Design and synthesis of tri-ring P3 benzamide-containing aminonitriles as potent, selective, orally effective inhibitors of cathepsin K

Article abstract of DOI:10.1021/jm058198r

We have prepared a series of achiral aminoacetonitriles, bearing tri-ring benzamide moieties and an aminocyclohexanecarboxylate residue at P₂. This combination of binding elements resulted in sub-250 pM, reversible, selective, and orally bioavailable cathepsin K inhibitors. Lead compounds displayed single digit nanomolar inhibition in vitro (of rabbit osteoclast-mediated degradation of bovine bone). The best compound in this series, 39n (CRA-013783/L-006235), was orally bioavailable in rats, with a terminal half-life of over 3 h. 39n was dosed orally in ovariectomized rhesus monkeys once per day for 7 days. Collagen breakdown products were reduced by up to 76% dose-dependently. Plasma concentrations of 39n above the bone resorption IC₅₀ after 24 h indicated a correlation between functional cellular and in vivo assays. Inhibition of collagen breakdown by cathepsin K inhibitors suggests this mechanism of action may be useful in osteoporosis and other indications involving bone resorption.

Full text of DOI:10.1021/jm058198r

7520
J. Med. Chem. 2005, 48, 7520-7534
Articles
Design and Synthesis of Tri-Ring P3 Benzamide-Containing Aminonitriles as
Potent, Selective, Orally Effective Inhibitors of Cathepsin K
James T. Palmer,*,† Clifford Bryant,† Dan-Xiong Wang,† Dana E. Davis,† Eduardo L. Setti,†
Robert M. Rydzewski,† Shankar Venkatraman,† Zong-Qiang Tian,† Leland C. Burrill,† Rohan V. Mendonca,†
Eric Springman,† John McCarter,† Tobee Chung,† Harry Cheung,† James W. Janc,† Mary McGrath,†
John R. Somoza,† Philip Enriquez,† Z. Walter Yu,† Robert M. Strickley,† Liang Liu,† Michael C. Venuti,†
M. David Percival,‡ Jean-Pierre Falgueyret,‡ Peppi Prasit,‡ Renata Oballa,‡ Denis Riendeau,‡ Robert N. Young,‡
Gregg Wesolowski,§ Sevgi B. Rodan,§ Colena Johnson,| Donald B. Kimmel,§ and Gideon Rodan§
Celera Genomics, Inc., 180 Kimball Way, South San Francisco, California 94080, Merck Frosst Centre for Therapeutic
Research, 16711 TransCanada Highway, Kirkland, Quebec, Canada H9H 3L1, Department of Bone Biology & Osteoporosis,
Merck Research Laboratories, West Point, Pennsylvania, and Department of Laboratory Animal Resources, Merck Research
Laboratories, West Point, Pennsylvania
Received February 28, 2005
We have prepared a series of achiral aminoacetonitriles, bearing tri-ring benzamide moieties
and an aminocyclohexanecarboxylate residue at P2. This combination of binding elements
resulted in sub-250 pM, reversible, selective, and orally bioavailable cathepsin K inhibitors.
Lead compounds displayed single digit nanomolar inhibition in vitro (of rabbit osteoclast-
mediated degradation of bovine bone). The best compound in this series, 39n (CRA-013783/
L-006235), was orally bioavailable in rats, with a terminal half-life of over 3 h. 39n was dosed
orally in ovariectomized rhesus monkeys once per day for 7 days. Collagen breakdown products
were reduced by up to 76% dose-dependently. Plasma concentrations of 39n above the bone
resorption IC50 after 24 h indicated a correlation between functional cellular and in vivo assays.
Inhibition of collagen breakdown by cathepsin K inhibitors suggests this mechanism of action
may be useful in osteoporosis and other indications involving bone resorption.
Introduction
tive location of this enzyme in the cells responsible for
bone resorption suggest its potential as a target for
osteoporosis therapeutics. Furthermore, a genetic defi-
ciency in this enzyme in humans results in pcynodys-
ostosis, a condition whose phenotype is characterized
by short, stubby, and dense bones (osteopetrosis).3 This
syndrome indicates a clear connection between an
imbalance in cathepsin K activity and the overall bone
turnover process. A similar genetic mutation introduced
experimentally in mice also results in an osteopetrotic
phenotype.4
Inhibitor Design Motif. A number of recent reports
on the inhibition of cathepsin K have focused on
carbonyl-containing, transition state mimic-based in-
hibitors, including aldehydes,5 aminomethyl ketones,6
and hydroxymethyl ketones.7 The challenges posed by
inhibitors of such classes include, but are not limited
to, high molecular weight, low oral bioavailability, and
metabolic or chemical instability. Several reports have
also issued describing the crystal structures of cathepsin
K with vinyl sulfones,8 substituted ketone-based inhibi-
tors,9 and ketobenzoxazoles.10 The active site of cathe-
psin K in said cocrystal structures has revealed distinct
opportunities for obtaining selectivity over other cys-
teine protease antitargets, such as cathepsins B, L, and
S. The S3 subsite of cathepsin K contains an aspartic
acid residue (Asp61) capable of forming ionic bonds/salt
bridges with an appropriately designed P3 moiety. Also,
the wide opening of the S2 pocket suggested accom-
Osteoporosis can be described as brittle bone disease,
manifested by loss of bone mass and an increased
tendency to fracture. The condition can be brought on
by an imbalance of the functions of osteoblasts, respon-
sible for bone formation, and osteoclasts, which are
responsible for bone resorption.1 The major structural
components of extracellular bone are hydroxyapatite
and protein, of which the latter is 90% type 1 collagen.
During the bone resorption process, the multinucleated
osteoclasts form resorption lacunae on the surface of the
bone, into which protons and proteases are secreted.
This acidic environment is conducive to the decalcifica-
tion of the bone and is also suitable for the proteolytic
degradation of the collagen component.
As a member of the papain family of cysteine pro-
teases, including cathepsins B, L, S, F, V, W, O, H, C,
and Z, cathepsin K’s distribution renders it an attractive
target for selective inhibition. Cathepsin K is the major
cysteine protease expressed in osteoclasts.2 Type 1
collagen is readily degraded by cathepsin K over a pH
range of approximately 4-7. The abundance and selec-
* To whom correspondence should be addressed. Tel.: (650) 866-
6641. Fax: (650) 866-6654. e-mail: jim.palmer@celera.com.
† Celera Genomics, Inc.
‡ Merck Frosst Centre for Therapeutic Research.
§ Department of Bone Biology & Osteoporosis, Merck Research
Laboratories.
| Department of Laboratory Animal Resources, Merck Research
Laboratories
10.1021/jm058198r CCC: $30.25 © 2005 American Chemical Society
Published on Web 10/29/2005

Tri-Ring P3 Benzamide-Containing Aminonitriles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7521
Table 1. Assay Conditions for Cathepsins K, B, L, and S. All Assays Were Performed with 8-Point Dose-Response Curves
Cathepsin K
Cathepsin B
Cathepsin L
Cathepsin S
substrate
Z-Phe-Arg-AMC, 40 µM
Boc-Leu-Lys-Arg-AMC, 190 µM
Z-Phe-Arg-AMC, 10 µM
Z-Val-Val-Arg-AMC, 60 µM
buffer, pH 50 mM MES, 2.5 mM EDTA,
50 mM BES, 2.5 mM EDTA,
50 mM MES, 2.5 mM EDTA, 50 mM MES, 2.5 mM EDTA,
2.5 mM DTT, 10% DMSO, pH 5.5 0.001% Tween-20, 2.5 mM DTT, 2.5 mM DTT, 10% DMSO,
100 mM NaCl, 0.001% BSA,
10% DMSO, pH 6.5
10% DMSO, pH 6.0
pH 5.5
Table 2. Inhibition Constants (Ki(app), µM) for CBZ-P2-Xaa-GlyCN Derivatives
Scheme 1. Synthesis of CBZ-P2-Xaa-GlyCN Derivatives
modation by a variety of P2 binding elements capable
of forming hydrophobic interactions. Last, the structures
thus far solved all pointed to the crucial backbone
interactions between the P2-P1 amide of the inhibitors
and key residues of the enzyme (Gly66, Asn158). With
these facts in mind, we commenced our inhibitor design
based on a modified dipeptide motif. We selected nitrile-
based inhibitors as the framework for the studies
described herein so as to lower molecular weight and
provide a facile chemotype for rapid analogue genera-
tion.
The inhibitors were then assayed against a panel of
cysteine proteases using conditions tailored for the
particular enzymes, cathepsins K, B, L, and S (Table
1). Table 2 shows the Ki(app) values (in µM units)
obtained for this series following a 15 min preincubation
of enzyme with inhibitor, prior to the addition of
substrate. Values were obtained via 8-point dose-
response curves. Apparent inhibition constants, Ki-
(app)’s, were calculated from the velocity data generated
at the various inhibitor concentrations using the soft-
ware package, BatchKi (obtained from Dr. Petr Kuzmic,
Biokin Ltd., Pullman, WA).18 BatchKi provides a para-
metric method for the determination of inhibitor potency
using a transformation of the tight binding inhibition
model described by Morrison19 and further refines the
Dipeptide-based nitriles have been recently described
in the preparation of cathepsin B11 and cathepsin
S-selective inhibitors,12 as well as for cathepsin K-
targeted inhibitors.13 Starting with aminoacetonitrile as
the reactive center, we prepared a series of carboben-
zyloxy (CBZ)-protected dipeptide nitriles in order to
probe the selectivity elements demanded by cathepsin
K’s active site. Using standard peptide-type coupling
methods,14 we converted CBZ-protected, commercially
available amino acids to their aminoacetonitrile deriva-
tives. Path A directly permitted us to couple the ami-
noacetonitrile moiety with the carboxylate, while path
B involved the N-hydroxysuccinimidoyl active ester,
which was readily displaced by aminoacetonitrile.

