Cyclic ketone inhibitors of the cysteine protease cathepsin K

Article abstract of DOI:10.1021/jm000320t

Cathepsin K (EC 3.4.22.38), a cysteine protease of the papain superfamily, is predominantly expressed in osteoclasts and has been postulated as a target for the treatment of osteoporosis. Crystallographic and structure-activity studies on a series of acyc

Full text of DOI:10.1021/jm000320t

J . Med. Chem. 2001, 44, 725-736
725
Cyclic Keton e In h ibitor s of th e Cystein e P r otea se Ca th ep sin K
Robert W. Marquis,*,† Yu Ru,† J in Zeng,† Robert E. Lee Trout,† Stephen M. LoCastro,† Andrew D. Gribble,‡
J ason Witherington,‡ Ashley E. Fenwick,‡ Benedicte Garnier,‡ Thaddeus Tomaszek,§ David Tew,§
Mark E. Hemling,| Chad J . Quinn,| Ward W. Smith, Baoguang Zhao, Michael S. McQueney,#
Cheryl A. J anson,# Karla D’Alessio,# and Daniel F. Veber†
Departments of Medicinal Chemistry (U.S.A.), Medicinal Chemistry (U.K.), Molecular Recognition, Physical and Structural
Chemistry, Structural Biology, and Protein Biochemistry, GlaxoSmithKline, New Frontiers Science Park, Third Avenue,
Harlow, Essex CM19 5AW, U.K., and 709 Swedeland Road, King of Prussia, Pennsylvania 19406
Received J uly 27, 2000
Cathepsin K (EC 3.4.22.38), a cysteine protease of the papain superfamily, is predominantly
expressed in osteoclasts and has been postulated as a target for the treatment of osteoporosis.
Crystallographic and structure-activity studies on a series of acyclic ketone-based inhibitors
of cathepsin K have led to the design and identification of two series of cyclic ketone inhibitors.
The mode of binding for four of these cyclic and acyclic inhibitors to cathepsin K is discussed
and compared. All of the structures are consistent with addition of the active site thiol to the
ketone of the inhibitors with the formation of a hemithioketal. Cocrystallization of the C-3
diastereomeric 3-amidotetrahydrofuran-4-one analogue 16 with cathepsin K showed the
inhibitor to occupy the unprimed side of the active site with the 3S diastereomer preferred.
This C-3 stereochemical preference is in contrast to the X-ray cocrystal structures of the
3-amidopyrrolidin-4-one inhibitors 29 and 33 which show these inhibitors to prefer binding of
the 3R diastereomer. The 3-amidopyrrolidin-4-one inhibitors were bound in the active site of
the enzyme in two alternate directions. Epimerization issues associated with the labile R-amino
ketone diastereomeric center contained within these inhibitor classes has proven to limit their
utility despite promising pharmacokinetics displayed in both series of compounds.
In tr od u ction
use of an antisense oligonucelotide, Yamamura and co-
workers have inhibited osteoclast bone resorption in a
dose-dependent manner.8 Finally, two groups have
independently generated cathepsin K-deficient mice
which were viable but displayed a distinct osteopetrotic
phenotype.9
The proteolytic processing of endogenous peptide
substrates by cysteine proteases has been implicated in
the pathology of a wide variety of diseases.1 These
diseases include inflammation,2 tumor progression,3
parasitic infections,4 and osteoporosis.5 Cathepsin K (EC
3.4.22.38), a 24-kDa cysteine protease of the papain
superfamily, is selectively and abundantly expressed
within osteoclasts.6 Upon expression, this protease is
secreted from the osteoclast into the resorption pit
where it degrades the protein matrix of bone. The
osteoclast-selective expression of cathepsin K suggests
that this enzyme may play a crucial role in the degra-
dative phase of the remodeling of the bone matrix. As
such, selective inhibition of this enzyme could prove to
be an effective strategy for the treatment of diseases
such as osteoporosis, which is characterized by an
imbalance between the formation and resorption of the
bone matrix. Several recently published studies support
this hypothesis. First, Gelb and J ohnson have shown
that mutations in the gene which encodes for cathepsin
K are associated with a rare autosomal disorder of bone
remodeling known as pycnodysostosis.7 This disease is
characterized by short stature, dwarfism, high bone
fracture rates, and osteosclerosis. Second, through the
Two of the key issues in the development of thera-
peutically useful inhibitors of proteases involve the
mechanism of protease inhibition as well as the opti-
mization of structural features and physiochemical
properties which may limit their oral bioavailabilty.
Cysteine protease inhibitors may be broadly divided
into two classes.10 The first are a series of active site
titrants whose mechanism for inhibition is to perma-
nently modify the target protease via an irreversible
alkylation of the active site cysteine. Notable examples
of this inhibitor class include the naturally derived
epoxide E-64,11 the (acyloxy)methyl ketone quiescent
affinity labels,12 peptidyl vinyl sulfones,13 and R,â-
unsaturated esters.14 The second class of inhibitor is
based on the formation of a covalent, yet fully reversible,
transition-state intermediate with the thiol of the active
site cysteine residue. Inhibitors in this series include
peptide aldehydes,15 cyclopropenones,16 peptide ni-
triles,17 R-keto amides,18 and diamino ketones.19
Our efforts have focused on the development of
reversible inhibitors of cathepsin K. In a disease such
as osteoporosis, which would most likely require chronic
administration of a therapeutic agent, reversible inhibi-
tion may avoid immunological responses which are
associated with the permanent, covalent modification
of proteins.20 We have recently disclosed a series of
* To whom correspondence should be addressed. Phone: 1-610-270-
6570. Fax: 1-610-270-4451. E-mail: Robert_W_Marquis@sbphrd.com.
† Department of Medicinal Chemistry (U.S.A.).
‡ Department of Medicinal Chemistry (U.K.).
§ Department of Molecular Recognition.
| Department of Physical and Structural Chemistry.
Department of Structural Biology.
#
Department of Protein Biochemistry.
10.1021/jm000320t CCC: $20.00 © 2001 American Chemical Society
Published on Web 01/24/2001

726 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
Marquis et al.
Sch em e 1. Synthesis of 3-Amidotetrahydrofuran-4-one
and 4-Amidotetrahydropyran-3-one Inhibitorsa
a
Reagents and conditions: (a) NaN3, NH4Cl, CH3OH, H2O; (b)
H2, 10% Pd/C, CH3OH; (c) Cbz-leucine, EDC, CH2Cl2; (d) (COCl)2,
DMSO, TEA; (e) 10% Pd/C, H2, EtOAc; (f) quinoline-2-carboxylic
acid, EDC; (g) SO3-pyridine, TEA, DMSO.
and piperidinone ring systems followed conceptually
from the furanone and pyranone systems, synthetic
expedience led first to the nitrogen-based ketones which
have been previously reported in this J ournal.25 Sub-
stitution of the oxygen or nitrogen in the heterocyclic
rings of these templates with a methylene group pro-
vides the carbocyclic analogues 7 and 8. These car-
bocycles were prepared in order to determine the role
the heteroatom plays in modulating the electrophilicity
of the carbonyl group contained within the cyclic inhibi-
tor templates described herein.
