Design of Potent, Selective, and Orally Bioavailable Inhibitors of Cysteine Protease Cathepsin K

Article abstract of DOI:10.1021/jm030373l

Osteoclast-mediated bone matrix resorption has been attributed to cathepsin K, a cysteine protease of the papain family that is abundantly and selectively expressed in osteoclast. Inhibition of cathepsin K could potentially be an effective method to preve

Full text of DOI:10.1021/jm030373l

588
J . Med. Chem. 2004, 47, 588-599
Design of P oten t, Selective, a n d Or a lly Bioa va ila ble In h ibitor s of Cystein e
P r otea se Ca th ep sin K
Francis X. Tavares,*,† Virginia Boncek,‡,§ David N. Deaton,† Anne M. Hassell,| Stacey T. Long, Aaron B. Miller,|
Alan A. Payne,‡ Larry R. Miller,‡ Lisa M. Shewchuk,| Kevin Wells-Knecht,4 Derril H. Willard J r.,X,O
Lois L. Wright,∇ and Hui-Qiang Zhou†
Department of Medicinal Chemistry, Discovery Research Biology, Department of Research Bioanalysis and Drug Metabolism,
Discovery Research CASS, Department of Molecular Pharmacology, Preclinical Development, and Department of Gene
Expression and Protein Biochemistry, GlaxoSmithKline, Research Triangle Park, North Carolina, 27709
Received August 5, 2003
Osteoclast-mediated bone matrix resorption has been attributed to cathepsin K, a cysteine
protease of the papain family that is abundantly and selectively expressed in osteoclast.
Inhibition of cathepsin K could potentially be an effective method to prevent osteoporosis.
Structure-activity studies on a series of reversible ketoamides based inhibitors of cathepsin
K have led to identification of potent and selective compounds. Crystallographic studies have
given insights into the mode of binding of these inhibitors. A series of ketoamides with varying
P1 moieties were first synthesized to find an optimum group that would fit into the S1 subsite
of the cysteine protease, cathepsin K. With a desired P1 group in place a variety of heterocyclic
analogues in the P′ region were synthesized to study their steric and electronic effects. In the
process of exploring these P′ heterocyclic variations, excellent selectivity was gained over other
highly homologous cysteine proteases, including cathepsins L, S, and V. The favorable
pharmacokinetic properties of some of these cathepsin K inhibitors in rats make them suitable
for evaluation in rodent osteoporosis models. A representative cathepsin K inhibitor was shown
to attenuate PTH-stimulated hypercalcemia in the TPTX rat model. These inhibitors provide
a viable lead series in the discovery of new therapies for the prevention and treatment of
osteoporosis
In tr od u ction
bone matrix. The expression of cathepsin K, a cysteine
protease of the papain superfamily, is abundant and
selective in osteoclasts, suggesting that this enzyme is
crucial for bone resorption.2a,b Cathepsin K antisense
nucleotide studies have implicated this enzyme in
osteoclast-mediated bone resorption.3 In addition, a
link between mutations in human cathepsin K and
pycnodysostosis, a rare osteopetrotic disease character-
ized by abnormal bone resorption, has been demon-
strated.4 Recently, it has also been shown that cathepsin
K-deficient mice exhibit a distinct osteopetrotic pheno-
type.5 Recent work has shown that cathepsin K, and not
cathepsin L, is the major protease responsible for
human osteoclastic bone resorption.6 The effects of a
small molecule inhibitor of cathepsin K has also been
evaluated on bone resorption in vivo using a nonhuman
primate model of postmenopausal bone loss in which
the active form of cathepsin K is identical to the human
ortholog. These studies suggest that selective inhibition
of cathepsin K could provide an effective therapy for the
treatment of osteoporosis.
Bone metabolism, consisting of bone formation and
resorption is a dynamic process in life, and balance of
these opposing processes is necessary for the preserva-
tion of skeletal mass and architecture. A shift in the
balance of this dynamic process can lead to abnormali-
ties in bone mass. Osteoporosis is a disease character-
ized by generalized skeletal fragility resulting in frac-
tures occurring with minimal trauma. Bone remodeling
involves osteoclast-mediated dissolution of bone mineral
as well as degradation of its protein matrix. Osteoclasts
form an extracellular compartment by firmly attaching
to the bone surface. This attachment allows the estab-
lishment of an acidic environment that facilitates de-
mineralization of the bone.1 Osteoclasts then secrete
proteolytic enzymes into this compartment to degrade
the organic matrix. Cysteine proteases have been im-
plicated in this osteoclast-mediated resorption on the
* To whom correspondence should be addressed. Phone: (919) 483-
7456; Fax: (919)-483-6053; e-mail: fxt66911@gsk.com.
† Department of Medicinal Chemistry.
Cysteine proteases make up the vast majority of
lysosomal proteases. There are currently 11 members
of the human cathepsin cysteine protease family. With
the exception of cathepsin K, the physiological role
and pathological implications of many cathepsins
are not well understood. Cysteine proteases such as
Cathepsins B, S, and L have been implicated in im-
‡ Department of Molecular Pharmacology.
§ Present address: 3040 Cornwallis Rd., 111 Hermann Building,
Research Triangle Park, NC 27709.
| Discovery Research CASS.
Preclinical Development.
4 Department of Research Bioanalysis and Drug Metabolism.
X Department of Gene Expression and Protein Biochemistry.
O Deceased.
∇ Discovery Research Biology.
10.1021/jm030373l CCC: $27.50 © 2004 American Chemical Society
Published on Web 12/24/2003

Inhibitors of Cysteine Protease Cathepsin K
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 3 589
Sch em e 1
vitro SAR studies, the evaluation of certain compounds
in pharmacokinetic and pharmacodynamic studies in
the rat will be described.
Ch em istr y. The general syntheses of ketoamide
inhibitors are shown in Scheme 2. The alcohol 3 was
treated with phosgene to afford a chloroformate, and
subsequent addition of an amino ester provided car-
bamates 4.18 Hydrolysis of esters 4 to acids followed by
coupling with the phosphorane afforded cyanoylides 5.
Oxidation of the ylide 5 following the Wassermann
procedure19 and trapping the acyl nitrile with amines
afforded the ketoamides 6-43. The 1-alkyl-1H-pyrazole-
5-ylamines (26a -32a ) and the amines 34a , 35a , and
36a were synthesized following literature proce-
dures.20-22
munological responses.8,9 Cathepsin B, for example, is
the most abundant lysosomal cysteine protease impli-
cated in the pathogenesis of rheumatoid arthritis,
muscular dystrophy, and tumor metastasis.7 Cathepsins
V and S are more tissue specific with cathepsin V
expressed in thymus, and cathepsin S in spleen and
lung.8-10 Cathepsins S and L have been implicated in
major histocompatibility complex (MHC) class II invari-
ant chain processing.8,9 In lieu of the fact that cathepsins
L, S, V, and K have a high degree of homology, design
of inhibitors that are selective for the inhibition of
cathepsin K is challenging. The difficulty in designing
selective cathepsin inhibitors results from the similarity
in their substrate recognition as well as their common
proteolytic mechanism.10b Since the treatment of osteo-
porosis would require chronic administration of a drug,
the development of a selective reversible inhibitor was
desired. This could potentially avoid antigenicity due
to covalent modification of proteins via irreversible
inhibition. Numerous warheads have been utilized in
reversible inhibitors of cysteine proteases including
peptidic aldehydes,11 nitriles,12 cyclopropenones,13 di-
amino ketones,14 and R-ketoamides.15 There has also
been a recent report on the use of noncovalent amides
as cathepsin K inhibitors.16 This group was particularly
interested in exploring R-ketoamides as reversible in-
hibitors of cathepsin K. The ketoamide class of inhibi-
tors has been widely employed in inhibiting serine
proteases.15,17 Herein is described its use in the inhibi-
tion of the cysteine protease, cathepsin K. Synthetic
efforts in these laboratories focused around the keto-
amide inhibitor 2 (cathepsin K IC50 ) 2.54 nM) which
was derived from the peptide aldehyde lead 1 (cathepsin
K IC50 ) 51 nM). The aim was to optimize on the
structural and physiochemical features of 2 by examin-
ing changes in the P1 amino acid of the ketoamide as
well as studying the role of P′ electron deficient hetero-
cycles in modulating the electrophilicity of the warhead.
