Selective nitrile inhibitors to modulate the proteolytic synergism of cathepsins S and F

Article abstract of DOI:10.1021/jm300734k

A series of dipeptide nitriles with different P3 substituents was designed to explore the S3 binding pocket of cathepsin S. Racemic 7-16 and the enantiopure derivative (R)-22 proved to be potent inhibitors of human cathepsin S and exhibited notable selectivity over human cathepsins L, K, and B. Inhibition of cathepsin F, the functional synergist of cathepsin S, was not observed. The azadipeptide analogue of 22, compound 26, was highly potent but nonselective.

Full text of DOI:10.1021/jm300734k

Brief Article
pubs.acs.org/jmc
Selective Nitrile Inhibitors To Modulate the Proteolytic Synergism of
Cathepsins S and F
Maxim Frizler, Janina Schmitz, Anna-Christina Schulz-Fincke, and Michael Gutschow*
̈
Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany
S
* Supporting Information
ABSTRACT: A series of dipeptide nitriles with different P3 substituents was designed to explore the S3 binding pocket of
cathepsin S. Racemic 7−16 and the enantiopure derivative (R)-22 proved to be potent inhibitors of human cathepsin S and
exhibited notable selectivity over human cathepsins L, K, and B. Inhibition of cathepsin F, the functional synergist of cathepsin S,
was not observed. The azadipeptide analogue of 22, compound 26, was highly potent but nonselective.
substituent.12 Although certain series of inhibitors exhibited
INTRODUCTION
Major histocompatibility complex class II molecules (MHC-II)
are expressed by only few specialized antigen-presenting cell
■
remarkable selectivity for cathepsin S over cathepsins L, S, and
B,12,14 it remained unclear whether they are also selective for
cathepsin S over the functionally synergistic species cathepsin
types including macrophages, B cells, and dendritic cells. They
F.
are αβ heterodimers synthesized and assembled in the
endoplasmic reticulum. The heterodimerization is assisted by
In this study, we have focused on the P3−S3 interaction of
peptidic inhibitors for cathepsin S. To explore the S3 pocket, a
the invariant chain chaperone molecule that additionally blocks
small library of dipeptide nitriles, containing a fixed S-
the MHC-II peptide-binding groove, preventing premature
(isobutyl)cysteine sulfone moiety at the P2 position and
peptide loading. Furthermore, the invariant chain is important
different P3 substituents, was synthesized and evaluated on
for the transport of assembled MHC class II molecules through
the Golgi apparatus.1,2 Two main proteolytic events occur
human cathepsins L, S, K, and B. Additionally, the inhibitors
were tested on cathepsin F to assess, for the first time, possible
differences of their inhibition profiles toward the invariant chain
processing enzymes cathepsins S and F.
during MHC-II-mediated antigen presentation: (i) cleavage of
the antigens to small antigenic peptides and (ii) degradation of
the invariant chain. Cathepsin S was described as the major
processing enzyme of the invariant chain in the MHC-II-
mediated antigen presentation. Cathepsin S is capable of
cleaving the invariant chain, leaving the short class II associated
RESULTS AND DISCUSSION
To obtain dipeptide nitriles with different P3 substituents, the
P2 building block 6 containing the S-(isobutyl)cysteine sulfone
was synthesized (Scheme 1). The thiol group of the Boc-
■
invariant chain peptide (CLIP) which can be replaced by
antigenic peptides.2,3
protected cysteine methyl ester 1 was alkylated with isobutyl
bromide in the presence of sodium methanolate to obtain 2.15
The thioether group of 2 was oxidized with MCPBA, leading to
the sulfone 3. The methyl ester was cleaved under basic
conditions, and 4 was obtained as a free acid. The carboxylic
group of 4 was coupled with aminoacetonitrile, leading to the
Boc-protected dipeptide nitrile 5. After deprotection with
methanesulfonic acid, building block 6 was obtained. Finally, 6
was coupled with various P3 substituents to obtain the targets
7−16 (Table 1). This final acylation step was performed with
the appropriate acyl chlorides or carboxylic acids. The
corresponding P3 building blocks for the preparation of 9−
12 were synthesized by separate multistep routes (Schemes S1
and S2 in Supporting Information).
