Structure-based design of cathepsin K inhibitors containing a benzyloxy- substituted benzoyl peptidomimetic

Article abstract of DOI:10.1021/jm980474x

Peptidomimetic cathepsin K inhibitors have been designed using binding models which were based on the X-ray crystal structure of an amino acid- based, active site-spanning inhibitor complexed with cathepsin K. These inhibitors, which contain a benzyloxybenzoyl group in place of a Cbz-leucine moiety, maintained good inhibitory potency relative to the amino acid-based inhibitor, and the binding models were found to be very predictive of relative inhibitor potency. The binding mode of one of the inhibitors was confirmed by X-ray crystallography, and the crystallographically determined structure is in close qualitative agreement with the initial binding model. These results strengthen the validity of a strategy involving iterative cycles of structure-based design, inhibitor synthesis and evaluation, and crystallographic structure determination for the discovery of peptidomimetic inhibitors.

Full text of DOI:10.1021/jm980474x

J . Med. Chem. 1998, 41, 3923-3927
3923
Expedited Articles
Str u ctu r e-Ba sed Design of Ca th ep sin K In h ibitor s Con ta in in g a
Ben zyloxy-Su bstitu ted Ben zoyl P ep tid om im etic†
,
§
‡
‡
§
∇
Scott K. Thompson,* Ward W. Smith, Baoguang Zhao, Stacie M. Halbert, Thaddeus A. Tomaszek,
∇
∇,|
⊥
⊥
⊥
⊥
David G. Tew, Mark A. Levy, Cheryl A. J anson, Karla J . D′Alessio, Michael S. McQueney, J eff Kurdyla,
Christopher S. J ones, Renee L. DesJ arlais, Sherin S. Abdel-Meguid, and Daniel F. Veber
⊥
#,X
‡
§
Departments of Medicinal Chemistry, Structural Biology, Molecular Recognition, Protein Biochemistry, and Physical and
Structural Chemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406
Received August 13, 1998
Peptidomimetic cathepsin K inhibitors have been designed using binding models which were
based on the X-ray crystal structure of an amino acid-based, active site-spanning inhibitor
complexed with cathepsin K. These inhibitors, which contain a benzyloxybenzoyl group in
place of a Cbz-leucine moiety, maintained good inhibitory potency relative to the amino acid-
based inhibitor, and the binding models were found to be very predictive of relative inhibitor
potency. The binding mode of one of the inhibitors was confirmed by X-ray crystallography,
and the crystallographically determined structure is in close qualitative agreement with the
initial binding model. These results strengthen the validity of a strategy involving iterative
cycles of structure-based design, inhibitor synthesis and evaluation, and crystallographic
structure determination for the discovery of peptidomimetic inhibitors.
Cysteine proteases have been broadly implicated as
targets for therapeutic intervention (e.g., cancer, ar-
thritis, viral and parasitic diseases).1 Cathepsin K, a
cysteine protease of the papain superfamily, has been
implicated in the process of bone resorption.2 Selective
inhibitors of cathepsin K could therefore be promising
therapeutic agents for the treatment of diseases char-
acterized by excessive bone loss, such as osteoporosis.
The structure-based design and X-ray crystallo-
graphic analysis of a new class of active site-spanning
cathepsin K inhibitors which contain a 1,5-diacylcar-
bohydrazide scaffold have recently been reported from
aspect of the design of therapeutically useful enzyme
inhibitors has been the replacement of amino acid
residues by isosteric elements. The design of such
elements has met with much success in the recent past,
owing greatly to the availability of crystallographic data
from enzyme/inhibitor complexes. The present report
describes the structure-based design and synthesis of
potent 1,5-diacylcarbohydrazides which contain a ben-
zyloxy-substituted benzoyl group as a mimic for a
benzyloxycarbonyl (Cbz) leucine residue.
