Identification of selective, nonpeptidic nitrile inhibitors of cathepsin S using the substrate activity screening method

Article abstract of DOI:10.1021/jm060701s

The substrate activity screening method, a substrate-based fragment identification and optimization method for the development of enzyme inhibitors, was previously applied to cathepsin S to obtain low nanomolar 1,4-disubstituted-1,2,3-triazole-based aldeh

Full text of DOI:10.1021/jm060701s

6298
J. Med. Chem. 2006, 49, 6298-6307
Identification of Selective, Nonpeptidic Nitrile Inhibitors of Cathepsin S Using the Substrate
Activity Screening Method
Andrew W. Patterson,† Warren J. L. Wood,† Michael Hornsby,‡ Scott Lesley,‡ Glen Spraggon,*,‡ and Jonathan A. Ellman*,†
Department of Chemistry, UniVersity of California, Berkeley, California 94720, and Genomics Institute of the NoVartis Research Foundation,
10675 John Jay Hopkins DriVe, San Diego, California 92121
ReceiVed June 10, 2006
The substrate activity screening method, a substrate-based fragment identification and optimization method
for the development of enzyme inhibitors, was previously applied to cathepsin S to obtain low nanomolar
1,4-disubstituted-1,2,3-triazole-based aldehyde inhibitors (Wood, W. J. L.; Patterson, A. W.; Tsuruoka, H.;
Jain, R. K.; Ellman, J. A. J. Am. Chem. Soc. 2005, 127, 15521-15527). Replacement of the metabolically
labile aldehyde pharmacophore with the nitrile pharmacophore provided inhibitors with moderate potency
for cathepsin S. The inhibitors showed good selectivity over cathepsins B and L but no selectivity over
cathepsin K. X-ray structures of two crystal forms (1.5 and 1.9 Å) of a complex between cathepsin S and
a triazole inhibitor incorporating a chloromethyl ketone pharmacophore guided the design of triazole substrates
with increased cleavage efficiency and selectivity for cathepsin S over cathepsins B, L, and K. Conversion
of select substrates to nitrile inhibitors yielded a low molecular weight (414 Da) and potent (15 nM) cathepsin
S inhibitor that showed >1000-fold selectivity over cathepsins B, L, and K.
Scheme 1. Outline of the Substrate Activity Screening Method
Introduction
We recently disclosed the first substrate-based fragment
identification and optimization approach for the development
of novel, nonpeptidic protease inhibitors called substrate activity
screening (SAS; Scheme 1).1,2,3 In the first step of the three-
step process, a library of N-acyl aminocoumarins are screened
to identify protease substrates using a high throughput, continu-
ous fluorescence-based assay. False positives due to aggregation,
protein precipitation, or nonspecific binding are not observed
because active enzyme and productive active site binding are
required for the protease-catalyzed amide bond hydrolysis that
releases the fluorescent coumarin group.4 In step 2, the activity
of the substrates is rapidly optimized by the synthesis and
subsequent assay of focused libraries of substrate analogues.
Step 3 then builds upon a key attribute of this mechanism-based
substrate screen, namely, that the N-acyl aminocoumarin must
be precisely oriented in the active site to enable productive
substrate cleavage, and therefore, the aminocoumarin can be
replaced with mechanism-based pharmacophores to directly
provide protease inhibitors. The choice and versatility of
pharmacophores allows reversible or irreversible inhibitors to
be rapidly obtained once efficient substrates are identified.
We applied the SAS method to the cysteine protease cathepsin
S, which is involved in the final step of the degradation of the
invariant chain necessary for antigen presentation and subse-
quent immune response and is consequently implicated in
autoimmune diseases such as rheumatoid arthritis and multiple
sclerosis,5 as well as tumor development and growth.6 Multiple
distinct classes of nonpeptidic substrates to cathepsin S were
identified upon screening a N-acyl aminocoumarin library. Select
substrates were optimized and then directly converted to highly
novel, low molecular weight, nonpeptidic aldehyde inhibitors
with nanomolar affinity to cathepsin S.1
The 1,4-disubstituted-1,2,3-triazole-based aldehyde inhibitors
1 and 2 (Figure 1) were the most potent compounds to be
identified toward cathepsin S using the SAS method and
represent promising inhibitor structures as a result of their
novelty and nonpeptidic character. However, in our initial
publication, we did not address whether selective triazole-based
inhibitors could be obtained. Moreover, the aldehyde pharma-
cophore is not desirable because it is a very reactive electrophile
that may lack selectivity in vivo and is rapidly metabolized.
Here we report on the development of triazole-based inhibitors
with greater than 1000-fold selectivity for cathepsin S over the
highly homologous papain family human cathepsins B, K, and
L7 and that incorporate the more desirable, clinically validated
nitrile8 rather than the aldehyde pharmacophore.
Chemistry
The synthesis of 1,4-disubstituted-1,2,3-triazole substrates and
inhibitors required the preparation of enantiomerically pure
chiral propargylamine intermediates, which was accomplished
by a 1,2-addition of lithiated (trimethylsilyl)acetylene to N-tert-
butanesulfinyl ketimines 3 (Scheme 2).9,10 The additions
proceeded with uniformly high diastereoselectivities (97:3 to
>99:1 d.r.) to generate the sulfinamide intermediates 4. Chro-
matography provided diastereomerically pure material, which
after silyl and sulfinyl group removal gave the enantiomerically
pure propargylamine salts 6.
* Corresponding authors. E-mail: jellman@uclink.berkeley.edu (J.A.E.);
gspraggon@gnf.org (G.S.). Tel.: 510-642-4488 (J.A.E.); 858-812-1567
(G.S.). Fax: 510-642-8369 (J.A.E.); 858-812-1746 (G.S.).
† The University of California-Berkeley.
‡ Genomics Institute of the Novartis Research Foundation.
10.1021/jm060701s CCC: $33.50 © 2006 American Chemical Society
Published on Web 09/12/2006

SelectiVe Nitrile Inhibitors of Cathepsin S by SAS
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21 6299
Scheme 5a Synthesis of Chloromethyl Ketone Inhibitor 13
Figure 1. Lead 1,2,3-triazole aldehyde inhibitors identified for
cathepsin S.
a Conditions: (a) isobutyl chloroformate, N-methylmorpholine, THF, -25
°C; (b) diazomethane, THF, -25 °C; (c) HCl, CH3CO2H, THF, rt.
Scheme 2a Synthesis of Chiral Propargylamines
Table 1. Inhibition of Cathepsins B, K, L, and S by 1,2,3-Triazole
Nitrile Inhibitors
a Conditions: (a) Ti(OEt)4, THF, reflux; (b) AlMe3, (trimethylsilyl)ethy-
nyllithium, -78 °C to rt; (c) TBAF, THF, rt; (d) HCl, CH3OH, rt.
Scheme 3a Synthesis of 1,2,3-Triazole Substrates
a IC50 of inhibitor with enzyme was >10 µM.
produced the desired 1,2,3-triazole nitrile inhibitors 11 as single
diastereomers.
The synthesis of chloromethyl ketone inhibitor 13 was
accomplished by forming the mixed anhydride of 1,2,3-triazole
carboxylic acid 12 and isobutyl chloroformate. Addition of
diazomethane to form a diazomethyl ketone, followed by the
addition of HCl, yielded chloromethyl ketone 13 as a mixture
of diastereomers due to epimerization at the carbon alpha to
the ketone during the reaction (Scheme 5).
a Conditions: (a) 6, CuI, i-Pr2EtN, THF, rt; (b) R2CO2H, triphosgene,
THF, rt; (c) CF3CO2H, CH2Cl2, rt.
Results and Discussion
Scheme 4a Synthesis of 1,2,3-Triazole Nitrile Inhibitors
To begin probing the activity of nitrile inhibitors, the nitrile
analogues 11a and 11b of the most potent aldehyde inhibitors
1 and 2 were assayed against cathepsin S, as well as the related
cathepsins B, K, and L (Table 1). Replacing the aldehyde with
the nitrile pharmacophore resulted in a ∼45-fold drop in potency
for compounds 11a and 11b. Moreover, compounds 11a and
11b selectively inhibited cathepsin S over cathepsins B and L,
but no selectivity over cathepsin K was observed. Therefore,
the goal of inhibitor optimization was to improve potency and
increase selectivity for cathepsin S over cathepsin K.
In collaboration with the Genomics Institute of the Novartis
Research Foundation, a crystal structure of the chloromethyl
ketone analogue of 1 (compound 13) in complex with cathepsin
S was obtained to guide inhibitor optimization (Figure 2).
Crystallographic statistics are summarized in Table 2. Cathepsin
S shows a high degree of sequence and structure homology with
other cathepsins, including cathepsins B, K, and L. As the
functional differences between many of the cathepsins can
largely be attributed to tissue distribution, achieving specificity
can be challenging. To investigate the binding mode and
structural differences between the molecules, the X-ray crystal
structure of a complex between cathepsin S and compound 13
was solved in two different crystal forms at 1.5 and 1.9 Å,
respectively.12,13 The structure of cathepsin S conforms to the
canonical papain-like fold consisting of two equal-sized do-
mains, denoted left (L) and right (R), connected by an extensive
valley-like cleft containing the catalytic triad, Cys 25, His 164,
and Asn 184.14 The butyl, isopropyl, and benzamide groups of
a Conditions: (a) 6, CuI, i-Pr2EtN, THF, rt; (b) R2CO2H, triphosgene,
THF, rt; (c) CF3CO2H, CH2Cl2, rt; (d) 2,4,6-trichlorotriazine, DMF, rt.
For the synthesis of 1,4-disubstituted-1,2,3-triazole-based
substrates, the support-bound aminocoumarin acylated with
L-norleucine azido acid (7) was reacted with chiral propargy-
lamines 6 via a regioselective Cu(I)-catalyzed 1,3-dipolar
cycloaddition.11 Acylation of the resulting support-bound R-amino-
1,2,3-triazoles, followed by cleavage from the solid support,
yielded the 1,2,3-triazole substrates 8 as single diastereomers
(Scheme 3).
