Synthesis and kinetic evaluation of ethyl acrylate and vinyl sulfone derived inhibitors for human cysteine cathepsins

Article abstract of DOI:10.1016/j.bmcl.2018.05.005

A series of inhibitors targeting human cathepsins have been designed and synthesized following a combinatorial approach. The compounds bear an α,β-unsaturated phenyl vinyl sulfone or ethyl acrylate warhead and a peptidomimetic portion aligned to the non-primed binding region. Biochemical evaluation toward four human cathepsins was carried out and the kinetic characterization confirmed an irreversible mode of inhibition. Compound 6c combining the most advantageous building blocks for cathepsin S inhibition was identified as a potent cathepsin S inactivator exhibiting a second-order rate constant of 30600 M^?1 s^?1.

Full text of DOI:10.1016/j.bmcl.2018.05.005

Accepted Manuscript
Synthesis and kinetic evaluation of ethyl acrylate and vinyl sulfone derived in-
hibitors for human cysteine cathepsins
Christian Breuer, Carina Lemke, Janina Schmitz, Ulrike Bartz, Michael
Gütschow
PII:
S0960-894X(18)30390-1
https://doi.org/10.1016/j.bmcl.2018.05.005
BMCL 25822
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
7 February 2018
30 April 2018
1 May 2018
Please cite this article as: 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, Bioorganic &
Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.05.005
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Graphical Abstract
Synthesis and kinetic evaluation of
Leave this area blank for abstract info.
ethyl acrylate and vinyl sulfone derived
inhibitors for human cysteine cathepsins
Christian Breuer, Carina Lemke, Janina Schmitz, Ulrike Bartz and Michael Gütschow*

Bioorganic & Medicinal Chemistry Letters
Synthesis and kinetic evaluation of ethyl acrylate and vinyl sulfone derived
inhibitors for human cysteine cathepsins
Christian Breuera,b,c, Carina Lemkea,c, Janina Schmitza,b, Ulrike Bartzb and Michael Gütschowa,*
a Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany
b Department of Natural Sciences, University of Applied Sciences Bonn-Rhein-Sieg, von-Liebig-Strasse 20, D-53359 Rheinbach, Germany
Article history:
Received
Revised
Accepted
Available online
A series of inhibitors targeting human cathepsins have been designed and
synthesized following a combinatorial approach. The compounds bear an
α,β-unsaturated phenyl vinyl sulfone or ethyl acrylate warhead and
a
peptidomimetic portion aligned to the non-primed binding region. Biochemical
evaluation toward four human cathepsins was carried out and the kinetic
characterization confirmed an irreversible mode of inhibition. Compound 6c
combining the most advantageous building blocks for cathepsin S inhibition was
identified as a potent cathepsin S inactivator exhibiting a second-order rate constant
of 30600 M-1s-1.
Keywords:
Cathepsins
Cysteine proteases
Horner-Wadsworth-Emmons olefination
Irreversible inhibition
Michael acceptors
2018 Elsevier Ltd. All rights reserved.
Cysteine cathepsins, an important group of proteases, belong
to the C1 family, of which the Carica papaya enzyme papain is
one representative.1 These cathepsins were first discovered in the
lysosomes and are ubiquitously expressed. Besides their role in
final protein degradation, cysteine cathepsins are involved in
physiological cell regulation processes, such as bone remodeling,
cell proliferation and immune response, but also act in
pathophysiological events, such as angiogenesis and tumor
invasion, arthritis, osteoporosis, atherosclerosis and neurological
disorders.2
Cysteine cathepsins consist of an L and R domain of
comparable size which are displaying a V-shaped configuration.
The catalytic cysteine is present on the L domain, while a
histidine residue, forming an ion pair with the cysteine, is located
on the R domain. Substrates bind along the active site to be
proteolytically degraded. The specificity pockets of the non-
primed (S1, S2…) or primed binding sites (S1’, S2’…) interact
with the corresponding amino acid residues (at positions P1,
P2… or P1’, P2’…), leading to preferences of the enzymes for
specific substrates. The nucleophilic attack of the deprotonated
cysteine residue at the carbonyl group of the scissile peptide bond
forms an enzyme-substrate thioester which is subsequently
hydrolyzed.2 This catalytic mechanism can be utilized for the
sulfones, which are susceptible to a nucleophilic addition of the
catalytic cysteine residue. Covalent irreversible cathepsin
inhibition can be achieved that way.3,4
Among such peptidic Michael acceptors, vinyl sulfones have
received most attention as cysteine protease inhibitors.5-10
Noteworthy, certain cathepsin inhibitors with this type of
warhead exhibited a selectivity for cathepsin S over other human
cathepsins, such as K, B and L.5-7 Further peptidic Michael
acceptors, which have been reported as cysteine protease
inhibitors contain a ketone, ester, carboxamide,8,9,11 or sulfonate
moiety in place of the sulfonyl structure.12 All these chemotypes
have, moreover, successfully been applied as activity-based
probes for human cysteine cathepsins.6-8,13
Herein we describe the synthesis and biological evaluation of
inhibitors derived from two different peptidic scaffolds, i.e. ethyl
acrylate or S-phenyl vinyl sulfone. The design was focused on
human cathepsin S, because it was intended to identify
appropriate structures for the subsequent development of
cathepsin S-selective tool compounds. For this purpose, we
combined the two aforementioned warheads with two different
amino acids in P2 position and spacers of different lengths which
are terminated by a Boc-protected amino group. The structure at
the P1 position of the so designed compounds corresponds to
glycine. Glycine, besides glutamine, serine, and glutamic acid
belongs to the amino acids which are preferred at the P1 position
of cathepsin S substrates.14
design of inhibitors, for example ,-unsaturated ketones or
________________
* Corresponding author. Tel.: +49 228 73 2317; fax: +49 228 73 2567.
E-mail: guetschow@uni-bonn.de.
c These authors contributed equally.

O
O
3000
2000
1000
0
H
H
O
N
O
N
O
a
OH
N
1
O
O
O
H
N
O
b
H
2
O
Scheme 1. Synthesis of N-Boc-Gly-H via Weinreb amide formation from
the corresponding Boc-protected amino acid. Reagents and conditions:
(a) 1. N-methylmorpholine (NMM), ClCO2iBu, CH2Cl2; 2. N,O-dimethyl-
hydroxylamine × HCl, -25 °C to rt; (b) LiAlH4, THF, argon, -78 °C.
