Dipeptide nitrile inhibitors of cathepsin K

Article abstract of DOI:10.1016/j.bmcl.2006.01.104

A series of dipeptidyl nitriles as inhibitors of cathepsin K have been explored starting from lead structure 1 (Cbz-Leu-NH-CH₂-CN, IC ₅₀ = 39 nM). Attachment of non-natural amino acid side chains in P1 and modification of the P3 subu

Full text of DOI:10.1016/j.bmcl.2006.01.104

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2549–2554
Dipeptide nitrile inhibitors of cathepsin K
Eva Altmann,* Reiner Aichholz, Claudia Betschart, Thomas Buhl, Jonathan Green,
´
Rene Lattmann and Martin Missbach
Novartis Institutes for BioMedical Research, CH-4002 Basel, Switzerland
Received 20 December 2005; revised 16 January 2006; accepted 18 January 2006
Available online 9 February 2006
Abstract—A series of dipeptidyl nitriles as inhibitors of cathepsin K have been explored starting from lead structure 1 (Cbz–Leu–
NH–CH2–CN, IC50 = 39 nM). Attachment of non-natural amino acid side chains in P1 and modification of the P3 subunit led to
inhibitors with higher potency and improved pharmacokinetic properties.
Ó 2006 Elsevier Ltd. All rights reserved.
Bone remodeling is a complex and important process in
the physiology of the skeleton. It is a tightly coupled
process which is based on a precisely tuned interplay be-
tween bone resorbing osteoclasts and bone forming
osteoblasts.1,2 An imbalance in bone remodeling due
to increased bone resorption will lead to reduced bone
mass and disturbed microarchitecture causing bone fra-
gility and fractures. Therapeutic inhibition of excessive
bone resorption can be achieved in multiple ways. One
possibility is to modulate the resorptive activity of the
osteoclasts without affecting bone formation. Bone
resorption by osteoclasts occurs in two distinct steps
namely by first demineralization of the bone matrix
via acid secretion and in a second phase by degradation
of the organic bone matrix via proteolytic enzymes.
Cathepsin K is the major osteoclastic protease3 and
has been proposed to play a central role in the degrada-
tion of type I collagen and other important components
of the bone matrix.4 Thus, specific inhibition of cathep-
sin K should substantially decrease bone resorption and
therefore may offer an efficacious treatment for diseases
characterized by excessive bone loss such as
osteoporosis.
co-workers.6 Three recent reports on peptidic nitrile
inhibitors for cathepsins S7, B,8 and K9 now prompt
us to disclose our results in this area.
O
H
O
N
N
H
N
O
1
Our initial studies aimed at exploring the effects of
attaching an extended P1-substituent on potency and
on the metabolic stability. These experiments were
driven on the assumption that non-natural amino acid
side chains in P1 would increase metabolic stability
without negatively impacting the inhibitory potency.
In a second round of optimization, we investigated
the influence of the replacement of the P3 benzyloxy-
carbonyl group by different acyl moieties on potency,
on the selectivity profile, and on physico-chemical
properties. Finally, in vitro metabolism and pharmaco-
kinetic properties of the most potent inhibitors were
assessed to have an integral understanding of the
drug-like properties of this series.
As part of our efforts to identify potent and reversible
inhibitors of cathepsin K, we have explored a series of
dipeptide nitriles starting from our lead structure 1
(Cbz–L-Leu–NH–CH2–CN, IC50 cathepsin K =
39 nM).5 Peptide nitriles as covalent, reversible inhibi-
tors of papain were first described by Hanzlik and
The synthesis of Cbz-protected dipeptide nitrile cathep-
sin K inhibitors is outlined in Schemes 1–3. Depending
on the commercial availability of the amino acid
precursors, different approaches were followed. Dipep-
tide nitriles 5a, 5b, and 5e were prepared starting from
the Boc-protected amino acids 2a, 2b and 2e (Scheme
1). The mixed anhydride of the Boc-protected amino
acids 2a, 2b, and 2e with isobutyl-chloroformate was
quenched with aqueous NH3 to provide the amide
*
Corresponding author. Tel.: +41 61 696 1227; fax: +41 61 696
6071; e-mail: eva.altmann@novartis.com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.01.104

