3,4-Disubstituted azetidinones as selective inhibitors of the cysteine protease cathepsin K. Exploring P3 elements for potency and selectivity

Article abstract of DOI:10.1016/j.bmcl.2004.12.088

The synthesis of a series of highly potent and selective inhibitors of cathepsin K based on the 3,4-disubstituted azetidin-2-one warhead is reported. A high degree of potency and selectivity was achieved by introducing a basic nitrogen into the distal par

Full text of DOI:10.1016/j.bmcl.2004.12.088

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1529–1534
3,4-Disubstituted azetidinones as selective inhibitors of the
cysteine protease cathepsin K. Exploring P3 elements for
potency and selectivity
Eduardo L. Setti,* Dana Davis, James W. Janc, Douglas A. Jeffery,
Harry Cheung and Walter Yu
Celera Genomics, 180 Kimball Way, South San Francisco, CA 94080, USA
Received 24 September 2004; revised 7 December 2004; accepted 20 December 2004
Available online 25 January 2005
Abstract—The synthesis of a series of highly potent and selective inhibitors of cathepsin K based on the 3,4-disubstituted azetidin-2-
one warhead is reported. A high degree of potency and selectivity was achieved by introducing a basic nitrogen into the distal part of
the P3 element of the molecule. Data from kinetic and mass spectrometry experiments are consistent with the interpretation that
compounds of this series transiently acylate the sulfhydrile of cathepsin K.
Ó 2005 Elsevier Ltd. All rights reserved.
Bone is a living tissue whose healthy structure depends
on the maintenance of an adequate balance between
the formation of the bone matrix by osteoblasts and
the degradation of bone matrix by osteoclasts. Osteo-
porosis is a disease that results from an imbalance in
activity between osteoblasts and osteoclasts and is char-
acterized by a progressive decrease in bone density.
There is mounting evidence that, at their site of at-
tachment, osteoclasts release cathepsin K (cat K) and
generate an acidic environment that facilitates
demineralization and matrix degradation.1 The fact that
cat K has been shown to be highly and selectively ex-
pressed in osteoclasts2 suggests that this specific protease
may play a crucial role in bone resorption and may be a
relevant target for therapeutic intervention to treat osteo-
porosis and related diseases.
cathepsin inhibitors. In a previous publication, we have
shown that structures containing cyclic moieties as P2
elements and a 3,4-disubstituted azetidin-2-ones war-
heads, give rise to molecules that behave as selective
and potent inhibitors of cat K.11 In our continuing stud-
ies on the development of novel therapeutic agents to
treat osteoporosis, we have synthesized a new series of
3,4-disubstituted azetidin-2-ones with the aim of identi-
fying inhibitors with increased potency and selectivity, a
good PK profile and suitability for in vivo evaluation.
Having identified the Ac612 moiety as the best P2 residue
for selectivity, we decided to incorporate this structural
motif into our inhibitors and, in an effort to further in-
crease the binding affinity for cat K, to exploit known
structural residues present in the S3 pocket of the
enzyme. The possibility of designing P3 elements tai-
lored to interact with Asp-61 has been one of our most
promising approaches to obtain improved potency. This
residue, located in the S3 subsite of the enzyme, is
known to form ionic interactions with positively charged
P3 moieties.13
Several chemotypes have been described as active-site
directed inhibitors of cat K. Whereas epoxides,3 vinyl
sulfones,4 chloromethyl,5 and acyloxymethyl ketones6
are reported to be irreversible inhibitors, nitriles,7 alde-
hydes,8 and ketones9 are known to bind via a reversible
covalent bond to the catalytic Cys-25. Recently, Zhou
et.al.10 have reported the synthesis of a series of 3-acyl-
amino-azetidin-2-one derivatives as non selective
In this article, we report the synthesis and inhibitory
activity of a new series of highly potent and selective
cat K inhibitors based on 3,4-disubstituted azetidin-2-
ones. We also present data to assess the potential of this
warhead in the development of new therapeutic agents
to treat osteoporosis.
*
Corresponding author. Fax: +1 650 866 6654; e-mail: eduardo.setti@
celera.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.12.088

1530
E. L. Setti et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1529–1534
The overall general strategy used for the synthesis of the
compounds presented in Table 1 was based on the re-
moval of the Cbz protecting group14 of intermediate 2
and coupling the free amine to the corresponding acids,
using HATU as an activating agent and diisopropyleth-
ylamine (DIPEA) as a base15 (see Scheme 1). The inter-
mediate 1 was obtained from the 6-aminopenicillanic
acid (6-APA) according to procedures reported
elsewhere.16
cule (see compounds 23, 25, 27, 28, 29, and 32).