For the synthesis of 1,4-disubstituted-1,2,3-triazole-based
nitrile inhibitors, the chiral propargylamines 6 were reacted with
L-norleucine azido acid derivatized Sieber Amide resin 9 to give
support-bound 1,2,3-triazole amides (Scheme 4). Acylation of
the resulting support-bound R-amino-1,2,3-triazoles, followed
by cleavage from the solid support, yielded the 1,2,3-triazole
primary amides 10. Dehydration with 2,4,6-trichlorotriazine then

6300 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21
Patterson et al.
Figure 2. Crystal structure of chloromethyl ketone inhibitor 13 with
cathepsin S. The cathepsin S structure is shown as a stick representation
above which the semitransparent solvent excluded surface is placed.
The atoms are shaded according to element, with the protein carbons
colored yellow, the inhibitor carbons are shaded green, nitrogens are
blue, and oxygens are red. The figure was produced using Pymol
(www.pymol.org).
Table 2. Crystallographic Data for Cathepsin S in Complex with 13
cathepsin S
space group
crystal form 1
crystal form 2
P4122
P6522
unit cell dimensions (Å)
a ) b
c
85.2
150.8
165.9
142.1
wavelength (Å)
resolution (Å)
outer shell (Å)
1.0
1.5
1.0
1.9
Figure 3. Structural alignment of the cathepsin S structure with
cathepsins B, K, and L (PDB entries 1CSB, 1ICF, and 1TU6,
respectively). Numbering is according to the cathepsin S structure.
Residues colored green are conserved across all cathepsins, while those
in yellow are positively conserved. Regions that are boxed are
structurally conserved. Alpha helices are represented as blue tubes, while
beta sheets are magenta. The figure was produced using STAMP and
ALSCRIPT.15
1.53-1.48
0.073 (0.642)
27.4 (2.2)
92 453
(422 837)
99.3 (95.3)
0.19 (0.20)
3906
350
76
0.008
1.12
1.97-1.90
0.089 (0.75)
28.8 (2.1)
90 365
(526 303)
99.7 (100.0)
0.23 (0.25)
5182
369
89
0.014
1.366
Rmerge (%) (outer shell)
I/sigI (σ) (outer shell)
unique refs
(obsd)
completeness (%) (outer shell)
R
factor (Rfree *) (%)
no. protein atoms
no. water atoms
no. hetero atoms
rmsd bonds (Å)
rmsd bond angles
mean B-factor (Å2)
12.13
25.78
the triazole inhibitor occupy the S1, S2, and S3 enzyme pockets,
respectively, and Cys 25 has displaced the chlorine atom of 13
to form a covalent sulfur-carbon bond characteristic of mech-
anism-based chloromethyl inhibitors of cysteine proteases
(Figure 2). The aliphatic P1 moiety (butyl group) projects into
the solvent and does not form close hydrophobic contacts with
the surrounding protein. The S2 binding site is a large pocket
lined with hydrophobic residues, while the S3 binding site is a
shallow pocket. The structures of cathepsins K, L, and S are
extremely structurally homologous with root-mean-squared
deviations (rmsds) of 0.64 and 0.55 Å on 209 and 211 aligned
CR atoms, respectively. Cathepsin B is a more distant homo-
logue but is still very similar, with an rmsd of 1.23 Å on 187
aligned CR atoms (Figure 3). In general, specificity determinants
of the cathepsins are largely confined to the S1′, S1, and S2
pockets, with lesser contributions from the S3 pocket.
Figure 4. Comparison of specificity pockets with the structure of
cathepsin K (PDB ID: 1TU6). Coloring conventions are the same as
in Figure 2a, with the exception that the carbons of cathepsin K are
shaded cyan.
Despite the very high homology across the cathepsin family
members, this structure provided valuable insight from which
to attain selectivity. The S3 pocket, occupied by the benzamide
moiety, is a small pocket that should not accommodate
substitution on the aromatic ring. The flexible nature of Lys 64
(as indicated by the temperature factor for the residue), may
allow some movement to accommodate small moieties. Our
prior inhibitor SAR verifies this conclusion.1 In addition, the
substitution of negatively charged aspartates and glutamates in
structurally identical positions in cathepsins K and L may
provide some differential specificity (Figure 4). The S2 pocket,
however, provides the greatest opportunity for achieving
selectivity. This pocket is rather large and left unfilled by the
isopropyl group. Therefore, replacement of the isopropyl group
with larger, extended hydrophobic substituents should result in
increased potency as well as selectivity over cathepsin K, which

SelectiVe Nitrile Inhibitors of Cathepsin S by SAS
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21 6301
Table 3. Cleavage Efficiencies of 1,2,3-Triazole Substrates with
Table 4. . Steady State Kinetic Constants for Cathepsin S Acting on
Cathepsins B, K, L, and S
1,2,3-Triazole Substrates
Full enzymatic assay analysis of 1,2,3-triazole substrates (8a-
8e) with cathepsin S is shown in Table 4. It is interesting that,
for this substrate series, the increase in substrate-cleavage
efficiency is primarily due to gains in Km. In the optimization
of peptide-based mechanism-based inhibitors, increases in kcat
often contribute considerably to increases in substrate-cleavage
efficiency, even when modifications are made at sites distant
from the mechanism-based pharmacophore.17 Indeed, in our
previously reported optimization of the triazole-based substrates,
contributions from both Km and kcat were observed.1
Nitrile inhibitor analogues of compounds 8c, 8d, and 8e were
subsequently synthesized (11c, 11d, and 11e, respectively) and
their inhibitory activities toward cathepsins B, K, L, and S were
determined (Table 5). The plot of log(Ki) versus log(Km/kcat)
for cathepsin S shows excellent correlation between substrate
activity and inhibitor potency (Figure 5), which is consistent
with transition state theory and with previous correlations
between substrates and their corresponding mechanism-based
inhibitors.18
Considerable improvements in both potency and selectivity
of cathepsin S inhibitors were achieved upon introducing the
trans-4-methylcyclohexyl substituent, as predicted by the sub-
strate assay results. Specifically, a 28-fold increase in inhibitor
potency against cathepsin S was observed, with 11b having a
Ki of 420 nM and the optimized inhibitor 11e having a Ki of 15
nM. No significant inhibition of cathepsin B or L was observed
when assayed against any of the nitrile inhibitors. Most
importantly, the Ki value for 11e with cathepsin K is signifi-
cantly greater than 15 µM. Consequently, compound 11e shows
>1000-fold inhibitory selectivity for cathepsin S over all of the
closely related human cathepsins that were investigated.
a Relative cleavage efficiency values are given as kcat/Km relative to the
activity of substrate 8a with cathepsin S, which is assigned a value of 1.
b No activity detected when assayed with [substrate] ) 50 µM and [enzyme]
) 50 nM.
has a slightly smaller S2 pocket relative to cathepsin S created
by the substitution of larger hydrophobic residues at positions
162 (a leucine instead of a valine) and 137 (an alanine instead
of a glycine).16
As previously reported in the development of inhibitors 1
and 2 (Figure 1), outstanding correlation between substrate-
cleavage efficiency and inhibitory activity for the triazole-based
substrates and their corresponding mechanism-based inhibitors
was observed.2 Due to the greater ease of synthesizing and
evaluating substrates relative to their corresponding inhibitors,
substrate analogues were first prepared to evaluate the effect
of introducing larger R1 substituents on binding and selectivity
(Table 3). For substrates with the benzamide moiety, replace-
ment of the isopropyl group (8a) with the cyclohexyl group
(8c) at R1 resulted in a 3-fold increase in substrate-cleavage
efficiency. The same replacement at R1 with thiophene-
containing substrates (8b-8d) resulted in a 7-fold increase in
activity. In contrast, this substitution resulted in a modest drop
in substrate-cleavage efficiency for cathepsins B and K and only
a very modest increase for cathepsin L. Further extending the
R1 substituent by introducing 4-methyl substitution on the
cyclohexyl ring (8e and 8f) resulted in no detectable substrate
cleavage by cathepsin K, with modest reductions also being
observed for cathepsins B and L. Moreover, the trans-4-
methylcyclohexyl substitution, but not the cis-4-methylcyclo-
hexyl substitution (8e and 8f, respectively), provided an
additional increase in cleavage efficiency by cathepsin S,
resulting in an overall 10-fold improvement in cleavage ef-
ficiency relative to 8b, which incorporated an isopropyl group
at R1.
Conclusion
The 1,2,3-triazole nitrile inhibitors 11a and 11b were designed
based upon the corresponding aldehyde inhibitors 1 and 2 and
were determined to have moderate inhibitory activity and poor
selectivity for cathepsin S over cathepsin K. A crystal structure

6302 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21
Patterson et al.