The role of cathepsin S in the invariant chain degradation
process in cells from cathepsin S deficient mice can be adopted
by cathepsin F, which is expressed at high levels in
macrophages but not in dendritic and B cells.4 This proteolytic
synergism of cathepsins S and F might be exploited for fine
modulation of the immune response, e.g., by taking advantage
of selective inhibitors for each of these two enzymes. Cathepsin
S deficient mice exhibit defects in immune function but are
healthy and normal in most other respects.2,5 For cathepsin F
deficient mice, however, severe neurological disorders were
observed.6
Cathepsin S was recognized as a promising target for the
treatment of autoimmunity. Inhibition of cysteine cathepsins
can be attained by low molecular weight compounds that
oxidize the active-site cysteine or undergo a nucleophilic
addition reaction.7−9 Thus, several types of peptidomimetic
compounds containing electrophilic groups covalently interact
with the active-site cysteine. Among them, nitrile-based
compounds have received much attention.9−13 Recently,
nitrile-based cathepsin S inhibitors were obtained by a
combination of a P2 cysteine sulfone attached to a large
group pointing to the S2 pocket, and a small aromatic P3
The S-alkylation of N-acetylcysteine in the presence of
sodium alcoholates under elevated temperature without
racemization was reported.15 However, dipeptide nitriles 7−
16 were obtained in racemic form, as shown for compound 7
using chiral HPLC (Figure S1 in Supporting Information). The
Received: March 31, 2012
Published: June 11, 2012
© 2012 American Chemical Society
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Journal of Medicinal Chemistry
Brief Article
a
Scheme 1. Synthesis of Dipeptide Nitriles 7−16
Table 1. Ki Values of Dipeptide Nitriles 7−16 and 22
a
Reagents and conditions: (a) isobutyl bromide, NaOMe, MeOH, Δ;
(b) MCPBA, CH2Cl2, rt; (c) LiOH, THF, H2O, rt; (d) (1) NMM,
ClCO2-i-Bu, THF, −25 °C; (2) H2NCH2CN·H2SO4, NaOH, H2O,
−25 °C to rt; (e) MeSO3H, THF, rt; (f) acyl chloride (RCl), Et3N,
THF, rt or carboxylic acid (ROH), Et3N, EDC, DMAP, rt.
synthetic route to the building block 6 (Scheme 1) contains
two steps with a risk of racemization: (i) alkylation of the thiol
group of the cysteine derivative 1 in the presence of sodium
methanolate and (ii) cleavage of the methyl ester 3 with lithium
hydroxide. To prevent the racemization, a modified route was
applied (Scheme 2). The thiol group of L-cysteine 17 was
alkylated under milder conditions and in the presence of the
weaker base sodium hydroxide,16 followed by protection of the
amino group using (Boc)2O to obtain 18. The thioether
derivative 18 was oxidized with KMnO4 instead of MCPBA
(Scheme 1), leading to the corresponding sulfone 19, which
was coupled with aminoacetonitrile to form the Boc-protected
dipeptide nitrile 20. After deprotection of 20, the resulting
building block 21 was coupled with benzoyl chloride, leading to
the enantiopure dipeptide nitrile 22 whose enantiopurity was
confirmed by chiral HPLC (Figure S1, Supporting Informa-
tion).
a
Data were calculated from duplicate experiments with at least five
different inhibitor concentrations. Mean values of steady-state rates
were used for nonlinear regression to obtain IC50. Standard errors
( SE) of these nonlinear regression analyses are given. Cathepsins L,
S, K, and B were assayed as described.29 For cathepsin F assay, see
b
Supporting Information. For cathepsin L, all limits of >40 μM relate
The nitrogens of the aza-amino nitrile moiety of azadipeptide
nitrile inhibitors are essentially alkylated for reasons of the
synthetic access.17 To synthesize the azadipeptide nitrile 26
(Scheme 2), the aza-analogue of dipeptide nitrile 22,
compound 19 was converted to the corresponding 1,2-
dimethylhydrazide 23. Deprotection of 23 under acidic
conditions and the following basic extraction led to 24 which
was treated with benzoyl chloride to obtain 25. The final
reaction with cyanogen bromide afforded the azadipeptide
nitrile 26.