Potency against cat K was also increased specially when
the basic nitrogen was incorporated into a large substi-
tuent, particularly evident in 29 and 32 since these inhibi-
tors exhibited subnanomolar potency against cat K. The
increased affinity of these two compounds toward cat K
is believed to be the result of the presence of a positive
charge combined with the particular shape of the P3 res-
idue. This moiety presumably allows the positive charge
of the nitrogen to perform an effective ionic binding
interaction with the Asp-61 residue of the S3 pocket.
Our assumption is based on the fact that crystallo-
graphic data obtained from compounds having the same
P3–P2 moieties as compounds 23, 28, and 27, attached
to a nitrile based warhead,7a have shown that the distal
nitrogen at the P3 elements is indeed interacting with the
Asp-61 of the S3 region of the enzyme.18 Unfortunately,
attempts to crystallize the complex of cat K with any of
the most soluble compounds of the azetidinone-based
series have failed.
While the intermediate acids used in the synthesis of the
biaryl analogues 24 and 25 were obtained from 4-fluoro-
benzonitrile (3) in two steps according to the sequence
described in Scheme 2, the corresponding acid used in
the synthesis of 27 was made following the synthetic
sequence shown in Scheme 3. The intermediate acids
required for the synthesis of the triaryl analogues 26,
13, 32, 33, and 34 were obtained according to reaction
conditions depicted in Schemes 4–6, respectively.
Cat K inhibition and selectivity data are presented in
Table 1.17 As can be seen in these Tables, there was a
significant improvement in selectivity profile, specially
against cat S and cat L, when a basic nitrogen was intro-
duced into the distal part of the P3 region of the mole-
b-Lactams are well known inhibitors of serine proteases.
Enzymes such as tryptase and elastase have been tar-
geted for inhibition with compounds based upon this
warhead.19 This class of compounds exerts its mecha-
H
H
O
H
N
H2N
R2
H
S
N
O
(a)
(b)
Cbz
N
(c)
(d)
R2
H
Cbz
N
H
R1
N
H
N
N
O
O
N
O
N
O
O
H
O
CO2H
O
1
2
6-APA
Scheme 1. Reagents: (a) Ref. 16a,b; (b) Ref. 11; (c) Pd/C*/H2, EtOAc; (d) R1CO2H, HATU, DIPEA, DMF.
CN
CO2H
(a)
CN
(b)
F
N
N
X
X
4
3
5 (X: NMe) 6 (X: O)
Scheme 2. Reagents and conditions: (a) N-methylpiperazine or morpholine, DMF, 100 °C; (b) HCl, 100 °C.
CO2H
CO2H
CO2H
(a)
(b)
N
N
Br
O
O
S
7
8
9
Scheme 3. Reagents and conditions: (a) AcOH, Br2, 60 °C; (b) Me2NCSNH2, EtOH, 70 °C.
CO2H
N
S
NH2
N
CN
(b)
(a)
N
O
N
O
S
O
12
11
10
Scheme 4. Reagents and conditions: (a) pyridine, H2S; (b) 8, EtOH, 70 °C.

E. L. Setti et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1529–1534
1531
CO2H
S
H
(a)
(b)
N
S
(c)
N
N
NH2
N
N
N
N
13
14
15
Scheme 5. Reagents and conditions: (a) thiophosgene, TEA, THF; (b) NH4OH/MeOH; (c) 8, EtOH, 70 °C.
H
NH2
N
CO2H
NH2
N
(a)
(b)
(c)
N
N
N
N
N
N
S
S
N
H
16
17
18
Scheme 6. Reagents and conditions: (a) thiophosgene, TEA, THF; (b) NH4OH/MeOH; (c) 8, EtOH, 70 °C.
