Potent and selective ketoamide-based inhibitors of cysteine protease, cathepsin K

Article abstract of DOI:10.1021/jm0400799

Cathepsin K, a lysosomal cysteine protease of the papain superfamily, is abundantly and selectively expressed in osteoclasts, suggesting that this enzyme is crucial for bone resorption. Prevention of osteoclast-mediated bone resorption via inhibition of c

Full text of DOI:10.1021/jm0400799

J . Med. Chem. 2004, 47, 5049-5056
5049
P oten t a n d Selective Ketoa m id e-Ba sed In h ibitor s of Cystein e P r otea se,
Ca th ep sin K
Francis X. Tavares,*,† David N. Deaton,† Aaron B. Miller,‡ Larry R. Miller,§ Lois L. Wright,| and
Hui-Qiang Zhou†
Department of Medicinal Chemistry, Discovery Research Biology, Department of Computational, Analytical and Structural
Sciences, and Department of Molecular Pharmacology, GlaxoSmithKline, Research Triangle Park, North Carolina 27709
Received April 5, 2004
Cathepsin K, a lysosomal cysteine protease of the papain superfamily, is abundantly and
selectively expressed in osteoclasts, suggesting that this enzyme is crucial for bone resorption.
Prevention of osteoclast-mediated bone resorption via inhibition of cathepsin K could be an
effective approach to prevent osteoporosis. Potent and selective reversible ketoamide-based
inhibitors have been identified in the present study. Using a known crystal structure of a
ketoamide-based inhibitor, information from residues that form the P2/P3 pocket was used in
the design of inhibitors that could allow for gains in selectivity and potency. Further,
incorporation of P′ selective heterocycles, along with the P2/P3 modifications, is also described.
These modifications have resulted in potent and selective cathepsin K inhibitors that allow
for improvements in their physiochemical properties and represent a viable lead series for the
discovery of new therapies for the prevention and treatment of osteoporosis
In tr od u ction
trotic phenotype.5 Recent work has shown that cathe-
psin K, and not cathepsin L, is the major protease
responsible for human osteoclastic bone resorption.6
Further, recent studies have shown that complex for-
mation with glycosaminoglycans specifically enhances
collagenase activity of cathepsin K in osteoclast, while
potently inhibiting collagenase activity of other cysteine
proteases such as cathepsins L and S.6c The irreversible
cysteine protease inhibitor E-64 has been shown to
attenuate osteoclastic bone resorption.7 The effects of a
small molecule inhibitor of cathepsin K have 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.
Cysteine proteases make up the vast majority of
lysosomal proteases. There are currently eleven mem-
bers of the human cathepsin cysteine protease family.
Compared to cathepsin K, the physiological role and
pathological implications of many cathepsins are less
well understood. Since cysteine proteases such as cathe-
psins B, S, and L have been implicated in immunological
responses, selectivity versus these proteases is desir-
able.8,9 In lieu of the fact that cathepsins K, L, S, and V
have a high degree of homology, design of inhibitors that
are selective for the inhibition of cathepsin K is chal-
lenging. The difficulty in designing selective cathepsin
inhibitors results from the similarity in their substrate
recognition as well as their common proteolytic mech-
anism.11b Various irreversible inhibitors such as ep-
oxides, peptidyl vinyl sulfones, and acyloxymethyl ke-
tones have been reported in the literature.10 Since the
treatment of osteoporosis would require chronic drug
administration, the development of a selective reversible
inhibitor is desired. This could potentially avoid anti-
genicity due to covalent modification of proteins via
Bone is a mechanically optimized connective tissue
providing structural integrity for movement and protec-
tion. It is composed mainly of inorganic matter with the
remainder consisting of organic or extracellular matrix
and water. Bone remodeling, consisting of bone forma-
tion and resorption, is a dynamic process that requires
proliferation and differentiation of osteoprogenitor cells.
Osteoclasts, members of the monocyte/macrophage fam-
ily are key players in the dissolution of bone mineral
as well as degradation of its protein matrix. Osteoclasts
contain membrane-bound receptors such as Rvâ3 inte-
grins that bind to bone matrix proteins containing an
RGD motif to from an extracellular, resorptive compart-
ment bordered by the ruffled membrane. This attach-
ment allows the establishment of an acidic environment,
a process mediated by a vacuolar H+-ATPase in the
ruffled membrane, to facilitate demineralization of the
bone. This is followed by the secretion of proteolytic
enzymes to degrade the organic matrix. Cysteine pro-
teases have been implicated in this osteoclast-mediated
resorption of the bone matrix.1 The expression of cathe-
psin 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
characterized by abnormal bone resorption, has been
demonstrated.4 Moreover, it has also been shown that
cathepsin K-deficient mice exhibit a distinct osteope-
* To whom correspondence should be addressed. Phone: (919) 483-
7456; Fax: (919)-483-6053; e-mail: francis.x.tavares@gsk.com.
† Department of Medicinal Chemistry.
‡ Department of Computational, Analytical and Structural Sciences.
§ Department of Molecular Pharmacology.
| Discovery Research Biology.
10.1021/jm0400799 CCC: $27.50 © 2004 American Chemical Society
Published on Web 09/08/2004

5050 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21
Tavares et al.
Sch em e 2a
F igu r e 1.
