E-64c-hydrazide: A lead structure for the development of irreversible cathepsinc inhibitors

Article abstract of DOI:10.1002/cmdc.201300093

CathepsinC is a papain-like cysteine protease with dipeptidyl aminopeptidase activity that is thought to activate various granule-associated serine proteases. Its exopeptidase activity is structurally explained by the so-called exclusion domain, which blocks the active-site cleft beyond the S2 site and, with its Asp1 residue, provides an anchoring point for the Nterminus of peptide and protein substrates. Here, the hydrazide of (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane (E-64c) (k₂/K_i=140±5M^-1s^-1) is demonstrated to be a lead structure for the development of irreversible cathepsinC inhibitors. The distal amino group of the hydrazide moiety addresses the acidic Asp1 residue at the entrance of the S2 pocket by hydrogen bonding while also occupying the flat hydrophobic S1′-S2′ area with its leucine-isoamylamide moiety. Furthermore, structure-activity relationship studies revealed that functionalization of this distal amino group with alkyl residues can be used to occupy the conserved hydrophobic S2 pocket. In particular, the n-butyl derivative was identified as the most potent inhibitor of the series (k₂/K_i=56000±1700M^-1s^-1).

Full text of DOI:10.1002/cmdc.201300093

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DOI: 10.1002/cmdc.201300093
E-64c-Hydrazide: A Lead Structure for the Development of
Irreversible Cathepsin C Inhibitors
Hanna Radzey,[a, b] Markus Rethmeier,[a, c] Dennis Klimpel,[a] Maresa Grundhuber,[d]
Christian P. Sommerhoff,[d] and Norbert Schaschke*[a]
Cathepsin C is a papain-like cysteine protease with dipeptidyl
aminopeptidase activity that is thought to activate various
granule-associated serine proteases. Its exopeptidase activity is
structurally explained by the so-called exclusion domain, which
blocks the active-site cleft beyond the S2 site and, with its
Asp1 residue, provides an anchoring point for the N terminus
of peptide and protein substrates. Here, the hydrazide of
(2S,3S)-trans-epoxysuccinyl-l-leucylamido-3-methylbutane (E-
The distal amino group of the hydrazide moiety addresses the
acidic Asp1 residue at the entrance of the S2 pocket by hydro-
gen bonding while also occupying the flat hydrophobic S1’–
S2’ area with its leucine-isoamylamide moiety. Furthermore,
structure–activity relationship studies revealed that functionali-
zation of this distal amino group with alkyl residues can be
used to occupy the conserved hydrophobic S2 pocket. In par-
ticular, the n-butyl derivative was identified as the most potent
64c) (k2/Ki =140ꢀ5mꢁ1
s
ꢁ1) is demonstrated to be a lead struc-
inhibitor of the series (k2/Ki =56000ꢀ1700mꢁ1
s
ꢁ1).
ture for the development of irreversible cathepsin C inhibitors.
Introduction
Cathepsin C, also known as dipeptidyl peptidase I (DPPI), is
a cysteine protease that belongs to family C1A of clan CA
(papain superfamily) according to the classification scheme of
the MEROPS database.[1,2] Using both peptide hormones[3,4]
and synthetic peptides as substrates,[5,6] it has been well docu-
mented that this protease acts virtually exclusively as a dipep-
tidyl aminopeptidase with broad substrate specificity. Only few
restrictions have been identified, that is, substrates with lysine
and arginine in the P2 position, isoleucine and proline in the
P1 position, and proline in the P1’ position are not processed
by cathepsin C.
is composed of three chains; the light and heavy chains to-
gether form the catalytic domain with a papain-like fold and
harbor the residues of the catalytic diad (i.e. Cys234(25) and
His381(159); papain numbering given in brackets). The third
chain, derived from a portion of the propeptide, constitutes
the so-called exclusion domain that blocks the active-site cleft
binding sites beyond the S2 pocket and thus is responsible for
the exopeptidase activity of the protease. Furthermore, the
side chain carboxylic acid of the Asp1 residue of the exclusion
domain, which is located near the entrance of the S2 pocket,
acts as an anchoring point for the N-terminal a-amino function
of a peptide or protein substrate. The role of Asp1 governing
substrate recognition and processing has been confirmed by
the X-ray crystal structure of cathepsin C in complex with the
dipeptide substrate-based inhibitor H-Gly-Phe-CHN2.[8] Recently,
a detailed picture of the catalytic mechanism of substrate
cleavage by cathepsin C was elucidated by Schneck et al. by
applying a combination of pre-steady-state and steady-state ki-
netics and solvent kinetic isotope effects using the dipeptide
substrate l-seryl-l-tyrosine 7-amido-4-methylcoumarin (H-Ser-
Tyr-AMC).[9]
From a structural point of view, cathepsin C is unique
among the currently known eleven human cysteine cathepsins.
The X-ray crystal structure revealed that this protease consists
of four identical catalytically active subunits that assemble to
a tetramer and are located, with their active-site clefts fully sol-
vent exposed, at the corners of a tetrahedron.[7] Each subunit
[a] H. Radzey, M. Rethmeier, D. Klimpel, Priv.-Doz. Dr. N. Schaschke
Fakultꢀt fꢁr Chemie, Universitꢀt Bielefeld
Universitꢀtsstr. 25, 33615 Bielefeld (Germany)
E-mail: norbert.schaschke@uni-bielefeld.de
Cathepsin C is abundantly expressed in almost all mammali-
an tissues,[10,11] reflecting its nonspecific function in general ly-
sosomal protein turnover. In addition, knock-out studies have
implicated the protease in the activation of a variety of gran-
ule-associated serine proteases, among them the neutrophil-
derived proteases cathepsin G, elastase, and proteinase 3,[12]
the cytotoxic T lymphocyte-derived proteases granzyme A
and B,[13] as well as the mast-cell-derived proteases chymase
and tryptase.[14] Evidence that cathepsin C plays a role in the
activation of tryptase was also obtained using the human mast
cell line HMC-1 in combination with the selective irreversible
[b] H. Radzey
Institut fꢁr Organische und Biomolekulare Chemie
Georg-August-Universitꢀt Gçttingen
Tammannstr. 2, 37077 Gçttingen (Germany)
[c] M. Rethmeier
Fakultꢀt fꢁr Chemie und Biochemie
Ruhr-Universitꢀt Bochum
Universitꢀtsstr. 150, 44801 Bochum (Germany)
[d] M. Grundhuber, Prof. Dr. C. P. Sommerhoff
Institut fꢁr Laboratoriumsmedizin
Klinikum der Ludwig-Maximilians-Universitꢀt
Nußbaumstr. 20, 80336 Mꢁnchen (Germany)
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inhibitor H-Gly-Phe-CHN2.[15] Based on the well-established role
of granule-associated serine proteases in a variety of pro-in-
flammatory and immunologic processes, cathepsin C as their
activator represents a potential therapeutic target for the treat-
ment of related diseases, such as chronic obstructive pulmona-
ry disease (COPD), cystic fibrosis, rheumatoid arthritis, and al-
lergic asthma. This hypothesis is supported by the finding that
cathepsin C knock-out mice seem to be protected against the
detrimental actions of granule-associated serine proteases in
several disease-related mouse models.[12,16–18] On the other
hand, however, deficiency in cathepsin C due to a point muta-
tion in the gene causes Papillion–Lefꢁvre syndrome (PLS),
a rare genetic disease[19,20] characterized by periodontitis and
hyperkeratosis on hands and feet.