F igu r e 1. C-O and C-C bond formation of acyclic alkoxy-
methyl ketone 2 to provide 3-amidotetrahydrofuran-4-one and
4-amidotetrahydropyran-3-one inhibitors 3 and 4. Substitution
of nitrogen and carbon for oxygen provides analogues 5-8.
potent, reversible alkoxymethyl ketone inhibitors of
cathepsin K (1; Figure 1)21 which were based upon the
alkoxymethyl ketone template originally developed by
Szelke and co-workers for the inhibition of the serine
protease thrombin.22 While examining substitutions of
the P1 amino acid of the lead methoxymethyl ketone
inhibitor 1 (cathepsin K Ki,app ) 80 nM; Figure 1)23 we
had shown that the isobutyl moiety 1 could be replaced
by a methyl group to afford inhibitor 2 (cathepsin K
Ki,app ) 60 nM) without loss of inhibitory potency. In
the course of designing cyclic conformational constraints
it became clear that simple incorporation of ketone-
based ring systems into this acyclic template could lead
to potent inhibitors. As outlined in Figure 1, cyclization
of inhibitor 2 along path A by the formation of a carbon-
oxygen bond between the alanine methyl group and the
oxygen of the methyl ether generates the 3-amidotet-
rahydrofuran-4-one 3.24 Alternatively, cyclization along
path B by the formation of a carbon-carbon bond
between the alanine methyl group and the methyl ether
carbon provides the 4-amidotetrahydropyran-3-one de-
rivative 4. Substitution of nitrogen for the oxygen of
inhibitors 3 and 4 provides the previously reported
3-amidopyrrolidin-4-one- and 4-amidopiperidin-3-one-
based analogues 5 and 6.25 Although the pyrrolidinone
Syn th esis Ch em istr y
The syntheses of the 3-amidotetrahydrofuran-4-one
and 4-amidotetrahydropyran-3-one inhibitors 3, 4, and
16 are detailed in Scheme 1. Opening of the com-
mercially available 3,4-epoxytetrahydrofuran (9) with
sodium azide followed by reduction of the intermediate
azido alcohol provided the amino alcohol 11. Acylation
of 11 with Cbz-leucine in the presence of 1-[3-(dimethyl-
amino)propyl]-3-ethylcarbodiimide hydrochloride (EDC)
provided alcohol intermediate 13 which was oxidized to
provide ketone 3 as a mixture of diastereomers. In a
similar fashion, opening of the epoxide 10 with sodium
azide followed by reduction using hydrogen in the
presence of 10% palladium on carbon provided the
amino alcohol 12 along with approximately 20% of the
regioisomeric amino alcohol (not shown).26 The undes-
ired regioisomer was separated by column chromatog-
raphy at a later stage of the synthesis. Acylation of 12
with Cbz-leucine in the presence of EDC provided
alcohol 14 which on Swern oxidation provided the
ketone 4 as a mixture of diastereomers. Hydrogenolysis

Cyclic Ketone Inhibitors of Cathepsin K
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 727
Sch em e 2. Synthesis of Pyrrolidinone and Piperidinone Inhibitors 22-29a
a
Reagents and conditions: (a) Cbz-leucine, EDC, HOBt, CH2Cl2; (b) HCl, EtOAc; (c) AcCl, DIPEA, CH2Cl2 or HOAc, EDC, CH2Cl2; (d)
(COCl)2, DMSO, TEA, CH2Cl2; (e) N-Boc-N-methylleucine, EDC, CH2Cl2; (f) N-Cbz-N-methylleucine, EDC, HOBt, CH2Cl2; (g) J one’s reagent,
acetone.
of the Cbz group of intermediate 13 followed by acyla-
tion of the resulting amine with quinoline-2-carboxylic
acid provided the alcohol intermediate 15 which was
oxidized under Parikh conditions27 to provide 16 as a
1:1 mixture of diastereomers.
Boc-leucine (Scheme 3). Hydrogenolysis of the benzyl-
oyxcarbonyl protecting group provided amino alcohol
intermediate 31. Reductive amination of 31 with Cbz-
leucinal in the presence of sodium triacetoxyborohydride
and subsequent removal of the N-Boc protecting group
gave the intermediate amine salt 32. Coupling of 32
with quinoline-7-carboxylic acid followed by SO3-pyri-
dine oxidation of the resulting alcohol provided the
diastereomeric ketones 33.
The syntheses of the carbocyclic analogues 7 and 8
are shown in Scheme 4. Acylation of the known amino
alcohols 34 and 35 with Cbz-leucine provided alcohols
36 and 37. Parikh oxidation of these alcohols provided
the ketone derivatives 7 and 8 as mixtures of diaster-
eomers.
The syntheses of the pyrrolidinone and piperidinone
inhibitors 22-29 are detailed in Scheme 2 and follow
the general procedures described in our earlier com-
munication of a portion of this work.25 Acylation of the
amino alcohols 1728and 1829 with either Cbz-leucine or
N-methyl-Cbz-leucine in the presence of EDC followed
by removal of the tert-butoxycarbonyl protecting group
under acidic conditions provided the amine salts 19-
21. Acylation of the amine salt 19 with acetic acid in
the presence of EDC followed by Swern oxidation of the
resulting alcohol provided 22. Alternatively, acylation
of 20 with acetyl chloride and oxidation gave 23.
Inhibitors 24-28 were synthesized by the acylation of
amine salts 19-21 with either Cbz-leucine or N-Cbz-
N-methylleucine followed by oxidation of the intermedi-
ate alcohols. Alternatively, acylation of amine salt 19
with Boc-N-methylleucine followed by oxidation of the
intermediate alcohol and removal of the tert-butoxycar-
bonyl protecting group under acidic conditions provided
inhibitor 29.
Resu lts a n d Discu ssion
Cathepsin K inhibition and selectivity data versus
human cathepsins B, S, and L are shown in Table 1.30
Cyclization of the alkoxymethyl ketone inhibitor 2
produced the diastereomeric 3-amidotetrahydrofuran-
4-one and the 4-amidotetrahydropyran-3-one analogues
3 and 4 which resulted in only a slight loss of activity
when tested as a 1:1 mixture of diastereomers. Both 3
and 4 are selective inhibitors of cathepsin K versus
cathepsins B and L but were less selective for cathepsin
K over cathepsin S. The alcohol precursors 13 and 14
Inhibitor 33 was synthesized beginning with the
EDC-mediated acylation of amino alcohol 30 with N-

728 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
Sch em e 3. Synthesis of Pyrrolidinone Inhibitor 33a
Marquis et al.
a
Reagents and conditions: (a) N-Boc-leucine, EDC; (b) H2, 10% Pd/C, CH3OH; (c) Cbz-leucinal, NaBH(OAc)3, CH2Cl2; (d) HCl, EtOAc,
CH3OH; (e) quinoline-7-carboxylic acid, EDC, HOBt; (f) SO3-pyridine, TEA, DMSO.
Sch em e 4. Synthesis of Carbocyclic Inhibitors 7 and 8a
cathepsin K for 2 h followed by LC-MS analysis of the
protein/inhibitor mixture showed no covalent modifica-
tion of the protein. Preincubation of excess of 3 or 4 with
cathepsin K for 2 h followed by addition of the fluores-
cent substrate Z-Phe-Arg-AMC revealed no loss of
enzyme activity. This experiment provides additional
evidence for the reversible mechanism of inhibition of
these compounds. The quinoline-2-carboxamide 16 is 4
times as potent as analogue 3 with a Ki,app ) 44 nM
when tested as a 1:1 mixture of diastereomers.32 X-ray
crystallographic analysis of the inhibitor 16/cathepsin
K complex at 2.5 Å resolution shows the inhibitor
oriented on the unprimed side of the active site (Figure
2). The leucine is seen bound within the hydrophobic
S2 pocket formed by residues Leu 160, Ala 134, and Met
68 of the protein. The quinoline group is bound in the
S3 binding pocket forming a π-π stack with Tyr 67.33
The direction of binding for this inhibitor is likely to be
a result of both the π-stacking interaction formed
between the quinoline carboxamide and Tyr 67 as well
as the preferred specificity of the S2 binding pocket of
cathepsin K for leucine.13b The X-ray cocrystal structure
is consistent with the formation of a hemithioketal
between Cys 25 of cathepsin K and the carbonyl group
of the tetrahydrofuran-3-one, confirming our initial
hypothesis for the design of reversible, transition-state
inhibitors of cysteine proteases. The oxygen of the
hemithioketal is stabilized by two hydrogen bonds
formed within the oxyanion hole of the enzyme with the
C(O)NH2 group of Gln 19 as well as the NH of Cys 25.