Table 1. Cat K, B, S, and L inhibitory activity and selectivity
R1
R2
Cat K, K0i (um)
Cat B, K0i (um)
(B/K)
Cat S, K0i (uM)
(S/K)
Cat L, K0i (uM)
(L/K)
O
O
0.057
4.3
(75)
0.95
(16)
6.8
(119)
O
O
19
20
O
0.092
2.1
(23)
3.4
(36)
8.2
(89)
Cl
O
O
O
0.063
0.012
0.0031
1.2
(19)
0.52
(8.2)
2.6
(41)
O
O
O
21
22
23
24
25
Cl
1.2
(100)
3.5
(291)
6.4
(533)
S
0.53
(171)
2.8
(903)
2.1
(677)
N
O
O
0.005
0.98
(196)
14
(2800)
19
(3800)
N
O
O
O
O
0.0011
0.49
(445)
8.8
(8000)
1.4
(1273)
N
N
O
O
N
0.0075
0.0026
1.7
(227)
12
(1600)
11
(1467)
N
O
O
S
26
27
N
1.4
(538)
7.7
(2962)
0.39
(150)
N
S
(continued on next page)
(conti ued on next page)

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E. L. Setti et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1529–1534
Table 1 (continued)
R1
R2
Cat K, K0i (um)
Cat B, K0i (um)
(B/K)
Cat S, K0i (uM)
(S/K)
Cat L, K0i (uM)
(L/K)
O
N
N
0.0011
0.94
(85)
8.5
(1636)
3.3
(3000)
N
O
O
S
28
N
O
0.00025
0.091
(364)
8.8
(35,200)
2.1
(8400)
N
N
N
H
N
29
30
S
N
OPh
OPh
0.018
25
(1389)
10.0
(556)
0.089
(5)
S
N
N
N
0.0036
6.0
(1667)
51.0
(14,167)
1.1
(306)
N
S
31
OPh
0.00026
6.0
(230,774)
140
(538,461)
0.31
(1192)
N
N
N
H
32
33
S
N
N
OPh
OPh
0.0048
0.0082
0.34
(71)
17.0
(3542)
2.4
(500)
N
S
N
H
5.2
(634)
150
(18,292)
8.2
(1000)
N
N
N
34
S
nism of inhibition through direct interaction of Ser-195
with the C-2 carbonyl of the b-lactam. The covalent par-
ticipation of cathepsin K through Cys-25 should result
in transient acylation of the enzyme as in Scheme 7.
We examined the kinetics of inhibition of cathepsin K
by CRA-13427 (Fig. 1) and representative compounds
from Table 1. We typically observed slow on-set of inhi-
bition consistent with a covalent mode of inhibition
rather than simple rapid-equilibrium competitive
inhibition. When cathepsin K was preincubated with
CRA-13427,
a
fully inhibited species could be
generated.17 Following gel filtration chromatography,
the inhibited species was isolated and the recovery of
activity was monitored. We observed complete, time
dependent recovery of cathepsin K activity. The reco-
very of activity was a first order process and in the case
of CRA-13427 the t1/2 was 26 min. This presumably
corresponds to the rate of hydrolysis of the Cys-25
thioacyl enzyme intermediate to yield free cathepsin K
(see Scheme 7).
H
H
R
H
N
O
R
H
N
O
H
N
R
N
+ H2O
- AcO-
R
NH
O
O
NH
O
O
N
N
O
H
HO
S
S
-
HO
Enzyme
S
Enzyme
Enzyme
Enzyme
Scheme 7. Proposed mechanism of inhibition of cat K by 3,4-disubstituted azetidin-2-ones.

E. L. Setti et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1529–1534
1533
Acknowledgements
O
H
N
The authors wish to thank Merck & Co. for financial
support.
O
H
O
N
H
O
O
N
O
References and notes
CRA-13427
Figure 1.
1. (a) Erlebacher, A.; Filvaroff, E. H.; Gitelman, S. E.;
Derynck, R. Cell 1995, 80, 371; (b) Gowen, M. Exp. Opin.
Invest. Drugs 1997, 6, 1999.
To further substantiate whether CRA-13427 and
cathepsin K formed a covalent adduct, we used mass
spectrometry to measure the molecular weight of the
enzyme before and after reaction with CRA-13427.20
Untreated cathepsin K consisted of two species of
approximate equal proportion with molecular weights
of 23,711 and 23,768 Da. After incubation with CRA-
13427, there were two additional species with molecular
weights of 24,043 and 24,100 Da. This shift in molecular
weight of 332 Da is consistent with the covalent attach-
ment of one molecule of CRA-13427 to cathepsin K
with the subsequent loss of acetic acid as outlined in
Scheme 7. Approximately two-thirds of the cathepsin
K was modified by CRA-13427 under these conditions.
