Identification of a nonbasic, nitrile-containing cathepsin K inhibitor (MK-1256) that is efficacious in a monkey model of osteoporosis

Article abstract of DOI:10.1021/jm800610j

Herein, we report on the identification of nonbasic, potent, and highly selective, nitrile-containing cathepsin K (Cat K) inhibitors that are built on our previously identified cyclohexanecarboxamide core structure. Subsequent to our initial investigation

Full text of DOI:10.1021/jm800610j

6410
J. Med. Chem. 2008, 51, 6410–6420
Identification of a Nonbasic, Nitrile-Containing Cathepsin K Inhibitor (MK-1256) that is
Efficacious in a Monkey Model of Osteoporosis
Jo¨el Robichaud,† W. Cameron Black,† Michel The´rien,† Julie Paquet,† Renata M. Oballa,† Christopher I. Bayly,†
Daniel J. McKay,† Qingping Wang,† Elise Isabel,† Serge Le´ger,† Christophe Mellon,† Donald B. Kimmel,‡ Gregg Wesolowski,‡
M. David Percival,† Fre´deric Masse´,† Sylvie Desmarais,† Jean-Pierre Falgueyret,† and Sheldon N. Crane*,†
Merck Frosst Centre for Therapeutic Research, 16711 Trans Canada Highway, Kirkland, Quebec, Canada, H9H 3L1, and Merck Research
Laboratories, West Point, PennsylVania 19486
ReceiVed May 22, 2008
Herein, we report on the identification of nonbasic, potent, and highly selective, nitrile-containing cathepsin
K (Cat K) inhibitors that are built on our previously identified cyclohexanecarboxamide core structure.
Subsequent to our initial investigations, we have found that incorporation of five-membered heterocycles as
P2-P3 linkers allowed for the introduction of a methyl sulfone P3-substitutent that was not tolerated in
inhibitors containing a six-membered aromatic P2-P3 linker. The combination of a five-membered
N-methylpyrazole linker and a methyl sulfone in P3 yielded subnanomolar Cat K inhibitors that were
minimally shifted (<10-fold) in our functional bone resorption assay. Issues that arose because of metabolic
demethylation of the N-methylpyrazole were addressed through introduction of a 2,2,2-trifluoroethyl
substituent. This culminated in the identification of 31 (MK-1256), a potent (Cat K IC50 ) 0.62 nM) and
selective (>1100-fold selectivity vs Cat B, L, S, C, H, Z, and V, 110-fold vs Cat F) inhibitor of cathepsin
K that is efficacious in a monkey model of osteoporosis.
Introduction
recombinant parathyroid hormone (PTHa) and strontium ranelate
target the osteoblast, while the antiresorptive therapies that target
the osteoclast include hormone-replacement therapy (HRT),
selective estrogen receptor modulators (SERMS), calcitonin, and
the bisphosphonates.3a,b The most widely prescribed therapy for
osteoporosis, the bisphosphonate class of antiresorptives, halts
osteoclast activity and allows for the existing resorption spaces
generated by the osteoclast to be filled in with new bone by the
unaffected osteoblast. Placebo-controlled studies with bispho-
sphonates in postmenopausal women have repeatedly demon-
strated fracture reduction efficacy of ∼40-50% for both
vertebral and nonvertebral fractures.1a,b The bisphosphonates
are currently the preferred therapy for osteoporosis by virtue
of their long-term safety (>10 years) and superior efficacy
relative to the other available treatments in fracture reduction
trials.3a,b The ideal therapeutic strategy would serve to rebuild
lost bone while re-establishing the physiological balance
between osteoclast and osteoblast activity. Thus, we have been
actively seeking to develop antiresorptive agents that, unlike
bisphosphonates, uncouple these two processes to favor bone
formation over resorption.
As the majority of individuals comprising the baby-boomer
generation reach retirement age and enter their golden years,
Western society will become increasingly burdened with the
demands of addressing the medical needs of its sizable elderly
population. Many of these challenges will arise as a result of
the metabolic changes inherent in the process of aging, such as
those experienced by postmenopausal women following the loss
of ovarian sex steroid production. One of the consequences of
this particular physiological alteration is increased rates of bone
resorption relative to bone formation. Left untreated, the
resulting loss in bone mass places the individual in a state of
skeletal fragility and increased susceptibility to hip, spine, and
wrist fractures. This condition, commonly known as osteoporo-
sis, currently afflicts about 8 million women in the U.S. and is
one of the leading causes of morbidity and mortality in women
over 50 years of age. A fact that is less appreciated by the
population at large is that this condition also affects ap-
proximately 2 million men in the U.S. Although males are less
vulnerable to osteoporosis because of their greater peak bone
mass and the fact that they do not experience an abrupt loss of
gonadal sex steroids, the mortality rate associated with hip
fracture for men is twice that for women. Thus, osteoporosis
represents a significant health challenge for elderly individuals
of both sexes.1a,b
A little over a decade ago, biochemical and genetic evidence
began to surface that implicated the cysteine protease cathepsin
K (Cat K) as a key player in the process of bone resorption by
osteoclasts.4 This stimulated a vigorous research effort within
the pharmaceutical industry aimed at the development of Cat
K inhibitors for the treatment of osteoporosis and other disorders
Regardless of the underlying causative factors, osteoporosis
ultimately arises in both males and females as a result of an
increased activity of the bone resorbing cell, the osteoclast,
relative to its bone forming counterpart, the osteoblast.2a,b
Currently available treatment regimens for osteoporosis can be
separated into two classes depending on which cell type they
exert their effect. The bone forming agents such as injectable
a Abbreviations: Cat, cathepsin; hrab, humanized rabbit; rab, rabbit; P1,
P2, P3, peptide subsites 1, 2, and 3; S1, S2, S3, protease subsites 1, 2, and
3; PTH, parathyroid hormone; HRT, hormone replacement therapy; SERMS,
selective estrogen receptor modulators; BRS, bone resorption shift; PK,
pharmacokinetic; CYP, cytochrome P450; HATU, 2-(1H-7-azabenzotriazol-
1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; OVX, ovariecto-
mized; ELISA, enzyme-linked immunosorbant assay; NTx, N-terminal
telopeptide collagen degradation fragment; u-NTx, urinary N-terminal
telopeptide collagen degradation fragment; CTx, C-terminal telopeptide
collagen degradation fragment; Cre, creatinine; qd, once daily; po, oral;
NS, not significant; SW, saline wash.
* To whom correspondence should be addressed. Phone: 514-428-3934.
Fax: 514-428-2624. E-mail: sheldon_crane@merck.com.
†
Merck Frosst Centre for Therapeutic Research.
Merck Research Laboratories.
‡
10.1021/jm800610j CCC: $40.75
2008 American Chemical Society
Published on Web 09/24/2008

Cathepsin K Inhibitor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6411
Figure 1. gem-Difluorocyclohexylcarboxamide cathepsin K inhibitors
of varying polarity.
characterized by pathophysiological bone loss. Indeed, efforts
from our own laboratories have led to the identification of
odanacatib, a potent and selective Cat K inhibitor, that is
currently in phase III clinical trials for oseteoporosis.5a We have
recently disclosed the results of our preliminary investigations
aimed at developing a Cat K inhibitor built upon the cyclohex-
anecarboxamide scaffold.6a,b Herein, we report on the successful
identification of an orally available, small molecule inhibitor
from this distinct structural class.
Figure 2. Modeled structures of inhibitors 1 (gray) and 4 (green) bound
to the active site of cathepsin K.
Explicit solvent molecular dynamics calculations, conducted
using the AMBER suite of programs,11 suggested that switching
from a six- to a five-membered heteroaromatic P2-P3 linker
such as an N-methylpyrazole in 4 could allow for improved
binding to humanized rabbit Cat K10a by virtue of reduced steric
contact of the inhibitor with Asn158 in S2 (dashed line in Figure
2). More importantly, this structural modification was expected
to lift the sulfur atom of the P3-methyl thioether substituent
away from the enzyme surface in S3. Thus, it was reasoned
that the more sterically demanding methyl sulfone functionality
would be better accommodated in P3 upon switching to a five-
membered heteroaromatic linker.
In order to test this idea, a methyl sulfone was incorporated
into P3 of 4. This structural modification resulted in a mere 2-
to 4-fold decrease in humanized rabbit Cat K potency in contrast
with the 85-fold loss observed for 3, thereby validating the
molecular modeling hypothesis. Compound 5, incorporating the
pyrazole linker and methyl sulfone in P3, represented the first
subnanomolar Cat K inhibitor from the cyclohexanecarboxamide
class that was shifted less than 10-fold in the bone resorption
assay (Table 1).
Compound 5 exhibited a half-life in rats comparable to its
progenitor 4 (T1/2 of 4.6 and 4.1 h, respectively). Exchange of
the P3-methyl thioether in 4 for a methyl sulfone in 5 and
introduction of a cyclopropyl in P1 circumvented oxidative
metabolism in those regions of the inhibitor. However, the
methyl of the N-methylpyrazole in 5 was found to be susceptible
to CYP-mediated oxidation. This generated the active metabolite
6 that was observed to be circulating during in vivo studies.
Owing to the success of the fluorination of the cyclohexyl ring
as a strategy to reduce oxidative metabolism,6a it seemed logical
to employ a similar tactic to address the issues associated with
the oxidative demethylation of pyrazole 5. It was anticipated,
based in Figure 2, that a wide variety of substituents would be
tolerated on the pyrazole nitrogen owing to orientation of this
region of the enzyme-bound inhibitor toward bulk solvent.
