Lysosomotropism of basic cathepsin K inhibitors contributes to increased cellular potencies against off-target cathepsins and reduced functional selectivity

Article abstract of DOI:10.1021/jm0504961

The lysosomal cysteine protease cathepsin K is a target for osteoporosis therapy. The arylpiperazine-containing cathepsin K inhibitor CRA-013783/L-006235 (1) displays greater than 4000-fold selectivity against the lysosomal/endosomal antitargets cathepsin

Full text of DOI:10.1021/jm0504961

J. Med. Chem. 2005, 48, 7535-7543
7535
Lysosomotropism of Basic Cathepsin K Inhibitors Contributes to Increased
Cellular Potencies against Off-Target Cathepsins and Reduced Functional
Selectivity
Jean-Pierre Falgueyret,† Sylvie Desmarais,† Renata Oballa,‡ W. Cameron Black,‡ Wanda Cromlish,†
Karine Khougaz,§ Sonia Lamontagne,† Frederic Masse´,† Denis Riendeau,† Sylvie Toulmond,† and
M. David Percival*,†
Departments of Biochemistry, Molecular Biology and Pharmacology, Medicinal Chemistry, and Pharmaceutical Research and
Development, Merck Frosst Centre for Therapeutic Research, Kirkland, Quebec, Canada
Received May 26, 2005
The lysosomal cysteine protease cathepsin K is a target for osteoporosis therapy. The aryl-
piperazine-containing cathepsin K inhibitor CRA-013783/L-006235 (1) displays greater than
4000-fold selectivity against the lysosomal/endosomal antitargets cathepsin B, L, and S.
However, 1 and other aryl-piperazine-containing analogues, including balicatib (10), are ∼10-
100-fold more potent in cell-based enzyme occupancy assays than against each purified enzyme.
This phenomenon arises from their basic, lipophilic nature, which results in lysosomal trapping.
Consistent with its lysosomotropic nature, 1 accumulates in cells and in rat tissues of high
lysosome content. In contrast, nonbasic aryl-morpholino-containing analogues do not exhibit
lysosomotropic properties. Increased off-target activities of basic cathepsin K inhibitors were
observed in a cell-based cathepsin S antigen presentation assay. No potency increases of basic
inhibitors in a functional cathepsin K bone resorption whole cell assay were detected. Therefore,
basic cathepsin K inhibitors, such as 1, suffer from reduced functional selectivities compared
to those predicted using purified enzyme assays.
Introduction
differences in pH, ionic strength, presence of other
proteins and membranes, and cellular permeability. On
the other hand, the absence of highly specific endog-
enous or peptide substrates to measure the cellular
activities of individual cathepsins has hindered the
development of whole cell cathepsin assays. More
recently, whole cell enzyme occupancy assays have been
used to determine cathepsin inhibitor potencies within
intact cells. These assays employ a radiolabeled ir-
reversible inhibitor which competes with the reversible
test compound for the active site of the target enzyme.7,8
The successful targeting of a cathepsin K inhibitor
to lysosomes by conjugation with poly(ethylene glycol)
polymers has been described.9 These conjugated inhibi-
tors enter the cell by endocytosis, leading to lysosomal
drug delivery. Lysosomotropism, which is defined as the
ability of compounds to accumulate in acidic compart-
ments, is also a property of weakly basic, lipophilic
molecules.10 Neutral species are able to freely diffuse
across the cell membrane, but become membrane im-
permeable once protonated within the acidic compart-
ment. The degree of compound accumulation is depend-
ent on its pKa and the pH difference between the cytosol
and the lysosome. In addition, compound lipophilicity
can affect lysosomal accumulation, due to aggregation
or interaction with lipid membranes.11,12 Many common
drugs are highly lysosomotropic, and this property has
often been linked, especially in animal safety studies,
to the development of phospholipidosis.13 Drug lysoso-
motropism may also result in an alteration of the
disposition of other basic compounds14 and has also been
linked to the formation of lysosomal lipofuscin-like
bodies.15
Cathepsin K is a member of the papain family of
cysteine proteases of which 11 human members have
been identified.1,2 With the exception of cathepsin W,
which is localized in the endoplasmic reticulum, all are
found in the acidic lysosomal or endosomal compart-
ments of the cell. Certain cysteine cathepsins are also
found associated with the cell membrane or are secreted.
Cathepsin K is a pharmaceutical target for the treat-
ment of osteoporosis, as it is highly and selectively
expressed in osteoclasts, the cells which degrade bone
during the continuous cycle of bone degradation and
formation.3 Type I collagen is the major organic con-
stituent of bone, and cathepsin K has a potent collage-
nase activity, especially at the acidic pH required to
destroy the inorganic calcium hydroxyapatite compo-
nent of bone.
In developing potent and selective cathepsin K inhibi-
tors, most attention has focused on cathepsins B, L, and
S as antitargets, as they have high degrees of homology
to cathepsin K and have been attributed important
functions in vivo.4-6 Inhibitory potencies against target
and antitarget cathepsins are generally determined
using purified enzymes at the optimal acidic pH for each
enzyme and can give reliable estimates of intrinsic
potencies. However, these assay conditions may not
always reflect those of the enzyme in the cell, due to
* Correspondence to M. David Percival, Department of Biochemistry
and Molecular Biology, Merck Frosst Centre for Therapeutic Research,
P.O. Box 1005, Pointe-Claire-Dorval, Quebec, Canada. H9R 4P8. Tel
514-428-3191. Fax 514-428-4930. E-mail: dave_percival@merck.com.
† Department of Biochemistry, Molecular Biology and Pharmacology.
‡ Department of Medicinal Chemistry.
§ Department of Pharmaceutical Research and Development.
10.1021/jm0504961 CCC: $30.25 © 2005 American Chemical Society
Published on Web 10/29/2005

7536 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Scheme 1. Synthesis of Compound 2
Falgueyret et al.
