Fluorescent nitrile-based inhibitors of cysteine cathepsins

Article abstract of DOI:10.1016/j.bmcl.2012.09.086

Cysteine cathepsins play an important role in many (patho)physiological conditions. Among them, cathepsins L, S, K and B are subjects of several drug discovery programs. Besides their role as drug targets, cysteine cathepsins are additionally considered t

Full text of DOI:10.1016/j.bmcl.2012.09.086

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7715–7718
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Fluorescent nitrile-based inhibitors of cysteine cathepsins
⇑
Maxim Frizler, Matthias D. Mertens, Michael Gütschow
Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Cysteine cathepsins play an important role in many (patho)physiological conditions. Among them,
cathepsins L, S, K and B are subjects of several drug discovery programs. Besides their role as drug targets,
cysteine cathepsins are additionally considered to be possible biomarkers for inflammation and cancer.
Herein, we describe the design, synthesis, biological evaluation and spectral properties of fluorescently
labeled dipeptide- and azadipeptide nitriles.
Received 28 August 2012
Revised 20 September 2012
Accepted 25 September 2012
Available online 3 October 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Fluorescent probes
Coumarins
Dipeptide nitriles
Azadipeptide nitriles
Cysteine cathepsins
Cysteine cathepsins belong to the subfamily of papain-like pro-
teases exhibiting a high degree of homology. Eleven human cys-
teine cathepsins have been described. They are involved in many
(patho)physiological processes such as bone remodeling, antigen
presentation, osteoporosis, autoimmune disorders, and cancer.1
Therefore, cathepsins represent important targets for new thera-
peutic strategies. Among them, cathepsins L, S, K and B are subjects
of several drug discovery programs.2
Inhibitors of cathepsins are mainly peptidic or peptidomimetic
structures containing electrophilic groups for covalent interactions
with the active-site cysteine. Nitrile-based inhibitors have received
the most attention in the current research.2,3 Recently, we have re-
ported on the development of azadipeptide nitriles as highly po-
tent and non-selective inhibitors of cysteine cathepsins with Ki
values in picomolar range, which additionally exhibited antimalar-
ial activities.4,5 Moreover, using the example of cathepsin K, an ap-
proach towards developing selective azadipeptide nitriles by
structural optimization of the inhibitor scaffold was demon-
strated.6,7 Loh et al. described peptide conjugates of azadipeptide
nitriles to achieve organelle-specific delivery.8
Herein we describe the synthesis, biological evaluation and
spectral properties of novel fluorescent dipeptide- and azadipep-
tide nitriles which can prospectively be used to label cysteine
cathepsins. The design was focused on cathepsin K as the target
protease, as this enzyme is of particular therapeutic and diagnostic
importance.3
First, the coumarin derivative 2 was synthesized as a fluorescent
reporter by the reaction of 4-diethylamino-2-hydroxy-benzaldehyde
with isopropylidene malonate in the presence of piperidinium
acetate (Scheme 1).22 Compound 1 and dansyl chloride 3 were
commercially available.
Next, the homocycloleucyl-glycine nitrile scaffold 6 was synthe-
sized as shown in Scheme 2. The Cbz-protected homocycloleucine
4 was reacted with oxalyl chloride in the presence of a catalytic
amount of DMF to obtain homocycloleucine-NCA 5 in high yield
and purity. Compound 5 was heated with aminoacetonitrile lead-
ing to the dipeptide nitrile 6. Finally, the free amino group of 6
was converted with dansyl chloride 3 and the coumarin derivative
2 to obtain the fluorescently labeled dipeptide nitriles 7 and 8,
respectively (Scheme 2). As expected, the amino group of 6 showed
a decreased reactivity due to the gem-dialkyl effect operative in
homocycloleucine derivatives.7,23 Accordingly, the target com-
pounds 7 and 8 were isolated by chromatographic separation only
in low yields of 36% and 20%, respectively.
Besides their role as possible drug targets, cysteine cathepsins
are considered to be possible biomarkers for certain pathological
conditions including inflammation and cancer.9–14 Therefore, there
is a need for sensitive methods to label cathepsins for diagnostic
and research proposals.15–20 Recently, ‘activity-based’ probes, con-
taining an azadipeptide nitrile scaffold, were developed for rhodes-
ain from Trypanosoma brucei.21
The fluorescently labeled compound 8 exhibited better kinetic
properties than 7 with respect to cathepsin K inhibition. In order
to extend the residence time of the inhibitor, 8 was chosen for a
Ca/N exchange in the P1 amino acid. From previous studies, it
could be expected that the resulting azadipeptide nitrile forms a
slowly dissociating enzyme-inhibitor complex.4,6 The design of
the corresponding azadipeptide nitrile 12 is shown in Scheme 3.
⇑
Corresponding author. Tel.: +49 228 732317; fax: +49 228 732567.
E-mail address: guetschow@uni-bonn.de (M. Gütschow).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.09.086

