Arylaminoethyl amides as novel non-covalent cathepsin K inhibitors

Article abstract of DOI:10.1021/jm010801s

A series of N_α-benzyloxycarbonyl- and N_α-acyl-L-leucine(2-phenylaminoethyl)amide derivatives were prepared and evaluated for their inhibitory activity against rabbit and human cysteine proteases cathepsins K, L, and S. These data ind

Full text of DOI:10.1021/jm010801s

2352
J . Med. Chem. 2002, 45, 2352-2354
Ta ble 1. Inhibition of Rabbit Cathepsin K
Ar yla m in oeth yl Am id es a s Novel
Non -Cova len t Ca th ep sin K In h ibitor s
Eva Altmann,*,† J ohanne Renaud,† J onathan Green,†
David Farley,† Brian Cutting,‡ and Wolfgang J ahnke‡
a
compd
R
IC50 (µM)
Arthrithis & Bone Metabolism Therapeutic Area and
Central Technologies, Novartis Pharma Research,
CH-4002 Basel, Switzerland
1a
1b
1c
1d
1e
1f
H
0.47
0.46
0.19
>1
0.35
>1
0.06
3-Me
4-Me
3-Cl
4-Cl
3-OMe
4-OMe
Received J anuary 2, 2001
Abstr a ct: A series of NR-benzyloxycarbonyl- and NR-acyl-L-
leucine(2-phenylaminoethyl)amide derivatives were prepared
and evaluated for their inhibitory activity against rabbit and
human cysteine proteases cathepsins K, L, and S. These data
indicate that NR-acyl-R-amino acid-(arylaminoethyl)amides
represent a new class of selective non-covalent inhibitors of
cathepsin K. Compounds 4b, 4e, and 4g exhibit high potency
toward rabbit and human cathepsin K (IC50 < 0.006 µM) and
are characterized by an excellent selectivity profile vs human
cathepsins L and S.
1g
a
Inhibition of recombinant rabbit cathepsin K in a fluorescence
assay. Data are means of two experiments performed in duplicate.
Sch em e 1a
Cathepsin K is a cysteine protease of the papain
superfamily that is highly and selectively expressed in
osteoclasts.1,2 Osteoclasts are highly specialized multi-
nuclear cells that solubilize bone through acid and
protease secretion. The fact that cathepsin K is the
predominant cathepsin in osteoclasts1,2 suggests that
this enzyme may play an essential role in bone resorp-
tion. Cathepsin K exhibits potent collagenolytic activity
against type I collagen and is involved in the degrada-
tion of other components of the extracellular bone
matrix.3,4 Cathepsin K deficient mice develop osteo-
petrosis,5,6 confirming the importance of this enzyme for
bone remodeling. Likewise, pycnodysostosis, a rare
human disease characterized by bone abnormalities due
to impaired osteoclastic bone resorption, has been linked
to defects in the gene encoding cathepsin K.7-9 Thus,
selective inhibitors of cathepsin K may offer an effica-
cious treatment for diseases characterized by excessive
bone loss such as osteoporosis.
a
Reagents: (a) 2-oxazolidinone, 2-(2-methoxyethoxy)ethanol,
160-175 °C, 16-21 h, 34-60%; (b) Z-Leu-OSu, DMF, DIEA, room
temp, 16 h, 41-83%. For specific structures, see Table 1.
Sch em e 2a
Investigations of arylaminoethyl amides as potential
cathepsin K inhibitors started with NR-benzyloxycar-
bonyl-L-leucine(2-phenylaminoethyl)amide (1a ) as a
lead structure (Table 1). Compound 1a inhibits rabbit
cathepsin K with an IC50 of 470 nM. We were intrigued
by the fact that this compound does not contain any
electrophilic group that could covalently interact with
the cysteine residue at the active site, in contrast to
other cysteine protease inhibitors described so far.10 We
thus embarked in the preparation of various aminoethyl
amide derivatives to get further insight into this series
of compounds.
The synthesis of cathepsin K inhibitors related to lead
structure 1a is summarized in Schemes 1 and 2.
An efficient entry into the preparation of the 2-ami-
noethylanilines 3a -g is the decarboxylative ring open-
ing of oxazolidin-2-one with an aromatic amine (Scheme
a
Reagents: (a) H2, Pd/C (10%), MeOH, room temp, 2 h, 100%;
(b) R-COOH, BOP (4a , 4b, 4g, 4h ), or HATU (4c-f), DIEA, DMF,
room temp, 3-16 h, 34-75%. For specific structures, see Table 2.
1).11 Subsequent reaction of 3 with Z-Leu-OSu furnished
NR-Z-protected amides 1a -g.
The preparation of 4a -h was achieved by catalytic
hydrogenation of 1g, followed by coupling of the result-
ing amine with the appropriate benzoic or phenyltri-
azole carboxylic acid (Scheme 2).
In the first optimization cycle, we investigated the
effects of substituents on the anilinophenyl ring on
inhibitory activity. As can be seen from Table 1, the 3-
and the 4-methyl derivatives 1b and 1c as well as the
4-chloro derivative 1e have approximately the same
potency as 1a , while compounds 1d and 1f, incorporat-
ing a 3-chloro- or a 3-methoxy substituent on the phenyl
ring, are only micromolar inhibitors of rabbit cathepsin
K. A significant improvement in potency is realized in
the 4-methoxy compound 1g, which exhibits an IC50 of
* To whom correspondence should be addressed. Phone:
+41 61 696 12 27. Fax: +41 61 696 60 71. E-mail: eva.altmann@
pharma.novartis.com.
† Arthrithis & Bone Metabolism Therapeutic Area.
‡ Central Technologies.
10.1021/jm010801s CCC: $22.00 © 2002 American Chemical Society
Published on Web 05/10/2002

