3-Cyano-3-aza-β-amino acid derivatives as inhibitors of human cysteine cathepsins

Article abstract of DOI:10.1021/ml500238q

Nitrile-type inhibitors are known to interact with cysteine proteases in a covalent-reversible manner. The chemotype of 3-cyano-3-aza-β-amino acid derivatives was designed in which the N-cyano group is centrally arranged in the molecule to allow for interactions with the nonprimed and primed binding regions of the target enzymes. These compounds were evaluated as inhibitors of the human cysteine cathepsins K, S, B, and L. They exhibited slow-binding behavior and were found to be exceptionally potent, in particular toward cathepsin K, with second-order rate constants up to 52 900 × 10³M^-1s^-1.

Full text of DOI:10.1021/ml500238q

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Letter
3-Cyano-3-aza-beta-amino Acid Derivatives
as Inhibitors of Human Cysteine Cathepsins
Janina Schmitz, Anna-Madeleine Beckmann, Adela Dudic,
Tianwei Li, Robert Sellier, Ulrike Bartz, and Michael Gütschow
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/ml500238q • Publication Date (Web): 11 Aug 2014
Downloaded from http://pubs.acs.org on August 25, 2014
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ACS Medicinal Chemistry Letters
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3-Cyano-3-aza-β-amino Acid Derivatives as Inhibitors of Human
Cysteine Cathepsins
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Janina Schmitz,†,§,‡ Anna-Madeleine Beckmann,†,‡ Adela Dudic,† Tianwei Li,† Robert Sellier,† Ulrike
Bartz,§ and Michael Gütschow†,*
† Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany
§ Department of Natural Sciences, University of Applied Sciences Bonn-Rhein-Sieg, von-Liebig-Strasse 20, D-53359
Rheinbach, Germany
KEYWORDS: Cysteine proteases, human cathepsins, β-amino acids, nitrile inhibitors
ABSTRACT: Nitrile-type inhibitors are known to interact with cysteine proteases in a covalent-reversibly manner. The chemotype
of 3-cyano-3-aza-β-amino acid derivatives was designed in which the N-cyano group is centrally arranged in the molecule to allow
for interactions with the non-primed and primed binding regions of the target enzymes. These compounds were evaluated as
inhibitors of the human cysteine cathepsins K, S, B, and L. They exhibited slow-binding behavior and were found to be
exceptionally potent, in particular towards cathepsin K, with second-order rate constants up to 52,900 × 103 M-1s-1.
Cysteine cathepsins, proteolytic enzymes located within the
lysosomes, belong to the family of papain-like cysteine
peptidases. The overexpression of theses enzymes has been
implicated in a multitude of pathological conditions, such as
tumor progression and metastasis, MHC class II antigen
presentation and bone remodeling.1,2 The cysteine protease
cathepsin K degrades several components of the bone matrix and
has emerged as a promising target for intervention by small
molecules on the basis of the inhibition of osteoclastic resorption
observed in human and knock-out animal models. Several
cathepsin K inhibitors have been developed to reduce the
excessive bone matrix degradation associated with osteoporosis.
Among them, the front-runner odanacatib (1, Figure 1), a non-
basic and non-lysosomotropic peptidomimetic nitrile, is currently
being developed as a once weekly treatment for osteoporosis.3-6
by a nitrogen atom.8,9 Azadipeptide nitriles form covalent-
reversible isothiosemicarbazide adducts upon being attacked by
the active site cysteine nucleophile. Representative examples for
azadipeptide nitriles are depicted in Figure 1. The nitrogen atoms
of the aza-amino nitrile moiety of azadipeptide nitriles are
essentially alkylated for reasons of synthetic access. Undesired
cyclization reactions have been observed when the nitrogen of the
P2-P1 peptide bond and/or the P1 α-nitrogen bear a hydrogen
substituent (-CO-NH-NH-CN or -CO-NH-NR-CN or -CO-NR-
NH-CN).8 However, carbamate-protected aza-amino nitriles have
been described as cathepsin K inhibitors which contain the open-
chain O-CO-NH-NR-CN motif.12
A systematic scan with respect to the P3 and P2 substituents was
performed to elucidate the strong impact of the non-covalent
interactions for cathepsin K inhibition by azadipeptide nitriles.13,14
The high electrophilic reactivity15 toward thiols is not a sufficient
condition for cathepsin inhibition, as for example Gly in P2
position in place of a preferred amino acid reduced the potency by
three orders of magnitude.13 The sulfone derivative 3 showed dual
inhibition of human cathepsins S and K with picomolar Ki
values.16 In further series of azadipeptide nitriles, leucine was
maintained at P2 position and several aryl moieties were
introduced in the N-terminal amide portion;17 a direct linkage of
these aryl groups to the α-carbon of the P2 building block caused
Odanacatib (1) exhibits characteristic features of low-molecular-
weight inhibitors of cysteine cathepsins which are mainly
peptidomimetic structures containing an electrophilic warhead to
allow for covalent interactions with the active-site cysteine.