7522 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Palmer et al.
Table 3. Inhibition Constants (Ki(app))of Human Cysteine Proteases by Amide-Ac6-aminoacetonitriles (all values in µM)
Scheme 2. Synthesis of P3 Amides in the Ac6-GlyCN
Series
Ki(app) values by nonlinear least-squares regression.
The apparent inhibition constant is related to the true
thermodynamic binding constant, Ki, by the following
relationship: Ki ) Ki(app)/(1 + [substrate]/Km),19 since
substrates are typically supplied in the assays at their
Km, Ki ) Ki(app)/2.
From the examples prepared in this series, we ob-
served that large P2 amino acids (3, CBZ-Nal-GlyCN)
favored cathepsins L and S, while simple P2-leucine
derivatives, such as 1, 2, 7, and 10, all afforded
submicromolar Ki(app) values versus cathepsin K. In-
terestingly, the P2-D-leucine derivative 6 retained mod-
est potency against cathepsins K and S, and the racemic
R-methylleucine derivative 8 also showed single digit
micromolar potency against both enzymes. This sug-
gested that cathepsin K’s S2 subsite might accommodate
a bulky, R-substituted amino acid, and therefore we
prepared the achiral cycloalkane-based derivatives 12-
14. We were gratified to discover that both cyclopentyl
and cyclohexyl (Ac5, Ac6 respectively) afforded both
potency and selectivity in this simple series. Armed with
14, 84 nM versus cathepsin K, and several 100-fold
selectivity over our antitargets, we embarked on a more
elaborate series designed to maximize interactions
between the S3 subsite of the enzyme and the P3 binding
moiety of the inhibitor.
amides to probe this area of binding, using standard
coupling procedures (Scheme 2).
Attempts to use deprotecting acids (TFA, HCl/diox-
ane, etc.) in removing the Boc group of 15 resulted in
substantial formation of unwanted tert-butyl amide
product. We proposed that this resulted from protona-
tion of the nitrile by the acid; subsequently, the short-
lived tert-butyl cation that results from Boc removal
could be trapped by trace amounts of water to give tert-
butyl alcohol, which could react with the highly elec-
trophilic nitrilium ion, followed by tautomerization. This
phenomenon was always observed with trace amounts
of water in the system; we avoided this by ensuring that
both the methanesulfonic acid and THF were scrupu-
lously dry. Following isolation of 16, we then coupled a
series of acids (or acyl chlorides) via HATU methodology,
or via simple acid chloride addition in the presence of
base to afford 18a-o. Table 3 shows inhibition data for
this series.
Initially, we explored the relevance of the linker group
between the Ac6 amino group and the binding moiety.
Since it was not clear at this point how important
interactions were between the P3-P2 amide bond (or
equivalent) and the active site, we prepared a series of
We observed a clear requirement for sp2 hybridization
at the atom adjacent to the P3 carbonyl (18a versus 14,
18b-o) and thus pursued regiochemical substitution

Tri-Ring P3 Benzamide-Containing Aminonitriles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7523
Table 4. Inhibition of Cathepsin K (Ki(app)) and in Vitro Potency (IC50) in Bone Resorption Assay Functional Assay by Extended P3
Biphenylamide Derivatives of the Ac6-GlyCN Motif (all values in µM)
Scheme 3. Access to Extended P3 Biphenylamidesa
about the P3 benzamide ring. On the basis of the 13-
fold increase in cathepsin K potency comparing the
3-bromobenzamide 18b versus the 4-bromobenzamide
18g, and an even greater increase in potency between
the m-biphenyl 18c and the p-biphenyl 18l (34-fold), we
concluded that para-substituted benzamides would af-
ford the most potent compounds. This conclusion was
consistent with the 4-pyridylthiazolyl substituent in 18d
displaying intermediate cathepsin K potency of 150 nM,
the geometry of the five-membered heterocycle placing
the substituent closer to the 3-substituted benzamides
than to the 4-substituted. By contrast, the 2-bro-
mothiophene 18h, considered to possess similar geom-
etry to a bromophenyl group, was equipotent to 18g.
The introduction of a “kink” in the 4-substituent
(amide or sulfonamide, 18i, 18j respectively) appeared
to offer no improvement in binding affinity. Substitution
with additional rings, particularly those bearing some-
what basic moieties (18m-o), increased potency to
single-digit nanomolar values, without significant loss
in selectivity.
We realized that an additional aryl ring spacer
between the P3 benzamide and the basic moiety of the
extended P3 group might enhance activity, particularly
by fixing the geometry of the lengthened group such that
the Asp61 residue might form a tighter interaction. To
this end we prepared a series of 4-substituted bipheny-
lamides, via Scheme 3 as the key step.
Transformation of 18g to the corresponding pinacol-
boronate ester intermediate boronate I was effected by
cross-coupling with pinacoldiborane using anhydrous
conditions.15 This material was isolated by precipitation
as an off-white solid and was not purified further, but
was subjected to Suzuki cross-coupling with appropriate
aryl bromides. Alternatively, treatment of 18g with
a (a) Pd(dppf)Cl2‚CH2Cl2, KOAc, DMF, 80 °C,
Pd(dppf)Cl2‚CH2Cl2, DMF, 2 M Na2CO3, 80-85 °C, 3 h.
6 h; (b)
phenylboronates or phenylboronic acids substituted at
the 4-position in the presence of palladium(II) catalyst
and aqueous base in DMF under Suzuki-type conditions
afforded the biphenylamides 19a-m; elaborated ex-
amples required additional transformation following
coupling.
In addition to assaying this series against cathepsins
K, B, L, and S, we also assayed these compounds in an
in vitro bone resorption assay that evaluates degrada-
tion of bovine bone by isolated rabbit osteoclasts (mea-
suring the degradation of collagen using ELISA).16 Table
4 shows the results comparing this series’s selectivity
as well as its potency against the isolated enzyme and
in the functional cellular assay.
Clearly, the presence of a basic nitrogen distal from
the point of attachment to the second aryl ring enhances
activity against cathepsin K, resulting in sub-250 pM
potency in most cases. This effect appeared to hold up
even in the case of highly hindered amines (19e, 19m).