,3
Compound 1 exhibits time-dependent kinetics con-
sistent with an essentially irreversible mechanism of
inhibition. Mass spectral and NMR analysis of the
complex of 1 with cathepsin K give results consistent
with formation of an acyl enzyme intermediate with
cleavage of one of the acylhydrazine bonds and loss of
Cbz-leucinylhydrazine as reported previously.4 None-
theless, X-ray crystallographic studies of the complex
of 1 with cathepsin K show both halves of the inhibitor
present in the active site with the inhibitor bound across
both S and S′ sides (nomenclature of Schechter and
4
these laboratories. Compound 1, for example, is a
potent, selective, kinetically irreversible inhibitor of
cathepsin K but contains many groups, such as amino
acid residues, with hydrogen-bond potential thought to
have a negative impact on oral bioavailability. A key
5
Berger). Examination of the X-ray crystal structure
of 1 bound to cathepsin K revealed a nearly coplanar
arrangement between the leucine carbonyl and the
CR-N bond in the half of the inhibitor bound on the S′
side of the active site. This particular arrangement of
bonds suggested that a benzoyl group could function
effectively as an achiral replacement for the leucine
residue. Furthermore, the aromatic portion of the Cbz
group, which appears to be involved in a π-π interaction
with the aromatic side chain of Trp-184, a residue that
is conserved throughout this family of proteases, was
in a position such that attachment of a benzyloxy
substituent in either the ortho- or meta-position of the
†
The refined coordinates for the complexes of compounds 1 and 3
with cathepsin K have been deposited at the Protein Data Bank under
the filenames 1AYU and 1AYW, respectively.
§
Department of Medicinal Chemistry.
Department of Structural Biology.
Department of Molecular Recognition.
Department of Protein Biochemistry.
Department of Physical and Structural Chemistry.
Presently in the Department of Preclinical Pharmacokinetics.
Present address: 3-Dimentional Pharmaceuticals, Inc., 665 Stock-
‡
∇
⊥
#
|
X
ton Dr, Suite 104, Exton, PA 19341.
S0022-2623(98)00474-9 CCC: $15.00 © 1998 American Chemical Society
Published on Web 09/17/1998

3
924 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21
Thompson et al.
F igu r e 1. Stereoviews of binding models for ortho-substituted (white), meta-substituted (magenta), and para-substituted (green)
benzoyl peptidomimetics superimposed with inhibitor 1 (cyan). Selected active site residues are colored by atom (C ) gray, O )
red, N ) blue, S ) yellow).
Sch em e 1a
group. A benzyloxy group in the para-position cannot
occupy this space due to geometric constraints. Indeed,
the unsubstituted benzoyl analogue 5 is equivalent in
potency to 4, suggesting that substitution of the ben-
zyloxy group in the para-position does not allow for any
interaction of this group with the enzyme. This result
is in contrast to that reported for a structurally similar
class of active site-spanning 1,3-diamino ketone cathep-
a
sin K inhibitors in which an inhibitor containing a
(
a) PhCH2OCOCl, Na2CO3, 1,4-dioxane; (b) H2NNH2‚H2O,
methanol; (c) COCl2, toluene, reflux; (d) H2NNH2‚H2O, ethanol;
e) RCO2H, EDC‚HCl, 1-HOBT, DMF.
p-phenoxybenzoyl peptidomimetic retained the potency
7
(
of the Cbz-leucine analogue, but this may be a conse-
quence of the inherent differences in the positions that
can be occupied by the terminal phenyl group in a
phenoxy substituent relative to a benzyloxy substituent.
The decrease in potency observed for compounds 2 and
3 relative to the Cbz-leucine analogue 1 appears to be
a result of the inability of the benzoyl group to mimic
the γ-methyl groups of the leucine side chain. Indeed,
a similar decrease in potency is observed when the
leucine residue in 1 is replaced with alanine (compound
6) or (S)-2-aminobutyric acid (compound 7), while
replacement with L-norvaline did not lead to a decrease
in potency (compound 8), indicating that only one of the
γ-methyl groups of the leucine side chain is necessary
to achieve the high potency of compound 1. The relative
binding affinities of compounds 1-8 as measured by
Ki,app, determined from initial rates of substrate hy-
drolysis (Table 1), indicate that the differences in
potency are largely a result of binding interactions but
appear to be influenced to some degree by rate differ-
ences in the time-dependent step in the inhibition
mechanism. For example, the 10-fold decrease in ap-
parent second-order rate constant for inhibition by
compound 7 relative to compound 8 appears to be
governed by a difference in the rate of the time-
dependent step in the inhibition mechanism as well as
binding affinity (Ki,app) since the difference between
Ki,app values for compounds 7 and 8 is only about 4-fold.