Among such inhibitors, dipeptide nitriles have attracted particular
attention. In the course of the drug development, a dipeptide has
typically been decorated with an N-terminal capping group and
the carboxylic function has been replaced by a cyano group. The
P2 and P1 amino acid side chains then allow for specific binding
to the S2 and S1 pockets within the active site of the target
enzymes, the capping group for an interaction with the S3 binding
pocket. The reaction of dipeptide nitriles with cysteine proteases
involves the nucleophilic attack of the thiolate moiety at the
inhibitors’ nitrile carbon and thus the reversible formation of
thioimidate adducts.7
a
strong reduction of the affinity to cathepsin K.18 The
azadipeptide nitrile chemotype was employed for the design of
activity-based probes for in situ proteome profiling of
Trypanosoma brucei,19 and was chemically converted into
fluorescent derivatives to be used for cathepsin labeling.20 The
fluorine-18 labeled inhibitor 4 was applied for positron emission
tomography to image tumor-associated cathepsin activity.21
Moreover, a cathepsin inhibitor cargo was linked by click
chemistry to the localization peptide Tat for organelle-specific
targeting, and subcellular delivery of the inhibitor-Tat conjugate 5
to the lysosomes was reported.22
For papain-like cysteine proteases, azadipeptide nitriles have been
introduced as a new inhibitor class whose representatives
exhibited Ki values toward human cysteine cathepsins in the nano
and even picomolar range. Such inhibitors have been designed
through an exchange of the α-CH moiety of the P1 amino nitrile
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Scheme 1. Synthesis of 3-Cyano-3-aza-β-amino Acid
F
Derivatives 22-27a
1
2
3
4
5
6
O
O
CF3
H
N
S
N
O
O
O
a
b
N
H
O
NH2
N
N
O
N
O
N
OH
O
N
N
H
N
R1
R1
O
R1 = H
R1 = Me
6
7
8 R1 = H
S
1
3
R1 = Me
O
O
9
7
8
9
F18
O
O
H
N
O
O
O
O
N
H
N
c
N
R2
R2
N
O
N
N
O
N
N
H
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
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42
43
44
45
46
47
48
49
50
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60
R1
R3
R1
R3
N
O
N
10 15
-
16 21
-
2
PrN
N
O
N
H
N
N
N
O
O
O
d
4
R2
O
N
R1
N
R3
O
N
22 27
-
N
N
H
N
R1 = H, NR2R3 = 4-morpholinyl
R1 = H, R2 = Me, R3 = Bn
R1 = H, R2 = R3 = Bn
R1 = Me, NR2R3 = 4-morpholinyl
R1 = Me, R2 = Me, R3 = Bn
R1 = Me, R2 = R3 = Bn
10, 16, 22
11, 17, 23
12, 18, 24
13, 19, 25
14, 20, 26
15, 21, 27
O
N
O
O
N
N
Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-ArgNH2
5
Figure 1. Peptidomimetic cathepsin inhibitors: structures of the
cathepsin K inhibitors odanacatib (1) and balicatib (2) and of
azadipeptide nitriles.
a Reagents and conditions: (a) glyoxylic acid monohydrate, EtOH, RT; (b)
(i) ClCO2i-Bu , NMM, THF, -25 °C; (ii) HNR2R3, THF, -25 °C to RT; (c)
H2, Pd/C (10%), MeOH, 3.3 bar, RT; (d) BrCN, NaOAc, MeOH, RT.