Scheme 2. Synthesis of 14C-1
Scheme 3. Synthesis of 14C-9
Here we describe examples of basic amine-containing
cathepsin K inhibitors which show lysosomotropic be-
havior. This feature of these inhibitors has important
consequences for their activities against antitargets as
well as their tissue distributions.
with 1-aminocyclohexanecarbonyl chloride to generate
the amine 3 which is then coupled with the acid 4
under amide bond forming conditions to afford 2, the
non-nitrile version of 1.
18
The synthesis of the radiolabeled cathepsin inhibitor
14C-1 involves the lithiation of the aryl bromide 5 with
n-BuLi and trapping of the resultant aryllithium with
14CO2 (Scheme 2). The final step is an amide bond
formation with HATU and 1-amino-N-(cyanomethyl)-
cyclohexanecarboxamide methanesulfonate (6).18
The synthesis of the nonbasic radiolabeled cathepsin
inhibitor 14C-9 involves the reaction of amide 7 with
formaldehyde and benzotriazole (Scheme 3). The result-
ant benzotriazole 8 is then treated with Na14CN at 90
°C which affords 14C-9.
Biochemistry. The conditions used to assess inhibi-
tor potencies against purified human cathepsins B, L,
and S and rabbit cathepsin K, as well as the whole cell
enzyme occupancy assays for human cathepsins B (in
HepG2 cells), L (in HepG2 cells), and S (in Ramos cells),
were previously described.8 The effects of buffer pH on
inhibitor potencies against cathepsins B, K, L, and S
were performed using constant ionic strength buffers
(50 mM) at pH 3.5 and 4.5 (formate), pH 5.5 (acetate),
and pH 6.5 and 7.5 (Bis-Tris). Ionic strength was
adjusted with NaCl. Buffer additives for each cathepsin
were as previously detailed.8 The enzyme assay for
mouse cathepsin S16 was performed under the same
conditions as that for the human enzyme. The rabbit
osteoclast bone resorption assay was also previously
described.17
Results and Discussion
We recently described the synthesis and biological
activities of the potent, reversible inhibitor of cathepsin
K, CRA-013783/L-006235 (1) (Figure 1).19a On the basis
of inhibitory potencies against purified human cathep-
sins, 1 is highly active against human cathepsin K (IC50
∼0.25 nM) and is over 4000-fold selective against
Chemistry. The synthesis of the non-nitrile 2 is
described in Scheme 1. Trifluoroethylamine is reacted

Lysosomotropism of Basic Cathepsin K Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7537
Figure 1. Structures of cathepsin inhibitors used in this study.
Table 1. Potencies for Basic and Nonbasic Cathepsin K Inhibitors Inhibitors against Humanized Rabbit Cathepsin K,20 Human
Cathepsins B, L, and S in Purified Enzyme and Whole Cell Enzyme Occupancy Assaysa
inhibition of cathepsins, IC50 (nM)
Cathepsin K
enzyme
Cathepsin B
Cathepsin L
Cathepsin S
enzyme
cell (HepG2)
enzyme
cell (HepG2)
enzyme
cell (Ramos)
(1)
0.2519
1.4 ( 0.4
7.8 ( 0.8
2.3 ( 0.04
19 ( 6
1100 ( 43
17 ( 3
6300 ( 1500
503 ( 80
2600 ( 250
24000 ( 3000
32000 ( 6000
55 ( 11
340 ( 35
48 ( 15
47000 ( 7000
65000 ( 4000
20000 ( 7000
27000 ( 3000
52000 ( 6000
5.3 ( 0.2
790 ( 420
2900 ( 1300
1340 ( 400
>10000
4800 ( 2600
10 ( 6
(10)
(11)
(12)
(13)
(14)
4800 ( 200
61 ( 22
86000 ( 14000
4200 ( 500
15400 ( 1000
19000 ( 4000
4400 ( 2000
2900 ( 1100
4400 ( 1900
940 ( 370
320 ( 170
>10000
>10000
1030 ( 308
230 ( 180
a The data represent averages ((SEM) of at least duplicate experiments.
cathepsins B, L, and S (Table 1). However, when tested
in human whole cell enzyme occupancy assays against
cathepsins B, L, and S, it is apparent that the potencies
against these enzymes in their native cellular environ-
ment are increased ∼20-60-fold (Table 1). Other pip-
erazine-thiazole analogues (which had modifications at
the P2 spirocyclohexyl group, data not shown), as well
as a N-propyl-piperazine analogue 10 (balicatib)19a,b and
a nonpeptidic biaryl piperazine-containing cathepsin K
inhibitor 11,20 were also significantly more potent in
cell-based assays than against purified cathepsins (Table
1). In contrast, analogues of these compounds containing
a morpholino or a phenyl functionality (12, 13, and
14),18,19a generally gave similar IC50 values in both cell
and purified enzyme assays for each cathepsin (Table
1).