0
1000 2000 3000 4000 5000
time (s)
The synthesis of the P1 building block is shown in Scheme 1.
Boc-protected glycine was converted to the O,N-dimethyl
hydroxamate 1,15 using the mixed anhydride procedure.
Subsequently, a Lewis acid-mediated reduction of the Weinreb
amide under inert atmosphere afforded Boc-glycine aldehyde 2.16
This aldehyde was reacted with triethyl phosphonoacetate (3,
Scheme 2) or diethyl (phenylsulfonyl)methylphosphonate (3),
Figure 1. Human cathepsin S-catalyzed hydrolysis of Z-Phe-Arg-AMC
(40 μM) in the presence of increasing concentrations of compound 6c (from
top to bottom: 0 nM, 20 nM, 40 nM, 60 nM, 80 nM, 100 nM).
8
6
4
2
respectively, in
a
Horner-Wadsworth-Emmons olefination
forming the central ,-unsaturated Michael-acceptor building
blocks 4 (with R1 = CO2Et or SO2Ph, Scheme 2).17,18 After Boc-
deprotection using acetyl chloride / ethanol as HCl source,19 the
resulting ammonium chlorides were coupled in an uronium salt-
mediated amide formation with Boc-leucine and Boc-
phenylalanine, respectively,20 generating 5a-d.21-24 Subsequent
Boc-deprotection with AcCl/EtOH yielded the ammonium
chloride intermediates which were directly coupled to Boc-
-alanine to afford compounds 6a-d.25-28 Compounds 7b and 7c
were prepared in analogous manner from compounds 5b and 5c,
respectively, through connection to the appropriate Boc-protected
polyethylenglycol-spacer.29,30 The allylic substructure in
0
20
40
60
80
100
[
] (nM)
6c
Figure 2. Plot of the first-order rate constants kobs vs. concentrations of 6c.
1
compounds 5-7 provided H NMR signals as follows,  3.8 – 4.1
ppm (m, 2H, -CH2CH=CH-),  5.6 – 5.9 ppm (d, 3J = 15.7 – 15.8
Next, the occurrence and the length of the spacer and its effect
on enzyme inhibition were analyzed. Introduction of the short
-alanine spacer caused the strongest cathepsin S inactivation (5c
vs. 6c vs. 7c as well as 5b vs. 6b vs. 7b). Accordingly, the
optimized structure for cathepsin S combines the aforementioned
structural features, i.e. the S-phenyl vinyl sulfone, leucine, and
the -alanine spacer. The so assembled compound 6c exhibited a
kinac/Ki value of 30600 M-1s-1. The influence of the spacer on
inhibitory activity against the three other cathepsins was
indistinct. Incorporation of the expanded PEG spacer was
generally not beneficial as none of the four cathepsins was most
efficiently inhibited by the corresponding compounds 7b and 7c.
This result might reflect inappropriate interactions of the PEG
structure and/or the Boc moiety with the non-primed binding
region. Nevertheless, 7c was still a potent (kinac/Ki = 11900 M-1s-1)
and somewhat selective cathepsin S inhibitor. We observed weak
selectivity for cathepsin S over the other related cathepsins in
several cases, including the potent inhibitors 5c, 6c and 7c.
Referring to the identification of a general inhibitor structure
targeting all the four cathepsins, at best 5a can be considered.
With kinac/Ki values higher than 1200 M-1s-1, 5a is possibly useful
as a biochemical tool compound.
Hz, 1H, -CH2CH=CHCO-) or  6.4 – 6.7 ppm (d, 3J = 15.0 – 15.1
3
Hz, 1H, -CH2CH=CHSO2-) and 6.7 – 6.9 ppm (dt, J = 15.1 –
3
15.8 Hz, J = 4.0 – 4.7 Hz, 1H, -CH2CH=CH-).21-30 The trans
configuration was confirmed by the large constants (15 – 16 Hz)
of the 3J coupling via the olefinic backbone.
The small library of the so assembled peptidic Michael
acceptors was subjected to biochemical investigations. Protease
assays in the presence of compounds 5-7 were performed with
four different human cysteine cathepsins, i.e. cathepsins S, K, B,
and L.31 The progress curves indicated time-dependent inhibition
in accordance with the expected irreversible mode of action of
the investigated peptidic Michael acceptors (Fig. 1). The second-
order rate constants of inactivation, kinac/Ki, reflect the inhibitory
potency of irreversible inhibitors. They were obtained from plots
of the first-order rate constants, kobs, vs. the inhibitor
concentrations (Fig. 2, Table 1). We also determined the effect of
the reference inactivator E-64 on the four cathepsins.
The kinetic data of our compound series allow drawing
structure-activity relationships by comparing analogs with one
divergent moiety. Interestingly, vinyl sulfones turned out to be
more potent inactivators of cathepsin S and K than their
analogous ethyl acrylates (e.g. 6a vs. 6c). In contrary, a stronger
cathepsin B inhibition was observed for ethyl acrylates (e.g. 5a
vs. 5c). These findings reflect a preference of the S1’ binding
pockets of cathepsins S and K for aromatic and of cathepsin B for
aliphatic moieties. Leucine at the P2 position was advantageous
for inhibition of cathepsins S and K (e.g. 5a vs. 5b), while
phenylalanine was more accepted by cathepsin B (e.g. 6a vs. 6b).
With respect to both substructures at P1’ and P2 position,
cathepsin L did not exhibit a clear preference.

R2
O
O
P
O
H
H
R1
a
O
N
R1
N
R1
b
O
O
N
O
H
3
4
5a-d
O
R1 = ethoxycarbonyl, phenylsulfonyl
R2 = Ph, iPr
c
d
R2
R2
O
O
O
O
O
H
H
H
R1
N
R1
O
N
O
N
O
N
N
H
N
H
H
O
O
7b,c
6a-d
Scheme 2. Synthesis of compounds 5-7. Reagents and conditions: (a) KOtBu, Boc- Gly-H (2), THF, 0 °C to rt; (b) 1. AcCl, EtOH, EtOAc, 0 °C; 2. N-Boc-Phe-
OH or N-Boc-Leu-OH, HATU or HBTU, DIPEA, CH2Cl2, rt; (c) 1. AcCl, EtOH, EtOAc, 0 °C; 2. N-Boc-β-Ala-OH, HATU or HBTU, DIPEA, CH2Cl2, rt; (d) 1.