7524 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Palmer et al.
Table 5. Intravenous PK Parameters for Extended
ence of base, followed by Suzuki coupling of the product-
(s) with boronate I, available in one step from 18g via
bis-pinacolatodiborane cross-coupling, afforded the de-
sired products.
Inhibition data for cathepsins K, B, L, and S, osteo-
clast-based in vitro bone resorption data, and pharma-
cokinetic data for this series of thiazolebenzamides
(39a-m) are described in Table 6. A clear requirement
for sub-nM potency appeared in the shape of a charged
moiety at the extended P3 group; tertiary amines were
favored over secondary and primary amines. In the bone
resorption assay, substituted piperazines tended to be
the most potent.
P3-Biphenylamide-Ac6 Nitriles
entry Cl (mL/min/kg) Vss (L/kg) MRT (min) ât1/2 (min) Cmax (µM)
19a
19b
19d
19e
19f
19g
19h
19i
71
231
97
7.23
25.3
11.1
11.2
3.29
104
110
114
172
40
84
123
114
144
78
0.5
0.22
0.32
0.24
1.0
67
82
37
1.76
1.55
8.46
3.27
48
47
0.66
1.2
0.25
0.37
0.36
40
39
81
117
130
46
90
121
116
104
43
19k
19m
84
10.7
entry dose (mg/kg) F (%) ât1/2 (min) Cmax (min) Tmax (min)
19a 10 21 113 0.14 120
Compounds in this series bearing uncharged extended
P3 elements (39a,h) retained sub-10nM potency, but
introduction of a charged moiety increased potency to
sub-nM Ki levels. Tertiary amines at the extended
position tended to be more potent than primary amines
(39e). Potency was apparently independent of the
distance or spacer atom (nitrogen, 39j, oxygen, 39g, or
carbon, 39d) between the piperazine/piperidine/mor-
pholine ring and the thiazole, However, in the bone
resorption assay, substituted piperazines tended to be
the most potent (39l,m). Compounds bearing secondary
and primary amine moieties, as well as uncharged
compounds, tended to show lower potency in the bone
resorption assay, possibly due to cell permeability
issues.
Within this series, several compounds (39f,i,k) dis-
played long half-lives/MRT’s when dosed intravenously,
suggesting possible suitability of this family for oral
dosing and subsequent once per day administration in
appropriate in vivo models. Having optimized the
structural elements of the thiazole-benzamide series for
potency (sub-nM versus Cat K), selectivity (.100-fold
versus cathepsins B, L, and S), and efficacy in in vitro
systems (sub-10 nM IC50 in the bone resorption assay),
we dosed the most potent compounds from this series
in iv/po PK experiments to determine oral bioavailabil-
ity. In an optimized series substituted at P3 with
piperidine and piperazine derivatives (Table 7) we were
able to obtain oral bioavailability values (measured in
rats) of 35-68%, with terminal half-lives ranging from
1.4 to over 5 h. All the compounds bore a tertiary amine
at the distal position relative to the thiazole; this led
us to conclude that cellular permeability, expected to
be greater in examples with this type of functionality,
was evident in both the cellular bone resorption assay
and in oral bioavailability. Coupled with the ease of
synthesis of members of this series, these parameters
allowed us to conclude that an appropriate example for
additional in vivo studies would be the methylpiperazine
derivative (39n), prepared according to Scheme 4, using
procedure E to generate the intermediate thiourea for
condensation with 4-bromoacetylbenzoic acid and sub-
sequent HATU-mediated coupling with MsOH.Ac6-
GlyCN (16).
a Abbreviations: CL ) clearance; Vss ) volume of distribution;
MRT ) mean residence time; ât1/2 ) terminal half-life; Cmax ) peak
concentration. Tmax ) time of peak concentration post-dose. F )
oral bioavailability. Compounds were dosed intravenously at 0.5
mg/kg (iv) and 10 mg/kg for the oral study.
Having established sub-nM levels of potency versus
cathepsin K in this series, along with sub-100 nM
cellular potency as measured in the bone resorption
functional assay, we turned our attention toward de-
veloping this series for in vivo efficacy studies. To this
end, we explored derivatives in a cassette dosing mode
in rats, whereby intravenous pharmacokinetic param-
eters (half-life, clearance, mean residence time (MRT),
and volume of distribution) were obtained for up to four
compounds per study. We used parameters obtained via
this route to guide selection of candidates to be dosed
in single compound, iv/po PK studies that followed. PK
parameters for the cassette-dosed compounds are sum-
marized in Table 5. Compounds were typically formu-
lated in DMSO (<20%), PEG 400 (<20%), and water at
0.5 mg/mL/compound. A group of three rats were used
in each study to obtain statistical data. Animals were
dosed intravenously at 1 mL/kg to reach a final dose of
0.5 mg/kg/compound. Blood samples were taken at
different time points, and plasma samples were pre-
pared and analyzed by LC/MS/MS. Pharmacokinetic
analysis was performed using WinNonlin Software.
Of this series, 19a, bearing the 4-piperazinebipheny-
lamide moiety, was dosed in a single compound oral
bioavailability experiment. The low peak concentration
of 0.14 µM, when dosed at 10 mpk, was only twice that
of the cellular potency as measured in the bone resorp-
tion assay and was not sustained over the course of
several hours. This suggested that this series was not
likely suitable for further development as a once-per-
day preclinical candidate for osteoporosis treatment at
this dose.
To improve oral bioavailability, we modified the tri-
ring series such that the distal phenyl ring of the
biphenyl array would be replaced with a thiazole moiety,
attached to the para-position of the proximal phenyl
through the 4-carbon of the thiazole. Syntheses of the
heterocycles were achieved according to Scheme 4,
wherein p-bromoacetylbenzoic acid 20 was coupled with
appropriately constructed thioamides, thioureas, or
thiocarbamates (Procedures A-E). The derivatized ben-
zoic acids were then coupled to 16 according to the
procedure earlier described (Scheme 2) giving inhibitors
39a-q. Alternatively, treatment of 1,4-dibromothiazole
with substituted piperazines (Procedure F) in the pres-
X-ray Structural Analysis of 39n. At this point, a
crystallographic analysis of 39n revealed several dis-
tinct features. Figures 1 (distinct hydrogen bonding
network) and 2 (surface plots) indicate that the extended
P3 moiety consisting of the methylpiperazine-thiazole-
benzamide portion of the molecule is sufficiently close
to the Asp61 of the S3 pocket to be capable of forming

Tri-Ring P3 Benzamide-Containing Aminonitriles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7525
Scheme 4. Synthesis of P3-Thiazolebenzoate Moieties General P3-Thiazolebenzoate-Ac6-GlyCN Preparationa
a (a) Et3N, DMF; (b) Pd(dppf)Cl2‚CH2Cl2, DMF, MeCOOK, 80-85 °C, 3 h; (c) Pd(dppf)Cl2‚CH2Cl2, DMF, 2 M Na2CO3, 80-85 °C, 3 h.
an ionic interaction. The P2 cyclohexyl ring is confor-
mationally positioned with the P3-P2 amide nitrogen
adopting an axial arrangement, with the carbonyl of the
corresponding P2-P1 amide oriented equatorially. Key
hydrogen bonds appear between the P2-P1 amide NH
and the carbonyl of Asn158 (3.28 Å), and the P2-P1
amide carbonyl oxygen and the Gly66 NH (3.22 Å). An
additional essential hydrogen bond between the P3-P2
amide NH and the carbonyl of Gly66 (2.91 Å) reveals
the importance of the P2 amino acid H-bond acceptors
and donors within the enzyme’s active site.
Kinetic Characterization of 39n. The reversibility
of inhibition of human cathepsin K by 39n was dem-
onstrated in experiments in which a 1:1 enzyme-
inhibitor mixture was preincubated for 15 min, followed
by a 100-fold dilution into substrate-containing buffer.
After dilution of the inhibitor to 0.05 nM, the cathepsin
K enzyme activity was initially inhibited, but slowly
recovered with a first-order rate constant of 0.023 s-1
(t1/2 ) 30 s). The final steady-state enzyme activity was
80-90% that of control, demonstrating the complete
reversibility of the 39n-cathepsin K complex. Consis-

7526 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Palmer et al.
Table 6. Inhibition (Ki(app)) Values, in Vitro Bone Resorption Inhibition Constants (IC50), and Intravenous PK Parameters for
Extended P3-Thiazolobenzamide-P3-Ac6-GlyCN Series
tent with previous studies with biaryl nitrile cathepsin
K inhibitors, 39n was not a substrate for the nitrilase
activity of Cat K.13a
centrations in each urine specimen were measured as
described.13a The results are expressed as uNTx/Cre
(nM/mM). As shown in Figure 3, the cathepsin K
inhibitor 39n dosed at 15 mg/kg po, qd decreased uNTx/
Cre by an average of 76% relative to vehicle-treatment
(p < 0.001). The suppression was detected after the
second dose, the first sampling time. At 3 days after the
last dose of 39n, uNTx/Cre remained suppressed by 65%
(p ) 0.007). However, by 10 days after the last dose,
uNTx/Cre in 39n-treated monkeys had returned to
control group levels. When 39n was dosed at 3 mg/kg
and 0.6 mg/kg po, qd, uNTx/Cre was decreased by an
average of 68% (p < 0.001) and 31% (p < 0.01) relative
to vehicle-treatment respectively (Figure 4). As shown
in the single dose pharmacokinetic studies in intact
rhesus monkeys (Figure 5), plasma levels of 39n re-
mained at or above the bone resorption IC50 for at least
24 h following the 15 or 3 mg/kg oral doses, but dropped
below the bone resorption IC50 at the 0.6 mg/kg dose.
Rhesus Monkey Bone Resorption Marker Study.
A rhesus monkey bone resorption marker model, in
which estrogen-deficiency bone loss was established by
surgical oophorectomy (OVX), was used to evaluate the
anti-bone resorptive activity of 39n.13a Compound-
treated monkeys (average age 15 y, all 3 y post OVX)
were treated po, qd for 2 days with 1% carboxymethyl-
cellulose (vehicle) and then treated with 39n po, qd for
8-11 days at 15, 3, or 0.6 mg/kg. At the end of drug
treatment, monkeys were treated for three additional
days with vehicle. Vehicle monkeys received vehicle
throughout the 13-day period. Twenty-four hour urine
collections of both groups were made on the day before
the start of drug dosing and during drug dosing. One
additional urine collection was made 10-17 days after
the last drug dose. The urine samples were frozen at
-70 °C and N-telopeptides (NTx) and creatinine con-