Table 5. Inhibition of Cathepsins B, K, L, and S by 1,2,3-Triazole
Nitrile Inhibitors
none, and i-Pr2EtN, CH2Cl2, pyridine, toluene, Et2O, and CH3OH
were distilled under N2 over CaH2 immediately prior to use. (S)-
tert-Butanesulfinamide was provided by Suven Life Sciences
U.S.A., LLC (Monmouth Junction, NJ), and by AllyChem Co., Ltd
(Dalian, China). Low amine content DMF was purchased from
Acros, Wang resin and Sieber amide resin were purchased from
Novabiochem (San Diego, CA), and O-(7-azabenzotriazol-1-yl)-
N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU)a was
purchased from PerSeptive Biosystems (Foster City, CA). Fmoc-
protected 7-amino-4-methyl coumarin acetic acid (Fmoc-AMCA)
was synthesized according to a method analogous to the synthesis
of 7-amino-4-carbamoylmethylcoumarin (AMC).19 Norleucine azide
(N3-Nle-OH) was prepared from L-norleucine, according to literature
procedure.20 Compounds 1, 2, 8a, 8b, 12, and Fmoc-AMCA-Wang
resin were synthesized as previously reported.1 Compounds 3a-d
and 4a-d were synthesized according to reported procedures.10 All
solution-phase reactions were carried out in flame-dried glassware
under an inert N2 atmosphere. Normal-phase HPFC purification
was carried out on a Biotage SP1 instrument (Charlottesville, VA)
equipped with a Biotage Si flash column. Reverse-phase HPLC
analysis and purification were conducted with an Agilent 1100 series
instrument. Solid-phase reactions were conducted in polypropylene
cartridges equipped with 70 mm PE frits (Applied Separations,
Allentown, PA) and Teflon stopcocks and rocked on an orbital
1
shaker. H and 13C NMR spectra were obtained with Bruker AV-
300 and AV-400 spectrometers. Unless otherwise specified, all
spectra were obtained in CDCl3 and chemical shifts are reported
in ppm relative to internal CHCl3. Coupling constants are reported
in Hz. Elemental analyses and high-resolution mass spectrometry
analyses were performed by the University of California at Berkeley
Micro Analysis and Mass spectrometry Facilities.
a IC50 of inhibitor with enzyme is >10 µM. b IC50 of inhibitor with
enzyme is >15 µM.
Synthesis of Propargylamines (Scheme 2). Trimethylsilyl
Deprotection (5). To a 0.1 M solution of 410 (1 equiv) in THF
cooled in an ice-water bath was added tetrabutylammonium
fluoride (TBAF; 3 equiv). The solution was stirred for 2-4 h at
room temperature and then the solution was poured into saturated
aq NH4Cl with rapid stirring. The resulting suspension was
transferred to a separatory funnel and extracted three times with
Et2O. The combined organic portions were dried (Na2SO4), filtered,
and concentrated.
(Ss,S)-N-(1-Isopropyl-1-methyl-prop-2-ynyl)-2-methylpro-
panesulfinamide (5a). A mixture of 5.10 g of 4a (17.7 mmol) and
16.8 g of TBAF (53.2 mmol) was reacted in 180 mL of THF. Silica
gel chromatography (hexane/EtOAc ) 1:1) provided 5a (3.77 g,
99%) as a white solid: 1H NMR (400 MHz) δ 0.92 (d, J ) 6.8
Hz, 3H), 0.95 (d, J ) 6.8 Hz, 3H), 1.10 (s, 9H), 1.37 (s, 3H), 1.82
(septet, J ) 6.8 Hz, 1H), 2.37 (s, 1H), 3.18 (s, 1H); 13C NMR (100
MHz) δ 16.9, 17.4, 22.3, 25.0, 38.1, 55.8, 56.6, 72.8, 85.8. HRMS
(FAB+) m/z: (MH+) calcd for C11H22NOS, 216.1422; found,
216.1421.
Figure 5. Plot of log(Ki) vs log(Km/kcat) for 1,2,3-triazole substrates
and corresponding nitrile inhibitors with cathepsin S.
(Ss,S)-N-(1-Cyclohexyl-1-methyl-prop-2-ynyl)-2-methylpro-
panesulfinamide (5b). A mixture of 2.73 g of 4b (8.34 mmol)
and 7.89 g of TBAF (25.0 mmol) was reacted in 83 mL of THF.
HPFC purification (hexane/EtOAc ) 94:6 for 3 min, 94:6 to 50:
50 over 33 min) provided 5b (1.58 g, 74%) as a white solid: 1H
NMR (400 MHz) δ 0.99-1.27 (m, 5H), 1.17 (s, 9H), 1.44 (s, 3H),
1.47-1.55 (m, 1H), 1.62-1.68 (m, 1H), 1.75-1.82 (m, 2H), 1.87-
1.97 (m, 2H), 2.43 (s, 1H), 3.24 (s, 1H); 13C NMR (100 MHz) δ
22.5, 25.5, 26.17, 26.24, 27.0, 27.6, 48.2, 55.9, 56.5, 73.0, 86.3.
HRMS (FAB+) m/z: (MH+) calcd for C14H26NOS, 256.1735; found,
256.1728.
of cathepsin S, irreversibly inhibited by chloromethyl ketone
13, guided analogue design to improve potency and selectivity.
1,2,3-Triazole substrates 8 were first optimized for cleavage
efficiency, followed by the preparation and analysis of inhibitors
11 corresponding to select substrates 8. The nitrile inhibitor 11e
is a potent (Ki ) 15 nM) and low molecular weight (414 Da)
nonpeptidic inhibitor of cathepsin S. Additionally, 11e showed
>1000-fold selectivity over cathepsins B, K, and L. These
results demonstrate that the SAS method, which entails the
identification of novel, low molecular weight nonpeptidic
fragments by substrate screening, followed by substrate opti-
mization and conversion to inhibitors, can successfully lead to
the development of inhibitors that are novel, potent, and
selective.
(Ss,S)-N-[1-Methyl-1-(trans-4-methyl-cyclohexyl)-prop-2-ynyl]-
2-methylpropanesulfinamide (5c). A mixture of 0.69 g of 4c (2.0
mmol) and 1.6 g of TBAF (6.1 mmol) was reacted in 20 mL of
THF. HPFC purification (hexane/EtOAc ) 94:6 for 3 min, 94:6 to
a Abbreviation: AMC, 7-amino-4-carbamoylmethylcoumarin; AMCA,
7-amino-4-methyl coumarin acetic acid; E-64, L-trans-epoxysuccinyl-Leu-
4-guanidinobutylamide; E-64c, L-trans-epoxysuccinyl-Leu-3-methylbutyl-
amide; HATU, O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetra-methyluronium
hexafluorophosphate; HPFC, high-performance flash chromatography.
Experimental Section
General Synthesis Methods. Unless otherwise noted, all
reagents were obtained from commercial suppliers and used without
purification. THF was distilled under N2 from sodium/benzophe-

SelectiVe Nitrile Inhibitors of Cathepsin S by SAS
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21 6303
50:50 over 33 min) provided 5c (0.53 g, 97%) as a white solid:
1H NMR (400 MHz) δ 0.85 (d, J ) 6.4 Hz, 3H), 0.87-0.96 (m,
2H), 1.06-1.34 (m, 3H), 1.18 (s, 9H), 1.44 (s, 3H), 1.44-1.52
(m, 1H), 1.70-1.77 (m, 2H), 1.86-1.96 (m, 2H), 2.43 (s, 1H),
3.25 (s, 1H); 13C NMR (100 MHz) δ 22.3, 22.5, 25.7, 26.8, 27.5,
32.5, 34.8, 47.9, 55.9, 56.4, 73.0, 86.33. HRMS (FAB+) m/z:
(MH+) calcd for C15H28NOS, 270.1892; found, 270.1890.
(Ss,S)-N-[1-Methyl-1-(cis-4-methyl-cyclohexyl)-prop-2-ynyl]-
2-methylpropanesulfinamide (5d). A mixture of 1.0 g of 4d (2.9
mmol) and 2.3 g of TBAF (8.8 mmol) was reacted in 29 mL of
THF. HPFC purification (hexane/EtOAc ) 94:6 for 3 min, 94:6 to
50:50 over 33 min) provided 5d (0.78 g, 99%) as a white solid:
1H NMR (400 MHz) δ 0.94 (d, J ) 7.6 Hz, 3H), 1.20 (s, 9H),
1.31-1.44 (m, 2H), 1.46-1.60 (m, 8H), 1.62-1.72 (m, 2H), 1.90-
1.98 (m, 1H), 2.46 (s, 1H), 3.27 (s, 1H); 13C NMR (100 MHz) δ
17.3, 20.9, 21.5, 22.5, 25.7, 26.6, 31.50, 31.53, 48.4, 56.0, 56.6,
73.0, 86.3. HRMS (FAB+) m/z: (MH+) calcd for C15H28NOS,
270.1892; found, 270.1893.
Sulfinyl Cleavage (6). To a 0.15 M solution of 5 (1 equiv) in
CH3OH was added HCl (3 equiv) as a 4 M solution in 1,4-dioxane.
The solution was stirred for 1-2 h and concentrated to yield 6, the
propargylamine hydrochloride salts.
(S)-1-Isopropyl-1-methyl-prop-2-ynylamine Hydrochloride
(6a). A mixture of 3.77 g of 5a (17.5 mmol) and 13.0 mL of 4 M
HCl in dioxane (52.5 mmol) was reacted in 120 mL of CH3OH to
afford propargylamine 6a (2.55 g, 99%) as a white solid: 1H NMR
(CD3OD, 400 MHz) δ 0.93 (d, J ) 6.8 Hz, 3H), 0.97 (d, J ) 6.8
Hz, 3H), 1.60 (s, 3H), 2.10 (septet, J ) 6.8 Hz, 1H), 3.30 (s, 1H);
13C NMR (CD3OD, 100 MHz) δ 17.8, 17.9, 24.5, 37.7, 56.9, 78.1,
81.5. HRMS (FAB+) m/z: (MH+) calcd for C7H14N, 112.1126;
found, 112.1125.
(S)-1-Cyclohexyl-1-methyl-prop-2-ynylamine Hydrochloride
(6b). A mixture of 1.58 g of 5b (6.19 mmol) and 4.75 mL of 4 M
HCl in dioxane (19.0 mmol) was reacted in 41 mL of CH3OH to
afford propargylamine 6b (1.15 g, 99%) as a white solid: 1H NMR
(CD3OD, 400 MHz) δ 1.15-1.40 (m, 5H), 1.61 (s, 3H), 1.65-
1.78 (m, 2H), 1.82-1.96 (m, 4H), 3.32 (s, 1H); 13C NMR (CD3-
OD, 100 MHz) δ 24.6, 26.97, 27.01, 27.1, 28.3, 28.5, 47.1, 56.3,
78.0, 82.0. HRMS (FAB+) m/z: (MH+) calcd for C10H18N,
152.1439; found, 152.1443.