2550
E. Altmann et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2549–2554
a method originally described by Kricheldorf,11 involv-
R
O
R
O
a, b
c
ing the in situ silylation of the C-terminal amino acids
9c, 9h and subsequent reaction with Cbz–Leu–OSu.
The conversion to the target nitriles 5c, 5h was carried
out utilizing the same strategy as depicted in Scheme 1.
OH
O
NH2
O
N
H2N
H
O
3a-b, 3e
2a-b, 2e
R
O
R
O
H
H
d
O
N
NH2
O
N
N
N
Preparation of compounds 16a–h starts with the in situ
silylation of O-benzyl-L-serine 9h, followed by reaction
with Boc–Leu–OSu to yield dipeptide 12. The mixed
anhydride of the Boc-protected dipeptide 12 with isobu-
tyl-chloroformate was quenched with aqueous NH3 to
furnish the amide derivative 13, which was converted
to the nitrile compound 14 by (CF3CO)2O-mediated
dehydration.10 Removal of the Boc group was carried
out with neat HCOOH and the resulting amine 15 was
coupled with the appropriate carboxylic acid to yield
the target dipeptide nitriles 16a–h (Scheme 4).
N
H
H
O
O
4a-b, 4e
5a-b, 5e
Scheme 1. Reagents and conditions: (a) 1—Isobutyl-chloroformate,
N-methylmorpholine, THF, ꢀ15 °C, 15 min, 2—NH3 (aq), 88–96%;
(b) 4 N HCl/dioxane, rt, 6 h, 88–93%; (c) Cbz–Leu–OSu, DIEA,
CH2Cl2, rt, 16 h, 65–88%; (d) (CF3CO)2O, Et3N, THF, ꢀ15 °C, 2 h,
61–78%.
R
O
R
O
H
O
N
O
a
b
N
H
O
We first investigated the effects of attaching a P1-substi-
tuent to the a-position of the C-terminal Gly-nitrile moi-
ety of our lead 1 on potency and selectivity for cathepsin
K inhibition. In an attempt to exploit hydrophobic
interactions within the lipophilic S1 subsite of cathepsin
K, a series of compounds containing phenylalanine-de-
rived P1 side chains were investigated. The biological
activity of these analogs is summarized in Table 1. A
4-substituent on the phenyl ring proved favorable in
terms of potency against cathepsin K and also had a po-
sitive effect on the specificity profile of the inhibitors.
Compounds 5a and 5f incorporating a 4-methyl or a
4-trifluoromethyl-phenylalanine moiety were the most
potent and selective inhibitors of cathepsin K within
H2N
O
O
7d, 7f-g
6d, 6f-g
R
O
R
O
O
c
H
H
O
N
NH2
O
N
N
H
N
H
N
O
O
8d, 8f-g
5d, 5f-g
Scheme 2. Reagents and conditions: (a) Cbz–Leu–OSu, DIEA,
CH2Cl2, rt, 16 h, 92–100%; (b) NH3/MeOH, rt, 18 h, 61–84%; (c)
(CF3CO)2O, Et3N, THF, ꢀ15 °C, 2 h, 52–78%.
R
O
R
H
O
N
OH
a
b
N
OH
H
H2N
O
O
O
9c, 9h
10c,10h
O
O
a
b
O
O
O
H
R
O
R
O
N
OH
O
O
c
N
H
H
H
OH
O
N
NH2
O
N
H2N
O
N
N
N
H
H
O
11c, 11h
O
9h
12
5c, 5h
Scheme 3. Reagents and conditions: (a) 1—TMSiCl, DIEA, CH2Cl2,
rt, 1 h, 2—Cbz–Leu–OSu, DIEA, rt, 6 h, 50–63%; (b) 1—Isobutyl-
chloroformate, N-methylmorpholine, THF, ꢀ15 °C, 15 min, 2—NH3
(aq), 62–96%; (c) (CF3CO)2O, Et3N, THF, ꢀ15 °C, 2 h, 42–55%.
O
O
O
d
c
O
O
H
H
N
O
N
NH2
O
N
H
N
H
N
O
O
13
O
14
derivatives which upon removal of the Boc group (4 N
HCl in dioxane), gave the amines 3a, 3b, and 3e. Subse-
quent reaction with the N-hydroxysuccinimide ester of
Cbz–Leu (Cbz–Leu–OSu) yielded the dipeptide amides
4a, 4b, and 4e. Conversion to the target nitriles 5a, 5b,
and 5e was achieved by (CF3CO)2O-mediated dehydra-
tion10 of 4a, 4b, and 4e. Scheme 2 summarizes the syn-
thesis of 5d and 5f–g. The dipeptides 7d, 7f–g were
obtained by coupling of Cbz–Leu–OSu with amino acid
esters 6d, 6f–g. Treatment of the methylester with
ammonia afforded amides 8d, 8f–g, which were dehy-
drated with (CF3CO)2O to produce the nitriles 5d, 5f–g.
O
e
O
O
H
H2N
R
N
N
N
H
N
N
H
O
15
16a - h
Scheme 4. Reagents and conditions: (a) 1—TMSiCl, DIEA, THF, rt,
1 h, 2—Boc–Leu–OSu, DIEA, rt, 6 h, 96%; (b) 1—Isobutyl-chloro-
formate, N-methylmorpholine, THF, ꢀ15 °C, 15 min, 2—NH3 (aq),
97%; (c) (CF3CO)2O, Et3N, THF, ꢀ15 °C, 2 h, 70%; (d) HCOOH, rt,
18 h, 54%; (e) RCOOH, (benzotriazol-1-yloxy)-tris-(dimethylamin)-
phosphonium-hexafluorophosphate (BOP), DIEA, CH2CL2, rt, 16 h,
41–87%.
The preparation of 5c and 5h is depicted in Scheme 3.
The dipeptides 10c, 10h were synthesized according to

