(4-Piperidinylphenyl)aminoethyl amides as a novel class of non-covalent cathepsin K inhibitors

Article abstract of DOI:10.1016/j.bmcl.2003.10.009

A series of (4-piperidinylphenyl)aminoethyl amides based on dipeptide anilines were synthesized and tested against cathepsin K, cathepsin L and cathepsin B. These new non-covalent inhibitors exhibited single-digit nM inhibition of the cysteine proteases. Compounds 3 and 7 demonstrated potency in both mouse and human osteoclast resorption assays.

Full text of DOI:10.1016/j.bmcl.2003.10.009

Bioorganic & Medicinal Chemistry Letters 14 (2004) 87–90
(4-Piperidinylphenyl)aminoethyl amides as a novel class
of non-covalent cathepsin K inhibitors
Tae-Seong Kim,* Andrew B. Hague, Tony I. Lee, Brian Lian, Christopher M. Tegley,
Xianghong Wang, Teresa L. Burgess, Yi-Xin Qian, Sandra Ross, Philip Tagari,
Chi-Hwei Lin, Carol Mayeda, Jennifer Dao, Steven Jordan, Christopher Mohr,
Janet Cheetham, Vellarkad Viswanadhan and Andrew S. Tasker*
Department of Chemistry Research and Development, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320-1799, USA
Received 10 June 2003; revised 4 October 2003; accepted 6 October 2003
Abstract—A series of (4-piperidinylphenyl)aminoethyl amides based on dipeptide anilines were synthesized and tested against
cathepsin K, cathepsin L and cathepsin B. These new non-covalent inhibitors exhibited single-digit nM inhibition of the cysteine
proteases. Compounds 3 and 7 demonstrated potency in both mouse and human osteoclast resorption assays.
# 2003 Elsevier Ltd. All rights reserved.
Cathepsin K is a recently discovered cysteine protease
that is highly and selectively expressed in bone, osteo-
clast cells, osteoclast resorbing cartilage, osteoarthritic
synovium, and ovary.1,2 This enzyme is found to effi-
ciently degrade both telopeptide and triple helical
regions of type I collagen as well as native type II col-
lagen,3 which strongly suggests its possible involvement
in bone and cartilage breakdown in diseases such as
osteoporosis, osteoarthritis, rheumatoid arthritis,
hypercalcemia of malignancy, and Paget’s disease.
Studies of knock-out mouse models and mutations in
the human cathepsin K gene have demonstrated its cri-
tical role in bone resorption.4,5 Slowing or blocking the
bone resorption process by inhibiting cathepsin K could
potentially treat human diseases.
a series of (4-piperidinylphenyl)aminoethyl amides as a
novel non-covalent class of cathepsin K inhibitors.8
Based on inhibitory activities of dipeptidylaldehydes
such as Cbz-Leu-Phe-CHO (Ki, 0.020 mM) and Cbz-
Leu-Met-CHO (Ki, 0.013 mM) against cathepsin L,9 we
investigated replacement of the aldehyde function with
non-reactive chemical moieties. A dipeptidyl derivative
1, where aniline replaced the aldehyde group, showed an
IC50 of 1.1 mM against human recombinant cathepsin K
and an IC50 of 0.230 mM against cathepsin L. Further
derivatization of the aniline group furnished more
potent cathepsin K inhibitors (Table 1). The 4-piper-
idinyl aniline derivative 3 had an IC50 of 0.010 mM
against cathepsin K and 0.002 mM against cathepsin L.
To our surprise, potency of 3, a non-covalent inhibitor
against cathepsin L is similar to that of the reactive and
covalent inhibitor Cbz-Leu-Phe-CHO (IC50, 0.005 mM).
Cysteine protease inhibitors have been studied for the
purpose of finding therapeutics for a variety of other
diseases.6,7 However, the majority of these inhibitors
form a covalent bond between the thiol, at the active
site cysteine, and the reactive functional groups of inhi-
bitors such as aldehydes, ketones, haloketones, diazo-
Potency against cathepsin K was retained when phenyl-
alanine (compound 3) was replaced for homo-
phenylalanine (compound 8). For further optimization,
we focused our attention on the amino acid component.
Subsequent replacement of leucine with other amino
acids resulted in discovering not only potent cathepsin
K and L inhibitors but also more importantly selective
cathepsin K inhibitors. In particular, alanine and pro-
line derivatives 6 and 14 showed greater than 200-fold
selectivities over cathepsins L and B. The results are
ketones,
epoxides,
nitriles,
vinylsulfones
and
ketoamides, which may be a potential liability in toxi-
city and delivery to the targeted area. Herein, we report
Keywords: Non-covalent; cathepsin; inhibitor.
* Corresponding author. Tel.: +1-805-447-8895; fax: +1-805-480-
3016; e-mail: tkim@amgen.com
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.10.009