Sch em e 1a
a
(a) 20% COCl2 in toluene, THF, rt; (b) L-norleucine methyl
ester hydrochloride, Hunig’s base, THF, rt; (c) 1M 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.
a
(a) LDA, THF, EtI.; (b) LAH, THF, reflux.
irreversible inhibition. Numerous warheads have been
utilized in reversible cysteine protease inhibitors includ-
ing peptidic aldehydes,12 nitriles,13 cyclopropenones,14
diamino ketones,15 and R-ketoamides.16 There has also
been a recent report on the use of noncovalent amides
as cathepsin K inhibitors.17,25b This group was particu-
larly interested in exploring R-ketoamides as reversible
inhibitors of cathepsin K. The ketoamide class of inhibi-
tors has been widely employed in inhibiting cysteine and
serine proteases.16,18 Herein is described its use in the
inhibition of the cysteine protease, cathepsin K.19
A
report on the development of potent, selective and orally
bioavailable inhibitors was recently described from this
laboratory.19a That paper was primarily focused on the
heterocyclic modifications at the prime region of ketoa-
mide-based inhibitors keeping the P2/P3 substituent
constant (Figure 1). In the present report the conse-
quences of making changes in the P2/P3 region, along
with the incorporation of prime substituents that were
found to be important for selectivity, is disclosed.
Further, incorporation of diverse P2/P3 groups that are
potent and selective ketoamide inhibitors would allow
for opportunities in improving upon physiochemical
properties of this class of cathepsin K inhibitors.
Ch em ist r y. The requisite four-, five-, and six-
membered alcohols were synthesized as shown in Scheme
1. Deprotonation with LDA followed by electrophilic
quench with ethyl iodide and subsequent reduction of
the ester moiety afforded the desired (1-ethylcycloalkyl)-
methanol derivatives 3. The general syntheses of ke-
toamide inhibitors are shown in Scheme 2. The alcohols
4 were treated with phosgene to afford chloroformates,
and subsequent addition of an amino ester provided
carbamates 5.20 Hydrolysis of esters 5 to acids followed
by coupling with the phosphorane afforded nitrile ylides
6. Oxidation of the ylide 6 following the Wassermann
procedure21 and trapping the acyl nitrile with amines
afforded the ketoamides 7-39. The 1-alkyl-1H-pyrazole-
5-ylamines (13a , 29a -39a ) were synthesized following
literature procedures.22-24
F igu r e 2. a. Closeup of the S2 pocket of cathepsins K, S, V,
and L: S2 pocket of cathepsins K (cyan), S (magenta), V
(purple), and L (orange) with the residues for cathepsin K
labeled. The cathepsin K backbone and ligand (diisopropyl
carbamate moiety, Figure 2b) are shown19a with side chains
from other cathepsins colored as listed above. All of the
structures used are deposited in the PDB database and have
the following codes: cathepsin K (1Q6K), cathepsin S (1NPZ),
cathepsin V (1FH0), and cathepsin L (1ICF). The figure was
made using PYMOL (www.pymol.org).
substituted pyrazoles. Modifications in the P2/P3 region,
along with P′ changes, are the focus of this paper. The
S2 pocket of cathepsin K is composed of 67Tyr, 68Met,
134Ala, 160Leu, 163Ala, and 209Leu. The homologous
residues for cathepsins L, V, and S are very similar and
listed in Table 4 and shown in Figure 2a. All the S2
pockets of these enzymes have hydrophobic residues and
would favor small hydrophobic residues or groups. Since
the S2 pockets of cathepsins L, S, and V are formed by
residues that differ slightly in steric bulk, opportunities
for additional gain in potency and/or selectivity could
be envisioned by making changes in the P2/P3 groups
of the ketoamide-based inhibitors. Recent published
reports that have disclosed selective cathepsin K inhibi-
tors have taken advantage of steric congestion in the
P2 pocket.25 Cathepsin K inhibition data for the P2/P3
analogues, along with P′ modifications, are shown in
Resu lts a n d Discu ssion
Previous efforts in this laboratory identified P′ het-
erocyclic groups that provided potent and very selective
cathepsin K inhibitors, while keeping the P2/P3 sub-
stituent constant. In our earlier report, achievement of
selectivity over cathepsins L, S, and V had proven
difficult which was overcome by incorporation of N-

Ketoamide-Based Inhibitors of Cysteine Protease
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21 5051
Ta ble 1. Inhibitory Potencies vs Human Cathepsin K
Table 1. The P′ five-membered heterocycles having
hydrogen bonding ability with the indole NH of 184Trp
were preferred in the present work. Incorporation of the
P2/P3 gem-dimethyl group with the previously reported
nonselective P′ groups afforded analogues 7, 8, and 10.
Since the N-methyl group was found to provide potent
and selective cathepsin K inhibitors, this moiety was
incorporated, along with the gem-dimethyl change to
afford compound 9 (IC50 ) 1200 nM). A dramatic loss
in potency was observed. The unsubstituted pyrazole
analogue 8 was the most potent inhibitor in the P2/P3
gem-dimethyl analogues. In agreement with our previ-
ous work, changes in the P2/P3 region of the inhibitor
did not affect selectivity but they had a dramatic effect
on the potency against cathepsin K. Constrained ana-
logues of the gem-dimethyl moiety such as the cyclobu-
tyl, cyclopentyl, and cyclohexyl afforded analogues
11-28. In every case, the N-unsubstituted pyrazole
analogues 12, 17, 21, and 25 were found to be the most
potent inhibitors in the constrained series. Having a
heteroatom, as in the oxetane analogues 16-19, did not
seem to affect the general trend in potency. Incorpora-
tion of a phenyl substituent on the pyrazole that might
π-π stack with the indole side chain of 184Trp gave
varied results. There was a 3-fold and 12-fold decrease
in potency in the cyclobutyl and cyclopentyl analogues,
respectively (12 vs 14 and 21 vs 23). Constraining the
phenyl ring afforded an increase by 11-fold in the
cyclohexyl series (25 vs 28). Incorporation of the known
selective N-methylpyrazole group on the P′ region
afforded analogues 13, 22, and 26. While analogue 13
(IC50 ) 62 nM) and 22 (IC50 ) 135 nM) were fairly
potent, there was a dramatic loss in potency in analogue
26 (IC50 ) 12 µM). Although some decrease in potency
on N-methyl substitution was expected (33-fold for
analogue 13, 75-fold for analogue 22 vs analogues 12
and 21) due to a loss in hydrogen bonding to the enzyme,
the complete loss in potency of analogue 26 suggests
that accommodation in the S′ subsite results in shifts
in binding at other subsites that is less favorable to
cathepsin K.