Nonetheless, these data are wholly consistent with
predictions based on the binding models.
benzoyl group could result in effective mimicry of the
entire Cbz-leucine moiety. Indeed, binding models of
o-benzyloxy-substituted (white) and m-benzyloxy-sub-
stituted (magenta) benzoyl groups show very good
overlap with the Cbz-leucine moiety when superimposed
with inhibitor 1, while para-substitution (green) does
not allow for effective overlap of the benzyloxy groups
(Figure 1). Note, however, that the flat nature of the
benzoyl group precludes mimicry of both of the γ-methyl
groups of the leucine side chain. These models therefore
predict that compounds 2 and 3 will be more potent
inhibitors of cathepsin K than is compound 4 or the
unsubstituted benzoyl analogue 5 but may be less potent
than the Cbz-leucine analogue 1 because of the failure
to fully mimic the leucine side chain.
Compounds 2-8 were prepared as shown in Scheme
1
. Compounds 2-5 are all time-dependent inhibitors
of cathepsin K, exhibiting apparent second-order rate
-
1
constants (kobs/[I]) in the range of 38 000-318 000 M
-
1
s
(Table 1). The ortho- and meta-substituted ana-
logues 2 and 3 are about 1 order of magnitude less
potent than 1, while the para-substituted analogue 4 is
about 2 orders of magnitude less potent. The equivalent
potency of compounds 2 and 3 suggests that phenyl
portions of the benzyloxy groups in the two inhibitors
are likely occupying similar positions in the cathepsin
K active site, and this is easily accommodated by
rotation about the C-O-C-C bonds of the benzyloxy

Structure-Based Design of Cathepsin K Inhibitors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21 3925
Ta ble 1. Inhibition of Cathepsin K by Compounds 1-6a
kobs/[I] (M- s-1)b
1
Ki,app (nM)b
compd
RCO
Cbz-L-leucine
o-benzyloxybenzoyl
m-benzyloxybenzoyl
p-benzyloxybenzoyl
benzoyl
Cbz-L-alanine
Cbz-(S)-2-aminobutyric acid
Cbz-L-norvaline
formula
1
2
3
4
5
6
7
8
C29H40N6O7
C29H33N5O6
C29H33N5O6
C29H33N5O6
C22H27N5O5
C26H34N6O7
C27H36N6O7‚0.5H2O
C28H38N6O7‚0.5H2O
3 100 000
318 000
224 000
44 000
0.7
12
27
100
120
1.0
38 000
685 000
490 000
4 000 000
3.4
0.8
a
Assays were conducted as previously described.6 Average standard errors for the kobs/[I] and Ki,app values were (3-10%.
b
F igu r e 2. (A) Stereoview of 3 (cyan) bound in the active site of cathepsin K. Side chains from neighboring cathepsin K molecules
in the crystal lattice are shown in magenta. Selected active site residues are colored by atom (C ) gray, O ) red, N ) blue, S )
yellow). (B) Overlay of the bound conformation of 3 (cyan) with the binding model for its peptidomimetic portion (white) shown
in stereoview with selected active site residues. (C) Overlay of the bound conformations of 3 (cyan) and 9 (white) shown in stereoview
with selected active site residues.
Because of the equivalent potency and the likelihood
of similar binding modes of compounds 2 and 3, only
one of them, compound 3, was selected initially for
cocrystallization with cathepsin K. The complex of
compound 3 with cathepsin K crystallized under condi-
tions similar to that reported for the cathepsin K:1
side chain is bound in the hydrophobic S2 pocket. The
phenyl portion of the Cbz group appears to participate
in an edge-face aromatic-aromatic interaction with
Tyr-67, although from the opposite face of the tyrosine
ring than is observed in the cathepsin K:1 complex
(Figure 1). The centroid separation and dihedral angle
for this interaction are 6.9 Å and 66°, respectively, both
within the range preferred for an aromatic-aromatic
interaction as defined by Burley and Petsko.8 In the
cathepsin K:3 complex, binding of the Cbz group in the
same site occupied by the Cbz group in the cathepsin
K:1 complex is precluded by the presence of the side
4
complex, except in a different crystal form. X-ray
analysis of this complex at 2.4 Å shows the inhibitor
bound across both S and S′ sides of the active site with
a mode of binding consistent with addition of the
catalytic cysteine thiol to the central urea carbonyl
(Figure 2A). On the S side of the active site, the leucine

3
926 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21
Thompson et al.