Compounds incorporating a pyrazole P2-P3 linker such as
5 could be prepared using either the Diels-Alder (first
generation)6a or Robinson annulation (second generation)6b
based strategies reported previously by us (Figure 3). However,
these synthetic approaches were limiting in that an intact
aromatic group was required in the key building block and
thereby lacked the end-game flexibility required to study a
Background and Key Issues
The major impasse hindering identification of a development
candidate from the cyclohexanecarboxamide series was a
substantial loss of potency (50- to 100-fold) in our bone
resorption assay utilizing rabbit osteoclasts7a relative to activity
against isolated rabbit Cat K. During the early stages of our
research program, we had observed that cathepsin K inhibitors
of comparable intrinsic potency could exhibit disparate levels
of efficacy in our in vivo animal models at equivalent plasma
exposure levels. Invariably, it was ascertained that higher levels
of in vivo efficacy were observed for those Cat K inhibitors
that were more potent in our bone resorption assay. Thus,
minimization of this potency loss, formally defined here as the
bone resorption shift (BRS ) (rabbit bone resorption IC50)/
(rabbit Cat K IC50)), represented a key facet of our research
effort. The BRS value was also utilized to calculate the projected
monkey and human bone resorption IC50 values in lieu of
monkey and human versions of the bone resorption assay.7b For
the cyclohexylcarboxamide series of compounds, it appeared
unlikely that nonspecific binding to serum proteins present in
the assay media was solely responsible for an increase in BRS,
as representative compound 1 (Figure 1) was found to be shifted
53-fold in the functional assay despite being only moderately
bound (85%) to rat, rabbit, and human plasma proteins as
determined by ultracentrifugation.8 In general, it was observed
that as the polarity of our Cat K inhibitors increased, the BRS
value decreased. Recently, we reported that replacement of the
P2-P39 phenyl linker in 1 with more polar functionality such
as a pyridine in 2 served to ameliorate the undesirable potency
shift, regrettably at the expense of Cat K potency, counterscreen
selectivity, and pharmacokinetic (PK) properties.6b Subsequent
to this, we discovered that a variety of Cat K inhibitors
incorporating a methyl sulfone (3) instead of a methyl thioether
(1) in P3 were shifted only 8- to 10-fold in our functional assay.
Unfortunately, these gains were offset by an 85-fold reduction
in potency in this instance (humanized rabbit Cat K10a,b IC50 of
0.28 and 24 nM, respectively).

6412 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Robichaud et al.
Table 1. Potency and Selectivity Profiles of Selected Cathepsin K Inhibitors
a Each value represents an average of at least three independent determinations. b Counterscreen assays were performed using human Cat L, B, and S.
c Bone resorption shift BRS ) (bone resorption IC50)/(rabCatK IC50).
Figure 3. Strategies for the synthesis of cyclohexylcarboxamide cathepsin K inhibitors.
variety of heteroaromatic P2-P3 linkers. In addition, our
previous generation syntheses often relied on cumbersome
HPLC-based resolution procedures to produce the desired Cat
K inhibitors in high enantiopurity. In order to effectively deal
with the issues related to the oxidative demethylation of 5, a
more flexible, third generation enantioselective synthetic route
was required.
Chemistry
A strategy was devised that would allow for access to a wide
variety of heterocyclic P2-P3 linkers, with a focus on N-
substituted pyrazoles, from a late-stage keto-ester intermediate
(boxed in Figure 3). At the same time, we were mindful to
incorporate the gem-difluoro substituent that proved crucial for
controlling oxidative metabolism on the P2-cyclohexane portion

Cathepsin K Inhibitor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6413
Scheme 1a
a Conditions: (a) BnOCH2CHO, i-Pr2NEt, LiCl, CH3CN; (b) 2-[(trimethylsilyl)oxy]-1,3-butadiene, Et2AlCl, CH2Cl2, -78°C; (c) 6 M HCl, -40°C to
room temp, 45% over three steps; (d) (MeOCH2CH2)2NSF3, CH2Cl2, 0°C, 43%; (e) BuLi, benzyl mercaptan, 0°C, then LiAlH4, 96%.
Scheme 2a
a Conditions: (a) 1,2-bis[trimethylsilyl(oxy)]ethane, TMSOTf, CH2Cl2, room temp, >99%; (b) BuLi, ethanethiol, 0°C, then LiAlH4, 86%; (c) AcOH,
H2O, 85°C, 95%; (d) H2NNH2 ·H2O, MeOH, 4 Å sieves; (e) CuCl2, Et3N, MeOH, 0°C, 76%.
Scheme 3a
a Conditions: (a) DMP, CH2Cl2, 86% for 16, 78% for 17; (b) ethyl 4-(methylthio)phenylacetate, LDA, THF, -78 °C, 56% for 16, 80% for 17; (c) oxalyl
chloride, DMSO, Et3N, CH2Cl2, -78 °C to room temp, 88% for 20, 97% for 21.
of these inhibitors. We also prepared the corresponding gem-
dichloro analogue, as molecular modeling studies suggested
that a gem-dichlorocyclohexyl in conjunction with a five-
membered heterocyclic linker should be tolerated in the active
site of Cat K.
method described above, after first protecting the ketone
functionality as a 1,3-dioxolane in 12. Cleavage of the ketal
protecting group with wet acetic acid at 85 °C liberated the
ketone in 14 that was subsequently transformed to a gem-
dichloro in 15 via treatment of the intermediate hydrazone with
CuCl2 and Et3N.15
Alcohols 11 and 15 were transformed into the corresponding
aldehydes 16 and 17 by oxidation with Dess-Martin periodi-
nane (Scheme 3). Reaction of the aldehydes with the carbanion
generated from ethyl 4-(methylthio)phenylacetate and LDA
yielded alcohols 18 and 19, which were converted via a Swern
oxidation to the key keto-ester intermediates 20 and 21 targeted
in Figure 3.
With keto-esters 20 and 21 in hand, we were in a position to
prepare a variety of heterocyclic analogues of 5 using classical
heterocyclic chemistry methodology.16 As mentioned above, the
corresponding N-trifluoroethylpyrazole analogues were initially
targeted for synthesis from keto-esters 20 and 21 (Scheme 4).
Heating of 20 and 21 with trifluoroethylhydrazine and MgSO4
in refluxing toluene produced hydroxypyrazoles 22 and 23. The
hydroxyl oxygen was subsequently activated for removal by
conversion to the corresponding triflate, and the methyl thioether
was oxidized to a methyl sulfone in 24 and 25 with m-CPBA.
Exposure 24 and 25 to an atmosphere of hydrogen gas in the
presence of 10% Pd/C in methanol containing triethylamine
effected the necessary deoxygenation to complete the construc-
tion of the N-trifluoroethylpyrazole P2-P3 linker. Removal of
the benzyl ether protecting group was smoothly effected with
hydrogen in the presence of Pearlman’s catalyst and acetic acid
Our third generation synthesis begins in Scheme 1 with
preparation of dienophile 812 for utilization in an Evan’s
auxiliary-controlled Diels-Alder reaction13 with 2-[(trimeth-
ylsilyl)oxy]-1,3-butadiene. This sequence yielded the desired
cyclohexanone 9a following in situ hydrolysis of the initially
formed enol-silyl ether. Some of the corresponding diastereo-
meric cyclohexanone 9b, epimeric at the both the imide and
benzyl ether bearing stereocenters, was also formed from this
reaction sequence (ratio of 9a/9b ) 7:1). However, the major
diastereomer 9a proved to be more crystalline than the minor
isomer and could be separated by precipitation from a mixture
of ethyl acetate and hexanes on a larger scale. On a smaller
scale, quantitative separation was readily achieved by conven-
tional flash chromatography. Introduction of the gem-difluoro
substituent in 10 was accomplished by treatment of 9a with
Deoxofluor. Subsequent cleavage of the Evan’s auxiliary with
lithium benzylmercaptide and reduction of the intermediate
thioester with LiAlH4 generated alcohol 11.14
Preparation of the corresponding gem-dichloro alcohol re-
quired an alternative sequence of events (Scheme 2), as the
acyloxazolidinone functionality did not tolerate the conditions
used below for conversion of a ketone into a gem-dichloro
group. Thus, the acyloxazolidinone in 9a was first converted
into the corresponding primary alcohol 13 according to the

6414 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Robichaud et al.
Scheme 4a
a Conditions: (a) CF3CH2NHNH2, PhMe, MgSO4, reflux, 81% for 22, 64% for 23; (b) Tf2O, pyr, CH2Cl2; (c) mCPBA, CH2Cl2, 0°C to room temp, 96%
for 24, 90% for 25; (d) H2, 10% Pd/C, Et3N, EtOAc, 3 Å molecular sieves; (e) H2, 10% Pd(OH)2/C, EtOAc, AcOH, 66% for 26, 60% for 27 over two steps;
(f) Jones’ reagent, acetone, 0°C; (g) 1-aminocyclopropylacetonitrile hydrochloride, HATU, Hu¨nig’s base, DMF, room temp, 82% for 30, 91% for 31 over
two steps.
Table 2. Pharmacokinetic Profiles of
N-Trifluoroalkylpyrazole-Containing Cathepsin K Inhibitors
Figure 4. Metabolites generated from 31 in rats.
lysosomal cysteine proteases including human cathepsins C
in ethyl acetate. Attempts to accomplish these two transforma-
(6000-fold), H (>140000-fold), Z (1700-fold), and V (1100-
tions in a single operation using either of the palladium catalysts
were not successful. Jones’ oxidation of the liberated primary
alcohols 26 and 27 and HATU-mediated coupling of the
fold). Compounds 30 and 31 were also selective against human
cathepsin F but to a lesser degree (110-fold).
carboxylic acid products 28 and 29 to 1-aminocyclopropylac-
etonitrile concluded the synthetic sequence and provided the
enantiopure N-trifluoroethylpyrazole analogues 30 and 31 for
evaluation in our barrage of in vitro and in vivo assays.