Having identified the pyrazole as a potency enhancing
P′ moiety, a selectivity study was undertaken to deter-
mine how modifications in the P2/P3 region would affect
the potency/selectivity of these ketoamide inhibitors. As
can be seen from Table 2, these unsubstituted pyrazole
analogues did not show significant selectivity over
cathepsins S, V, and L. Although exploitation of steric
differences for selectivity/potency had been fruitful for
some known cathepsin K inhibitors,25 this was not found
to be the case for these ketoamide inhibitors. These
inhibitors contain a carbamate linker that forces the P2
substituent one atom further away compared to the
corresponding dipeptides-based inhibitors. This shift
seems to have a dramatic effect on the placement of the
P2 group and affects the selectivity ratios.
Although, addition of a N-methyl group resulted in
some loss in potency, it was gratifying to find that
changes in the P2/P3 pocket, along with N-methyl
pyrazole in the P′ region of ketoamide inhibitors, af-
forded a fairly potent and selective inhibitor 13. Encour-
aged by the enhanced selectivity of analogue 13, a series
of N-substituted ketoamides were synthesized. Increase
in the steric bulk at the P′ position afforded analogues

5052 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21
Tavares et al.
Ta ble 2. Inhibitory Potencies (IC50) vs Human Cathepsins K, S, V, and L
* Represents selectivity ratios over the other cathepsins (e.g. L/K ) IC50 cat L/IC50 cat K).
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.
Gen er a l P r oced u r e for th e Syn th esis of Eth yl 1-Eth -
ylcycloa lk ylca r boxyla tes. To a solution of diisopropylamine
(6.44 mL, 46 mmol) in THF (90 mL) at -20 °C was added a
2.5 M solution (15.81 mL, 50.6 mmol) of n-BuLi in hexanes,
and the contents were stirred for 15 min. The reaction mixture
was then cooled to -78 °C for 15 min followed by the addition
of ethyl cycloalkylcarboxylates (46 mmol). The reaction was
then warmed to -30 °C for 15 min and then cooled back to
-78 °C. Ethyl iodide (4.42 mL, 55.2 mmol) was then added,
and the contents were allowed to warm to room temperature
over 1 h. After stirring for another 3 h, the reaction mixture
was quenched with sat. NH4Cl (100 mL) followed by the
addition of EtOAc (200 mL). After separation of the layers,
the organic layer was dried with MgSO4 and then concentrated
under vacuum to afford the crude product which was purified
with hexane:ethyl acetate (95:5) to afford the desired product
in 60-75% yield as a colorless oil.
29-39. The N-alkyl analogues 13, 30-39 maintain
relatively constant cathepsin K inhibitory activity with
increases in selectivity compared to the N-unsubstituted
pyrazole 12. Increasing the steric bulk from methyl 13
to ethyl 30 to isopropyl 31 resulted in a gradual loss in
selectivity against cathepsin S (Table 3). Constrained
analogues of the isopropyl 31, particularly analogues
36-38, did not result in an increase in selectivity
against cathepsin S. It is clear that even though the S′
pockets of cathepsins K, L, V, and S are very similar,
the P′ groups have a dramatic effect on the selectivity
of the compounds. This suggests that either the orienta-
tion of the P′ groups of the compounds influences the
placement of the P2 groups conferring selectivity or that
the P′ groups somehow confer selectivity on their own
in the S′ pockets of the enzymes in a yet undefined
manner. Interestingly, the unsaturated version 39 of
analogue 38 resulted in a gain in selectivity over the
cathepsins tested. Movement of the substituent to the
adjacent nitrogen (analogue 29, IC50 ) 3.0) resulted in
a loss in selectivity but enhanced potency compared to
analogue 13.
Con clu sion
Based on the successful implementation of N-substi-
tuted pyrazoles with a fixed P2/P3 moiety for achieving
potency and selectivity of ketoamide inhibitors of cathe-
psin K in our previous work, efforts in the present work
deal with combining P2/P3 changes with N-substituted
pyrazoles. Although, differences in the P2 pocket were
identified from homology modeling, changes in the steric
bulk in this region did not afford selective cathepsin K
inhibitors. However, addition of P′ selective moieties
along with P2/P3 changes did afford selective cathepsin
K inhibitors. Such a change in the P2/P3 region broad-
ens the scope for designing cathepsin K inhibitors
allowing for improvements in their physiochemical
properties.
Eth yl 1-eth ylcyclobu ta n eca r boxyla te: 1H NMR (300
MHz, CDCl3) ppm: 4.13 (q, J ) 7.2 Hz, 2H), 2.41 (m, 2H),
1.88-1.71 (m, 6H), 1.23 (t, J ) 7.1 Hz, 3H), 0.78 (t, J ) 7.3
Hz, 3H).
E t h yl 1-et h ylcyclop en t a n eca r boxyla t e: 1H NMR (300
MHz, CDCl3) ppm: 4.12 (q, J ) 7.1 Hz, 2H), 2.40 (m, 2H),
1.90-1.69 (m, 8H), 1.21 (t, J ) 7.1 Hz, 3H), 0.80 (t, J ) 7.2
Hz, 3H).