Tri-Ring P3 Benzamide-Containing Aminonitriles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7527
Table 7. Inhibition, in Vitro, and Pharmacokinetic Data for Selected Extended P3-thiazolobenzamide-P3-Ac6-GlyCN Compounds
Figure 3. Ratio of N-telopeptides (NTx) to creatinine (Cr)
Figure 1. Hydrogen bonding network between Cat K active
during dosage of 39n in adult OVX monkeys.
site residues and 39n.
Figure 2. Surface plot depicting 39n bound in active site of
Figure 4. Dose response of 39n in OVX monkey model.
Cat K, covalently bound to catalytic cysteine.
afforded us single-digit nanomolar potency. By extend-
ing the benzamide further, through incorporation of an
Conclusions
Starting with a simple dipeptide nitrile scaffold (Z-
Leu-GlyCN), a relatively potent (35 nM) but nonselec-
tive cathepsin K inhibitor, we were able to tune selec-
tivity for cathepsin K versus the other antitargets of
interest (cathepsins B, L, and S) by replacing the
natural amino acid with an achiral, conformationally
restricted aminocyclohexanecarboxylate (Ac6) moiety.
By manipulating the P3 binding elements, we discovered
that para-substituted benzamides bearing basic groups
additional aromatic spacer, we were able to increase
potency to sub-nanomolar levels against the enzyme.
Furthermore, by retaining the basic amine group, again
optimally substituted on the distal aromatic ring, we
could translate enzyme potency to reasonable (10-100
nM) potency in the functional cellular assay of bone
resorption. By exchanging the distal phenyl ring for a
more stable thiazole heterocycle, we not only increased
the cellular potency of this series but also the pharma-

7528 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Palmer et al.
Synthesis of 2. A mixture composed of benzyloxycarbon-
ylnorleucine (0.425 g, 1.60 mmol), aminoacetonitrile hydro-
chloride (0.148 g, 1.60 mmol), PyBOP.RTM. (0.836 g, 1.60
mmol), N,N-diisopropylethylamine (0.84 mL, 4.83 mmol), and
DMF (10 mL) was stirred at room temperature for 2.5 h. The
mixture was concentrated, and the residue was taken up into
dichloromethane. The dichloromethane mixture was washed
with 1 N hydrochloric acid, water, and aqueous sodium
bicarbonate, dried (MgSO4), filtered, and concentrated. The
crude material was purified from the residue by silica gel
chromatography using 5% methanol in dichloromethane to
provide the final product (435 mg, 1.52 mmol, 95%) as an oil.
1H NMR (300 MHz, DMSO-d6): 8.65 (bt, 1H), 7.55 (d, J ) 7
Hz, 1H), 7.41 (bs, 5H), 5.00 (Abq, 2H), 4.08 (d, J ) 6 Hz, 2H),
3.97 (m, 1H), 1.52 (m, 2H), 1.24 (m, 4H), 0.81 (bs, 3H). Analysis
(C16H21N3O3): (C, H, N).
Figure 5. Day 8 PK from OVX monkey model.
Synthesis of 3. This material was produced using the same
procedure as for 2 starting with commercially available Z-Nal-
OH. 1H NMR (300 MHz, DMSO-d6): 8.81 (bt, 1H), 7.84 (m,
4H), 7.48 (m, 3H), 7.21 (m, 5H), 4.95 (Abq, 2H), 4.40 (m, 1H),
4.12 (d, J ) 7 Hz, 2H), 3.08 (m, 2H). Analysis (C23H21N3O3):
(C, H, N).
Synthesis of 4. This material was produced using the same
procedure as for 2 starting with commercially available Z-Thr-
OH. 1H NMR (300 MHz, DMSO-d6): 8.55 (t, 1H), 7.42 (m, 5H),
7.08 (d, 1H), 5.03 (ABq, 2H), 4.18 (m, 2H), 3.95 (d, 2H), 1.04
(d, 3H). MS (APCI): MH+ 292.
cokinetic suitability of the chemotype. Several examples
of sub-250 pM inhibitors bearing the charged alicyclic
ring-thiazole-benzamide P3 array proved to have high
oral bioavailability in the rat, with half-lives of 3-5 h.
The compound bearing the overall most favorable
attributes, 39n, was dosed in a primate bone resorption
marker model and indicated a clear dose-response in
reducing collagen breakdown products as a ratio with
creatinine, as measured in the urine. PK analysis at the
end of the study indicated a correlation between plasma
levels of the compound and the NTx/creatinine ratio;
furthermore, it was clear that the in vitro bone resorp-
tion assay IC50 offered a highly predictive measure of
the potential utility of this compound series in the in
vivo model. This study suggests that inhibition of
cathepsin K may be a viable therapeutic approach for
the treatment of osteoporosis and other bone disorders
displaying excessive levels of resorption.
Synthesis of 5. This material was produced using the same
procedure as for 1 starting with commercially available
1
Z-IleOSu. H NMR (300 MHz, DMSO-d6): 8.72 (bt, 1H), 7.50
(d, J ) 8 Hz, 1H), 7.35 (s, 5H), 5.02 (s, 2H), 4.13 (m, 2H), 3.85
(apparent t, 1H), 1.75 (m, 1H), 1.42 (m, 1H), 1.14 (m, 1H), 0.80
(m, 6H). Analysis (C16H21N3O3): (C, H, N).
Synthesis of 6. This material was produced using the same
procedure as for 2 starting with commercially available Z-D-
Leu-OH. mp 120-121°C.; 1H NMR (300 Mz, DMSO-d6): 8.68
(t, J ) 6 Hz, 1H), 7.54 (d, J ) 7 Hz, 1H), 7.33 (bs, 5H), 4.99
(ABq, 2H), 4.09 (d, J ) 5 Hz, 2H), 4.00 (m, 1H), 1.28-1.65 (m,
3H), 0.83 (apparent t, J ) 7 Hz, 6H); MS (electrospray): MH+
304 (100%);
Benzyl 1-cyanomethylcarbamoyl-4,4,4-trifluoro-3-meth-
ylbutylcarbamate (Z-F3C(D,L-Leu)-GlyCN, 7): 1H NMR
(DMSO-d6): 9.21 (s, 1 H); 8.01 (s, 1 H); 7.21 (m, 5 H); 5.12 (m,
1 H); 5.01 (s, 2 H); 4.21 (s, 2 H); 3.02 (m, 3 H); 1.01 (m, 3 H).
MS (electrospray): MH+ 358.2.
Benzyl 1-cyanomethylcarbamoyl-1,3-dimethylbutyl-
carbamate (Z-R-MeLeu-GlyCN, 8): 1H NMR (DMSO-d6):
9.21 (s, 1 H); 8.01 (s, 1 H); 7.21 (m, 5 H); 5.01 (s, 2 H); 4.01 (m,
3 H); 1.81 (m, 2 H); 0.86 (m, 6 H). MS (electrospray): MH+
318.2.
Benzyl (S)-1-cyanomethylcarbamoyl-2,2-dimethylpro-
pylcarbamate (Z-t-Leu-GlyCN, 9): 1H NMR (DMSO-d6):
9.21 (s, 1 H); 8.01 (s, 1 H); 7.21 (m, 5 H); 5.01 (s, 2 H); 4.01 (s,
2 H); 3.88 (m, 1 H); 0.96 (s, 9 H). MS (electrospray): MH+
304.2.
Experimental Section
Unless otherwise noted, all nonaqueous reactions were
carried out under a nitrogen atmosphere using commercial
1
grade solvents and reagents. H NMR spectra were recorded
on a JEOL-270 or a General Electric QE-300 FT spectropho-
tometer. Chemical shifts are reported in ppm relative to
tetramethylsilane, using the following abbreviations: s, sin-
glet; d, doublet; t, triplet; dd, doublet of doublets, etc.; m,
multiplet; b, broad. An asterisk (*) signifies an unresolved
peak. Coupling constants (J) are reported in hertz (Hz) where
relevant. Mass spectrometric analyses were obtained on an
ABI-Sciex LC/MS, with electrospray ionization methodology.
Flash chromatography was performed using EM silica gel 60
(230-400 mesh). Reverse phase preparative HPLC was per-
formed using Varian (Rainin) Dynamax, or Gilson instruments
using C18 columns. Elemental analyses were performed by
Robinson Microlit Laboratories, Inc., Madison, NJ.
Benzyl (S)-1-cyanomethylcarbamoyl-3,3-dimethylbu-
tylcarbamate (Z-t-BuAla-GlyCN, 10): 1H NMR (DMSO-
d6): 9.21 (s, 1 H); 8.01 (s, 1 H); 7.27 (m, 5 H); 5.01 (m, 2 H);
4.01 (m, 3 H); 1.55 (m, 2 H); 0.96 (s, 9 H). MS (electrospray):
MH+ 318.2.
Synthesis of 1. A mixture composed of 2,5-dioxopyrrolidin-
1-yl 2-benzyloxycarbonylamino-4-methylvalerate (Z-LeuOSu,
39.6 g, 0.109 mol), aminoacetonitrile hydrochloride (10.1 g,
0.109 mol), triethylamine (61 mL, 0.436 mol), DMF (40 mL),
and acetonitrile (360 mL) was stirred at room temperature for
27 h. The mixture was filtered, concentrated to a volume of
100 mL, and poured into ice-water (1000 mL). The mixture
was stirred until a precipitate had formed. The precipitate was
collected, washed with water, and dried. The dry product was
recrystallized from 55% ethanol/water (80 mL). The crystals
were collected and recrystallized from 65% ethanol/water (70
mL). The crystals were collected and dried to provide benzyl
(S)-1-cyanomethylcarbamoyl-3-methylbutylcarbamate (21.1 g,
Benzyl (S)-1-cyanomethylcarbamoyl-phenylmethyl-
1
carbamate (Z-Phg-GlyCN, 11): H NMR (DMSO-d6): 9.21
(s, 1 H); 8.01 (s, 1 H); 7.27 (m, 5 H); 5.01 (m, 2 H); 4.01 (m, 3
H); 1.55 (m, 2 H); 0.96 (s, 9 H). MS (electrospray): MH+ 324.2.
Analysis (C18H17N3O3): (C, H, N).
Benzyl [1-(cyanomethylcarbamoyl)-cyclopropyl]-car-
bamate (Z-Ac3-GlyCN, 12): Analysis (C14H15N3O3): (C, H,
N).
Benzyl [1-(cyanomethylcarbamoyl)-cyclopentyl]-car-
bamate (Z-Ac5-GlyCN, 13): Analysis (C16H19N3O3): (C, H,
N).
Benzyl [1-(cyanomethylcarbamoyl)-cyclohexyl]-car-
bamate (Z-Ac6-GlyCN, 14): Analysis (C17H21N3O3): (C, H,
N).
1
0.067 mol, 61%) as white needles, mp 120-121 °C. H NMR
(300 MHz, DMSO-d6): 8.68 (t, J ) 6 Hz, 1H), 7.54 (d, J ) 8
Hz, 1H), 7.33 (m, 5H), 5.00 (Abq, 2H), 4.09 (d, J ) 6 Hz, 2H),
4.03 (m, 1H), 1.24-1.64 (m, 3H), 0.84 (apparent t, J ) 7 Hz,
6H) MS (electrospray): MH+ 303.9 (100%). Analysis (C16H21-
N3O3): C, H, N.