Most of the cathepsin C inhibitors investigated so far are
mimics of dipeptide substrates functionalized with an electro-
philic moiety addressing the catalytic residue Cys234 either in
a reversible or irreversible fashion.[21,22] Bondebjerg et al. have
extended this strategy for inhibitor design.[23] Thus, based on
a semicarbazide scaffold, the authors developed inhibitors that
allow for addressing the S1’ and S2’ binding pockets in addi-
tion to the S sites in a substrate-like fashion. Although (2S,3S)-
oxirane-2,3-dicarboxylic acid has been extensively used as
a thiol-reactive group for the development of irreversible cys-
teine cathepsin inhibitors,[24,25] until now, little is known about
cathepsin C inhibitors based on this privileged electrophile.
Here, we report the structure-based design, synthesis and eval-
uation of a series of (2S,3S)-oxirane-2,3-dicarboxylic acid hydra-
zide-based cathepsin C inhibitors. Moreover, the structure–
activity relationships of this novel class of inhibitors were
established.
Figure 1. a) Chemical structure of E-64c. The arrow indicates the position
where the active-site cysteine attacks the oxirane ring yielding the corre-
sponding thioether upon ring opening. b) Covalent binding mode of E-64c
along the active-site cleft of cathepsin B as determined by X-ray crystallogra-
phy (PDB code: 1ITO).[26] Cathepsin B is shown in a transparent Connolly sur-
face representation colored according to the electrostatic potential. E-64c
and the catalytic cysteine are shown in stick representation (color code: C:
gray, N: blue, and O: red; S: yellow; H atoms are omitted for clarity). Panel b)
was prepared using Accelrys DS Visualizer v1.7.
Results and Discussion
Figure 2. Schematic representation of the proposed covalent binding mode
of E-64c along the active-site cleft of cathepsin C.
E-64c-hydrazide as a lead structure for the development of
irreversible cathepsin C inhibitors
The binding mode of (2S,3S)-trans-epoxysuccinyl-l-leucylami-
do-3-methylbutane (E-64c) along the active-site cleft of cathe-
psin B was elucidated in detail by X-ray crystallography
(Figure 1).[26] The complex shows that the leucine residue of E-
64c addresses the affinity-generating hydrophobic S2 binding
pocket, which is highly conserved among the cysteine cathe-
psins, whereas the iso-amyl moiety interacts with the S3 bind-
ing pocket. In addition, the thiol-reactive epoxide moiety
modifies the catalytic cysteine in a covalent manner. Because
of the high degree of structural similarity, this binding mode is
thought to be conserved for most of the known cysteine cath-
epsins. In the case of the structurally unique cathepsin C, how-
ever, the S3 pocket is blocked by the exclusion domain and
thus E-64c is predicted to address the S’ sites (Figure 2). Ac-
cordingly, the leucine residue and the iso-amyl moiety should
interact with the S1’ and S2’ pockets, respectively, while leav-
ing the affinity-generating S2 pocket unoccupied. This binding
mode should result in a rather low second-order rate constant
for the inhibition of cathepsin C by E-64c. Indeed, our kinetic
measurements revealed that E-64c is a very weak irreversible
cathepsin C inhibitor with a k2/Ki value of only 23mꢁ1 sꢁ1
(Table 1). For the structurally related 1-[N-[(l-3-trans-carboxyox-
irane-2-carbonyl)-l-leucyl]amino]-4-guanidinobutane
(E-64),
a comparably low second-order rate constant for the inhibition
of cathepsin C has been reported by Barrett et al. (k2/Ki =
100mꢁ1 sꢁ1).[27] According to the proposed binding mode of E-
64c along the active-site cleft of cathepsin C, the carboxylic
acid group of the (2S,3S)-oxirane-2,3-dicarboxylic acid portion
should be positioned in near proximity to Asp1 of the exclu-
sion domain. Thus, this functional group should be suitable for
grafting a mimic of the substrate’s N terminus that addresses
Asp1 through hydrogen bonding. Using a structure-based ap-
proach, hydrazine was identified as an appropriate chemical
entity that fulfills the structural requirements to act as such
a mimic. To verify this concept, E-64c-hydrazide (1) was synthe-
sized as outlined in Scheme 1. Briefly, E-64c, synthesized essen-
tially following a procedure as described previously,[28] was
coupled with tert-butyloxycarbonyl (Boc)-protected hydrazine.
Upon cleavage of the protecting group using 95% aqueous tri-
fluoroacetic acid (TFA), E-64c-hydrazide (1) was obtained. The
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Synthesis of E-64c-hydrazide-
based cathepsin C inhibitors
To identify an optimal hydrazine-
based ligand for the S2 binding
pocket, a set of alkylhydrazine
building blocks with systemati-
cally varying steric demand was
synthesized. To allow for selec-
Scheme 1. Synthesis of E-64c-hydrazide (1). Reagents and conditions: a) Boc-NH-NH2·HCl, EDC, HOBt, DIEA, CHCl3,
3 h, RT; b) TFA/H2O (95:5, v/v), 0.5 h, RT, 22% over two steps.
second-order rate constant for the inhibition of cathepsin C by
1 is reported in Table 1. A comparison of the k2/Ki value of E-
64c (23mꢁ1 sꢁ1) with that of corresponding hydrazide
tive coupling of these S2 ligands with E-64c, Boc protection at
the alkylated hydrazine nitrogen was employed. In particular,
methylhydrazine could be directly Boc-protected following
a procedure described by Busnel et al. yielding 1-methylhydra-
zinecarboxylic acid tert-butyl ester (2, Scheme 2).[29] However,
1 (140mꢁ1 ꢁ1) reveals a sixfold improvement in inhibitory po-
s
tency induced by this modification. These findings are consis-
tent with the design concept and classify E-64c-hydrazide (1)
as a promising first-generation lead structure for the develop-
ment of irreversible cathepsin C inhibitors.