chain of Lys-44 from a neighboring cathepsin K mol-
ecule in the crystal lattice. There are also differences
on the S′ side of the active site where, in the complex of
partitioned between ethyl acetate and water. The organic
layer was washed with saturated brine, dried (MgSO
filtered, and concentrated to give 3.1 g (100%) of 11 as a
colorless oil: 1H NMR (400 MHz, CDCl
4
),
3
) δ 7.34 (m, 5H), 5.27
3
with cathepsin K, the side chains of two arginine
(
0
d, 1H), 5.12 (s, 2H), 4.41 (s, 2H), 3.75 (s, 3H), 1.65 (m, 3H),
residues (Arg-123 and Arg-127), also from a neighboring
cathepsin K molecule, occupy a portion of the active site.
Nonetheless, an apparent π-π interaction with a cen-
troid separation and dihedral angle of 4.9 Å and 39°,
respectively, is observed between the benzyloxy portion
of the peptidomimetic and the side chain of Trp-184, as
was qualitatively predicted from the binding model.
Superposition of the bound conformation of 3 (cyan)
with the binding model of its peptidomimetic portion
+
.96 (m, 6H); MS (ESI) 302.1 (M + Na) .
N-Ben zyloxyca r bon yl-L-leu cin eh yd r a zid e (12). To a
stirring solution of 3.1 g (11.0 mmol) of 11 in 15 mL of
methanol was added 5.9 g (118 mmol, 5.73 mL) of hydrazine
hydrate. The solution was allowed to stir at room temperature
for 16 h and then concentrated to give 3.1 g (100%) of 12 as
an off-white solid: 1H NMR (400 MHz, CDCl
7
) δ 8.36 (bs, 1H),
.30 (m, 5H), 5.77 (d, 1H), 5.06 (dd, 2H), 4.19 (m, 1H), 3.80 (b
s, 2H), 1.62 (m, 2H), 1.54 (m, 1H), 0.89 (m, 6H); MS (ESI) 280.2
3
+
(
M + H) .
(white) shows a good, but not perfect, match between
1
-(N-Ben zyloxycar bon ylam in o)-3-m eth yl-1-(5-oxo-1,3,4-
them (Figure 2B). In this overlay, a number of close
contacts (1.9-3 Å carbon-carbon distance) are observed
between the Arg-127 side chain and various points on
the benzyloxy portion of the peptidomimetic model. One
such contact is between Nω of Arg-127 and the benzylic
carbon of the benzyloxy group (1.9 Å) and is highlighted
in Figure 2B (yellow dash). In the cathepsin K:3
complex, the corresponding nonbonded distance is 3.4
Å, and this may be a result of a slight movement in the
position and/or conformation of the inhibitor to accom-
modate the presence of the arginine side chains. Thus,
the differences between the bound conformation of 3 as
observed in its complex with cathepsin K and the
binding model may be at least partially a consequence
of the particular crystal form in which the complex
crystallized. Indeed, superposition of the bound con-
formation of 3 with that of a structurally similar Cbz-
oxa d ia zol-2-yl)bu ta n e (13). To a solution of 0.5 g (1.79
mmol) of 12 in 10 mL of toluene was added 9.27 mL (17.9
mmol) of a 1.93 M solution of phosgene in toluene. After
heating for 4 h at reflux, the solution was concentrated to an
oily residue which was purified by column chromatography
(
silica gel, ethyl acetate/hexane) to give 0.427 g (78%) of 13 as
1
an off-white foam: H NMR (400 MHz, CDCl
7
(dd, 6H); MS (ESI) 306.1 (M + H) .
