3,4-Disubstituted azetidinones as selective inhibitors of the cysteine protease cathepsin K. exploring P2 elements for selectivity

Article abstract of DOI:10.1016/S0960-894X(03)00304-4

A novel series of 3,4-disubstituted azetidinones based inhibitors of the cysteine protease cathepsin K (Cat K) has been identified. Although not optimized, some of these compounds show at least 100-fold selectivity against other cathepsins. The use of cyc

Full text of DOI:10.1016/S0960-894X(03)00304-4

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2051–2053
3,4-Disubstituted Azetidinones as Selective Inhibitors of
the Cysteine Protease Cathepsin K. Exploring P2 Elements
for Selectivity
Eduardo L. Setti,* Dana Davis, Tobee Chung and John McCartery
Department of Medicinal Chemisty, Celera, 180 Kimball Way, South San Francisco, CA 94080, USA
Received 6 November 2002; accepted 7 February 2003
Abstract—A novel series of 3,4-disubstituted azetidinones based inhibitors of the cysteine protease cathepsin K (Cat K) has been
identified. Although not optimized, some of these compounds show at least 100-fold selectivity against other cathepsins. The use of
cyclic moieties as P2 elements has proven to be crucial to achieve a high degree of selectivity.
# 2003 Elsevier Science Ltd. All rights reserved.
Osteoporosis, the most common form of metabolic
bone disease, is a consequence of an imbalance between
bone resorption and bone formation.1 Bone resorption
is carried out by multinuclear cells known as osteoclasts
while osteoblasts are responsible for bone formation.
Cathepsin K (Cat K) is a cysteine protease of the papain
super family that is highly expressed in osteoclasts and
is believed to play a key role in the degradation of the
organic matrix of bone.2 Consequently, there has been
significant interest in the development of drugs to control
bone loss by limiting the proteolytic activity of Cat K.
Figure 1.
As part of our studies on the design and synthesis of Cat
K inhibitors we recently reported the X-ray crystal
structure of human Cat K complexed with APC-3328
(CRA-03328), a potent irreversible inhibitor of this
enzyme.3 APC-3328 (CRA-03328) is a vinyl sulphone
which was found to bind to the active site in a manner
that mimics substrate binding from P1 to P3 (Fig. 1).
cell wall synthesis inhibition, respectively.4 Recently,
3,4-disubstituted azetidin-2-one derivatives have been
reported to have excellent inhibitory properties of Cat
B, L and S.5 Based on this fact, we decided to investi-
gate the usefulness of this warhead in the development
of novel Cat K inhibitors.
Knowing that leucine is a P2 residue preferred by Cat
K,6 our first approach to achieve selectivity was to fur-
ther investigate the structural differences of the S2
binding pocket among the different cathepsins by syn-
thesizing non-natural P2 containing inhibitors. Therefore,
here, we describe the synthesis and structure–activity
relationship (SAR) of a series of 3,4-disubstituted azetidin-
2-ones having different unnatural amino acid as P2
elements.
Having succeeded in finding a highly active irreversible
inhibitor of human Cat K, we focused our attention on
the study of new moieties as suitable warheads to
develop reversible and selective Cat K inhibitors. Cer-
tain 3,4-disubstituted azetidin-2-ones are known to bind
to the active site of bacterial serine b-lactamases and
d-d-transpeptidases leading to b-lactamase and bacterial
*Corresponding author. Fax: +1-650-866-6654; e-mail: eduardo.
setti@celera.com
yCurrent address: Amgen Inc., Thousand Oaks, CA 91320-1799, USA.
The designed inhibitors were synthesized as outlined in
Scheme 1. Intermediate 1 was obtained from the
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00304-4