Structure-based considerations to improve the cathepsin C
inhibitory potency
According to the proposed binding mode, the distal nitrogen
of the hydrazide moiety addresses key residue Asp1 at the en-
trance of the S2 site but still leaves the important affinity-gen-
erating pocket unoccupied. Similarly, as elucidated by the X-
ray crystal structure of cathepsin C in complex with the dipep-
tide substrate-based inhibitor H-Gly-Phe-CHN2, the P2 glycine
does not properly address the S2 binding pocket.[8] Rather, in-
spection of the structure reveals that the hydrophobic pocket
is occupied by a series of structurally well-defined water mole-
cules.[8] Therefore, for both enthalpic and entropic reasons, it
was important to occupy this S2 pocket with an appropriate
hydrophobic moiety to improve the inhibitory potency of our
lead structure. This prompted us to build a model of E-64c-hy-
drazide (1) bound along the active-site cleft of cathepsin C
(Figure 3). Inspection of this model predicts that the distal ni-
trogen of the hydrazide moiety is suited for functionalization
with unbranched and branched alkyl residues in order to
occupy the S2 binding pocket.
Scheme 2. Synthesis of alkylhydrazine-based S2 ligand building block 2 and
7a–g. Reagents and conditions: a) Boc2O, CHCl3, 0.5 h at 08C, then 24 h at RT,
84%; b) ZHN-NH2, toluene, 2 h at 608C, then 24 h at RT, 72–99%; c) DIBAL,
THF, 2 h at ꢁ788C, then 2 h at ꢁ408C!ꢁ108C (aldimines) or NaBH4, THF,
2 h at RT (ketimine); d) Boc2O, NaOH, dioxane/H2O (1.6:1, v/v), 16 h, RT, 17–
87% over two steps; e) H2, 10% Pd/C, EtOH, 16 h, RT, 21–94%.
our attempts to extend this strategy on other alkylhydrazines
were not successful. Therefore, an alternative approach was
chosen. Essentially according to a route described by Bailey
et al.,[30] benzyloxy-carbonyl (Z)-protected hydrazine was first
reacted with selected aldehydes 3a–e,g and ketone 3 f
(Scheme 2). Next, a reductive amination was performed on the
imine intermediates using diisobutylaluminium hydride (DIBAL)
for aldimines 4a–e,g and sodium borohydride for ketimine 4 f.
Subsequent Boc protection and cleavage of the Z protecting
group yielded a set of structurally diverse 1-alkylhydrazinecar-
Figure 3. Model of the covalent binding mode of E-64c-hydrazide (1) along
the active-site cleft of a subunit of the cathepsin C tetramer. The cathepsin C
subunit is shown in a transparent Connolly surface representation colored
according to the electrostatic potential. E-64c-hydrazide (1), Asp1, and
Cys234 are shown in stick representation (color code: C: gray, N: blue, and
O: red; S: yellow; H atoms are omitted for clarity). Figure 3 was prepared
using Accelrys DS Visualizer v1.7.
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cathepsins, cathepsin L was selected for initial selectivity stud-
ies. The second-order rate constants for the inhibition of cathe-
psin L by E-64c, lead compound 1, and optimized inhibitor 11
were determined (Table 2). Comparison of the k2/Ki value of E-
64c with that of inhibitor 11 clearly reveals that the 2400-fold
increase in affinity for cathepsin C is simultaneously accompa-
nied by a 24-fold loss in affinity for cathepsin L. Thus, imple-
mentation of the n-butylhydrazine-based S2 ligand in E-64c
has shifted the inhibitory profile from a virtually selective cath-
epsin L inhibitor into a remarkable potent cathepsin C inhibitor
with threefold selectivity for cathepsin C.
Scheme 3. Synthesis of E-64c-hydrazide-based cathepsin C inhibitors. Re-
agents and conditions: a) E-64c, TBTU, DIEA, CH2Cl2, 3 h, RT; b) TFA/H2O (95:5,
v/v), 0.5 h, RT, 19–83% over two steps.
Table 2. Affinity/selectivity profile of inhibitor 11.
[a]
Compd
k2/Ki [mꢁ1 sꢁ1
]
boxylic acid tert-butyl esters (7a–g). Finally, E-64c was coupled
with the S2 ligand building blocks, yielding upon cleavage of
the Boc group E-64c-hydrazide-based cathepsin C inhibitors 8–
15 (Scheme 3).
Cathepsin C
Cathepsin L
11
1
E-64c
56000ꢀ1700
140ꢀ5
17000ꢀ600
33000ꢀ800
23ꢀ1
410000ꢀ16000
[a] Data represent the mean ꢀ standard error (SE), n ꢂ10.
Inhibitory profiles of E-64c-alkylhydrazide-based cathepsin
C inhibitors
The second-order rate constants for the inhibition of cathe-
psin C by E-64c-alkylhydrazides 8–15 are summarized in
Table 1. First, a comparison of the k2/Ki values of these inhibi-
tors with lead compound 1 reveals that almost all of them ex-
hibit improved inhibitory potency. This finding is in full agree-
Conclusions
Using a structure-based approach, we have identified E-64c-hy-
drazide (1) as a lead compound for the development of irrever-
sible cathepsin C inhibitors. Furthermore, a focused structure–
activity relationship study revealed that functionalization of
the distal nitrogen of the hydrazide moiety with a n-butyl resi-
due allows the addressing of the affinity-generating hydropho-
bic S2 pocket in an optimal manner. The resulting inhibitor
(11), which in comparison to lead compound 1 has a superior
affinity/selectivity profile, is currently in use as a starting point
for the identification of optimized S1’–S2’ ligands.
Table 1. Inhibition of cathepsin C by E-64c and E-64c-hydrazides.
[a]
[a]
Compd
k2/Ki [mꢁ1 sꢁ1
]
Compd
k2/Ki [mꢁ1 sꢁ1
]
E-64c
1
8
9
23ꢀ1
140ꢀ5
50ꢀ1
11
12
13
14
15
56000ꢀ1700
29000ꢀ1000
1300ꢀ70
2100ꢀ50
9600ꢀ200
10
23000ꢀ600
21000ꢀ400
[a] Data represent the mean ꢀ standard error (SE), n ꢂ10.
Experimental Section
Solvents and reagents were of the highest purity commercially
available and were used without further purification. 1-Ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDC), N,N,N’,N’-tetramethyl-O-
(benzotriazol-1-yl)uronium tetrafluoroborate (TBTU), and hydroxy-
benzotriazole (HOBt) were purchased from Iris Biotech (Marktred-
witz, Germany), and methylhydrazine, hydrazinecarboxylic acid tert-
butyl ester, hydrazinecarboxylic acid benzyl ester, and di-tert-butyl
dicarbonate (Boc2O) from Sigma–Aldrich. E-64c was synthesized fol-
lowing a procedure described previously,[28] and 1-methylhydra-
zine-carboxylic acid tert-butyl ester (2) was prepared as described
by Busnel et al.[29] Precipitated compounds were collected by cen-
trifugation using a Labofuge I from Heraeus Christ GmbH (Hanau,
Germany).
ment with the applied structure-based approach to augment
the potency of lead compound 1 by addressing the hydropho-
bic S2 pocket in a proper manner (see above). Furthermore,
the obtained data show a clear structure–activity relationship
for the investigated S2–P2 interaction. In particular, starting
from inhibitor 8 (Me residue, k2/Ki =52mꢁ1 ꢁ1), the inhibitory
s
potency increase significantly with increasing length of the n-
alkyl chain reaching its maximum with inhibitor 11 (n-Bu resi-
s
due, k2/Ki =56000mꢁ1 ꢁ1). Further elongation of the n-alkyl
chain (12 and 13), however, is accompanied with a loss in in-
hibitory potency. Moreover, comparison of the inhibitor pairs
10 (n-Pr) and 14 (i-Pr) as well as 11 (n-Bu) and 15 (i-Bu), respec-
tively, reveals that unbranched alkyl residues are superior to
the structurally related branched analogues as S2 ligands.
