J = 243.9 Hz), 170.81. One carbon signal is missing due to
+
6
2
3
2
5. (Biphenyl-4-yl)sulfonyl-(S)-leucyl-phenylalanine-nitrile (10): Yield 56%.
1
3
4
1
Mp 181-182 °C. H NMR (500 MHz, DMSO-d
3
1
6
) δ 0.62 (d, J = 6.5 Hz,
3
+
H), 0.70 (d, J = 6.6 Hz, 3H), 0.96-1.01 (m, 1H), 1.11-1.18 (m, 1H),
2
3
.25-1.33 (m, 1H), 2.89 (dd, J = 13.5 Hz, J = 9.3 Hz, 1H), 3.09-3.03
33. 2-Chlorophenylsulfonyl-(S)-leucyl-aminocyclopropane-nitrile (18): Yield
3
3
1
(m, 1H), 3.71 (dd, J = 9.0 Hz, J = 5.8 Hz, 1H), 4.75-4.82 (m, 1H), 7.18-
60%. Mp 170-172 °C. H NMR (500 MHz, DMSO-d
6
) δ 0.62-0.66 (m,
3
3
3
7
1
2
.28 (m, 6H), 7.34-7.52 (m, 4H), 7.59-7.72 (m, 4H), 8.07 (d, J = 8.5 Hz,
1H), 0.68 (d, J = 6.6 Hz, 3H), 0.81 (d, J = 6.7 Hz, 3H), 0.84-0.88 (m,
1H), 1.25 (ddd, J = 13.8 Hz, J = 8.8 Hz, J = 5.1 Hz, 1H), 1.32-1.40 (m,
2H), 1.48 (ddd, J = 13.6 Hz, J = 9.9 Hz, J = 5.1 Hz, 1H), 1.56-1.64 (m,
3
13
2
3
3
H), 8.90 (d, J = 8.1 Hz, 1H); C NMR (125 MHz, DMSO-d
6
) δ 21.39,
2
3
3
2.59, 23.61, 37.47, 40.85, 41.41, 54.43, 118.89, 126.75, 127.06, 127.22,

1
H), 3.61-3.66 (m, 1H), 7.45-7.51 (m, 1H), 7.58-7.62 (m, 2H), 7.89-7.92
(125 MHz, DMSO) δ 15.45, 15.74, 19.40, 38.30, 57.62, 120.30, 126.68,
3
13
(
m, 1H), 8.17 (d, J = 5.3 Hz, 1H), 8.79 (s, 1H); C NMR (125 MHz,
DMSO-d ) δ 15.38, 15.80, 19.56, 21.13, 22.91, 23.93, 41.14, 54.55,
20.48, 127.48, 130.48, 131.22, 131.81, 134.14, 138.23, 172.43. LC/MS
127.32, 128.20, 129.36, 130.03, 131.03, 131.79, 133.90, 136.60, 138.27,
+
6
171.49. LC/MS (ESI): 97% purity, m/z [M+H] 404.0.
1
4,19,46,47
1
41. Assay conditions were as reported elsewhere.
Stock solutions of
+
(ESI): >99% purity, m/z [M+H] 369.9.
substrates and inhibitors were prepared in DMSO; the final DMSO
concentration was 2%. Fluorimetric (Cat K, Cat S) or photometric assays
(Cat B, Cat L) were performed in duplicate on a Fluostar OPTIMA plate
reader and a Varian Cary 50/100Bio UV-Visible spectrophotometer,
respectively. IC50 values were determined via non-linear regression using
3
4. 3-Chlorophenylsulfonyl-(S)-phenylalanyl-aminocyclopropane-nitrile
1
(19): Yield 66%. Mp 193-194 °C. H NMR (500 MHz, DMSO-d
6
2
) δ 0.50-
0
.55 (m, 1H), 0.77–0.80 (m, 1H), 1.30-1.37 (m, 2H), 2.71 (dd, J = 13.5
3
2
3
Hz, J = 8.5 Hz, 1H), 2.82 (dd, J = 13.5 Hz, J = 6.5 Hz, 1H), 3.82-3.89
3
(m, 1H), 7.07-7.12 (m, 2H), 7.16-7.23 (m, 3H), 7.50 (t, J = 8.0 Hz, 1H),
the equation v
correcting the IC50 value using the Cheng-Prusoff equation IC50
×(1+[S]/K ).