The above observations suggest that the purified
enzyme assay conditions for cathepsins B, L, and S do
not reflect the cellular enzyme environment, resulting
in inhibitor potencies for compounds 1, 10, and 11 (but
not 12, 13, and 14) which do not match those obtained
in whole cells. This phenomenon may be due to the basic
piperazine-containing compounds 1, 10, and 11 concen-
trating in the acidic lysosomal/endosomal compartments
containing the target cathepsins, a process known as
lysosomotropism. Alternatively, the differences in cel-
lular and enzyme potencies of 1, 10, and 11 may be
related to pH-dependent differences in the piperazine
group protonation state of these inhibitors.21 This may
lead to pH-dependent changes in inhibitor potencies
that do not correspond to those found with neutral
inhibitors. However, the titration of compounds 1, 10,
11, 12, 13, and 14 against purified cathepsins B, K, L,
and S over the pH range 3.5 to 7.5 revealed only small
decreases in inhibitory potencies at both pH extremes
(e4-fold), as compared to the potencies determined
under the conditions used in Table 1 (pH 5.5 to 6.5). In
no cases were inhibitor potencies significantly increased
over the pH range 3.5 to 7.5 compared to those obtained
at pH 5.5 to 6.5, consistent with previous studies.21
The piperazine-thiazole-phenyl group of 1 is intrinsi-
cally fluorescent which enabled its subcellular distribu-
tion to be examined in whole live cells by two-photon
confocal microscopy. Following incubation with 10 µM
1 for 1 h, HepG2 cells show a punctate fluorescence
throughout the cytoplasm (Figure 2A). A similar pattern
and intensity of cellular fluorescence was obtained with
the analogue 2, in which the electrophilic aminoaceto-
nitrile warhead, which forms a thioimidate linkage with
the active site cysteine, is replaced by an unreactive
trifluoroethylamino group (Figure 2B). Compound 2 is
devoid of activity against cathepsins B, L, K, and S (IC50
> 10 µM), demonstrating that the accumulation of 1
within the cell is not due to binding to cysteine cathe-
psins or other activated thiol-containing proteins. Little
cellular autofluorescence was observed with vehicle-
treated cells (Figure 2C). The cellular fluorescence was
blocked when cells were pretreated with 10 mM NH4Cl
which causes the alkalinization of acidic subcellular
compartments (data not shown).22 The punctate cyto-
solic fluorescence obtained with 1 colocalizes with that
of the fluorescent lysosomal probe LysoTracker Red
DND-99 (Figure 2D-F). Similar results were obtained
with human umbilical vein endothelial cells, another cell

7538 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Falgueyret et al.
Figure 2. Accumulation of 1 within lysosomes of HepG2 cells as observed by two-photon confocal microscopy. HepG2 cells were
incubated for 1 h with 10 µM of either 1 (A) or 2 (B) or DMSO vehicle (C) (all measured at an original magnification of 20×). To
assess the accumulation of 1 in lysosomes, colocalization with LysoTracker Red DND-99 was performed. The figures show
fluorescence observed with either 1 (D) or Red DND-99 (E) and when the two images are merged (F). Arrows indicate lysosomes
containing both 1 and Red DND-99 (original magnification 20×).
Figure 3. Accumulation of 14C-labeled cathepsin K inhibitors in HepG2 cells. A: HepG2 cells were grown to ∼90% confluency
in a Cytostar-T scintillation microplate and were incubated at time -10 min with 30 µM (0.02 µCi) 14C-1 (0) or 30 µM (0.02 µCi)
14C-9 (×), prior to determination of the accumulation of compound in cells by scintillation counting. After 50 min incubation, the
media containing 14C-1 was replaced by media without compound (O). Controls were performed without cells in the presence of
30 µM 14C-1 (]) and 30 µM 14C-9 (+). B: HepG2 cells were incubated in the presence of 30 µM of 14C-1 alone (0), after a 2 h
preincubation with 10 µM monensin (O), or after a 2 h preincubation with 10 mM NH4Cl ( × ). Control without cells in the
presence of 30 µM 14C-1 (]).
type which contains large numbers of lysosomes. Com-
pounds 12 and 13 do not have an appropriate intrinsic
fluorescence, therefore precluding similar cell-based
studies with these nonbasic, morpholino-containing
cathepsin K inhibitors.
The accumulation of piperazine and morpholino-
containing cathepsin K inhibitors in cells was further
investigated using 14C-labeled 1 and 9 (Schemes 2 and
3). HepG2 cells were grown as a monolayer in the wells
of a Cytostar-T scintillation microplate. Either 14C-
labeled 1 or 9 (30 µM) were added to the cell media,
and the signal, resulting from collision of the short
travelling â-particles with the scintillant in the base of
the plate, was determined over time. A signal is only
produced when â-particles are emitted in close proximity
to the plate base, i.e., associated with attached cells and
not when â-particles are emitted in the media above the
cells. The radioactivity signal from 14C-labeled 1 in-
creased rapidly over the first 40 min of the incubation
with the cells and then was constant until 120 min
(Figure 3A). In contrast, no time-dependent signal
increase was observed with the morpholino-containing
14C-labeled 9 in the presence of HepG2 cells, and the
signal obtained was similar to that of 14C-labeled 1 and
9 in the absence of cells. The apparent accumulation of
14C-1 in the HepG2 cells was reversible, since replace-
ment with fresh media (not containing 14C-1) after an
incubation time of 50 min resulted in a rapid loss of
signal (Figure 3A). The uptake of 14C-1 into the cells
was not saturated at 30 µM, as the incubation with 60
µM 14C-1 resulted in an approximate doubling of both
the rate of signal increase and the maximal intensity

Lysosomotropism of Basic Cathepsin K Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7539
Table 2. Experimentally determined Piperazine Group pKa
and Log P Values of Cathepsin K Inhibitors
rapidly eliminated via the biliary and renal excretion
routes. The whole body distribution study identified
other organs, including lung, spleen, and salivary,
lachrymal, adrenal, pituitary, and thyroid glands as
other sites of accumulation of 1 and/or its metabolites.
Low concentrations of radioactivity were detected in the
muscle and white fat.
Log P
pKa
(1)
2.30 ( 0.04
2.9 ( 0.3
1.81 ( 0.05
3.46 ( 0.03
0.98 ( 0.03
7.01 ( 0.05
6.84 ( 0.03
7.76 ( 0.05
8.77 ( 0.03
-
(2)
(10)
(11)
(13)
Rat tissue distribution studies further confirmed the
accumulation of the parent compound 1 in kidney,
spleen, lung and liver, and showed that concentrations
of both 1 and 10 in these tissues are ∼6-12 times
greater than those found in plasma (Table 4). These
tissues have high lysosome contents and are well-known
as the sites of concentration of lysosomotropic drugs.23,24
In contrast, the concentrations of 1 and 10 in white fat
and muscle, two tissues with low lysosome contents, are
similar to their plasma concentrations. The nonbasic
inhibitor 13 does not show any accumulation in tissues
with high lysosome content, and the tissue concentra-
tions in liver, kidney, and lung are similar to that in
plasma (Table 4). In general, lysosomotropic drugs have
a high volume of distribution (Vd) and have tissue levels
higher than that in plasma. The Vd of 1 is ∼2-fold higher
than that of 10 which may be related to its higher Log
P value (Table 2), whereas the Vd of 13 is close to unity.