(S)-1-Methyl-1-(trans-4-methyl-cyclohexyl)-prop-2-ynyl-
amine Hydrochloride (6c). A mixture of 0.37 g of 5c (1.4 mmol)
and 1.0 mL of 4 M HCl in dioxane (4.1 mmol) was reacted in 9
mL of CH3OH to afford propargylamine 6c (0.27 g, 96%) as a
white solid: 1H NMR (CD3OD, 400 MHz) δ 0.92 (d, J ) 6.4 Hz,
3H), 0.94-1.06 (m, 2H), 1.23-1.41 (m, 3H), 1.59 (s, 3H), 1.60-
1.69 (m, 1H), 1.80-1.95 (m, 4H), 3.29 (s, 1H); 13C NMR (CD3-
OD, 100 MHz) δ 22.8, 24.7, 28.2, 28.4, 33.5, 35.58, 35.64, 46.8,
56.2, 78.1, 82.0. HRMS (FAB+) m/z: (MH+) calcd for C11H20N,
166.1596; found, 166.1590.
(S)-1-Methyl-1-(cis-4-methyl-cyclohexyl)-prop-2-ynylamine Hy-
drochloride (6d). A mixture of 0.75 g of 5d (2.8 mmol) and 2.1
mL of 4 M HCl in dioxane (8.3 mmol) was reacted in 19 mL of
CH3OH to afford propargylamine 6d (0.55 g, 97%) as a white
solid: 1H NMR (CD3OD, 400 MHz) δ 0.89 (d, J ) 7.6 Hz, 3H),
1.31-1.59 (m, 12H), 1.83-1.92 (m, 1H), 3.20 (s, 1H); 13C NMR
(CD3OD, 100 MHz) δ 17.6, 22.3, 22.4, 24.7, 27.8, 32.2, 32.3, 47.3,
56.3, 78.0, 81.9. HRMS (FAB+) m/z: (MH+) calcd for C11H20N,
166.1596; found, 166.1593.
Synthesis of 1,4-Disubstituted-1,2,3-triazole AMCA Sub-
strates (Scheme 3). Synthesis of N3-Nle-AMCA-Wang Resin (7).
A 20% solution of piperidine in DMF (30 mL) was added to a
cartridge containing Fmoc-AMCA-Wang resin (4.3 g, 0.49 mmol/
g). The mixture was shaken for 5 min, the solution was removed,
and the resin was washed with DMF (30 mL). The process was
repeated once, and the resin was washed with three portions of
DMF (30 mL). A 0.4 M solution of HATU (4.0 g, 11 mmol) in 26
mL of DMF with collidine (1.4 mL, 11 mmol) and N3-Nle-OH
(1.7 g, 11 mmol) was added to the resin, and the mixture was shaken
for 48 h. After removal of the solution, the resin was washed with
three portions (30 mL) each of DMF, THF, CH3OH, THF, and
CH2Cl2, and the solvent was removed in vacuo. Resin 7 was stored
at -20 °C until further use.
General Synthesis of Substrates (8). To resin 7 (0.60-0.66
mmol/g, 1 equiv), preswollen in THF, was added a 0.02 M solution
of propargylamine 6 (1-2 equiv) in THF, with i-Pr2EtN (100 equiv)
and CuI (3 equiv). The mixture was shaken for 20-48 h. After
removal of the solution, the resin was washed with three portions
(20 mL) each of THF, CH3OH, CH3CN, and THF. After solvent
removal, i-Pr2EtN (8 equiv) was added to the derivatized resin. To
a 0.1 M solution of carboxylic acid (3.5 equiv) in THF with
triphosgene (1.1 equiv) was added 2,4,6-collidine (10 equiv). The
resulting slurry was stirred for about 1 min and then added to the
cartridge containing the derivatized resin. The resulting mixture
was shaken for 4-12 h. After removal of the solution, the resin
was washed with THF (20 mL) and the coupling was repeated two
more times. After removal of the solution, the resin was washed
with three portions (20 mL) each of THF, CH3OH, THF, and CH2-
Cl2, and then the product was cleaved from the solid support [see
general procedure for cleavage]. The crude product mixture was
purified by HPLC [preparatory reverse-phase C18 column (24.1
× 250 mm), CH3CN/H2O-0.1% CF3CO2H ) 5:95 over 50 min;
10 mL/min; 254 nm detection for 60 min], extracted into EtOAc,
dried (Na2SO4), filtered, and concentrated. Due to the presence of
CF3CO2H in the purification solvents, AMCA substrates, 8, were
1
isolated with 0-1 equiv of CF3CO2H (by H NMR). Purity of all
substrate products was determined to be >95%, analyzed by HPLC.
General Procedure for Support Cleavage. The resin was
swollen in CH2Cl2. To the swollen resin was added a solution of
9:1 CH2Cl2/(95% CF3CO2H, 2.5% H2O, 2.5% triisopropylsilane),
and the mixture was shaken for 1-2 h. Upon removal of the
solution, the resin was washed with one portion of the cleavage
solution and three portions of CH2Cl2. The combined washes were
concentrated under reduced pressure.
Substrate 8c. A mixture of 75 mg of propargylamine 6b (0.40
mmol), 0.19 g of CuI (1.0 mmol), and 6.0 mL of i-Pr2EtN (33
mmol) in 34 mL of THF was added to 0.50 g of resin 7 (0.33
mmol). The support-bound amine intermediate was then acylated
using 0.15 g of benzoic acid (1.2 mmol), 0.11 g of triphosgene
(0.36 mmol), 0.44 mL of 2,4,6-collidine (3.3 mmol), and 0.45 mL
of i-Pr2EtN (2.6 mmol) in 12 mL of THF. Preparative HPLC
purification gave 30 mg of 8c (14%) as a sticky yellow solid: 1H
NMR (DMSO-d6, 400 MHz) δ 0.76-1.36 (m, 12H), 1.42-1.50
(m, 1H), 1.54-1.66 (m, 2H), 1.68-1.75 (m, 1H), 1.76 (s, 3H),
1.82-1.89 (m, 1H), 2.12-2.24 (m, 2H), 2.26-2.33 (m, 1H), 2.34
(s, 3H), 3.52 (s, 2H), 5.52 (dd, J ) 7.0, 9.2 Hz, 1H), 7.41-7.46
(m, 2H), 7.47-7.53 (m, 2H), 7.73-7.79 (m, 4H), 8.05 (s, 1H),
8.13 (s, 1H), 11.28 (s, 1H); 13C NMR (DMSO-d6, 100 MHz) δ
13.7, 14.9, 20.8, 21.5, 26.1, 26.2, 27.0, 27.2, 27.4, 31.6, 33.7, 45.0,
57.4, 63.4, 106.0, 115.7, 116.1, 119.3, 121.7, 126.1, 127.3, 128.1,
130.9, 135.7, 140.9, 148.0, 150.9, 152.2, 160.9, 166.1, 167.5, 171.9.
HRMS (FAB+) m/z: (MH+) calcd for C35H42N5O6, 628.3135;
found, 628.3145.
Substrate 8d. A mixture of 56 mg of propargylamine 6b (0.30
mmol), 0.11 g of CuI (0.58 mmol), and 5.2 mL of i-Pr2EtN (30
mmol) in 15 mL of THF was added to 0.34 g of resin 7 (0.20
mmol). The support-bound amine intermediate was then acylated
using 90 mg of thiophene-3-carboxylic acid (0.70 mmol), 65 mg
of triphosgene (0.22 mmol), 0.26 mL of 2,4,6-collidine (2.0 mmol),
and 0.28 mL of i-Pr2EtN (1.6 mmol) in 7 mL of THF. Preparative
HPLC purification gave 66 mg of 8d (52%) as a sticky yellow
solid: 1H NMR (DMSO-d6, 400 MHz) δ 0.78-1.36 (m, 12H),
1.38-1.44 (m, 1H), 1.55-1.66 (m, 2H), 1.68-1.74 (m, 4H), 1.80-
1.87 (m, 1H), 2.11-2.24 (m, 2H), 2.25-2.34 (m, 1H), 2.37 (s,
3H), 3.59 (s, 2H), 5.47 (dd, J ) 6.6, 9.0 Hz, 1H), 7.44 (dd, J )
1.2, 5.2 Hz, 1H), 7.49-7.55 (m, 2H), 7.75 (d, J ) 2.0 Hz, 1H),
7.79-7.83 (m, 2H), 8.10 (s, 1H), 8.16 (dd, J ) 1.2, 2.8 Hz), 11.12
(s, 1H), 12.55 (br s, 1H); 13C NMR (DMSO-d6, 100 MHz) δ 13.7,
14.9, 20.7, 21.5, 26.1, 26.2, 26.9, 27.2, 27.5, 31.6, 32.9, 45.0, 57.3,
63.4, 106.0, 115.7, 115.9, 118.3, 118.8, 121.7, 126.3, 127.2, 128.5,

6304 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21
Patterson et al.
138.6, 141.0, 148.6, 151.0, 152.2, 160.8, 161.7, 167.5, 171.4. HRMS
(FAB+) m/z: (MH+) calcd for C33H40N5O6S, 634.2699; found,
634.2682.
Cl2, and then the product was cleaved from the solid support [see
general procedure for cleavage]. Primary amides, 10, were purified
by silica gel chromatography (hexane/EtOAc ) 1:7) or by HPFC
(hexane/EtOAc ) 88:12 for 2 min, 88:12 to 0:100 over 19 min).