Eth yl 1-eth ylcycloh exa n eca r boxyla te: 1H NMR (300
MHz, CDCl3) ppm: 4.12 (q, J ) 7.3 Hz, 2H), 2.42 (m, 2H),
1.91-1.57 (m, 10H), 1.25 (t, J ) 7.2 Hz, 3H), 0.78 (t, J ) 7.2
Hz, 3H).
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

Ketoamide-Based Inhibitors of Cysteine Protease
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21 5053
Ta ble 3. Inhibitory Potencies (IC50) vs Human Cathepsins K, L, S, V, H, and B
* Represents selectivity ratios over the other cathepsins (e.g. L/K ) IC50 cat L/IC50 cat K).
Ta ble 4. S2 Pocket Residues of Cathepsins K, L, S, and V
at reflux for 6 h. After the reaction was quenched with 1 N
HCl (200 mL), ether (200 mL) was added and the organic layer
separated. The aqueous layer was extracted with ether (3 ×
100 mL), and the combined organic layers were dried with
MgSO4 to afford after concentration under vacuum the crude
product. Column chromatography with hexane:ethyl acetate
(90:10) afforded the alcohols in 80-85% yield as a colorless
oil.
(1-Eth ylcyclobu tyl)m eth a n ol: 1H NMR (300 MHz, CDCl3)
ppm: 3.36 (s, 2H), 2.41 (br s, 1H), 1.78-1.21 (m, 8H), 0.80 (t,
J ) 7.3 Hz, 3H).
(1-Eth ylcyclopen tyl)m eth an ol: 1H NMR (300 MHz, CDCl3)
ppm: 3.68 (br s, 1H), 3.34 (s, 2H), 1.82-1.28 (m, 10H), 0.82
(t, J ) 7.5 Hz, 3H).
K
L
S
V
67Tyr
Leu
Met
Ala
Met
Gly
Ala
Phe
Met
Gly
Val
Gly
Phe
Phe
Met
Ala
Leu
Gly
Ala
68Met
134Ala
160Leu
163Ala
209Leu
Gen er a l P r oced u r e for th e Syn th esis of (1-Eth ylcy-
cloa lk yl)m et h a n ol. To a solution of ethyl 1-ethylcycloal-
kylcarboxylate (5.73 g, 36.7 mmol) in ether (75 mL) was
added a 1.0 M solution of LAH in THF (73.4 mL, 73.4 mmol)
at room temperature. The reaction contents were then heated

5054 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21
Tavares et al.
(1-Eth ylcycloh exyl)m eth an ol: 1H NMR (300 MHz, CDCl3)
ppm: 3.36 (s, 2H), 2.87 (br s, 1H), 1.81-1.20 (m, 12H), 0.81
(t, J ) 7.5 Hz, 3H).
(3-Eth yloxetan -3-yl)m eth yl (1S)-1-[cyan o(tr iph en ylph os-
p h or a n ylid en e)a cetyl]p en tylca r ba m a te: 1H NMR (300
MHz, CDCl3) ppm: 7.64-7.39 (m, 15H), 5.49 (d, J ) 7.9 Hz,
1H), 4.89 (m, 1H), 4.87 (s, 2H), 4.44 (s, 2H), 4.14 (s, 2H), 1.97-
1.64 (m, 4H), 1.36-1.14 (m, 4H), 0.90-0.82 (m, 6H).
Meth yl N-[(2,2-d im eth ylbu toxy)ca r bon yl]-L-n or leu ci-
1
n a te: H NMR (300 MHz, CDCl3) ppm: 5.20 (d, J ) 7.6 Hz,
1H), 4.40 (m, 1H), 3.83 (s, 2H), 3.78 (s, 3H), 1.87-1.65 (m, 2H),
1.40-1.29 (m, 6H), 0.95-0.84 (m, 12H).
(3-Eth yloxeta n -3-yl)m eth yl (1S)-1-(oxo{[(1R)-1-p h en yl-
1
eth yl]a m in o}a cetyl)p en tylca r ba m a te (16): H NMR (300
MHz, CDCl3) ppm: 7.42-7.30 (m, 5H), 7.17 (brs, 1H), 5.41 (d,
J ) 8.0 Hz, 1H), 5.12 (m, 2H), 4.53 (s, 2H), 4.43 (s, 2H), 4.21
(s, 2H), 2.08 (m, 1H), 1.81-1.73 (m, 3H), 1.59 (d, J ) 6.8 Hz,
3H), 1.54-1.27 (m, 4H), 0.96-0.88 (m, 6H). Anal. C, H, N.
(3-Eth yloxeta n -3-yl)m eth yl (1S)-1-[oxo(1H-p yr a zol-5-
yla m in o)a cetyl]p en tylca r ba m a te (17): 1H NMR (300 MHz,
CDCl3) ppm: 11.41 (s, 1H), 10.71 (brs, 1H), 7.51 (s, 1H), 6.87
(s, 1H), 6.16 (d, J ) 7.9 Hz, 1H), 5.30 (m, 1H), 4.56 (s, 2H),
4.40 (s, 2H), 4.09 (s, 2H), 1.99 (m, 1H), 1.72-1.64 (m, 3H),
1.33-1.21 (m, 4H), 0.89-0.81 (m, 6H). Anal. C, H, N.
2,2-Dim eth ylbu tyl (1S)-1-[cya n o(tr ip h en ylp h osp h or a -
n ylid en e)a cetyl]p en tylca r ba m a te: 1H NMR (300 MHz,
CDCl3) ppm: 7.62-7.22 (m, 15H), 5.44 (d, J ) 7.7 Hz, 1H),
4.88 (m, 1H), 3.73 (s, 2H), 1.89 (m, 1H), 1.68 (m, 1H), 1.32 (m,
4H), 1.23 (t, J ) 7.1 Hz, 2H), 0.86-0.74 (m, 12H).