2052
E. L. Setti et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2051–2053
Scheme 1.
Table 1.
commercially available 6-APA in four steps following
the procedure described by Arnould and Pasquet.7
R
Cat K Cat B
(mM)
Cat L
(mM)
Ki
Cat S
(mM)
Ki
B/K L/K
S/K
(mM)
Ki
Ki
A series of 3,4-disubstituted azetidinones was obtained
by removing the Cbz protecting group from the inter-
mediate 1,8 and coupling the free amine to various
N-protected amino acids, using HATU as an acti-
vating agent and diisopropylethyl–amine (DIPEA) as
a base.9
0.0022
2.1
0.088
0.0092 950
40
4
0.0057
0.16
4.3
7.7
1.6
6.8
0.95
750 1,190
160
175
15
Cat K inhibition and selectivity data are presented in
Table 1.10 Compounds 5–8 were tested as a 1:1 mix-
ture of diastereomers. Attempts to synthesize the
mixture of the corresponding trans-isomers of 5 and
6 have failed. Compound 25 was a potent and non-
selective inhibitor of Cat K. Here, we have shown
that the use of cyclic P2 elements have a dramatic
influence on the selectivity of the compounds, espe-
cially against Cat L and Cat S. From the P2 elements
investigated, the compound bearing a P2-leucine (2)
displayed the greatest potency, while those bearing the
Ac6 and cis-Cy611 (3 and 5) moieties were the most
selective (see Table 1). Since 3 was marginally more
selective than 5, the Ac6 moiety was chosen as a
structural element in the synthesis of more potent and
selective Cat K inhibitors.12
140
28
48
870
420
0.0088
3.7
0.13
180
2.1
52
65
>15011
>15023020
>150
24
>705
The lack of potency of 7, compared to 2 and 3, is prob-
ably due to the loss of hydrogen bonding of the P1–P2
amide bond with the oxygen of Gly-66, as was reported
for several ketone-based Cat K inhibitors that bind to
the non-prime side of the active site.13
0.2
>705010
0.95
120
>150120
>160 >160
Moreover, preliminary results with the phenoxy group
as a substituent in the 4-position of the azetidin-2-one
ring (see Table 2), showed that the a (trans) orientation
is preferred over the b (cis) orientation for potency and,
to a lesser extent, for selectivity. Compounds 9 and 10
were obtained following synthetic methods described by
Singh et al.5
Table 2.
Compd
Cat K Cat B Cat L Cat S B/K L/K S/K
(mM) (mM) (mM) (mM)
Ki
Ki
Ki
Ki
In this paper, we have disclosed a novel series of selec-
tive 3,4-disubstituted azetidinones based inhibitors of
the cysteine protease Cat K. The use of cyclic moieties
as P2 elements have proven to be crucial to achieve
selectivity over other cathepsins, specially against Cat L
and Cat S.
0.38
140 3.4 33 368
9
87
Acknowledgements
3.6 >15016
53
>404 15
The authors would like to acknowledge Merck for
financial support and Dr. James T. Palmer for his cri-
tical review of this manuscript.