7716
M. Frizler et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7715–7718
SO2Cl
O
O
a
OH
H
N
O
O
N
OH
N
1
2
3
Scheme 1. Synthesis of the coumarin derivative 2. (a) isopropylidene malonate, piperidinium acetate, EtOH, rt to
D.
O
H
N
a
b
OH
CN
O
BnO
N
HN
O
H2N
H
O
O
O
4
6
5
c
d
O
O
O
O
H
H
S
N
CN
N
CN
N
N
N
H
H
O
O
N
O
7
8
Scheme 2. Synthesis of fluorescently labeled dipeptide nitriles 7 and 8. (a) (COCl)2, CH2Cl2, DMF, rt; (b) H2NCH2CN Â H2SO4, THF, DIPEA, 100 °C; (c) compound 3, Et3 N, THF, rt
then
D
; (d) compound 2, EDC, DIPEA, THF, rt.
O
c
a
b
N
N
4
BnO
N
N
H2N
N
H
H
H
O
O
10
9
O
O
N
CN
N
d
N
N
N
N
H
H
H
O
O
N
O
O
N
O
O
12
11
Scheme 3. Synthesis of the fluorescently labeled azadipeptide nitrile 12. (a) 1. EDC, DMAP, THF, rt; 2. (NHMe)2 Â 2HCl, Et3N, rt; (b) Pd/C, H2, MeOH, rt; (c) compound 2, EDC,
DMAP, THF, rt; (d) BrCN, NaOAc, MeOH, rt.
(a)
(b)
0.24
0.24
0.2
0.16
0.12
0.08
0.04
0
0.2
0.16
0.12
0.08
0.04
0
E
E
0
20
40
60
80
0
2
4
6
8
10
t (min)
t (min)
Figure 1. (a) Monitoring of the human cathepsin B-catalyzed hydrolysis of Z-Arg-Arg-pNA (500
M; , 4 M; , 6 M; , 8 M; , 10 M). The linear progress curves indicate a fast-binding behavior of the inhibitor; (b) Monitoring of the human cathepsin B-catalyzed
hydrolysis of Z-Arg-Arg-pNA (500 M) in the presence of increasing concentrations of the slow-binding inhibitor 12 (d, 0; , 2 M; , 4 M; , 6 M; , 8 M; , 10 M).
l
M) in the presence of increasing concentrations of compound 7 (d, 0;
,
2
l
l
l
l
l
l
l
l
l
l
l