Scheme 2. Alternative Synthesis of the 3-Aza-β-amino Acid
In the present study we aimed at synthesizing cysteine protease
inhibitors with an N-cyano group which is centrally arranged in
the molecule. This group was expected to interact with the active
site cysteine and the flanking parts with the non-primed and
primed subsites of the target protease. Such type of nitrile-based
inhibitors has not been described so far and can be considered as a
structural expansion of cathepsin inhibiting alkyl 2-cyano-2-
methylhydrazinecarboxylates.12 It was intended to introduce the
cyano group via electrophilic cyanation.23 Incorporation of an aza-
α-amino acid would lead to a -CO-NH-NαH-CO- substructure, in
which the α-nitrogen would not be susceptible to electrophilic
cyanation. Therefore, we envisaged a 3-aza-β-amino acid as the
core element of the desired compounds. The corresponding -CO-
NH-NβH-CαH2-CO- substructure should then allow the
preparation of the 3-cyano-3-aza-β-amino acid derivatives.
Derivative 19a
O
O
O
O
H
N
a
N
O
N
OH
O
N
OH
R1
R1
R1 = H
R1 = H
8
28
9 R1 = Me
29 R1 = Me
O
H
O
b
N
O
N
N
Me
O
19
a Reagents and conditions: (a) H2, Pd/C (10%), MeOH, 3.3 bar, RT; (b) (i)
ClCO2i-Bu , NMM, THF, -25 °C; (ii) morpholine, THF, -25 °C to RT.
The synthesis of the final compounds is illustrated in Scheme 1.
Condensation of the carbazates 6 and 7 with glyoxylic acid gave
the hydrazones 8 and 9. Amide coupling of 8 and 9 with
morpholine, N-benzylmethylamine and N,N-dibenzylamine,
respectively, furnished 10-15. These compounds were
catalytically hydrogenated to hydrazinoacetic acid derivatives 16-
21 in order to make the β-nitrogen accessible to cyanation.
Reactions with cyanogen bromide produced the Boc-protected
final compounds 22-27. At the C-terminus, this first series of 3-
Concerning the structural elucidation we have initially paid
attention to the intermediates 10-15. Signals for two isomers
occurred in the 1H and 13C NMR spectra of the N-
benzylmethylamino derivatives 11 and 14. This could result from
two different functionalities. E/Z isomerism is possible (i) because
of the C=N double bond, or (ii) because of the partial double bond
character of the CCO-N bond. A corresponding E/Z isomerism
about the carbamate OCO-N bond was ruled out as this would
only be valid in case of 14. Thus, the amide bond in 11 and 14
was suspected to be responsible for the mixtures. This was also
based on the findings that (i) hydrogenation to 17 and 20 did not
lead to a disappearance of mixtures and (ii) such mixtures have
neither been observed in case of the morpholine-derived
intermediates 10 and 13 and hydrazinoacetic acid derivatives 16
and 19, nor in case of the N-dibenzylamine-derived intermediates
12 and 15 and hydrazinoacetic acid derivatives 18 and 21. Hence,
the appearance of the isomers resulted from the E/Z isomerism
about the amide bond in the unequally substituted tertiary
carboxamides 11, 14, 17, and 20; it was also observed in the
corresponding final products 23 and 26.
cyano-3-aza-β-amino acid derivatives contains
a
tertiary
carboxamide moiety. Whether it would also possible to exchange
R2 and/or R3 by hydrogen without eliciting a concomitant
cyclization was not examined in the course of this study.
An alternative order of reactions was followed as outlined in
Scheme 2. Starting with 9, the hydrogenation step was carried out
prior to the amide coupling. Indeed, we obtained 29 and
successively 19, the precursor to the final product 25. However,
when applying the hydrogenation-coupling sequence to the
demethylated analog 8 and then obtaining 28, the subsequent
coupling reaction with morpholine failed.
