K. K. Adkison et al. / Bioorg. Med. Chem. Lett. 16 (2006) 978–983
983
inhibitor concentration, suggesting that the added
semicarbazide did not affect the rate of hydrolysis. In
contrast, at pH 7.0, the semicarbazones were significant-
ly poorer inhibitors of cathepsin K under similar assay
minal adduct of the semicarbazone with cathepsin K
was detected. It was therefore concluded that, although
it is possible that some of these semicarbazones inhibit
cathepsin K directly, they most likely function as deliv-
ery vehicles for the aldehyde inhibitors.
conditions. Cathepsin
K hydrolyzes Cbz-Phe-Arg-
AMC at a slower rate at pH 7.0 than at pH 5.5, but it
still rapidly cleaves the substrate.15 These results can
be explained by a model in which hydrolysis of the
semicarbazones to generate the active inhibitor alde-
hydes is rapid at pH 5.5, and not influenced by the pres-
ence of excess semicarbazide, whereas the acid catalyzed
hydrolysis is slowed at pH 7.0, decreasing the concentra-
tion of active aldehyde and therefore the apparent
potency of the inhibitor. Thus, these data support the
hypothesis that semicarbazones inhibit cathepsin K by
acting as prodrugs of their aldehydes.
In summary, this report details the discovery and inves-
tigation of the mechanism of action of semicarbazone-
based inhibitors of cathepsin K. Semicarbazone hydro-
lysis rates at acidic pH, cathepsin K inhibition assays
at pH 7.0, and 13C NMR experiments support a theory
that these semicarbazones serve as prodrugs for the
actual aldehyde inhibitors. Although these semicarbaz-
ones were more water soluble than the corresponding
aldehydes, they offer only slightly enhanced pharmaco-
kinetic profiles than the parent aldehydes, presumably
due to their instability under acidic conditions. Never-
theless, based on surrogate markers, these semicarbaz-
ones were able to attenuate bone resorption in an
ex vivo model of osteoporosis, most likely via in situ
conversion to known aldehyde cathepsin K inhibitors
in the acidic resorption lacunae of osteoclasts.
To further explore this hypothesis, a 13C labeled inhibi-
tor was prepared for a NMR experiment with cathepsin
K. The carbonyl labeled Boc-Leu-H aldehyde 1e and
semicarbazone 2aj were synthesized as shown in Scheme
3. The amine of commercially available 13C labeled leu-
cine was converted to its tert-butyl carbamate. Then,
in situ generation of the mixed anhydride, from the acid,
followed by sodium borohydride-mediated reduction
provided the alcohol. The alcohol was subsequently oxi-
dized to the aldehyde 1e. Then, employing a similar
method as detailed in Scheme 1, the aldehyde was con-
verted into the semicarbazone 2aj.
References and notes
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Both the aldehyde 1e (IC50 = 31 nM) and the semicar-
bazone 2aj (IC50 = 110 nM) were potent inhibitors of
cathepsin K. The 13C NMR spectrum of aldehyde 1e
in D2O showed a small 13C resonance (ꢀ5%) at
206 ppm for the carbonyl carbon and a large resonance
(ꢀ95%) at 93 ppm for the hydrate, whereas the 13C
NMR spectrum of semicarbazone 2aj in D2O displayed
a large 13C resonance (ꢀ98%) at 150 ppm for the imine
carbon and a small resonance (ꢀ2%) at 92 ppm for the
hydrate. When added to cathepsin K at pH = 5.5, the
imine carbon of the semicarbazone inhibitor 2aj showed
a resonance at 149 ppm. The spectrum also exhibited a
small resonance at 204 ppm and a large resonance at
91 ppm representing the free aldehyde and hydrate,
respectively, from hydrolysis of the semicarbazone cata-
lyzed by the acidic buffer, as well as a new resonance at
80 ppm corresponding to the hemithioketal adduct with
cathepsin K. Importantly, no new resonance for a thioa-
8. Galpin, I. J.; Wilby, A. H.; Place, G. A.; Beynon, R. J. Int.
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Pharm. Res. 1998, 15, 11.
O
O
13C
H
H2N 13C
O
N
a-c
OH
H
O
5
1e
11. Kostewicz, E. S.; Brauns, U.; Becker, R.; Dressman, J. B.
Pharm. Res. 2002, 19, 345.
H
13C
O
H
H
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N.; Chiba, K.; Ishizaki, T.; Green, C. E.; Tyson, C. A.;
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nology 1984, 114, 1864.
d
O
N
N
N
N
O
O
2aj
Scheme 3. Reagents and conditions: (a) (t-BuOCO)2O, NaOH, diox-
ane, H2O, 99%; (b) i-PrOCOCl, NEt3, THF, ꢁ10 ꢁC; NaBH4, H2O,
0 ꢁC, 88%; (c) pyridine Æ SO3, NEt3, DMSO, CH2Cl2, ꢁ10 ꢁC, 90%; (d)
3y, PPTS, THF, 45%.
14. Conaway, H. H.; Grigorie, D.; Lerner, U. H. J. Endocri-
nol. 1997, 155, 513.
15. Bromme, D.; Okamoto, K.; Wang, B. B.; Biroc, S. J. Biol.
Chem. 1996, 271, 2126.