These results confirm that CRA-13427 forms a covalent
bond with cathepsin K likely through transient acylation
of Cys-25 during hydrolysis of the b-lactam.
2. (a) Shi, G.; Chapman, H. A.; Bhairi, S. M.; DeLeeuw, C.;
Reddy, V. Y.; Weiss, S. W. FEBS Lett. 1995, 357, 129; (b)
Drake, F. H.; Dodds, R. A.; James, I. A.; Connor, J. R.;
Debouck, C.; Richardson, S.; Lee-Rykaczewski, L.; Cole-
man, L.; Riemann, D.; Barthlow, R.; Hastings, G.;
Gowen, M. J. Biol. Chem. 1996, 271, 12511.
3. Barrett, A. J.; Hanada, K. Biochem. J. 1982, 201, 189.
4. Palmer, J. T.; Rasnick, D.; Klaus, J. L.; Bro¨mme, D.
J. Med. Chem. 1995, 38, 3193.
5. Shaw, E. In The Enzymes; Boyer, P. D., Ed.; 3rd ed.;
Academic: New York, 1970; Vol. 1, p 91.
6. Dai, Y.; Hedstrom, L.; Abeles, R. H. Biochemistry 2000,
39, 6498.
7. (a) Robichaud, J.; Oballa, R.; Prasit, P.; Falgueyret, J.-P.;
Percival, M. D.; Wesolowski, G.; Rodan, S. B.; Kimmel,
D.; Johnson, C.; Bryant, C.; Venkatraman, S.; Setti, E.;
Mendonca, R.; Palmer, J. T. J. Med. Chem. 2003, 46,
3709; (b) Falgueyret, J.-P.; Oballa, R. M.; Okamoto, O.;
Wesolowski, G.; Aubin, Y.; Rydzewski, R. M.; Prasit, P.;
Riendeau, D.; Rodan, S. B.; Percival, M. D. J. Med.
Chem. 2001, 44, 94.
Having identified several potent and selective cat K
inhibitors, we decided to modify their structures in order
to obtain molecules with potentially improved PK prop-
erties. To achieve this goal, we pursued the replacement
of the acetyl group in the 4 position of the azetidin-2-one
with a phenoxy group. In average, this structural change
produced compounds with reduced cat K potency (com-
pare compounds 27 and 30, and 28 and 31), being this
change more noticeable in b (cis)-4-substituted ana-
logues (compare compounds 28 and 33, and 29 and
34). This finding is opposite to what was reported by
other researchers where the b-phenoxy-4-substituted
analogues were more potent than their a-isomers.10 Pre-
liminary rat plasma stability studies showed that 4b-iso-
mers were far more stable than the corresponding
4a-isomer. Therefore, rat pharmacokinetic parameters
were measured for 33. This compound when given intra-
venously, possessed high clearance (92 mL/min/kg),
moderate volume of distribution (1.6 L/kg) and a short
MRT (17 min) with a b t1/2 (50%) of 21 min and a t1/2
(50%) of 2 min. Consequently, considering their sub-
strate-like mode of inhibition along with their poor
PK profiles, we believe that 3,4-azetidin-2-ones moieties
are unlikely to be suitable electrophiles to develop new
therapeutic agents to treat osteoporosis.
8. Hanzlik, R. P.; Jacober, S. P.; Zygmunt, J. Biochim.
Biophys. Acta 1991, 1073, 33.
9. (a) Marquis, R.; Yamashita, D.; Ru, Y.; Lo Castro, S.;
Oh, H.; Erhard, K.; Des Jarlais, R.; Head, M.; Smith, W.;
Zhao, B.; Janson, C.; Abdel-Meguid, S.; Tomaszek, T.;
Levy, M.; Veber, D. J. Med. Chem. 1998, 41, 3563; (b)
Marquis, R.; Ru, Y.; Lo Castro, S.; Zeng, J.; Yamashita,
D.; Oh, H.; Erhard, K.; Davis, L.; Tomaszek, T.; Tew, D.;
Salyers, K.; Proksch, J.; Ward, K.; Smith, B.; Levy, M.;
Cummings, M.; Haltiwanger, R.; Trescher, G.; Wang, B.;
Hemling, M.; Quinn, C.; Cheng, H.; Lin, F.; Smith, W.