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Chart 1. Atropisomeric structures of azadipeptides 30 and 31
1
2
3
4
5
6
7
8
8
O
N
Me
Me
O
O
N
N
O
N
N
NH2
Me
6
4
2
0
O
N
H
Me
O
N
H
O
31
30
In previous studies, it has been shown, that the azadipeptide
amide 30 forms atropisomers (Chart 1).32,33 The diastereotopic
protons of the glycine fragment indicated a restricted rotation
around the N-N single bond. This rotation was enforced with a
variable temperature (VT) NMR experiment and a value of 117
kJ/mol was determined for the rotational barrier. As atropisomers
have rotational barriers higher than 95 kJ/mol, compound 30
exhibits chirality as a result of its atropisomerism.32 Moreover, the
azadipeptide nitrile 31 showed diastereotopic glycine methylene
protons,13 and is structurally similar to the final compounds 25-27
of this study. Therefore, we addressed the question whether 25-27
would also form atropisomers. However, diastereotopic protons in
the corresponding 1H NMR spectra were not observed. For
example, in the 1H NMR spectrum of 27, three distinct singlets for
the methylene protons appear. It could therefore be concluded that
in 25-27 a free rotation around the N-N single bond is possible.
With respect to such a bond rotation, the significant difference
between the azadipeptides 30 and 31, on the one hand, and the 3-
cyano-3-aza-β-amino acid derivatives 25-27, on the other hand, is
the position next to the N-N-CO carbonyl group. In comparison to
the methylene group (in 30 and 31) the oxygen atom (in 25-27) is
less voluminous. Moreover, it is the methylation of the peptide
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
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39
40
41
42
43
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50
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58
59
60
0
1000
2000
t (s)
3000
4000
0.004
0.003
0.002
0.001
0
0.003
0.002
0.001
0
0
400 800 1200 1600 2000
[22] (pM)
0
400
800
1200
1600
2000
bond in azadipeptides 30 and 31 which leads to the
E
[
] (pM)
22
configuration (Chart 2) and hence to the aforementioned
atropisomerism.32 In 3-cyano-3-aza-β-amino acid derivatives 25-
27, one more mesomeric structure is possible and the contribution
of the third limiting structure to the resonance reduces the double
bond character of the CO-N bond (Chart 2).34-36 Thus, the E
configuration in 25-27 is supposed to be less defined and
consequentially the N-N single bond rotation is not hindered.
Figure 2. Inhibition of human cathepsin K by 22. Top: Monitoring of the
cathepsin K-catalyzed hydrolysis of Z-Leu-Arg-AMC in the presence of
increasing concentrations of compound 22 (closed circles, 0 pM; open
circles 400 pM; closed squares 800 pM; open squares 1200 pM; closed
triangles 1600 pM; open triangles 2000 pM. Fluorescence emission at 440
nm was measured after excitation at 360 nm. Bottom: Plot of the rates
obtained in duplicate measurements versus concentrations of 22.
Nonlinear regression gave an IC50 value of 158 ( 14) pM. Inset: Plot of
the first-order rate constants kobs versus concentrations of 22. Linear
regression gave a value kon/(1+[S]/Km) of 1.33 ( 0.15) × 10-6 pM-1s-1.
The results of the biochemical evaluation at four human cysteine
cathepsins are outlined in Table 1. Fluorogenic or chromogenic
peptide substrates were employed in the activity assays. Our
inhibitors showed time-dependent inhibition of the target
cathepsins. This behavior was in agreement with the expected
covalent and reversible enzyme-inhibitor interaction. In some
cases, the progress curves were analyzed after achievement of
steady-state by linear regression. Plots of the steady-state rates
versus inhibitor concentration gave IC50 values. Assuming an
active-site directed mode of action, those were corrected using the
Cheng-Prusoff equation to obtain the true inhibition constants, Ki.
In other cases, the progress curves were analyzed by means of the
slow binding equation,37 and non-linear regression analysis of the
progress curves provided the first-order rate constants kobs, besides
the steady-state rates. The kobs values were used to obtain second-
order rate constants, kon, governing the enzyme-inhibitor
association. As Ki values were also determined as noted above,
first-order rate constants for the dissociation of the enzyme-
inhibitor complex, koff, were also accessible (Ki = koff/kon). A
representative kinetic analysis is illustrated in Figure 2.