AcCl, EtOH, EtOAc, 0 °C; 2. dicyclohexylammonium 1-(tert-butyloxycarbonylamino)-3,6-dioxa-octanoate, HBTU, DIPEA, CH2Cl2, rt.
Table 1
Enzyme inhibitory properties of the prepared inhibitors 5–7 and half-lives for the reaction with N-acetylcysteine
kinac/Ki (M-1s-1)a
Compd
R1
R2
t1/2 (h)f
hCat Sb
hCat Kc
hCat Bd
hCat Le
iPr
Ph
iPr
Ph
iPr
Ph
iPr
Ph
6600
11600
3090
1280
26
32
14
15
24
23
5.2
8.6
5a
5b
5c
5d
6a
6b
6c
6d
525
28400
7960
2590
879
752
18300
1320
2890
759
3660
85.1
378
1120
879
5880
554
248
2660
58.3
214
3540
671
30600
13100
12500
2230
12800
Ph
557
297
348
652
35
7b
iPr
11900
25.5
150
3310
6.5
7c
n.d.g
142000
192000
258000
98100
E-64
a
The values for kinac/Ki (M-1s-1) were determined via non-linear regression using the equation [P] = vi × (1 - exp(-kobs × t) / kobs + d, where [P] is the product
concentration, vi the initial rate, kobs the observed first-order rate constant and d is the offset, and the equation kobs = (kinac × [I]) / ([I] + Ki × (1 + [S]/Km)), where
kinac is the first-order inactivation rate constant, [I] the inhibitor concentration, Ki the dissociation constant of the enzyme-inhibitor complex, [S] the substrate
concentration and Km is the Michaelis constant.
b 25 °C, λex = 360 nm, λem = 440 nm, pH 6.0, substrate: Z-Phe-Arg-AMC (40 µM = 0.74 Km).
c 25 °C, λex = 360 nm, λem = 440 nm, pH 5.0, substrate: Z-Leu-Arg-AMC (40 µM = 13.33 Km).
d 37 °C, λmax = 405 nm, pH 6.0, substrate: Z-Arg-Arg-pNA (500 µM = 0.45 Km).
e 37 °C, λmax = 405 nm, pH 6.0, substrate: Z-Phe-Arg-pNA (100 µM = 5.88 Km).
f Calculated from pseudo-first order rate constants of the reactions of the compound (0.1 mM) and N-acetylcysteine (2 mM) at pH 7.4 and 37 °C.
g not determined.

M.; Váchová, J.; Fanfrlík, J.; Brynda, J.; McKerrow, J. H.; Caffrey, C.
R.; Mareš, M. J. Biol. Chem. 2011, 286, 35770. (e) Wiggers, H. J.;
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McKerrow, J. H.; Montanari, C. A. PLoS Negl. Trop. Dis. 2013, 7,
e2370.
The cysteine reactivity of our ten electrophilic inhibitors was
examined. For this purpose, we investigated the reactions with
N-acetylcysteine under physiological conditions,32 and analyzed
the reactions by HPLC at several time points. In each case, only
one product peak of increasing area under curve (AUC) was
observed. The half-lives of 5a-7c are listed in Table 1.33 As an
example, the reaction product of 5a and N-acetylcysteine was
independently prepared, subjected to LC/MS analysis and this
material was used to confirm that the corresponding
HPLC product peak refers to the Michael addition product of 5a
with N-acetylcysteine.34 For our ten inactivators, half-lives
between 5 and 35 hours were obtained. Expectedly, vinyl
sulfones exhibited a higher cysteine reactivity than the analogous
ethyl acrylates.35
Our test compounds bear a Boc group serving as a placeholder
for any functional moiety. Following Boc deprotection of 6c or
7c, the amino group can be connected, for example, with a
fluorophore or biotin to generate activity-based probes for
cathepsin S. Heterobifunctional compounds might be designed to
develop PROTACs (proteolysis-targeting chimeras) recruiting
cathepsin S for ubiquitination and subsequent proteasomal
degradation. Moreover, the free amino group can be utilized to
attach the inactivator to an affinity chromatography matrix.
11. (a) Ettari, R.; Micale, N.; Schirmeister, T.; Gelhaus, C.; Leippe, M.; Nizi,
E.; Di Francesco, M. E.; Grasso, S.; Zappalà, M. J. Med. Chem. 2009, 52,
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McKerrow, J. H.; Hansell, E. J. Am. Chem. Soc. 1998, 120, 10994. (b)
Dana, D.; De, S.; Rathod, P.; Davalos, A. R.; Novoa, D. A.; Paroly, S.;
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Am. Chem. Soc. 2006, 128, 5616. (b) Uttamachandani, M.; Liu, K.;
Panicker, R. C.; Yao, S. Q. Chem. Commun. 2007, 1518.
14. (a) Rückrich, T.; Brandenburg, J.; Cansier, A.; Müller, M.; Stevanović,
S.; Schilling, K.; Wiederanders, B.; Beck, A.; Melms, A.; Reich, M.;
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5363.
15. Boc-Gly-N(Me)-OMe (1): N-Boc-Gly-OH (20 mmol) was dissolved in
CH2Cl2 (100 mL) and cooled to -25 °C. NMM (44 mmol) and isobutyl
chloroformate (22 mmol) were added. After stirring for 30 min, N,O-
dimethylhydroxylamine hydrochloride (22 mmol) was added at -25 °C.