The lysosmotropic properties of 1 may contribute to its
favorable pharmacokinetic profile, as its terminal half-
life is increased 7-fold over that of the morpholino
analogue 12.19a
The possibility was considered that the lysosomotropic
properties of 1 may also contribute to its functional
efficacy, since cathepsin K-mediated degradation of
collagen occurs in the acidic lacunae between the
osteoclasts and bone, as well as in lysosomes within the
osteoclasts.25,26 Thus, one may expect the increased
potencies of basic compounds such as 1, 10, and 11
against antitarget cathepsins B, L, and S in whole cell
assays (Table 1), to be compensated by a corresponding
increased potency against cellular cathepsin K activity.
However, a comparison of the potencies of basic and
nonbasic inhibitors against purified rabbit cathepsin K
with those from a functional rabbit osteoclast bone
resorption assay does not confirm this prediction (Table
5). Although the intrinsic potencies of the basic com-
pounds 1 and 10 against rabbit cathepsin K are greater
than their nonbasic analogues 12 and 13 (due to a
favorable electrostatic interaction with a S3 aspartic
acid),19a the potency shifts between the purified enzyme
assay and the functional cell-based assay are roughly
equivalent for both basic and nonbasic inhibitors. It
should be emphasized that the cathepsin K bone resorp-
tion assay is a functional assay that measures type I
collagen degradation after a three day incubation of
osteoclasts and bone with test compound. The IC50 value
from the osteoclast assay for a given compound should
not therefore be directly compared with those from the
cathepsins B, L, and S whole cell assays, which measure
the degree of inhibitor occupancy of the active site of
each enzyme in their native cellular environments.
Although whole cell enzyme occupancy inhibitor poten-
cies may not be equivalent to those obtained in func-
tional assays, the same rank order of potencies would
be expected for a series of inhibitors in the two types of
assays.
Figure 4. Whole-body autoradiogram illustrating the distri-
bution of 14C-1 in the female Sprague-Dawley rat. 14C-1 whole-
body distribution was assessed 1 h after iv injection of the
radiolabeled compound.
Table 3. Rat Whole Body Distribution of 14C-1a
tissue
concn (µM)
tissue
concn (µM)
small intestine
content
bladder content
salivary glands
lung
289
spinal marrow
15
148
54
49
thyroid
14
11
9
bone marrow
large intestine
content
lachrymal glands
liver
45
26
23
22
21
19
15
thymus
heart
pancreas
muscle
blood
white fat
brain
6
6
5
4
4
2
1
spleen
adrenal glands
brown fat
pituitary
kidney
a Tissue concentrations were determined by analysis of whole
body sections 1 h following an iv dose using â Vision+ software.
(data not shown). The pretreatment of the HepG2 cells
with either monensin or NH4Cl, both agents which
result in the alkalinization of lysosomes, inhibited the
time dependent signal increase obtained with 14C-1
(Figure 3B).
The above data is consistent with the aryl-piperazine
groups of 1, 10, and 11 conferring lysosomotropic
properties on these cathepsin K inhibitors, thus provid-
ing them with increased lysosomal concentrations and,
in turn, increased apparent whole cell potencies. The
experimentally determined pKa and Log P values of the
cathepsin inhibitors (Table 2) reveal that 1, 2, 10, and
11 have pKa values around neutrality or higher and are
relatively lipophilic, two features required for a high
degree of lysosomotropism.11 The calculated pKa values,
by ACD software, for the terminal piperazine nitrogen
of 1, 2, 10, and 11 gave values ranging from 6.1 to 8.9.
By comparison, the nitrogen proximal to the aromatic
group gave calculated pKa values in the range of -1.3
to 2.2 for these four compounds. A second experimental
pKa value of 1.76 ( 0.22 was obtained for 11 (calculated
value 2.2 ( 0.4), consistent with the above assignment.
A study of the whole body distribution of 14C-1 in rats
by autoradiography (Figure 4, Table 3) 1 h after iv
injection of the radiolabeled compound showed high
concentrations in the liver, kidney, and small intestine
and urinary bladder contents, indicating that 14C-1 is

7540 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Table 4. Drug Tissue Concentrations in Ratsa
Falgueyret et al.
tissue concentration (µM)
compound
liver
kidneyb
lung
spleen
muscle
brain
fat
plasma
Vd (L/kg)
1c
20
15
16
20
35
-
5
1.1
_
2.4
11
4.7
1.9
10c
37
41
31
5
0.6
2.5
3.6
13d
0.18
0.22
0.18
0.22
0.02
0.03
0.17
a Compounds were dosed either po or iv and tissues taken for analysis 2 h following dosing. b Renal cortex. c Dosed at 10 mg/kg po.
d Dosed at 5 mg/kg iv.