In order to assess the effects of these modifications to E-64c
on the inhibitory profile within the group of human cysteine
1H NMR spectra were recorded on a DRX 500 (500 MHz) or an
Avance 600 (600 MHz) spectrometer from Bruker. Electrospray ioni-
zation (ESI) mass spectrometry (MS) was performed on a Bruker Es-
quire 3000 quadrupole ion trap mass spectrometer, high-resolution
mass spectrometry (HRMS) was performed on a APEX III FT-ICR
(MALDI) or a micrOTOF (ESI) mass spectrometer from Bruker Dal-
tonics. Thin layer chromatography (TLC) was performed on silica
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gel 60 plates with fluorescence indicator F254, and flash chromatog-
raphy was carried out using silica gel 60 (230–400 mesh) both from
VWR International GmbH (Darmstadt, Germany). The compounds
on the TLC plates were visualized with UV light (l=254 nm) using
a MinUVIS lamp from Desaga (Heidelberg, Germany) with ninhy-
drin (hydrazines) or with 4-(4-nitrobenzyl)pyridine (oxiranes).[31]
2-(2-Methylpropyliden)hydrazinecarboxylic acid benzyl ester
(4g): Colorless solid (3.42 g, 99%); 1H NMR (500 MHz, CDCl3): d=
1.07 (d, J=6.8 Hz, 6H, 2ꢂCH3), 2.57 (m, 1H, CH), 5.20 (br s, 2H,
OCH2), 7.01 (br m, 1H, N=CH), 7.33 (m, 5H, aryl-H), 7.93 ppm (br s,
1H, NH), exclusively the E isomer is visible; MS (ESI): m/z (%): 243.0
[M+Na]+ (66).
Analytical reverse phase (RP)-HPLC was performed with a system
from Thermo Separation Products using a column from Macherey–
Nagel (type ET 125/4 Nucleosil 100-5 C18 PPN, column size 125ꢂ
4 mm) and eluting at a flow rate of 1.5 mLminꢁ1 with the gradient:
0–1 min, isocratic A/B (100:0); 1–14 min, linear gradient from A/B
(100:0) to A/B (0:100) (eluent A: H2O/MeCN/TFA, 95:5:0.1 v/v/v,
eluent B: MeCN/H2O/TFA, 95:5:0.1 v/v/v). Preparative RP-HPLC was
performed with a LaChrome Type HPLC system from Merck Hitachi
using a column from Phenomenex (Type Jupiter 10 mm, C18,
300 ꢃ, 250ꢂ21.20 mm), eluting at a flow rate of 10 mLminꢁ1 with
the following gradient: 0–5 min, isocratic A/B (100:0); 5–50 min,
linear gradient from A/B (100:0) to A/B (50:50) (eluent A: H2O/
MeCN/TFA, 95:5:0.1 v/v/v, eluent B: MeCN/H2O/TFA, 95:5:0.1 v/v/v).
1-Alkylhydrazine-1,2-dicarboxylic acid 2-benzyl 1-tert-butyl esters
6a–g were synthesized according to the procedure described by
Bailey et al.[30] In the case of ketimine 4 f, NaBH4 was used instead
of DIBAL for the reduction.
1-Ethylhydrazine-1,2-dicarboxylic acid 2-benzyl 1-tert-butyl ester
(6a): Colorless oil (1.15 g, 37% over two steps); Rf =0.8 (n-hexane/
1
EtOAc 2:1, v/v); H NMR (500 MHz, CDCl3): d=1.13 (br t, J=6.8 Hz,
3H, CH3), 1.41 (br s, 9H, C(CH3)3), 3.51 (br s, 2H, NCH2), 5.16 (s, 2H,
OCH2), 6.36 (br s, 1H, NH, Z isomer), 6.59 (br s, 1H, NH, E isomer),
7.34 ppm (m, 5H, aryl-H); MS (ESI): m/z (%): 317.1 [M+Na]+ (100).
1-Propylhydrazine-1,2-dicarboxylic acid 2-benzyl 1-tert-butyl
ester (6b): Colorless oil (1.31 g, 29% over two steps); Rf =0.8 (n-
1
hexane/EtOAc 3:1, v/v); H NMR (500 MHz, CDCl3): d=0.88 (m, 3H,
CH3), 1.40 (br s, 9H, C(CH3)3), 1.56 (m, 2H, CH2CH3), 3.42 (m, 2H,
NCH2), 5.15 (s, 2H, OCH2), 6.31 (br s, 1H, NH, Z isomer), 6.54 (br s,
1H, NH, E isomer), 7.34 ppm (m, 5H, aryl-H); MS (ESI): m/z (%):
331.2 [M+Na]+ (100).
Synthesis
2-Alkylidenhydrazinecarboxylic acid benzyl esters 4a–g were syn-
thesized according to the procedure described by Bailey et al.[30]
and used without further purification in the next step.
1-Butylhydrazine-1,2-dicarboxylic acid 2-benzyl 1-tert-butyl
ester (6c): Colorless oil (935 mg, 23% over two steps); Rf =0.6 (n-
1
2-Ethylidenhydrazinecarboxylic acid benzyl ester (4a): Colorless
solid (2.27 g, 98%); 1H NMR (500 MHz, CDCl3): d=1.77 (d, J=
5.6 Hz, 3H, CH3, Z isomer), 1.95 (d, J=5.3 Hz, 3H, CH3, E isomer),
5.20 (br s, 2H, OCH2), 6.80 (br s, 1H, N=CH, Z isomer), 7.17 (br s,
1H, N=CH, E isomer), 7.34 (m, 5H, aryl-H), 8.05 ppm (br s, 1H, NH),
E/Z ratio: 70:30; MS (ESI): m/z (%): 214.9 [M+Na]+ (100).
hexane/EtOAc 3:1, v/v); H NMR (500 MHz, CDCl3): d=0.90 (m, 3H,
CH3), 1.26–1.56 (m, 13H, C(CH3)3, CH2CH2), 3.45 (m, 2H, NCH2), 5.15
(s, 2H, OCH2), 6.32 (br s, 1H, NH, Z isomer), 6.54 (br s, 1H, NH, E
isomer), 7.34 ppm (m, 5H, ArH); MS (ESI): m/z (%): 345.2 [M+Na]+
(83).