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T.-S. Kim et al. / Bioorg. Med. Chem. Lett. 14 (2004) 87–90
summarized in Table 2. These data support that
cathepsin K allows residues with a small side chain for
hydrophobic interaction, but cathepsin L prefers resi-
dues with a branched or larger side chain.
To address the ability of these compounds to inhibit
bone resorption, 3 and 7 were evaluated in murine10 and
human osteoclast resorption assays.11 These compounds
showed inhibitory activities in both mouse (3, 7: 1 mM,
0.330 mM) and human (3, 7: 1 mM, 0.560 mM) bone
resorption assays, and IC50 values were determined by
The synthesis of the compounds 1–14 is straightforward
and shown in Scheme 1. Anilines were reacted with
t-Boc-phenylalanine or t-Boc-homophenylalanine under
standard amide coupling conditions to give amide
15a–e. Selective reduction of the amide carbonyl group
in 15a–e with borane provided the corresponding ani-
lines. Exposure of the anilines to acidic conditions
furnished 16a–e, which were then coupled with Cbz-
protected amino acids to afford dipeptidyl anilines 1–14.
Table 2. Inhibition of dipeptidyl 4-piperidylanilines against cathep-
sins
Table 1. Inhibition of dipeptidyl anilines against recombinant human
cathepsins
Compd
AA
(amino acid)
Cat K
(IC50, mM)a
Cat L
(IC50, mM)b
Cat B
(IC50, mM)c
5
6
7
8
Gly
Ala
Val
Leu
Met
Phe
Tyr
HPhe
Trp
Pro
>10.00
0.009
0.004
0.005
0.003
0.003
0.008
0.014
0.048
0.012
>10.00
4.110
0.001
0.002
0.036
0.001
0.001
0.009
0.001
7.844
>10.00
2.094
0.007
0.658
0.012
0.167
0.106
9.664
3.362
>10.00
9
10
11
12
13
14
Compd
R
Cat K (IC50, mM)a
Cat L (IC50, mM)b
1
2
3
4
H
Benzene
Piperidine
Morpholine
1.100
0.706
0.010
0.015
0.230
0.032
0.002
0.002
a Recombinant human cathepsins K (3.3 nM) was used in a fluores-
cence assay using 20 mM Cbz-Phe-Arg-AFC as substrate in 0.45 M
NaOAc (pH 5.2), 4.8 mM EDTA, 0.1% Triton X-100, 8 mM DTT.
b Recombinant human cathepsins L (1.2 nM): was used in a fluores-
cence assay using 20 mM Cbz-Phe-Arg-AFC as substrate in 0.45 M
NaOAc (pH 5.2), 4.0 mM EDTA, 0.1% Triton X-100, 8 mM DTT.
c Recombinant human cathepsins B was used in a fluorescence assay
using 20 mM Cbz-Phe-Arg-AFC as substrate in 0.45 M NaOAc (pH
5.2), 4.0 mM EDTA, 0.1% Triton X-100, 8 mM DTT.
a Recombinant human cathepsins K (3.3 nM) was used in a fluores-
cence assay using 20 mM Cbz-Phe-Arg-AFC as substrate in 0.45 M
NaOAc (pH 5.2), 4.8 mM EDTA, 0.1% Triton X-100, 8 mM DTT.
b Recombinant human cathepsins L (1.2 nM): was used in a fluores-
cence assay using 20 mM Cbz-Phe-Arg-AFC as substrate in 0.45 M
NaOAc (pH 5.2), 4.0 mM EDTA, 0.1% Triton X-100, 8mM DTT.
Scheme 1.

T.-S. Kim et al. / Bioorg. Med. Chem. Lett. 14 (2004) 87–90
89
Figure 1. Effect of cathepsin K inhibitor 3 on mouse bone resorption assay: (a) untreated bone slice (BMC); (b) DMSO; (c) inhibitor at 1 mM;
(d) inhibitor at 5 mM. Toluidine blue stained bone slices show inhibition of resorption pit formation (Â10 magnification).
reading the C-terminal telopeptides (CTX) of type I
collagen released. As shown in Figure 1, compound 3
partially inhibited pit formation at 1 mM and completely
inhibited at 5 mM.
Acknowledgements
We acknowledge Dr. Yining Zhao for high resolution
mass spectrometry analysis and thank Dr. Randy Hun-
gate and Dr. Nuria Tamayo for helpful discussion.
With potent and selective cathepsin K inhibitors in
hand, we investigated their kinetic profiles. Simple
Lineweaver–Burk analyses of 3 using Cbz-Phe-Arg-
AFC as a substrate showed it to be a competitive inhi-
bitor. In addition, reversibility of these inhibitors was
demonstrated by the recovery of activity of inhibited
enzyme after dialysis and dilution.
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