After 5 h, the reaction mixture was washed with 10% KHSO4 (2 ×
100 mL), sat. NaHCO3 (2 × 100 mL) and H2O (1 × 100 mL), dried over
Na2SO4 and evaporated. The obtained yellowish solid was purified by
column chromatography using CH2Cl2/EtOAc (1+1) to give 1 as a white
solid (80%). mp 102-104 °C (lit. 102-103 °C);36 1H NMR (500 MHz,
DMSO-d6) δ 1.37 (s, 9H), 3.08 (s, 3H), 3.66 (s, 3H), 3.81 (d, 3J = 6.0 Hz,
2H), 6.79 (t, 3J = 6.0 Hz, 1H); 13C NMR (125 MHz, DMSO-d6) δ 28.31,
32.23, 40.95, 61.21, 78.05, 156.00, one carbon signal (CH2CO) is
missing; LC-MS (ESI) (90% H2O to 100% MeOH in 10 min, then 100%
MeOH for 10 min, DAD 200.0–400.0 nm) tr = 8.16 min, 96% purity,
m/z: 219 ([M+H]+).
Acknowledgments
C.B. was supported by a grant from the Department of Natural
Sciences, University of Applied Sciences Bonn-Rhein-Sieg. The
authors thank Lina Tschinse and Duyen Dao for support.
References and notes
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16. Boc-Gly-H (2): Boc-Gly-N(Me)-OMe (1, 5.00 mmol) was dissolved in
THF (10 mL) and injected through a septum to a suspension of LiAlH4
(6.25 mmol) in THF (10 mL) at -78 °C under argon atmosphere. TLC
control after 10 min using CH2Cl2/EtOAc (8.5+1.5) indicated the
completion of the reaction. Subsequently, the reaction mixture was
quenched with 10% KHSO4 (1 × 20 mL) and extracted with diethyl ether
(30 mL). The organic layer was washed with 10% KHSO4 (1 × 20 mL)
and sat. NaHCO3 (2 × 20 mL), dried over Na2SO4 and evaporated
1
yielding 2 as a colorless resin (50%). H NMR (500 MHz, DMSO-d6) δ
1.38 (s, 9 H), 3.72 (d, 3J = 5.7 Hz, 2 H), 7.16 (br t, 1 H), 9.44 (s, 1 H). 13
NMR (125 MHz, DMSO-d6) δ 28.26, 50.48, 78.46, 156.09, 200.74.
C
17. (E)-Ethyl 4-(tert-butoxycarbonylamino)but-2-enoate (4, R = CO2Et):
KOtBu (4.40 mmol) was added at 0 °C to a solution of triethyl
phosphonoacetate (3, R = CO2Et, 4.40 mmol) in dry THF (50 mL) and
stirred for 30 min. Boc-Gly-H (2, 4.40 mmol) was dissolved in dry THF
(10 mL) and added to the reaction mixture that was stirred at rt for 2 h.
The solvent was evaporated under reduced pressure. The obtained crude
red oil was dissolved in diethyl ether (100 mL) and washed with sat.
NaHCO3 (2 × 100 mL), H2O (100 mL) and 10% KHSO4 (2 × 100 mL).
The organic layer was dried over Na2SO4 and evaporated. The obtained
orange oil was purified by column chromatography using CH2Cl2/EtOAc
(8.5+1.5) to obtain a yellowish oil (46%).37 1H NMR (500 MHz, DMSO-
d6) δ 1.20 (t, 3J = 7.1 Hz, 3H), 1.38 (s, 9 H), 3.72 (t, 3J = 4.3 Hz, 2H),
3
3
4
4.11 (q, J = 7.0 Hz, 2H), 5.82 (dt, J = 15.8 Hz, J = 1.7 Hz, 1H), 6.79
(dt, J = 15.5 Hz, 3J = 4.7 Hz, 1H), 7.14 (s, 1H); 13C NMR (125 MHz,
3
DMSO-d6) δ 14.24, 28.31, 40.82, 59.98, 78.13, 120.17, 146.62, 155.64,
165.58; LC-MS (ESI) (90% H2O to 100% MeOH in 10 min, then 100%
MeOH for 10 min, DAD 220.0 – 400.0 nm): tr = 9.94 min, 100% purity,
m/z = 230.2 ([M+H]+).
9. Previti, S.; Ettari, R.; Cosconati, S.; Amendola, G.; Chouchene, K.;
Wagner, A.; Hellmich, U. A.; Ulrich, K.; Krauth-Siegel, R. L.; Wich, P.
R.; Schmid, I.; Schirmeister, T.; Gut, J.; Rosenthal, P. J.; Grasso, S.;
Zappalà, M. J. Med. Chem. 2017, 60, 6911.
10. (a) Ettari, R.; Nizi, E.; Di Francesco, M. E.; Dude, M. A.; Pradel, G.;
Vicík, R.; Schirmeister, T.; Micale, N.; Grasso, S.; Zappalà, M. J. Med.
Chem. 2008, 51, 988. (b) Mendieta, L.; Picó, A.; Tarragó, T.; Teixidó,
M.; Castillo, M.; Rafecas, L.; Moyano, A.; Giralt, E. ChemMedChem,
2010, 5, 1556. (c) Newton, A. S.; Glória, P. M.; Gonçalves, L. M.; dos
Santos D. J.; Moreira, R.; Guedes, R. C.; Santos, M. M. Eur. J. Med.
Chem. 2010, 45, 3858. (d) Jílková, A.; Rezácová, P.; Lepsík, M.; Horn,
18. (E)-tert-Butyl 3-(phenylsulfonyl)allylcarbamate (4, R = SO2Ph): KOtBu
(6.00 mmol) was added at 0 °C to
(phenylsulfonyl)methyl)phosphonate (3,
a
solution of diethyl
R
= SO2Ph, 5.00 mmol,
prepared as described in dry THF (10 mL),6 cooled to 0 °C and stirred for
30 min. Boc-Gly-H (2, 5.00 mmol) was dissolved in dry THF (5 mL) and
added dropwise leading to an orange color of the solution. After stirring
at rt for 2 h the solvent was evaporated under reduced pressure. The

3
3
residue was dissolved in EtOAc (50 mL), washed with sat. NaHCO3
(2 × 50 mL), H2O (50 mL) and brine (50 mL) and dried over Na2SO4.