As a first result of the kinetic investigations, a major difference in
the inhibitory potency of the non-methylated and the methylated
morpholine derivatives was ascertained (22 versus 25) which was
particularly striking in case of cathepsin K (Ki of 11 pM versus Ki
of 31 nM). It has been described that a non-methylated amide
bond in nitrile-type cysteine protease inhibitors (-CO-NH-X-CN)
resulted in a much stronger inhibition than the methylated analogs
(-CO-NMe-X-CN).8,12
Chart 2. Mesomeric structures of the E configured hydrazide
and carbazate fragments of 30, 31 and 25-27
Thus, we focused on the three non-methylated 3-cyano-3-aza-β-
amino acid derivatives (22-24) to elucidate structure-activity
relationships. Noteworthy, these three derivatives exhibited a
gradual decrease in affinity towards cathepsins K, S, B and L. The
stepwise introduction of one or two bulky aromatic moieties
resulted in an improved inhibitory potency (22 versus 23 versus
24), which was determined for the four enzymes. Obviously, the
benzyl groups allow for hydrophobic or cation-π interactions with
the cathepsins’ active sites.
N
N
N
N
Me
Me
O
O
N
N
N
N
N
N
O
O
O
Me
Me
Me
O
O
O
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Table 1. Kinetic Parameters of Inhibition of Cathepsins by 3-Cyano-3-aza-β-amino Acid Derivatives 22-25 and Balicatib
1
2
3
Human Cathepsin Ka b
Human Cathepsin Sa c
Human Cathepsin Ba d
Human Cathepsin La e
,
,
,
,
Compound
4
5
6
7
22
Ki =
0.0110 ( 0.0010) nMf
Ki =
0.777 ( 0.033) nMf
Ki =
3.56 ( 0.21) nMg
Ki =
573 ( 36) nMg
N
O
O
N
kon
=
kon =
757 ( 28) × 103 M-1s-1
O
N
H
N
19,000 ( 2,100) × 103 M-1s-1
O
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
23
24
Ki =
0.00420 ( 0.00048) nMf
Ki =
0.176 ( 0.083) nMf
Ki =
0.459 ( 0.023) nMg
Ki =
228 ( 20) nMg
N
N
O
O
Me
kon
=
kon =
1,590 ( 320) × 103 M-1s-1
O
N
H
N
28,900 ( 3,100) × 103 M-1s-1
Bn
Ki =
0.00143 ( 0.00020) nMf
Ki =
0.130 ( 0.044) nMf
Ki =
0.406 ( 0.010) nMg
Ki =
185 ( 7) nMg
N
N
O
O
Bn
kon
=
kon =
2,580 ( 440) × 103 M-1s-1
O
O
N
H
N
52,900 ( 7,700) × 103 M-1s-1
Bn
N
N
Ki =
31.0 ( 2.1) nMf
Ki =
793 ( 155) nMf
Ki =
194 ( 10) nMf
Ki =
1,690 ( 70) nMf
25
O
O
N
N
kon
=
kon
=
kon
=
kon =
4.05 ( 0.52) × 103 M-1s-1
0.461 ( 0.115) × 103 M-1s-1
2.92 ( 0.28) × 103 M-1s-1 0.301 ( 0.039) × 103 M-1s-1
Me
O
2
balicatib
Ki = 1.29 ( 0.14) nMh
Ki = 30,100 ( 3,800) nMh
Ki = 3,800 ( 220) nMh
Ki = 3,260 ( 250) nMh
a
IC50 values were obtained from duplicate measurements in the presence of five different inhibitor concentrations. IC50 values were
determined by nonlinear regression using equation vs = v0/(1+[I]/IC50), were vs is the steady-state rate, v0 is the rate in the absence of the
inhibitor, and [I] is the inhibitor concentration. Ki values were calculated from IC50 values by applying the equation Ki = IC50/(1+[S]/Km),
where [S] is the substrate concentration. b The cathepsin K assay was performed with the fluorogenic substrate Z-Leu-Arg-AMC at a final
concentration of 40 µM (13.3 × Km). The reaction was followed over 60 min.13
The cathepsin S assay was performed with the
d
c
fluorogenic substrate Z-Phe-Arg-AMC at a final concentration of 40 µM (0.74 × Km). The reaction was followed over 60 min.38 The
cathepsin B assay was performed with the chromogenic substrate Z-Arg-Arg-pNA at a final concentration of 500 µM (0.45 × Km). The
e
reaction was followed over 30 min.13 The cathepsin L assay was performed with the chromogenic substrate Z-Phe-Arg-pNA at a final