All final compounds (7−16, 22, and 26) were evaluated on
human cathepsins L, S, K, B, and F in the presence of
chromogenic or fluorogenic peptide substrates. In general,
these inhibitors showed two different kinetic modes. As
expected,10 the dipeptide nitriles 7−16 and 22 were “fast-
binding” inhibitors indicated by linear progress curves (Figure
S2, Supporting Information). This behavior reflects a relatively
fast formation of covalent enzyme−inhibitor thioimidate
adducts. The corresponding rates were plotted versus inhibitor
to the maximum possible inhibitor concentrations of >30 μM; all
limits of >4 μM relate to maximum possible inhibitor concentrations
c
of ≤30 μM. For cathepsins K and F, all limits of >40 μM relate to the
maximum possible inhibitor concentrations of >10 μM; all limits of >4
μM relate to the maximum possible inhibitor concentrations of ≤10
μM.
concentrations to obtain IC50 values by nonlinear regression,
which were corrected to zero substrate concentrations leading
to inhibition constants Ki. The azadipeptide nitrile 26 exhibited
a time-dependent inhibition of cathepsins L, S, K, and B, which
implies a relatively slow approach to steady state with an
equilibrium between free and inhibitor-bound enzyme (Figure
S2, Supporting Information). Besides Ki, the kinetic parameters
of association to a covalent isothiosemicarbazide adduct and its
dissociation, kon and koff, become accessible.
The Ki values of dipeptide nitriles 7−16 and 22 are listed in
Table 1. On the one hand, they were all potent inhibitors of
human cathepsin S with Ki values between 33 and 200 nM. On
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Journal of Medicinal Chemistry
Brief Article
P2 position of peptidic substrates or inhibitors might occupy
the S2 pocket. Obviously, the P2 residue of inhibitors 7−16
and 22 is too extended for a favorable accommodation within
the S2 pocket of cathepsin F.
Scheme 2. Synthesis of the Enantiopure Dipeptide Nitrile 22
and the Azadipeptide Nitrile 26
a
We introduced structural diversity into the P3 group of the
peptide scaffold to perform an active-site mapping with respect
to the S3 pocket of cathepsin S. The S3 binding site is defined
by three Gly motifs (62, 68, 69) and the backbone structure of
Asn63. The sides of this shallow pocket are shaped by the
residues Lys64 and Phe70. The flexible nature of Lys64 was
considered to allow for a less restricted interaction with a P3
group.18,20 High affinity for cathepsin S was obtained with
peptidic or peptidomimetic ligands containing monocyclic aroyl
moieties at P3 position able to interact with Lys64 and
Phe70.12,20 However, the P3 portion could be substantially
expanded without a loss of activity.14
Thus, we introduced not only monocyclic (hetero)aroyl
groups (7, 8) but also acyl substitutents composed of two
(hetero)aromatic rings (9−14) and fused aromatic systems
(15, 16). The strongest cathepsin S inhibition was achieved
with the nonextended (hetero)aroyl compounds 7 and 8,
exhibiting Ki values of 40 and 45 nM, respectively. Compounds
9−14 showed only a somewhat lower activity, provided that the
proximal aromatic ring is thiophene; the linear motif as present
in 13 was less advantageous. Noteworthy, a distal acidic
tetrazole moiety (in 12) did not produce an effect distinguish-
able from that of the phenyl or thienyl groups (in 9−11 and
14). In general, the inhibitors listed in Table 1 are selective for
cathepsin S over the other cathepsins L, K, B, and F;
compounds 7 and 8 are characterized by a selectivity ratio of
more than 500. The structure of the benzoyl derivative was
chosen to prepare the enantiopure 22.
a
Reagents and conditions: (a) (1) isobutyl bromide, tetrabutylammo-
nium iodide, NaOH, H2O, EtOH, rt; (2) (Boc)2O, NaOH, H2O,
EtOH, rt; (b) KMnO4, AcOH, H2O, rt; (c) (1) NMM, ClCO2-i-Bu,
THF, −25 °C; (2) H2NCH2CN·H2SO4, NaOH, H2O, −25 °C to rt;
(d) MeSO3H, THF, rt; (e) benzoyl chloride, DIPEA, THF, rt; (f) (1)
NMM, ClCO2-i-Bu, THF, −25 °C; (2) (NHMe)2·2HCl, NaOH, H2O,
−25 °C to rt; (g) AcCl, EtOH, MeCO2Et, rt, basic extraction; (h)
BrCN, NaOAc, MeOH, rt.