The stereochemistry of the C-3 diastereomeric center
is consistent with the S configuration. Since the rate of
epimerization of the C-3 chiral center is rapid relative
to the rate of crystallization, this stereochemical prefer-
ence may be a reflection of the more potent of the two
diastereomers or may be a result of crystal packing
forces. Assuming that the S diastereomer of 16 has
formed the initial complex with cathepsin K, then the
active site cysteine has added to the diastereotopically
more hindered si face of the ketone. Two hydrogen bonds
were observed between Gly 66 of the enzyme with the
C-3 leucinamide portion of the inhibitor. The C-2
methylene group of the tetrahydrofuran-3-one ring is
directed out toward solvent with no apparent interac-
tions with the protein. The orientation of the C-2
methylene group toward solvent suggests that this
portion of the molecule may serve as a suitable position
a
Reagents and conditions: (a) Cbz-leucine, EDC; (b) SO3-
pyridine, TEA, DMSO.
Ta ble 1. Cathepsin K, B, S, and L Inhibition Dataa-c
Ki,app (nM)
inhibitor cathepsin K cathepsin B cathepsin S cathepsin L
2
3
4
7
8
13
14
16
22
23
24
25
26
27
28
29
33
60
140
150
>1000
>10000
>10000
>10000
>10000
513
2600
850
310
530
>10000
>10000
>1000
>1000
44
>10000
>10000
>1000
>1000
1100
>10000
>10000
>1000
>1000
250
7800
250
230
480
580
2.3
0.6
180
2.6
1.9
>1000
90
51
39
2600
440
49
16
8.0
77
1.6
7300
46
290
400
a
With the exception of inhibitor 2, all compounds were tested
b
as mixtures of diastereomers. See ref 30 for enzyme assay
conditions. c Average standard errors for Ki,app values were 3-10%.
were significantly less active than the corresponding
ketone derivatives 3 and 4 with Ki,app’s > 1000 nM when
tested as a mixture of diastereomers. This result is in
agreement with the transition state mechanism of
inhibition for ketone-based inhibitors.31 The Lineweaver-
Burk plot of 3 displays a 1/v axis intercept which is
constant with increasing inhibitor concentration, indica-
tive of competitive inhibition (see Supporting Informa-
tion). Competitive inhibition was also observed for 4.
Neither compound displayed time-dependent inhibition
over a 30-min progress curve analysis. Ketones 3 and 4
were also shown to be reversible inhibitors of cathepsin
K. In separate experiments, incubation of 3 and 4 with

Cyclic Ketone Inhibitors of Cathepsin K
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 729
F igu r e 3. Schematic representation of the 2.5 Å X-ray
cocrystal structure of the inhibitor 38 bound within the active
site of cathepsin K (see ref 21).
has added to the re face of the carbonyl group in a
Felkin-Anh manner, opposite to the bulky P1′ isobutyl
group. The n-propyl alkyl side chain was oriented on
the unprimed side of the active site partially penetrating
the hydrophobic S2 binding pocket.
Replacement of the ring oxygen atom of 3 and 4 with
an N-acetyl moiety provided 22 and 23 which were 250
and 230 nM inhibitors of cathespin K, respectively
(again tested as a 1:1 mixture of diastereomers). Inhibi-
tor 23 showed a 2-fold selectivity for cathepsin K versus
cathepsins L and S. Replacement of the N-acetyl group
with Cbz-leucine in order to introduce additional bind-
ing elements which would permit access to both sides
of the active site provided analogues 24 and 27 which
are 2.3 and 2.6 nM inhibitors of cathepsin K, respec-
tively. The approximately 100-fold increase in potency
upon incorporation of the C-3 leucinamide reflects the
additional binding energies associated with the inhibitor
occupying the S3 and S3′ binding pockets of the active
site of the protein. Inhibitor 24 showed good selectivity
for cathepsin K over cathepsins B, L, and S, while 27
was somewhat less selective for cathepsin K versus
cathepsins L and S. These cyclic diamino ketones were
also characterized as competitive, reversible inhibitors
of cathepsin K. Incubation of 24 or 27 with cathepsin K
followed by LC-MS analysis showed that there had
been no irreversible covalent modification of the enzyme.
The N-methylated analogues 25 and 28 were also potent
inhibitors of cathepsin K with Ki,app’s of 0.6 and 1.9 nM,
respectively. N-Methylation has also served to elucidate
the relative importance of the hydrogen bond which is
formed between the inhibitor and the amide carbonyl
of Gly 66 of the protein. The bis-N-methyl analogue 26
was significantly less potent than 25 indicating that the
methylation of the C-3 leucinamide has either elimi-
nated a critical hydrogen bond between the inhibitor
and the enzyme and/or imparted a highly unfavorable
conformation of the urethane which precluded effective
inhibitor binding. Removal of the benzyloxycarbonyl
moiety from 25 provided 29 which was a 77 nM inhibitor
of cathepsin K. The loss in activity of 29 relative to 25
highlights the role of the carbonylbenzyloxy moiety for
potent inhibition. The 2.4 Å resolution X-ray cocrystal
structure of 29 shows that this inhibitor spans both the
primed and unprimed sides of the active site (Figure
4). The direction of binding of inhibitor 29 is the same
F igu r e 2. The 2.5 Å X-ray cocrystal structure of inhibitor 16
(yellow) seen bound within the active site of cathepsin K (blue
and orange). The inhibitor is oriented on the unprimed side
of the active site with the isobutyl group of the inhibitor bound
within the hydrophobic S2 binding pocket The extended
π-aromatic of the quinoline moiety is seen bound in the S3
pocket of the active site. A tetrahedral hemithioketal is formed
between the ketone carbonyl of the inhibitor and the active
site Cys 25 of cathepsin K. The thiol group of Cys 25 has added
to the sterically more congested si face of the carbonyl group
of 16. The inhibitor has crystallized within the enzyme active
site with the S stereochemistry at the C-3 stereocenter.
from which to optimize the physiochemical properties
of these inhibitors which may play a role in determining
their pharmacokinetic behavior.
The direction of binding for inhibitor 16 in the active
site of cathepsin K is opposite to the previously pub-
lished X-ray cocrystal structure of the weakly time-
dependent acyclic n-propyloxymethyl ketone inhibitor
38 (Kinact/I ) 4100 M-1 s-1; Figure 3).21 In this X-ray
cocrystal structure, 38 was oriented on the primed side
of the active site with the isobutyl groups bound in the
S1′ and S2′ pockets of the protein. The phenyl moiety of
the benzyloxycarbonyl group was seen bound in the S3′
pocket forming an edge-face interaction with Trp 184.
This edge-face aromatic interaction between the phenyl
moiety of the inhibitor and the indole of Trp 184 may
be the most critical for determining the direction of
binding for this inhibitor. The N-H of Trp 184 forms a
hydrogen bond with the urethane carbonyl of the
inhibitor, while the amide carbonyl of 38 forms a
hydrogen bond with the N-H group of His 162. The
X-ray cocrystal structure was consistent with the ad-
dition of the thiol group of the active site Cys 25 to the
carbonyl of 38, forming a hemithioketal. The oxygen of
the hemithioketal is stabilized in the oxyanion hole by
hydrogen bonds to the Cys 25 NH and the H2NC(O) of
Gln 19. Here, the thiol group of the active site cysteine

730 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
Marquis et al.
Here again, the oxygen of the hemithioketal is stabilized
within the oxyanion hole by two hydrogen bonds with
the backbone N-H of Cys 25 and the C(O)NH2 of Gln
19. The inhibitors in the cocrystal structures of both 24
and 29 have crystallized within the active site with the
R configuration of the labile C-3 stereocenter. As in the
case of the inhibitor 16/cathepsin K complex, this
stereochemical preference for the R diastereomer may
be a reflection of the more active of the two diastereo-
mers or may be a result of crystal packing forces. The
sulfur nucleophile of Cys 25 has added to the sterically
more congested si face of 29. The S3 pocket of the
inhibitor 29/cathepsin K complex is vacant, while the
S2 pocket is occupied by the isobutyl group. The C-2
methylene group of the pyrrolidinone ring in the crystal
structures of both 24 and 29 is oriented away from the
protein backbone toward solvent making no significant
contacts with the protein. As in the X-ray cocrystal
structure of furanone derivative 16, the orientation of
the C-2 methylene group toward solvent suggests that
this position may be used to alter the pharmacokinetic
properties of these inhibitors or to slow the rate of
enolization of the C-3 methine hydrogen. The isobutyl
group of the C-3 leucinamide is bound in the S2′ pocket
with the aryl group of the carbonylbenzyloxy group
located in the S3′ pocket forming a π-π stacking
interaction with Trp 184. Again, we believe that it is
this interaction which has dominated the direction of
binding for this inhibitor.