Table 5. Shift in Inhibitor Potencies between Rabbit
Cathepsin K Enzyme Assay and Functional Rabbit Bone
Resorption Assay for Basic and Nonbasic Inhibitorsa
containing inhibitors alone. A possible additional func-
tional effect of lysosomal/endosomal alkalinization does
not appear to be a major contributor to the potency of
the basic compounds, as 2 (which is basic, but only
weakly active versus isolated cathepsins) is inactive in
the functional cathepsin S assay. The requirement for
a high degree of cathepsin S inhibition for functional
activity by the potent, neutral inhibitor 14, is consistent
with observations of cathepsin S genetic deletion in
mice. In these animals, the cathepsin S substrate Ii p10
accumulates in splenocytes of homozygous null mice, but
not to any detectable degree in heterozygotes.29 Al-
though the potencies of the basic cathepsin K inhibitors
(1, 10, and 11) in the functional cathepsin S antigen
presentation assay are relatively weak, it demonstrates
the affect of their accumulation in the acidic subcellular
compartment that contains an antitarget enzyme. It is
expected that lysosomal cathepsin B activity would be
inhibited by relatively low concentrations of 1, 10, and
11, as predicted by the potencies of these compounds
in the cathepsin B whole cell enzyme occupancy assay
(Table 1).
inhibition of cathepsin K, IC50 (nM) ((SEM)
rabbit bone
rabbit cathepsin K resorption assay potency shift (fold)
(1)
0.5
5 ( 1
97 ( 6
149 ( 20
41 ( 12
580 ( 230
10
35
5
(10)
(11)
(12)
(13)
2.7 ( 0.6
33 ( 2
9.5 ( 0.6
72 ( 27
4
8
a The data represent averages ((SEM) of at least duplicate
experiments.
Table 6. Potencies for Basic and Nonbasic Cathepsin K
Inhibitors against Mouse Cathepsin S in Purified Enzyme,
Whole Cell Mouse Splenocyte Enzyme Occupancy, and
Cell-based Functional Mouse Antigen Presentation Assaysa
inhibition of mouse cathepsin S, IC50 (nM) (( SEM)
enzyme
cell (splenocyte) antigen presentation
318 ( 150 6200 ( 1800
>10000
(1)
5400 ( 150
33000 ( 5000
6350 ( 180
(2)
>100000
(10)
480 ( 100
740 (n)1)
6700 ( 900
8500 ( 1500
40 ( 18
6180 ( 1400
8600 ( 5000
(11) 34100 ( 500
(12)
(13)
(14)
8500 ( 3000
4500 ( 900
13 ( 4
>100000
>100000
1220 ( 300
Conclusion
In summary, we have demonstrated that the basic
nitrogen-containing lipophilic cathepsin K inhibitors
described here exhibit lysosomotropic properties. This
feature results in their accumulation within acidic
subcellular organelles and an apparent increase in
inhibitor potency against the antitarget enzymes present
in these compartments. Interestingly, their lysosomo-
tropic properties do not result in increased potencies in
a functional cathepsin K assay of bone resorption, but
do so against the antitarget cathepsin S. The reason for
this anomaly is unknown, but practically, this results
in a reduction in the effective selectivity of these basic
inhibitors. This phenomenon of increased potency of
basic compounds against lysosomal cathepsins is not
likely a cathepsin-specific effect and should manifest
itself for other lysosomal targets as well. Thus, the
cellular potencies of lysosomotropic inhibitors which
selectively target lysosomal cathepsins (e.g. B, L, and
S) would be predicted to be increased over those
determined using purified enzymes. The techniques
described here, as well as others in the literature,11,22
should facilitate the identification of lysosomotropic
molecules and aid in the understanding of the inhibition
of their targets in their native subcellular compart-
ments.
a The data represent averages ((SEM) of at least duplicate
experiments (except where noted).
The effect of inhibitor basicity on off-target cathepsin
S functional activity was investigated using a cell-based
mouse antigen presentation assay (Table 6). In a subset
of antigen-presenting cells, cathepsin S cleaves the
invariant chain (Ii) to allow efficient peptide antigen
loading of the type II major histocompatibility complex
and antigen presentation.27 The nonbasic cathepsin S
selective inhibitor 14,28 which is highly potent in both
purified mouse cathepsin S enzyme and mouse spleno-
cyte whole cell cathepsin S enzyme occupancy assays,
is significantly shifted to lower potency in the cell-based
functional assay of antigen presentation. In contrast,
the basic inhibitors (1, 10, and 11), which are relatively
weak against purified mouse cathepsin S (but show
increased activity in the splenocyte whole cell enzyme
occupancy assay) exhibit unexpected activities in the
cathepsin S-dependent cell-based antigen presentation
assay. Cathepsin K is not known to play a role in
antigen presentation. These results suggest that the
lysosomotropic properties of 1, 10, and 11 contribute to
their potencies in the functional cell-based assay. By
comparison, the neutral analogues 12 and 13, which
have similar potencies to 1, 10, and 11 against purified
mouse cathepsin S, are inactive in the functional assay.
The mouse splenocyte cathepsin S enzyme occupancy
assay results (Table 6) are consistent with those ob-
tained in human Ramos cells (Table 1) and show
increased cellular potencies for the basic nitrogen-
Experimental Section
General. Proton (1H NMR) magnetic resonance spectra
were recorded on a Bruker instrument operating at either 400
or 500 MHz. All spectra were recorded using residual solvent
(DMSO or acetone) as internal standard. Signal multiplicity
was designated according to the following abbreviations: s )

Lysosomotropism of Basic Cathepsin K Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7541
singlet, d ) doublet, dd ) doublet of doublets, t ) triplet, m )
multiplet, br s ) broad singlet. Elemental analyses were
provided by Onieda Research Services Inc., Whitesboro, NY.
High-resolution mass spectra (HRMS-FAB+) were obtained
at the Biomedical Mass Spectrometry Unit, McGill University,
Montreal, Quebec, Canada. All substrates and reagents were
obtained commercially and used without further purification.
Reactions were carried out with continuous stirring under a
positive pressure of nitrogen except where noted. Flash chro-
matography was carried out with silica gel 60, 230-400 mesh.