E. Altmann et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2549–2554
2551
Table 1. Inhibition of homologous cathepsins by compounds 5a–h
in nature and therefore polar substituents should be tol-
erated as part of the P3 subunit, which might in fact al-
low to fine-tune the physico-chemical properties of our
inhibitors. As illustrated by the data summarized in
Table 2 this strategy afforded several highly potent
cathepsin K inhibitors (16c, 16e, and 16f). Replacement
of the benzyloxycarbonyl group by a 4-phenyl-piperi-
R
O
H
O
N
N
H
N
O
Compound
R
IC50 (nM)
din-1-yl)-acetyl moiety led to
a loss in potency
Cat Ka Cat Lb
Cat Sc
(5h ! 16a). The introduction of a 4-pyridin-4-yl-pipera-
zin-1-yl)-acetyl moiety resulted in a 9 nM inhibitor, but
16b is significantly less specific for cathepsin K than
5a
5b
5c
5d
5e
5f
48
9900
7700
1100
1400
>10,000
120
63
2300
Table 2. Inhibition of homologous cathepsins by compounds 16a–h
O
O
700
O
O
H
R
N
398
>1000
43
>10,000
N
H
N
O
O
> 10,000 >10,000
Compound
R
IC50 (nM)
Cat Lb
F
F
F
Cat Ka
Cat Sc
140
8000
>10,000
N
16a
16b
83
1200
330
O
O
5g
5h
240
9
2800
1700
900
969
N
N
9
180
N
a Inhibition of recombinant human (rh) cathepsin K activity in a
fluorescence assay using 48 lM Cbz–Phe-Arg–AMC as substrate in
100 mM NaH2PO4, 1 mM EDTA, 20 lM Tween 20, and 2 mM
DTT, pH 7.
b Inhibition of rh cathepsin L activity using 3 lM Cbz–Phe-Arg–AMC
as substrate in 100 mM NaOAc, 1 mM EDTA, 0.005% Brig 35, and
1 mM DTT, pH 5.5.
c Inhibition of rh cathepsin S activity using 11 lM Cbz–Leu-Leu-Arg–
AMC as substrate in 100 mM NaOAc, 1 mM EDTA, 0.01% Triton
X-100, 1 mM DTT, pH 5.5. Data represent means of two experi-
ments performed in duplicate, individual data points in each exper-
iment were within a 3-fold range of each other.
Cl
16c
16d
16e
<1
430
<1
<30
190
154
52
41
29
O
Cl
N
H
N
H
16f
<1
83
47
N
the phenylalanine series. However, increasing the steric
bulk of the 4-substituent led to a gradual loss in potency
for compounds 5a ! 5c ! 5d ! 5e. A 3-substituent
resulted in a less potent analog 5a ! 5b. Compounds
5g and 5h are inhibitors incorporating O-protected L-
serine derivatives as P1-subunit. Whereas the O-tert-
butyl-ether derivative 5g is not very active, compound
5h, incorporating an O-benzyl serine P1 moiety, is the
most potent inhibitor investigated within the Cbz-pro-
tected dipeptide nitrile series. In addition, 5h exhibits
good specificity for cathepsin K over the two highly
homologous cathepsins L (K/L = 190) and S (K/S = 107).
16g
16h
24
110
660
130
240
HN
N
N
N
190
a Inhibition of recombinant human (rh) cathepsin K activity in a
fluorescence assay using 48 lM Cbz–Phe-Arg–AMC as substrate in
100 mM NaH2PO4, 1 mM EDTA, 20 lM Tween 20, and 2 mM
DTT, pH 7.
b Inhibition of rh cathepsin L activity using 3 lM Cbz–Phe-Arg–AMC
as substrate in 100 mM NaOAc, 1 mM EDTA, 0.005% Brig 35, and
1 mM DTT, pH 5.5.
c Inhibition of rh cathepsin S activity using 11 lM Cbz-Leu-Leu-Arg-
AMC as substrate in 100 mM NaOAc, 1 mM EDTA, 0.01% Triton
X-100, and 1 mM DTT, pH 5.5. Data represent means of two
experiments performed in duplicate, individual data points in each
experiment were within a 3-fold range of each other.
Based on the most potent inhibitor 5h we embarked on a
second round of optimization that involved replacement
of the benzyloxycarbonyl group by different acyl moie-
ties. The S3 subsite of cathepsin K is rather hydrophilic