3
) δ 9.18 (s, 1H),
.38 (m, 5H), 5.13 (m, 3H), 4.79 (m, 1H), 1.71 (m, 3H), 0.98
+
2-(N-Ben zyloxyca r b on yl-L-leu cin yl)ca r b oh yd r a zid e
(14). To a solution of 0.427 g (1.4 mmol) of 13 in 2 mL of
ethanol was added 0.7 g (14 mmol, 0.68 mL) of hydrazine
hydrate. After stirring for 24 h at room temperature, the
solution was concentrated to give 0.472 g (100%) of 14 as an
1
off-white foam: H NMR (400 MHz, CDCl
3
) δ 9.25 (bs, 1H),
8
1
.03 (bs, 1H), 7.29 (m, 6H), 6.12 (d, 1H), 5.13 (d, 1H), 4.97 (d,
H), 4.33 (m, 1H), 3.87 (m, 2H), 1.61 (m, 3H), 0.89 (m, 6H);
+
MS (ESI) 338.2 (M + H) .
Gen er a l P r oced u r e for th e P r ep a r a tion of Ben zyloxy-
ben zoa te Ester s. To a suspension of 395 mg (9.87 mmol) of
NaH (60% in mineral oil) in 20 mL of DMF was added 1.0 g
7
leucine-containing active site-spanning inhibitor (com-
pound 9) which crystallizes in the same crystal form
shows a very good match between all elements of the
two inhibitors (Figure 2C). The bound conformation of
(
6.58 mmol) of the hydroxybenzoate methyl ester. After
stirring for 15 min at room temperature, 1.1 g (6.58 mmol,
.76 mL) of benzyl bromide was added. After stirring at room
0
3
in its complex with cathepsin K is nonetheless in close
temperature for 3 h, the solution was partitioned between ethyl
acetate and water. The organic layer was washed with water
qualitative agreement with the initial binding model.
(
2 × 75 mL), saturated aqueous sodium bicarbonate, and
saturated brine, then dried (MgSO
to give the desired product.
4
), filtered, and concentrated
Gen er a l P r oced u r e for th e P r ep a r a tion of Ben zyloxy-
ben zoic Acid s. To a solution of 400 mg (1.65 mmol) of the
benzoate methyl ester in 2 mL of THF and 2 mL of water was
added 76 mg (1.82 mmol) of lithium hydroxide monohydrate.
After stirring at reflux for 5 h, the solution was partitioned
between ethyl acetate and 3 N HCl. The organic layer was
In conclusion, we have used the crystal structure of
a cathepsin K/inhibitor complex to effectively design
peptidomimetic inhibitors containing a benzyloxyben-
zoyl mimic of Cbz-leucine through the use of binding
models. The models were subsequently validated by
SAR studies and crystallographic analysis of the mod-
eled inhibitor complexed with cathepsin K. The strat-
egy involving iterative cycles of structure-based design,
inhibitor synthesis and evaluation, and crystallographic
structure determination has thus proven to be a viable
approach to obtain potent cathepsin K inhibitors wherein
an amino acid element has been replaced with an
achiral surrogate.
washed with saturated brine, dried (MgSO
concentrated to give the desired product.
4
), filtered, and
Gen er a l P r oced u r e for th e Cou p lin g of 14 w ith Ca r -
boxylic Acid s. To a stirring solution of 100 mg (0.3 mmol)
of 14, a carboxylic acid (0.315 mmol), and 8 mg (0.06 mmol) of
1-hydroxybenzotriazole in 3 mL of DMF was added 60 mg
(0.312 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiim-
ide hydrochloride. After stirring for 16 h at room temperature,
the solution was poured into water and filtered to give the
desired product.
Spectral data for compounds 2-8 are as follows.
2
-(2-Be n zyloxyb e n zoyl)-2′-(N -b e n zyloxyca r b on yl-L-
1
leu cin yl)ca r boh yd r a zid e (2): H NMR (400 MHz, CDCl
CD OD) δ 7.89 (d, 1H), 7.34-7.16 (m, 11H), 6.94 (d, 2H), 5.14
m, 2H), 4.95 (dd, 2H), 4.08 (m, 1H), 1.60-1.43 (m, 3H), 0.81
3
/
3
(
(
0
Exp er im en ta l Section
+
m, 6H); MS (ESI) 548.1 (M + H) . Anal. Calcd for C29
.5H O: C, 62.58; H, 6.16; N, 12.58. Found: C, 62.41; H, 5.44;
2
H
33
N
5
O
6
‚
N-Ben zyloxyca r bon yl-L-leu cin e Meth yl Ester (11). To
a stirring solution of 2.0 g (11 mmol) of L-leucine methyl ester
hydrochloride in 20 mL of 1,4-dioxane was added 12.1 mL (24.2
N, 13.29.