1-Pentylhydrazine-1,2-dicarboxylic acid 2-benzyl 1-tert-butyl
ester (6d): Colorless oil (578 mg, 24% over two steps); Rf =0.6 (n-
hexane/EtOAc 3:1, v/v); 1H NMR (500 MHz, CDCl3): d=0.83 (t, J=
6.7 Hz, 3H, CH3), 1.21–1.54 (m, 15H,C(CH3)3, CH2CH2CH2), 3.40 (s,
2H, NCH2), 5.11 (s, 2H, OCH2), 6.32 (br s, 1H, NH, Z isomer), 6.53 (s,
1H, NH, E isomer), 7.30 ppm (m, 5H, aryl-H); MS (ESI): m/z (%):
359.2 [M+Na]+ (68).
2-Propylidenhydrazinecarboxylic acid benzyl ester (4b): Color-
less solid (3.57 g, 72%); 1H NMR (500 MHz, CDCl3): d=1.06 (t, J=
7.6 Hz, 3H, CH3), 2.28 (m, 2H, CH2), 5.19 (br s, 2H, OCH2), 7.15 (br
m, 1H, N=CH), 7.32 (m, 5H, aryl-H), 8.13 ppm (br s, 1H, NH), data
given are for the E isomer (E/Z ratio: 81:19); MS (ESI): m/z (%):
229.0 [M+Na]+ (100).
2-Butylidenhydrazinecarboxylic acid benzyl ester (4c): Colorless
solid (3.38 g, 98%); H NMR (500 MHz, CDCl3): d=0.93 (t, J=7.5 Hz,
3H, CH3), 1.50 (m, 2H, CH2CH3), 2.25 (m, 2H, N=CHCH2), 5.20 (br s,
2H, OCH2), 7.12 (br s, 1H, N=CH), 7.33 (m, 5H, aryl-H), 7.93 ppm
(br s, 1H, NH), data given are for the E isomer (E/Z ratio: 79:21);
MS (ESI): m/z (%): 243.0 [M+Na]+ (39).
1-Hexylhydrazine-1,2-dicarboxylic acid 2-benzyl 1-tert-butyl
ester (6e): Colorless oil (960 mg, 87% over two steps); Rf =0.5 (n-
hexane/EtOAc 3:1, v/v); H NMR (500 MHz, CDCl3): d=0.84 (m, 3H,
CH3), 1.18–1.52 (m, 17H,C(CH3)3, CH2CH2CH2CH2), 3.42 (s, 2H, NCH2),
5.13 (s, 2H, OCH2), 6.52–6.92 (br m, 1H, NH, E and Z isomer),
7.32 ppm (m, 5H, aryl-H); MS (ESI): m/z (%): 373.2 [M+Na]+ (18).
1
1
2-Pentylidenhydrazinecarboxylic acid benzyl ester (4d): Pale
yellow solid (3.68 g, 83%); H NMR (500 MHz, CDCl3): d=0.82 (t, J=
7.3 Hz, 3H, CH3), 1.25 (m, 2H, CH2), 1.43 (m, 2H, CH2), 2.24 (m, 2H,
N=CHCH2), 5.17 (br s, 2H, OCH2), 7.14 (br m, 1H, N=CH), 7.31 (m,
5H, aryl-H), 8.30 ppm (br s, 1H, NH), data given are for the E
isomer (E/Z ratio: 81:19); MS (ESI): m/z (%): 257.1 [M+Na]+ (100).
1
1-Isopropylhydrazine-1,2-dicarboxylic acid 2-benzyl 1-tert-butyl
ester (6 f): Colorless oil (769 mg, 17% over two steps); Rf =0.7 (n-
hexane/EtOAc 3:1, v/v); 1H NMR (500 MHz, CDCl3): d=1.10 (br s,
6H, CH3), 1.40 (br s, 9H, C(CH3)3), 4.36 (br s, 1H, NCH), 5.15 (s, 2H,
OCH2), 6.04 (br s, 1H, NH, Z isomer), 6.26 (br s, 1H, NH, E isomer),
7.35 ppm (m, 5H, aryl-H); MS (ESI): m/z (%): 331.2 [M+Na]+ (100).
2-Hexylidenhydrazinecarboxylic acid benzyl ester (4e): Colorless
solid (7.20 g, 99%); H NMR (500 MHz, CDCl3): d=0.84 (t, J=6.7 Hz,
3H, CH3), 1.29 (m, 4H, 2ꢂCH2), 1.47 (m, 2H, CH2), 2.27 (m, 2H,N=
CHCH2), 5.20 (br s, 2H, OCH2), 7.14 (br m, 1H, N=CH), 7.33 (m, 5H,
aryl-H), 7.99 ppm (br s, 1H, NH), data given are for the E isomer (E/
Z ratio: 79:21); MS (ESI): m/z (%): 271.1 [M+Na]+ (100).
1
1-(2-Methylpropyl)hydrazine-1,2-dicarboxylic acid 2-benzyl 1-
tert-butyl ester (6g): Colorless oil (3.26 g, 66% over two steps);
Rf =0.7 (n-hexane/EtOAc 3:1, v/v); 1H NMR (500 MHz, CDCl3): d=
0.88 (m, 6H, CH3), 1.32–1.53 (br m, 9H, C(CH3)3, E and Z isomer),
1.88 (m, 1H, CH), 3.27 (br s, 2H, NCH2), 5.15 (s, 2H, OCH2), 6.24–
6.70 (br m, 1H, NH, E and Z isomer), 7.34 ppm (m, 5H, aryl-H); MS
(ESI): m/z (%): 345.2 [M+Na]+ (100).
2-(Propan-2-yliden)hydrazinecarboxylic acid benzyl ester (4 f):
1
Colorless solid (3.21 g, 99%); H NMR (500 MHz, CDCl3): d=1.78 (s,
3H, CH3), 2.03 (s, 3H, CH3), 5.22 (s, 2H, OCH2), 7.35 (m, 5H, aryl-H),
7.62 ppm (br s, 1H, NH); MS (ESI): m/z (%): 207.0 [M+H]+ (73).
1-Alkylhydrazinecarboxylic acid tert-butyl esters 7a–g were synthe-
sized according to the procedure described by Bailey et al.[30] When
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necessary, Boc-protected alkylhydrazines were purified by flash
chromatography after cleavage of the Z protecting group.
100 mL). The obtained precipitate was collected by centrifugation,
washed with petroleum ether (3ꢂ10 mL), and dried in vacuo. If
necessary, the inhibitor was further purified by RP-HPLC.