2552
E. Altmann et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2549–2554
compound 5h. A 4-chlorophenoxy-2-methyl-propionyl
P3 group afforded the sub-nanomolar inhibitor 16c.
However, this compound features a poor specificity pro-
file. Surprisingly, compound 16d, which differs from 16c
only by the replacement of the phenoxy- by a phenyla-
mino moiety, shows a reversed selectivity profile in favor
of cathepsin S. An unsubstituted and 1-methyl-indolyl
moiety provide analogs 16e and 16f, both of which show
also sub-nanomolar potency. These two compounds
exhibit moderate specificity, in particular with regard
to cathepsin S.12
administration of a potential drug candidate. Com-
pounds with promising in vitro potencies were thus
evaluated in in vitro metabolism assays and their phar-
macokinetic properties were determined in a cassette
dosing experiment. The focus of these studies was to
reveal potential weak points within the series to guide
further modifications.
Metabolic stability of compounds 5h, 16b, 16c, 16f, and
16g was assessed in vitro in rat or human liver micro-
somes as well as in rat plasma. Capillary HPLC/MS–
MS was used to determine remaining parent compound
and characterize potential metabolites.13 Figure 1 shows
the selected ion chromatogram of 5h after 1 h incubation
with rat liver microsomes.
The two imidazolyl-acetyl-based analogs 16g and 16h
are essentially non-specific with a maximal specificity
ratio of 5-fold in favor of cathepsin K.
One of the main challenges in the optimization of pep-
tidic lead structures is to generate compounds with good
pharmacokinetic properties, in order to allow for oral
The metabolism of all five compounds investigated
followed a similar pattern which is exemplified in
Figure 1 for compound 5h. In all cases, O-debenzylation
OH
O
H
N
O
N
H
N
O
c
a
m3
OH
O
H
N
O
O
O
H
N
H
N
O
N
O
N
H
N
O
HO
m1, m2
b
5h
d
O
O
H
N
O
N
H
N
O
HO
m4, m5
+
Figure 1. Proposed metabolic pathways of 5h and selected ion chromatogram over M+NH4 of 5h (m/z 441), m4/m5 (m/z 457), m3 (m/z 351), and
m1/m2 (m/z 367), 1 h after incubation of 5h in rat liver microsomes (* peaks are not related to 5h).