The substitution pattern of the azadipeptide nitrile 26 was
accordingly derived from 7/22. The replacement of CH by
NMe and the concomitant methylation of the P2−P1 peptide
bond resulted in an enhanced enzyme inhibition (Table 2),
which can be attributed to the formation of stabilized
isothiosemicarbazide adducts.17 However, 26 was no longer
selective for cathepsin S. We observed a fast association of
cathepsin S and inhibitor, as governed by the second-order rate
constant kon of 2 600 000 M−1 s−1. This finding indicates that
suitable P2 substituents in cathepsin S inhibiting azadipeptide
the other hand, none of them showed a notable inhibition of
cathepsin F, the functional synergist of cathepsin S. Thus, the
selected amino acid in P2 position, S-(isobutyl)cysteine sulfone,
accounts for the affinity to cathepsin S and can favorably be
accommodated in its S2 binding pocket. Gauthier et al.14
demonstrated this interaction by molecular dynamics calcu-
lations based on the crystal structure of human cathepsin S.18
The rather large S2 binding pocket of cathepsin S is lined by
the side chain of Met71 and two Gly methylene groups (137
and 165). The pocket is shaped by the side chains of Phe70 and
Val162. The S2 subsite is terminated by Phe211. Its side chain
is able to perform a conformational switch leading to a
remarkable plasticity of the S2 pocket of cathepsin S.18−20
Therefore, besides the side chains of aliphatic amino acids
preferred in P2 position of cathepsin S substrates,21,22 several
hydrophobic moieties of inhibitors can be accommodated in
the S2 pocket. Examples include fluorinated phenyl or
alkylphenyl,23,24 cyclohexyl, and 4-methylcyclohexyl groups20
and the residues of phenylalanine,18,25 (naphthalen-2-yl)-
alanine,26 leucine,18,25 cyclohexylalanine25,26 methyl-branched
cycloalkylalanines,14 (1,2,3,4-tetrahydronaphthalen-2-yl)-
alanine,27 and S-(isobutyl)cysteine sulfone.12 The last amino
acid was a particular suitable building block to achieve strong
cathepsin S inhibition and selectivity over cathepsins L, K, and
B.12
a
Table 2. Kinetic Parameters of (R)-26
cathepsin
Ki (nM)
kon (103 M−1 s−1
)
koff (10−3 s−1
)
b
c
c
L
S
15
1
nd
nd
d
0.55 0.03
0.66 0.06
2600 400
1.4 0.2
K
B
F
59
2
0.039 0.004
0.75 0.12
d
5.8 0.2
130 20
e
c
c
21
3
nd
nd
a
Data were calculated from duplicate experiments with at least five
different inhibitor concentrations. Mean values of steady-state rates
were used for nonlinear regression to obtain IC50. Mean values of
observed first-order rate constants were used for linear regression to
obtain kon/(1 + [S]/Km) values. The standard errors ( SE) of the
linear and nonlinear regression analyses are given. koff was calculated as
koff = Kikon. Cathepsins L, S, K, and B were assayed as described.29 For
Among the amino acid residues that are defining the S2
pocket, Phe70 and Phe211 of cathepsin S are replaced by Leu
and Met, respectively, in cathepsin F. The Met residue points
toward the Leu residue but does not block the pocket
entirely.28 Thus, medium-sized hydrophobic amino acids at
b
cathepsin F assay, see Supporting Information. The progress curves
were analyzed by linear regression in a time interval between 8 and 16
c
d
min. Not determined. The progress curves were analyzed by
e
nonlinear regression in a time interval of 20 min. The progress curves
were analyzed by linear regression in a time interval of 30 min.
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Journal of Medicinal Chemistry
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(R)-N-(Benzoyl)-S-(isobutyl)cysteinylsulfone-glycine-nitrile
(22). Compound 20 (0.68 g, 1.96 mmol) was dissolved in dry THF
(10 mL). Under ice-cooling, dry methanesulfonic acid (1.14 g, 11.9
mmol) was added, and the resulting mixture was stirred for 14 h at
room temperature. The precipitated product was filtered off, washed
with petroleum ether, and dried to obtain 21 as a white solid without
further purification (0.33 g). Compound 21 (0.33 g, 0.96 mmol) was
dissolved in dry THF (30 mL). DIPEA (0.35 g, 2.71 mmol) and
benzoyl chloride (0.19 g, 1.35 mmol) were added consecutively. The
solution was stirred for 24 h at room temperature. The solvent was
evaporated, and the resulting white solid was extracted with ethyl
aceate (3 × 30 mL). The combined organic layers were washed with
10% KHSO4 (30 mL) and brine (30 mL). The solvent was dried
(Na2SO4) and evaporated. The crude product was recrystallized from
ethyl acetate to obtain 22 as a white solid (0.28 g, 41% from 20).
nitriles give rise to an accelerated association. For
N‑(benzyloxycarbonyl)cyclohexylalanyl-methylazaalanine-ni-
trile a likewise high kon value was reported.29
CONCLUSION
■
We have performed a systematic scan to explore the S3 binding
pocket of cathepsin S and designed highly potent and selective
dipeptide nitrile inhibitors for this protease. Furthermore, the
optimized structure (22) was applied in the synthesis of an
analogous azadipeptide representative (26). A substrate
specificity profiling of papain-like cysteine proteases with
respect to the amino acids at positions P4−P1 revealed a
high similarity of the two functionally synergistic proteases
cathepsins S and F.21 Therefore, the selectivity profile of our
inhibitors was unexpected. Inhibition of cathepsin F was
practically not achieved by the nitrile compounds of this study,
with the exception of the unselective azadipeptide nitrile 26.