i 0 i
= v /(1+([I]/IC50). K values were determined by
3
4
4
7
.56 (dt, J = 8.0 Hz, J = 1.5 Hz, 1H), 7.60 (t, J = 1.8 Hz, 1H), 7.64
=
3
4
4
(ddd, J = 7.0 Hz, J = 2.5 Hz, J = 1.0 Hz, 1H), 8.54 (s, 1H), 8.88 (s,
K
i
M
1
3
1
5
1
4
H); C NMR (125 MHz, DMSO-d
6
) δ 15.72, 16.03, 19.66, 38.90,
42. (a) Choe Y, Leonetti F, Greenbaum DC, Lecaille F, Bogyo M, Brömme
D, Ellman JA, Craik CS. Substrate profiling of cysteine proteases using a
combinatorial peptide library identifies functionally unique specificities.
J Biol Chem. 2006;281:12824–12832. (b) Lecaille F, Chowdhury S,
Purisima E, Brömme D, Lalmanach G. The S2 subsites of cathepsins K
and L and their contribution to collagen degradation. Protein Sci.
2007;16:662–670.
7.63, 120.50, 125.31, 126.44, 127.07, 128.51, 129.64, 131.42, 132.68,
+
33.94, 136.74, 143.35, 171.68. LC/MS (ESI): 99% purity, m/z [M+Na]
26.4.
3
5. (Biphenyl-3-yl)sulfonyl-(S)-phenylalanyl-aminocyclopropane-nitrile (20):
1
6
Yield 71%. Mp 112-114 °C. H NMR (500 MHz, DMSO-d ) δ 0.58-0.65
2
3
(m, 1H), 0.77-0.87 (m, 1H), 1.22-1.25 (m, 2H), 2.70 (dd, J = 13.5 Hz, J
2
3
=
1
=
7
7
8
3
1
8.0 Hz, 1H), 2.82 (dd, J = 13.5 Hz, J = 7.1 Hz, 1H), 3.86-3.92 (m,
43. (a) Gauthier JY, Chauret N, Cromlish W, Desmarais S, Duong LT,
Falgueyret JP, Kimmel DB, Lamontagne S, Léger S, LeRiche T, Li CS,
Massé F, McKay DJ, Nicoll-Griffith DA, Oballa RM, Palmer JT,
Percival MD, Riendeau D, Robichaud J, Rodan GA, Rodan SB, Seto C,
Thérien M, Truong VL, Venuti MC, Wesolowski G, Young RN,
Zamboni R, Black WC. The discovery of odanacatib (MK-0822), a
selective inhibitor of cathepsin K. Bioorg Med Chem Lett. 2008;18:923–
928. (b) Breuer C, Lemke C, Schmitz J, Bartz U, Gütschow M. Synthesis
and kinetic evaluation of ethyl acrylate and vinyl sulfone derived
inhibitors for human cysteine cathepsins. Bioorg Med Chem Lett.
2018;28:2008–2012. (c) Cianni L, Feldmann CW, Gilberg E, Gütschow
M, Juliano L, Leitão A, Bajorath J, Montanari CA. J Med Chem.
2019;62:10497–10525.
3
H), 7.07-7.10 (m, 2H), 7.10-7.14 (m, 1H), 7.15-7.19 (m, 2H), 7.43 (tt, J
7.4 Hz, J = 1.4 Hz, 1H), 7.49-7.54 (m, 2H), 7.56 (t, J = 7.7 Hz, 1H),
.62 (dt, J = 7.8 Hz, J = 1.3 Hz, 1H), 7.65-7.68 (m, 2H), 7.85 (dt, J =
.6 Hz, J = 1.4 Hz, 1H), 7.90 (t, J = 1.7 Hz, 1H), 8.32-8.38 (m, 1H),
4
3
3
4
3
4
4
1
3
6
.78 (s, 1H), C NMR (125 MHz, DMSO-d ) δ 15.26, 15.74, 19.30,
8.76, 57.29, 120.20, 124.70, 125.33, 126.71, 127.05, 128.22, 128.35,
29.26, 129.34, 129.81, 130.65, 136.48, 138.87, 140.98, 141.89, 171.44.
