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F. X. Tavares et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3891–3895
yl group as in analog 12, that could potentially form a
favorable hydrophobic interaction with 184Trp, resulted
in no additional gain in potency. Incorporation of elec-
tron-withdrawing groups to increase the electrophilicity
of the carbonyl group was then studied. Addition of an
ester to the 5-position of the thiazole, analog 13, resulted
in a decrease in inhibitory activity. In contrast, the 4-
substituted thiazole 14 (IC50 = 230 nM) gave a 10-fold
boost in potency.
shown in Table 2. Analog 14 was found to be over 40-
fold selective over cathepsins L, V, H, and B.
In conclusion, the present work describes the use of a
ketoheterocyclic warhead for the inhibition of cysteine
protease, cathepsin K. In addition to the use of standard
protocols for their synthesis, a novel route using the thio-
amide functionality as a handle to get into ketothiazoles
has been described. Access to other heterocyclic groups
from the thioamide moiety using known methods (e.g.,
S-alkylation, followed by subsequent displacement with
appropriate synthons) can be easily envisaged. The use
of this mild method allows for the presence of diverse
functional groups, such as amide and carbamate func-
tionalities, commonly found in protease inhibitors that
have peptidomimetic scaffolds. In addition, this method
allows for incorporation of peptidomimetic scaffolds
extending into the S0 region of proteases.
Replacement of the 4-substituted ester with an electroneg-
ative trifluoromethyl substituent as in analog 16 resulted
in a loss of potency. Thus, the enhanced activity of the es-
ter 14 may be partly attributed to its ability to accept a
hydrogen bond, possibly through a bridging water mole-
cule versus the enhanced electrophilicity of the carbonyl
thiol reactive group. Alternately, the trifluoromethyl
group may not be accommodated by the enzyme active
site. Analog 15 lacks norleucine-derived P1 substituent.
In the aldehyde series, 100-fold decrease in inhibitory
activity was seen when the P1 moiety was changed from
n-butyl to hydrogen.12 The loss of conformational bias
combined with the loss of S1 subsite van der Waals inter-
actions also led to a loss of activity in the ketoheteroaryl
series (analogs 15, 20, and 23). Replacement of the thia-
zole with oxadiazoles, as in examples 17 and 18, resulted
in analogs that were as potent as the thiazoles. As in the
thiazole 12, no additional gain in potency was observed
by having a phenyl substituent (18).
Acknowledgments
The authors thank Randy Rutkowski, Robert Johnson,
and Peter Kitrinos for analytical support.
References and notes
1. Einhorn, T. A. In Osteoporosis; Marcus, R., Feldman,
D., Kelsey, J., Eds.; Academic Press: San Diego, CA,
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To increase the diversity of these inhibitors, various
aldehyde inhibitor-derived P1, P2 and P3 groups were
used, keeping the ketoheterocycle group constant, as
in examples 19–24, 27. The dipeptide, as well as the
other P1, P2, and P3 groups, were all found to be less ac-
tive against cathepsin K. Since the di-iso-propyl moiety
was known to enhance potency against cathepsin K,10,16
a few ketoaryl/heteroaryl analogs were also made, as in
examples 25 and 26. Interestingly, the phenyl analog 29
was found to be inactive, whereas the thiophene isostere
26 was a low micromolar inhibitor of cathepsin K.
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Table 2. Inhibitory potencies (IC50) versus human cathepsins K, L, V,
S, and B
Compound
K (IC50 nM)
230
L/Ka
>40
V/Kb
>40
S/Kc
>40
B/Kd
>40
14
a Inhibition of recombinant human cathepsin L activity in a fluores-
cence assay using 5 lM Cbz-Phe-Arg-AMC as a substrate in 100 mM
NaOAc, 10 mM DTT, and 120 mM NaCl, pH 5.5.
b Inhibition of recombinant human cathepsin V activity in a fluores-
cence assay using 2 lM Cbz-Phe-Arg-AMC as a substrate in 100 mM
NaOAc, 10 mM DTT, and 120 mM NaCl, pH 5.5.
c Inhibition of recombinant human cathepsin S activity in a fluores-
cence assay using 10 lM Cbz-Val-Val-Arg-AMC as a substrate in
100 mM NaOAc, 10 mM DTT, and 120 mM NaCl, pH 5.5.
d Inhibition of recombinant human cathepsin B activity in a fluores-
cence assay using 10 lM Cbz-Phe-Arg-AMC as a substrate in
100 mM NaOAc, 10 mM DTT, and 120 mM NaCl, pH 5.5. The IC50
values are means of two or three inhibition assays, individual data
points in each experiment within a 2-fold range of each other.
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M.; McFadyen, R. B.; Miller, A. B.; Miller, L. R.;
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Shewchuk, L. M.; Thompson, J. B.; Willard, D. H.;
Wright, L. L. Bioorg. Med. Chem. Lett. 2004, 14, 3425.