F igu r e 4. The 2.4 Å X-ray cocrystal structure of inhibitor 29
(yellow) seen bound within the active site of cathepsin K (blue
and orange). The inhibitor spans S2 to S3′ of the active site. A
tetrahedral hemithioketal is formed between the ketone car-
bonyl of the inhibitor and the active site Cys 25 of cathepsin
K with the thiol moiety adding from the more hindered si face
of the carbonyl group. The inhibitor has crystallized within
the enzyme active site with the R stereochemistry at the C-3
diastereomeric center.
Analogue 33, in which a methylene group has re-
placed the N-1 tertiary amide and a quinoline-7-car-
boxamide has replaced the carbonylbenzyloxy group, is
as potent as the parent cyclic diamino ketone 24 with a
Ki,app ) 1.6 nM as a diastereomeric mixture. These
changes have served to reduce the peptidic nature of
these inhibitors while also increasing their overall
aqueous solubility. As seen in Figure 6, the 1.9 Å
resolution cocrystal structure of analogue 33/cathepsin
K reveals that this inhibitor again spans both sides of
the active site. In contrast to the direction of binding of
inhibitors 24 and 29, analogue 33 binds in the opposite
direction, with the N-1 tertiary amine of the pyrrolidi-
none ring oriented on the primed side of the active site
and the C-3 leucinamide on the unprimed side. The C-3
diastereomeric center with the R configuration has
crystallized within the active site of the protein. The
active site cysteine has added to sterically more acces-
sible re face of the ketone, opposite to the C-3 leucin-
amide moiety. The quinoline-7-carboxamide is bound in
the S3 pocket of cathepsin K, forming an edge-face
electrostatic interaction with Tyr 67. The isobutyl
moiety of the C-3 leucinamide lies within the S2 pocket.
On the primed side of the active site the isobutyl group
of the N-1 tertiary amine is bound within the S2′ pocket
with the phenyl of the Cbz group in the S3′ pocket
forming a π-π stack with Trp 184. The orientation of
binding for 33 is likely a result of the preferred interac-
tions of the phenyl moiety of the inhibitor with the S3′
tryptophan of the protein and the extended π aromatic
group of the quinoline-7-carboxamide with Tyr 67 of the
S3 binding pocket. In this example, the S2 binding pocket
may be irrelevant to the direction of inhibitor binding
due to the presence of two isobutyl groups in the
inhibitor.
F igu r e 5. Schematic representation of the 2.3 Å resolution
X-ray cocrystal structure of inhibitor 24 bound within the
active site of cathepsin K (see ref 25). The inhibitor spans the
active site of the protein with the thiol of Cys 25 adding to
the sterically more congested si face of the carbonyl group of
the inhibitor. The inhibitor has crystallized within the active
site with the R stereochemistry at the C-3 chiral center of the
pyrrolidinone ring.
as that of the previously published cocrystal structure
of inhibitor 24 (see Figure 5).25 In both structures the
N-1 tertiary amide of the pyrrolidinone ring is bound
on the unprimed side while the C-3 leucinamide is seen
bound on the primed side of the active site. The X-ray
cocrystal structure of 29 is consistent with the formation
of a covalent bond between the ketone carbonyl of the
inhibitor and the thiol group of Cys 25 of the protein.

Cyclic Ketone Inhibitors of Cathepsin K
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 731
potency relative to the positioning of the heteroatom
within the cyclic ketone framework reveals the dramatic
difference between a through-bond effect and a through-
space electrostatic effect in altering the reactivity of the
ketone carbonyl group of these inhibitors.34 These
results highlight the essential requirement of these
heteroatoms for the potent inhibition of cysteine pro-
teases by cyclic ketone-based inhibitors.
Having established the cyclic ether and the cyclic
amine classes of compounds as potent, reversible inhibi-
tors of cathepsin K, the pharmacokinetics of several
analogues in each of these series where evaluated in
order to determine if the introduction of a conforma-
tional constraint has improved the bioavailability of
these compounds relative to their acyclic counterparts.
Their pharmacokinetic behavior was profiled in the rat
as multicomponent mixtures utilizing LC-MS-MS
analysis following modified protocols described by Shaf-
fer and co-workers.35 Several analogues from each class
displayed promising pharmacokinetics in the rat. How-
ever due to the facile epimerization of the R-amino
ketone chiral center, these compounds were unsuitable
for further development or for full pharmacokinetic
evaluation of the active diastereomer.
Con clu sion s
F igu r e 6. The 1.9 Å X-ray cocrystal structure of inhibitor 33
(yellow) seen bound within the active site of cathepsin K (blue
and orange). The inhibitor is bound within the S3 to S3′ pockets
of the active site. A tetrahedral hemithioketal is formed
between the ketone carbonyl of the inhibitor and the active
site Cys 25 of cathepsin K with addition of the thiol from the
sterically more accessible re face of the ketone. The inhibitor
has crystallized within the enzyme active site with the R
stereochemistry at the R-amino carbon stereocenter.
In this paper we have disclosed a new series of
3-amidotetrahydrofuran-4-one- and 4-amidotetrahydro-
pyran-3-one-based inhibitors of the cysteine protease
cathepsin K. These inhibitors were characterized as
reversible and competitive. The binding of one of these
cyclic inhibitors was demonstrated by X-ray cocrystal-
lization with the protein, to bind on the unprimed side
of the active site. This mode of binding was opposite to
the orientation of an acyclic analogue which was seen
bound on the primed side of the active site. X-ray
crystallographic analysis of two 3-amidopyrrolidin-4-
one-based inhibitors reveals these analogues to bind in
opposite directions in the active site. The X-ray crystal-
lographic analysis of several protein/inhibitor complexes
was instrumental in delineating the contribution of the
electrostatic interactions which determine the direction
of binding for both the cyclic ether and the cyclic
diamino ketone inhibitor classes. Furthermore, all the
cocrystal structures presented herein revealed that the
C-2 methylene group in all of the bound inhibitors is
oriented away from the protein toward solvent. This
observation suggests that this group may be used to
attenuate the rate of epimerization of this class of cyclic
ketone inhibitors. The introduction of a conformational
constraint was seen to provide potent inhibitors which
displayed promising, yet inconclusive, pharmacokinet-
ics.
F igu r e 7. Effect of positioning of the heteroatom on inhibitor
potencies.
Removal of the electron-withdrawing oxygen or ni-
trogen heteroatoms from the cyclic ketone templates
provided the carbocyclic ketones 7 and 8 which were
not active up to a concentration of 10 µM when tested
against cathepsins K, B, L, and S. Analogues 39 and
40 were evaluated as inhibitors of cathepsin K in order
to determine the effect that the positioning of the
heteroatom within a cyclic framework has on inhibitor
potency. As shown in Figure 7, the 4-amidotetrahydro-
pyran-3-one 39 is an 11 nM inhibitor of cathepsin K
when tested as a mixture of diastereomers. Analogue
40, in which the oxygen is now positioned γ to the
ketone, is a 560 nM inhibitor. This loss in inhibitor
Exp er im en ta l P r oced u r es
Gen er a l. All materials and reagents were used as supplied.
Nuclear magnetic resonance spectra were recorded at 400 MHz
using a Bruker AC 400 spectrometer. Chemical shifts are
reported in parts per million (δ). Mass spectra were taken on
either VG 70 FE, PE Sciex API III, or VG ZAB HF instru-
ments, using electrospray ionization (ESI) techniques. El-
emental analyses were obtained using a Perkin-Elmer 240C
elemental analyzer. Reactions were monitored by TLC analysis
using Analtech silica gel GF or E. Merck silica gel 60 F-254
thin layer plates. Flash chromatography was carried out on
E. Merck Kieselgel 60 (230-400 mesh) silica gel.

732 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
Marquis et al.
tr a n s-4-Am in o-3-h yd r oxytetr a h yd r ofu r a n (11). 3,4-Ep-
oxytetrahydrofuran (9 g, 105 mmol) was added to a stirred
solution of sodium azide (27 g, 415 mmol) and ammonium
chloride (9 g, 159 mmol) in aqueous methanol (95%, 200 mL).