Compounds 1, 10, 11, 12, 13, and 14 were prepared as
previously described.19a,20,28
4-[2-(-Methylpiperazin-1-yl)-1,3-thiazol-4-yl]-N-(1-{[(2,2,2-
trifluoroethyl)amino]-carbonyl}cyclohexyl)benzamide
(2). To a solution of PCl5 (27.6 g, 132 mmol) in CH3CN (400
mL) was added 1-aminocyclohexanecarboxylic acid hydrochlo-
ride (20 g, 111 mmol). The resultant slurry was stirred at room
temperature for 3 h followed by filtration. The filter cake was
washed with CH3CN and dried under a N2 flow followed by
drying under high vacuum overnight. 1-Aminocyclohexanecar-
bonyl chloride hydrochloride was obtained in 91% yield (20
g). To a mixture of 1-aminocyclohexanecarbonyl chloride
hydrochloride (10.2 g, 51.5 mmol) in CH3CN (400 mL) was
added trifluoroethylamine (9.0 mL, 113 mmol) over 5 min. The
resultant slurry was stirred at room temperature for 30 min
before partitioning between EtOAc and water. The organic
layer was separated, dried over MgSO4, and concentrated to
yield 1-amino-N-(2,2,2-trifluoroethyl)cyclohexane-carboxamide
(3) (7.79 g, 67% yield) which was used as such in the next
reaction. 1H NMR (acetone-d6, 500 MHz) δ 8.45 (1H, br s), 3.42
(2H, m), 1.85 (3H, m), 1.59 (4H, m), 1.38 (2H, br d), 1.25 (1H,
m).
To a solution of 4-[2-(4-methylpiperazin-1-yl)-1,3-thiazol-4-
yl]benzoic acid hydrobromide (4)18 (13.42 g, 34.9 mmol) and
the amine 3 (7.79 g, 34.7 mmol) in DMF (65 mL) were added
Et3N (12.1 mL, 86.8 mmol), HOBT‚H2O (5.58 g, 36.4 mmol),
and EDC‚H2O (7.3 g, 38.1 mmol). The reaction mixture was
stirred at room temperature for 2 days and then partitioned
between EtOAc and water. The organic layer was separated,
dried over Na2SO4, and concentrated. The resultant beige solid
was stirred with boiling EtOAc (400 mL) followed by the
addition of hexane (400 mL). A light beige solid was collected
by filtration to afford 4-[2-(4-methylpiperazin-1-yl)-1,3-thiazol-
4-yl]-N-(1-{[(2,2,2-trifluoroethyl)amino]-carbonyl}cyclohexyl)-
benzamide (2) (7.89 g, 45% yield). The product was determined
to be >99.5% pure by reverse phase HPLC. 1H NMR (acetone-
d6, 500 MHz) δ 7.97 (2H, d), 7.95 (1H, m), 7.91 (2H, d), 7.43
(1H, br s), 7.23 (1H, s), 3.94 (2H, m), 3.52 (4H, t), 2.48 (4H, t),
2.29 (2H, m), 2.27 (3H, s), 1.94 (2H, m), 1.62 (5H, m), 1.35
(1H, m). HRMS (+FAB): calcd for C24H31N5O2SF3 [MH+]
510.21528, found 510.21506. Anal. (C24H30N5O2SF3) C: calcd,
56.57; found, 56.04; H: calcd, 5.93; found, 5.25; N: calcd, 13.74;
found, 13.59; S: calcd, 6.29; found, 6.13.
14C-N-(1-{[(Cyanomethyl)amino]carbonyl}cyclohexyl)-
4-[2-(4-methylpiperazin-1-yl)-1,3-thiazol-4-yl]benzam-
ide (14C-1). To a suspension of 2-bromo-1-(4-bromophenyl)-
ethanone (1.96 g, 7.05 mmol) in EtOH (50 mL) was added
4-methylpiperazine-1-carbothioamide (1.12 g, 7.03 mmol). The
mixture was heated to reflux for 1.5 h, then cooled and filtered
to give 2.82 g (95%) of 4-[4-(4-bromophenyl)-1,3-thiazol-2-yl]-
1-methylpiperazin-1-ium bromide. 1H NMR (DMSO-d6, 400
MHz) δ 9.9 (1H, br s), 7.82 (2H, d), 7.60 (2H, d), 7.48 (1H, s),
4.1 (2H, br s), 3.5 (2H, br s), 3.4 (2H, br s), 3.2 (2H, br s), 2.85
(3H, s). This salt (585 mg) was partitioned between ethyl
acetate and aq NaHCO3. The organic phase was washed with
brine, dried over MgSO4 and evaporated to give 400 mg of 1-[4-
(4-bromophenyl)-1,3-thiazol-2-yl]-4-methylpiperazine (5). 1H
NMR (DMSO-d6, 400 MHz) δ 7.80 (2H, d), 7.55 (2H, d), 7.35
(1H, s), 3.45 (4H, m), 2.43 (4H, m), 2.21 (3H, s).
mg, 3.6 mCi/mmol), and both flasks were swept with N2(g).
The aryllithium flask was cooled in liquid nitrogen and the
N2 (g) flow was stopped when the solvent solidified. Concen-
trated sulfuric acid (0.8 mL) was then added dropwise to the
Ba14CO3, liberating 14CO2. After 5 min, both the cannula and
the liquid nitrogen bath were removed, and the flask was
allowed to warm to -78 °C and stir for 1 h. The mixture was
then warmed to room temperature, where TLC analysis
indicated formation of lithium 4-[2-(4-methylpiperazin-1-yl)-
1,3-thiazol-4-yl]benzoate. The THF was removed under a flow
of N2 (g). To the residue were added HATU (220 mg, 0.58
mmol), 1-amino-N-(cyanomethyl)cyclohexanecarboxamide meth-
anesulfonate (6)18 (167 mg, 0.60 mmol), and DMF (4 mL).