In the process of these P′ heterocyclic replacements, the
achievement of selectivity over other cysteine proteases
was obtained and will be discussed. In addition to in
Resu lts a n d Discu ssion
Previous efforts in these laboratories had identified
the P1 n-butyl substituent, derived from commercially
available norleucine, as a potency enhancing group for
aldehyde-based inhibitors. Since changing the thiol
reactive group might modify the P1 SAR, P1 analogues
in the ketoamide series were synthesized. The IC50
values are the mean of two or three inhibition assays,
individual data points in each experiment were within
3-fold range of each other. Cathepsin K inhibition data
for the P1 modification are shown in Table 1. The
glycine-derived analogue 6, lacking a chiral center (R
) H), was found to be a weak inhibitor (IC50 > 12 µM).
Introduction of a substituent on the carbon adjacent to
the ketone can bias the preferred rotational conformer
of the inhibitor as well as providing opportunities for
additional binding interactions. The methyl analogue
7 exhibits enhanced potency (IC50 ) 360 nM). Some of
this increase may arise from a lower entropic cost of
binding due to stabilization of the active rotamer.
Increasing the size of the substituent at P1 with amino
acids valine, leucine, isoleucine, phenylalanine, and
norleucine gave analogues 8-10 and 12 and 13, respec-
tively. These modifications gave a boost in potency, with
compound 13 being the most potent (IC50 ) 9.2 nM) in
this series of ketoamides. The cyclohexylmethyl ana-
logue 11, the saturated version of the phenylalanine
derivative, was also a potent inhibitor of cathepsin K
(IC50 ) 14 nM). On the basis of these results and
desiring to control cost of goods of any potential drug
candidate by limiting synthetic complexity, the nor-
leucine-derived n-butyl group was selected as an ap-
propriate P1 moiety.
Sch em e 2a
a
(a) 20% COCl2 in toluene, THF, rt; (b) L-norleucine methyl ester hydrochloride, Hunig’s base, THF, rt; (c) 1 M LiOH/THF (1:1), rt; (d)
(triphenylphosphoranylidene)acetonitrile, EDCI, DMAP, DCM, rt; (e) ozone, DCM, -78 °C, 15 min.; (f) amine, 1 M AgNO3/THF (1:5), rt.

590 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 3
Ta ble 1. Inhibitory Potencies vs Human Cathepsin K
Tavares et al.
amide inhibitors with the hope that they might π-π
stack with the indole side chain of 184Trp or form
hydrogen bonds with its indole NH.
The six-membered P′ heterocycles, compounds 14-
16 exhibited less inhibitory activity than the lead
compound 2, with the pyridine analogue 14 (IC50 ) 32
nM) being the most potent. Incorporation of another
nitrogen as in the pyrazine analogue 16 did not boost
activity. Fusion of another aryl ring as in the isoquino-
line 18 provided a 4-fold enhancement in potency. The
site of ring attachment proved crucial with the quinoline
17 being 30-fold less active. This increase in inhibitory
activity with the bicyclic system 18 could result from
interactions with the tryptophan moiety.
The five-membered P′ heterocycles 19-22 proved to
be more promising than their corresponding six-
membered congeners 14-16. In the five-membered
heterocyclic series it was found that heterocycles hav-
ing a potential hydrogen bond acceptor, as in com-
pounds 19-22, were very potent reversible inhibitors
of cathepsin K. The isoxazole 21 was as potent as the
lead 2 and the pyrazole 22 was an even better inhibitor.
The thiazole 19 and the thiadiazole 20 were significantly
less potent. It is difficult rationalizing this potency on
the steric or electronic properties of these heterocycles.
Insights into the binding of these heterocycles was
obtained from an X-ray cocrystal structure of compound
22 bound to cathepsin K at 2.2 Å resolution (Figure 1).
The pyrazole has two interactions with the protein: one
through a two water interaction to 18Gln and another
with 184Trp. The S1 pocket forms a “face” using back-
bone atoms as well as 64Gly and 65Gly. The norleucine
is in the S1 groove with one face and the end exposed
to solvent. One of the isopropyl groups is bound in the
S2 pocket formed by residues 67Tyr, 68Met, 134Ala, 163Ala,
and 209Leu of cathepsin K. From the residues that make
up this pocket, it is clear why hydrophobic groups are
well tolerated. The other isopropyl group projects in the
direction of S3 pocket but does not occupy it. A covalent
interaction is made between pyrazole 22 and the
protein. The formation of a hemithioketal between 25Cys
and the carbonyl of the ketoamide is observed. This is
consistent with a reversible transition state inhibitor.
However, what is interesting is that the resulting OH
of the hemithioketal is not directed into the anionic
hole of the protein. The OH interacts with 162His that
is part of the catalylic triad, as well as the backbone
carbonyl of 161Asn. This represents attack of the thiol
in the opposite stereochemistry from previously reported
cathepsin K cocrystal structures.14b,d The oxyanionic
hole is filled by the amide carbonyl of the ketoamide
and interacts with the backbone NH of 25Cys and the
side chain of 19Gln. Peptidic recognition site interactions
are formed between the NH and carbonyl of the inhibi-
tor carbamate group to the backbone carbonyl of 161Asn
and the backbone HN of 66Gly, respectively.
Extrapolating the X-ray data from pyrazole 22, the
enhanced potency of analogues 14 and 16 compared to
pyridine 15 may be explained by the possible hydrogen
bond formation with 184Trp.
The cysteine cathepsins contain a highly conserved
tryptophan in the S′ pocket of the active site. Designing
ketoamide inhibitors that would take advantage of the
interactions with 184Trp in the cathepsin K substrate
binding groove would be highly desirable to enhance
potency. To this end, various five- and six-membered
heterocyclic P′ groups were incorporated into the keto-
From X-ray information of 22, one could also specu-
late that in compounds 19-21 the lone pair on the
nitrogen forms a hydrogen bond with 184Trp. The fact
that isoxazole 21 is over 8-fold more active than thiazole

Inhibitors of Cysteine Protease Cathepsin K
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 3 591
F igu r e 1. X-ray cocrystal structure of compound 22 bound to cathepsin K at 2.2 Å. Cathepsin K is colored orange, compound 22
by atom type, and critical waters are cyan colored spheres. (a) Active site with hydrogen bonds of the complex highlighted in
yellow bonds and (b) surface of active site of cathepsin K. Figure made with the PYMOL program (Delano Scientific,
www.pymol.org).
19 and thiadiazole 20 may reflect its ability to form
multiple or stronger hydrogen bonds than its congeners,
but their drop in potency could also arise from detri-
mental lone pair interactions of their differentially
substituted rings.
Prior to the solution of the X-ray cocrystal structure
of pyrazole 22, the N-methylpyrazole analogues 23 and
24 were synthesized to explore the importance of the
pyrazole NH in its enhanced inhibitory activity. The 40-
fold loss in potency of the N-methyl analogue 23 relative
to 22 (Table 2) can be readily explained from the crystal
structure as resulting from the loss of hydrogen bonding
to the enzyme. Analogue 24 was found to be equipotent
to analogue 22. The striking difference between ana-
logues 23 and 24 was that N-methylpyrazole 23, al-
though not as potent, was found to be quite selective
over all the other cathepsins B, H, L, S, and V.
Encouraged by the results of enhanced selectivity of
analogue 23, a series of N-substituted ketoamides were
prepared. Increase in the steric bulk at this position
gave analogues 25 to 35. These analogues maintain
relatively constant cathepsin K inhibitory activity (Table
2). All the analogues exhibited increased selectivity
relative to pyrazole 22. Constrained analogues of the
isopropyl 26, particularly cyclobutyl 30, cyclopentyl 31,
and cyclohexyl 32 gave us highly selective inhibitors
with analogues 30 and 32 being over a 100 fold selective
over all the other cathepsins tested. The six-membered
aryl analogues 33 to 35 are not as broadly selective as
the saturated rings such as cyclohexyl analogue 32.