Letters
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12 2353
Ta ble 2. Inhibition of Rabbit Cathepsin K
F igu r e 1. 13C NMR spectra (150.9 MHz 13C frequency) of
compound 4g(13C) in the absence (top) and presence (bottom)
of cathepsin K, indicating noncovalent binding of 4g(13C) to
cathepsin K. The resonance at 184 ppm is an artifact of the
instrument.
inhibitors of rh cathepsin K, profound differences in
their selectivity profile vs rh cathepsins L and/or S are
observed depending on the specific nature of the sub-
stituents present on the NR-benzoyl- or -phenyltriazolyl
moieties, respectively. In the NR-benzoyl series, replace-
ment of a linear alkyl chain (4c) by a branched alkyl
group (4d ) improves selectivity toward rh cathepsin L,
while a phenoxy substituent (4b) dramatically improves
selectivity toward rh cathepsin L and S, making 4b a
particularly selective, highly potent inhibitor of cathe-
psin K. Replacement of the N-terminal 4-isopropylben-
zoyl moiety in 4d with a corresponding pyridine car-
boxylic acid residue (4e) results in a 30-fold increase in
selectivity toward rh cathepsin L, thus making 4e
completely selective for cathepsin K. In the NR-phenyl-
triazolyl series, the ortho chlorophenyl derivative 4g
proved to be a potent and highly specific cathepsin K
inhibitor, while the dichlorophenyl analogue 4h is
inactive (IC50 > 10 µM) against rh cathepsin L but
exhibits low micromolar activity against rh cathepsin
S.
a
Inhibition of recombinant rabbit cathepsin K in a fluorescence
assay. Data are means of two experiments performed in duplicate.
TFA salt.
b
Ta ble 3. Selectivity Data on the Inhibition of Homologous
Cathepsins
IC50 (µM)
compd
rh cath Ka
rh capth Lb
rh cath Sc
4b
4c
4d
4e
4g
4h
0.006
<0.003
<0.003
<0.003
0.006
>10
<0.03
0.05
1.5
2.3
>10
9.5
1.2d
5.7
7.2
1.9
0.19
0.009
The kinetics profiles of these novel cathepsin K
inhibitors were investigated. The equilibrium constants
for inhibition were determined using progress curves
(changes in fluorescence versus time), following the
hydrolysis of Z-Phe-Arg-AMC in the absence and pres-
ence of inhibitor. When standard assay conditions for
rabbit cathepsin K were used, the Ki values for 4b and
4h were obtained and are given in Table 2. In addition,
Lineweaver-Burk analysis of 4b and 4h demonstrated
that these compounds are purely competitive inhibitors.
Reversible binding was established by an experiment
in which the inhibitors 4b and 4h were preincubated
with cathepsin K at high concentration to ensure
complete loss of enzyme activity. After dialysis and
dilution into assay buffer containing substrate, the
entire enzyme activity was restored.
a
b
Inhibition of recombinant rh cathepsin K. Inhibition of rh
cathepsin L. c Inhibition of rh cathepsin S. Data represent the
mean of two independent experiments each performed in duplicate.
d
The reported IC50 for rh cath S of 1.2 µM represents the mean of
four experiments carried out in duplicate (SEM ) -0.52 µM).
64 nM and is thus 7-fold more potent as an inhibitor of
rabbit cathepsin K than the lead structure 1a .
On the basis of this latter result, we embarked on a
second optimization round that involved replacement
of the benzyloxycarbonyl group in 1g by different acyl
moieties. This strategy resulted in a number of highly
potent cathepsin K inhibitors such as 4a -h , all of which
are characterized by the presence of a N-terminal para-
substituted benzoic acid, a 5-substituted pyridine-2-
carboxylic acid, or a phenyltriazole carboxylic acid
residue 4g,h (Table 2).
For a selected number of compounds, activity against
recombinant human (rh) cathepsin K and selectivity
against rh cathepsins L and S were evaluated. The
results of these studies are summarized in Table 3.
While all compounds in Table 3 are also low nanomolar
The 13C spectrum of 4g(13C) is shown in Figure 1A.
Incubation of 4g(13C) with rh cathepsin K results in a
broadening of the 13C resonance of the labeled carbonyl
carbon and a minor change in chemical shift from 177.2
to 175.6 ppm (Figure 1B). Both effects are consistent
with a noncovalent binding mode of the inhibitor.