Amide 10a. A mixture of 0.11 g of propargylamine 6a (0.75
mmol), 0.29 g of CuI (1.5 mmol), and 8.7 mL of i-Pr2EtN (50
mmol) in 38 mL of THF was added to 0.42 g of resin 9 (0.50
mmol). The support-bound amine intermediate was then acylated
using 0.21 g of benzoic acid (1.8 mmol), 0.16 g of triphosgene
(0.55 mmol), 0.66 mL of 2,4,6-collidine (5.0 mmol), and 0.70 mL
of i-Pr2EtN (4.0 mmol) in 18 mL of THF. HPFC purification
provided 10a (62 mg, 33%) as a yellow solid: 1H NMR (400 MHz)
δ 0.79-0.88 (m, 6H), 0.97 (d, J ) 6.8 Hz, 3H), 1.06-1.16 (m,
1H), 1.20-1.38 (m, 3H), 1.88 (s, 3H), 2.10-2.28 (m, 2H), 2.85
(septet, J ) 6.8 Hz, 1H), 5.09 (dd, J ) 6.0, 9.6 Hz, 1H), 6.12 (br
s, 1H), 6.63 (br s, 1H), 7.19 (s, 1H), 7.38-7.42 (m, 2H), 7.47 (t,
J ) 7.2 Hz, 1H), 7.74 (d, J ) 7.6 Hz, 1H), 7.78 (s, 1H); 13C NMR
(100 MHz) δ 13.7, 17.3, 17.6, 21.4, 21.9, 27.9, 32.4, 34.9, 58.3,
64.4, 122.3, 126.8, 128.6, 131.4, 135.2, 150.7, 166.9, 170.8. HRMS
(FAB+) m/z: (MH+) calcd for C20H30N5O2, 372.2400; found,
372.2393.
Amide 10b. A mixture of 0.30 g of propargylamine 6a (2.0
mmol), 0.57 g of CuI (3.0 mmol), and 17 mL of i-Pr2EtN (100
mmol) in 100 mL of THF was added to 0.90 g of resin 9 (0.50
mmol). The support-bound amine intermediate was then acylated
using 0.64 g of thiophene-3-carboxylic acid (5.0 mmol), 0.33 g of
triphosgene (1.1 mmol), 1.3 mL of 2,4,6-collidine (10 mmol), and
1.4 mL of i-Pr2EtN (8.0 mmol) in 50 mL of THF. Chromatography
provided 10b (144 mg, 38%) as a yellow solid: 1H NMR (400
MHz) δ 0.77 (d, J ) 6.8 Hz, 3H), 0.81 (t, J ) 7.0 Hz, 3H), 0.93
(d, J ) 6.8 Hz, 3H), 1.03-1.15 (m, 1H), 1.16-1.35 (m, 3H), 1.84
(s, 3H), 2.02-2.25 (m, 2H), 2.79 (septet, J ) 6.8 Hz, 1H), 5.19
(dd, J ) 6.2, 9.4 Hz, 1H), 6.45 (br s, 1H), 7.00 (br s, 1H), 7.24-
7.28 (m, 2H), 7.37 (dd, J ) 1.2, 5.2 Hz, 1H), 7.78 (s, 1H), 7.85
(dd, J ) 1.2, 3.0 Hz, 1H); 13C NMR (100 MHz) δ 13.6, 17.2, 17.5,
21.3, 21.8, 27.7, 32.4, 34.9, 58.2, 64.0, 121.7, 126.1, 126.3, 128.0,
138.2, 150.6, 162.4, 170.7; HRMS (FAB+) m/z: (MH+) calcd for
C18H28N5O2S, 378.1964; found, 378.1961.
Substrate 8e. A mixture of 61 mg of propargylamine 6c (0.30
mmol), 0.11 g of CuI (0.58 mmol), and 5.2 mL of i-Pr2EtN (30
mmol) in 15 mL of THF was added to 0.34 g of resin 7 (0.20
mmol). The support-bound amine intermediate was then acylated
using 90 mg of thiophene-3-carboxylic acid (0.70 mmol), 65 mg
of triphosgene (0.22 mmol), 0.26 mL of 2,4,6-collidine (2.0 mmol),
and 0.28 mL of i-Pr2EtN (1.6 mmol) in 7 mL of THF. Preparative
HPLC purification gave 48 mg of 8e (37%) as a sticky yellow
1
solid: H NMR (DMSO-d6, 400 MHz) δ 0.74-0.85 (m, 7H), 0.85-
1.34 (m, 8H), 1.36-1.44 (m, 1H), 1.55-1.63 (m, 1H), 1.65-1.71
(m, 1H), 1.73 (s, 3H), 1.78-1.84 (m, 1H), 2.09-2.28 (m, 3H),
2.31 (s, 3H), 3.41 (s, 2H), 5.47-5.54 (m, 1H), 7.44 (dd, J ) 1.2,
5.2 Hz, 1H), 7.48 (dd, J ) 2.0, 8.8 Hz, 1H), 7.54 (dd, J ) 2.8, 5.2
Hz, 1H), 7.68-7.72 (m, 2H), 7.82 (s, 1H), 8.11 (s, 1H), 8.17 (dd,
J ) 1.2, 2.8 Hz, 1H), 11.34 (s, 1H); 13C NMR (DMSO-d6, 100
MHz) δ 14.2, 15.3, 21.2, 21.9, 22.9, 27.1, 27.4, 27.9, 32.0, 32.7,
35.3, 35.7, 45.1, 57.7, 63.7, 106.4, 116.0, 116.8, 121.4, 122.2, 126.2,
126.8, 127.7, 129.0, 139.0, 140.9, 147.3, 151.4, 152.5, 161.5, 162.1,
167.9, 173.0. HRMS (FAB+) m/z: (MH+) calcd for C34H42N5O6S,
648.2856; found, 648.2855.
Substrate 8f. A mixture of 61 mg of propargylamine 6d (0.30
mmol), 0.11 g of CuI (0.58 mmol), and 5.2 mL of i-Pr2EtN (30
mmol) in 15 mL of THF was added to 0.34 g of resin 7 (0.20
mmol). The support-bound amine intermediate was then acylated
using 90 mg of thiophene-3-carboxylic acid (0.70 mmol), 65 mg
of triphosgene (0.22 mmol), 0.26 mL of 2,4,6-collidine (2.0 mmol),
and 0.28 mL of i-Pr2EtN (1.6 mmol) in 7 mL of THF. Preparative
HPLC purification gave 72 mg of 8f (56%) as a sticky yellow
solid: 1H NMR (DMSO-d6, 400 MHz) δ 0.77 (d, J ) 7.2 Hz, 3H),
0.81 (t, J ) 7.2 Hz, 3H), 0.93-1.62 (m, 12H), 1.76 (s, 3H), 1.80-
1.88 (m, 1H), 2.13-2.30 (m, 3H), 2.32 (s, 3H), 3.47 (s, 2H), 5.48-
5.54 (m, 1H), 7.44 (d, J ) 1.2, 5.2 Hz, 1H), 7.49 (dd, J ) 1.8, 8.4
Hz, 1H), 7.54 (dd, J ) 2.8, 5.2 Hz, 1H), 7.71-7.76 (m, 2H), 7.83
(s, 1H), 8.12 (s, 1H), 8.16 (dd, J ) 1.2, 2.8 Hz, 1H), 11.27 (s, 1H);
13C NMR (DMSO-d6, 100 MHz) δ 14.2, 15.3, 17.5, 21.1, 21.3,
21.5, 21.9, 26.7, 27.9, 31.9, 34.8, 45.6, 57.9, 63.8, 106.4, 116.0,
116.6, 120.4, 122.2, 126.4, 126.8, 127.6, 129.0, 139.1, 141.2, 148.0,
151.1, 152.5, 161.4, 162.1, 167.9, 172.7. HRMS (FAB+) m/z:
(MH+) calcd for C34H42N5O6S, 648.2856; found, 648.2849.
Synthesis of 1,4-Disubstituted-1,2,3-triazole Inhibitors (Scheme
4). Synthesis of N3-Nle-Sieber Amide Resin (9). A 20% solution
of piperidine in DMF (30 mL) was added to a cartridge containing
Sieber Amide resin (1.9 g, 100-200 mesh, 0.52 mmol/g). The
mixture was shaken for 5 min, the solution was removed, and the
resin was washed with DMF (30 mL). The process was repeated
once, and the resin was washed with three portions of DMF (30
mL). A 0.4 M solution of HATU (1.9 g, 5.0 mmol) in 13 mL of
DMF with collidine (0.66 mL, 5.0 mmol) and N3-Nle-OH (0.79 g,
5.0 mmol) was added to the resin, and the mixture was shaken for
48 h. After removal of the solution, the resin was washed with
three portions (30 mL) each of DMF, THF, CH3OH, THF, and
CH2Cl2, and the solvent was removed in vacuo. Resin 9 was stored
at -20 °C until further use.
General Synthesis of Primary Amides (10). To resin 9 (0.83
mmol/g, 1 equiv), preswollen in THF, was added a 0.02 M solution
of propargylamine 6 (1-2 equiv) in THF, with i-Pr2EtN (100 equiv)
and CuI (3 equiv). The mixture was shaken for 20-48 h. After
removal of the solution, the resin was washed with three portions
(20 mL) each of THF, CH3OH, CH3CN, and THF. After solvent
removal, i-Pr2EtN (8 equiv) was added to the derivatized resin. To
a 0.1 M solution of carboxylic acid (3-5 equiv) in THF with
triphosgene (1.1 equiv) was added 2,4,6-collidine (10 equiv). The
resulting slurry was stirred for about 1 min and then added to the
cartridge containing the derivatized resin. The resulting mixture
was shaken for 4-12 h. After removal of the solution, the resin
was washed once with THF (20 mL), and the coupling was repeated
two more times. After removal of the solution, the resin was washed
with three portions (20 mL) each of THF, CH3OH, THF, and CH2-
Amide 10c. A mixture of 0.38 g of propargylamine 6b (2.0
mmol), 0.57 g of CuI (3.0 mmol), and 17 mL of i-Pr2EtN (100
mmol) in 100 mL of THF was added to 0.83 g of resin 9 (1.0
mmol). The support-bound amine intermediate was then acylated
using 0.43 g of benzoic acid (3.5 mmol), 0.33 g of triphosgene
(1.1 mmol), 1.3 mL of 2,4,6-collidine (10 mmol), and 1.4 mL of
i-Pr2EtN (8.0 mmol) in 35 mL of THF. Chromatography provided
1
10c (147 mg, 37%) as a yellow solid: H NMR (400 MHz) δ 0.76-
1.39 (m, 12H), 1.56-1.84 (m, 5H), 1.87 (s, 3H), 2.05-2.25 (m,
2H), 2.33-2.42 (m, 1H), 5.13 (app t, J ) 7.6 Hz, 1H), 6.39 (br s,
1H), 6.93 (br s, 1H), 7.31 (s, 1H), 7.38 (app t, J ) 7.4 Hz, 2H),
7.45 (t, J ) 7.4 Hz, 1H), 7.74 (d, J ) 7.6 Hz, 2H), 7.76 (s, 1H);
13C NMR (100 MHz) δ 13.7, 21.7, 21.9, 26.2, 26.3, 27.5, 27.8,
32.4, 45.3, 58.0, 64.1, 122.1, 126.8, 128.5, 131.4, 135.2, 151.0,
166.9, 171.0. HRMS (FAB+) m/z: (MH+) calcd for C23H34N5O2,
412.2713; found, 412.2703.