Tri-Ring P3 Benzamide-Containing Aminonitriles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7529
Synthesis of 1-Amino-N-cyanomethylcyclohexanecar-
boxamide Methanesulfonate (MsOH‚Ac6-GlyCN, 16).
1-tert-Butoxycarbonylaminocyclohexanecarboxylic acid (Boc-
Ac6OH, 5.0 g, 21.0 mmol) was taken up in DMF (40 mL). The
mixture was cooled in an ice bath and then sequentially
treated with aminoacetonitrile hydrochloride (3.8 g, 42 mmol),
HATU (8.25 g, 21 mmol), and triethylamine (8.0 mL, 63 mmol).
The reaction was allowed to proceed for 4 h, and then the
mixture was concentrated under vacuum. The residue was
treated with saturated NaHCO3 solution (40 mL) and ethyl
acetate (150 mL). The organic layer was separated, sequen-
tially washed with water, 1 M hydrochloric acid (20 mL),
water, and brine, dried over anhydrous MgSO4, and concen-
trated under reduced pressure. The product was purified from
the residue using a plug of silica with ethyl acetate as eluent,
giving 15 (Boc-Ac6-GlyCN). 1H NMR (DMSO-d6) δ 8.09 (s, 1H);
6.76 (s, 1H); 0.4.00 (d, 2H); 1.88-1.07 (m, 19H). MS: M + H+
282.0. This material was dissolved in anhydrous THF (20 mL)
under N2 and dry methanesulfonic acid (6.0 equiv) was added
and the mixture stirred for approximately 12 h. The mixture
was concentrated, and the residue was triturated with ether
(100 mL) and dried under vacuum to provide 16 (5.5 g, >99%
yield). MS (M + H)+: 182.2.
hydrochloride (225 mg,1.16 mmol), and HBTU (493 mg, 1.3
mmol) were dissolved in 5 mL N,N′-dimethylformamide with
excess triethylamine (1.0 mL, 7.17 mmol) and allowed to stir
overnight. The reaction was quenched by the addition of excess
1.0 N HCl and transferred to a separatory funnel. The mixture
was extracted twice with ethyl ether. The combined volume
of ether was washed one time each with water, saturated
aqueous sodium bicarbonate, and brine. The ether was dried
over magnesium sulfate and then filtered through sintered
glass. The filtrate was concentrated in vacuo to give methyl
1-(4-bromobenzoylamino)cyclohexanecarboxylate as a clear,
colorless oil that solidified under continued vacuum. 363 mg,
92% yield. MS (electrospray): MH+ 341.2 (100%). To a solution
of this material (363 mg, 1.07 mmol) in 5 mL dry DMF in a
sealed tube reaction vessel was added dichlorobis(triph-
enylphosphine)palladium(II) (25 mg, 0.036 mmol), CuI (10 mg,
0.053 mmol) triethylamine(0.3 mL, 2.15 mmol), and (trimeth-
ylsilyl)acetylene (0.158 mL, 1.12 mmol). The reaction mixture
was placed under a blanket of nitrogen, sealed (to prevent
escape of volatile (trimethylsilyl)acetylene), and stirred over-
night at 75 °C. The reaction mixture was cooled to ambient
temperature and transferred to a separatory funnel and
extracted thrice with dichloromethane. The combined volume
of dichloromethane was extracted thrice with water and once
with brine. The dichloromethane solution was then dried over
magnesium sulfate and filtered over sintered glass. The filtrate
was then flushed through a short plug of Celite washing with
more dichloromethane. The combined volume of the dichlo-
romethane solution was evaporated to dryness. 1H NMR
showed presence of expected TMS protons. 290 mg yellow oil,
76% crude yield of methyl 1-[4-(trimethylsilanylethynyl)-
benzoylamino]cyclohexanecarboxylate. This material was taken
up with vigorous stirring in 10 mL tetrahydrofuran and 10
mL water. To the stirring solution was added excess lithium
hyrdoxide. The opaque white reaction solution immediately
turned dark brown. The reaction as allowed to stir 2 h. The
reaction solution was quenched by the addition of excess 1.0
N HCl and transferred to a separatory funnel. The mixture
was extracted thrice with ethyl acetate. The combined ethyl
acetate volume was washed once each with 1.0 N HCl, water,
and brine. The ethyl acetate solution was dried over magne-
sium sulfate and filtered over sintered glass. The filtrate was
concentrated under reduced pressure. LCMS of residual oils
showed the desired product (1-(4-ethynylbenzoylamino)-cyclo-
hexanecarboxyclic acid) as the major species. MS (electro-
spray): MH+ 271.7 (100%). The crude 1-(4-ethynylbenzoylami-
no)-cyclohexanecarboxyclic acid (110 mg, 0.406 mmol), HBTU
(161 mg, 0.425 mmol), and aminoacetonitrile (50 mg, 0.540
mmol) was dissolved with vigorous stirring in 10 mL DMF and
2 mL triethylamine. The reaction was allowed to stir overnight.
The reaction was quenched by the addition of 1.0 N HCl and
transferred to a separatory funnel. The mixture was extracted
thrice with ethyl ether. The combined ethereal volume was
washed once each with 1.0 N HCl, water, saturated aqueous
sodium bicarbonate, and brine. The ether solution was dried
over magnesium sulfate and filtered through sintered glass.
The filtrate was concentrated under reduced pressure. The
residual oils were purified by reversed phase preparative
HPLC to give the title compound. 36 mg, 29% yield. Proton
NMR (400 MHz, DMSO-d6): δ 8.2 (s, 1H) 8.0 (d, 1H) δ 7.8 (d,
2H) δ 7.6 (d, 2H) δ 4.2 (d, 2H) δ 2.1 (s, 1H) δ 1.8-0.8 (m, 10H).
MS (electrospray): MH+ 309.1 (100%).
Synthesis of P3-Ac6-GlyCN Derivatives 18a-c, 18e-j,
18l-o (General Procedure). To a mixture of 16 (1 equiv),
HATU (1 equiv), and P3-CO2H (1 equiv) in DMF (0.2 M
concentration) at 0 °C was added diisopropylethylamine (3
equiv). The solution was allowed to warm to room temperature
over 2 h. The reaction was quenched with an equal volume of
saturated aqueous NaHCO3, extracted with ethyl acetate,
washed with water, saturated aqueous NaHCO3, and brine,
dried over MgSO4, filtered, evaporated to dryness, and purified
by crystallization, flash chromatography, or reverse-phase
HPLC as required. Yields ranged from 30 to 80%.
18a: 1H NMR (DMSO-d6): δ 8.04 (s, 1H); 0.7.65 (s, 1H); 7.20
(m, 5H); 4.00 (d, 2H); 4.02 (s, 2H); 2.77 (t, 2H); 2.45 (s,br,
2H);1.95-1.07 (m, 10H); MS: M + H+ 314.0.
18b. 1H NMR (DMSO-d6): δ 8.27-8.14 (m, 2H); 8.08 (s, 1H);
7.82 (d, 1H); 7.71 (d, 1H); 7.40 (t, 1H); 4.02 (d, 2H); 2.15-1.10
(m, 10H). MS: M + H+ 364.0 and 366.2.
Analysis (C16H18BrN3O2): (C, H, N).
1
18c. H NMR (DMSO-d6): δ 8.22 (s,br, 1H); 8.14 (d, 2H);
7.81 (t, 2H); 7.72 (d, 2H); 7.58-7.33 (m, 4H); 4.01 (d, 2H); 2.17-
1.13 (m, 10H). MS: M + H+ 362.0. Analysis (C22H23N3O2): (C,
H, N).
Synthesis of 2-Pyridin-4-yl-thiazole-4-carboxylic Acid
[1-(Cyanomethyl-carbamoyl)-cyclohexyl]-amide (18d).
2-(4-Pyridyl)-thiazole-4-carboxylic acid (209 mg, 1.01 mmol)
and 16 (279 mg, 1.01 mmol) were dissolved in 3.5 mL DMF.
DIPEA (870 µL, 5.0 mmol) was added, and the mixture was
stirred for 5 min. HATU (460 mg, 1.21 mmol) was added, and
the solution was stirred at room-temperature overnight. The
volatiles were removed under reduced pressure. The residue
was dissolved in 50 mL EtOAc and stirred with 25 mL
saturated aqueous NaHCO3 for 30 min. The organic phase was
washed with 3 × 15 mL water and 1 × 25 mL brine, dried
over anhydrous MgSO4, and filtered through a short plug of
silica gel. The solvent was concentrated and dried under high
vacuum to obtain 361 mg of the title compound as a tan solid
in 97% crude yield. 1H NMR (300 MHz, DMSO-d6): 8.77 (d, J
) 7 Hz, 2H), 8.50 (s, 1H), 8.46 (t, J ) 6 Hz, 1H), 8.04 (d, J )
7 Hz, 2H), 7.91 (s, 1H), 4.07 (d, J ) 6 Hz, 2H), 2.17 (apparent
d, J ) 14 Hz, 2H), 1.80 (apparent t, J ) 13 Hz, 2H) 1.2-1.7
(m, 6H). MS (electrospray): M + H+ 370.0.
18l: Analysis (C22H23N3O2): (C, H, N).
18m. 1H NMR (DMSO-d6): δ.8.13 (s, 1H); 7.73 (d, 2H) 7.57
(s, 1H) 6.68 (d, 2H) 4.23-4.05 (s,br, 6H) 4.02 (s, 2H) 2.93 (s,
br, 6H) 2.17-1.03 (m, 10H). MS: M + H+ 329.4.
18e. Analysis (C16H20N4O2): (C, H, N).
1
18f. H NMR (DMSO-d6): δ 8.11 (s, 1H); 7.76(d, 2H), 7.65
18n: Analysis (C24H28N4O2): (C, H, N).
18o: Analysis (C21H29N5O2): (C, H, N).
(s, 1H); 6.93 (d, 2H); 4.02 (d, 2H); 3.70 (s, br, 4H); 3.17 (s, br,
4H); 2.15-1.04 (m, 10H): MS: M + H+ 370.6. Analysis
(C20H26N4O3): (C, H, N).
Synthesis of Biphenyl Amide Derivatives 19a-m (il-
lustrative procedure). To a solution of 18g (3.9 g, 10.7 mmol)
in degassed DMF (50 mL) were added 4,4,4′,4′,5,5,5′,5′-
octamethyl-2,2′-bi-1,3,2-dioxaborolane (Frontier Scientific, 3.0
g, 11.8 mmol), potassium acetate (3.15 g, 32.1 mmol), and
PdCl2(dppf).CH2Cl2 (Strem Chemical, 0.3 g, 0.37 mmol). The
18g. Analysis (C14H16BrN3O2S): (C, H, N).
18h. Analysis (C14H16BrN3O2S): (C, H, N).
Synthesis of N-[1-(Cyanomethylcarbamoyl)cyclohexyl]-
4-ethynylbenzamide (18k). 4-Bromobenzoic acid (233 mg,
1.16 mmol), 1-aminocyclohexanecarboxyclic acid methyl ester