Thus, it could be demonstrated for the first time that prototype
cathepsin S inhibitors may not necessarily affect cathepsin F.
Treatment with such cathepsin S inhibitors would only partially
reduce antigen presentation, inasmuch as the cathepsin F-
catalyzed processing of the invariant chain, particularly in
macrophages, would be maintained. Future medicinal chemistry
studies will have to focus on selective inhibitors for cathepsin F,
as well as dual inhibitors for both proteases, cathepsins S and F.
ASSOCIATED CONTENT
■
S
* Supporting Information
Preparation of compounds, cathepsin F assay, chiral chromato-
grams, representative kinetic plots, H and 13C NMR spectra.
1
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +49 228 732317. E-mail: guetschow@uni-bonn.de.
■
Notes
The authors declare no competing financial interest.
EXPERIMENTAL SECTION
General methods and materials are given in the Supporting
Information. All tested compounds possessed a purity of not less
than 95% except for 12, which had 94% purity.
■
ACKNOWLEDGMENTS
■
This work was supported by the NRW International Research
School Biotech-Pharma and the German Research Foundation
(Grant GRK 804).
N-(tert-Butyloxycarbonyl)-S-(isobutyl)cysteine (18). L-Cys-
teine (17, 5.00 g, 41.3 mmol) was dissolved in a 1:1 EtOH/2 N
NaOH mixture (82 mL). Isobutyl bromide (6.22 g, 45.4 mmol) and
tetrabutylammonium iodide (0.46 g, 1.25 mmol) were added. The
mixture was stirred for 3 days at room temperature. (Boc)2O (9.91 g,
45.4 mmol) was added, and the mixture was additionally stirred for 1
day at room temperature. EtOH was evaporated under reduced
pressure. The aqueous residue was acidified with concentrated HCl
(pH ≈ 1) and extracted with ethyl acetate (3 × 30 mL). The
combined organic layers were washed with 10% KHSO4 (30 mL) and
saturated NaCl (30 mL). The solvent was dried over Na2SO4 and
evaporated to obtain 18 as an oily yellow product (10.9 g, 95%).
N-(tert-Butyloxycarbonyl)-S-(isobutyl)cysteinylsulfone-gly-
cine-nitrile (20). Compound 18 (10.8 g, 38.9 mmol) was dissolved in
AcOH (80 mL). KMnO4 (12.3 g, 77.8 mmol) was dissolved in H2O
(130 mL) and slowly added to the reaction mixture. It was stirred for
2.5 h, followed by the addition of saturated KHSO3 solution until the
mixture became colorless. It was concentrated under reduced pressure,
and the aqueous suspension was extracted with ethyl acetate (3 × 100
mL). The combined organic layers were washed with H2O (30 mL),
brine (30 mL) and dried over Na2SO4. The solvent was evaporated to
obtain 19 as a colorless oily product (11.1 g, 92%). Compound 19
(2.00 g, 6.46 mmol) was dissolved in dry THF (40 mL), and the
mixture was cooled to −25 °C. N-Methylmorpholine (0.72 g, 7.12
mmol) and isobutyl chloroformate (0.97 g, 7.10 mmol) were added
consecutively. Aminoacetonitrile monosulfate (1.47 g, 9.54 mmol) was
dissolved in H2O (2 mL), treated with 2 N NaOH (5 mL), and added
to the mixture when the precipitation of N-methylmorpholinium
chloride occurred. It was allowed to warm to room temperature within
30 min and stirred for additional 90 min. THF was evaporated, and the
resulting aqueous suspension was extracted with ethyl acetate (3 × 30
mL). The combined organic layers were washed with 10% KHSO4 (30
mL), saturated NaHCO3 (30 mL), H2O (30 mL), and saturated NaCl
(30 mL). The solvent was dried (Na2SO4) and evaporated. The
product was recrystallized from ethyl acetate to obtain 20 as a white
solid (0.94 g, 42% from 19).
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