Since selectivity versus cathepsins S and V had proven
difficult in this ketoamide series, these results were
quite gratifying. The additional selectivity exhibited by
these compounds is difficult to rationalize from struc-
tural data based on the similarities of the S′ subsites
in these cathepsins. The enzymes would have to breathe
to allow these inhibitors in a similar binding mode.
Alternatively, they may bind in a different rotamer
conformation. It is also possible that accommodation of
these inhibitors in the S′ subsites results in shifts in
binding in other subsites that are less favorable for
cathepsin L, S, and V. Substitution at other positions
of the pyrazole ring were also investigated. The steric
probes 36 to 39 maintained similar activity to the parent
pyrazole 22, but did not offer increases in selectivity.
Indazole 40, a conformationally constrained analogue
of compound 38, did not give any significant boost in
potency, indicating that favorable entropic effects of
restricting internal rotors might be offset by unfavorable
enthalpic effects that could not be attributed to specific
ligand-protein interactions or lack thereof.23 Although
a thorough electronic effects study on analogues of
pyrazole 22 was not done, several analogues (41 to 43)
were synthesized. No dramatic improvements over
pyrazole 22 were evident.
P h a r m a cok in etic An a lysis. One of the major chal-
lenges in optimizing a peptidomimetic lead is achieving
the desired physiochemical properties which will enable
delivery of an orally bioavailable drug having good
pharmacokinetic properties. Most of these inhibitors
met Lipinski’s criteria and were profiled in several in
vitro pharmacokinetic predictive assays.24 The phar-
macokinetic and efflux properties of the P′ heterocyclic
analogues are presented in Table 3. These compounds
were characterized by excellent cell permeability in the
Madin-Darby canine kidney cell monolayer transport
assay. Many also had acceptable solubilities in the
simulated intestinal fluid (SIF) to allow absorption.
Unfortunately, analogues 19, 20, and 23 showed higher
rates of in vitro S9 instability. Some of these analogues
also exhibited plasma instability, the exception being
the N-substituted pyrazoles such as 23. Despite these
limitations, pharmacokinetic properties of several ana-
logues were evaluated in rats (Table 4). The more
soluble analogues such as pyridine 14 and pyrazole 22
exhibited good oral bioavailabilities (%F ) 58 and 41,
respectively), while the other analogues had moderate
bioavailabilities (%F ) 15-28). Pyrazole 22 had a very
high rate of clearance (exceeding the hepatic blood flow
for rat), extremely long terminal half-life (700 min) and
very high volume of distribution (Vss ) 15 L/kg).
Introduction of an N-methyl group (analogue 23) low-
ered the clearance rate (CL ) 26 mL/min/kg) by blocking
the cleavage of the amide bond next to the pyrazole and/
or preventing phase II conjugation. The half-life and
volume were also lower. Analogues 14, 19, and 20 were
more moderately cleared than pyrazole 22.

592 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 3
Tavares et al.
Ta ble 2. Inhibitory Potencies (IC50) vs Human Cathepsins K, L, S, V, H, B and Rat Cathepsin K

Inhibitors of Cysteine Protease Cathepsin K
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 3 593
Ta ble 2 (Continued)
* Represents selectivity ratios over the other cathepsins (e.g. L/K ) IC50 cat L/IC50 cat K).
Ta ble 3. In Vitro Data for P′ Ketoamides
rodent pharmacodynamic model of osteoporosis. These
ketoamide inhibitors were found to be less potent
inhibitors of the rat cathepsin K ortholog despite high
amino acid sequence identity (88%). Presumably, this
difference arises from a substitution of 133Ser in the rat
ortholog for 133Ala in the human S2 pocket.25 Pyrazole
22, the most potent rat cathepsin K inhibitor with good
pharmacokinetic parameters, was selected for evalua-
tion. Analogue 22 was prescreened in an ex vivo model
before its evaluation in an in vivo setting. Several
methods have been described in the literature to study
bone resorption in an ex vivo model.26 Since neonatal
rat calvaria are an excellent source of live bone tissue
and have been well documented as a bone organ culture
model system, analogue 22 was evaluated in the rat
calvaria assay. In Figure 2, bone resorption was stimu-
lated with the addition of parathyroid hormone (PTH)
followed by the addition of compound 22 at 1, 3, and 10
µM. A decrease in the efflux of calcium and deoxypyri-
dinoline was obtained in a dose-dependent manner. In
fact, the 10 µM dose of compound 22 completely reversed
the stimulation of resorption brought about by PTH,
bringing the efflux of calcium and deoxypyridinoline
back to around the level of nonstimulated calvaria.
Having proved the effective anti-resorptive properties
of compound 22 ex vivo, it was decided to test this
compound in our in vivo assay.
a
Simulated intestinal fluid solubility assay, pH ) 6.8 phosphate
buffer with isoosmotic KCl, lethicin, and sodium taurocholate.
b Madin-Darby canine kidney cell monolayer transport assay.c Rat
S9 liver slice metabolism assay, % metabolized after 1 h incubation
at 37 °C in duplicates.
A PTH-stimulated hypercalcemia model, the thyro-
parathyroidectomized (TPTX) rat assay, was used to
monitor inhibition of osteoclastic resorption. In this
model, removal of the parathyroid gland results in a
precipitous drop in serum calcium levels. Blood ionized
Encouraged by the acceptable pharmacokinetic pa-
rameters for orally dosing several of these ketoamide
inhibitors, it was decided to profile some in a suitable

594 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 3
Ta ble 4. Rat Pharmacokinetic Data for P′ Ketoamides
Tavares et al.
a
Compounds were dosed in solutol/citrate buffer, pH 3.5 (concn ) 2.5 mg/mL): Plasma drug levels were determined by LC-MS/MS.
F igu r e 2. Deoxypyridinoline (a) and ionized calcium (b) levels from calvaria of 1-2 day old Wistar rats incubated with h-PTH
(1-34), 10 ng/mL or vehicle only, and compound 22 added at 1, 3, and 10 µM concentrations. Error bars represent standard error
of the mean.
calcium levels are increased significantly by infusion of
30 µg/kg/h hPTH (1-34). Codosing cathepsin K inhibitor
22 (25 mg/kg po) with PTH (30 µg/kg/h infusion)
attenuates the PTH-stimulated increase in serum cal-
cium by blocking collagen matrix degradation and
subsequently inhibiting dissolution of hydroxyapatite
from bone (Figure 3).
cathepsins tested. Exploitation of this region resulted
in very selective (>100 fold) and potent cathepsin K
inhibitors such as analogues 30 and 32. A set of these
ketoamides exhibited good oral bioavailabilities. En-
couraged by the good oral pharmacokinetics properties
of these ketoamides, a representative example, com-
pound 22, was used in PTH-stimulated rat calvaria bone
resorption assay to monitor inhibition of osteoclastic
resorption. Pyrazole 22 was also found to be efficacious
in attenuating hypercalcemia in the TPTX rat model.
The excellent in vitro potency and selectivity profile of
several compounds described, combined with their
pharmacokinetic profile, suggests that these compounds
may represent a viable lead series in the discovery of
new therapies for the treatment of osteoporosis.
Con clu sion
Modifications of the P1 and P′ regions were explored
starting from a potent, ketoamide cathepsin K inhibitor.
In the process of exploring these heterocyclic P′ varia-
tions, it was discovered that N-substituted pyrazoles
such as analogue 23 had increased selectivity over other

Inhibitors of Cysteine Protease Cathepsin K
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 3 595
mixture was extracted with ether three times, and the
combined ether layer was washed with brine. After removal
of the solvent, the residue was redissolved in 30 mL of
dichloromethane, and 5.2 g (27 mmol) of EDC, 6.6 g (22 mmol)
of (triphenylphosphoranylidene)acetonitrile, and 256 mg (2.1
mmol) of DMAP were added. The reaction was stirred at rt
overnight. The reaction was diluted with 100 mL of dichloro-
methane, and this organic layer was washed three times with
brine and dried over magnesium sulfate. After removal of
solvent, column chromatography with hexane:ethyl acetate
1
(1:1) gave 8.5 g of product 5 in 73% yield. H NMR (300 MHz,
CDCl3) ppm: 7.52-7.70 (m, 15H), 5.40 (d, J ) 8.0 Hz, 1H),
4.90 (m, 1H), 4.42 (t, J ) 6.0 Hz, 1H), 1.20-2.08 (m, 8H), 0.86-
0.95 (m, 15H).