7530 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Palmer et al.
mixture was heated at 80 °C for 6 h. The solution was cooled,
diluted with a 4:1 solution of ethyl acetate:THF (500 mL),
washed with 1:1 brine:water (500 mL) and brine (500 mL),
and then rapidly filtered through a short plug containing
layers (top:bottom) of Celite:activated charcoal:silica gel:
MgSO4). The solution was concentrated, and the residue was
dissolved in CH2Cl2. Hexane was added, and the resulting
precipitate was allowed to stir for 2 h. The intermediate N-[1-
(cyanomethyl-carbamoyl)-cyclohexyl]-4-(4,4,5,5-tetramethyl-
[1,3,2]dioxaborolan-2-yl)-benzamide (37) was collected by fil-
tration as a slightly off-white solid, weight 3.1 g (70%). MS
(M + H): 412. The material was used without further
purification.
To a solution of 37 (0.21 g, 0.5 mmol), 4-piperazin-1-yl-
phenyl bromide hydrochloride (0.28 g, 1.0 mmol), and 2 M
aqueous Na2CO3 (1.5 mL) in degassed DMF (5 mL) was added
PdCl2 (dppf)‚CH2Cl2 (25 mg, 0.03 mmol). The mixture was
stirred at 80 °C in a sealed tube for 3 h, cooled to room
temperature, diluted with 4:1 ethyl acetate/THF (100 mL),
washed with 1:1 brine:water (50 mL) and brine (50 mL),
filtered through a short plug containing layers (top:bottom) of
Celite:activated charcoal:silica gel:MgSO4), concentrated under
reduced pressure, and purified by preparative reverse phase
HPLC to yield the product (157 mg, 35%) as the trifluoroac-
etamide salt. A small portion was converted to the free base
by stirring with K2CO3 in acetonitrile, filtering through Celite,
from which white crystals were obtained as a monohydrate.
19a. 1H NMR (DMSO-d6) (400 MHz): δ 8.23 (1H, t, J ) 4.5
Hz), 8.01 (1H, s), 7.96 (2H, d, J ) 6.6 Hz), 7.26 (2H, d, J ) 6.6
Hz), 7.31 (2H, d, J ) 6.6 Hz), 7.04 (2H, d, J ) 6.6 Hz), 4.08
(2H, d, J ) 4 Hz), 3.14 (4H, 2xd, J ) 3, 3 Hz), 2.87 (4H, 2xd,
J ) 3, 3 Hz), 2.15 (2H, m), 1.78 (2H, m), 1.56 (5H, m), 1.28
(1H, m). 13C NMR (DMSO-d6) (500 MHz): δ 174.8, 165.9,
151.23, 142.5, 132.04, 128.6, 128.2, 127.1, 124.8, 117.6, 115.1,
59.1, 48.7, 45.3, 31.5, 27.4, 24.9, 21.0. LC/MS: M + 1: 446.
Analysis: Calcd for C26H31N5O2‚H2O, C, 67.36; H, 7.18; N,
15.11. Found: C, 67.54, H, 6.86, N, 15.14.
19b. 1H NMR (DMSO-d6) (270 MHz): δ 8.24 (1H, t, J ) 5.5
Hz), 8.02 (1H, s), 7.95 (2H, d), 7.70 (3H, m), 7.14 (2H, bs), 5.34
(1H, br), 4.05 (2H, d, J ) 8.4 Hz), 3.78 (2H, m), 3.20 (6H),
2.18 (6H, m), 1.77 (2H, m), 1.53 (5H, m), 1.25 (1H, m). MS: M
+ H+: 505.6.
2.00 (2H, m), 1.70 (6H, m), 1.53 (5H, m), 1.26 (1H, m). MS: M
+ 1: 474.4. Analysis (C28H34N4O3): (C, H, N).
General Synthesis of 4-N-Alkylpiperazines, Illustrated
for 4-N-tert-Butylpiperidine. To a mixture of tert-butyl-4-
oxopiperidine-1-carboxylate (7.0 g, 35.13 mmol) and tert-
butylamine (7.41 mL, 70.26 mmol) under nitrogen was added
titanium isopropoxide (12.55 mL, 43.91 mmol) at room tem-
perature and stirred. A white precipitate was observed after
15 min. After 2 h and 30 min, 50 mL ethanol was added
followed by 700 mg PtO2. This was then hydrogenated over-
night using a Parr reactor at 50 psi hydrogen pressure. The
reaction was then filtered and concentrated to give 5.81 g of
tert-butyl 4-tert-butylaminopiperidine-1-carboxylate. 1H
NMR (DMSO-d6) (270 MHz): δ 3.79 (4H, m), 2.78 (1H, t), 2.61
(1H, m), 1.61 (4H, m), 1.36 (9H, s), 1.03 (9H, s). MS: M + H+:
256.6. Note: In the above procedure 2 equiv of the amine was
used because the excess amine could be evaporated. In the case
of higher boiling point amines 1.2 stoichiometric equivalents
of the amine to 1 stoichiometric equivalent of the ketone is
used.
4-tert-Butylamino-piperidine-1-carboxylic acid tert-butyl es-
ter (5.81 g) was treated with 100 mL 4 M HCl in dioxane at
room temperature and stirred for 1 h. LC/MS shows reaction
complete. The reaction was concentrated under reduced pres-
sure and the residue triturated with ether (3 × 100 mL). This
was then dried under vacuum to yield 5.63 g of tert-butylami-
nopiperidin-4-yl-amine. 1H NMR (DMSO-d6) (270 MHz): δ 3.31
(2H, m), 2.92 (3H, m), 2.06 (4H, m), 1.39 (9H, s). MS: M +
H+: 256.6. This material was then coupled with 37 as
described earlier to provide 19m.
General P3-Thiazolebenzoate-Ac6-GlyCN Procedure.
Synthesis of Substituted Thiazolebenzoic Acids. 4-Acetyl-