Triethylamine (0.1 mL, 0.7 mmol) was then added, the mixture
was stirred overnight at room temperature and then parti-
tioned between ethyl acetate and water. The organic phase
was washed with dilute NaHCO3 and brine and then dried
over Na2SO4 and concentrated. The experiment was repeated
with 267 mg of 1-[4-(4-bromophenyl)-1,3-thiazol-2-yl]-4-meth-
ylpiperazine (5), and the combined crude products were
purified by flash chromatography (5 to 7% MeOH in CH2Cl2
+ 0.5% NH4OH) to give 155 mg of 92% pure 14C-N-(1-{-
[(cyanomethyl)amino]carbonyl}cyclohexyl)-4-[2-(4-methylpip-
erazin-1-yl)-1,3-thiazol-4-yl]benzamide (14C-1). Further puri-
fication was accomplished using preparative HPLC (RP C18
column, gradient: 27% to 31% CH3CN/0.2% aq formic acid over
10 min, TR ) 4.4 min) to provide 101 mg of the title compound
(0.197 mmol, 0.71 mCi).
14C-N1-(Cyanomethyl)-N2-(4-morpholin-4-ylbenzoyl)-
leucinamide (14C-9). To a solution of 4-morpholin-4-ylbenzoic
acid (0.73 g, 1 equiv) and PyBOP (1.6 g, 1 equiv) in DMF (25
mL) was added triethylamine (1.3 mL, 3.1 equiv). The reaction
mixture was stirred at room temperature for 10 min followed
by the addition of L-leucinamide hydrochloride (0.5 g, 1 equiv).
The resultant suspension was stirred at room temperature
under an atmosphere of nitrogen overnight. Saturated aqueous
NaHCO3 was added followed by EtOAc and water. The
aqueous layer was separated and extracted with EtOAc (7×).
The combined organic extracts were washed with water (1×),
dried, and concentrated. The residue was stirred with a
minimal amount of diethyl ether and then filtered to provide
0.91 g (95% yield) of the desired N2-(4-morpholin-4-ylbenzoyl)-
leucinamide (7). 1H NMR (400 MHz, DMSO-d6) δ 8.05 (d, J )
8.2 Hz, 1H), 7.78 (d, J ) 8.9 Hz, 2H), 7.31 (br s, 1H), 6.95 (d,
J ) 8.9 Hz, 2H), 6.93 (br s, 1H), 4.41 (m, 1H), 3.73 (t, J ) 4.8
Hz, 4H), 3.20 (t, J ) 4.8 Hz, 4H), 1.48-1.69 (m, 3H), 0.87 (dd,
J ) 16, 6.3 Hz, 6H).
To a suspension of N2-(4-morpholin-4-ylbenzoyl)leucinamide
(7) (500 mg, 1 equiv) in toluene (10 mL) were added formal-
dehyde (0.25 mL of a 37% aq solution, 2 equiv) and 1H-1,2,3-
benzotriazole (375 mg, 2 equiv). pTsOH‚H2O (30 mg, 0.1 equiv)
was added, and the resultant suspension was heated to reflux
for 2 h with a dean-stark apparatus to remove water. The
toluene was then removed using rotary evaporation, and the
crude residue was dissolved in diethyl ether and washed with
0.2 N NaOH (3×) and brine (1×), dried, and concentrated. The
residue was purified by flash column chromatography on silica
gel (gradient elution: 60% EtOAc/hexane to 100% EtOAc) to
afford 255 mg (37% yield) of N1-(1H-1,2,3-benzotriazol-1-
ylmethyl)-N2-(4-morpholin-4-ylbenzoyl)leucinamide (8). 1H
NMR (400 MHz, DMSO-d6) δ 9.37 (t, J ) 6.4 Hz, 1H), 8.19 (d,
J ) 8.33 Hz, 1H), 8.02 (d, J ) 8.5 Hz, 1H), 7.93 (d, J ) 8.4 Hz,
1H), 7.77 (d, J ) 8.8 Hz, 2H), 7.53 (t, J ) 7.3 Hz, 1H), 7.39 (t,
J ) 7.4 Hz, 1H), 6.94 (d, J ) 8.9 Hz, 2H), 5.99 (d, J ) 6.6 Hz,
2H), 4.47 (m, 1H), 3.73 (t, J ) 4.4 Hz, 4H), 3.19 (t, J ) 4.4 Hz,
4H), 1.36-1.64 (m, 3H), 0.78 (dd, J ) 17, 6.4 Hz, 6H).
To a solution of N1-(1H-1,2,3-benzotriazol-1-ylmethyl)-N2-
(4-morpholin-4-ylbenzoyl)leucinamide (8) (28.8 mg, 1 equiv) in
DMSO-d6 (0.2 mL) was added Na14CN (4.9 mg from ARC 140A,
lot # 980422, 5 mCi). The brown solution was heated to 95 °C
for 4 h. EtOAc (40 mL) was added, and the solution was
washed with 0.2 N NaOH (6 × 10 mL) and brine (1 × 10 mL),
dried, and concentrated to yield 21.1 mg (92% yield) of N1-
To a -78 °C solution of 1-[4-(4-bromophenyl)-1,3-thiazol-2-
yl]-4-methylpiperazine (5) (195 mg, 0.576 mmol) in THF (8 mL)
was added n-BuLi (1.6 M, 0.43 mL, 0.69 mmol), and the
solution was stirred for 15 min. A cannula was connected
between this flask and a 5 mL flask containing Ba14CO3 (114

7542 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Falgueyret et al.
(C14cyanomethyl)-N2-(4-morpholin-4-ylbenzoyl)leucinamide (14C-
9) which was 95% pure by radioactive reverse phase HPLC.
Compound 14C-9 was purified by flash column chromatography
on silica gel (gradient elution: 50% EtOAc/hexanes to 60%
EtOAc/hexanes) to provide >99% purity by radioactive reverse
phase HPLC (2 mCi). The specific activity of 14C-9 was 42.6
mCi/mmole.
onto Scotch tape and then dried overnight in a vacuum-
desiccator at room temperature. 14C-1 standards were obtained
by serial dilution in rat blood. Calibrated drops of the blood/
14C-1 standards were placed on Scotch tape strips, which were
dried as above. The simultaneous, quantitative determination
of the radioactivity in whole-body sections and standards was
carried out using a beta-imager 2000 (Biospace Measures,
Paris, France). Data from whole-body sections and standards
were collected for 21 h. Data analysis was performed using a
[â vision +] software (Biospace Measures).