The reaction was heated to 75 °C and stirred for 20 h. The
reaction was cooled, filtered and evaporated under reduced
pressure. The residue was diluted with water and extracted
with ethyl acetate, dried and evaporated under reduced
pressure to afford 10g (74%) of trans-4-azido-3-hydroxytet-
rahydrofuran as a clear oil: 1H NMR (400 MHz, CDCl3) δ 4.32
(m, 1H), 4.09 (dd, 1H, J ) 4.8, 9.9 Hz), 3.99 (dd, 1H, J ) 4.3,
10.1 Hz), 3.94 (m, 1H), 3.81 (dd, J ) 2.1, 9.9 Hz), 3.73 (dd,
1H, J ) 1.8, 10.1 Hz), 2.72 (d, 1H, J ) 4.6 Hz).
To a solution of trans-4-azido-3-hydroxytetrahydrofuran
(13.2 g, 102 mmol) in methanol (100 mL) was added 10%
palladium on charcoal (2 g). The reaction was stirred under
an atmosphere of hydrogen overnight whereupon it was
filtered and concentrated to give 10.2 g of 11 as a brown solid.
This material was of sufficient purity to carry on to the next
step with no further purification.
[(S)-1-((3R,S),(4R,S)-4-H yd r oxyt et r a h yd r ofu r a n -3-yl-
ca r ba m oyl)-3-m eth ylbu tyl]ca r ba m ic Acid Ben zyl Ester
(13). To a solution of amino alcohol 11 (1.0 g, 9.7 mmol) in
CH2Cl2 (20 mL) were added Cbz-leucine (2.83 g, 10.7 mmol)
and EDC (2.83 g, 21.3 mmol). The reaction was maintained
at room temperature for 2 h whereupon it was concentrated.
The residue was dissolved in ethyl acetate and washed with 1
N HCl, saturated NaHCO3, brine, dried (MgSO4), filtered and
concentrated. Column chromatography (2:1 hexanes:ethyl
acetate) of the residue provided 1.7 g (50%) of 13 as a white
powder: 1H NMR (400 MHz, DMSO-d6, reported as a mixture
of diastereomers) δ 8.08 (app d, 1H), 7.34 (m, 5H), 5.24 (br s,
1H), 5.01 (app s, 2H), 4.03-3.78 (m, 5H), 3.49-3.32 (m, 3H),
1.58-1.33 (m, 3H), 0.87-0.83 (m, 6H); MS(ESI) 351.1 (M +
H)+, 372.6 (M + Na)+. Anal. (C18H26N2O5‚0.15EtOAc) C, H, O.
[(S)-1-((3R,S)-4-Oxotetr a h yd r ofu r a n -3-ylca r ba m oyl)-3-
m eth ylbu tyl]ca r ba m ic Acid Ben zyl Ester (3). To a -78
°C solution of oxalyl chloride (0.11 mL, 1.3 mmol) in CH2Cl2
was added DMSO (0.18 mL, 2.6 mmol). The solution was
maintained at -78 °C for 10 min whereupon a solution of 13
(0.30 g, 0.86 mmol) in CH2Cl2 (5.0 mL) was added dropwise.
The reaction was stirred at -78 °C for 30 min whereupon TEA
(0.60 mL, 4.3 mmol) was added. The reaction was warmed to
room temperature, diluted with ethyl acetate and washed
sequentially with 1 N HCl, saturated NaHCO3, brine, dried
(MgSO4), filtered and concentrated. Column chromatography
(1:1 hexanes:ethyl acetate) provided 0.24 g (80%) of 3 as a
white powder: 1H NMR (400 MHz, DMSO-d6, reported as a
mixture of diastereomers) δ 8.45 and 8.35 (d, J ) 5 Hz, 1 H),
7.35-7.32 (m, 5H), 5.04 (ABq, J ) 7.9 Hz, 2H), 4.33-3.76 (m,
6H), 1.61-1.39 (m, 3H), 0.88-0.84 (m, 6H); MS(ESI) 349.0
(M + H)+, 371.0 (M + Na)+. Anal. (C18H24N2O5) C, H, N, O.
matography (3:1 ethyl acetate:hexanes) of the residue provided
0.45 g (52%) of 15 as an off-white solid: 1H NMR (400 MHz,
CDCl3, reported as a mixture of diastereomers) δ 8.64-7.31
(m, 7H), 4.72 (m, 1H), 4.36-3.62 (m, 8H), 1.90-1.66 (m, 3H),
0.96-0.83 (m, 6H); MS(ESI) 372 (M + H)+. Anal. (C20H25N3O4‚
55H2O) C, H, N.
Qu in olin e-2-ca r boxylic Acid [(S)-1-((3R,S)-4-Oxotet-
r a h yd r ofu r a n -3-ylca r ba m oyl)-3-m eth ylbu tyl]a m id e (16).
To a solution of 15 (0.2 g, 0.54 mmol) in DMSO (10 mL) were
added sulfur trioxide-pyridine complex (0.26 g, 1.62 mmol)
and TEA (0.45 mL, 3.24 mmol). The reaction was maintained
at room temperature for 2 h whereupon it was diluted with
ethyl acetate and washed with water, saturated NaHCO3,
brine, dried (MgSO4), filtered and concentrated. Column
chromatography (1:1 ethyl acetate:hexanes) of the residue
provided 0.14 g (70%) of ketone 16 as a white powder: 1H NMR
(400 MHz, CDCl3, reported as a mixture of diastereomers) δ
8.55 (m, 1H), 8.27-8.10 (m, 3H), 7.84-7.58 (m, 3H), 7.26 (br
s, 1H), 4.78-4.75 (m, 1H), 4.62-4.56 (m, 1H), 4.29-43.86 (m,
4H), 1.92-1.74 (m, 3H), 0.99-0.95 (m, 6H); MS(ESI) 370
(M + H)+. Anal. (C20H23N3O4) C, H, N.
(3R,S),(4R,S)-4-Am in otetr a h yd r op yr a n -3-ol (12). To a
solution of the epoxide 10 (1.0 g, mmol) in methanol (40 mL)
and water (5 mL) were added NH4Cl (1.12 g, mmol) and NaN3
(3.25 g). The reaction was heated to 78 °C until complete
consumption of the starting material was observed by TLC
analysis. The mixture was concentrated in vacuo to ap-
proximately 1/3 the original volume. The mixture was then
diluted with ether and washed with saturated NaHCO3, brine,
dried (MgSO4), filtered and concentrated to provide (3R,S),-
(4R,S)-4-azidotetrahydropyran-3-ol which was of sufficient
purity to carry onto the following step with no further
purification: 1H NMR (400 MHz, CDCl3) δ 3.96 (m, 2H), 3.57
(m, 1H), 3.46 (m, 2H), 3.22 (m,1H) 2.03 (m, 1H), 1.70 (m, 1H).
To a solution of (3R,S),(4R,S)-4-azidotetrahydropyran-3-ol
(0.20 g, 1.4 mmol) in methanol (3 mL) was added 10% Pd/C
(0.1 g). The reaction was stirred rapidly under an atmosphere
of hydrogen overnight whereupon it was filtered through a pad
of Celite. The filtrate was concentrated to provide the amino
alcohol 12: 1H NMR (400 MHz, CDCl3) δ 3.90 (m, 2H), 3.29
(m, 2H), 3.08 (m, 1H), 2.66, 1H); MS(ESI) 117.8 (M + H)+.