2,2-Dim eth ylbu tyl (1S)-1-(oxo{[(1R)-1-p h en yleth yl]-
a m in o}a cetyl)p en tylca r ba m a te (7): 1H NMR (300 MHz,
CDCl3) ppm: 7.42-7.23 (m, 5H), 7.15 (d, J ) 7.0 Hz, 1H), 5.25
(m, 3H), 3.82 (s, 2H), 1.90 (m, 1H), 1.63 (m, 4H), 1.43 (m, 6H),
0.96-0.84 (m, 12H). Anal. C, H, N.
(3-Eth yloxeta n -3-yl)m eth yl (1S)-1-[(isoxa zol-3-yla m i-
n o)(oxo)a cetyl]p en tylca r ba m a te (18): 1H NMR (300 MHz,
CDCl3) ppm: 9.70 (s, 1H), 8.33 (s, 1H), 7.06 (s, 1H), 5.43 (d, J
) 7.8 Hz, 1H), 5.20 (m, 1H), 4.46 (s, 2H), 4.34 (s, 2H), 4.14 (s,
2H), 1.99 (m, 1H), 1.77-1.55 (m, 4H), 1.36-1.19 (m, 3H), 0.87-
0.84 (m, 6H). LC-MS (C17H25N3O6 + H) 369.
2,2-Dim eth ylbu tyl (1S)-1-[oxo(1H-p yr a zol-3-yla m in o)-
a cetyl]p en tylca r ba m a te (8): 1H NMR (300 MHz, CDCl3)
ppm: 12.09 (s, 1H), 10.76 (brs, 1H), 7.61 (s, 1H), 6.94 (s, 1H),
5.49 (brs, 1H), 5.30 (m, 1H), 3.99 (brs, 1H), 3.81 (s, 2H), 2.08
(m, 1H), 1.65-1.42 (m, 7H), 0.93-0.88 (m, 11H). Anal. C, H,
N.
(3-Eth yloxeta n -3-yl)m eth yl (1S)-1-{oxo[(3-p h en yl-1H-
pyr azol-5-yl)am in o]acetyl}pen tylcar bam ate (19): 1H NMR
(300 MHz, CDCl3) ppm: 11.10 (s, 1H), 7.66 (s, 1H), 7.49-7.40
(m, 5H), 7.20 (s, 1H), 6.42 (d, J ) 8.2 Hz, 1H), 5.42 (m, 1H),
4.52 (s, 2H), 4.47 (s, 2H), 4.11 (s, 2H), 2.13 (m, 1H), 1.81-1.64
(m, 3H), 1.37-1.26 (m, 4H), 0.96-0.88 (m, 6H). Anal. C, H,
N.
2,2-Dim eth ylbu tyl (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 (9): 1H NMR (300
MHz, CDCl3) ppm: 8.00 (s, 1H), 5.93 (brs, 2H), 5.35 (d, J )
7.4 Hz, 1H), 5.24 (m, 1H), 3.83 (s, 3H), 3.65 (s, 2H), 2.08 (m,
1H), 1.65 (m, 1H), 1.59-1.27 (m, 12H), 0.94-0.83 (m, 6H).
2,2-Dim et h ylbu t yl (1S)-1-[(isoxa zol-3-yla m in o)(oxo)-
1
a cetyl]p en tylca r ba m a te (10): H NMR (300 MHz, CDCl3)
Meth yl N-{[(1-eth ylcyclop en tyl)m eth oxy]ca r bon yl}-L-
n or leu cin a te: 1H NMR (300 MHz, CDCl3) ppm: 5.11 (d, J )
7.9 Hz, 1H), 4.32 (m, 1H), 3.83 (s, 2H), 3.70 (s, 3H), 1.78 (m,
1H), 1.64 (m, 5H), 1.43-1.21 (m, 10H), 0.87 (m, 6H).
(1-E t h ylcyclop en t yl)m et h yl (1S)-1-[cya n o(t r ip h en yl-
ph osph or an yliden e)acetyl]pen tylcar bam ate: 1H NMR (300
MHz, CDCl3) ppm: 7.71-7.53 (m, 15H), 5.51 (d, J ) 7.8 Hz,
1H), 4.96 (m, 1H), 4.14 (s, 2H), 1.77-1.40 (m, 6H), 1.39-1.27
(m, 10H), 0.97-0.82 (m, 6H).
ppm: 9.61 (s, 1H), 8.41 (s, 1H), 7.14 (s, 1H), 5.28 (m, 2H), 3.83
(s, 2H), 1.99 (m, 1H), 1.67-1.28 (m, 7H), 0.93-0.88 (m, 12H).
Anal. C, H, N.
Meth yl N-{[(1-eth ylcyclobu tyl)m eth oxy]ca r bon yl}-L-
n or leu cin a te: 1H NMR (300 MHz, CDCl3) ppm: 5.14 (d, J )
7.9 Hz, 1H), 4.32 (m, 1H), 3.97 (s, 2H), 3.70 (s, 3H), 1.81-1.43
(m, 9H), 1.32-1.18 (m, 5H), 0.87-0.77 (m, 6H).
(1-Eth ylcyclobu tyl)m eth yl (1S)-1-[cyan o(tr iph en ylph os-
p h or a n ylid en e)a cetyl]p en tylca r ba m a te: 1H NMR (300
MHz, CDCl3) ppm: 7.70-7.30 (m, 15H), 5.52 (d, J ) 7.7 Hz,
1H), 4.97 (m, 1H), 4.11 (s, 2H), 1.85-1.73 (m, 7H), 1.54-1.26
(m, 7H), 0.95-0.80 (m, 6H).