Pharmacokinetics and Metabolism
Both 30 and 31 displayed acceptable PK profiles in multiple
animal species (Table 2). gem-Dichloro analogue 31 was
ultimately selected for metabolism and excretion profiling on
the basis of its superior bioavailability and half-life.
Potency and Counterscreen Selectivity
Rat plasma samples from PK studies and the assay medium
from compound incubations conducted with freshly isolated rat
hepatocytes were analyzed by LC-MS. From these experiments,
it was ascertained that 31 was subject to less than 1% oxidative
metabolism. Similar observations were also made during human
hepatocyte incubations. In order to fully characterize the
metabolic fate of 31 in rats, excretion studies were conducted
with radiolabeled compound17 introduced orally in bile-cannu-
lated animals. During these experiments, it was demonstrated
that after 96 h, the total amount of recovered radioactivity was
quite high, with 92% of the dosed radioactivity being recovered
from the bile (16%), urine (18%), and feces (58%). In bile
samples collected between 2 and 4 h postdosing, the parent drug
As shown in Table 1, compounds 30 and 31 were ascertained
to be subnanomolar inhibitors of humanized rabbit Cat K and,
like other inhibitors possessing a methyl sulfone in P3,5a,b were
less than 10-fold shifted in the functional bone resorption assay
(BRS ) 6 and 8, respectively). No appreciable loss of potency
or selectivity was observed by switching from a gem-difluoro
in 30 to a gem-dichloro in 31, in contrast to the situation
described previously with 1.6a Relative to N-methylpyrazole 5,
the N-(2,2,2-trifluoroethyl) analogues exhibited similar potencies
against humanized rabbit Cat K and comparable selectivities
against human cathepsins B, L, and S. Compounds 30 and 31
were also quite selective against a wider panel of human

Cathepsin K Inhibitor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6415
(68% u-NTx reduction at 3 mpk/qd),5b which is structurally
similar to our clinical candidate odanacatib.5a
Kinetic Characterization
The reversibility of inhibition of humanized rabbit Cat K by
31wasdemonstratedinexperimentsinwhicha1:1enzyme-inhibitor
mixture (0.12 µM) was preincubated for 15 min, followed by a
500-fold dilution into substrate-containing buffer (0.2 µM
citrate). After dilution, the Cat K enzyme activity was initially
>99% inhibited and slowly recovered with a t1/2 ) 215 s to a
velocity similar to that of the control incubation, demonstrating
the reversibility of the formation of the 31-cathepsin K
complex.
Figure 5. Urinary NTx/Cre during 1 mpk compound 31 po, q.d. adult
OVX rhesus monkeys. Compound 31 reduces bone resorption in OVX
rhesus monkeys. Bone resorption in OVX monkeys treated with
compound 31 (b) or vehicle (2) was evaluated using the urine
biochemical marker NTx. Data are reported as uNTx/creatine (nM/
mM).
Discussion
It is widely accepted that bisphosphonate antiresorptives exert
their effects on osteoclasts via disruption of the mevalonic acid
pathway following selective uptake of the bone-immobilized
bisphosphonate.3b The ensuing dysfunction and ultimate apo-
ptotic demise of the osteoclast then effect the management of
excessive bone resorption. The intricate process that constitutes
the maintenance of normal bone structure relies on the coor-
dinated action of both the osteoclast and the osteoblast.2a,b The
observed levels of clinical efficacy associated with bisphospho-
nate treatment have been attributed to, and appear to be limited
by, osteoblast-mediated refilling of the existing resorption spaces
left by the osteoclast.1a Since its initial discovery over a decade
ago, the key role of cathepsin K in osteoclast-mediated bone
resorption has been firmly established.4 Compared to bisphos-
phonates, cathepsin K inhibition blocks bone resorption to a
similar degree but reduces bone formation to a lesser extent.20
Thus, a Cat K inhibitor would be a welcome addition to a
physician’s armamentarium by virtue of this unique mode of
action. However, it still remains to be determined during ongoing
clinical trials21 if a small molecule Cat K inhibitor will achieve
greater fracture reduction efficacy than currently available
therapies.
and three main metabolites were detected by radio-HPLC
analysis. These metabolites were identified by LC-MS as the
dealkylated pyrazole 32, the nitrile hydrolysis product 33, and
the Cys-Gly conjugate 34 (Figure 4). The latter two metabolites
were likely generated through the mercapturic acid biotrans-
formation pathway.18a,b Of the total recovered radioactivity, 90%
was attributed to unchanged parent 31 with the metabolites
constituting the remaining 10%. The dealkylated pyrazole 32
was found to comprise only 0.1% of the total recovered
radioactivity. Thus, oxidative metabolism did not significantly
contribute to the clearance of 31 during rat PK studies, consistent
with the observations made during hepatocyte incubations. This
was not surprising given the fact that the three sites susceptible
to oxidative metabolism in earlier generation inhibitors, namely,
the cyclohexyl ring, the substituent on nitrogen of the pyrazole,
and the methylene adjacent to the nitrile warhead, have all been
suitably modified to circumvent this in 31.
In summary, our efforts in this area have led to the identi-
fication of 31 (MK-1256), a nitrile-based inhibitor of Cat K
built on a trans-2-substituted cyclohexanecarboxamide scaffold.
Compound 31 is a reversible, potent (humanized rabbit Cat
K10a,b IC50 ) 0.62 nM), and selective (>1100-fold vs human
cathepsins B, L, S, C, H, V, and Z, 110-fold vs human cathepsin
F) inhibitor of cathepsin K. This compound demonstrated an
excellent pharmacokinetic profile across several animal species
and achieved efficacy comparable to other known Cat K
inhibitors in an OVX rhesus model of osteoporosis.
Pharmacology
Ovariectomized (OVX) monkeys experience estrogen defi-
ciency and accelerated bone loss analogous to postmenopausal
women.19a Assessment of the magnitude of bone turnover can
be achieved through ELISA-monitoring of the bone resorption
biomarkers CTx and NTx in serum and/or urine. Thus, evalu-
ation of the effect of compound treatment on bone resorption
biomarkers relative to untreated control animals constitutes a
rapid measure of biochemical efficacy of the pharmacological
agent of interest in an osteoporotic context.19b On the basis of
data obtained during a study of the pharmacokinetic behavior
of 31 in rhesus monkeys (Table 2), it was predicted that a dose
of 1 mpk would provide trough levels (t ) 24 h) near the
projected monkey bone resorption IC50 value of 2 nM.7b Under
the above experimental paradigm, once-daily (qd) oral admin-
istration of 31 at 1 mpk for a period of 1 week achieved a mean
72% reduction (Figure 5) of urinary-N-telopeptide fragment
levels (u-NTx, normalized to creatine output). The mean plasma
concentrations of 31 measured on the final day of dosing for
this study were as follows: Cmax ) 400 nM (Tmax ) 4 h), C24h
) 80 nM, AUC ) 4.2 µM·h. This magnitude of bone turnover
marker supression is comparable to that seen for other nitrile-
based inhibitors of Cat K, such as our first generation dipeptide
inhibitor L-006,235 (76% u-NTx reduction at 15 mpk/qd)5c and
our nonbasic trifluoroethylamine based inhibitor L-873,724
Experimental Section
General. m-Chloroperoxybenzoic acid (m-CPBA) was purified
by washing a benzene solution of commercial material with two
portions of pH 7.4 phosphate buffer followed by recrystallization
from ether/hexanes. All other reagents were obtained commercially
and were used without further purification. Proton (1H NMR)
magnetic resonance spectra were recorded on Bruker 400 and 500
MHz instruments. All spectra were recorded in acetone-d6 (unless
otherwise indicated) using residual solvent as the internal standard.
Signal multiplicity was designated according to the following
abbreviations: s ) singlet, d ) doublet, dd ) doublet of doublets,
ddd ) doublet of doublet of doublets, t ) triplet, q ) quartet, dt
) doublet of triplets, td ) triplet of doublets, dq ) doublet of
quartets, m ) multiplet, br ) broad. Elemental analyses were
provided by Prevalere Life Sciences Inc., Whitesboro, NY. Rota-
tions were measured on a Perkin-Elmer model 241 polarimeter.
Reactions were carried out with continuous stirring under a positive

6416 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Robichaud et al.
pressure of nitrogen except where noted. Flash chromatography was
carried out with silica gel 60, 230-400 mesh.
t, J ) 8.2 Hz), 4.64 (2H, s), 4.43 (1H, t, J ) 8.5 Hz), 4.32 (2H,
m), 4.29 (1H, dd, J ) 2.9, 8.9 Hz), 3.24 (1H, dd, J ) 3.1, 14 Hz),
3.03 (1H, dd, J ) 8.4, 14 Hz).
Enzyme Expression and Purification. See ref 10b.
Enzyme Activity Assays. See ref 10b.
(4R)-4-Benzyl-3-({(1R,2R)-2-[(benzyloxy)methyl]-4-oxocyclohexyl}-
carbonyl)-1,3-oxazolidin-2-one (9a). A 1.8 M solution of Et2AlCl in
toluene (812 mL, 1460 mmol, 1.4 equiv) was added slowly to a
-78 °C solution of dienophile 8 (367 g, 1044 mmol) in CH2Cl2
(1100 mL). The resulting yellow solution was stirred at this
temperature for 10 min prior to the slow introduction of 2-[(trim-
ethylsilyl)oxy]-1,3-butadiene (520 g, 3.5 equiv) as a solution in
CH2Cl2 (500 mL). After 16 h at -78 °C, the temperature was raised
to -40 °C and a 1:1 mixture of 6 M HCl and THF (500 mL) was
carefully added dropwise followed by an additional portion of 1:1
THF/6 M HCl (500 mL) after gas evolution had ceased. The mixture
was then stirred at room temperature until TLC analysis indicated
that hydrolysis of the enol-ether was complete (about 30 min).