2-(3-Be n zyloxyb e n zoyl)-2′-(N -b e n zyloxyca r b on yl-L-
1
mmol) of a 2 M aqueous solution of Na
2
CO
3
followed by 1.96
leu cin yl)ca r boh yd r a zid e (3): H NMR (400 MHz, CDCl
CD OD) δ 7.46 (s, 1H), 7.39-7.26 (m, 12H), 7.07 (dd, 1H), 5.02
(m, 4H), 4.14 (m, 1H), 1.61 (m, 2H), 1.50 (m, 1H), 0.86 (m,
3
/
g (11.5 mmol, 1.64 mL) of benzyl chloroformate. The mixture
was allowed to stir at room temperature for 4 h and then
3

Structure-Based Design of Cathepsin K Inhibitors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21 3927
+
6
0
H); MS (ESI) 548.1 (M + H) . Anal. Calcd for C29
H
33
N
5
O
6
‚
(2) Drake, F. H.; Dodds, R. A.; J ames, I. E.; Connor, J . R.; Debouck,
C.; Richardson, S.; Lee-Rykaczewski, E.; Coleman, L.; Rieman,
D.; Barthlow, R.; Hastings, G.; Gowen, M. Cathepsin K, But Not
Cathepsins B, L or S, Is Abundantly Expressed in Human
Osteoclasts. J . Biol. Chem. 1996, 271, 12511-12516.
3) Bossard, M. J .; Tomaszek, T. A.; Thompson, S. K.; Amegadzie,
B. Y.; Hanning, C. R.; J ones, C.; Kurdyla, J . T.; McNulty, D. E.;
Drake, F. H.; Gowen, M.; Levy, M. A. Proteolytic Activity of
Human Osteoclast Cathepsin K. J . Biol. Chem. 1996, 271,
2
.1H O: C, 63.40; H, 6.09; N, 12.75. Found: C, 63.02; H, 5.37;
N, 13.27.
-(4-Be n zyloxyb e n zoyl)-2′-(N -b e n zyloxyca r b on yl-L-
2
1
leu cin yl)ca r boh yd r a zid e (4): H NMR (400 MHz, CDCl
CD OD) δ 7.77 (d, 2H), 7.37-7.23 (m, 10H), 6.95 (d, 2H), 5.03
m, 4H), 4.17 (m, 1H), 1.61 (m, 2H), 1.50 (m, 1H), 0.88 (m,
3
/
(
3
(
6
+
H); MS (ESI) 548.1 (M + H) . Anal. Calcd for C29
C, 63.60; H, 6.07; N, 12.79. Found: C, 63.45; H, 5.87; N, 13.37.
-Ben zoyl-2′-(N-ben zyloxyca r bon yl-L-leu cin yl)ca r bo-
33 5 6
H N O :
1
2517-12524.
2
(
4) Thompson, S. K.; Halbert, S. M.; Bossard, M. J .; Tomaszek, T.
A.; Levy, M. A.; Zhao, B.; Smith, W. W.; Abdel-Meguid, S. S.;
J anson, C. A.; D′Alessio, K. J .; McQueney, M. S.; Amegadzie, B.
Y.; Hanning, C. R.; DesJ arlais, R. L.; Briand, J .; Sarkar, S. K.;
Huddleston, M. J .; Ijames, C. F.; Carr, S. A.; Garnes, K. T.; Shu,
A.; Heys, J . R.; Bradbeer, J .; Zembryki, D.; Lee-Rykaczewski,
L.; J ames, I. E.; Lark, M. W.; Drake, F. H.; Gowen, M.; Gleason,
J . G.; Veber, D. F. Design of Potent and Selective Human
Cathepsin K Inhibitors Which Span the Active Site. Proc. Natl.
Acad. Sci. U.S.A. 1997, 94, 14249-14254.