1-Ethylhydrazinecarboxylic acid tert-butyl ester (7a): Colorless oil
(287 mg, 66%); Rf =0.4 (n-hexane/EtOAc 3:1, v/v); 1H NMR
(500 MHz, CDCl3): d=1.10 (t, J=7.1 Hz, 3H, CH3), 1.45 (s, 9H,
C(CH3)3), 3.38 (q, J=7.1 Hz, 2H, NCH2), 3.69 ppm (br s, 2H, NH2);
MS (ESI): m/z (%): 182.9 [M+Na]+ (61).
(2S,3S)-3-(Hydrazinecarbonyl)-N-((S)-1-(isopentylamino)-4-
methyl-1-oxopentan-2-yl)oxirane-2-carboxamide (1): Colorless
solid (21 mg, 22% over two steps); HPLC: tR =6.9 min; 1H NMR
(500 MHz, [D6]DMSO): d=0.80–0.91 (m, 12H, d-CH3 Leu, d-CH3 i-
amyl), 1.27 (m, J=5 Hz, 2H, b-CH2 i-amyl), 1.45 (m, 2H, b-CH2 Leu),
1.54 (m, 2H, g-CH Leu, g-CH i-amyl), 3.01 (m, 1H, a-CH2 i-amyl),
3.07 (m, 1H, a-CH2 i-amyl), 3.60 (m, 1H, CH oxirane), 3.71 (m, 1H,
CH oxirane), 4.30 (m, 1H, a-CH Leu), 8.04 (t, J=5.6 Hz, 1H, NH i-
amyl), 8.63 ppm (d, J=8.2 Hz, 1H, NH Leu), signals for NHNH2 are
not visible; HRMS (MALDI): m/z [2M+Na]+ calcd for
(C15H28N4O4)2Na: 679.41133, found: 679.41196.
1-Propylhydrazinecarboxylic acid tert-butyl ester (7b): Colorless
oil (204 mg, 79%); Rf =0.4 (n-hexane/EtOAc 3:1, v/v); 1H NMR
(500 MHz, CDCl3): d=0.85 (t, J=7.4 Hz, 3H, CH3), 1.45 (s, 9H,
C(CH3)3), 1.56 (m, 2H, CH2CH3), 3.30 (t, J=7.1 Hz, 2H, NCH2),
3.90 ppm (br s, 2H, NH2); MS (ESI): m/z (%): 197.0 [M+Na]+ (100).
Data are in agreement with that reported by Park et al.[32]
1-Butylhydrazinecarboxylic acid tert-butyl ester (7c): Colorless oil
(288 mg, 82%); Rf =0.3 (n-hexane/EtOAc 3:1, v/v); 1H NMR
(500 MHz, CDCl3): d=0.91 (t, J=7.4 Hz, 3H, CH3), 1.28 (m, 2H,
CH2CH2CH3), 1.45 (s, 9H, C(CH3)3), 1.52 (m, 2H, CH2CH2CH3), 3.34 (t,
J=7.2 Hz, 2H, NCH2), 3.94 ppm (br s, 2H, NH2); MS (ESI): m/z (%):
211.1 [M+Na]+ (79).
(2S,3S)-N-((S)-1-(Isopentylamino)-4-methyl-1-oxopentan-2-yl)-3-
(2-methylhydrazinecarbonyl)oxirane-2-carboxamide (8): Colorless
solid (79 mg, 60% over two steps); HPLC: tR =6.9 min; 1H NMR
(500 MHz, [D6]DMSO): d=0.82–0.91 (m, 12H, d-CH3 Leu, d-CH3 i-
amyl), 1.26 (m, 2H, b-CH2 i-amyl), 1.44 (m, 2H, b-CH2 Leu), 1.54 (m,
2H, g-CH Leu, g-CH i-amyl), 2.62 (s, 3H, NHCH3), 3.01 (m, 1H, a-CH2
i-amyl), 3.07 (m, 1H, a-CH2 i-amyl), 3.54 (d, J=1.5 Hz, 1H, CH oxir-
ane), 3.71 (d, J=1.6 Hz, 1H, CH oxirane), 4.30 (m, 1H, a-CH Leu),
8.05 (t, J=5.5 Hz, 1H, NH i-amyl), 8.62 (d, J=8.4 Hz, 1H, NH Leu),
10.86 ppm (br s, 1H, NHNHCH3), signal for NHNHCH3 is not visible;
HRMS (MALDI): m/z [M+Na]+ calcd for C16H30N4O4Na: 365.21593,
found: 365.21631.
1-Pentylhydrazinecarboxylic acid tert-butyl ester (7d): Colorless
oil (212 mg, 94%); Rf =0.3 (petroleum ether/EtOAc 5:1, v/v);
1H NMR (500 MHz, CDCl3): d=0.87 (t, J=7.2 Hz, 3H, CH3), 1.22 (m,
2H, CH2CH2CH2CH3), 1.30 (m, 2H, CH2CH2CH2CH3), 1.44 (s, 9H,
C(CH3)3), 1.53 (m, 2H, CH2CH2CH2CH3), 3.32 (t, J=7.2 Hz, 2H, NCH2),
3.88 ppm (br s, 2H, NH2); MS (ESI): m/z (%): 225.0 [M+Na]+ (7).
(2S,3S)-3-(2-Ethylhydrazinecarbonyl)-N-((S)-1-(isopentylamino)-4-
methyl-1-oxopentan-2-yl)oxirane-2-carboxamide (9): Colorless
solid (95 mg, 83% over two steps); HPLC: tR =6.9 min; 1H NMR
(500 MHz, [D6]DMSO): d=0.82–0.91 (m, 12H, d-CH3 Leu, d-CH3 i-
amyl), 1.11 (t, J=7.2 Hz, b-CH3 Et), 1.27 (m, 2H, b-CH2 i-amyl), 1.44
(m, 2H, b-CH2 Leu), 1.52 (m, 2H,g-CH Leu, g-CH i-amyl), 2.96–3.13
(m, 4H, a-CH2 i-amyl, a-CH2 Et), 3.63 (d, J=1.5 Hz, 1H, CH oxirane),
3.74 (d, J=1.5 Hz, 1H, CH oxirane), 4.31 (m, 1H, a-CH Leu), 8.07 (t,
J=5.5 Hz, 1H, NH i-amyl), 8.68 (d, J=8.4 Hz, 1H, NH Leu),
11.30 ppm (br s, 1H, NHNHCH2), signal for NHNHCH2 is not visible;
HRMS (MALDI): m/z [M+Na]+ calcd for C17H32N4O4Na: 379.23158,
found: 379.23138.
1-Hexylhydrazinecarboxylic acid tert-butyl ester (7e): Colorless
oil (142 mg, 29%); Rf =0.3 (petroleum ether/EtOAc 5:1, v/v);
1H NMR (500 MHz, CDCl3): d=0.87 (t, J=6.8 Hz, 3H, CH3), 1.28 (m,
6H, CH2CH2CH2CH2CH3), 1.45 (s, 9H,C(CH3)3, E isomer), 1.48 (s,
9H,C(CH3)3, Z isomer), 1.64 (m, 2H, CH2CH2CH2CH2CH3), 3.50 ppm (t,
J=7.5 Hz, 2H, NCH2), signal for NH2 not visible; MS (ESI): m/z (%):
239.1 [M+Na]+ (58).