The crude product was purified by column chromatography using
1H), 6.99 (d, 1H, J = 7.8 Hz), 7.18 – 7.26 (m, 5H), 7.64 (t, J = 7.5 Hz,
3 3 3
2H), 7.72 (t, J = 7.1 Hz, 1H), 7.82 (d, J = 7.8 Hz, 2H), 8.22 (t, J = 5.6
Hz, 1H); 13C NMR (125 MHz, DMSO-d6) δ 28.27, 37.23, 39.04, 56.15,
78.26, 126.36, 127.18, 128.16, 129.25, 129.69, 133.71, 138.15, 140.56,
144.66, 155.48, 171.97; LC-MS(ESI) (90% H2O to 100% MeOH in 10
min, then 100% MeOH for 10 min, DAD 220.0 – 400.0 nm): tr = 10.52
min, 99 % purity, m/z = 462.0 ([M+NH4]+).
1
petroleum ether/EtOAc (2+1) to obtain a colorless resin (27%); H NMR
(500 MHz, DMSO-d6) δ 1.38 (s, 9H), 3.97 (d, 3J = 7.8 Hz, 2H), 4.79 –
4.83 (m, 1H), 6.43 – 6.48 (m, 1H), 7.63 (t, 3J = 7.7 Hz, 2H), 7.72 (t, 3J =
7.5 Hz, 1H), 7.79 – 7.84 (m, 2H), 9.29 (br s, 1H); 13C NMR (125 MHz,
DMSO-d6) δ 28.33, 56.88, 79.54, 128.00, 129.29, 129.73, 132.82,
133.71, 138.99, 156.33; LC-ESI/MS LC-MS (ESI) (90% H2O to 100%
MeOH in 10 min, then 100% MeOH for 10 min, DAD 200.0 – 400.0
nm): tr = 9.31 min, 95 % purity, m/z = 298.3 ([M+H+]).
25. (S,E)-Ethyl 10-isobutyl-2,2-dimethyl-4,8,11-trioxo-3-oxa-5,9,12-triaza-
hexadec-14-en-16-oate (6a): The compound was synthesized by
successively following the general procedures I with 5a (1.00 mmol) and
II using HBTU (1 mmol), the deprotected 5a (0.80 mmol) and Boc-β-
Ala-OH (0.80 mmol). Yield: 46%; mp = 106-108 °C; 1H NMR (500
MHz, DMSO-d6) δ 0.85 (2 × d, 3J = 6.6 Hz, 3J = 6.5 Hz, 6H), 1.19 (t, 3J =
19. General procedure for Boc-deprotection (I): The N-Boc-protected amine
(1 equiv) was dissolved in EtOAc. Acetyl chloride (2-10 equiv) and
ethanol (2-10 equiv) were added at 0 °C and the reaction mixture was
stirred and allowed to reach rt during 18 h. The solvent was either
evaporated or filtered off to obtain the corresponding hydrochloride salt.
20. General procedure for amide formation (II): N-Boc-protected acid
(1 equiv) was dissolved in CH2Cl2, DIPEA (1 equiv) and HBTU (1-2
equiv) or HATU (1-1.1 equiv) were added and the reaction mixture was
stirred at rt for 30 min. An ammonium hydrochloride (1 equiv) and
DIPEA (1 equiv) were added and the solution was stirred at rt for 5-18 h.
After evaporation of the solvent, the crude product was dissolved in
EtOAc and extracted with 10% KHSO4, H2O, sat. NaHCO3 and brine.
The solvent was dried with Na2SO4 and evaporated. The product was
purified either by silica gel column chromatography or recrystallization.
21. (S,E)-Ethyl 4-(2-(tert-butoxycarbonylamino)-4-methylpentanamido)but-
2-enoate (5a): The compound was synthesized following the general
procedures I with 4 (R = CO2Et, 4.00 mmol) and II using HBTU (2.40
mmol), the deprotected intermediate (2.40 mmol) and Boc-Leu-
3
3
7.1 Hz, 3H), 1.36 (s, 9H), 1.45 (t, J = 7.2 Hz, 2H), 1.57 (hept, J = 6.7
Hz, 1H), 2.28 (t, 3J = 7.3 Hz, 2H), 3.11 (dt, 3J = 7.0 Hz, 3J = 6.5 Hz, 2H),
3.77 – 3.91 (m, 2H), 4.10 (q, 3J = 7.1 Hz, 2H), 4.26 (q, 3J = 7.7 Hz, 1H),
5.84 (dt, 3J = 15.8 Hz, 4J = 2.0 Hz, 1H), 6.64 (br t, 3J = 5.0 Hz, 1H), 6.80
(dt, 3J = 15.8 Hz, 3J = 4.6 Hz, 1H), 7.98 (d, 3J = 8.1 Hz, 1H), 8.16 (t, 3J =
5.8 Hz, 1H); 13C NMR (125 MHz, DMSO) δ 14.23, 21.63, 23.09, 24.38,
28.34, 35.72, 36.84, 40.82, 51.11, 59.96, 77.68, 120.32, 145.98, 155.56,
165.61, 170.58, 172.33. One signal is obscured by the DMSO solvent
signal; LC-MS (ESI) (90% H2O to 100% MeOH in 10 min, then 100%
MeOH for 10 min, DAD 220.0 – 400.0 nm): tr = 10.35 min, 97% purity,
m/z = 414.2 ([M+H]+).
10-benzyl-2,2-dimethyl-4,8,11-trioxo-3-oxa-5,9,12-triaza-
hexadec-14-en-16-oate (6b): The compound was synthesized by
successively following the general procedures I with 5b (1.00 mmol) and
II using HBTU (0.75 mmol), the deprotected 5b (0.75 mmol) and Boc--
Ala-OH (0.75 mmol). Yield: 41%; mp 153-156 °C; 1H NMR (600 MHz,
DMSO-d6) δ 1.20 (t, 3J = 7.1 Hz, 3H), 1.35 (s, 9H), 2.14 – 2.27 (m, 2H),
26. (S,E)-Ethyl
1
OH × H2O (2.40 mmol). Yield: 60%. H NMR (500 MHz, DMSO-d6) δ
3
3
0.86 (2 × d, J = 6.7 Hz, J = 6.6 Hz, 6H), 1.18 (t, 3J = 7.1 Hz, 3H), 1.37
(s, 9H), 1.40 – 1.47 (m, 2H), 1.54 – 1.62 (m, 1H), 3.77 – 3.89 (m, 2H),
3.90 – 3.96 (m, 1H), 4.09 (q, 3J = 7.1 Hz, 2H), 5.87 (d, 3J = 15.7 Hz, 1H),
6.80 (dt, 3J = 15.8 Hz, 3J = 4.4 Hz, 1H), 6.88 (d, 3J = 8.1 Hz, 1H), 8.09 (t,
3J = 5.9 Hz, 1H); 13C NMR (125 MHz, DMSO) δ 14.24, 21.72, 23.02,
24.44, 28.33, 40.72, 53.06, 59.92, 78.17, 120.21, 146.10, 155.52, 165.68,
172.83. One signal is obscured by the DMSO solvent signal; LC-MS
(ESI) (90% H2O to 100% MeOH in 10 min, then 100% MeOH for 10
min, DAD 200.0 – 400.0 nm): tr = 10.53 min, 99% purity, m/z = 343.0
([M+H]+).