M. Frizler et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7715–7718
7717
Table 1
The Cbz-protected homocycloleucine 4 was activated with EDC
and DMAP and reacted with 1,2-dimethylhydrazine to obtain the
dimethylhydrazide 9. The Cbz protecting group was hydrogenolyt-
ically removed producing the building block 10 which was coupled
with the fluorescent reporter 2 using an EDC/DMAP procedure. In
the final step, the obtained 1,2-dimethylhydrazide derivative 11
was converted to the azadipeptide nitrile 12 by the reaction with
cyanogen bromide. Compound 12 was obtained in 19% yield after
column chromatography.
The fluorescent probes 7, 8 and 12 were evaluated on human
cathepsins L, S, K and B using chromogenic or fluorogenic peptides
as substrates. While the dipeptide nitriles 7 and 8 exhibited a fast-
binding behavior, the aza-analogous counterpart 12 was a slow-
binding inhibitor (Fig. 1). In general, compounds 7, 8 and 12
showed a slight preference for cathepsin K over cathepsins L, S
and B (Table 1). Such a profile was expected, since homocycloleu-
cine was established to be an optimized P2 moiety prone to favor-
able interaction with the S2 binding pocket of cathepsin K.2,3,24
The Ki values of dipeptide nitriles 7 and 8 on cathepsin K were
in the nanomolar range. Compound 7 was ca. 8–45-fold selective
for cathepsin K over cathepsins L, S and B. Compound 8 showed
a better selectivity (ca. 40–170-fold). Furthermore, the replace-
Ki values of compounds 7, 8, and 12
Compd
Ki (
l
M)a
Cat K
Cat L
>40
Cat S
Cat B
7
8
12
6.6 1.3
8.4 1.5
0.88 0.18
0.23 0.02
0.019 0.002
>40
>40
0.14 0.01
18
2
0.76 0.08b
0.75 0.03
a
For cathepsin assays, see the Supplementary data. Compounds 7, 8 and 12 were
tested in duplicate measurements with five different inhibitor concentrations. The
reactions were followed over 10 min for fast-binding inhibitors 7 and 8. Reactions
with compound 12 displaying a slow-binding behavior were followed over 80 min.
b
The enzymatic reaction was started by addition of chromogenic substrate after
30 min preincubation of cathepsin L with compound 12. The progress curves were
analyzed by linear regression over 10 min.
Table 2
kon and koff values of 12
kon (Â103 MÀ1 sÀ1
)
koff (Â10À3 sÀ1
)
Cat L
Cat S
Cat K
Cat B
n.d.a,b
n.d.
0.49 0.09
9.3 1.7
0.32 0.03
0.37 0.08
0.18 0.04
0.045 0.005
ment of the a-carbon in compound 8 by a nitrogen atom, leading
to the corresponding azadipeptide nitrile 12, resulted in a ca. 10-
fold improvement of the inhibitory activity toward cathepsin K
(0.23 lM versus 0.019 lM). The fluorescent probe 12 exhibited
a
Not determined.
b
For [12] = 30 lM, a kobs value could not be obtained by non-linear regression.
Therefore, a limit kobs(1+[S]/Km)/[I] < 1.0 Â 103 MÀ1 sÀ1 was estimated.
an approximately 40-fold selectivity for cathepsin K over cathep-
sins L and S and was only moderately selective for cathepsin K over
cathepsin B. Due to the slow-binding behavior of 12, kon and koff
values could be additionally calculated for this compound (Table
2). From the koff values, half life periods of the corresponding cova-
lent isothiosemicarbazide adducts of 12 with cathepsins S, K und B
were calculated to be in the range of 0.5–4 h.
The absorption and emission spectra of the fluorescently la-
beled dipeptide nitriles 7 and 8 and the corresponding azadipep-
tide nitrile 12 were recorded in different solvents to study their
spectral properties (Table 3). The fluorescence of compounds 7, 8
and 12 was quenched by methanol and water, as it is exemplarily
Table 3
kex and kem of compounds 7, 8 and 12 (10
l
M, 1% DMSO)
Compd
kex(kem) (nm)
CH2Cl2
MeOH
H2O
7
8
12
343(494, 522)
423(458)
423(458)
340(534)
422(470)
423(468)
328(566)
433(486)a
434(482)
a
The absorption and emission spectra of 8 in H2O + 1% DMSO were recorded with
a final concentration of 1
l
M (see the Supplementary data).
0.8
0.6
0.4
0.2
0
16
14
12
10
8
300
400
500
λ (nm)
600
700
120
100
80
60
40
20
0
6
4
2
350
400
450
500
550
λ (nm)
0
300
400
500
600
700
800
λ (nm)
Figure 2. Emission spectra of 12 (10
emission spectra in MeOH and H2O. In the insert below, normalized excitation (d) and emission ( ) spectra of 12 (10
l
M, 1% DMSO) in CH2Cl2
(
, kex = 423 nm), MeOH ( , kex = 423 nm) and H2O ( , kex = 434 nm). The insert above shows the magnified
M, 1% DMSO) in CH2Cl2 are depicted.
l

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M. Frizler et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7715–7718
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Supplementary data
Supplementary data associated with this article can be found, in
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