E. Altmann et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2549–2554
2553
70.0
of the serine side chain was observed (pathway a) and
0.15
for 16b this was the only transformation detected. For
5h, 16c, 16f, and 16g additional metabolites were formed
through hydroxylation of the isobutyl side chain of leu-
cine (pathway b; benzyl group still intact). A metabolite
arising from both debenzylation and leucine hydroxyl-
ation was only observed for 5h (pathways c or d).
Metabolite patterns were similar between microsomes
from rat and human.
60.0
50.0
40.0
30.0
20.0
10.0
0.0
0.10
0.05
0.00
In addition to liver metabolism, plasma stability plays
an important role in drug discovery, as compounds with
insufficient plasma stability tend to exhibit rapid clear-
ance and short half-lives, which are associated with poor
in vivo performance. We have thus investigated the sta-
bility of compounds 1, 5h, 16b, 16c, 16f, and 16g in
freshly prepared rat plasma.14 Lead compound 1 was
degraded to a significant extent after 1 h (>50%).15 In
contrast, the optimized inhibitors proved to be com-
pletely stable, which validates our initial working
hypothesis about the beneficial effects of extended
P1-substituents on metabolic stability.
30 60
120 180 240
360
480
1440
time (min)
Figure 2. Blood levels of unchanged 5h (d) and 16g (s) after po
administration (3.0 mg kgꢀ1) to conscious rats (n = 4). Dosing was in a
cassette format together with four other compounds.
absorption and a reduced first-pass effect, also led to a
substantial improvement in oral bioavailability for these
two compounds (F = 26.4% and 30.1%, respectively),
compared to our lead 1 (F = 5%).
Pharmacokinetic profiles in rats were determined in a
cassette dosing experiment with six different com-
pounds, including 1, which had been previously tested
separately in a discrete dosing experiment, thus serving
as a reference compound (no significant difference in F
and other pharmacokinetic parameters between cassette
and discrete dosing was observed). Pharmacokinetic
data obtained in the cassette dosing experiment16 are
summarized in Table 3. The highly soluble compounds
16b and 16g are extremely rapidly cleared from blood
after iv administration and exhibit very low oral bio-
availability (3.5% and 4.6%, respectively). We assume
that after po dosing, due to their high solubility in the
acidic gastric environment, 16b and 16g may be partially
degraded by proteolytic enzymes already in the stomach
and upper gastrointestinal tract. Data on blood levels
and the resulting very low Cmax po for 16g are shown
in comparison to 5h in Figure 2. High total clearance
and low oral bioavailability are also observed for the
less soluble compound 16c, however, in contrast to
16b and 16g, this might occur via first-pass metabolism.
A distinct reduction in total clearance resulted in a
significant prolongation of terminal half-lives for inhib-
itors 5h and 16f. This, probably together with improved
In summary, we have identified a series of potent dipep-
tide nitrile inhibitors of cathepsin K with considerable
metabolic stability. Starting from lead structure 1 varia-
tions in the P1 and P3 substituents led to new analogs,
which proved to be highly potent enzyme inhibitors.
For several of these compounds, their in vitro metabolism
was assessed in liver microsomes and in plasma, and the
major metabolites were identified by HPLC/MS–MS.
All optimized compounds proved to be completely stable
in rat plasma. The pharmacokinetic properties of the best
analogs were determined and for 16f oral bioavailability
was improved to 30% up from 5% for our initial lead
structure 1. The data presented in this study set the stage
for additional medicinal chemistry efforts on dipeptide ni-
trile inhibitors of cathepsin K, and also other cathepsins,
with even further enhanced properties.
Acknowledgments
We thank Werner Gertsch, Doris Huerzeler, Philippe
Ramstein, Jean-Louis Runser, Gerard Schreiber, and
Hans Zihlmann for excellent technical assistance.
Table 3. Rat pharmacokinetic data for compounds 1, 5h, 16b–c, and 16f–g
CLa (mL minꢀ1 kgꢀ1
)
Vss (L kgꢀ1
)
Fe (%)
b
c
t1/2 term (h)
d
Cmax po (nM)
Compound
1
5h
243 27
45 12
3.5 0.5
4.6 1.4
18.0 1.2
13.6 3.0
7.7 3.0
4.3 0.9
0.2 0.03
2.2 0.4
1.0 0.1
3.1 0.5
1.4 0.4
0.2 0.1
5.4 2.5
44.3 12.0
2.5 0.6
5.2 2.9
26.4 12.7
3.5 0.9
16b
16c
16f
16g
320 78
141 26
82 37
3.5 1.0
51.1 26.1
11.3 2.0
10.1 5.2
30.1 9.5
4.6 1.6
396 94
a Total clearance.
b Volume of distribution at steady state.
c Terminal half life for elimination (non-compartmental estimate).
d Maximal blood concentration (dose-normalized to 1 mg kgꢀ1).
e Oral bioavailability.

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E. Altmann et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2549–2554
10. Campagna, F.; Carotti, A.; Casini, G. Tetrahedron Lett.
1977, 21, 1813.
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IC50 values were also determined against cathepsin B
and found to be >100 nM (selectivity ratio CatK/CatB
ꢁ100).
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our experimental conditions.
16. PK cassette-dosing in rats. The experiment was performed
in conscious, fed, permanently cannulated rats kept under
standard conditions. For iv bolus administration, all
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PEG200 (30:70, v/v; 0.5 mL kgꢀ1) and dosed at 1 mg kgꢀ1
.
For oral administration compounds were dissolved/sus-
pended and sonicated in a solution of tartaric acid (1%)
with carboxymethyl cellulose (1%) in bidistilled water
(2.5 mL kgꢀ1) and administered at 3 mg kgꢀ1. Blood
samples were collected from the femoral artery for 24 h
after iv and after oral dosing. Iv and po administration
was carried out in the same animals with a 48 h washout
period between administrations. After acetonitrile precip-
itation of plasma samples, parent drug concentrations in
extracts were measured using
method.
a specific HPLC/MS