[(S)-1-((4R,S)-3-Oxot et r a h yd r op yr a n -4-ylca r b a m oyl)-
3-m eth ylbu tyl]ca r ba m ic Acid Ben zyl Ester (4). To a
solution of 12 (0.094 g, 0.8 mmol) in CH2Cl2 (10 mL) were
added Cbz-leucine (0.21 g, 0.8 mmol) and EDC (0.15 g, 0.85
mmol). The reaction was maintained at room temperature
overnight whereupon it was concentrated and the residue
chromatographed (100% ethyl acetate) to provide 0.13 g of the
alcohol 14: 1H NMR (400 MHz, CDCl3, reported as a mixture
of diastereomers) δ 7.29-7.24 (m, 5H), 6.97-6.84 (m, 1H),
5.81-5.60 (m, 1H), 5.08-4.97 (m, 2H), 4.20-3.83 (m, 4H),
3.42-3.27 (m, 3H), 3.08 (t, 1H), 1.88-1.51 (m, 5H), 0.90-0.86
(m, 6H); MS(ESI) 365.0 (M + H)+, 387.0 (M + Na)+.
To a solution of the alcohol 14 (0.07 g, 0.02 mmol) in DMSO
(1.5 mL) were added TEA (0.17 mL, 0.012 mmol) and sulfur
trioxide-pyridine complex (0.09 g, 0.06 mmol). The reaction
was maintained at room temperature for 2 h whereupon it was
diluted with ethyl acetate and washed with saturated NaH-
CO3, brine, dried (MgSO4), filtered and concentrated to provide
ketone 4 as a mixture of diastereomers: 1H NMR (400 MHz,
DMSO-d6, reported as a mixture of diastereomers) δ 8.15 (d,
1H), 7.39-7.29 (m, 5H),5.02 (d, 2H), 4.63 (m, 1H), 4.14-4.10
(m, 2H), 3.97-3.83 (m, 3H), 2.10 (m, 1H), 1.61 (m, 1H), 1.45
(m, 1H), 0.95 (m, 6H); MS(ESI) 363.0 (M + H)+.
[(S)-1-((3R,S),(4R,S)-4-Hyd r oxyp yr r olid in -3-ylca r ba m -
oyl)-3-m eth ylbu tyl]ca r ba m ic Acid Ben zyl Ester Hyd r o-
ch lor id e (19). To a solution of 17 (0.20 g, 1.14 mmol) in
CH2Cl2 (5.0 mL) were added Cbz-leucine (0.30 g, 1.14 mmol),
HOBt (0.15 g, 1.14 mmol) and EDC (0.26 g, 1.37 mmol). The
reaction was allowed to stir until complete by TLC analysis
whereupon it was diluted with EtOAc and washed sequentially
with pH 4 buffer, saturated K2CO3, water and brine. The
organic layer was dried (MgSO4), filtered and concentrated.
Column chromatography of the residue (3:1 EtOAc:hexanes)
Qu in olin e-2-ca r boxylic Acid [(S)-1-((3R,S),(4R,S)-4-Hy-
d r oxyt et r a h yd r ofu r a n -3-ylca r b a m oyl)-3-m et h ylb u t yl]-
a m id e (15). To a solution of 13 (1.0 g, 2.86 mmol) in methanol
(25 mL) was added 10% Pd/C (0.50 g). A balloon of hydrogen
gas was attached and the reaction was stirred rapidly for 1 h
whereupon it was filtered through
a pad of Celite and
concentrated to provide 0.6 g (97%) of (S)-2-amino-4-methyl-
pentanoic acid ((4R,S),(3R,S)-4-hydroxytetrahydrofuran-3-yl)-
amide as a clear, light green oil. This material was of sufficient
purity to use in the following procedure with no further
purification: 1H NMR (400 MHz, CDCl3, reported as a mixture
of diastereomers) δ 7.66 (br s, 1H), 4.19-3.99 (m, 4H), 3.68
(m, 2H), 3.85 (m, 1H), 2.65 (br s, 3H), 1.65 (m, 2H), 1.31 (m,
1H), 0.94-0.89 (m, 6H): MS(ESI); 216.8 (M + H)+.
To a solution of (S)-2-amino-4-methylpentanoic acid ((4R,S),-
(3R,S)-4-hydroxytetrahydrofuran-3-yl)amide (0.50 g, 2.31 mmol)
in CH2Cl2 (5.0 mL) were added quinoline-2-carboxylic acid (0.4
g, 2.31 mmol) and EDC (0.48 g, 2.54 mmol). The reaction was
maintained at room temperature for approximately 2 h
whereupon it was concentrated. The residue was diluted with
ethyl acetate and washed with water, saturated NaHCO3,
brine, dried (MgSO4) filtered and concentrated. Column chro-

Cyclic Ketone Inhibitors of Cathepsin K
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 733
provided 0.32 g (62%) of (3R,S)-((S)-2-benzyloxycarbonylamino-
4-methylpentanoylamino)-(4R,S)-4-hydroxypyrrolidine-1-car-
boxylic acid tert-butyl ester: 1H NMR (400 MHz, DMSO-d6,
reported as a mixture of diastereomers) δ 8.11 (m, 1H), 7.44-
7.37 (m, 5H), 5.29 (br s, 1H), 5.01 (app s, 2H), 3.97-3.87 (m,
3H), 3.48-3.11 (m, 6H), 1.56 (m, 1H), 1.40 (s, 9H), 0.86-0.83
(m, 6H); MS(ESI) 450.3 (M + H)+, 472.2 (M + Na)+. Anal.
(C23H35N3O6) C, H, N.
(3% CH3OH:CH2Cl2) provided 0.015 g (35%) of 22 as an off
white powder: 1H NMR (400 MHz, CDCl3, reported as a 1:1
mixture of diastereomers) δ 7.22 (m, 5H), 6.15-5.87 (m, 1H),
4.97 (m, 2H), 4.19-3.16 (m, 7H), 1.99-1.81 (m, 3H), 1.56-
1.46 (m, 3H), 1.19-1.31.09 (m, 2H), 0.89-0.83 (m, 6H); MS-
(ESI) 390.1 (M + H)+, 779.3 (2M + H)+. Anal. (C20H27N3O5‚
0.25EtOAc) C, H, N, O.
((S)-1-{[(3R,S)-((S)-2-Ben zyloxyca r bon yla m in o-4-m eth -
ylp en t a n oyla m in o)-4-oxop yr r olid in -1-yl]m et h a n oyl}-3-
m et h ylb u t yl)ca r b a m ic Acid Ben zyl E st er (24). To a
solution of 19 (0.25 g, 0.64 mmol) in CH2Cl2 (10 mL) were
added Cbz-leucine (0.17 g, 0.64 mmol), HOBt (0.08 g, 0.64
mmol), NMM (0.300 mL) and EDC (0.15 g, 0.77 mmol). The
reaction was allowed to stir at room temperature for 2 h
whereupon it was diluted with ethyl acetate and washed
sequentially with 1 N HCl, saturated K2CO3, water and brine.
The organic layer was dried (MgSO4), filtered and concen-
trated. Column chromatography of the residue (3:1 EtOAc:
hexanes) gave 0.1 g (26%) of ((S)-1-{1-[(3R,S)-((S)-2-benzyloxy-
carbonylamino-4-methylpentanoylamino)-(4R,S)-hydroxypyrroli-
din-1-yl]methanoyl}-3-methylbutyl)carbamic acid benzyl ester:
1H NMR (400 MHz, CDCl3; reported as a mixture of diaster-
eomers) δ 7.31-7.26 (m, 12H), 5.76-5.44 (m, 3H), 5.13-4.90
(m, 5H), 4.37-4.12 (m, 8H), 1.69-1.46 (m, 6H), 0.96-0.87 (m,
12H); MS(ESI) 597.1 (M + H)+, 619.1 (M + Na)+. Anal.
(C32H46N407‚0.5H2O) C, H, N, O.