1-Isopropylhydrazinecarboxylic acid tert-butyl ester (7 f): Color-
1
less oil (179 mg, 63%); Rf =0.6 (n-hexane/EtOAc 4:1, v/v); H NMR
(500 MHz, CDCl3): d=1.09 (d, J=6.6 Hz, 6H, CH3), 1.45 (s, 9H,
C(CH3)3), 3.49 (br s, 2H, NH2), 4.19 ppm (br m, 1H, CH); MS (ESI):
m/z (%): 197.0 [M+Na]+ (55).
(2S,3S)-N-((S)-1-(Isopentylamino)-4-methyl-1-oxopentan-2-yl)-3-
(2-propylhydrazinecarbonyl)oxirane-2-carboxamide (10): Color-
1-(2-Methylpropyl)hydrazinecarboxylic acid tert-butyl ester (7g):
Colorless oil (124 mg, 21%); Rf =0.3 (n-hexane/EtOAc 5:1, v/v);
1H NMR (500 MHz, CDCl3): d=0.85 (d, J=6.7 Hz, 6H, C(CH3)2), 1.44
(s, 9H, C(CH3)3), 1.97 (m, 1H, CH), 3.15 (d, J=7.3 Hz, 2H, NCH2),
3.94 ppm (br s, 2H, NH2); MS (ESI): m/z (%): 211.0 [M+Na]+ (100).
1
less solid (86 mg, 80% over two steps); HPLC: tR =7.5 min; H NMR
(500 MHz, [D6]DMSO): d=0.81–0.92 (m, 15H, d-CH3 Leu, d-CH3 i-
amyl, g-CH3 n-Pr), 1.27 (m, 2H, b-CH2 i-amyl), 1.44 (m, 2H, b-CH2
Leu), 1.53 (m, 4H, g-CH Leu, g-CH i-amyl, b-CH2 n-Pr), 2.91 (m, 2H,
a-CH2 n-Pr), 3.01 (m, 1H, a-CH2 i-amyl), 3.07 (m, 1H, a-CH2 i-amyl),
3.60 (d, J=1.5 Hz, 1H, CH oxirane), 3.73 (d, J=1.5 Hz, 1H, CH oxir-
ane), 4.31 (m, 1H, a-CH Leu), 8.06 (t, J=5.5 Hz, 1H, NH i-amyl),
8.65 (d, J=8.5 Hz, 1H, NH Leu), 11.11 ppm (br s, 1H, NHNHCH2),
signal for NHNHCH2 is not visible; HRMS (MALDI): m/z [M+H]+
calcd for C18H35N4O4: 371.26528, found: 371.26567.
General procedure for the synthesis of inhibitors 1, 8–15: A solu-
tion of E-64c (75 mg, 0.24 mmol), TBTU (77 mg, 0.24 mmol), and
DIPEA (41 mL, 0.24 mmol) in CH2Cl2 (5 mL) was treated with hydrazi-
necarboxylic acid tert-butyl ester (0.36 mmol, 1.5 equiv) dissolved
in CH2Cl2 (2 mL). In the case of inhibitor 1, EDC and HOBt were
used instead of TBTU for the coupling step. The solution was
stirred at RT for 3 h. Then, the solvent was removed in vacuo, and
the obtained residue distributed between EtOAc (30 mL) and H2O
(10 mL). The resulting organic phase was washed with 5% aq
KHSO4 (3ꢂ10 mL), 5% aq NaHCO3 (5ꢂ10 mL), and brine (1ꢂ
10 mL), and then dried (Na2SO4), and concentrated in vacuo. The
protected inhibitor was isolated as a colorless powder by precipita-
tion from EtOAc/n-hexane and subjected to deprotection without
further purification. For this, the protected inhibitor was dissolved
at RT in TFA/H2O (95:5, v/v; 2 mL). After 30 min, the inhibitor was
isolated as the TFA salt by adding the cleavage solution dropwise
to ice-cold tert-butyl methyl ether/petroleum ether (1:2, v/v;
(2S,3S)-3-(2-Butylhydrazinecarbonyl)-N-((S)-1-(isopentylamino)-4-
methyl-1-oxopentan-2-yl)oxirane-2-carboxamide (11): Colorless
solid (67 mg, 57% over two steps); HPLC: tR =8.0 min; 1H NMR
(500 MHz, [D6]DMSO): d=0.81–0.92 (m, 15H, d-CH3 Leu, d-CH3 i-
amyl, d-CH3 nBu), 1.27 (m, 2H, b-CH2 i-amyl), 1.31 (m, 2H, g-CH2
nBu), 1.45 (m, 4H, b-CH2 Leu, b-CH2 nBu), 1.54 (m, 2H,g-CH Leu, g-
CH i-amyl), 2.87 (m, 2H, a-CH2 nBu), 3.01 (m, 1H,a-CH2 i-amyl), 3.07
(m, 1H, a-CH2 i-amyl), 3.56 (d, J=1.8 Hz, 1H, CH oxirane), 3.71 (d,
J=1.8 Hz, 1H, CH oxirane), 4.30 (m, 1H, a-CH Leu), 8.05 (t, J=
5.6 Hz, 1H, NH i-amyl), 8.63 (d, J=8.4 Hz, 1H, NH Leu), 10.79 ppm
(br s, 1H, NHNHCH2), signal for NHNHCH2 is not visible; HRMS
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(MALDI): m/z [M+Na]+ calcd for C19H36N4O4Na: 407.26288, found:
407.26279.
at excitation and emission wavelengths of 380 and 460 nm, respec-
tively. After 5–10 min, that is, when a constant rate was estab-
lished, test inhibitor was added, and the reaction was followed for
up to 60 min. The pseudo-first-order rate constant (kobs) was ob-
tained by fitting the observed pre-steady-state progress curve to
the integrated equation of Morrison[33,34] with pro-Fit (Quantum-
Soft, Uetikon am See, Switzerland) using a Levenberg–Marquardt
algorithm. Apparent second-order rate constants were subsequent-
ly calculated as k2/Ki =kobs/It from several independent experiments
by linear regression and corrected for substrate competition.