Ethyl acetate and Celite were added (to precipitate and capture
polymeric material) with stirring at room temperature for 10-15
min followed by suction filtration and washing of the pad with ethyl
acetate. The filtrate was washed with brine and dried over Na2SO4/
MgSO4. Concentration in vacuo and chromatography on silica gel
(3 kg in a sintered glass funnel), eluting with 20% ethyl acetate/
hexanes, afforded material that was swished with 15% ether in
hexanes to afford 220 g of solid that was further purified by
recrystallization from ethyl acetate/hexanes (250/650 mL) to afford
the desired product as a colorless powder (160 g). Purification of
the mother liquor concentrate by flash chromatography eluting with
an increasing proportion of ethyl acetate in hexanes afforded an
additional 42.1 g of product (202.1 g total, 45% yield over three
Pharmacokinetic and Bioavailability Experiments. Single
compounds were dosed orally as suspensions in 1% methocel and
intravenously via the jugular vein as solutions in PEG-200/H2O
(3:2). Dosing volumes used for oral administration were 10 mL/kg
for rats, 1 mL/kg for monkeys, and 5 mL/kg for dogs. Intravenous
dosing volumes of 1 mL/kg were used for all species. Animals
receiving the oral suspensions were fasted overnight prior to a.m.
dosing. Plasma samples were collected at regular intervals and
analyzed by LC-MS following a protein precipitation with 1.5
volumes of acetonitrile and centrifugation. All animal procedures
were conducted in accordance with Institutional Animal Care and
Use Committee guidelines.
Hepatocyte Incubations. For hepatocyte incubations, 1 × 106
cells diluted in 0.5 mL of Krebs-Henseleit buffer were first
prepared at 37 °C for 20 min under 95:5 O2/CO2 (BOC gases;
Montreal, Canada) in a 48-well plate, and then an amount of 5 µL
of 10 mM solution of compound dissolved in acetonitrile was added
to each well to a final concentration of 50 µM. After 2 h of
incubation at 37 °C under 95:5 O2/CO2 atmosphere, one volume
of acetonitrile was added in each well. A quenched incubation
spiked with the parent compound and a blank were also prepared
as controls. Once transferred, samples were centrifuged for 10 min
at 14 000 rpm using an Eppendorf 5415C centrifuge (Hamburg,
Germany), and the supernatant was used for LC/UV/MS analysis.
Diethyl {2-[(4R)-4-Benzyl-2-oxo-1,3-oxazolidin-3-yl]-2-oxoethyl}-
phosphonate (7). n-BuLi (2.5 M in hexanes, 622 mL, 1550 mmol,
1 equiv) was added over 10-15 min to a -78 °C solution of (4R)-
4-benzyl-1,3-oxazolidin-2-one (275 g, 1550 mmol) in THF (3.7 L)
at a rate such that the internal temperature was maintained below
-65 °C. The resulting yellow solution was stirred at this temper-
ature for an additional 10 min prior to the rapid introduction of
bromoacetyl bromide (135 mL, 1550 mmol, 1 equiv) over a period
of 15 min. The mixture was then allowed to slowly attain room
temperature, followed by stirring at room temperature overnight.
Water (370 mL) was added, and the majority of the THF was
removed by rotary evaporation. The residue was partitioned between
ethyl acetate and water, and the layers were separated. The aqueous
phase was extracted with an additional portion of ethyl acetate,
and the combined organics were washed with water and brine, dried
over Na2SO4, and concentrated. The residue was purified by
filtration through a pad of silica gel in a large sintered glass funnel
eluting with 20% ethyl acetate in hexanes to afford (4R)-4-benzyl-
3-(bromoacetyl)-1,3-oxazolidinon-2-one as a waxy solid (316 g,
68% yield). This material (316 g, 1060 mmol) and triethylphosphite
(204 mL, 1170 mmol, 1.1 equiv) were heated together in refluxing
toluene (740 mL) for 20 h. Concentration by rotary evaporation
followed by hi-vac treatment with heating afforded the desired keto-
phosphonate as a thick, yellow syrup (377 g) that was used directly
in the next step.
(4R)-4-Benzyl-3-[(2E)-4-(benzyloxy)-but-2-enoyl]-1,3-oxazolidin-
2-one (8). Hu¨nig’s base (204 mL, 1170 mmol, 1.1 equiv) was added
at 0 °C to a mixture of keto-phosphonate 7 (377 g, 1060 mmol)
and anhydrous LiCl (49.4 g, 1170 mmol, 1.1 equiv) in CH3CN (1
L). The mixture was mechanically stirred at this temperature for
10 min prior to the slow introduction of benzyloxyacetaldehyde
(150 mL, 1060 mmol, 1 equiv) followed by stirring at room
temperature overnight (12 h). Water was then added followed by
extraction with ether (2×). The combined extracts were then
vigorously stirred at room temperature with an equal volume of 1
M HCl for 1 h. Stirring with HCl is required to isomerize the (Z)-
isomer, which is also produced in the reaction, to the desired (E)-
isomer. The organic phase was then washed with brine and saturated
NaHCO3 solutions and dried over Na2SO4/MgSO4. Concentration
in vacuo and passage of the residue through a silica plug eluting
with 1:1 ethyl acetate/hexanes yielded a colorless solid (372 g) upon
trituration with ether. 1H NMR (500 MHz, acetone-d6) δ 7.57 (1H,
d, J ) 16 Hz), 7.43-7.27 (10H, m), 7.21-7.15 (1H, m), 4.84 (1H,
1
steps) as a single diastereoisomer. H NMR (400 MHz, acetone-
d6) δ 7.38-7.32 (6H, m), 7.31-7.25 (4H, m), 4.59-4.53 (1H, m),
4.47 (2H, q, J ) 8.5 Hz), 4.19 (1H, dd, J ) 3.0, 9.0 Hz), 4.15-4.09
(1H, m), 4.03 (1H, t, J ) 8.6 Hz), 3.53-3.47 (2H, m), 3.13 (1H,
dd, J ) 3.4, 14 Hz), 2.97 (1H, dd, J ) 8.2, 14 Hz), 2.70-2.60
(1H, m), 2.54-2.46 (1H, m), 2.44 (2H, d, J ) 9.3 Hz), 2.41-2.29
(2H, m), 1.92-1.82 (1H, m). For the minor diastereomer: 1H NMR
(400 MHz, acetone-d6) δ 7.40-7.22 (10H, m), 4.76 (1H, m), 4.55
(2H, m), 4.35 (1H, t, J ) 8.4 Hz), 4.24-4.15 (2H, m), 3.56 (1H,
dd, J ) 9.4, 5.0 Hz), 3.49 (1H, dd, J ) 9.4, 4.5 Hz), 3.16 (1H, dd,
J ) 14, 3.3 Hz), 2.70-2.57 (2H, m), 2.51 (1H, m), 2.48-2.39
(2H, m), 2.36-2.23 (2H, m), 1.78 (1H, m).
(4R)-4-Benzyl-3-({(1R,2R)-2-[(benzyloxy)methyl]-4,4-difluorocy-
clohexyl}carbonyl)-1,3-oxazolidin-2-one (10). Deoxofluor (21 mL,
110 mmol) was added slowly over 5 min to a 0 °C solution of
ketone 9a (18.7 g, 44.4 mmol) in CH2Cl2 (100 mL). After 2 h at
this temperature, the reaction was quenched by the careful addition
of an equal volume of ice-water. After separation of the phases,
the aqueous layer was extracted with CH2Cl2 and the combined
organics were washed with water and saturated aqueous NaHCO3
and dried (Na2SO4). The methylene chloride solution containing
the crude product was concentrated to a volume of 100 mL and
treated with m-chloroperoxybenzoic acid (6.0 g, 35 mmol) with
stirring at room temperature for 3 h to effect conversion of the
fluoroalkene byproduct to the corresponding epoxide. Additional
CH2Cl2 (100 mL) was then added followed by Me2S (3 mL) and
Ca(OH)2 (20 g) with stirring at room temperature for 15 min. The
slurry was filtered through Celite, and the filtrate was concentrated
in vacuo. Flash chromatography of the residue on silica gel eluting
with 1/3 ethyl acetate/hexanes yielded the title compound as a thick,
1
colorless syrup (8.52 g, 43%). H NMR δ (500 MHz, acetone-d6)
7.36-7.32 (6H, m), 7.26 (4H, dd, J ) 7.3, 24 Hz), 4.54-4.50 (1H,
m), 4.48-4.42 (2H, m), 4.19-4.15 (1H, m), 3.99 (1H, t, J ) 8.6
Hz), 3.80-3.74 (1H, m), 3.47 (2H, d, J ) 5.5 Hz), 3.13-3.09 (1H,
m), 2.94 (1H, dd, J ) 8.2, 14 Hz), 2.56-2.48 (1H, m), 2.26-2.12
(3H, m), 1.95-1.71 (3H, m).
{(1R,2R)-2-[(Benzyloxy)methyl]-4,4-difluorocyclohexyl}methanol (11).
A solution of n-BuLi (2.5 M in hexanes, 10 mL) was added slowly
to a solution of benzylmercaptan (3.6 mL, 40 mmol) in THF (40
mL) at 0 °C. The resulting pale-yellow solution was stirred at 0 °C
for 10 min prior to the introduction of acyloxazolidinone 10 (8.52
g, 19.2 mmol) as a solution in THF (30 mL). After 10 min, a

Cathepsin K Inhibitor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6417
solution of LiAlH4 (1.0 M in THF, 22 mL) was slowly added to
the reaction mixture with continued stirring at 0 °C for 45 min.