W.; Janson, C.; Zhao, B.; McQueney, M.; DꢀAlessio, K.;
Lee, C.; Marzulli, A.; Dodds, R.; Blake, S.; Hwang, S.;
James, I.; Gress, C.; Bradley, B.; Lark, M.; Gowen, M.;
Veber, D. J. Med. Chem. 2001, 44, 1380; (c) McGrath, M.
E.; Sprengeler, P. A.; Hill, C. M.; Martichonok, V.;
Cheung, H.; Somoza, J. R.; Palmer, J. T.; Janc, J. A.
Biochemistry 2003, 42, 15018.
10. Zhou, N. E.; Guo, D.; Thomas, G.; Reddy, A. V. N.;
Kaleta, J.; Purisima, E.; Menard, R.; Micetich, R. G.;
Singh, R. Bioorg. Med. Chem. Lett. 2003, 13, 139.
11. Setti, E. L.; Davis, D. Bioorg. Med. Chem. Lett. 2003, 13,
2051.
12. Ac6 stands for alicyclic 1,1-disubstituted cyclohexyl.
13. McGrath, M. E.; Klaus, J. L.; Barnes, M. G.; Bro¨mme, D.
Nat. Struct. Biol. 1996, 4, 105.
14. Standard procedure for hydrogenolysis: to a solution of 2
(1 mmol) in ethyl acetate (15 mL), 10% Pd/C* (200 mg)
was added. The mixture was hydrogenated at 50 psi 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.
15. Standard procedure for coupling: to a solution of the
corresponding acid (1 mmol) in DMF (10 mL), HATU
(380 mg, 1 mmol), a solution of the free amine in ethyl
acetate (15 mL) and diisopropylethylamine (209 lL,
In summary, the synthesis and evaluation of a novel ser-
ies of potent and selective cat K inhibitors based on 3,4-
azetidin-2-ones as warheads have been described. Com-
bination of an Ac6 moiety in P2 and the presence of a
basic nitrogen in the distal portion of the P3 of the mole-
cule are essential to achieve a high degree of selectivity
and potency. Initial data indicates that this class of com-
pounds binds reversibly to the catalytic Cys-25 of cat K.

1534
E. L. Setti et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1529–1534
1.2 mmol) were added at rt. The reaction mixture was
inhibitor concentrations using the software package,
Batch Ki (Biokin Ltd, Pullman, WA) [Kuzmic, P. Anal.
Biochem. 1996, 237, 260, and; Kuzmic, P.; Sideris, S.;
Cregar, L. M.; Elrod, K. C.; Rice, K. D.; Janc, J. W. Anal.
Biochem. 2000, 281, 62]. Batch Ki provides a parametric
method for the determination of inhibitor potency using a
transformation of the tight binding inhibition model
described by Morrison [Morrison, J. F. Biochem. Biophys.
Acta 1969, 185, 269]. The apparent inhibition constant is
related to the true thermodynamic binding constant, Ki, by
the following relationship: Ki ¼ K0i=ð1 þ ½substrateꢀ=KmÞ.
Since substrates are typically supplied in the assays at their
Km, Ki ¼ K0i=2. The reversibility of rabbit cathepsin K
inhibition by CRA-13427 was carried out by pre-forming
the enzymeÆinhibitor complex during a 30 min incubation
phase. CRA-13427 was supplied at 25 nM during the
incubation phase. Following the incubation phase, the
reaction was diluted 50-fold into buffer containing sub-
strate such that the final concentration of CRA-13427 was
0.5 nM. Alternatively, the enzymeÆinhibitor complex was
subjected to size exclusion chromatography using a
BioRad BioSpin column according to the manufactures
specifications. The recovery of activity of rabbit cathepsin
K was monitored as a function of time and the t1/2 for the
recovery of enzyme activity (presumably corresponding to
deacylation of Cysteine-25) was obtained by standard
kinetic methods.
stirred for 18 h at rt. The mixture was washed with satd
NaHCO3, dried over Na2SO4, evaporated, and the crude
was purified on silica gel column, using a mixture of ethyl
acetate/methanol. Satisfactory 1HNMR and mass spectral
data were obtained for all compounds.
16. (a) Arnould, J. C.; Pasquet, M. J. Eur. J. Med. Chem.
1992, 27, 131; (b) Singh, R.; Zhou, N. E.; Guo, D.;
Micetich, R. Int. Pat. WO 98/12176, 1998.