(1-Eth ylcyclopen tyl)m eth yl (1S)-1-(oxo{[(1R)-1-ph en yl-
1
eth yl]a m in o}a cetyl)p en tylca r ba m a te (20): H NMR (300
MHz, CDCl3) ppm: 7.42-7.33 (m, 5H), 7.16 (d, J ) 7.2 Hz,
1H), 5.25 (brs, 1H), 5.19 (m, 2H), 3.90 (s, 2H), 1.95 (m, 1H),
1.86-1.2 (m, 18H), 0.99-0.84 (m, 6H). Anal. C, H, N.
(1-Eth ylcyclobu tyl)m eth yl (1S)-1-(oxo{[(1R)-1-p h en yl-
1
eth yl]a m in o}a cetyl)p en tylca r ba m a te (11): H NMR (300
(1-Eth ylcyclop en tyl)m eth yl (1S)-1-[oxo(1H-p yr a zol-5-
yla m in o)a cetyl]p en tylca r ba m a te (21): 1H NMR (300 MHz,
CDCl3) ppm: 11.93 (s, 1H), 10.71 (brs, 1H), 7.61 (s, 1H), 6.96
(s, 1H), 5.41 (brs, 1H), 5.29 (m, 1H), 3.90 (s, 2H), 2.08 (m, 1H),
1.62-1.22 (m, 15H), 0.96-0.88 (m, 6H). Anal. C, H, N.
(1-Eth ylcyclopen tyl)m eth yl (1S)-1-[[(1-m eth yl-1H-pyr a-
zol-5-yl)am in o](oxo)acetyl]pen tylcar bam ate (22): 1H NMR
(300 MHz, CDCl3) ppm: 7.99 (s, 1H), 5.92 (brs, 2H), 5.31 (m,
2H), 3.89 (s, 2H), 3.64 (s, 3H), 1.95 (m, 1H), 1.61-1.40 (m,
15H), 0.96-0.84 (m, 6H). Anal. C, H, N.
(1-Eth ylcyclop en tyl)m eth yl (1S)-1-{oxo[(3-p h en yl-1H-
pyr azol-5-yl)am in o]acetyl}pen tylcar bam ate (23): 1H NMR
(300 MHz, CDCl3) ppm: 11.75 (s, 1H), 7.69 (s, 1H), 7.52-7.41
(m, 5H), 7.21 (s, 1H), 5.38 (brs, 1H), 5.24 (m, 1H), 3.91 (s, 2H),
2.08 (m, 1H), 1.65-1.22 (m, 15H), 0.97-0.85 (m, 6H). Anal.
C, H, N.
Meth yl N-{[(1-eth ylcycloh exyl)m eth oxy]ca r bon yl}-L-
n or leu cin a te: 1H NMR (300 MHz, CDCl3) ppm: 5.11 (d, J )
7.9 Hz, 1H), 4.31 (m, 1H), 3.86 (s, 2H), 3.69 (s, 3H), 1.80-1.61
(m, 2H), 1.39-1.20 (m, 15H), 0.84 (m, 4H), 0.77 (t, J ) 7.5 Hz,
3H).
MHz, CDCl3) ppm: 7.42-7.30 (m, 5H), 7.15 (d, J ) 7.7 Hz,
1H), 5.25 (m, 3H), 4.04 (s, 2H), 1.94-1.71 (m, 6H), 1.63-1.30
(m, 11H), 0.98-0.82 (m, 6H). Anal. C, H, N.
(1-E t h ylcyclob u t yl)m et h yl (1S)-1-[oxo(1H-p yr a zol-3-
yla m in o)a cetyl]p en tylca r ba m a te (12): 1H NMR (300 MHz,
CDCl3) ppm: 11.98 (s, 1H), 10.79 (brs, 1H), 7.61 (s, 1H), 6.96
(s, 1H), 5.47 (d, J ) 7.5 Hz, 1H), 5.28 (m, 1H), 4.04 (s, 2H),
1.88-1.29 (m, 13H), 0.96-0.85 (m, 6H). Anal. C, H, N.
(1-Eth ylcyclobu tyl)m eth yl (1S)-1-[[(1-m eth yl-1H-p yr a -
zol-5-yl)am in o](oxo)acetyl]pen tylcar bam ate (13): 1H NMR
(300 MHz, CDCl3) ppm: 8.05 (s, 1H), 5.88 (brs, 2H), 5.33 (m,
2H), 4.04 (s, 2H), 3.65 (s, 3H), 1.89-1.27 (m, 14H), 0.96-0.85
(m, 6H). Anal. C, H, N
(1-Eth ylcyclobu tyl)m eth yl (1S)-1-{oxo[(3-p h en yl-1H-
pyr azol-5-yl)am in o]acetyl}pen tylcar bam ate (14): 1H NMR
(300 MHz, CDCl3) ppm: 7.66 (s, 1H), 7.65 (s, 1H), 7.63-7.22
(m, 5H), 7.11 (s, 1H), 5.34 (brs, 1H), 5.17 (m, 1H), 4.17 (s, 2H),
2.04 (m, 1H), 1.85-1.44 (m, 7H), 1.40-1.16 (m, 6H), 0.89-
0.81 (m, 6H). Anal. C, H, N.
(1-Eth ylcyclobu tyl)m eth yl (1S)-1-[(isoxazol-3-ylam in o)-
(oxo)a cetyl]p en tylca r ba m a te (15): 1H NMR (300 MHz,
CDCl3) ppm: 9.42 (s, 1H), 8.41 (s, 1H), 7.14 (s, 1H), 5.26
(brs, 2H), 4.05 (s, 2H), 2.08-1.27 (m, 14H), 0.97-0.82 (m, 6H).