To a 0 °C solution of of ((S)-1-{1-[(3R,S)-((S)-2-benzyloxy-
carbonylamino-4-methylpentanoylamino)-(4R,S)-hydroxypyr-
rolidin-1-yl]methanoyl}-3-methylbutyl)carbamic acid benzyl
ester (0.10 g, 0.17 mmol) in acetone (5.0 mL) was added J one’s
reagent dropwise until the brown color persisted. The reaction
was allowed to warm to room temperature and stirred ap-
proximately 48 h whereupon it was quenched with 2-propanol,
diluted with EtOAc and washed sequentially with saturated
K2CO3, water and brine. The organic layer was dried (MgSO4),
filtered and concentrated. Column chromatography of the
residue (3:1 EtOAc:hexanes) gave 0.03 g of 24 as a white
powder: MS(ESI) 595.1 (M + H)+, 617.0 (M + Na)+. Anal.
(C32H44N4O7) C, H, N, O.
To a solution of (3R,S)-((S)-2-benzyloxycarbonylamino-4-
methylpentanoylamino)-(4R,S)-4-hydroxypyrrolidine-1-carbox-
ylic acid tert-butyl ester (0.31 g, 0.69 mmol) in dry EtOAc (5.0
mL) was bubbled HCl gas for approximately 5 min. The
reaction was stirred until TLC analysis indicated the complete
consumption of the starting material. The reaction was then
concentrated in vacuo to provide 0.25 g (94%) of 19 as a white
solid: 1H NMR (400 MHz, DMSO-d6, reported as a mixture of
diastereomers) δ 8.39 (m, 1H), 7.46 (m, 1H), 7.35 (m, 5H), 5.77
(br s, 1H), 5.01 (m, 2H), 4.16-3.95 (m, 3H), 3.49-3.23 (m, 3H),
3.03 (m, 2H), 1.63-1.45 (m, 3H), 0.89-0.83 (m, 6H); MS(ESI)
350.3 (M + H)+. Anal. (C18H27N3O4) C, H, N, O.
[(S)-1-((3R,S),(4R,S)-4-Hyd r oxyp ip er id in -3-ylca r ba m -
oyl)-3-m eth ylbu tyl]ca r ba m ic Acid Ben zyl Ester (20). To
a solution of 18 (1.0 g, 4.62 mmol) in CH2Cl2 were added Cbz-
leucine (1.22 g, 4.62 mmol), EDC (1.07 g, 5.58 mmol) and HOBt
(0.62 g, 4.62 mmol). The reaction was allowed to stir until
complete as indicated by TLC analysis. The reaction was
concentrated and the residue dissolved in ethyl acetate,
washed with 1 N HCl, saturated NaHCO3, brine, dried, filtered
and concentrated. Column chromatography (1:1 hexanes:
EtOAc) of the residue provided 0.88 g (42%) of (4R,S)-((S)-2-
benzyloxycarbonylamino-4-methylpentanoylamino)-(3R,S)-hy-
droxypiperidine-1-carboxylic acid tert-butyl ester: 1H NMR
(400 MHz, DMSO-d6, reported as a mixture of diastereomers)
δ 7.82-7.72 (m, 1H), 7.33 (m, 5H), 4.99 (m, 2H), 4.90 (m, 1H),
4.02-3.51 (m, 4H), 3.49-3.31-3.25 (m, 1H), 2.66 (br s, 1H),
1.71-1.58 (m, 2H), 1.48-1.36 (m, 2H), 1.37 (s, 9H), 0.86-0.82
(m, 6H); MS(ESI) 464.4 (M + H)+, 486.2 (M + Na). Anal.
(C24H37N3O6) C, H, N.
To a solution of (4R,S)-((S)-2-benzyloxycarbonylamino-4-
methylpentanoylamino)-(3R,S)-hydroxypiperidine-1-carboxyl-
ic acid tert-butyl ester (0.88 g, 1.96 mmol) in dry EtOAc (10
mL) was bubbled HCl gas for approximately 5 min. The
reaction was stirred until TLC analysis indicated complete
consumption of the starting material. The reaction was then
concentrated in vacuo to give 0.74 g (98%) of 20 as a white
powder which was of sufficient purity to carry on to the
next step with no further characterization: MS(ESI) 364.3
(M + H)+.
[(S)-1-((3R,S)-1-Acetyl-4-oxop yr r olid in -3-ylca r ba m oyl)-
3-m eth ylbu tyl]ca r ba m ic Acid Ben zyl Ester (22). To a
solution of 19 (0.20 g, 0.57 mmol) in CH2Cl2 were added EDC
(0.12 g, 0.57 mmol), HOBt (0.077 g, 0.57 mmol), TEA (0.14 g,
1.43 mmol) and acetic acid (0.034 g, 0.57 mmol). The reaction
was stirred until complete as indicated by TLC analysis
whereupon it was concentrated, the residue dissolved in EtOAc
and washed with 0.5 N HCl, saturated NaHCO3, brine, dried
(MgSO4), filtered and concentrated. Column chromatography
(10% CH3OH:CH2Cl2) of the residue provided 0.096 g (57%) of
[(S)-1-((3R,S),(4R,S)-1-acetyl-4-hydroxypyrrolidin-3-ylcarbam-
oyl)-3-methylbutyl]carbamic acid benzyl ester as an off-white
powder: MS(ESI) 392.2 (M + H)+. Anal. (C20H29N3O5‚0.1H2O)
C, H, N, O.
((S)-1-{[(3R,S)-((S)-2-(Ben zyloxycar bon ylm eth ylam in o)-
4-m et h ylp en t a n oyla m in o)-4-oxop yr r olid in -1-yl]m et h a -
n oyl}-3-m eth ylbu tyl)ca r ba m ic Acid Ben zyl Ester (25). To
a solution of 19 (0.1 g, 0.26 mmol) in CH2Cl2 (5.0 mL) were
added N-methyl-Cbz-leucine (0.07 g, 0.26 mmol), HOBt (0.03
g, 0.64 mmol), NMM (0.30 mL) and EDC (0.06 g, 0.31 mmol).
The reaction was allowed to stir at room temperature for 2 h
whereupon it was diluted with ethyl acetate and washed
sequentially with 1 N HCl, saturated K2CO3, water and brine.
The organic layer was dried (MgSO4), filtered and concen-
trated. Column chromatography of the residue (3:1 EtOAc:hex-
anes) gave 0.13 g (82%) of ((S)-1-{[(3R,S)-((S)-2-(benzyloxy-
ca rbon ylm et h yla m ino)-4-m et hylpen t a n oyla m in o)-(4R,S)-
hydroxypyrrolidin-1-yl]methanoyl}-3-methylbutyl)carbamic acid
benzyl ester: 1H NMR (400 MHz, CDCl3; reported as a mixture
of diastereomers) δ 7.33-7.26 (m, 10H), 5.13-4.99 (m, 4H),
4.31-3.25 (m, 7H), 3.03-2.84 (m, 4H), 1.79-1.40 (m, 5H),
0.93-0.83 (m, 12H); MS(ESI) 611.3 (M + H)+, 632.9 (M + Na)+.
To a -78 °C solution of oxalyl chloride (0.013 mL, 0.13
mmol) in CH2Cl2 (5.0 mL) was added DMSO (0.018 mL, 0.26
mmol). The reaction was maintained at -78 °C for 10 min
whereupon a solution of ((S)-1-{[(3R,S)-((S)-2-(benzyloxycar-
bonylmethylamino)-4-methylpentanoylamino)-(4R,S)-hydroxy-
pyrrolidin-1-yl]methanoyl}-3-methylbutyl)carbamic acid ben-
zyl ester (0.075 g, 0.12 mmol) in CH2Cl2 (2.0 mL) was added
in a dropwise fashion. The reaction was maintained at -78
°C for 30 min whereupon TEA (0.083 mL, 0.6 mmol) was
added. The reaction was warmed to room temperature, diluted
with ethyl acetate and washed with 1 N HCl, saturated K2-
CO3, water and brine. The organic layer was dried (MgSO4),
filtered and concentrated. Column chromatography (2:1 ethyl
acetate: hexanes) of the residue provided 0.04 g (55%) of 25
as a white solid: 1H NMR (400 MHz, CDCl3, reported as a
mixture of diastereomers) δ 7.35-7.25 (m, 10H), 5.15-5.10
To a -78 °C solution of oxalyl choride (0.01 mL, 0.11 m mol)
in CH2Cl2 (0.3 mL) was added DMSO (0.016 mL, 0.23 mmol)
in CH2Cl2 (0.05 mL). The reaction was maintained at -78 °C
for approximately 10 min whereupon a solution of [(S)-1-
((3R,S),(4R,S)-1-acetyl-4-hydroxypyrrolidin-3-ylcarbamoyl)-3-
methylbutyl]carbamic acid benzyl ester (0.04 g, 0.10 mmol)
in CH2Cl2 (0.10 mL) was added. The reaction was maintained
at -78 °C for another 20 min whereupon TEA (0.07 mL) was
added. The mixture was warmed to 0 °C then diluted with
EtOAc and washed with saturated NaHCO3, brine, dried
(MgSO4) filtered and concentrated. Column chromatography

734 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
Marquis et al.