Ice-water was then slowly added to destroy excess hydride
followed by 6 M HCl (20 mL) to pH 1. The mixture was extracted
with two portions of ethyl acetate, and the combined organics were
washed with saturated aqueous NaCl and dried over Na2SO4/
MgSO4. Concentration in vacuo and flash chromatography of the
residue on silica gel, eluting with 45/55 ethyl acetate/hexanes,
afforded the title compound as a thick, colorless syrup (5.01 g,
96%). 1H NMR (500 MHz, acetone-d6) δ 7.36 (4H, m), 7.34-7.28
(1H, m), 4.56-4.49 (2H, m), 3.67-3.53 (5H, m), 2.20-2.12 (1H,
m), 2.09-2.02 (1H, m), 1.91-1.71 (4H, m), 1.58-1.50 (2H, m).
(1R,2R)-2-[(Benzyloxy)methyl]-4,4-difluorocyclohexanecarbal-
dehyde (16). Dess-Martin periodinane (9.84 g, 23.2 mmol) was
added to a solution of alcohol 11 (5.01 g, 20.2 mmol) in CH2Cl2
(50 mL) at 0 °C. The mixture was stirred at 0 °C for 5 min, then
at room temperature for 1 h. Ethyl acetate (100 mL), water (100
mL), and a saturated aqueous solution of NaHCO3 containing
Na2S2O3 ·5H2O (28.9 g, 120 mmol) were added with stirring at room
temperature for 30 min. The layers were separated, and the aqueous
phase was extracted with additional ethyl acetate. The combined
organics were washed with saturated NaCl aqueous solution and
dried over Na2SO4/MgSO4. Concentration in vacuo and flash
chromatography on silica gel, eluting with 16/84 ethyl acetate/
hexanes, gave the title compound as a colorless liquid (4.64 g, 86%).
1H NMR (500 MHz, acetone-d6) δ 9.66 (1H, d, J ) 3.0 Hz),
7.37-7.33 (4H, m), 7.31-7.29 (1H, m), 4.52 (2H, m), 3.55 (1H,
dd, J ) 4.8, 9.3 Hz), 3.51-3.45 (1H, m), 2.43-2.33 (2H, m),
2.20-2.12 (2H, m), 1.98-1.66 (4H, m).
Ethyl 3-{(1R,2R)-2-[(Benzyloxy)methyl]-4,4-difluorocyclohexyl}-
3-hydroxy-2-[4-(methylthio)phenyl]propanoate (18). A 2.1 M solu-
tion of n-BuLi (11 mL, 23 mmol) in hexanes was added slowly to
a 0 °C solution of diisopropylamine (4.0 mL, 28 mmol) in THF
(10 mL). Stirring was continued at this temperature for an additional
10 min prior to cooling to -78 °C. A solution of ethyl [4-(meth-
ylthio)phenyl]acetate (4.64 g, 18.9 mmol) in THF (15 mL) was
then introduced, and the mixture was stirred at -78 °C for 10 min,
-40 °C for 10 min, and finally 0 °C for 5 min. The solution was
recooled to -78 °C, and a solution of aldehyde 16 (4.64 g, 18.9
mmol) in THF (15 mL) was added down the flask wall over 5 min.
Stirring was continued at -78 °C for 30 min and then at -40 °C
for 1 h. A saturated aqueous solution of NH4Cl was added, and the
reaction mixture was partitioned between ethyl acetate and water.
The organic phase was washed with saturated aqueous NaCl
solution and dried over Na2SO4/MgSO4. Concentration in vacuo
and flash chromatography on silica gel, eluting with 3/7 ethyl
acetate/hexanes, gave the title compound as a single diastereomer
in the form of a faint-yellow, thick syrup (5.09 g, 56%). 1H NMR
(400 MHz, acetone-d6) δ 7.43-7.33 (5H, m), 7.33-7.29 (2H, m),
7.15-7.11 (2H, m), 4.66 (1H, m), 4.57-4.49 (2H, m), 4.16-4.00
(2H, m), 3.74 (2H, m), 3.45 (1H, m), 2.46 (3H, s), 2.13-1.95 (4H,
overlapped m), 1.84-1.75 (2H, m), 1.73-1.63 (2H, m), 1.45 (1H,
m), 1.17 (3H, t, J ) 7.1 Hz).
Ethyl 3-{2-[(Benzyloxy)methyl]-4,4-difluorocyclohexyl}-2-[4-
(methylthio)phenyl]-3-oxopropanoate (20). Oxalyl chloride (2.9 mL,
34 mmol) was added dropwise to a -78 °C solution of dimethyl
sulfoxide (2.9 mL, 41 mmol) in CH2Cl2 (40 mL). Stirring was
continued at -78 °C for an additional 10 min prior to the slow
introduction down the flask wall of a solution of alcohol 18 (5.09
g, 10.6 mmol) and triethylamine (16 mL, 120 mmol) in CH2Cl2
(40 mL). The mixture was then held at -78 °C for 10 min followed
by rapid warming to room temperature. The reaction vessel contents
were then poured into 2 M HCl in a separatory funnel, the layers
were shaken and separated, and the aqueous phase was extracted
with an additional portion of CH2Cl2. The combined organics were
washed with water, dried (Na2SO4), and concentrated. Flash
chromatography of the residue on silica gel, eluting with 1/4 ethyl
acetate/hexanes, afforded the desired compound as a thick, tan
colored syrup (4.43 g, 88%).
3-{(1R,2R)-2-[(Benzyloxy)methyl]-4,4-difluorocyclohexyl}-4-[4-
(methylthio)phenyl]-1-(2,2,2-trifluoroethyl)-1H-pyrazol-5-ol (22). A
slurry composed of keto-ester 20 (4.43 g, 9.31 mmol), 2,2,2-
trifluoroethylhydrazine (70 wt % solution in water, 13 mL, 100
mmol, 10 equiv), 1,1,2,2-tetrachloroethane (100 mL), and MgSO4
(50 g) was heated at 120 °C for 3 days. The mixture was then
cooled in ice, and water (200 mL) was added followed by 6 M
HCl to pH 1. The layers were separated, and the aqueous phase
was extracted with two portions of CH2Cl2. The combined organics
were washed with water, dried (Na2SO4), and concentrated. Flash
chromatography on silica gel, eluting with 15/85 acetone/benzene,
1
yielded the title compound as a yellow foam (3.95 g, 81%). H
NMR (400 MHz, acetone-d6) δ 9.66 (1H, br s), 7.36-7.26 (5H,
m), 7.20 (4H, m), 4.78 (1H, m), 4.37 (1H, d, J ) 11 Hz), 4.30
(1H, d, J ) 11 Hz), 3.38 (2H, m), 2.75-2.92 (3H, m), 2.50 (3H,
s), 2.41 (1H, m), 2.23 (1H, m), 2.00-1.75 (4H, m).
3-{(1R,2R)-2-[(Benzyloxy)methyl]-4,4-difluorocyclohexyl}-4-[4-
(methylsulfonyl)phenyl]-1-(2,2,2-trifluoroethyl)-1H-pyrazol-5-yl Tri-
fluoromethanesulfonate (24). A solution of hydroxypyrazole 22
(3.95 g, 7.50 mmol) and pyridine (0.95 mL, 12 mmol) in CH2Cl2
(30 mL) was cooled to 0 °C and treated with trifluoromethane-
sulfonic acid anhydride (1.5 mL, 9.2 mmol). After completion of
the reaction, as indicated by TLC analysis (15 min at 0 °C), the
reaction vessel contents were partitioned between water and CH2Cl2,
and the layers were separated. The aqueous layer was extracted
with an additional portion of CH2Cl2, and the combined organics
were washed with water, dried (Na2SO4), and concentrated. Flash
chromatography on silica gel, eluting with 15/85 ethyl acetate/
hexanes, gave the corresponding triflate as a faint-yellow solid (4.36
g, 96%). This material was dissolved in CH2Cl2 (40 mL) and treated
with m-chloroperoxybenzoic acid (2.62 g, 15.2 mmol) at 0 °C. The
resulting slurry was stirred at 0 °C for 5 min and then at room
temperature for 30 min before being diluted with CH2Cl2 (40 mL)
and treated with dimethyl sulfide (0.25 mL) and Ca(OH)2 (9.0 g,
160 mmol). After being stirred at room temperature for 10 min,
the mixture was filtered (Celite) and the filtrate was concentrated
in vacuo. Flash chromatography on silica gel, eluting with 3/7 ethyl
acetate/hexanes, gave the title compound as a colorless foam (4.54
1
g, 99%). H NMR (400 MHz, acetone-d6) δ 7.97 (2H, d, J ) 8.1
Hz), 7.70 (2H, d, J ) 8.1 Hz), 7.36-7.26 (3H, m), 7.19 (2H, m),
5.17-5.07 (2H, m), 4.35 (1H, d, J ) 12 Hz), 4.28 (1H, d, J ) 12
Hz), 3.37-3.27 (2H, m), 3.13 (3H, s), 2.97 (1H, m), 2.41 (1H, m),
2.22 (1H, m), 2.10-2.02 (2H, m), 2.00-1.92 (3H, m).