17. Determination of apparent inhibition constants (K0i) were
performed as follows. All enzymes used in these studies
were produced by Celera Genomics with the exception of
human cathepsin B. Human cathepsin B isolated from
liver was obtained from Athens Research and Technology
(Athens, GA). The substrates used in these studies were
purchased from the following vendors: Z-Phe-Arg-AMC,
Boc-Leu-Lys-Arg-AMC, and Z-Val-Val-Arg-AMC were
from Bachem (Torrance, CA, USA) and Z-Leu-Arg-AMC
was from Calbiochem-Novabiochem (San Diego, CA,
USA). Typical rabbit cathepsin K, and human cathepsin L
inhibition studies were performed in 50 mM MES
(pH 5.5), 2.5 mM EDTA, 2.5 mM DTT, and 10% DMSO.
The substrate used to monitor cathepsin K and L activity
was Cbz-Phe-Arg-AMC. In both cases the substrate
concentration was fixed at the Km (40 lM for cathepsin
K and 10 lM for cathepsin L). Cathepsin B inhibition
studies were performed in 50 mM MES (pH 6.0), 2.5 mM
EDTA, 2.5 mM DTT, 0.001% Tween-20, and 10%
DMSO. The substrate used to monitor cathepsin B
activity was Boc-Leu-Lys-Arg-AMC (supplied at the Km,
190 lM). Cathepsin S inhibition studies were performed in
50 mM MES (pH 6.5), 2.5 mM EDTA, 100 mM NaCl,
2.5 mM 2-mercaptoethanol, 0.001% BSA, and 10%
DMSO. The substrate used to monitor cathepsin S activity
was Cbz-Val-Val-Arg-AMC (supplied at the Km, 60 lM).
Typically, the evaluation of a given inhibitorꢀs potency
was determined with the cathepsin of interest supplied at
1 nM (active-site concentration determined by titration
with E-64). Enzyme was incubated with inhibitor, present
at varying concentrations, for 30 min at room temperature
(21–24 °C) in 96-well microtiter plates to allow for
equilibrium to be achieved for slow binding inhibitors.
After preincubation, reactions were initiated with the
addition of the fluorogenic substrate specified above. The
hydrolysis of this substrate yields AMC, which was
monitored fluorometrically (filter pair: excitation 355 nm,
emission 460 nm) using an FMAX Kinetic Microplate
Reader (Molecular Devices, Sunnyvale, CA). The velocity
of the cathepsin-catalyzed reaction was obtained from the
linear portion of the progress curves using a response
factor of 275 RFU/lM (determined experimentally under
the standard assay conditions with freshly prepared AMC
stock solutions). Apparent inhibition constants, K0i, were
calculated from the velocity data generated at the various
18. Unpublished results.
19. (a) Sykes, N. O.; Macdonald, S. J. F.; Page, M. I. J. Med.
Chem. 2002, 45, 2850; (b) Cainelli, G.; Galletti, P.;
Garbisa, S.; Giacomini, D.; Sator, L.; Quintavalla, A.
Bioorg. Med. Chem. 2003, 11, 5391; (c) Sutton, J. C.;
Bolton, S. A.; Davis, M. E.; Hartl, K. S.; Jacobson, B.;
Mathur, A.; Ogletree, M. L.; Sulsarchyk, W. A.; Zahler,
R.; Seiler, S. M.; Bisacchi, G. S. Bioorg. Med. Chem. Lett.
2004, 14, 2233.
20. Recombinant rabbit cathepsin K (16 lg) was denatured
with 2.5 M guanadinium HCl and purified by standard
reversed-phase HPLC on a Phenomenex Jupiter C5
column in water/acetonitrile plus 0.1% TFA. There was
a single peak of absorbance at 280 nm that was collected.
The molecular weight was measured by direct injection at
300 nL/min into an ABI QStar mass spectrometer. Data
acquisition was performed in the multiple channel aver-
aging (MCA) mode for 15–30 min. Rabbit cathepsin K
that had been inhibited with CRA-13427 was treated
exactly the same way and reversed-phase purification was
carried out within 10 min of quenching the incubation
with the addition of guanadinium HCl. The standard
deviation among the charge species for the molecular
weight measurements was 10 ppm. The difference in
mass for the two species in untreated cathepsin K is likely
due to differential proteolytic processing that occurs
during purification.