+
LC/MS (ESI): 96% purity, m/z [M+H] 445.9.
3
6. (Biphenyl-3-yl)sulfonyl-(R)-phenylalanyl-aminocyclopropane-nitrile
1
(21):Yield 89%. Mp 120-122 °C. H NMR (500 MHz, DMSO-d
6
2
) δ 0.38-
0
.43 (m, 1H), 0.60-0.65 (m, 1H), 1.22-1.25 (m, 2H), 2.70 (dd, J = 13.5
3
2
3
3
Hz, J = 7.9 Hz, 1H), 2.81 (dd, J = 13.5 Hz, J = 7.1 Hz, 1H), 3.89 (t, J
7.5 Hz, 1H), 7.07-7.10 (m, 2H), 7.10-7.18 (m, 3H), 7.41-7.45 (m, 1H),
th
=
44. RCSB Protein Data Bank. http://www.rcsb.org (accessed December 5 ,
3
3
4
7
1
7
.49-7.53 (m, 2H), 7.56 (t, J = 7.7 Hz, 1H), 7.62 (dt, J = 7.9 Hz, J =
2018)
3
4
.5 Hz, 1H), 7.65-7.68 (m, 2H), 7.85 (dt, J = 7.6 Hz, J = 1.5 Hz, 1H),
.90 (t, J = 1.8 Hz, 1H), 8.34 (s, 1H), 8.78 (s, 1H); C NMR (125 MHz,
45. Molecular Operating Environment (MOE), 2018.01; Chemical
Computing Group ULC, 1010 Sherbooke St. West, Suite #910, Montreal,
QC, Canada, H3A 2R7, 2018.
4
13
DMSO-d
6
) δ 15.28, 15.76, 19.32, 38.77, 57.31, 120.22, 124.71, 125.35,
1
1
4
26.73, 127.06, 128.23, 128.38, 129.28, 129.35, 129.83, 130.67, 136.49,
38.88, 141.00, 141.90, 171.46. LC/MS (ESI): 99% purity, m/z [M+H]
46.0.
46. Cianni L, Lemke C, Gilberg E, Feldmann C, Rosini F, Rocho FDR,
Ribeiro JFR, Tezuka DY, Lopes CD, de Albuquerque S, Bajorath J,
Laufer S, Leitão A, Gütschow M, Montanari CA. Mapping the S1 and
S1’ subsites of cysteine proteases with new dipeptidyl nitrile inhibitors as
trypanocidal agents. PLoS Negl Trop Dis. 2020;14:e0007755.
47. Mertens MD, Schmitz J, Horn M, Furtmann N, Bajorath J, Mareš M,
Gütschow M. A coumarin-labeled vinyl sulfone as tripeptidomimetic
activity-based probe for cysteine cathepsins. ChemBioChem.
2014;15:955–959.
+
3
7. (4'-Fluorobiphenyl-3-yl)sulfonyl-(S)-phenylalanyl-aminocyclopropane-
1
nitrile (22): Yield 41%. Mp 122-124 °C. H NMR (500 MHz, DMSO-d
δ 0.38-0.45 (m, 1H), 0.58-0.66 (m, 1H), 1.21-1.26 (m, 2H), 2.70 (dd, J =
1
6
)