{(1R,2R)-5,5-Difluoro-2-[4-[4-(methylsulfonyl)phenyl]-1-(2,2,2-
trifluoroethyl)-1H-pyrazol-3-yl]cyclohexyl}methanol (26). A mix-
ture of triflate 24 (4.54 g, 6.58 mmol), triethylamine (1.3 mL, 9.3
mmol, 1.4 equiv), and 10% Pd/C (2.20 g) was stirred together in
ethyl acetate (60 mL) under an atmosphere of hydrogen gas at room
temperature for 16 h. Filtration through Celite, concentration of
the filtrate, and flash chromatography on silica gel, eluting with
45/55 ethyl acetate/hexanes, yielded a colorless, thick syrup (2.15
g, 72%). This material (2.15 g, 3.97 mmol) and Pearlman’s catalyst
(1.0 g) were stirred together in ethyl acetate (50 mL) containing
acetic acid (5 mL) under an atmosphere of hydrogen at room
temperature for 20 h. The reaction mixture was then filtered through
a Celite pad, and the filtrate was washed with saturated aqueous
solutions of NaHCO3 and NaCl before drying over Na2SO4. Solvent
removal in vacuo yielded the desired compound as a faint-yellow,
thick syrup (1.64 g, 91%). 1H NMR (400 MHz, acetone-d6) δ 8.06
(1H, s), 7.96 (2H, d, J ) 8.4 Hz), 7.79 (2H, d, J ) 8.4 Hz), 5.08
(2H, q, J ) 8.8 Hz), 3.70 (1H, m), 3.47 (1H, m), 3.36 (1H, m),
3.16 (3H, s), 3.09 (1H, m), 2.38 (1H, m), 2.25 (1H, m), 2.15-1.30
(5H, m).
(1R,2R)-5,5-Difluoro-N-(1-cyanocyclopropyl)-2-[4-[4-(methylsul-
fonyl)phenyl]-1-(2,2,2-trifluoroethyl)-1H-pyrazol-3-yl]cyclohexan-
ecarboxamide (30). A solution of alcohol 26 (1.64 g, 3.63 mmol)
in acetone (40 mL) was treated at 0 °C with Jones’ reagent (3.8
mL) followed by stirring at room temperature for 30 min. Water
was added, and the mixture was extracted with two portions of
ethyl acetate. The combined organics were washed with water until
no orange coloration was visible in the aqueous phase, followed

6418 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Robichaud et al.
by washing with a saturated aqueous solution of NaCl before drying
over Na2SO4/MgSO4. Concentration in vacuo gave the desired
carboxylic acid as a faint-yellow foam (1.69 g, 99%). This material
was then combined with 1-aminocyclopropanecarbonitrile hydro-
chloride (1.16 g, 9.83 mmol) and HATU (1.72 g, 4.53 mmol) in
DMF (10 mL), and the mixture was treated with diisopropylethy-
lamine (3.2 mL, 18 mmol) with stirring at room temperature for
2.5 h. The reaction vessel contents were then partitioned between
ethyl acetate and water, and the layers were separated. The aqueous
phase was extracted with additional ethyl acetate, and the combined
organics were washed with 2 M HCl, 10% Na2CO3, and saturated
NaCl aqueous solutions, followed by drying over Na2SO4. Con-
centration in vacuo and flash chromatography on silica gel, eluting
with 22.5/77.5 acetone/benzene, provided the title compound as a
colorless powder (1.57 g, 82%) upon trituration with a mixture of
hexanes and ether. [R]D +2.2° (c 0.5, acetone). 1H NMR (400 MHz,
acetone-d6) δ 8.03 (1H, s), 7.99 (2H, d, J ) 8.5 Hz), 7.83 (1H,
overlapped m), 7.81 (2H, d, J ) 8.5 Hz), 5.05 (2H, q, J ) 8.8 Hz),
3.36 (1H, m), 3.18 (3H, s), 3.12 (1H, m), 2.23 (1H, m), 2.15-1.95
(4H, m), 1.79 (1H, m), 1.37 (2H, m), 1.05 (1H, m), 0.81 (1H, m).
vac, and the residue was dissolved in methanol (300 mL) and treated
with K2CO3 (78 g) with stirring at room temperature. Water was
added until the salts dissolved, and the methanol was removed by
rotary evaporation under reduced pressure. The residue was
partitioned between water and ethyl acetate. After separation of
the layers, the aqueous phase was extracted with additional ethyl
acetate and the combined organics were washed with brine, dried
(Na2SO4/MgSO4), and concentrated to afford a tan syrup (34.36 g,
95% yield). 1H NMR (500 MHz, acetone-d6) δ 7.36 (4H, m), 7.30
(1H, m), 4.56-4.50 (2H, m), 3.70 (1H, m), 3.66-3.60 (2H, m),
3.56 (1H, dd, J ) 5.4, 9.3 Hz), 3.54-3.48 (1H, dd, J ) 4.0, 9.3
Hz), 2.44-2.26 (4H, m), 2.13-2.07 (2H, m), 1.91 (1H, m), 1.67
(1H, m).
{(1R,2R)-2-[(Benzyloxy)methyl]-4,4-dichlorocyclohexyl}methanol (15).
Hydrazine hydrate (150 mL, 20 equiv) was added to a slurry of
powdered 4 Å molecular sieves (activated grade, Sigma-Aldrich)
in anhydrous methanol (500 mL) with stirring at room temperature
for 15 min. A solution of ketone 14 (34.46 g, 140 mmol) in
methanol (300 mL) was then added with stirring at room temper-
ature until the reaction was judged complete by TLC analysis (2
h). The mixture was filtered (Celite), and the pad was washed with
methanol. The filtrate was then concentrated by rotary evaporation
under hi-vac with heating at 50 °C. Meanwhile, Et3N (69 mL, 500
mmol) was slowly added to a room temperature solution of
anhydrous CuCl2 (132.8 g, 990 mmol) in methanol (500 mL) with
stirring for 30 min prior to cooling to 0 °C and additional stirring
at this temperature for 2 h. The crude hydrazone from above was
taken up in methanol (400 mL) and slowly added to this mixture
over 25 min. Stirring was continued for an additional 45 min after
warming the reaction flask contents to room temperature. Saturated
NH4Cl and 2 M HCl were added to dissolve the copper salts,
followed by the addition of water and extraction with ethyl acetate
(3×). The combined organics were washed with saturated NH4Cl
solution (3×) and brine, followed by drying over Na2SO4/MgSO4.
Concentration in vacuo and flash chromatography of the residue
on silica gel (18 cm × 22 cm), eluting with 45/55 EtOAc/hexanes,
gave the desired compound as a yellow oil (32.16 g, 76% yield).
1H NMR (500 MHz, acetone-d6) δ 7.37 (4H, m), 7.31 (1H, m),
4.51 (2H, q, J ) 12 Hz), 3.65-3.51 (5H, m), 2.66 (1H, dt, J )
3.9, 14 Hz), 2.54 (1H, dq, J ) 3.3, 14 Hz), 2.29-2.19 (2H, m),
1.86 (1H, m), 1.75 (1H, m), 1.56 (1H, m).
(1R,2R)-2-[(Benzyloxy)methyl]-4,4-dichlorocyclohexanecarbal-
dehyde (17). Dess-Martin periodinane (52.63 g, 124.1 mmol, 1.2
equiv) was added to a 0 °C solution of the alcohol 15 (32.16 g,
106.5 mmol) in CH2Cl2 (250 mL) followed by stirring at room
temperature. TLC analysis indicated the reaction was complete after
1.5 h. Ethyl acetate (500 mL) and a solution composed of saturated
NaHCO3 (500 mL), water (200 mL), and NaS2O3 ·5H2O (170 g)
were then added with vigorous stirring at room temperature for 30
min. The layers were then separated, the aqueous phase was
extracted with additional ethyl acetate, and the combined organics
were washed with brine and dried (Na2SO4/MgSO4). Concentration
in vacuo and flash chromatography under nitrogen pressure on silica
gel (18 cm × 22 cm), eluting with 15/85 ethyl acetate/hexanes,
gave the desired aldehyde as a colorless syrup (24.87 g, 78% yield).
1H NMR (500 MHz, acetone-d6) δ 9.65 (1H, d, J ) 3.07 Hz),
7.39-7.32 (4H, m), 7.30 (1H, m), 4.50 (2H, m), 3.55 (1H, dd, J )
9.4, 4.9 Hz), 3.48 (1H, dd, J ) 9.5, 5.5 Hz), 2.66 (1H, dt, J ) 14,
3.1 Hz), 2.61 (1H, dq, J ) 14, 3.2 Hz), 2.52-2.38 (2H, m), 2.36
(1H, m), 2.22 (1H, dd, J ) 14, 12 Hz), 1.93-1.79 (2H, m).
Ethyl 3-{(1R,2R)-2-[(Benzyloxy)methyl]-4,4-dichlorocyclohexyl}-
3-hydroxy-2-[4-(methylthio)phenyl]propanoate (19). n-BuLi (2.5
M in hexanes, 36 mL, 90 mmol) was added dropwise to a 0 °C
solution of diisopropylamine (15 mL, 110 mmol) in THF (44 mL)
with stirring at this temperature for 15 min. This solution was then
cooled to -78 °C, and a solution of ethyl 4-(methylthio)pheny-
lacetate (20.05 g, 95.5 mmol) in THF (80 mL) was added dropwise.
The mixture was then warmed to 0 °C for 10 min prior to recooling
to -78 °C. The aldehyde 17 (24.87 g, 82.9 mmol) was then added
dropwise as a solution in THF (80 mL). Stirring was continued at
-78 °C for 2 h with slow warming to -40 °C over 2 h. The reaction
MS (+ESI) 531.0 [M + H]+ Anal. (C23H23F5N4O3S) C: calcd,
.
52.07; found, 51.97. H: calcd, 4.37; found, 4.34. N: calcd, 10.56;
found, 10.10.