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
6-43. Ozone was bubbled through a solution of 1-isopropyl-
2-methylpropyl (1S)-1-[cyano(triphenylphosphoranylidene)-
acetyl]pentylcarbamate 5 (1 mmol) dissolved in 25 mL of
dichloromethane at -78 °C for 15 min until the solution turned
blue. The solution was purged with a stream of nitrogen for 5
min, and then the amine as a solution in 2 mL of dichloro-
methane was added. The solution was stirred at -78 °C for
15 min and then warmed to room temperature. It was
concentrated, and 5 mL of a 1 M solution of silver nitrate in
tetrahydrofuran:water (4:1) was added. The resulting mixture
was stirred for 16 h at room temperature and then extracted
with dichloromethane. The extract was washed with saturated
aqueous sodium chloride. It was then dried over anhydrous
magnesium sulfate, filtered, and concentrated. The residue
was purified by silica gel column chromatography.
1-Isopropyl-2-methylpropyl 2,3-dioxo-3-[(thien-2-ylmethyl)-
amino]propylcarbamate (6): 1H NMR (300 MHz, CDCl3)
ppm: 7.26 (d, J ) 6 Hz, 1H), 6.93-7.01 (m, 2H), 4.65 (m, 3H),
4.42 (m, 1H), 1.87 (m, 2H), 0.88(m, 12H). Anal. C, H, N.
1-Isop r op yl-2-m eth ylp r op yl (1S)-1-m eth yl-2,3-d ioxo-3-
[(th ien -2-ylm eth yl)a m in o]p r op ylca r ba m a te (7): 1H NMR
(300 MHz, CDCl3) ppm: 7.26 (d, J ) 4.1 Hz, 1H), 7.18 (br,
1H), 6.95-7.01 (m, 2H), 5.20 (br, 1H), 4.66 (br, 2H), 4.41 (m,
1H), 1.89 (m, 2H), 1.47 (d, J ) 7.0 Hz, 3H), 0.88 (m, 12H).
Anal. C, H, N.
1-Isop r op yl-2-m eth ylp r op yl (1S)-1-isop r op yl-2,3-d ioxo-
3-[(th ien -2-ylm eth yl)am in o]pr opylcar bam ate (8): 1H NMR
(400 MHz, CDCl3) ppm: 7.26 (d, J ) 5.9 Hz, 1H), 7.19 (br,
1H), 6.96-7.01(m, 2H), 5.27(d, J ) 9.0 Hz, 1H), 5.08 (dd, J )
9.2, 4.8 Hz, 1H), 4.65 (d, J ) 6.0 Hz, 2H), 4.40 (t, J ) 6.0 Hz,
1H), 2.42 (m, 1H), 1.87 (m, 2H), 1.05 (d, J ) 6.8 Hz, 3H), 0.87
(m, 15H). Anal. C, H, N.
1-Isopr opyl-2-m eth ylpr opyl (1S)-3-m eth yl-1-{oxo[(th ien -
2-ylm eth yl)a m in o]a cetyl}bu tylca r ba m a te (9): 1H NMR
(400 MHz, CDCl3) ppm: 7.25 (d, J ) 6.4 Hz, 1H), 7.17 (br,
1H), 6.95-7.01 (m, 2H), 5.15 (br, 1H), 4.64 (d, J ) 5.7 Hz, 2H),
4.38 (br, 1H), 1.76-1.88 (m, 4H), 1.42 (m, 1H), 1.04(d, J ) 6.0
Hz, 3H), 0.96 (d,J ) 6.4 Hz, 3H), 0.87 (m, 12H). Anal. C, H, N.
1-Isop r op yl-2-m eth ylp r op yl (1S,2S)-2-m eth yl-1-{oxo-
[(th ien -2-ylm eth yl)am in o]acetyl}bu tylcar bam ate (10): 1H
NMR (400 MHz, CDCl3) ppm: 7.26 (d, J ) 5.5 Hz, 1H), 7.19
(br, 1H), 6.95-7.00 (m, 2H), 5.28 (d, J ) 8.8 Hz, 1H), 5.04 (dd,
J ) 9.3, 5.5 Hz, 1H), 4.66 (m, 2H), 4.39 (br, 1H), 2.17 (br, 1H),
1.87 (m, 2H), 1.33 (br, 1H), 1.13 (m, 1H), 1.00 (d, J ) 6.8 Hz,
3H), 0.87 (m, 15H). Anal. C, H, N.
F igu r e 3. Ionized calcium levels of male CD TPTX rats
receiving h-PTH (1-34), 30 µg/kg/h, sc via minipump or h-PTH
and compound 22 administered orally 1 h prior to PTH.
Compound 22 was dosed orally at 25 mg/kg in 0.5% methyl
cellulose suspension at a concentration of 5 mg/mL. Error bars
represent standard error of the mean.
Exp er im en ta l Section
Ch em istr y. Gen er a l Meth od s. Melting points were de-
termined using a Thomas-Hoover melting point apparatus and
are uncorrected. Unless stated otherwise, reagents were
obtained from commercial sources and were used directly.
Reactions involving air- or moisture-sensitive reagents were
carried out under a nitrogen atmosphere. If not specified,
reactions were carried out at ambient temperature. Silica gel
(EM Science, 230-400 mesh) was used for chromatographic
purification unless otherwise indicated. Anhydrous solvents
were obtained from Aldrich (Sure Seal). 1H NMR spectra were
recorded on a Varian spectrometer; chemical shifts are re-
ported in parts per million (ppm) relative to TMS. The
following abbreviations are used to describe peak patterns
when appropriate: b ) broad, s ) singlet, d ) doublet, t )
triplet, q ) quartet, m ) multiplet. High performance liquid
chromatography (HPLC) was performed on a Beckman 126
with a Beckman 166 UV Detector (monitoring at 215 nm) with
a Rainin Dynamax-60A column using a gradient consisting of
20/80 A:B to 10/90 A/B over 20 min, where A ) 1% aqueous
trifluoroacetic acid (TFA), B ) 1% TFA in CH3CN. Elemental
analyses, performed by Atlantic Microlab, Inc. Norcross, GA,
were within 0.4% of the theoretical values calculated for C,
H, and N.
Meth yl N-[(1-Isop r op yl-2-m eth ylp r op oxy)ca r bon yl]-L-
n or leu cin a te (4). To a solution of 18 mL (129 mmol) of 2,4-
dimethyl-3-pentanol 3 in 150 mL of THF was added 81.5 mL
(155 mmol) of 20% phosgene in toluene at 0 °C. The reaction
was stirred at rt overnight. THF was removed and the
chloroformate intermediate in toluene was directly used in the
next step.
To the intermediate chloroformate was added 50 mL of THF
followed by 23.4 g (129 mmol) of norleucine methyl ester
hydrochloride and 67.5 mL (387 mmol) of Hunig’s base at 0
°C. The reaction was stirred at rt overnight. Solvent was
removed and water was added. The aqueous layer was
extracted with ether three times, and the combined ether layer
was washed with brine and dried over magnesium sulfate.
After removal of the solvent, column chromatography with
hexane:ethyl acetate (10:1) as eluent provided 32 g of product
4 in 86% yield. 1H NMR (300 MHz, CDCl3) ppm: 5.12 (br, 1H),
4.36 (m, 2H), 3.71 (s, 3H), 1.23-1.89 (m, 8H), 0.84-0.92 (m,
15H).
1-Isop r op yl-2-m eth ylp r op yl (1S)-1-cycloh exylm eth yl-
2,3-d ioxo-3-[(th ien -2-ylm eth yl)a m in o]p r op ylca r ba m a te
(11): 1H NMR (300 MHz, CDCl3) ppm: 7.25 (d, J ) 6.0 Hz,
1H), 7.19 (br, 1H), 6.95-7.00 (m, 2H), 5.18 (m, 2H), 4.64 (d, J
) 5.5 Hz, 2H), 4.39 (m, 1H), 1.03-1.98 (m, 15H), 0.88 (m, 12H).
Anal. C, H, N.