LC-MS (C18H27N3O5 + H) 366.
Meth yl N-{[(3-eth yloxeta n -3-yl)m eth oxy]ca r bon yl}-L-
n or leu cin a te: 1H NMR (300 MHz, CDCl3) ppm: 5.23 (d, J )
8.0 Hz, 1H), 4.47 (s, 2H), 4.42 (s, 2H), 4.36 (s, 2H), 4.32 (m,
1H), 3.71 (s, 3H), 1.80-1.60 (m, 5H), 1.28 (m, 3H), 0.89-0.84
(m, 6H).
(1-Eth ylcycloh exyl)m eth yl (1S)-1-[cyan o(tr iph en ylph os-
p h or a n ylid en e)a cetyl]p en tylca r ba m a te: 1H NMR (300
MHz, CDCl3) ppm: 7.66-7.50 (m, 15H), 5.48 (d, J ) 7.0 Hz,
1H), 4.91 (m, 1H), 3.88 (s, 2H), 2.03 (m, 1H), 1.71 (m, 1H),
1.56-1.25 (m, 16H), 0.91-0.79 (m, 6H).
(1-Ethylcyclohexyl)methyl(1S)-1-(oxo{[(1R)-1-phenylethyl]-
1
a m in o}a cetyl)p en tylca r ba m a te (24): H NMR (300 MHz,
CDCl3) ppm: 7.39-7.32 (m, 5H), 7.18 (d, J ) 7.3 Hz, 1H), 5.26

Ketoamide-Based Inhibitors of Cysteine Protease
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21 5055
(brs, 1H), 5.19 (m, 2H), 3.92 (s, 2H), 2.01 (m, 1H), 1.59 (d, J )
6.9 Hz, 3H), 1.50-1.33 (m, 17H), 0.95-0.79 (m, 6H). Anal. C,
H, N.
NMR (300 MHz, CDCl3) ppm: 7.98 (s, 1H), 5.88 (brs, 2H), 5.33
(d, J ) 7.9 Hz, 1H), 5.21 (m, 1H), 4.01 (s, 2H), 3.74 (m, 1H),
1.89-1.24 (m, 22H), 0.89-0.78 (m, 6H). Anal. C, H, N.
(1-Eth ylcyclobu tyl)m eth yl (1S)-1-[[(1-cycloh exyl-1H-
p yr a zol-5-yl)a m in o](oxo)a cetyl]p en tylca r ba m a te (38): 1H
NMR (300 MHz, CDCl3) ppm: 7.97 (s, 1H), 5.88 (brs, 2H), 5.33
(d, J ) 7.7 Hz, 1H), 5.20 (m, 1H), 4.31 (m, 1H), 4.01 (s, 2H),
1.89-1.24 (m, 24H), 0.89-0.78 (m, 6H). Anal. C, H, N.
(1-Eth ylcyclobu tyl)m eth yl (1S)-1-{oxo[(1-p h en yl-1H-
pyr azol-5-yl)am in o]acetyl}pen tylcar bam ate (39): 1H NMR
(300 MHz, CDCl3) ppm: 8.17 (s, 1H), 7.56-7.42 (m, 5H), 6.14
(brs, 2H), 5.34 (d, J ) 7.7 Hz, 1H), 5.21 (m, 1H), 4.01 (s, 2H),
1.89-1.24 (m, 14H), 0.89-0.78 (m, 6H). Anal. C, H, N.
(1-Eth ylcycloh exyl)m eth yl (1S)-1-[oxo(1H-p yr a zol-5-
yla m in o)a cetyl]p en tylca r ba m a te (25): 1H NMR (300 MHz,
CDCl3) ppm: 11.93 (s, 1H), 10.78 (brs, 1H), 7.62 (s, 1H), 6.96
(s, 1H), 5.44 (brs, 1H), 5.30 (m, 1H), 3.93 (s, 2H), 2.09 (m, 1H),
1.64-1.23 (m, 17H), 0.98-0.84 (m, 6H). Anal. C, H, N.
(1-Eth ylcycloh exyl)m eth yl (1S)-1-[[(1-m eth yl-1H-p yr a -
zol-5-yl)am in o](oxo)acetyl]pen tylcar bam ate (26): 1H NMR
(300 MHz, CDCl3) ppm: 8.00 (s, 1H), 5.88 (brs, 2H), 5.31 (brs,
1H), 5.23 (m, 1H), 3.93 (s, 2H), 3.65 (s, 3H), 2.08 (m, 1H), 1.94
(m, 1H), 1.44-1.34 (m, 16H), 0.97-0.79 (m, 6H). Anal. C, H,
N.
(1-Eth ylcycloh exyl)m eth yl (1S)-1-[(isoxa zol-3-yla m i-
n o)(oxo)a cetyl]p en tylca r ba m a te (27): 1H NMR (300 MHz,
CDCl3) ppm: 9.52 (s, 1H), 8.42 (s, 1H), 7.14 (s, 1H), 5.26 (brs,
2H), 3.95 (s, 2H), 2.09 (m, 1H), 1.68-1.34 (m, 17H), 0.97-0.82
(m, 6H). Anal. C, H, N.
(1-Eth ylcycloh exyl)m eth yl (1S)-1-[[(6-ch lor o-1H-in d a -
zol-3-yl)am in o](oxo)acetyl]pen tylcar bam ate (28): 1H NMR
(300 MHz, CDCl3) ppm: 11.01 (s, 1H), 10.90 (brs, 1H), 8.16
(d, J ) 7.7 Hz, 1H), 7.48 (s, 1H), 7.12 (d, J ) 7.8 Hz, 1H), 5.48
(d, J ) 7.9 Hz, 1H), 5.28 (m, 1H), 5.30 (m, 1H), 3.79 (s, 2H),
2.05 (m, 1H), 1.93 (m, 2H), 1.44-1.27 (m, 14H), 0.96-0.80 (m,
6H). Anal. C, H, N.