Amide 10d. A mixture of 0.38 g of propargylamine 6b (2.0
mmol), 0.57 g of CuI (3.0 mmol), and 17 mL of i-Pr2EtN (100
mmol) in 100 mL of THF was added to 0.90 g of resin 9 (0.50
mmol). The support-bound amine intermediate was then acylated
using 0.64 g of thiophene-3-carboxylic acid (5.0 mmol), 0.33 g of
triphosgene (1.1 mmol), 1.3 mL of 2,4,6-collidine (10 mmol), and
1.4 mL of i-Pr2EtN (8.0 mmol) in 50 mL of THF. Chromatography
provided 10d (72 mg, 17%) as a yellow solid: 1H NMR (400 MHz)
δ 0.72-1.03 (m, 6H), 1.08-1.40 (m, 6H), 1.58-1.85 (m, 5H), 1.88
(s, 3H), 2.12-2.20 (m, 1H), 2.23-2.34 (m, 1H), 2.38-2.47 (m,
1H), 5.09 (dd, J ) 6.0, 9.2 Hz, 1H), 5.86 (br s, 1H), 6.49 (br s,
1H), 7.14 (s, 1H), 7.28-7.34 (m, 1H), 7.38 (d, J ) 5.2 Hz, 1H),
7.66 (s, 1H), 7.85 (d, J ) 2.0 Hz, 1H); 13C NMR (100 MHz) δ
13.7, 21.9, 22.3, 26.29, 26.32, 26.4, 27.68, 27.71, 27.9, 32.5, 45.1,
58.2, 64.6, 121.9, 126.1, 126.4, 127.9, 138.5, 151.2, 162.2, 170.4.
HRMS (FAB+) m/z: (MH+) calcd for C21H32N5O2S, 418.2276;
found, 418.2275.

SelectiVe Nitrile Inhibitors of Cathepsin S by SAS
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21 6305
Amide 10e. A mixture of 0.15 g of propargylamine 6c (0.75
mmol), 0.29 g of CuI (1.5 mmol), and 8.7 mL of i-Pr2EtN (50
mmol) in 38 mL of THF was added to 0.42 g of resin 9 (0.50
mmol). The support-bound amine intermediate was then acylated
using 0.22 g of thiophene-3-carboxylic acid (1.8 mmol), 0.16 g of
triphosgene (0.55 mmol), 0.66 mL of 2,4,6-collidine (5.0 mmol),
and 0.70 mL of i-Pr2EtN (4.0 mmol) in 18 mL of THF. HPFC
purification provided 10e (70 mg, 32%) as a yellow solid: 1H NMR
(400 MHz) δ 0.77-0.85 (m, 6H), 0.86-1.05 (m, 4H), 1.06-1.36
(m, 5H), 1.54-1.76 (m, 3H), 1.79-1.87 (m, 4H), 2.12-2.27 (m,
2H), 2.28-2.37 (m, 1H), 5.04-5.13 (m, 1H), 6.40 (br s, 1H), 6.80
(br s, 1H), 7.04 (s, 1H), 7.27-7.33 (m, 2H), 7.77-7.84 (m, 2H);
13C NMR (100 MHz) δ 13.7, 21.6, 21.9, 22.3, 27.3, 27.5, 27.9,
32.3, 32.6, 34.8, 34.9, 45.0, 57.9, 64.3, 122.7, 125.9, 126.5, 128.5,
137.9, 151.0, 162.6, 171.8. HRMS (FAB+) m/z: (MH+) calcd for
C22H34N5O2S, 432.2433; found, 432.2428.
General Synthesis of Nitrile Inhibitors (11). A 0.15 M solution
of amide 10 (1 equiv) in DMF was cooled in an ice-water bath
and 2,4,6-trichlorotriazine (1 equiv) was added. The solution was
stirred for 2 h at room temperature before being added to 25 mL
of EtOAc. The solution was transferred to a separatory funnel and
washed three times with H2O, dried (Na2SO4), filtered, and
concentrated. The inhibitors, 11, were purified by HPLC [prepara-
tory reverse-phase C18 column (24.1 × 250 mm), CH3CN/H2O-
0.1% CF3CO2H ) 5:95 over 50 min; 10 mL/min; 254 nm detection
for 60 min] or by HPFC (hexane/EtOAc ) 94:6 for 1 min, 94:6 to
0:100 over 10 min).
Inhibitor 11a. A mixture of 45 mg of amide 10a (0.12 mmol)
and 22 mg of 2,4,6-trichlorotriazine (0.12 mmol) was reacted in
0.8 mL of DMF. HPFC purification provided 11a (27 mg, 64%)
as a yellow solid: 1H NMR (400 MHz) δ 0.81 (d, J ) 6.8 Hz,
3H), 0.91 (t, J ) 7.0 Hz, 3H), 0.97 (d, J ) 6.8 Hz, 3H), 1.33-1.49
(m, 4H), 1.89 (s, 3H), 2.19-2.33 (m, 2H), 2.90 (septet, J ) 6.8
Hz, 1H), 5.52 (app t, J ) 7.6 Hz, 1H), 7.11 (s, 1H), 7.38-7.44 (m,
2H), 7.45-7.51 (m, 1H), 7.74-7.79 (m, 3H); 13C NMR (100 MHz)
δ 13.6, 17.3, 17.5, 21.4, 21.6, 27.2, 34.1, 34.8, 51.1, 58.3, 115.5,
120.5, 126.8, 128.5, 131.4, 135.3, 151.7, 166.6. HRMS (FAB+)
m/z: (MH+) calcd for C20H28N5O, 354.2294; found, 354.2294. Anal.
(C20H27N5O) C, H, N.
Inhibitor 11b. A mixture of 40 mg of amide 10b (0.11 mmol)
and 20 mg of 2,4,6-trichlorotriazine (0.11 mmol) was reacted in
0.8 mL of DMF. HPFC purification provided 11b (32 mg, 81%)
as a yellow solid: 1H NMR (DMSO-d6, 400 MHz) δ 0.72 (d, J )
6.8 Hz, 3H), 0.82 (t, J ) 7.1 Hz, 3H), 0.90 (d, J ) 6.8 Hz, 3H),
1.09-1.34 (m, 4H), 1.68 (s, 3H), 2.12-2.27 (m, 2H), 2.60-2.70
(m, 1H), 6.08 (app t, J ) 7.6 Hz, 1H), 7.44 (dd, J ) 1.2, 5.0 Hz,
1H), 7.55 (dd, J ) 2.9, 5.0 Hz, 1H), 7.87 (s, 1H), 8.16 (dd, J )
1.3, 2.9 Hz, 1H), 8.19 (s, 1H); 13C NMR (DMSO-d6, 100 MHz) δ
13.8, 17.2, 17.6, 19.8, 21.2, 26.8, 33.0, 34.8, 50.0, 57.3, 117.2,
122.7, 126.5, 127.4, 128.8, 138.5, 151.5, 162.0. HRMS (FAB+)
m/z: (MH+) calcd for C18H26N5OS, 360.1858; found, 360.1859.
Anal. (C18H25N5OS) C, H, N.
1H); 13C NMR (100 MHz) δ 13.6, 21.6, 22.2, 26.3, 26.4, 27.3,
27.6, 27.7, 29.7, 34.2, 45.1, 51.2, 58.2, 115.5, 120.3, 126.1, 126.4,
127.9, 138.4, 152.2, 162.2. HRMS (FAB+) m/z: (MH+) calcd for
C21H29N5OS, 400.2171; found, 400.2170.
Inhibitor 11e. A mixture of 52 mg of amide 10e (0.12 mmol)
and 22 mg of 2,4,6-trichlorotriazine (0.12 mmol) was reacted in
0.8 mL of DMF. HPFC purification provided 11e (20 mg, 40%) as
a yellow solid: 1H NMR (400 MHz) δ 0.83 (d, J ) 6.4 Hz, 3H),
0.86-1.05 (m, 7H), 1.12-1.24 (m, 1H), 1.34-1.50 (m, 4H), 1.55-
1.85 (m, 4H), 1.88 (s, 3H), 2.18-2.33 (m, 2H), 2.37-2.46 (m,
1H), 5.52 (app t, J ) 7.6 Hz, 1H), 7.01 (s, 1H), 7.31 (dd, J ) 2.8,
5.2 Hz, 1H), 7.38 (dd, J ) 1.2, 5.2 Hz, 1H), 7.71 (s, 1H), 7.84 (dd,
J ) 1.2, 2.8 Hz, 1H); 13C NMR (100 MHz) δ 13.6, 21.6, 22.2,
22.3, 27.2, 27.4, 32.6, 34.2, 34.8, 34.9, 44.7, 51.1, 58.0, 115.5,
120.4, 126.0, 126.4, 127.9, 138.4, 152.1, 162.2. HRMS (FAB+)
m/z: (MH+) calcd for C22H32N5OS, 414.2328; found, 414.2335.