2354 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12
Letters
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In conclusion, we have discovered a novel class of
potent and selective cathepsin K inhibitors whose
activity does not depend on the covalent interaction with
the cysteine residue at the active site.
(5) Saftig, P.; Hunziker, E.; Wehmeyer, O.; J ones, S.; Boyde, A.;
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Ack n ow led gm en t. The authors thank M. Bisping,
D. Huerzeler, M. Kindler, P. Lerch, B. Mathys, G.
Schreiber, Y. Seltenmeyer, and A. Wigg for their excel-
lent technical assistance. Dr. T. Buhl, Dr. K. Gohda, Dr.
M. Missbach, and Dr. R. Lattmann are acknowledged
for helpful discussions.
(6) Gowen, M.; Lazner, F.; Dodds, R.; Kapadia, R.; Feild, J .; Tavaria,
M.; Bertoncello, I.; Drake, F.; Zavarselk, S.; Tellis, I.; Hertzog,
P.; Debouck, C.; Kola, I. Cathepsin K knockout mice develop
osteopetrosis due to a deficit in matrix degradation but not
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Genet. 1995, 10, 235-237.
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Su p p or tin g In for m a tion Ava ila ble: Description of the
inhibition assays, details of kinetics and NMR experiments,
and characterization (1H NMR and HRMS) of compounds 1a -
g, 4a -h , a n d 4g(13C). This material is available free of charge
via the Internet at http://pubs.acs.org.
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J M010801S