2
3
2
3
3.5 Hz, J = 7.9 Hz, 1H), 2.81 (dd, J = 13.5 Hz, J = 7.0 Hz, 1H), 3.89
3
(
app. q, J = 8.0 Hz, 1H), 7.06-7.20 (m, 5H), 7.32-7.37 (m, 2H), 7.56 (t,
3
3
4
J = 7.7 Hz, 1H), 7.61 (dt, J = 8.0 Hz, J = 1.4 Hz, 1H), 7.68-7.73 (m,
3
4
4
2
8
1
1
1
2
H), 7.84 (dt, J = 7.7 Hz, J = 1.5 Hz, 1H), 7.87 (t, J = 1.8 Hz, 1H),
3
13
6
.35 (d, J = 9.0 Hz, 1H), 8.78 (s, 1H); C NMR (125 MHz, DMSO-d ) δ
5.29, 15.72, 19.30, 38.75, 57.29, 116.10 (d, J = 21.4 Hz), 120.19,
24.62, 125.30, 126.71, 128.21, 129.15 (d, J = 8.2 Hz), 129.34, 129.83,
30.61, 135.36 (d, J = 3.1 Hz), 136.48, 139.94, 141.90, 162.45 (d, J =
2
3
4
1
+
43.8 Hz), 171.43. LC/MS (ESI): 99% purity, m/z [M+H] 464.0.
3
8. (Biphenyl-4-yl)sulfonyl-(S)-phenylalanyl-aminocyclopropane-nitrile (23):
1
6
Yield 76%. Mp 186-188 °C. H NMR (500 MHz, DMSO-d ) δ 0.48-0.55
2
3
(
=
m, 1H), 0.72-0.78 (m, 1H), 1.20-1.34 (m, 2H), 2.72 (dd, J = 13.6 Hz, J
2
3
3
8.2 Hz, 1H), 2.82 (dd, J = 13.6 Hz, J = 6.8 Hz, 1H), 3.88 (app. q, J =
.2 Hz, 1H), 7.08-7.14 (m, 2H), 7.12-7.24 (m, 3H), 7.42 (tt, J = 7.3 Hz,
J = 1.6 Hz, 1H), 7.47-7.52 (m, 2H), 7.64-7.71 (m, 4H), 7.70-7.76 (m,
H), 8.26-8.34 (m, 1H), 8.78-8.84 (m, 1H); C NMR (125 MHz, DMSO-
) δ 15.42, 15.67, 19.37, 38.69, 57.34, 120.31, 126.68, 127.14, 127.20,
3
7
4
1
3
2
d
1
1
6
27.28, 128.26, 128.56, 129.26, 129.42, 136.59, 138.85, 139.97, 143.97,
+
71.55. LC/MS (ESI): 98% purity, m/z [M+H] 445.9.
3
9. (4'-Fluorobiphenyl-4-yl)sulfonyl-(S)-phenylalanyl-aminocyclopropane-
1
nitrile (24): Yield 46%. Mp 212-214 °C. H NMR (500 MHz, DMSO-d
6
)
2
δ 0.48-0.57 (m, 1H), 0.68-0.78 (m, 1H), 1.23-1.33 (m, 2H), 2.72 (dd, J =
3
2
3
1
(
3.5 Hz, J = 8.2 Hz, 1H), 2.81 (dd, J = 13.5 Hz, J = 6.9 Hz, 1H), 3.88
3
3
dt, J = 8.4 Hz, J = 6.7 Hz, 1H), 7.08-7.14 (m, 2H), 7.12-7.23 (m, 3H),
.29-7.35 (m, 2H), 7.64-7.69 (m, 2H), 7.69-7.77 (m, 4H), 8.31 (d, J =
.9 Hz, 1H), 8.81 (s, 1H); C NMR (125 MHz, DMSO-d
5.63, 19.35, 38.66, 57.32, 116.09 (d, J = 21.4 Hz), 120.28, 126.65,
27.12, 127.21, 128.23, 129.30 (d, J = 8.4 Hz), 129.40, 135.30 (d, J =
.0 Hz), 136.56, 139.94, 142.87, 162.56 (d, J = 244.3 Hz), 171.51.
LC/MS (ESI): 98% purity, m/z [M+H] 464.0.
3
7
8
1
1
3
1
3
6
) δ 15.43,
2
3
4
1
+
4
0. 2-Chlorosulfonyl-(S)-phenylalanyl-aminocyclopropane-nitrile
(25):
1
Yield 74%. Mp 130-132 °C. H NMR (500 MHz, DMSO-d
6
) δ 0.55-0.60
2
3
(
m, 1H) 0.72-0.77 (m, 1H), 1.30-1.36 (m, 2H), 2.79 (dd, J = 13.6 Hz, J
2
3
3
=
8.6 Hz, 1H), 2.85 (dd, J = 13.6 Hz, J = 6.5 Hz, 1H), 3.87 (t, J = 7.3
Hz, 1H), 7.11-7.21 (m, 5H), 7.35-7.40 (m, 1H), 7.49-7.56 (m, 2H), 7.72
3
4
13
(
dd, J = 7.9 Hz, J = 1.5 Hz, 1H), 8.34 (s, 1H), 8.77 (s, 1H); C NMR