(1-Eth ylcyclobu tyl)m eth yl (1S)-1-[[(1-m eth yl-1H-p yr a -
zol-3-yl)am in o](oxo)acetyl]pen tylcar bam ate (29): 1H NMR
(300 MHz, CDCl3) ppm: 9.12 (s, 1H), 7.29 (brs, 1H), 6.71 (s,
1H), 5.27 (m, 2H), 4.00 (s, 2H), 3.83 (s, 3H), 2.01-1.25 (m,
14H), 0.91-0.75 (m, 6H). Anal. C, H, N.
(1-Eth ylcyclobu tyl)m eth yl (1S)-1-[[(1-eth yl-1H-p yr a -
zol-5-yl)am in o](oxo)acetyl]pen tylcar bam ate (30): 1H NMR
(300 MHz, CDCl3) ppm: 7.99 (s, 1H), 5.88 (brs, 2H), 5.32 (d, J
) 7.8 Hz, 1H), 5.21 (m, 1H), 4.00 (s, 2H), 3.95 (q, J ) 7.2 Hz,
2H), 1.90-1.62 (m, 7H), 1.59-1.24 (m, 10H), 0.93-0.79 (m,
6H). Anal. C, H, N.
(1-E t h ylcyclob u t yl)m et h yl (1S)-1-[[(1-isop r op yl-1H -
p yr a zol-5-yl)a m in o](oxo)a cetyl]p en tylca r ba m a te (31): 1H
NMR (300 MHz, CDCl3) ppm: 8.01 (s, 1H), 5.90 (brs, 2H), 5.29
(brs, 1H), 5.21 (m, 1H), 4.19 (m, 1H), 3.99 (s, 2H), 2.03-1.25
(m, 20H), 0.89-0.77 (m, 6H). Anal. C, H, N.
(1-Eth ylcyclobu tyl)m eth yl (1S)-1-[[(1-isobu tyl-1H-pyr a-
zol-5-yl)am in o](oxo)acetyl]pen tylcar bam ate (32): 1H NMR
(300 MHz, CDCl3) ppm: 8.00 (s, 1H), 5.89 (brs, 2H), 5.32 (brs,
1H), 5.22 (m, 1H), 4.00 (s, 2H), 3.67 (d, J ) 7.3 Hz, 2H), 2.26
(m, 1H), 1.82-1.17 (m, 14H), 0.96-0.79 (m, 12H). Anal. C, H,
N.
(1-Eth ylcyclobu tyl)m eth yl (1S)-1-[{[1-(cyclopr opylm eth -
yl)-1H-p yr a zol-5-yl]a m in o}(oxo)a cetyl]p en tylca r ba m a te
(33): 1H NMR (300 MHz, CDCl3) ppm: 7.99 (s, 1H), 5.94 (brs,
2H), 5.32 (d, J ) 7.5 Hz, 1H), 5.22 (m, 1H), 4.00 (s, 2H), 3.81
(d, J ) 6.8 Hz, 2H), 1.83-1.17 (m, 15H), 0.92-0.79 (m, 10H).
Anal. C, H, N.
(1-Eth ylcyclobu tyl)m eth yl (1S)-1-[[(1-ben zyl-1H-p yr a -
zol-5-yl)am in o](oxo)acetyl]pen tylcar bam ate (34): 1H NMR
(300 MHz, CDCl3) ppm: 8.06 (s, 1H), 7.53-7.20 (m, 5H), 5.70
(brs, 2H), 5.36 (d, J ) 7.5 Hz, 1H), 5.33 (m, 1H), 5.16 (s, 2H),
4.00 (s, 2H), 1.83-1.61 (m, 7H), 1.58-1.24 (m, 7H) 0.92-0.79
(m, 6H). Anal. C, H, N.
Ack n ow led gm en t. The authors wish to thank Ran-
dy Rutkowski, Robert J ohnson and Peter Kitrinos for
analytical support.
Su p p or tin g In for m a tion Ava ila ble: Elemental analyses.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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(1-Eth ylcyclobu tyl)m eth yl (1S)-1-[{[1-(3,3-d im eth yl-
b u t yl)-1H -p yr a zol-5-yl]a m in o}(oxo)a ce t yl]p e n t ylca r -
1
ba m a te (35): H NMR (300 MHz, CDCl3) ppm: 7.98 (s, 1H),
5.79 (brs, 2H), 5.32 (d, J ) 7.9 Hz, 1H), 5.20 (m, 1H), 4.01 (s,
2H), 3.89 (t, J ) 8.2 Hz, 2H), 1.84-1.25 (m, 16H), 0.99 (s, 9H),
0.98-0.79 (m, 6H). Anal. C, H, N.
(1-Eth ylcyclobu tyl)m eth yl (1S)-1-[[(1-cyclobu tyl-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 (300 MHz, CDCl3) ppm: 8.00 (s, 1H), 5.84 (brs, 2H), 5.32
(d, J ) 8.1 Hz, 1H), 5.19 (m, 1H), 4.46 (m, 1H), 4.00 (s, 2H),
2.68 (m, 2H), 2.44 (m, 2H), 2.04-1.23 (m, 16H), 0.92-0.77 (m,
6H). Anal. C, H, N.
(1-Eth ylcyclobu tyl)m eth yl (1S)-1-[[(1-cyclop en tyl-1H-
p yr a zol-5-yl)a m in o](oxo)a cetyl]p en tylca r ba m a te (37): 1H

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