Mouse Cathepsin S Whole Splenocyte Enzyme Oc-
cupancy Assay. Mouse splenocytes were prepared from 8 to
10-week old C57Bl/6 mice spleens in PBS, 2 mM glutamine,
10 mM glucose, and 1% (v/v) FBS using a Medimachine tissue
disaggregator (DakoCytomation) with a 50 µm Medicons
chamber and passed through a 50 µm Filcons filter. Following
red blood cells lysis in 140 mM NH4Cl, 17 mM Tris-HCl pH
7.5, splenocytes were resuspended in media (RPMI 1640, 10
mM HEPES, pH 7, 1 mM sodium pyruvate, 100 U/mL
penicillin, 100 µg/mL streptomycin) at 2.5 × 107 cells/mL. The
splenocytes (200 uL) were preincubated with test compound
for 60 min at 37 °C in a CO2 incubator in a 96-well plate. The
iodinated probe BIL-DMK8 (1 nM) was then added for 20 min,
the reaction was stopped with 10 µM E-64d for 10 min, and
cells were centrifuged at 300g. The splenocytes were washed
with PBS and labeled proteins separated by 12% Tris-Glycine
PAGE and quantified as described.8
Mouse Antigen Presentation Assay. The mouse antigen
presentation assay was performed using a modification of an
established protocol.30 Briefly, compounds were preincubated
with A20µPC cells (A20 cells transfected with PC-specific
mIgM, obtained from Dr J. R. Drake, Albany Medical College)
in a 96-well plate (1 × 105 cells) for 60 min at 37 °C in a CO2
incubator in RPMI 1640, 10% FBS, 2 mM glutamine, 50 µM
â-mercaptoethanol, and 100 U/mL penicillin, 100 µg/mL strep-
tomycin. DO.11.10 cells (1 × 105) (provided by the National
Jewish Medical and Research Center, Denver, CO) and the
antigen PC-OVA30 (30 µg/mL) in a final volume of 200 µL were
added. IL-2 sretion into culture media, as measured using a
mouse IL-2 ELISA kit (Pierce), was determined following an
overnight incubation. IC50 values were determined with the
SOFTmax Pro software using DMSO as the control for no
inhibition.
Confocal Microscopy of HepG2 Cells. HepG2 cells were
grown in SlideFlasks in culture media containing MEM Earle’s
salt supplemented with L-glutamine (Invitrogen); 10% FBS; 1
mM sodium pyruvate; 0.1 mM nonessential amino acids; 100
units/mL penicillin-streptomycin at 37 °C in the presence of
5% CO2. At 50% confluency, cells were washed with fresh
media and then incubated with media containing the test
compound added from a 1000-fold stock in DMSO. Control
experiments contained only DMSO vehicle. Cells were washed
with Hanks’s balanced salt solution supplemented with 15 mM
HEPES (HHBSS) and then observed by laser scanning mi-
croscopy using a Zeiss LSM 510 equipped with an inverted
Zeiss Axiovert 100M microscope. Compound 1 (10 µM) was
detected using a 2-photon Mira 900 Ti/sapphire (Coherent)
laser tuned to 710 nm, with a pulse width of ∼3 ps and a
repetition rate of 76 MHz. Emitted light was captured with
one photomultiplier for wavelengths below 680 nm. To visual-
ize the fluorescent amine Red DND-99 (10 nM), the illumina-
tion source was the 543-nm line from a HeNe 1-mW laser
passing through the 488/543-nm main dichroic mirror. Fluo-
rescence was recorded using one photomultiplier and a 560-
nm long-pass filter. Pinhole and laser intensity were adjusted
for the control experiment to avoid saturation of the signal.
Accumulation of 14C-Labeled Cathepsin K Inhibitors
within HepG2 Cells. HepG2 cells (grown as above) were
seeded in a 96-well Cytostar-T scintillation microplate (Am-
ersham) the day before the experiment and grown as mono-
layers to ∼90% confluency. The cells were washed with 200
µL of HHBSS. Then, 200 µL of HHBSS containing 30 µM of
either 14C-1 (0.02 µCi) or 14C-9 (0.02 µCi) was added to the
cells. Immediately after this addition, the uptake of 14C in to
the cells was monitored using a Wallac MicroBeta plate
counter. In each well, the radioactivity was read as CPM every
10 min over a 120 min period.
pKa and Log P Measurements. The pKa and octanol-
water partition coefficients were measured using the GLpKa
(Sirius Analytical Instruments) at 25 °C (0.15M KCl, argon)
according to the manufacturer’s instructions. Potentiometric
titrations for 1 and 10 were carried out in dioxane/water and
in DMSO/water for 2. The pKa measurement for 11 was
evaluated in water using a spectroscopic probe. The Log P
value for 13 was evaluated by the shake flask method
described as follows. The partition coefficients were determined
by adding 50 µL of the compound dissolved in acetonitrile (2
mg/mL) into 0.75 mL of water and 0.75 mL of 1-octanol which
have been mutually saturated with each other. The sample
was then vortexed (16000 rpm, 60 s) and allowed to separate
(6 h). The concentrations in each phase were determined by
HPLC.
Tissue Level Extraction Procedure. Tissue levels were
determined by taking a known weight of tissue, adding 2
volumes of water and homogenizing to a frothy suspension
using a tissue homogenizer. To this suspension was added 3
volumes of acetonitrile, containing an internal standard. This
was then further homogenized, followed by centrifugation (10
min at 14000 rpm). The supernatant was analyzed by LC-
MSMS using an internal standard and levels obtained were
quantified by comparing them to a standard curve generated
by spiking rat plasma with compound following the same
procedure as detailed above.
Acknowledgment. We thank Matthew Zrada and
Cynthia Bazin for performing the Log P and pKa
analyses and Gregg Wesolowski for performing the bone
resorption assays.
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JM0504961