concentration of 100 µM (5.88 × Km). The reaction was followed over 30 min.13 f Progress curves were analyzed by nonlinear regression
using the slow-binding equation [P] = vst+(vi-vs)(1-exp(-kobst))/kobs+d to give vs, vi and kobs, where [P] is the product concentration, vi is the
initial rate, kobs is the observed first-order rate constant, and d is the offset. To obtain IC50 values, vs values from reactions in the presence
of the inhibitor and v0 values obtained by linear regression of the progress curves in the absence of the inhibitor, were used. The second-
order rate constants kon were obtained by linear regression according to equation kobs = kon[I]/(1+[S]/Km)+koff, where koff is the first-order
g
h
rate constant of dissociation.
Progress curves were analyzed by linear regression after steady-state was reached (5-30 min).
reaction was followed over 20 min and progress curves were analyzed by linear regression. For literature IC50 values and corresponding
assay conditions, see refs.39,40
The
For human cathepsins K and S, inhibitors 22-24 exhibited very
low Ki values down to 1.43 pM (24 at cathepsin K). This strong
affinity is mainly due to an accelerated association to form the
enzyme-inhibitor complex(es), as it can be inferred from the high
second-order rate constants (e.g. kon = 52,900 × 103 M-1 s-1 for 24
at cathepsin K). For 22-24, half-lives of the cathepsin K-inhibitor
and cathepsin S-inhibitor complexes could be calculated from the
corresponding koff values and were in the range between 10 and
150 min (22 at cathepsin S and 24 at cathepsin K).
209Leu. Hydrogen bonding interactions of the carbamate occur
with the backbone NH of 66Gly and the backbone carbonyl
oxygen
of
161Asn.12
Although
alkyl
2-cyano-2-
methylhydrazinecarboxylates exhibit a pronounced cathepsin K
inhibition, the cathepsin K selectivity of the inhibitors described
herein is even stronger and is probably caused by hydrophobic or
cation-π interactions with the non-primed binding region of
cathepsin K. In future studies, it is intended to expand the
inhibitor structure to both directions. This will enable to better
define the binding mode of this new type of cysteine protease
inhibitors. On the one hand, prior to cyanation and hydrogenation,
a Boc-deprotection would allow for the incorporation of moieties
to interact with the S1 and S2 pockets. On the other hand, the
introduction of amino acid derivatives, in place of the secondary
amines used in this study, might direct the so modified amide
We propose that the Boc capping group of the 3-cyano-3-aza-β-
amino acid derivatives is bound in the non-primed binding region
of the target enzymes and the amide part is accommodated in the
primed binding region. This assumption is based on the co-crystal
structure of tert-butyl 2-cyano-2-methylhydrazinecarboxylate
with cathepsin K.12 In this complex, the tert-butyl group occupies
the S2 pocket formed by residues 67Tyr, 68Met, 134Ala, 163Ala, and
part to the S1’ and S2’ pockets.
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ACS Medicinal Chemistry Letters
Willard, D. H. Jr.; Wright, L. L. Acyclic cyanamide-based inhibitors
AUTHOR INFORMATION
of cathepsin K. Bioorg. Med. Chem. Lett. 2005, 15, 3039-3043.
(13) Frizler, M.; Lohr, F.; Furtmann, N.; Kläs, J.; Gütschow, M.
Structural optimization of azadipeptide nitriles strongly increases
association rates and allows the development of selective cathepsin
inhibitors. J. Med. Chem. 2011, 54, 396-400.