3
3
2.76 (dt, J = 9.8 Hz, J = 3.4 Hz, 1H), 2.96 – 3.05 (m, 3H), 3.79 – 3.86
(m, 2H), 4.11 (q, 3J = 7.0 Hz, 2H), 4.47 (dt, 3J = 8.4 Hz, 3J = 5.6 Hz, 1H),
5.77 (d, 3J = 15.7 Hz, 1H), 6.59 (br t, 1H), 6.76 (dt, 3J = 15.7 Hz, J =
3
4.1 Hz, 1H), 7.15 – 7.27 (m, 5H), 8.15 (d, 3J = 8.2 Hz, 1H), 8.23 (br t,
1H); 13C NMR (150 MHz, DMSO) δ 14.27, 28.37, 35.73, 36.71, 37.71,
40.24, 54.15, 59.98, 77.73, 120.40, 126.43, 128.20, 129.22, 138.04,
145.80, 155.56, 165.62, 170.46, 171.31; LC-MS (ESI) (90% H2O to
100% MeOH in 10 min, then 100% MeOH for 10 min, DAD 200.0 –
400.0 nm): tr = 10.34 min, 95% purity, m/z = 448.1 ([M+H]+).
27. (S,E)-tert-Butyl
3-(4-methyl-1-oxo-1-(3-(phenylsulfonyl)allylamino)-
22. (S,E)-Ethyl 4-(2-(tert-butoxycarbonylamino)-3-phenylpropanamido)but-
2-enoate (5b): The compound was synthesized by successively following
the general procedures I with 4 (R = CO2Et, 4.00 mmol) and II using
HBTU (1.50 mmol), the deprotected intermediate (1.50 mmol) and Boc-
Phe-OH (1.50 mmol). Yield: 61%; mp 56-58 °C; 1H NMR (500 MHz,
pentan-2-ylamino)-3-oxopropylcarbamate (6c): The compound was
synthesized by successively following the general procedures I with 5c
(0.60 mmol) and II using HATU (0.50 mmol), the deprotected 5c (0.50
mmol) and Boc--Ala-OH (0.50 mmol). Yield: 61%; 1H NMR
3
(500 MHz, DMSO-d6) δ 0.82 (2 × d, J = 6.6 Hz, 6H), 1.36 (s, 9H), 1.40
3
DMSO-d6) δ 1.19 (t, J = 7.1 Hz, 3H), 1.31 (s, 9H), 2.76 (dd, 2J = 13.7
3
(t, J = 7.3 Hz, 2H), 1.54 (hept, 3J = 6.7 Hz, 1H), 2.20 – 2.30 (m, 2H),
3
Hz, J = 9.8 Hz, 1H), 2.94 (dd, 2J = 13.7 Hz, 3J = 5.1 Hz, 1H), 3.78 –
3.00 – 3.11 (m, 2H), 3.80 – 3.87 (m, 1H), 3.90 – 4.02 (m, 1H), 4.15 (q,
3J = 7.50 Hz, 1H), 6.65 (t, 3J = 4.9 Hz, 1H), 6.69 (dt, 3J = 15.1 Hz,
4J = 1.9 Hz, 1H), 6.84 (dt, 3J = 15.1 Hz, 3J = 4.1 Hz, 1H), 7.60 – 7.66 (m,
2H), 7.68 – 7.73 (m, 1H), 7.81 – 7.85 (m, 2H), 8.00 (d, 3J = 7.6 Hz, 1H),
8.24 (t, 3J = 5.7 Hz, 1H); 13C NMR (125 MHz, DMSO-d6) δ 21.70, 22.96,
24.33, 28.35, 35.66, 36.79, 38.95, 51.42, 77.70, 127.14, 129.69, 129.97,
133.69, 140.54, 144.32, 155.58, 170.77, 172.46; LC-MS (ESI) (90% H2O
to 100% MeOH in 10 min, then 100% MeOH for 10 min, DAD 220.0 –
400.0 nm): tr = 10.81 min, 99% purity, m/z = 482.2 ([M+H]+).
3
3
3.93 (m, 2H), 4.10 (q, J = 7.0 Hz, 2H), 4.11 – 4.17 (m, 1H), 5.84 (dt, J
= 15.7 Hz, 4J = 2.0 Hz, 1H), 6.78 (dt, 3J = 15.7 Hz, 3J = 4.5 Hz, 1H), 6.96
(d, 3J = 8.4 Hz, 1H), 7.15 – 7.20 (m, 1H), 7.21 – 7.28 (m, 4H), 8.16 (t, J
3
= 5.7 Hz, 1H); 13C NMR (125 MHz, DMSO) δ 14.25, 28.27, 37.49,
56.06, 59.91, 78.20, 120.31, 126.34, 128.16, 129.28, 138.22, 145.89,
155.40, 165.66, 171.84. One signal is obscured by the DMSO solvent
signal; LC-MS (ESI) (90% H2O to 100% MeOH in 10 min, then 100%
MeOH for 10 min, DAD 220.0 – 400.0 nm): tr = 10.57 min, 99% purity,
m/z = 377.3 ([M+H]+).