benzoic acid (20, 20 g, 128.2 mmol) was taken up in 200 mL
of acetic acid. The acid was dissolved by heating the solution
to 80 °C. Bromine (7.0 mL, 128.2 mmol) was added (exothermic
reaction) and stirred for 1 h while the solution cooled to room
temperature. The product, 21, was filtered and washed with
ether and dried. Yield: 20 g (67%). 1H NMR (DMSO-d6, ppm):
δ 10.1 (br. s. 1 H), 7.81 (m, 4 H), 5.51 (s, 2 H).
21 was condensed with thioamides, thioureas, thiocarbam-
ates (25, 28, 30, 32, and 34 as described in procedures A-E
below) to form the requisite thiazolebenzoic acids.
19d. 1H NMR (DMSO-d6) (270 MHz): δ 8.20 (1H, t, J ) 8.4
Hz), 8.00(1H, s), 7.95 (2H, d, J ) 12.4 Hz), 7.71 (2H, d, J )
12.4 Hz), 7.65 (2H, d, J ) 12.8 Hz), 7.06 (2H, d, J ) 12.8 Hz),
4.44 (1H, m), 4.05 (2H, d, J ) 8.4 Hz), 2.65 (2H, m), 2.20 (5H,
m), 1.95 (2H, m), 1.70 (6H, m), 1.53 (5H, m), 1.26 (1H, m).
LC/MS: M + 1: 475.4.
General Synthesis Procedure A, Illustrated for Prepa-
ration of 39d. Potassium carbonate (31.1 g, 225 mmol),
1-methylpiperazine (23, R) Me; 18.3 mL, 165 mmol), and
bromoacetonitrile (10.4 mL, 150 mmol) were stirred in aceto-
nitrile (450 mL) for 75 h. The solids were removed by filtration,
and the filtrate was concentrated in vacuo. The residue was
purified by flash chromatography on silica gel using 9:1 CH2-
Cl2:MeOH eluent to obtain 11.41 g (55%) of 24 as a pale amber
oil.
24 (13.04 g, 93.7 mmol) was stirred in 400 mL of 3:1
triethylamine:pyridine for 30 min. The stirred suspension was
saturated with hydrogen sulfide via a gas dispersion frit. A
white precipitate formed during introduction of the H2S. The
flask was sealed and stirred overnight, concentrated, and the
residue was boiled in ethyl acetate (250 mL) for 15 min. The
supernatant was decanted and cooled, yielding 4.20 g small
brown crystals by filtration. A second crop of 0.94 g was
obtained (37% yield of 25.
Synthesis of 19i. To a solution of 1-methylpiperidin-3-ol
(2.2 g, 0.0191 mol) and 4-bromophenol (3.3 g, 0.0191 mol) in
dry THF (20 mL), at 0 °C, were added a solution of triph-
enylphosphine (5.0 g, 0.0191 mol) in THF (15 mL) and diethyl
azodicarboxylate (3.0 mL, 0.0191 mL). After stirring for 18 h
at room temperature, under nitrogen, the mixture was diluted
with diethyl ether (50 mL) and treated with 1 N HCl (50 mL).
The aqueous solution was further extracted with diethyl ether
(2 × 20 mL), and the pH was adjusted to about 10 by adding
1 N NaOH. This mixture was extracted several times (3×) with
DCM (50 mL), and the combined organic extracts were dried
over magnesium sulfate. After evaporation of the solvent, the
product was obtained as a thick oil (3.1 g, 60%), LC/MS: M +
1: 271. This material was coupled with 37 using the pal-
ladium-assisted cross-coupling procedure illustrated for 19a.
1H NMR (DMSO-d6) (270 MHz): δ 8.21 (1H, t, J ) 5.4 Hz),
8.00 (1H, s), 7.95 (2H, d, J ) 8.4 Hz), 7.72 (2H, d, J ) 8:4 Hz),
7.67 (2H, d, J ) 8.8 Hz), 7.05 (2H, d, J ) 8.8 Hz), 4.05 (2H,
m), 3.88 (1H, m), 2.96 (1H, m), 2.60 (1H, m), 2.37 (3H, s), 2.20
(2H, m), 1.95 (2H, m), 1.70 (6H, m), 1.53 (5H, m), 1.26 (1H,
m). Analysis: C, 70.86%; H, 7.22%; N, 11.81%. Found: C,
68.35%; H, 7.79%; N, 11.43%. LC/MS: M + 1: 475.4.
25 (1.73 g, 10.0 mmol) was dissolved in anhydrous THF (40
mL). A solution of 4-(2-bromoacetyl)benzoic acid (2.43 g, 10.0
mmol in anhydrous THF (20 mL) was added. The mixture was
stirred at 70 °C for 5 h, cooled, and filtered. The solids were
washed with Et2O and dried under high vacuum over P2O5 to
obtain 3.40 g (85%) of 4-[2-(4-methyl-piperazin-1-ylmethyl)-
thiazol-4-yl]-benzoic acid hydrobromide as an orange solid.
This material was coupled with 16 in the presence of HATU
and diisopropylethylamine as described previously, in 7% yield
after reverse phase HPLC using a 20 f 65% MeCN/H2O +
19j. 1H NMR (DMSO-d6) (270 MHz): δ 8.20 (1H, t, J ) 5.4
Hz), 8.00 (1H, s), 7.95 (2H, d, J ) 8.4 Hz), 7.71 (2H, d, J ) 8.4
Hz), 7.65 (2H, m), 7.05 (2H, d, J ) 8.9 Hz), 4.05 (2H, J ) 5.4
Hz), 3.90 (2H, m), 2.95 (1H, m), 2.37 (3H, s), 2.10 (2H, m),
0.05% TFA gradient elution. H NMR (300 MHz, DMSO-d6):
1
δ 9.58 (br s, 1H), 8.26* (s, 1H), 8.24 (t, J ) 6 Hz, 1H), 8.05* (s,
1H), 8.03 (d, J ) 9 Hz, 2H), 7.96 (d, J ) 9 Hz, 2H), 4.06 (m,
2H), 3.38-3.48 (m, 2H), 3.03-3.17 (m, 4H), 2.82 (s, 3H), 2.52-