(4R)-4-Benzyl-3-({(7R,8R)-7-[(benzyloxy)methyl]-1,4-dioxaspiro-
[4.5]dec-8-yl}carbonyl)-1,3-oxazolidin-2-one (12). TMSOTf (8.7 mL,
48 mmol, 10 mol %) was added to a 0 °C solution of ketone 9a
(202.1 g, 479.0 mmol) and 1,2-bis[trimethylsilyl(oxy)]ethane (147
mL, 600 mmol, 1.25 equiv) in CH2Cl2 (1 L). The mixture was
stirred at room temperature for 12 h prior to the addition of Et3N
(11 mL, 80 mmol, 0.17 equiv) to quench the reaction. The mixture
was then washed with saturated aqueous NaHCO3 and dried over
Na2SO4. Concentration by rotary evaporation followed by hi-vac
application at 55 °C afforded the desired acetal as a thick, tan
1
colored syrup (235.2 g, >99%). H NMR (500 MHz, acetone-d6)
δ 7.36-7.22 (10H, m), 4.48 (1H, m), 4.42 (2H, s), 4.11 (1H, dd,
J ) 3.1, 8.9 Hz), 3.97 (4H, m), 3.94-3.90 (1H, t, J ) 9.0 Hz),
3.67 (1H, m), 3.44-3.38 (2H, m), 3.09 (1H, dd, J ) 3.3, 14 Hz),
2.92 (1H, dd, J ) 8.3, 14 Hz), 2.52 (1H, m), 1.97 (1H, m),
1.86-1.76 (3H, m), 1.57-1.50 (1H, m), 1.44 (1H, t, J ) 13 Hz).
{(7R,8R)-7-[(Benzyloxy)methyl]-1,4-dioxaspiro[4.5]dec-8-
yl}methanol (13). n-BuLi (2.5 M in hexanes, 248 mL, 620 mmol,
1.3 equiv) was added dropwise to a 0 °C solution of ethanethiol
(53 mL, 720 mmol, 1.5 equiv) in THF (900 mL). The resulting
pale-yellow solution was stirred at 0 °C for 15 min prior to the
slow introduction of the acyloxazolidinone 12 (222 g, 477 mmol)
as a solution in THF (700 mL). The mixture was stirred at 0 °C
until judged complete by TLC (20 min). A 1.0 M THF solution of
LiAlH4 (525 mL, 1.1 equiv) was then added dropwise with
continued stirring at 0 °C. TLC analysis indicated that reduction
of the thioester intermediate was complete within 1 h. Water (50
mL) was carefully added in a dropwise manner until gas evolution
ceased and a gelatinous mass formed. Then 6 M HCl (380 mL, 1
mol of HCl per mol of hydride in LiAlH4) was then carefully added
dropwise to pH 3-4. Additional water was added, and the layers
were separated. The aqueous phase was extracted with ethyl acetate,
and the combined organics were washed with brine, dried over
Na2SO4/MgSO4, and concentrated. The residue was triturated with
ether, and the resulting solid (recovered Evan’s auxiliary) was
collected by filtration (55.6 g, 66% recovery). The filtrate was
concentrated, and the residue was chromatographed on silica gel
(3 kg in a sintered glass funnel), eluting with 25/75 acetone/hexanes
to afford the desired alcohol as a faint yellow syrup (119 g, 86%
yield). 1H NMR (500 MHz, acetone-d6) δ 7.36 (4H, m), 7.29 (1H,
m), 4.50 (2H, q, J ) 9.6 Hz), 3.90 (4H, s), 3.61 (1H, m), 3.52-3.42
(3H, m), 3.43 (1H, t, J ) 5.5 Hz), 1.88-1.72 (4H, m), 1.55-1.41
(4H, m).
(3R,4R)-3-[(Benzyloxy)methyl]-4-(hydroxymethyl)cyclohex-
anone (14). The acetal 13 (42.96 g, 147.1 mmol) was heated at 85
°C in a mixture of acetic acid (280 mL) and water (100 mL). TLC
analysis revealed the reaction was complete after 2 h. The majority
of the acetic acid was removed by rotary evaporation under hi-

Cathepsin K Inhibitor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6419
vessel contents were finally warmed to 0 °C with stirring at this
temperature for 10 min prior to quenching of the reaction with
saturated NH4Cl solution. The mixture was partioned between ethyl
acetate and water, and the layers were separated. The aqueous phase
was extracted with additional ethyl acetate, and the combined
organics were washed with brine before drying over Na2SO4/
MgSO4. Concentration in vacuo and flash chromatography on silica
gel (18 cm × 24 cm), eluting with an increasing proportion of ethyl
acetate in hexanes (1/4, 35/65, then 2/3), afforded the desired
product as a single diastereomer at the hydroxyl and ester bearing
(7H, m), 7.20-7.15 (2H, m), 5.12-5.03 (2H, m), 4.36 (1H, d, J )
12 Hz), 4.27 (1H, d, J ) 12 Hz), 3.35 (1H, m), 3.28 (1H, m), 2.93
(1H, m), 2.82 (1H, m), 2.70 (1H, m), 2.43-2.28 (2H, m), 2.57
(1H, m), 2.52 (3H, s), 2.43-2.28 (2H, m). m-CPBA (14.3 g, 83.0
mmol, 2.1 equiv) was added portionwise to a solution of product
from above (28.01 g, 40.5 mmol) in CH2Cl2 (200 mL) at 0 °C
followed by stirring at room temperature until the reaction was
judged to be complete by TLC analysis (1.5 h). Calcium hydroxide
(54 g, 730 mmol) was added with stirring at room temperature for
20 min prior to filtration through Celite. Concentration in vacuo
gave the title compound as a faint-yellow solid (26.95 g, 92% yield).
1H NMR (400 MHz, acetone-d6) δ 7.98 (2H, d, J ) 8.2 Hz), 7.70
(2H, d, J ) 8.2 Hz), 7.38-7.26 (3H, m), 7.24-7.17 (2H, m),
5.21-5.07 (2H, m), 4.36 (1H, d, J ) 12 Hz), 4.28 (1H, d, J ) 12
Hz), 3.37 (1H, dd, J ) 9.4, 3.0 Hz), 3.31 (1H, dd, J ) 9.4, 4.8
Hz), 3.14 (3H, s), 2.98 (1H, td, J ) 12, 4.1 Hz), 2.82 (1H, narrow
m), 2.70 (1H, dt, J ) 14, 3.0 Hz), 2.63-2.52 (2H, m), 2.44 (1H,
m), 2.36 (1H, m), 2.02 (1H, m).
{(1R,2R)-5,5-Dichloro-2-[4-[4-(methylsulfonyl)phenyl]-1-(2,2,2-
trifluoroethyl)-1H-pyrazol-3-yl]cyclohexyl}methanol (27). To a
mixture of triflate 25 (28.95 g, 39.13 mmol), 3 Å activated
molecular sieve powder (54 g), and 10% Pd/C (27 g) under N2
was added ethyl acetate (400 mL) with stirring at room temperature
for 10 min. Et3N (7.8 mL, 1.4 equiv) was then added, and the
mixture was placed under an atmosphere of hydrogen (balloon)
with stirring at room temperature overnight. The solids were
removed by filtration through Celite, and the filtrate was concen-
trated. Flash chromatography of the residue on silica gel, eluting
with 2/3 ethyl acetate/hexanes and then with pure ethyl acetate,
afforded 3-{(1R,2R)-2-[(benzyloxy)methyl]-4,4-dichlorocyclohexyl}-
4-[4-(methylsulfonyl)phenyl-2-(2,2,2-trifluoroethyl)-1H-pyrazole as
a colorless foam (14.15 g, 66% yield). 1H NMR (400 MHz, acetone-
d6) δ 8.09 (1H, s), 7.89 (2H, d, J ) 8.1 Hz), 7.74 (2H, d, J ) 8.1
Hz), 7.34-7.23 (3H, m), 7.18-7.12 (2H, m), 5.10 (2H, q, J ) 8.8
Hz), 4.34 (1H, d, J ) 12 Hz), 4.22 (1H, d, J ) 12 Hz), 3.37 (1H,
dd, J ) 9.4, 2.7 Hz), 3.31 (1H, dd, J ) 9.3, 4.8 Hz), 3.19 (1H, td,
J ) 12, 3.8 Hz), 3.12 (3H, s), 2.74 (1H, m), 2.70-2.53 (2H, m),
2.52-2.38 (2H, m), 2.16 (1H, m), 2.02 (1H, m). Ethyl acetate (250
mL) and acetic acid (50 mL) were added to a flask containing
Pearlman’s catalyst (3.6 g) and the benzyl ether from above (14.15
g, 24.6 mmol) under an atmosphere of nitrogen. The atmosphere
was then switched to hydrogen with stirring at room temperature
overnight. Filtration (Celite) and concentration in vacuo provided
the title alcohol as a colorless syrup (10.72 g, 90% yield). 1H NMR
(400 MHz, acetone-d6) δ 8.06 (1H, s), 7.96 (2H, d, J ) 8.3 Hz),
7.78 (2H, d, J ) 8.3 Hz), 5.08 (2H, q, J ) 8.8 Hz), 3.67 (1H, dd,
J ) 5.3, 4.3 Hz), 3.47 (1H, m), 3.34 (1H, m), 3.15 (3H, s), 3.09
(1H, td, J ) 12, 3.9 Hz), 2.79 (1H, narrow m), 2.76 (1H, dt, J )
14, 3.0 Hz), 2.60-2.51 (2H, m), 2.38 (1H, m), 2.11 (1H, m), 1.92
(1H, m).
1
stereocenters (33.85 g, 80% yield). H NMR (400 MHz, acetone-
d6) δ 7.45-7.29 (7H, m), 7.17-7.11 (2H, m), 4.64 (1H, m),
4.58-4.52 (2H, m), 4.21 (1H, d, J ) 6.06 Hz), 4.13 (1H, m), 4.06
(1H, m), 3.78-3.71 (2H, m), 3.48 (1H, m), 2.57-2.48 (2H, m),
2.47 (3H, s), 2.26 (1H, m), 1.97-1.88 (2H, m), 1.77 (1H, m), 1.24
(1H, m), 1.23-1.15 (3H, t, J ) 7.0 Hz).