(14) Frizler, M.; Lohr, F.; Lülsdorff, M.; Gütschow, M. Facing the
gem-dialkyl effect in enzyme inhibitor design: preparation of
homocycloleucine-based azadipeptide nitriles. Chem. Eur. J. 2011,
17, 11419-11423.
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2
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Corresponding Author
* E-mail: guetschow@uni-bonn.de. Phone: +49 228 732317.
Author Contributions
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the
manuscript. ‡ J.S. and A.M.B contributed equally.
8
9
(15) Oballa, R. M.; Truchon, J. F.; Bayly, C. I.; Chauret, N.; Day,
S.; Crane, S.; Berthelette, C. A generally applicable method for
assessing the electrophilicity and reactivity of diverse nitrile-
containing compounds. Bioorg. Med. Chem. Lett. 2007, 17, 998-1002.
(16) Frizler, M.; Schmitz, J.; Schulz-Fincke, A. C.; Gütschow, M.
Selective nitrile inhibitors to modulate the proteolytic synergism of
cathepsins S and F. J. Med. Chem. 2012, 55, 5982-5986.
(17) Ren, X. F.; Li, H. W.; Fang, X.; Wu, Y.; Wang, L.; Zou, S.
Highly selective azadipeptide nitrile inhibitors for cathepsin K:
design, synthesis and activity assays. Org. Biomol. Chem. 2013, 11,
1143-1148.
(18) Yuan, X. Y.; Fu, D. Y.; Ren, X. F.; Fang, X.; Wang, L.; Zou,
S.; Wu, Y. Highly selective aza-nitrile inhibitors for cathepsin K,
structural optimization and molecular modeling. Org. Biomol. Chem.
2013, 11, 5847-5852.
(19) Yang, P. Y.; Wang, M.; Li, L.; Wu, H.; He, C. Y.; Yao, S. Q.
Design, synthesis and biological evaluation of potent azadipeptide
nitrile inhibitors and activity-based probes as promising anti-
Trypanosoma brucei agents. Chem. Eur. J. 2012, 18, 6528-6541.
(20) Frizler, M.; Mertens, M. D.; Gütschow, M. Fluorescent
nitrile-based inhibitors of cysteine cathepsins. Bioorg. Med. Chem.
Lett. 2012, 22, 7715-7718.
(21) Löser, R.; Bergmann, R.; Frizler, M.; Mosch, B.;
Dombrowski, L.; Kuchar, M.; Steinbach, J.; Gütschow, M.; Pietzsch,
J. Synthesis and radiopharmacological characterisation of a fluorine-
18-labelled azadipeptide nitrile as a potential PET tracer for in vivo
imaging of cysteine cathepsins. ChemMedChem 2013, 8, 1330-1344.
(22) Loh, Y.; Shi, H.; Hu, M.; Yao, S. Q. "Click" synthesis of
small molecule-peptide conjugates for organelle-specific delivery and
inhibition of lysosomal cysteine proteases. Chem. Commun. 2010, 46,
8407-8409.
(23) For examples of electrophilic cyanations, see refs 24-28. For
recent examples of nucleophilic metal-catalyzed or photoinduced
direct cyanations, see refs 29-31.
(24) Hoshikawa, T.; Yoshioka, S.; Kamijo, S.; Inoue, M.
Photoinduced direct cyanation of C(sp3)-H bonds. Synthesis 2013, 45,
874-887.
Notes
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The authors declare no competing financial interest.
ACKNOWLEDGMENT
J.S. is supported by the Gender Equality Center of the Bonn-
Rhein-Sieg University of Applied Sciences.
ASSOCIATED CONTENT
Supporting Information Available
Synthetic procedures and characterization of compounds.
1H NMR and 13C NMR spectra. Enzyme inhibition assays. This
material is available free of charge via the Internet at
http://pubs.acs.org.
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Table of Contents artwork
1
2
3
Cat K
Cat K
4
5
6
7
SH
O
N
N
HN
S
= 52,900 x 10-3 M-1 s-1
= 75.6 x 10-6 s-1
kon
O
O
O
N
O
N
H
N
O
N
H
N
koff
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21
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