(2S,3S)-N-((S)-1-(Isopentylamino)-4-methyl-1-oxopentan-2-yl)-3-
(2-pentylhydrazinecarbonyl)oxirane-2-carboxamide (12): Color-
less lyophilisate (19 mg, 19% over two steps); HPLC: tR =8.5 min;
1H NMR (500 MHz, [D6]DMSO): d=0.81–0.90 (m, 15H, d-CH3 Leu, d-
CH3 i-amyl, e-CH3 n-Pn), 1.21–1.31 (m, 6H, b-CH2 i-amyl, g-CH2 n-Pn,
d-CH2 n-Pn), 1.36–1.48 (m, 4H, b-CH2 Leu, b-CH2 n-Pn), 1.54 (m,
2H,g-CH Leu, g-CH i-amyl), 2.72 (t, J=7.1 Hz, 2H, a-CH2 n-Pn),
2.96–3.12 (m, 2H, a-CH2 i-amyl), 3.48 (d, J=1.8 Hz, 1H, CH oxirane),
3.67 (d, J=1.8 Hz, 1H, CH oxirane), 4.29 (m, 1H, a-CH Leu), 8.03 (t,
J=5.6 Hz, 1H, NH i-amyl), 8.59 (d, J=8.4 Hz, 1H, NH Leu),
10.13 ppm (s, 1H, NHNHCH2), signal for NHNHCH2 is not visible;
HRMS (ESI): m/z [M+H]+ calcd for C20H39N4O4: 399.29710, found:
399.2966.
Model of E-64c-hydrazide (1) bound along the active-site cleft
of cathepsin C
Using HyperChem (release 8.0.6), the X-ray crystal structures of
cathepsin C in complex with the peptide-derived inhibitor H-Gly-
Phe-CHN2 (PDB code: 2DJF)[8] and the E-64-derived inhibitor NS-
134 in complex with cathepsin B (PDB code: 1SP4)[35] were super-
imposed based on the conserved residues of the catalytic diad
(Cys25 and His159; papain numbering). Upon removal of H-Gly-
Phe-CHN2, E-64c-hydrazide (1) was positioned along the active-site
cleft of cathepsin C utilizing NS-134 as a molecular scaffold. Finally,
the geometry of the covalently bound E-64-hydrazide (1) was opti-
mized using an AMBER force field (algorithm: steepest descent).
(2S,3S)-3-(2-Hexylhydrazinecarbonyl)-N-((S)-1-(isopentylamino)-
4-methyl-1-oxopentan-2-yl)oxirane-2-carboxamide (13): Colorless
lyophilisate (36 mg, 38% over two steps); HPLC: tR =9.2 min;
1H NMR (500 MHz, [D6]DMSO): d=0.81–0.91 (m, 15H, d-CH3 Leu, d-
CH3 i-amyl, x-CH3 n-Hex), 1.20–1.32 (m, 8H, b-CH2 i-amyl, g-CH2 n-
Hex, d-CH2 n-Hex, e-CH2 n-Hex), 1.37–1.49 (m, 4H,b-CH2 Leu, b-CH2
n-Hex), 1.54 (m, 2H, g-CH Leu, g-CH i-amyl), 2.74 (m, 2H, a-CH2 n-
Hex), 2.97–3.12 (m, 2H, a-CH2 i-amyl), 3.49 (d, J=1.5 Hz, 1H, CH
oxirane), 3.67 (d, J=1.6 Hz, 1H, CH oxirane), 4.30 (m, 1H, a-CH
Leu), 8.02 (t, J=5.5 Hz, 1H, NH i-amyl), 8.58 (d, J=8.4 Hz, 1H, NH
Leu), 10.21 ppm (s, 1H, NHNHCH2), signal for NHNHCH2 is not visi-
ble; HRMS (ESI): m/z [M+H]+ calcd for C21H41N4O4: 413.31275,
found: 413.3124.
Acknowledgements
(2S,3S)-N-((S)-1-(Isopentylamino)-4-methyl-1-oxopentan-2-yl)-3-
(2-isopropylhydrazinecarbonyl)oxirane-2-carboxamide (14): Col-
orless solid (93 mg, 79% over two steps); HPLC: tR =7.2 min;
1H NMR (500 MHz, [D6]DMSO): d=0.82–0.90 (m, 12H, d-CH3 Leu, d-
CH3 i-amyl), 1.06 (d, J=6.2 Hz, 6H, CH3 iPr), 1.27 (m, 2H, b-CH2 i-
amyl), 1.45 (m, 2H, b-CH2 Leu), 1.55 (m, 2H,g-CH Leu, g-CH i-amyl),
3.01 (m, 1H, a-CH2 i-amyl), 3.07 (m, 1H, a-CH2 i-amyl), 3.20 (m, 1H,
CH iPr), 3.59 (d, J=1.8 Hz, 1H, CH oxirane), 3.72 (d, J=1.8 Hz, 1H,
CH oxirane), 4.31 (m, 1H, a-CH Leu), 8.06 (d, J=5.5 Hz, 1H, NH
i-amyl), 8.65 (d, J=8.4 Hz, 1H, NH Leu), 10.71 ppm (br s, 1H,
NHNHCH2), signal for NHNHCH2 is not visible; HRMS (MALDI): m/z
[M+Na]+ calcd for C18H34N4O4Na: 393.24723, found: 393.24752.
Funding of N.S. by a Heisenberg Fellowship and of C.P.S. by proj-
ect SO 249/1–1, priority program 1394 ‘Mast cells — promoters
of health and modulator of diseases’, of the Deutsche For-
schungsgemeinschaft (DFG) is gratefully acknowledged.
Keywords: cathepsin C · dipeptidyl peptidase I (DPPI) · E-64 ·
hydrazines · papain-like cysteine proteases · structure–activity
relationships
[1] MEROPS, the Peptide Database, Sanger Institute, Wellcome Trust, UK;
http://merops.sanger.ac.uk/.
(2S,3S)-3-(2-Isobutylhydrazinecarbonyl)-N-((S)-1-(isopentylami-
no)-4-methyl-1-oxopentan-2-yl)oxirane-2-carboxamide (15): Col-
orless solid (36 mg, 30% over two steps); HPLC: tR =8.0 min;
1H NMR (600 MHz, [D6]DMSO): d=0.82–0.91 (m, 18H, d-CH3 Leu, d-
CH3 i-amyl, g-CH3 iBu), 1.27 (m, 2H, b-CH2 i-amyl), 1.44 (m, 2H, b-
CH2 Leu), 1.54 (m, 2H, g-CH Leu, g-CH i-amyl), 1.72 (m, 1H, b-CH
iBu), 2.59 (m, 2H, a-CH2 iBu), 3.01 (m, 1H,a-CH2 i-amyl), 3.07 (m,
1H, a-CH2 i-amyl), 3.49 (d, J=1.8 Hz, 1H, CH oxirane), 3.67 (d, J=
1.8 Hz, 1H, CH oxirane), 4.29 (m, 1H, a-CH Leu), 8.02 (d, J=5.6 Hz,
1H, NH i-amyl), 8.57 (d, J=8.4 Hz, 1H, NH Leu), 10.25 ppm (br s,
1H, NHNHCH2), signal for NHNHCH2 is not visible; HRMS (MALDI):
m/z [M+Na]+ calcd for C19H36N4O4Na: 407.26288, found:
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Received: February 26, 2013
Published online on June 18, 2013
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ChemMedChem 2013, 8, 1314 – 1321 1321