1
h yd r a zid e (5): H NMR (400 MHz, CDCl
H), 7.48-7.17 (m, 8H), 5.00 (d, 2H), 4.12 (m, 1H), 1.54 (m,
H), 0.86 (m, 6H); MS (ESI) 442.1 (M + H) . Anal.
) C, H, N.
-(N-Ben zyloxyca r bon yl-L-a la n yl)-2′-(N-ben zyloxyca r -
3
/CD
3
OD) δ 7.75 (d,
2
3
+
22 27 5 5
(C H N O
2
1
bon yl-L-leu cin yl)ca r boh yd r a zid e (6): H NMR (400 MHz,
CDCl /CD OD) δ 7.22 (m, 10H), 5.00 (m, 4H), 4.13 (m, 2H),
.54 (m, 3H), 1.31 (d, 3H), 0.87 (m, 6H); MS (ESI) 543.1 (M +
3
3
1
+
H) . Anal. Calcd for C26
Found: C, 57.48; H, 5.85; N, 15.04.
-(N-Ben zyloxyca r b on yl-(S)-2-a m in ob u t yr oyl)-2′-(N-
34 6 7
H N O : C, 57.55; H, 6.32; N, 15.49.
(
5) Schechter, I.; Berger, A. On the Size of the Active Site in
Proteases. Biochem. Biophys. Res. Commun. 1967, 27, 157-162.
6) Votta, B.; Levy, M. A.; Badger, A.; Bradbeer, J .; Dodds, R. A.;
J ames, I. E.; Thompson, S.; Bossard, M. J .; Carr, T.; Connor, J .
R.; Tomaszek, T. A.; Szewczuk, L.; Drake, F. H.; Veber, D. F.;
Gowen, M. Peptide Aldehyde Inhibitors of Cathepsin K Inhibit
Bone Resorption Both In Vitro and In Vivo. J . Bone Miner. Res.
2
(
1
ben zyloxycar bon yl-L-leu cin yl)car boh ydr azide (7): H NMR
(400 MHz, CDCl
3 3
/CD OD) δ 7.26 (m, 10H), 5.00 (q, 4H), 4.12
(
9
m, 1H), 4.04 (m, 1H), 1.78 (m, 1H), 1.55 (m, 4H), 0.86 (m,
+
H); MS (ESI) 579.3 (M + Na) . Anal. (C27
C, H, N.
-(N-Ben zyloxyca r b on yl-L-leu cin yl)-2′-(N-b en zyloxy-
H
36
N
6
O
7
2
‚0.5H O)
1
997, 12, 1396-1406.
2
(7) Yamashita, D. S.; Smith, W. W.; Zhao, B.; J anson, C. A.;
Tomaszek, T. A.; Bossard, M. J .; Levy, M. A.; Oh, H.-J .; Carr, T.
J .; Thompson, S. K.; Ijames, C. F.; Carr, S. A.; McQueney, M.
S.; D′Alessio, K. J .; Amegadzie, B. Y.; Hanning, C. R.; Abdel-
Meguid, S. S.; DesJ arlais, R. L.; Gleason, J . G.; Veber, D. F.
Structure and Design of Potent and Selective Cathepsin K
Inhibitors. J . Am. Chem. Soc. 1997, 119, 11351.
1
ca r bon yl-L-n or va lin yl)ca r boh yd r a zid e (8): H NMR (400
MHz, CDCl /CD OD) δ 7.24 (m, 10H), 5.00 (q, 4H), 4.12 (m,
H), 1.68 (m, 1H), 1.55 (m, 4H), 1.31 (m, 2H), 0.87 (m, 9H);
3
3
2
+
MS (ESI) 571.3 (M + H) . Anal. (C28
H
38
N
6
O
7
2
‚0.5H O) C, H,
N.
(
8) Burley, S. K.; Petsko, G. A. Aromatic-Aromatic Interaction: A
Ack n ow led gm en t. We thank Edith A. Reich for
conducting elemental analyses.
Mechanism of Protein Structure Stabilization. Science 1985, 229,
2
3-28.
Refer en ces
(
1) McKerrow, J . H., J ames, M. N. G., Eds. Perspect. Drug Discovery
Des. 1997, 6, 1-125.
J M980474X