1-Isop r op yl-2-m eth ylp r op yl (1S)-2,3-d ioxo-1-p h en yl-3-
[(th ien -2-ylm eth yl)am in o]pr opylcar bam ate (12): 1H NMR
(300 MHz, CDCl3) ppm: 6.97-7.29 (m, 8H), 5.44 (m, 1H), 5.22
(br, 1H), 4.70 (m, 2H), 4.37 (t, J ) 6.5 Hz, 1H), 1.84 (m, 2H),
0.85 (d, J ) 7.0 Hz, 6H), 3.14-3.40 (m, 2H), 0.83(d, J ) 6.9
Hz, 6H). Anal. C, H, N.
1-Isop r op yl-2-m et h ylp r op yl (1S)-1-[cya n o(t r ip h en yl-
p h osp h or a n ylid en e) a cetyl]p en tylca r ba m a te (5). To a
solution of 6 g (21 mmol) of methyl N-[(1-isopropyl-2-methyl-
propoxy)carbonyl]-L-norleucinate 4 in 30 mL of THF was added
28 mL (28 mmol) of 1 M lithium hydroxide. The reaction was
stirred at rt overnight. Water was added, and the solution was
acidified with 1 N hydrochloric acid to around pH ) 3. The
1-Isop r op yl-2-m et h ylp r op yl (1S)-1-{oxo[(t h ien -2-yl-
m eth yl)a m in o]a cetyl}p en tylca r ba m a te (13): 1H NMR

596 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 3
Tavares et al.
(400 MHz, CDCl3) ppm: 6.92-7.30 (m, 4H), 5.21 (br, 1H), 5.13
(br, 1H), 4.65 (s, 2H), 4.39 (br, 1H), 1.80-2.05 (m, 3H), 1.30-
1.66 (m, 5H), 0.88 (m, 15H). Anal. C, H, N.
1-Isop r op yl-2-m et h ylp r op yl (1S)-1-[oxo(p yr id in -2-yl-
a m in o)a cetyl]p en tylca r ba m a te (14): 1H NMR (300 MHz,
CDCl3) ppm: 9.30 (s, 1H), 8.39 (d, J ) 4.4 Hz, 1H), 8.27 (d J
) 8.2 Hz, 1H), 7.79 (m, 1H), 7.16 (m, 1H), 5.30 (br, 2H), 4.43
(t, J ) 5.9 Hz, 1H), 1.38-2.08 (m, 8H), 0.91 (m, 15H). Anal.
C, H, N.
1-Isop r op yl-2-m et h ylp r op yl (1S)-1-[oxo(p yr id in -3-yl-
a m in o)a cetyl]p en tylca r ba m a te (15): 1H NMR (300 MHz,
CDCl3) ppm: 8.91 (s, 1H), 8.77 (s, 1H), 8.45 (br, 1H), 8.26 (d,
J ) 7.5 Hz, 1H), 7.35 (br, 1H), 5.21-5.29 (m, 2H), 4.42 (br,
1H), 1.43-2.07 (m, 8H), 0.90 (br, 15H). Anal. C, H, N.
1-Isop r op yl-2-m et h ylp r op yl (1S)-1-[oxo(p yr a zin -2-yl-
a m in o)a cetyl]p en tylca r ba m a te (16): 1H NMR (400 MHz,
CDCl3) ppm: 9.53 (s, 1H), 9.22 (br, 1H), 8.39 (s, 1H), 8.30 (s,
1H), 5.18 (m, 2H), 4.35 (br, 1H), 1.32-2.00 (m, 8H), 0.86 (m,
15H). Anal. C, H, N.
1-Isop r op yl-2-m et h ylp r op yl (1S)-1-[oxo(q u in olin -2-
yla m in o)a cetyl]p en tylca r ba m a te (17): 1H NMR (400 MHz,
CDCl3) ppm: 9.37 (s, 1H), 8.39 (d, J ) 9.0 Hz, 1H), 8.19 (d,J
) 9.0 Hz, 1H), 7.85 (d, J ) 8.6 Hz, 1H), 7.78 (d, J ) 8.2 Hz,
1H), 7.67 (m, 1H), 7.58 (m, 1H), 5.26 (m, 1H), 5.21 (br, 1H),
4.37 (t, J ) 5.9 Hz, 1H), 1.20-2.10 (m, 8H), 0.86 (m, 15H).
Anal. C, H, N.
1-Isop r op yl-2-m eth ylp r op yl (1S)-1-[(isoqu in olin -3-yl-
a m in o)(oxo)a cetyl]p en tylca r ba m a te (18): 1H NMR(300
MHz, CDCl3) ppm: 9.40 (s, 1H), 9.08 (s, 1H), 8.66 (s, 1H), 7.98
(d, J ) 8.0 Hz, 1H), 7.90 (d, J ) 8.1 Hz, 1H), 7.73 (br, 1H),
7.58 (br, 1H), 5.33 (br, 2H), 4.46 (br, 1H), 1.20-2.20 (m, 8H),
0.93 (m, 15H). Anal. C, H, N.
1-Isop r op yl-2-m eth ylp r op yl (1S)-1-[oxo(1,3-th ia zol-2-
yla m in o)a cetyl]p en tylca r ba m a te (19): 1H NMR (300 MHz,
CDCl3) ppm: 12.48 (br, 1H), 7.80 (d, J ) 2.2 Hz, 1H), 7.14(d,
J ) 2.2 Hz, 1H), 5.29 (m, 2H), 4.43 (m, 1H), 1.29-2.08 (m,
8H), 0.91 (m, 15H). Anal. C, H, N.
1-Isop r op yl-2-m et h ylp r op yl (1S)-1-[oxo(1,3,4-t h ia d i-
a zol-2-yla m in o)a cetyl]p en tylca r ba m a te (20): 1H NMR
(300 MHz, CDCl3) ppm: 8.99 (s, 1H), 7.30 (s, 1H), 5.23 (br,
2H), 4.41 (br, 1H), 1.40-2.05 (m, 8H), 0.90 (m, 15H). Anal. C,
H, N.
1-Isop r op yl-2-m eth ylp r op yl (1S)-1-[(isoxa zol-3-yla m i-
n o)(oxo)a cetyl]p en tylca r ba m a te (21): 1H NMR (300 NMR,
CDCl3) ppm: 9.72 (br, 1H), 8.40 (s, 1H), 7.13 (s, 1H), 5.24 (br,
2H), 4.42 (br, 1H), 1.42-2.01 (m, 8H), 0.90 (m, 15H). Anal. C,
H, N.
1-Isop r op yl-2-m eth ylp r op yl (1S)-1-[oxo(1H-p yr a zol-5-
yla m in o)a cetyl]p en tylca r ba m a te (22): 1H NMR (300 MHz,
DMSO-d6) ppm: 10.86 (s, 1H), 7.68 (s, 1H), 7.50 (d, J ) 8.0
Hz, 1H), 6.53 (s, 1H), 4.85 (m, 1H), 4.27 (t, J ) 6.1 Hz, 1H),
1.34-1.82 (m, 8H), 0.80 (m, 15H). Anal. C, H, N.
1-Isop r op yl-2-m eth ylp r op yl (1S)-1-[[(1-m eth yl-1H-p yr -
a zol-5-yl)a m in o](oxo)a cetyl]p en tylca r ba m a te (23): 1H
NMR (300 MHz, CDCl3) ppm: 7.97 (s, 1H), 5.98 (br, 2H), 5.27
(br, 1H), 5.18 (br, 1H), 4.40 (m, 1H), 3.63 (s, 3H), 1.21-2.00
(m, 8H), 0.90 (m, 15H). Anal. C, H, N.
1-Isop r op yl-2-m eth ylp r op yl (1S)-1-[[(1-m eth yl-1H-p yr -
a zol-3-yl)a m in o](oxo)a cetyl]p en tylca r ba m a te (24): 1H
NMR (300 MHz, CDCl3) ppm: 9.24 (s, 1H), 7.32 (d, J ) 2.0
Hz, 1H), 6.75 (d, J ) 2.0 Hz, 1H), 5.27 (m, 2H), 4.43 (m, 1H),
1.29-2.08 (m, 8H), 0.91 (m, 15H). Anal. C, H, N.