E. L. Setti et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2051–2053
2053
References and Notes
10. The enzyme active site concentrations were measured by
titration with the either E-64 or an in-house vinyl-sulfone.
Inhibition assays: Inhibitor potency measurements were per-
formed at room temperature using 96-well kinetic plate read-
ers. Reaction velocities were monitored at varying inhibitor
concentrations by following the hydrolysis of amino-
methylcoumarin substrates (ex355, em460) as indicated. All
substrates were added at a concentration equal to their Km.
Control reactions in the absence of inhibitor were performed
in parallel. The Ki apparent (Ki0) values were determined by a
non-linear least squares regression fit of the experimentally
derived data to the Morrison equation for tight-binding inhi-
bitors as described (3) or by least squared regression fit of the
Henderson equation for tight-binding inhibitors (4). Enzyme
and inhibitor were incubated 30min prior to initiation of
reaction by the addition of substrate. Cathepsin B: Enzyme
(5.0nM) was mixed with inhibitor in 50mM MES or BES (pH
6.0), 2.5 mM DTT, 2.5 mM EDTA, 0.05% Tween 20 and 10%
DMSO. The substrate was Z-Phe-Arg-AMC (300 mM).
Cathepsin K: Enzyme (3.6 nM) was mixed with inhibitor in 50
mM MES (pH 5.5), 2.5 mM DTT, 2.5 mM EDTA, 0.05%
1. Baron, R. Anatomy and Ultrastructure of Bone. In Primer
on the Metabolic Bone Disorders of Mineral Metabolism, 1st
ed.; Favus, M. J., Ed.; American Society for Bone and Mineral
Research: Kelseyville, CA, 1990, p 3.
2. (a) Drake, F. H.; Dodds, R. A.; James, I. A.; Connor, J. R.;
Debouck, C.; Richardson, S.; Lee-Rykaczewski, L.; Coleman,
L.; Riemann, D.; Barthlow, R.; Hasting, G.; Gowen, M.
J. Biol. Chem. 1996, 271, 12511. (b) Bossard, M. J.; Tomaszek,
T. A.; Thompson, S. K.; Amegadzie, B. Y.; Hanning, C. R.;
Jones, C.; Kurdyla, J. T.; McNulty, D. E.; Drake, F. H.;
Gowen, M.; Levy, M. A. J. Biol. Chem. 1996, 271, 12517.
3. McGrath, M. E.; Klaus, J. L.; Barnes, M. G.; Broemme, D.
Nat. Struct. Biol. 1996, 4, 105.
4. Gelleni, M.; Lamotte-Brasseur, J.; Raquet, X.; Dubus, A.;
Monnaire, D.; Knox, J. R.; Frere, J.-M. Biochem. Pharmacol.
1995, 49, 1171.
5. Singh, R.; Zhou, N. E.; Guo, D.; Micetich, R. Int. Pat. WO
98/12176, 1998.
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Tween-20and 1%0 DMSO. The substrate was
Z-Phe-Arg-
7. Arnould, J. C.; Pasquet, M. J. Eur. J. Med. Chem. 1992, 27,
131.
AMC (40 mM). Cathepsin L: Enzyme (1.3 nM) was mixed with
inhibitor in 50mM MES (pH 5.5), 2.0mM EDTA, 2 mM DTT,
0.05% Tween-20 and 10% DMSO. The substrate was Z-Phe-
Arg-AMC (10 mM). Cathepsin S: Enzyme (1.0nM) was mixed
with inhibitor in 50mM MES (pH 6.5), 100mM NaCl, 2.5 mM
EDTA, 2.5 mM 2-mercaptoenthanol, 0.001% bovine serum
albumin and 10% DMSO. The substrate was Z-Val-Val-AMC.
11. Ac6 stands for alicyclic 1,1-disbustituted cyclohexyl and
Cy6 for 1,2-disubstituted cyclohexyl.
8. Standard procedure for hydrogenolysis: To a solution 1
(1 mmol) in ethyl acetate (15 mL), 10% Pd/C (200 mg) was
added. The mixture was hydrogenated at 50psi for 8 h. The
catalyst was separated by filtration through a short plug of
Celite and the solution of the amine was used for coupling.
9. Standard procedure for coupling. To a solution of the corres-
ponding acid (1 mmol) in THF (10mL), HATU (380mg, 1
mmol), a solution of the free amine in ethyl acetate (15 mL)
and diisopropylethylamine (209 mL, 1.2 mmol) were added at
rt. The reaction mixture was stirred for 18 h at rt. The mixture
was washed with satd NaHCO3, dried over Na2SO4, evapo-
rated and the crude was purified by silica gel column, using a
mixture of ethyl acetate/hexane (1:1). Satisfactory spectral
data was obtained for all compounds.
12. Unpublished results.
13. Marquis, R. W.; Ru, Y.; Zeng, J.; Lee Trout, R. E.;
LoCastro, S. M.; Gribble, A. D.; Witherington, J.; Fenwick,
A. E.; Garnier, B.; Tomaszek, T.; Tew, D.; Hemling, M. E.;
Quinn, C. J.; Smith, W. W.; Zhao, B.; McQueney, M. S.;
Janson, C. A.; D’Alessio, K.; Veber, D. F. J. Med. Chem.
2001, 44, 725.