Anal. (C22H31N5OS) C, H, N.
Synthesis of Chloromethyl Ketone Inhibitor (13). According
to literature procedure,22 a 0.2 M solution of 1,2,3-triazole car-
boxylic acid 12 (90 mg, 0.24 mmol) in 1.2 mL of THF was stirred
at -25 °C. To this solution were added 39 µL of N-methylmor-
pholine (0.34 mmol) and then 41 µL of isobutyl chloroformate (0.31
mmol). After addition of the chloroformate, a white solid formed.
After 1 h, the reaction mixture was filtered and the solid was rinsed
with 4 mL of ice-cold THF. The reaction mixture was again stirred
at -25 °C until filtration resulted in a clear, colorless solution.
Excess diazomethane, prepared from p-toluenesulfonylmethylnit-
rosamide (Diazald), was introduced in situ, according to literature
procedure,23 while the flask was maintained at -25 °C. After the
addition of diazomethane, the solution was stirred at -25 °C for
0.5 h and then at room temperature for 0.5 h. In an ice-water bath,
a 1:1 solution of concentrated hydrochloric acid and glacial acetic
acid was then added dropwise to the solution until the evolution of
N2 was no longer observed. The solution was diluted with EtOAc
(15 mL) and transferred to a separatory funnel where it was washed
with water (5 mL), satd NaHCO3 (5 mL), and water (5 mL) until
the final water wash was pH neutral. The organic layer was dried
(Na2SO4), concentrated, and purified by silica gel chromatography
(Et2O/CH2Cl2 ) 3:97 to 20:80). The 1H NMR data corresponds to
a 1:1 mixture of diastereomers, with about 5% of the methyl ester
product observed. 1H NMR (300 MHz) δ 0.78-1.02 (m, 9H), 1.10-
1.23 (m, 1H), 1.23-1.47 (m, 3H), 1.94 (s, 3H), 2.08-2.20 (m,
1H), 2.22-2.36 (m, 1H), 2.90-3.05 (m, 1H), 4.17-4.22 (m, 2H),
5.54 (dd, J ) 5.1, 10.1 Hz, 1H), 7.29-7.38 (m, 1H), 7.40-7.55
(m, 3H), 7.68-7.76 (m, 1H), 7.77-7.89 (m, 2H). HRMS (FAB+)
m/z: (MH+) calcd for C21H30ClN4O2, 405.2057; found, 405.2052.
General Assay Procedure. Cathepsins B, K, L, and S were
purchased from Calbiochem (San Diego, CA). Cbz-Leu-Arg-AMC,
Cbz-Phe-Arg-AMC, l-trans-epoxysuccinyl-Leu-4-guanidinobuty-
lamide (E-64), and l-trans-epoxysuccinyl-Leu-3-methylbutylamide
(E-64c) were purchased from Bachem (Torrance, CA). The pro-
teolytic cleavage of N-acyl aminocoumarins by cathepsins B, K,
L, and S was conducted in Dynatech Microfluor fluorescence 96-
well microtiter plates, and readings were taken on a Molecular
Devices Spectra Max Gemini XS instrument. The excitation
wavelength was 370 nm and the emission wavelength was 455 nm,
with a cutoff of 435 nm for AMCA substrates; the excitation
wavelength was 355 nm and the emission wavelength was 450 nm
for peptidyl-AMC substrates. The assay buffer consisted of a 100
mM solution of pH 6.1 sodium phosphate buffer with 100 mM of
sodium chloride, 1 mM of DTT, 1 mM of EDTA, and 0.001% of
Tween-20.
Inhibitor 11c. A mixture of 100 mg of amide 10c (0.24 mmol)
and 44 mg of 2,4,6-trichlorotriazine (0.24 mmol) was reacted in
1.7 mL of DMF. HPLC purification provided 11c (39 mg, 41%)
as a yellow solid: 1H NMR (400 MHz) δ 0.82-1.11 (m, 6H),
1.15-1.59 (m, 7H), 1.63-1.75 (m, 2H), 1.77-1.88 (m, 2H), 1.89
(s, 3H), 2.20-2.33 (m, 2H), 2.38-2.48 (m, 1H), 5.53 (app t, J )
7.4 Hz, 1H), 7.21 (s, 1H), 7.42 (app t, J ) 7.4 Hz, 2H), 7.50 (t, J
) 7.4 Hz, 1H), 7.74 (d, J ) 7.6 Hz, 2H), 7.77 (s, 1H); 13C NMR
(100 MHz) δ 13.6, 21.3, 21.6, 26.2, 26.3, 27.2, 27.5, 27.6, 34.1,
45.3, 51.3, 58.1, 115.4, 120.9, 126.8, 128.6, 131.7, 134.7, 151.9,
167.5. HRMS (FAB+) m/z: (MH+) calcd for C24H32N5O, 394.2607;
found, 394.2611. Anal. (C21H30N5OS) C, H, N.
Inhibitor 11d. A mixture of 50 mg of amide 10d (0.12 mmol)
and 22 mg of 2,4,6-trichlorotriazine (0.12 mmol) was reacted in
0.8 mL of DMF. HPLC purification provided 11d (19 mg, 39%)
as a yellow solid:21 1H NMR (DMSO-d6, 300 MHz) δ 0.77-1.46
(m, 13H), 1.56-1.90 (m, 7H), 2.13-2.33 (m, 3H), 6.07 (app t, J
) 10.0 Hz, 1H), 7.44 (dd, J ) 1.2, 4.8 Hz, 1H), 7.54 (dd, J ) 2.8,
4.8 Hz, 1H), 7.86 (s, 1H), 8.16 (dd, J ) 1.2, 2.8 Hz, 1H), 8.18 (s,
Assay Procedure for 1,2,3-Triazole AMCA Substrates. Assays
were conducted at 37 °C in duplicate with and without the enzyme.
In each well was placed 38 µL of enzyme solution and 2 µL of a
DMSO substrate solution. Assays were performed at substrate
concentrations that were at minimum 10-times less than the Km
for that substrate. Relative fluorescent units (RFU) were measured
at regular intervals over a period of time (maximum 15 min). A
plot of RFU versus time was made for each substrate with and
without cathepsin S. The slope of the plotted line gave Vmax/Km of

6306 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21
Patterson et al.
each substrate for each cathepsin. Using the RFU per µM for
AMCA, substrate concentration, and enzyme concentration (deter-
mined by E-64 titration for cathepsins B, K, and L or E-64c
titration for cathepsin S),24 kcat/Km was determined.
in the Protein Data Bank; PDB ID: 2H7J and 2HXZ. This material
is available free of charge via the Internet at http://pubs.acs.org.
References
To determine the Km and kcat of substrates with cathepsin S,
assays were conducted at 37 °C in duplicate with and without the
enzyme at six substrate concentrations above and below the Km of
each substrate. In each well was placed 190 µL of enzyme solution
and 10 µL of a DMSO substrate solution. RFU were measured at
regular intervals over a period of time (maximum 15 min). A plot
of RFU/s versus substrate concentration, analyzed using Kaleida-
Graph, was used to determine Km and Vmax. Using the RFU per
µM for AMCA and the cathepsin S concentration (determined by
E-64c titration),24 kcat was determined.
(1) Wood, W. J. L.; Patterson, A. W.; Tsuruoka, H.; Jain, R. K.; Ellman,
J. A. Substrate Activity Screening: A fragment-based method for
the rapid identification of nonpeptidic protease inhibitors. J. Am.
Chem. Soc. 2005, 127, 15521-15527.
(2) Salisbury, C. M.; Ellman, J. A. Rapid identification of potent
nonpeptidic serine protease inhibitors. ChemBioChem 2006, 7, 1034-
1037.
(3) For recent reviews on fragment-based methods, see: (a) Erlanson,
D. A.; McDowell, R. S.; O’Brien, T. Fragment-based drug discovery.
J. Med. Chem. 2004, 47, 3463-3482. (b) Carr, R. A. E.; Congreve,
M.; Murray, C. W.; Rees, D. C. Fragment-based lead discovery:
Leads by design. Drug DiscoVery Today 2005, 10, 987-992. (c)
Hartshorn, M. J.; Murray, C. W.; Cleasby, A.; Frederickson, M.;
Tickle, I. J.; Jhoti, H. Fragment-based lead discovery using X-ray
crystallography. J. Med. Chem. 2005, 48, 403-413.
(4) (a) McGovern, S. L.; Caselli, E.; Grigorieff, N.; Shoichet, B. K. A
common mechanism underlying promiscuous inhibitors from virtual
and high-throughput screening. J. Med. Chem. 2002, 45, 1712-1722.
(b) McGovern, S. L.; Helfand, B. T.; Feng, B.; Shoichet, B. K. A
specific mechanism of nonspecific inhibition. J. Med. Chem. 2003,
46, 4265-4272. (c) McGovern, S. L.; Shoichet, B. K. Kinase
inhibitors: Not just for kinases anymore. J. Med. Chem. 2003, 46,
1478-1483.
(5) (a) Leroy, V.; Thurairatnam, S. Cathepsin S inhibitors. Expert Opin.
Ther. Pat. 2004, 14, 301-311. (b) Yasuda, Y.; Kaleta, J.; Broemme,
D. The role of cathepsins in osteoporosis and arthritis: Rationale
for the design of new therapeutics. AdV. Drug DeliVery ReV. 2005,
57, 973-993. (c) Leung-Toung, R.; Zhao, Y.; Li, W.; Tam, T. F.;
Karimian, K.; Spino, M. Thiol proteases: inhibitors and potential
therapeutic targets. Curr. Med. Chem. 2006, 13, 547-581. (d) Tully,
D. C.; Liu, H.; Alper, P. B.; Chatterjee, A. K.; Epple, R.; Roberts,
M. J.; Williams, J. A.; Nguyen, K. T.; Woodmansee, D.; Tumanut,
C.; Li, J.; Spraggon, G.; Chang, J.; Tuntland, T.; Harris, J. L.;
Karanewsky, D. S. Synthesis and evaluation of arylaminoethyl amides
as noncovalent inhibitors of cathepsin S. Part 3: Heterocyclic P3.