1-Isop r op yl-2-m et h ylp r op yl (1S)-1-[[(1-et h yl-1H -p yr -
a zol-5-yl)a m in o](oxo)a cetyl]p en tylca r ba m a te (25): 1H
NMR (400 MHz, CDCl3) ppm: 7.90 (s, 1H), 5.86 (br, 2H), 5.21
(d, J ) 8.1 Hz, 1H), 5.1(m, 1H), 4.33 (t, J ) 6.0 Hz, 1H), 3.87
(q, J ) 7.3 Hz, 2H), 1.36 (t, J ) 7.3 Hz, 3H), 1.20-2.00 (m,
8H), 0.83 (m, 15H). Anal. C, H, N.
1-Isop r op yl-2-m et h ylp r op yl (1S)-1-[[(1-Isop r op yl-1H-
pyr azol-5-yl)am in o](oxo)acetyl]pen tylcar bam ate (26): 1H
NMR (300 MHz, CDCl3) ppm: 8.00 (s, 1H), 5.92 (br, 2H), 5.28
(br, 1H), 5.17 (br, 1H), 4.42 (t, J ) 6.0 Hz, 1H), 4.21 (m, 1H),
1.51 (d, J ) 7.4 Hz, 6H), 0.90 (m, 15H). Anal. C, H, N.
1-Isop r op yl-2-m et h ylp r op yl (1S)-1-[[(1-isob u t yl-1H -
pyr azol-5-yl)am in o](oxo)acetyl]pen tylcar bam ate (27): 1H
NMR (400 MHz, CDCl3) ppm: 7.92 (s, 1H), 5.82 (br, 2H), 5.21
(d, J ) 8.0 Hz, 1H), 5.10 (m, 1H), 4.34 (t, J ) 6.0 Hz, 1H),
3.61 (d, J ) 7.3 Hz, 2H), 2.19 (m, 1H), 1.20-2.00 (m, 8H), 0.90
(m, 18H). Anal. C, H, N.
1-Isop r op yl-2-m eth ylp r op yl (1S)-1-[{[1-(cyclop r op yl-
m et h yl)-1H -p yr a zol-5-yl]a m in o}(oxo)a cet yl]p en t ylca r -
ba m a te (28): 1H NMR (300 MHz, CDCl3) ppm: 7.98 (s, 1H),
6.13 (s, 1H), 5.29 (m, 1H), 5.20 (m, 1H), 4.40 (t, J ) 5.8 Hz,
1H), 3.82 (d, J ) 6.6 Hz, 2H), 1.20-2.00 (m, 9H), 0.89 (m, 15H),
0.66 (m, 2H), 0.42 (m, 2H). Anal. C, H, N.
1-Isop r op yl-2-m eth ylp r op yl (1S)-1-[{[1-(3,3-d im eth yl-
bu tyl)-1H-p yr a zol-5-yl]a m in o}(oxo)a cetyl]p en tylca r ba m -
a te (29): 1H NMR (300 MHz, CDCl3) ppm: 7.97 (s, 1H), 5.92
(br, 2H), 5.31 (d, J ) 8.2 Hz, 1H), 5.17 (br, 1H), 4.41 (m, 1H),
3.90 (t, J ) 8.4 Hz, 2H), 1.2-2.0 (m, 10H), 1.01 (s, 9H), 0.89
(m, 15H). Anal. C, H, N.
1-Isop r op yl-2-m eth ylp r op yl (1S)-1-[[(1-cyclobu tyl-1H-
pyr azol-5-yl)am in o](oxo)acetyl]pen tylcar bam ate (30): 1H
NMR (400 MHz, CDCl3) ppm: 7.93 (s, 1H), 5.79 (br, 2H), 5.21
(d, J ) 8.0 Hz, 1H), 5.09 (br, 1H), 4.37 (m, 1H), 2.64 (m, 2H),
2.37 (m, 2H), 1.2-2.0 (m, 10H), 0.83 (m, 15H). Anal. C, H, N.
1-Isop r op yl-2-m eth ylp r op yl (1S)-1-[[(1-cyclop en tyl-1H-
pyr azol-5-yl)am in o](oxo)acetyl]pen tylcar bam ate (31): 1H
NMR (300 MHz, CDCl3) ppm: 7.98 (s, 1H), 5.86 (s, 2H), 5.30
(d, J ) 8.4 Hz, 1H), 5.17 (m, 1H), 4.42 (t, J ) 6.0 Hz, 1H),
4.32 (m, 1H), 1.23-2.20 (m, 16H), 0.90 (m, 15H). Anal. C, H,
N.
1-Isop r op yl-2-m eth ylp r op yl (1S)-1-[[(1-cycloh exyl-1H-
pyr azol-5-yl)am in o](oxo)acetyl]pen tylcar bam ate (32): 1H
NMR (400 MHz, CDCl3) ppm: 7.91 (s, 1H), 5.76 (s, 2H), 5.21
(d, J ) 8.2 Hz, 1H), 5.08 (m, 1H), 4.34 (t, J ) 6.1 Hz, 1H),
3.66 (m, 1H), 1.20-1.99 (m, 18H), 0.82 (m, 15H). Anal. C, H,
N.
1-Isop r op yl-2-m eth ylp r op yl (1S)-1-{oxo[(1-p h en yl-1H-
pyr azol-5-yl)am in o]acetyl}pen tylcar bam ate (33): 1H NMR
(300 MHz, CDCl3) ppm: 8.16 (s, 1H), 7.30-7.60 (m, 5H), 6.17
(s, 2H), 5.33 (d, J ) 8.0 Hz, 1H), 5.17 (m, 1H), 4.42 (m, 1H),
1.28-2.07 (m, 8H), 0.90 (m, 15H). Anal. C, H, N.
1-Isop r op yl-2-m et h ylp r op yl (1S)-1-{oxo[(1-p yr id in -4-
yl-1H-p yr a zol-5-yl)a m in o]a cetyl}p en tylca r ba m a te (34):
1H NMR (400 MHz, CDCl3) ppm: 8.73 (d, J ) 5.3 Hz, 2H),
8.16 (s, 1H), 7.51 (d, J ) 5.3 Hz, 2H), 6.38 (s, 2H), 5.20 (d, J
) 7.7 Hz, 1H), 5.06 (br, 1H), 4.33 (br, 1H), 1.20-2.00 (m, 8H),
0.83 (m, 15H). Anal. C, H, N.
1-Isop r op yl-2-m et h ylp r op yl (1S)-1-{oxo[(1-p yr id in -2-
yl-1H-p yr a zol-5-yl)a m in o]a cetyl}p en tylca r ba m a te (35):
1H NMR (400 MHz, CDCl3) ppm: 8.33 (d, J ) 4.4 Hz, 1H),
8.09 (s, 1H), 7.90 (d, J ) 8.2 Hz, 1H), 7.79 (m, 1H), 7.14 (m,
1H), 5.27 (d, J ) 7.9 Hz, 1H), 5.10 (m, 1H), 4.34 (t, J ) 6.9
Hz, 1H), 1.20-2.00 (m, 8H), 0.83 (m, 15H). Anal. C, H, N.
1-Isop r op yl-2-m eth ylp r op yl (1S)-1-[[(4-m eth yl-1H-p yr -
a zol-5-yl)a m in o](oxo)a cetyl]p en tylca r ba m a te (36): 1H
NMR (400 MHz, CDCl3) ppm: 9.21 (s, 1H), 7.30 (s, 1H), 5.26
(br, 1H), 5.16 (br, 1H), 4.36 (br, 1H), 2.01 (s, 3H), 1.33-1.83
(m, 8H), 0.84 (m, 15H). Anal. C, H, N.
1-Isop r op yl-2-m eth ylp r op yl (1S)-1-[[(3-m eth yl-1H-p yr -
a zol-5-yl)a m in o](oxo)a cetyl]p en tylca r ba m a te (37): 1H
NMR (400 MHz, CDCl3) ppm: 11.51 (br, 1H), 6.61 (s, 1H), 5.23
(br, 1H), 5.14 (br, 1H), 4.36 (br, 1H), 2.32 (s, 3H), 1.34-2.01
(m, 8H), 0.85 (m, 15H). Anal. C, H, N.