Ethyl 3-{(1R,2R)-2-[(Benzyloxy)methyl]-4,4-dichlorocyclohexyl}-
3-[4-(methylthio)phenyl]-3-oxopropanoate (21). Oxalyl chloride (20
mL, 3.2 equiv) was added dropwise over 20 min to a -78 °C
solution of DMSO (20 mL) in CH2Cl2 (250 mL). Stirring was
continued at this temperature for an additional 40 min prior to the
rapid introduction, via cannula over 5 min, of a -78 °C solution
of 19 (36.76 g, 72.1 mmol) and Et3N (100 mL, 10 equiv) in CH2Cl2
(250 mL). Stirring at -78 °C was continued for 45 min before
allowing the reaction vessel contents to warm to 0 °C over 1 h,
followed by the addition of a 2 M solution of HCl (200 mL) to
quench the reaction. The mixture was transferred to a separatory
funnel, and the layers were separated. The aqueous phase was
extracted with CH2Cl2, and the combined organics were washed
with water and dried over Na2SO4. Concentration in vacuo and flash
chromatography on silica gel (11 cm × 23 cm), eluting with 1/9
and then 1/3 ethyl acetate/hexanes, gave the keto-ester product as
a thick, yellow syrup (35.53 g, 97% yield).
3-{(1R,2R)-2-[(Benzyloxy)methyl]-4,4-dichlorocyclohexyl}-4-[4-
(methylthio)phenyl]-1-(2,2,2-trifluoroethyl)-1H-pyrazol-5-ol (23). A
mixture of keto-ester 21 (35.53 g, 70 mmol) and trifluoroethylhy-
drazine (70 wt % in water, 110 g, 10 equiv) in toluene (700 mL)
was heated at 125 °C in a three-neck flask fitted with a pressure
equalizing addition funnel containing 3 Å molecular sieves (215
g, round pellets, activated grade from Acros) and a reflux condenser
placed on top of the addition funnel. Heating at reflux, at a rate
sufficient to force the vapor phase up the entire length of the foil
wrapped funnel sidearm, was continued until no more water was
evident in the reaction flask (35 min). The reaction vessel contents
were then cooled to 60 °C, and anhydrous MgSO4 powder (36 g)
was added directly to the reaction flask. Heating at reflux (oil bath
temp ) 125 °C) was then continued for 3.5 days before cooling to
room temperature and filtering to remove the MgSO4. Concentration
of the filtrate and flash chromatography on silica gel (18 cm × 23
cm), eluting with 15/85 acetone/benzene, yielded the title compound
as a yellow foam (25.16 g, 64% yield). 1H NMR (500 MHz,
acetone-d6) δ 9.80 (1H, br s), 7.37 (1H, s), 7.35-7.24 (5H, m),
7.19 (4H, m), 4.75 (2H, m), 4.37 (1H, d, J ) 12 Hz), 4.28 (1H, d,
J ) 12 Hz), 3.41-3.32 (2H, m), 2.80 (1H, overlapped m), 2.70
(1H, m), 2.59-2.49 (2H, m), 2.50 (3H, s), 2.34 (1H, m), 2.29 (1H,
m), 1.91 (1H, m).
(1R,2R)-5,5-Dichloro-2-[4-[4-(methylsulfonyl)phenyl]-1-(2,2,2-tri-
fluoroethyl)-1H-pyrazol-3-yl]cyclohexanecarboxylic Acid (29).
Jones’ reagent (2.7 M, 29 mL, 2.7 equiv) was added dropwise at 0
°C to a solution of alcohol 27 (13.75 g, 28.30 mmol) in acetone
(250 mL) followed by stirring at room temperature until the reaction
was judged complete by TLC (1 h). The mixture was then
partitioned between ethyl acetate and water, and the layers were
separated. The aqueous phase was extracted with additional ethyl
acetate, and the combined organics were washed with water until
no color was visible in the organic phase (two to three washings).
This was followed by washing with brine and drying of the organics
over Na2SO4/MgSO4. Concentration in vacuo gave the desired acid
as a faint-yellow foam (13.45 g, 95% yield). 1H NMR (400 MHz,
acetone-d6) δ 11.50 (1H, br), 8.03 (1H, s), 7.98 (2H, d, J ) 8.3
Hz), 7.81 (2H, d, J ) 8.3 Hz), 5.04 (2H, q, J ) 8.8 Hz), 3.50 (1H,
m), 3.42 (1H, m), 3.16 (3H, s), 2.91 (1H, dt, J ) 14, 3.0 Hz),
2.60-2.44 (3H, m), 2.12-1.92 (2H, overlapped m).
3-{(1R,2R)-2-[(Benzyloxy)methyl]-4,4-dichlorocyclohexyl}-4-[4-
(methylsulfonyl)phenyl]-1-(2,2,2-trifluoroethyl)-1H-pyrazol-5-yl-tri-
fluoromethanesulfonate (25). Trifluoromethanesulfonic anhydride
(9.1 mL, 54 mmol, 1.2 equiv) was added dropwise to a solution of
hydroxypyrazole 23 (25.16 g, 45 mmol) and pyridine (5.5 mL, 68
mmol, 1.5 equiv) in CH2Cl2 (200 mL) at 0 °C, followed by stirring
at room temperature for 30 min. The mixture was then partitioned
between ether and water, and the layers were separated. The aqueous
phase was extracted with additional ether, and the combined
organics were dried over Na2SO4/MgSO4. Concentration in vacuo
and flash chromatography of the residue on silica gel, eluting with
1/4 ethyl acetate/hexanes, gave 3-{(1R,2R)-2-[(benzyloxy)methyl]-
4,4-dichlorocyclohexyl}-4-[4-(methylthio)phenyl]-1-(2,2,-trifluroro-
ethyl)-1H-pyrazol-5-yl trifluoromethanesulfonate as a colorless foam
(28.01 g, 90% yield). 1H NMR (500 MHz, acetone-d6) δ 7.37-7.22
(1R,2R)-5,5-Dichloro-N-(1-cyanocyclopropyl)-2-[4-[4-(methylsul-
fonyl)phenyl]-1-(2,2,2-trifluoroethyl)-1H-pyrazol-3-yl]cyclohexan-
ecarboxamide (31). A mixture of carboxylic acid 29 (13.45 g, 26.95
mmol), HATU (11.27 g, 29.65 mmol, 1.1 equiv), and cyclopro-

6420 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Robichaud et al.
Disubstituted Aromatic Core. Bioorg. Med. Chem. Lett. 2007, 17,
3146–3151.
pylaminoacetonitrile (7.03 g, 59.3 mmol, 2.2 equiv) in DMF (150
mL) was treated with Hu¨nig’s base (18.8 mL, 4 equiv) with stirring
at room temperature for 2 days. The mixture was then added to
10% HCl and extracted with ether (2×). The combined ether
extracts were washed with saturated brine and sodium bicarbonate
solutions, dried over Na2SO4, and concentrated. Flash chromatog-
raphy on silica gel, eluting with 1/1 ethyl acetate/hexanes, afforded
the final compound as a colorless powder (14.01 g, 96% yield).
(7) (a) Falgueyret, J.-P.; Oballa, R.; Okamoto, O.; Wesolowski, G.; Aubin,
Y.; Rydzewski, R. M.; Prasit, P.; Riendeau, D.; Rodan, S. B.; Percival,
M. D. Novel, Nonpeptidic Cyanamides as Potent and Reversible
Inhibitors of Human Cathepsins K and L. J. Med. Chem. 2001, 44,
94–104. (b) The projected monkey and human bone resorption assay
values were calculated by multiplying the bone resorption shift (BRS)
by the humanized rabbit Cat K IC50 value. The human, humanized
rabbit, and monkey Cat K IC50 values would be expected to be very
similar (see refs 10a and 19b).
(8) Wright, J. D.; Boudinot, F. D.; Ujhelyi, M. R. Measurement and
Analysis of Unbound Drug Concentrations. Clin. Pharmacokinet. 1996,
30, 445–462.
(9) Schechter, I.; Berger, A. On the Size of the Active Site in Proteases.
Biochem. Biophys. Res. Commun. 1967, 27, 157–162.
1
[R]D +5.6° (c 0.5, acetone). H NMR (500 MHz, acetone-d6) δ
8.03 (1H, s), 8.01 (1H, overlapped br s), 7.98 (2H, d, J ) 8.4 Hz),
7.80 (2H, d, J ) 8.4 Hz), 5.06 (2H, q, J ) 8.8 Hz), 3.43 (1H, m),
3.29 (1H, dt, J ) 3.3, 12 Hz), 3.19 (3H, s), 2.71 (1H, m), 2.58-2.68
(3H, m), 2.00-1.94 (2H, m), 1.43-1.31 (2H, m), 1.10 (1H, m),
0.86 (1H, m). MS (+ESI) 563.1 [M + H]+ Anal. (C23H23Cl2-
.
(10) (a) Humanized rabbit cathepsin K (hrab Cat K) was obtained by
mutation of two active site amino acid residues of the rabbit enzyme
F3N4O3S). C: calcd, 49.03; found, 48.92. H: calcd, 4.11; found,
4.01. N: calcd, 9.94; found 9.68.
to the corresponding human residues (Tyr61 f Asp61 and Val157
f
Leu157). See ref 10b for expression and purification procedures.
Cyclohexanecarboxamide based inhibitors were equipotent against
humanized-rabbit and human Cat K. (b) Robichaud, J.; Oballa, R.;
Prasit, P.; Falgueyret, J.-P.; Percival, M. D.; Wesolowski, G.; Rodan,
S. B.; Kimmel, D.; Johnson, C.; Bryant, C.; Venkatraman, S.; Setti,
E.; Mendonca, R.; Palmer, J. T. A Novel Class of Nonpeptidic Biaryl
Inhibitors of Human Cathepsin K. J. Med. Chem. 2003, 46, 3709–
3737.
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