28. (S,E)-tert-Butyl
3-oxo-3-(1-oxo-3-phenyl-1-(3-(phenylsulfonyl)allyl-
23. (S,E)-tert-Butyl 4-methyl-1-oxo-1-(3-(phenylsulfonyl)allylamino)pentan-
2-ylcarbamate (5c): The compound was synthesized by successively
following the general procedures I with 4 (R = SO2Ph, 2.00 mmol) and II
using HATU (0.90 mmol), the deprotected intermediate (0.90 mmol) and
Boc-Leu-OH × H2O (0.90 mmol). Yield: 67%. 1H NMR (500 MHz,
amino)propan-2-ylamino)propylcarbamate (6d): The compound was
synthesized by successively following the general procedures I with 5d
(0.60 mmol) and II using HATU (0.50 mmol), the deprotected 5d (0.50
mmol) and Boc--Ala-OH (0.50 mmol). Yield: 43%; mp = 178-180 °C;
1H NMR (500 MHz, DMSO-d6) δ 1.36 (s, 9H), 2.14 – 2.25 (m, 2H), 2.76
3
2
2
(dd, J = 13.7 Hz, 3J = 9.1 Hz, 1H), 2.94 (dd, J = 13.7 Hz, 3J = 5.8 Hz,
DMSO-d6) δ 0.82 (2 × d, J = 6.6 Hz, 6H), 1.33 (s, 9H), 1.35 – 1.43 (m,
2H), 1.54 (hept, 3J = 6.6 Hz, 1H), 3.80 – 3.88 (m, 2H), 3.96 – 4.01 (m,
3
3
1H), 3.01 (dt, J = 7.3 Hz, J = 6.0 Hz, 2H), 3.83 – 3.97 (m, 2H), 4.37 –
4.43 (m, 1H), 6.54 (dt, 3J = 15.1 Hz, 4J = 1.9 Hz, 1H), 6.60 (br s), 6.81
(dt, 3J = 15.1 Hz, 3J = 4.2 Hz, 1H), 7.13 – 7.24 (m, 5H), 7.62 – 7.66 (m,
2H), 7.69 – 7.74 (m, 1H), 7.80 – 7.85 (m, 2H), 8.14 (d, 3J = 8.0 Hz, 1H),
8.26 (t, 3J = 5.7 Hz, 1H); 13C NMR (125 MHz, DMSO-d6) δ 28.36, 35.67,
36.67, 37.42, 39.01, 54.34, 77.73, 126.43, 127.21, 128.18, 129.16,
129.69, 129.85, 133.72, 137.92, 140.52, 144.28, 155.55, 170.59, 171.41;
LC-MS (ESI) (90% H2O to 100% MeOH in 10 min, then 100% MeOH
for 10 min, DAD 220.0 – 400.0 nm): tr = 7.71 min, 97 % purity, m/z =
516.3 ([M+H]+).
3
3
1H), 6.67 (d, J = 15.1 Hz, 1H), 6.87 (dt, J = 15.1 Hz, 3J = 4.0 Hz, 1H),
3
3
6.93 (d, J = 7.4 Hz, 1H), 7.63 (t, J = 7.6 Hz, 2H), 7.68 – 7.74 (m, 1H),
7.79 – 7.83 (m, 2H), 8.16 (t, 3J = 5.6 Hz, 1H); 13C NMR (125 MHz,
DMSO-d6) δ 21.75, 22.92, 24.36, 28.32, 38.97, 39.53, 53.15, 78.19,
127.15, 129.69, 129.77, 133.71, 140.57, 144.75, 155.63, 172.94; LC-MS
(ESI) (60% H2O to 100% MeOH in 10 min, then 100% MeOH for 10
min, DAD 220.0 – 400.0 nm): tr = 7.87 min, 99% purity, m/z = 428.1
([M+NH4]+).
24. (S,E)-tert-Butyl 1-oxo-3-phenyl-1-(3-(phenylsulfonyl)allylamino)propan-
2-ylcarbamate (5d): The compound was synthesized by successively
following the general procedures I with 4 (R = SO2Ph, 2.00 mmol) and II
using HATU (0.90 mmol), the deprotected intermediate (0.90 mmol) and
Boc-Phe-OH (0.90 mmol). Yield: 64%; mp = 170-172 °C; 1H NMR (500
MHz, DMSO-d6) δ 1.28 (s, 9H), 2.74 (dd, 2J = 13.4 Hz, 3J = 9.5 Hz, 1H),
2.90 (dd, 2J = 13.7 Hz, 3J = 5.3 Hz, 1H), 3.81 – 3.88 (m, 1H), 4.00 – 4.12
29. (S,E)-Ethyl 15-benzyl-2,2-dimethyl-4,13,16-trioxo-3,8,11-trioxa-5,14,17-
triazahenicos-19-en-21-oate (7b): The compound was synthesized by
successively following the general procedures I with 5b (1.00 mmol) and
II using HBTU (1.00 mmol), the deprotected 5b (0.50 mmol) and
dicyclohexylammonium
1-(tert-butyloxycarbonylamino)-3,6-dioxa-
octanoate (0.50 mmol). Yield: 40%. 1H NMR (500 MHz, DMSO-d6) δ
1.21 (t, 3J = 7.1 Hz, 3H), 1.36 (s, 9H), 2.88 (dd, 2J = 13.7 Hz, 3J = 9.0 Hz,
3
3
3
(m, 2H), 6.59 (d, J = 15.0 Hz, 1H), 6.85 (dt, J = 15.1 Hz, J = 4.0 Hz,

2
3
3
1H), 3.03 (dd, J = 13.7 Hz, J = 5.5 Hz, 1H), 3.07 (q, J = 6.1 Hz, 2H),
3.38 (t, 3J = 6.1 Hz, 2H), 3.41 – 3.51 (m, 4H), 3.78 (d, 2J = 15.3 Hz, 1H),
3.85 – 3.88 (m, 2H), 3.87 (d, 2J = 15.3 Hz, 1H), 4.11 (q, 3J = 7.1 Hz, 2H),
4.58 (dt, 3J = 8.7 Hz, 3J = 5.4 Hz, 1H), 5.79 (dt, 3J = 15.8 Hz, 4J = 1.9 Hz,
3
3
1H), 6.73 (br t, 1H), 6.77 (dt, J = 15.8 Hz, J = 4.7 Hz, 1H), 7.16 – 7.22
3
3
(m, 3H), 7.23 – 7.27 (m, 2H), 7.69 (d, J = 8.5 Hz, 1H), 8.33 (t, J = 5.8
Hz, 1H); 13C NMR (125 MHz, DMSO) δ 14.26, 28.35, 37.84, 40.29,
53.50, 59.99, 69.38, 69.43, 69.82, 70.27, 77.71, 120.53, 126.53, 128.24,
129.28, 137.57, 145.60, 155.71, 165.56, 169.16, 170.90. One signal is
obscured by the DMSO solvent signal; LC-MS (ESI) (90% H2O to
100% MeOH in 10 min, then 100% MeOH for 10 min, DAD 220.0 –
400.0 nm): tr = 10.66 min, 96% purity, m/z = 522.4 ([M+H]+).