1-Isop r op yl-2-m eth ylp r op yl (1S)-1-{oxo[(4-p h en yl-1H-
pyr azol-5-yl)am in o]acetyl}pen tylcar bam ate (38): 1H NMR
(400 MHz, CDCl3) ppm: 9.14 (s, 1H), 7.66 (s, 1H), 7.28-7.43
(m, 5H), 5.31 (s, 1H), 5.13 (br, 1H), 4.34 (br, 1H), 1.20-2.02
(m, 8H), 0.82 (m, 15H). Anal. C, H, N.
1-Isop r op yl-2-m eth ylp r op yl (1S)-1-{oxo[(3-p h en yl-1H-
pyr azol-5-yl)am in o]acetyl}pen tylcar bam ate (39): 1H NMR
(300 MHz, CDCl3) ppm: 7.70 (d, J ) 7.4 Hz, 2H), 7.46 (m,
3H), 7.19 (s, 1H), 5.33 (d, J ) 7.5 Hz, 1H), 5.19 (br, 1H), 4.41
(m, 1H), 1.38-2.15 (m, 8H), 0.87 (m, 15H). Anal. C, H, N.
1-Isop r op yl-2-m eth ylp r op yl (1S)-1-[[(4-flu or o-1H-in d -
a zol-3-yl)a m in o](oxo)a cetyl]p en tylca r ba m a te (40): 1H

Inhibitors of Cysteine Protease Cathepsin K
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 3 597
NMR (300 MHz, CDCl3) ppm: 9.43 (s, 1H), 7.36 (br, 2H), 6.81
(m, 1H), 5.36 (br, 2H), 4.47 (br, 1H), 1.20-2.20 (m, 8H), 0.94
(m, 15H). Anal. C, H, N.
1-Isop r op yl-2-m eth ylp r op yl (1S)-1-[[(4-cya n o-1H-p yr -
a zol-5-yl)a m in o](oxo)a cetyl]p en tylca r ba m a te (41): 1H
NMR (300 MHz, CDCl3) ppm: 9.80 (s, 1H), 7.92 (s, 1H), 5.31
(d, J ) 7.7 Hz, 1H), 5.12 (m, 1H), 4.40 (br, 1H), 1.30-2.10 (m,
8H), 0.93 (m, 15H). Anal. C, H, N.
Eth yl 5-[((3S)-3-{[(1-Isop r op yl-2-m eth ylp r op oxy)ca r -
bon yl]am in o}-2-oxoh eptan oyl)am in o]-1H-pyr azole-4-car -
boxyla te (42): 1H NMR (400 MHz, CDCl3) ppm: 10.65 (s,
1H), 7.85 (s, 1H), 5.26 (br, 1H), 5.14 (m, 1H), 4.32 (m, 3H),
1.36 (t, J ) 7.0 Hz, 3H), 1.20-2.00 (m, 8H), 0.83 (m, 15H).
Anal. C, H, N.
1-Isop r op yl-2-m eth ylp r op yl (1S)-1-[[(4-br om o-1H-p yr -
a zol-5-yl)a m in o](oxo)a cetyl]p en tylca r ba m a te (43): 1H
NMR (400 MHz, CDCl3) ppm: 8.93 (s, 1H), 7.5 (s, 1H), 5.15
(m, 2H), 4.35 (br, 1H), 1.23-2.05 (m, 8H), 0.84 (m, 15H). Anal.
C, H, N.
Ra t Ca lva r ia Assa y. Calvaria was surgically removed from
1 to 2 day old Wistar rats and placed in a sterile centrifuge
tube containing BGJ b media (Fitton-J ackson Modification),
GIBCO BRL with penicillin (10 U/mL)/streptomycin (10 µg/
mL). In a tissue culture hood, calvaria were washed 2× with
fresh media, trimmed of any excess tissue attached to the
calvaria, and placed in fresh media. The calvaria were cut
using a 8 mm keyhole punch to give circular calvaria pieces
of uniform size and were then placed into wells of a 48-well
tissue culture plate containing 0.4 mL of BGJ b media contain-
ing 1 mg/mL BSA and penicillin (10 U/mL)/streptomycin (10
µg/mL). Calvaria were then incubated for 24 h at 37 °C, 5%
CO2 on a Nutator rotator prior to treatment. Media was
aspirated and replaced with 0.4 mL of fresh media containing
designated concentration of PTH (10 ng/mL media) or vehicle
only. Vehicle is 4mM acetic acid/1 mg/mL BSA. Cultures were
incubated for 24 h, retreated daily as described, for a total
treatment time of 48 h. Upon completion of the assay, the 24-
48 h media is collected in tubes and stored at -20 °C until
analysis is performed. Calvaria were air-dried for several days,
and dry weight was quantitated on a Mettler balance if needed.
PTH, IL1, and PGE2 have been shown to stimulate bone
resorption in in vitro rat neonatal calvaria assays and are
utilized as controls in this assay. Resorption is measured in
three ways: (1) quantitation of calcium in the supernatant by
atomic emission spectroscopy (ICPE); (2) quantitation of
calcium in the supernatant by a colorimetric assay; (3)
quantitation of deoxypyridinoline in the supernatant by ELISA
(Metra DPD EIA Kit, Catalog #8007, Quidel Corp.). Deoxy-
pyridinoline is released as a byproduct of collagen degradation
by the osteoclast.
method at room temperature. Protein was mixed with an equal
volume of reservoir buffer containing 0.2 M (NH4) SO4 and
2
30% PEG8000. Prior to data collection, glycerol was added to
25%, and the crystals were flash frozen in a stream of liquid
nitrogen.
Diffraction data was collected on an RAXIS4 area detector
operating on a Rigaku rotating anode generator. The crystals
diffracted to 2.2 Å resolution and belonged to the space group
P1 with cell dimensions a ) 31.63 Å, b ) 72.86 Å, c ) 78.47
Å, R ) 90.42°, â ) 90.28°, γ ) 90.16°. The data were processed
with DENZO and SCALEPACK. The data were 83% complete
with a Rmerge of 6.5%. The structure was solved by molecular
replacement using coordinates 1EF7 from the PDB databank
and CNX. The structure was refined to a Rfactor of 20.9%. In
the final model, all residues lie within the allowed regions of
the Ramachandran plot with rms deviations in the bonds and
angle of 0.008 Å and 1.6°, respectively.
Biologica l Da ta . All assays for cathepsin K were carried
out with human and rat recombinant enzyme. Assays for
cathepsins S and V were also carried out with human
recombinant enzyme. Assays for human cathepsins B, H, and
L were carried out with enzyme, purchased from Athens
Research and Technology, Inc., prepared from human liver
tissue. Standard assay conditions for the determination of
kinetic constants used a fluorogenic peptide substrate, typi-
cally (5S,8S)-13-amino-5-benzyl-13-imino-3-methylene-N-(4-
methyl-2-oxo-2H-chromen-7-yl)-6-oxo-1-phenyl-2-oxa-4,7,12-
triazatridecane-8-carboxamide (Cbz-Phe-Arg-AMC) and were
determined in 100 mM sodium acetate at pH 5.5 containing
10 mM dithiothreitol and 120 mM sodium chloride. A stock
substrate solution of Cbz-Phe-Arg-AMC was prepared at a
concentration of 50 mM in dimethyl sulfoxide. This substrate
was diluted into the assay for a final substrate concentration
of 10 µM in the rat cathepsin K, human cathepsin K, and
human cathepsin B assays; a final substrate concentration of
5 µM in the human cathepsin L assay; and a final substrate
concentration of 2 µM in the human cathepsin V assay. The
Km value for Cbz-Phe-Arg-AMC on rat cathepsin K is 27 µM,
on human cathepsin K is 70 µM, on human cathepsin B is 400
µM, on human cathepsin L is 2.7 µM and on human cathepsin
V is 32 µM.