(m, 6H), 4.95-3.40 (m, 6H), 2.93-2.83 (m, 3H), 1.67-1.36 (m,
6H), 0.97-0.91 (m, 12H); MS(ESI) 609.1 (M + H)+, 630.8
(M + Na)+.
complexed with the inhibitor were grown by the vapor diffusion
method from a solution of 18% PEG 8000, 0.06 M sodium
acetate at pH 4.5 containing 0.12 M Li2SO4. Crystals of the
complex are tetragonal, space group P43212, with cell constants
of a ) 57.9 Å and c ) 130.3 Å. The structure was determined
by molecular replacement with a model consisting of all protein
atoms from the previously determined cathepsin K/E-64
complex.36 The structure was refined at 2.4 Å resolution. The
final Rc was 0.218.
((S)-1-{[(4R,S)-((S)-2-Ben zyloxyca r bon yla m in o-4-m eth -
ylp en t a n oyla m in o)-3-oxop ip er id in -1-yl]m et h a n oyl}-3-
m et h ylbu t yl)ca r ba m ic Acid Ben zyl E st er (27). To a
solution of 20 (0.15 g, 0.43 mmol) in CH2Cl2 were added Cbz-
leucine (0.11 g, 0.43 mmol), EDC (0.1 g, 0.52 mmol), HOBt
(0.058 g, 0.43 mmol) and NMM (0.14 mL, 1.28 mmol). The
reaction was stirred until complete as indicated by TLC
analysis. Workup and column chromatography (2:1 EtOAc:
hexanes) gave 0.22 g (84%) of ((S)-1-{[(4R,S)-((S)-2-benzyloxy-
carbonylamino-4-methylpentanoylamino)-(3R,S)-hydroxypip-
eridin-1-yl]methanoyl}-3-methylbutyl)carbamic acid benzyl
ester: MS(ESI) 611.2 (M + H)+, 633.2 (M + Na)+. Anal.
(C33H46N4O7) C, H, N, O.
3. Cr ysta lliza tion of th e Com p lex of Ca th ep sin K w ith
In h ibitor 33. Crystals of mature activated cathepsin
K
complexed with the inhibitor were grown by the vapor diffusion
method from a solution of 30% MPD, 0.1 M MES, 0.1 M Tris
at pH 7.0. Crystals of the complex are orthorhombic, space
group P212121, with cell constants of a ) 38.2 Å, b ) 50.6 Å,
and c ) 102.3 Å. The structure was determined as described
above for the complex of cathepsin K with inhibitor 29 and
refined at 1.9 Å. The final Rc was 0.253.
To a -78 °C solution of oxalyl chloride (0.043 mL, 0.49
mmol) in CH2Cl2 (5.0 mL) was added DMSO (0.07 mL, 0.98
mmol). The reaction was maintained at -78 °C for 10 min
whereupon a solution of ((S)-1-{[(4R,S)-((S)-2-benzyloxycar-
bonylamino-4-methylpentanoylamino)-(3R,S)-hydroxypiperidin-
1-yl]methanoyl}-3-methylbutyl)carbamic acid benzyl ester (0.19
g, 0.32 mmol) in CH2Cl2 (3 × 2.0 mL) was added in a dropwise
fashion. The reaction was maintained at -78 °C for 30 min
whereupon TEA (0.23 mL, 1.63 mmol) was added. The reaction
was warmed to room temperature, diluted with ethyl acetate
and washed with 1 N HCl, saturated K2CO3, water and brine.
The organic layer was dried (MgSO4), filtered and concen-
trated. Column chromatography (2:1 ethyl acetate:hexanes)
of the residue provided 0.11 g (54%) of 27 as a white solid:
MS(ESI) 611.2 (M + H)+, 633.2 (M + Na)+. Anal. (C33H44N4O7)
C, H, N, O.
Ma ss Sp ectr a l An a lysis of Ca th ep sin K/In h ibitor Com -
p lexes. The inhibitors were prepared at
a 5-fold molar
concentration to the enzyme in water (Milli-Q 18 MΩ) with a
20% final volume of DMSO making sure that the inhibitor was
completely dissolved. Human cathepsin K was provided in
buffer (100 mM sodium acetate, 100 mM NaCl, 2 mM L-
cysteine, pH 5.5). The enzyme, 1 nmol, was mixed with
inhibitor solution 1:1 in a 0.65-mL plastic centrifuge tube,
vortexed and a minimum of 120-min incubation time was
allowed before analysis by LC-MS. The entire reaction
mixture was injected onto the peptide trap. The trap was
washed with 1 mL of water (Milli-Q 18 MΩ) manually before
being back-eluted onto the analytical column.
[(S)-1-(3RS,4RS)-4-Hyd r oxy-tetr a h yd r o-fu r a n -3-ylca r -
ba m oyl)-3-m eth ylbu tyl]ca r ba m ic Acid Ben zyl Ester (36).
To a solution of trans-2-aminocyclopentanol 34 (1.00 g, 6.59
mmol) in CH2Cl2 (20 mL) were added Cbz-leucine (1.75 g, 6.59
mmol), EDC (1.23 g, 6.59 mmol) and N-methylmorpholine. The
reaction was maintained at room temperature overnight
whereupon it was concentrated in vacuo and the residue
dissolved in ethyl acetate and washed with 1 N HCl, saturated
NaHCO3, water and brine. The organic layer was dried
(MgSO4), filtered and concentrated. Column chromatography
(2:1 ethyl acetate:hexanes provided 1.16 g of 36 as a white
solid: 1H NMR (400 MHz, CDCl3, reported as a mixture of
diastereomers) δ 7.38-7.32 (m, 5H), 6.41 (br s, 1H), 5.25 (m,
1H), 5.11 (app s, 2H), 4.17 (m, 1H), 3.92 (m, 1H), 3.79 (m, 1H),
3.49 (m, 1H), 2.11-1.53 (m, 8H), 0.95 (m, 6H); MS(ESI) 349.1
(M + H)+.
[(S)-1-((R,S)-2-Oxocyclop en tylca r ba m oyl)-3-m eth ylbu -
tyl]ca r ba m ic Acid Ben zyl Ester (8). To a solution of 37
(0.50 g, 1.37 mmol) in DMSO (5.0 mL) was added sulfur
trioxide-pyridine complex (0.66 g, 4.13 mmol). The reaction
was stirred for approximately 2 h whereupon it was diluted
with ethyl acetate and washed with 1 N HCl, water and brine.
The organic layer was dried (MgSO4), filtered and concen-
trated. Column chromatography (1:1 ethyl acetate:hexanes)
provided 0.48 g of 8 as a white solid: 1H NMR (400 MHz,
CDCl3, reported as a mixture of diastereomers) δ 7.31 (m, 5H),
6.87 (m, 1H), 5.65 (m, 1H), 5.28 (m, 2H), 4.50 (m, 1H), 4.09
(m, 1H), 2.49-1.53 (m, 8H), 0.91 (m, 6H); MS(ESI) 347.3
(M + H)+.
Su p p or tin g In for m a tion Ava ila ble: Lineweaver-Burk
plots for 3 and 24. This material is available free of charge
via the Internet at http://pubs.acs.org.
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)
3
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