30. (S,E)-tert-Butyl
10-isobutyl-8,11-dioxo-15-(phenylsulfonyl)-3,6-dioxa-
9,12-diazapentadec-14-enylcarbamate (7c): The compound was
synthesized by successively following the general procedures I with 5c
(3.00 mmol) and II using HBTU (3.30 mmol), the deprotected 5c (2.00
mmol) and dicyclohexylammonium 1-(tert-butyloxycarbonylamino)-3,6-
dioxa-octanoate (2.20 mmol). Yield: 27%. 1H NMR (600 MHz, CDCl3) δ
3
0.89 (2 x d, J = 6.4 Hz, 6H), 1.40 (s, 9H), 1.50 – 1.60 (m, 2H), 1.64 –
1.70 (m, 1H), 3.22 – 3.35 (m, 2H), 3.48 – 3.66 (m, 6H), 3.88 (d, 2J =
16.0 Hz, 1H), 4.01 (d, 2J = 16.0 Hz, 1H), 4.01 – 4.12 (m, 2H), 4.47 (dt, 3J
= 8.3 Hz, 3J = 6.5 Hz, 1H), 5.41 (br s, 1H), 6.45 (d, 3J = 15.1 Hz, 1H),
6.89 (dt, 3J = 15.1 Hz, 3J = 4.5 Hz, 1H), 6.93 (br s, 1H), 7.26 (d, 3J =
3
3
8.0 Hz, 1H), 7.51 (t, J = 7.7 Hz, 2H), 7.59 (t, J = 7.4 Hz, 1H), 7.84 (d,
3J = 7.4 Hz, 2H); 13C NMR (150 MHz, CDCl3) δ 22.07, 22.74, 24.71,
28.37, 38,77, 39.20, 40.34, 51.10, 69.88, 70.07, 70.69, 71.12, 79.17,
127.62, 129.28, 131.15, 133.47, 139.98, 142.11, 156.06, 170.56, 171.81;
LC-MS (ESI) (90% H2O to 100% MeOH in 10 min, then 100% MeOH
for 10 min, DAD 220.0 – 400.0 nm): tr = 10.56 min, 98% purity, m/z =
556.2 ([M+H]+).
31. Assay conditions were as reported elsewhere.7,38 Stock solutions of
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.
32. Affini, A.; Hagenow, S.; Zivkovic, A.; Marco-Contelles, J.; Stark, H.
Eur. J. Med. Chem. 2018, 148, 487.
33. The respective compound (0.1 mM) and N-acetylcysteine (2 mM) were
incubated at 37 °C in a 9+1 mixture of phosphate-buffered saline (137
mM NaCl, 2.7 mM KCl, 12 mM total phosphate, pH 7.4) and MeCN.
Aliquots of 20 µL were injected into an HPLC device (Jasco HPLC
2000, Machery-Nagel RP-18 4 µm Nucleosil 125-4 column) at least at
six different time points. Chromatograms were recorded with H2O/MeCN
(6+4) containing 0.1% F3CCO2H at a flow rate of 1 mL/min for 25 min.
Oven temperature was 37 °C. Detection wavelength was 220 nm.
Pseudo-first order rate constants for the consumption of the electrophilic
compound and the formation of the product, respectively, were obtained
from plotting the AUCs versus the incubation time, using equations of
the exponential decay and product formation, respectively. In case of 5a
and 6b, the concentration of the electrophilic compound did not approach
zero, and an offset value was implemented in the equation of the
exponential decay. From means of the so-obtained pseudo-first order rate
constants, half-lives were calculated.
34. To compound 5a (0.5 mmol) in THF (25 mL) N-acetylcysteine (2.5
mmol) and NEt3 (3.25 mmol) were added. The mixture was stirred at rt
for 7 days under argon atmosphere. After evaporation, the residue was
partitioned between EtOAc (30 mL) and sat. NaHCO3 (30 mL). The
aqueous layer was acidified (2 M HCl) and extracted with EtOAc (2 × 50
mL). The solvent was removed in vacuo and the residue was subjected to
column chromatography using petroleum ether/EtOAc (1+1) and
CH2Cl2/MeOH (9+1) with 1% AcOH each. A partly purified material
was obtained. LC-MS (ESI) (90% H2O to 100% MeOH in 10 min, then
100% MeOH for 10 min, DAD 220.0 – 400.0 nm): tr = 9.75 min, m/z =
506.4 ([M+H]+). This material (1 mg) was dissolved in 2 mL of the 9+1
buffer/MeCN mixture.33
A solution of 5a (0.25 mM) and N-
acetylcysteine (5 mM) in the buffer/MeCN mixture was incubated for 28
h at 37 °C. This solution was diluted (i) 1+1 with the buffer/MeCN
mixture and (ii) 1+1 with the aforementioned solution of the material.
Volumes of 20 µL each were injected into the HPLC. The second
chromatogram showed a single product peak of a significantly increased
AUC.
35. Reddick, J. J.; Cheng, J.; Roush, W. R. Org. Lett. 2003, 5, 1967.
36. Coffin, A. R.; Roussell, M. A.; Tserlin, E.; Pelkey, E. T. J. Org. Chem.
2006, 71, 6678.
37. Becht, J. M.; Meyer, O.; Helmchen, G. Synthesis 2003, 2805.
38. Frizler, M.; Lohr, F.; Lülsdorff, M.; Gütschow, M. Chem. Eur. J. 2011,
17, 11419.

Ten inhibitors of the phenyl vinyl sulfone or ethyl acrylate
chemotype were prepared.
The potency against four human cathepsins was examined.
The thiol reactivity under physiological conditions was
estimated.
Compound 6c was identified as a potent cathepsin S
inactivator.
39.