A stock substrate solution of benzyl (1S)-1-{[((1S)-1-{[((1S)-
4-{[a m in o(im in o)m et h yl]a m in o}-1-{[(4-m et h yl-2-oxo-2H -
chromen-7-yl)amino]carbonyl}butyl)amino]carbonyl}-2-meth-
ylpropyl)amino]carbonyl}-2-methylpropylcarbamate (Cbz-Val-
Val-Arg-AMC) was prepared at a concentration of 10 mM in
dimethyl sulfoxide. This substrate was diluted into the assay
for a final substrate concentration of 10 µM in the human
cathepsin S assay. The Km value for Cbz-Val-Val-Arg-AMC on
human cathepsin S is >100 µM.
A stock substrate solution of (2S)-2-amino-5-{[amino(imino)-
methyl]amino}-N-(2-naphthyl)pentanamide hydrochloride (L-
Arg-â-naphthalamide‚HCl) was prepared at a concentration
of 10 mM in dimethyl sulfoxide. This substrate was diluted
into the assay for a final substrate concentration of 50 µM in
the cathepsin H assay. The Km value for L-Arg-b-naphthalamide‚
HCl on cathepsin H is 195 µM.
All assays contained 10% dimethyl sulfoxide. Independent
experiments found that this level of dimethyl sulfoxide had
no effect on kinetic enzymatic constants. All assays were
conducted at 30 °C. Product fluorescence (excitation at 360 nm;
emission at 440 nm (except cathepsin H which used excitation
at 340 nm; emission at 420 nm)) was monitored with a
PerSeptive Biosystems Cytofluor II fluorescence plate reader.
Product progress curves were generated over 2.3 h monitoring
the formation of 7-amino-4-methylcoumarin product (or â-naph-
thalamide for cathepsin H).
Ca th ep sin K Activa tion . The proform of cathepsin K was
converted to mature cathepsin K by brief exposure to pH 4 in
the presence of 5 mM L-cysteine. Typically, 5 mM L-cysteine
was added to 10 mL of approximately 1 mg/mL procathepsin
K. One milliliter of this solution was diluted 10-fold into 450
mM sodium acetate at pH 4.0 containing 5 mM L-cysteine. This
solution was reacted at 23°C for 2 min before neutralization
TP TX P r otocol. Male CD rats weighing 275-300 g,
Charles River Research Laboratory (Raleigh, NC), were re-
ceived and allowed one week to acclimate to the facility.
Animals had free access to water and were maintained on a
normal diet and light cycle. Following the acclimation period,
animals were anesthetized and a thyroparathyroidectomy
performed. Forty-eight hours post TPTX, animals were anes-
thetized and a blood sample collected via the tail vein to
measure serum calcium concentration to verify successful
TPTX and randomize the animals into treatment groups. For
administration of PTH, 30 µg/kg/h, animals were anesthetized
and an osmotic pump implanted sc to initiated PTH infusion.
To evaluate the anti-resorptive effect of test compounds, the
compounds were given at different times depending upon route
of administration. Compounds administered orally were given
an hour prior to implantation. Compounds administered sc,
iv, or ip were either coadministered or administered 2 h
postimplant. To measure the compound’s ability to block the
increase in serum calcium, rats were anesthetized 2, 4, and 6
h postinfusion and a blood sample collected via tail vein. After
the 6 h bleed, animals were euthanized.
Cr ysta lliza tion . Activated cathepsin K was complexed with
a 10-fold molar excess of inhibitor and concentrated to ∼2 mg/
mL. Crystals were grown by the hanging drop vapor diffusion

598 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 3
Tavares et al.
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with 2 mL of 1.8 M sodium acetate at pH 6.0. The neutralized
sample was added to the remaining 9 mL of procathepsin K.
The mixture was incubated at 4 °C for 2-3 days. The activated
cathepsin K was chromatographed on a Poros HS II column
as described above.
In h ibition Stu d ies. Potential inhibitors were evaluated
using the progress curve method. Assays were carried out in
the presence of variable concentrations of test compound.
Reactions were initiated by addition of buffered solutions of
inhibitor and substrate to enzyme. Data analysis was con-
ducted according to one of two procedures depending on the
appearance of the progress curves in the presence of inhibitors.
For those compounds whose progress curves were linear, the
enzymatic activity (RATE) was plotted against the concentra-
tion of test compound, including inhibitor concentration of zero
([I] ) 0), and the IC50 determined from a fit of eq 1 to the data,
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rate Vmax/(1 + ([I]/ IC50))
(1)
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Rommerskirch, W.; Detlev, J . D.; Schu, P.; von Figura, K.
Impaired osteoclastic bone resorption leads to osteopetrosis in
cathepsin K deficient mice. Proc. Natl. Acad. Sci. U.S.A. 1998,
95, 13453-13458. (b) Gowen, M.; Lazner, F.; Dodds, R.; Kapadia,
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where Vmax is the best fit estimate of the maximal enzymatic
activity. Ki values were calculated from IC50 values using eq
2 assuming a competitive model.
S
Ki ) IC50 × 1 -
(2)
[
]
(S + Km)
For those compounds whose progress curves showed down-
ward curvature characteristic of time-dependent inhibition, the
data from individual sets was analyzed using the computer
program DynaFit (Kuzmic, P. Anal. Biochem. 1996, 237, 260-
273) to give Ki values according to the following kinetic
mechanism:
E + S S ES
ES f E + P
E f EX
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Gress, C. J .; Ru, Y.; Zembryki, D.; Yamashita, D. S.; McQueney,
M. S.; Tomaszek, T. A.; Oh, H. J .; Gowen, M.; Veber, D. F.; Lark,
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S. J .; J ames, I. E.; Marquis, R. W.; Ru, Y.; Vasko-Moser, J . A.;
Smith, B. R.; Tomaszek, T.; Gowen, M. Potent and selective
inhibition of human cathepsin K leads to inhibition of bone
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E + I S EI
P h a r m a cok in etic Stu d ies in Ra ts. Male Han Wistar rats
(weight range: 225-300 g) were anesthetized with isofluorane
and surgically prepared with jugular vein and femoral vein
cannulas (oral rats had only jugular cannulas) 2 days before
dosing. On the prior day of dosing, each compound was
dissolved in 15% solutol/citrate buffer/0.1% ascorbic acid, pH
3.5. Rats were dosed intravenously into the femoral vein at a
level of 5 mg/kg or orally at 10 mg/kg. Blood samples were
collected prior to dosing and at 0.03, 0.25, 0.5, 0.75, 1, 1.5,
2.5, 4, 6, 8, and 24 h postdosing from the jugular vein cannula
into unheparinized syringes. The blood was clotted on ice and
centrifuged to obtain serum and frozen until analysis. Serum
samples were prepared by protein precipitation with aceto-
nitrile containing an internal standard. Compound analysis
was by turboionspray on a Sciex API III+ using multiple
reaction monitoring. Samples were chromatographed using a
Phenomenex Luna C18 column (30 × 2 mm, 3m) at ambient
temperature using a gradient (20-95%) with 10 mM am-
monium acetate (pH 4.0) and methanol mobile phases at a flow
rate of 0.20 mL/min. Data reduction was performed using Sciex
MacQuan software. Noncompartmental methods were used to
calculate pharmacokinetic parameters from the resulting
serum concentration vs time profiles using WinNonLin.
(7) Hashimoto, Y.; Kakegawa, H.; Narita, Y.; Hachiya, Y.; Haya-
kawa, T.; Kos, J .; Turk, V.; Katunuma, N. Significance of
cathepsin
B accumulation in synovial fluid of rheumatoid
arthritis. Biochem. Biophys. Res. Commun. 2001, 283 (2), 334-
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of Cathepsin B contributes to invasiveness in cancer. Cancer Res.
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Tommasi, R. A.; Cowen, S. D.; McQuire, L. W.; Farley, D. L.;
Van Duzer, J . H.; Goldberg, R. L.; Zhou, H.; Du, Z.; Fitt, J . J .;
Coppa, D. E.; Fang, Z.; Macchia, W.; Zhu, L.; Michael, P.
Identification of dipeptidyl nitriles as potent and selective
inhibitors of cathepsin B through structure-based drug design.
J . Med. Chem. 2001, 44, 4524-4534.
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proteases: more than scavengers. Biochim. Biophys. Acta 2000,
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Ack n ow led gm en t . The authors wish to thank
Randy Rutkowski, Robert J ohnson, and Peter Kitrinos
for analytical support.
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