Bioorg. Med. Chem. Lett. 2006, 16, 1975-1980.
(6) Gocheva, V.; Zeng, W.; Ke, D.; Klimstra, D.; Reinheckel, T.; Peters,
C.; Hanahan, D.; Joyce, J. A. Distinct roles for cysteine cathepsin
genes in multistage tumorigenesis. Genes DeV. 2006, 20, 543-556.
(7) (a) Rawlings, N. D.; Barrett, A. I. Families of cysteine proteases.
Methods Enzymol. 1994, 244, 461-486. (b) Cygler, M.; Mort, J. S.
Proregion structure of members of the papain superfamily: Mode
of inhibition of enzymic activity. Biochimie 1997, 79, 645-652.
(8) Villhauer, E. B.; Brinkman, J. A.; Naderi, G. B.; Burkey, B. F.;
Dunning, B. E.; Prasad, K.; Mangold, B. L.; Russell, M. E.; Hughes,
T. E. 1-[[(3-Hydroxy-1-adamantyl)amino]acetyl]-2-cyano-(S)-pyrro-
lidine: A potent, selective, and orally bioavailable dipeptidyl
peptidase IV inhibitor with antihyperglycemic properties. J. Med.
Chem. 2003, 46, 2774-2789.
Assay Procedure for Nitrile Inhibitors. The dissociation
constants (Ki) were calculated by the method of Dixon.25 Cathepsin
B concentration in the assays was 2 nM; cathepsin K concentration
in the assays was 10 nM; cathepsin L concentration in the assays
was 0.03 nM; and cathepsin S concentration in the assays was 0.6
nM. Two concentrations of Cbz-Phe-Arg-AMC (5 µM and 2.5 µM
for cathepsins B and K, 0.6 µM and 0.3 µM for cathepsin L) or
Cbz-Leu-Arg-AMC (5 µM and 2.5 µM for cathepsin S) were used.
Assays were conducted in duplicate with and without inhibitor at
five inhibitor concentrations to provide 10-90% enzyme inhibition.
In each well was placed 180 µL of enzyme solution and 10 µL of
a DMSO inhibitor solution. The resulting solutions were incubated
for 5 min at 37 °C, and then 10 µL of the peptidyl-AMC substrate
was added and generation of AMC was monitored over 5 min.
Crystallization and Data Collection. Cathepsin S was expressed
in the baculovirus system and its complex with inhibitor 13,
produced by adding 13 to the protein in a 2:1 molar ratio prior to
concentration of the protein to 15 mg/mL. A coarse screen of 480
crystallization conditions was set up in low profile 96-well Greiner
plates at 4 °C in a sitting drop format containing 250 nL of protein
combined with an equal volume of crystallization solution. Crystals
appeared after 1-2 weeks in two distinct crystal forms. Data were
collected at beamline 5.0.3 of the Advanced Light Source (ALS)
in Berkeley, CA. The two crystal forms belong to space groups
P4122 and P6522 and diffracted to 1.5 and 1.9 Å, respectively (data
statistics are presented in Table 2). The tetragonal crystal form was
isomorphous with a previously determined crystal form,5d while
that of the hexagonal crystal form was solved by molecule
replacement using this model as a search probe with MOLREP.26
Both structures were then iteratively refined until convergence with
coot27 and refmac5,28 all other crystallographic manipulations were
carried out with the CCP4 program package.29 Both structures
exhibited excellent geometry, with no residues in disallowed regions
of the Ramachandran plot. The two crystals contained two and three
molecules per asymmetric unit, providing five separate structures
of compound binding that were essentially identical. The structures
were deposited with PDB code 2H7J and 2HXZ.
(9) Cogan, D. A.; Liu, G.; Ellman, J. A. Asymmetric synthesis of chiral
amines by highly diastereoselective 1,2-additions of organometallic
reagents to N-tert-butanesulfinyl imines. Tetrahedron 1999, 55,
8883-8904.
(10) Patterson, A. W.; Ellman, J. A. Asymmetric synthesis of R,R-
dibranched propargylamines by acetylide additions to N-tert-butane-
sulfinyl ketimines. J. Org. Chem. 2006, 71, 7110-7112.
(11) Tornoe, C. W.; Christensen, C.; Meldal, M. Peptidotriazoles on solid
phase: [1,2,3]-Triazoles by regiospecific copper(I)-catalyzed 1,3-
dipolar cycloadditions of terminal alkynes to azides. J. Org. Chem.
2002, 67, 3057-3064.
Acknowledgment. J.A.E., W.J.L.W., and A.W.P. gratefully
acknowledge the NIH (GM54051) for support of this work.
A.W.P. greatly appreciates an American Chemical Society
Medicinal Chemistry Graduate Student Fellowship sponsored
by Bristol Myers Squibb. The authors would also like to thank
Andreas Kreusch, Mike Didonato, and Christian Lee for help
with data collection and Peter Schultz for continued support
and encouragement. The work in this paper is based on
experiments conducted at beamline 5.0.3 of the advanced light
source (ALS). The ALS is supported by the Director, Office of
Science, Office of Basic Energy Sciences, Material Sciences
Division of the U.S. Department of Energy under Contract No.
DE-AC03-76SF00098 at Lawrence Berkeley National Labora-
tory. We would like to thank all of the staff at these beamlines
for their continued support.
(12) PDB ID: 2H7J
(13) PDB ID: 2HXZ
(14) McGrath, M. E. The lysosomal cysteine proteases. Annu. ReV.
Biophys. Biomol. Struct. 1999, 28, 181-204.
(15) (a) Russell, R. B.; Barton, G. J. Multiple protein sequence alignment
from tertiary structure comparison. Proteins: Struct., Funct., Genet.
1992, 14, 309-323. (b) Barton, G. J. ALSCRIPT: A tool to format
multiple sequence alignments. Protein Eng. 1993, 6, 37-40.
(16) (a) Bromme, D.; Klaus, J. L.; Okamoto, K.; Rasnick, D.; Palmer, J.
T. Peptidyl vinyl sulfones: A new class of potent and selective
cysteine protease inhibitors. S2P2 specificity of human cathepsin O2
in comparison with cathepsins S and L. Biochem. J. 1996, 315, 85-
89. (b) Marquis, R. W.; James, I.; Zeng, J.; Trout, R. E. L.;
Thompson, S.; Rahman, A.; Yamashita, D. S.; Xie, R.; Ru, Y.; Gress,
C. J.; Blake, S.; Lark, M. A.; Hwang, S.-M.; Tomaszek, T.; Offen,
P.; Head, M. S.; Cummings, M. D.; Veber, D. F. Azepanone-based
Supporting Information Available: Purity data of compounds
8 and 11. Data for the X-ray crystal structures have been deposited

SelectiVe Nitrile Inhibitors of Cathepsin S by SAS
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21 6307
inhibitors of human cathepsin L. J. Med. Chem. 2005, 48, 6870-
6878. (c) Loser, R.; Schilling, K.; Dimmig, E.; Gutschow, M.
Interaction of papain-like cysteine proteases with dipeptide-derived
nitriles. J. Med. Chem. 2005, 48, 7688-7707.
(23) Lombardi, P. A rapid, safe, and convenient procedure for the
preparation and use of diazomethane. Chem. Ind. (London) 1990,
708.
(24) Barrett, A. J.; Kembhavi, A. A.; Brown, M. A.; Kirschke, H.; Knight,
C. G.; Tamai, M.; Hanada, K. L-trans-Epoxysuccinyl-leucylamido-
(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine
proteinases including cathepsins B, H, and L. Biochem. J. 1982, 201,
189-198.
(25) Dixon, M. Determination of enzyme-inhibitor constants. Biochem.
J. 1953, 55, 170-171.
(26) Vagin, A.; Teplyakov, A. An approach to multi-copy search in
molecular replacement. Acta Crystallogr., Sect. D 2000, 56, 1622-
1624.
(27) Emsley, P.; Cowtan, K. Coot: Model-building tools for molecular
graphics. Acta Crystallogr., Sect. D 2004, 60, 2126-2132.
(28) Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Refinement of
macromolecular structures by the maximum-likelihood method. Acta
Crystallogr., Sect. D 1997, 53, 240-255.
(29) Collaborative; Computing; Project; Number 4 The CCP4 suite:
Programs for protein crystallography, Version 3.1. Acta Crystallogr.,
Sect. D 1994, 50, 760-763.
(17) Phillips, M. A.; Kaplan, A. P.; Rutter, W. J.; Bartlett, P. A. Transition-
state characterization: A new approach combining inhibitor analogues
and variation in enzyme structure. Biochemistry 1992, 31, 959-963.
(18) Mader, M. M.; Bartlett, P. A. Binding energy and catalysis: The
implications for transition-state analogues and catalytic antibodies.
Chem. ReV. 1997, 97, 1281-1301.
(19) Maly, D. J.; Leonetti, F.; Backes, B. J.; Dauber, D. S.; Harris, J. L.;
Craik, C. S.; Ellman, J. A. Expedient solid-phase synthesis of
fluorogenic protease substrates using the 7-amino-4-carbamoylm-
ethylcoumarin (ACC) fluorophore. J. Org. Chem. 2002, 67, 910-
915.
(20) Lundquist, J. T., IV; Pelletier, J. C. Improved solid-phase peptide
synthesis method utilizing R-azide-protected amino acids. Org. Lett.
2001, 3, 781-783.
(21) Purity was determined to be >95%, analyzed by HPLC, as described
for substrates 8.
(22) Krantz, A.; Copp, L. J.; Coles, P. J.; Smith, R. A.; Heard, S. B.
Peptidyl (acyloxy)methyl ketones and the quiescent affinity label
concept: the departing group as a variable structural element in the
design of inactivators of cysteine proteinases. Biochemistry 1991,
30, 4678-4687.
JM060701Sz