Tri-Ring P3 Benzamide-Containing Aminonitriles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7531
2.60 (m, 2H), 2.07-2.19 (m, 2H), 1.70-1.84 (m, 2H), 1.46-
1.60 (m, 5H), 1.2-1.36 (m, 1H). MS (ES): MH+ 480.6.
for 3 h, filtered, and concentrated. Trituration of the white
solid residue with 3 × 15 mL acetone and drying under high
vacuum gave 1.45 g (51%) of 1-methylpiperidine-4-carboxam-
ide as a white solid.
Synthesis of 39a. General synthesis procedure A (starting
with morpholine and bromoacetonitrile) was employed. The
final step (HATU-mediated condensation between 16 and 4-(2-
morpholin-4-ylmethylthiazol-4-yl)-benzoic acid) proceeded in
11% isolated yield. 1H NMR (300 MHz, DMSO-d6): δ 8.23* (t,
J ) 6 Hz, 1H), 8.22* (s, 1H), 8.05* (s, 1H), 8.03 (d, J ) 9 Hz,
2H), 7.96 (d, J ) 9 Hz, 2H), 4.06 (d, J ) 6 Hz, 2H), 3.90 (s,
2H), 3.63 (t, J ) 5 Hz, 4H), 2.56 (t, J ) 5 Hz, 4H), 2.08-2.20
(m, 2H), 1.68-1.84 (m, 2H), 1.48-1.62 (m, 5H), 1.20-1.38 (m,
1H). Calcd exact mass: 467.1991. MS (ES) MH+ 467.4.
General Synthesis Procedure B, Illustrated for Prepa-
ration of 39c. Boc-isonipecotic acid (26, 3.07 g, 13.4 mmol)
and 1,1-carbonyldiimidazole (2.53 g, 15.6 mmol) were dissolved
in 50 mL THF and stirred at room temperature for 1.5 h. The
solution was cooled in an ice bath, and 15 mL (30 mmol) of a
2.0 M solution of NH3 in MeOH was added. The solution was
stirred 30 min at 0 °C and then overnight at room temperature.
A white precipitate formed. The solvent was removed under
reduced pressure. The residue was dissolved in EtOAc (150
mL) and washed with 2 × 50 mL 10% aqueous citric acid, 1 ×
50 mL 10% aqueous NaHCO3, and 1 × 75 mL brine. The
solution was dried over anhydrous MgSO4 and concentrated
under vacuum to give 2.50 g of BOC-isonipecotamide, 27, as
a white solid. This material was dissolved in THF (50 mL).
Lawesson’s reagent (4.47 g (11 mmol) was added. The mixture
was heated at reflux under nitrogen for 3 h, cooled, and
allowed to stand overnight, before being concentrated under
reduced pressure. The residue was dissolved in EtOAc (200
mL) and washed with 1 × 75 mL 10% aqueous citric acid, 1 ×
75 mL 10% aqueous NaHCO3, 2 × 75 mL water, and 1 × 75
mL brine. The solution was dried over anhydrous MgSO4 and
concentrated. The residue was purified by flash chromatog-
raphy to obtain 1.26 g of tert-butyl 4-thiocarbamoylpiperidine-
1-carboxylate, 28, as a white solid in 38% yield.
28 (1.26 g, 5.16 mmol) and 1.25 g (5.14 mmol) 4-(2-
bromoacetyl)benzoic acid (1.25 g, 5.14 mmol) were dissolved
in anhydrous THF (100 mL) and heated at 70 °C for 3 h. The
solvent was removed under reduced pressure to obtain 2.55 g
of a white solid, which was dissolved in DMF (20 mL). 16 (1.40
g, 5.0 mmol) and DIPEA (4.5 mL, 26 mmol) were added and
stirred for 5 min. HATU (2.08 g, 5.47 mmol) was added. The
mixture was stirred at room temperature for 3 h. The solvents
were removed under reduced pressure. The residue was
dissolved in EtOAc (300 mL), washed with 1 × 75 mL 10%
aqueous citric acid, 1 × 75 mL water, 1 × 75 mL 10% aqueous
Na2CO3, 1 × 75 mL water, and 1 × 75 mL brine, dried over
anhydrous MgSO4, and concentrated. The residue was purified
by radial chromatography using EtOAc eluent to obtain 1.11
g of 4-(4-{4-[1-(cyanomethyl-carbamoyl)-cyclohexylcarbamoyl]-
phenyl}-thiazol-2-yl)-piperidine-1-carboxylic acid tert-butyl es-
ter in 39% yield.
To a rapidly stirred solution of this material (5.65 g, 39.7
mmol) in anhydrous CHCl3 (100 mL) was added thionyl
chloride (29.0 mL, 398 mmol) at a fast dropping rate. A lumpy
precipitate formed during addition. The mixture was then
heated at reflux for 20 h. Excess solvent and SOCl2 were
removed by distillation. The residue was dissolved in CH2Cl2
(50 mL), and then 5% aqueous NH4OH (150 mL) was added
and stirred for 15 min. The phases were separated. The
aqueous phase was extracted further with CH2Cl2 (2 × 50 mL).
The organics were combined, washed with water (2 × 50 mL)
and brine (75 mL), dried over MgSO4, filtered, and concen-
trated. The residue was distilled (72-74 °C/1.3 kPa) to obtain
2.54 g (52%) of 1-methylpiperidine-4-carbonitrile as a pale
yellow oil.
A solution of 2.5 g (20 mmol) of this material in anhydrous
THF was added dropwise to 15 mL (30 mmol) of a clear orange
2.0 M solution of lithium diisopropylamide in THF/heptane/
ethylbenzene at -10 °C, stirred at 0 °C for 1.5 h, cooled to
-10 °C, and treated with CH3I (1.5 mL, 24 mmol) dropwise.
The solution was stirred at 0 °C for 1.5 h, quenched with
ethanol (1.5 mL), diluted with water (25 mL), stirred for 15
min, and filtered through Celite, washing the Celite with ether
(50 mL). The aqueous phase was extracted with ether (3 × 25
mL). The combined organic extracts were washed with brine
(75 mL), dried over MgSO4, filtered, and concentrated to obtain
2.2 g of a clear yellow liquid. This material was dissolved in
Et2O and converted to its hydrochloride salt with 4.0 M HCl/
dioxane. The precipitate was filtered and dried under high
vacuum to obtain 1.51 g (43%) of 1,4-dimethylpiperidine-4-
carbonitrile hydrochloride. The synthesis of 39f was then
accomplished via the general synthesis procedure B outlined
above. 1H NMR (300 MHz, DMSO-d6): 8.23 (t, J ) 6 Hz, 1H),
8.17 (s, 1H), 8.04 (d, J ) 9 Hz, 2H), 8.02* (s, 1H), 7.96 (d, J )
9 Hz, 2H), 4.06 (d, J ) 6 Hz, 2H), 2.40-2.50 (m, 2H), 2.04-
2.38 (m, 6H), 2.14* (s, 3H), 1.70-1.86 (m, 4H), 1.46-1.62 (m,
5H), 1.36* (s, 3H), 1.20-1.40 (m, 1H). Calcd exact mass:
479.2355. MS (ES): MH+ 479.8 (100%).
General Synthesis Procedure C, Illustrated for Prepa-
ration of 39g. Di-tert-butyl dicarbonate (75.5 g, 346 mmol)
was dissolved in 250 mL anhydrous CH2Cl2 under N2. The
solution was cooled in an ice bath, and 4-hydroxypiperidine
(25.1 g, 248 mmol) dissolved in 200 mL anhydrous CH2Cl2 was
added, followed by Et3N (35 mL,250 mmol), keeping the
reaction temperature below 10 °C. The mixture was then
stirred at room temperature for 4 h, diluted with methanol
(100 mL), and stirred for 60 min. The volatiles were removed
on a rotary evaporator and the residue dried under reduced
pressure. The residue was dissolved the residue in EtOAc (300
mL), washed with 1 × 100 mL 10% aqueous citric acid, 1 ×
100 mL water, 1 × 100 mL 10% aqueous NaOH, 1 × 100 mL
water, and 1 × 100 mL brine, dried over anhydrous MgSO4,
filtered, and concentrated to give 38.0 g (76%) of Boc-4-
hydroxypiperidine (29: R) Boc) as a white solid.
29 (18.47 g, 91.8 mmol) and Et3N (14.1 mL, 101 mmol) were
dissolved in THF (250 mL) and cooled in an ice bath. Thio-
phosgene (7.0 mL, 92 mmol) was added dropwise, maintaining
a reaction temperature below 15 °C. The mixture was stirred
at room temperature for 1 h, cooled again in an ice bath, and
treated with 35 mL (252 mmol) of 28% aqueous NH4OH. The
solution was stirred at room temperature for 3 h, concentrated,
dissolved in EtOAc (300 mL), washed with 2 × 100 mL 10%
aqueous citric acid, 1 × 100 mL 10% saturated aqueous
NaHCO3, 1 × 100 mL water, and 1 × 100 mL brine, dried
over anhydrous MgSO4, filtered, and concentrated. The product
was purified by flash chromatography on silica gel using 3:1
CH2Cl2:EtOAc eluent to obtain 1.57 g of 30 R ) Boc, 7% yield.
This material (208 mg, 0.376 mmol) was dried under high
vacuum, dissolved in 1.0 mL anhydrous THF, and stirred until
homogeneous. The solution was cooled in an ice bath, and
anhydrous methanesulfonic acid (74 µ, 1.1 mmol) was added.
The mixture was allowed to stand at room-temperature
overnight. The solution was concentrated at 20 °C, and the
residue was triturated three times with Et2O. The product,
39c, was purified by reverse phase preparative TLC using 7:3
1
MeCN:H2O eluent giving 116 mg (56%) as a white solid. H
NMR (300 MHz, DMSO-d6): δ 8.3-8.6 (br s, 1H), 8.26 (t, J )
6 Hz, 1H), 8.21 (s, 1H), 8.07 (s, 1H), 8.04 (d, J ) 9 Hz, 2H),
7.97 (d, J ) 9 Hz, 2H), 4.06 (d, J ) 6 Hz, 2H), 3.35-3.50 (m,
2H), 3.07 (t, J ) 13, 2H), 1.85-2.30 (m, 6H), 1.68-1.84 (m,
2H), 1.48-1.60 (m, 5H), 1.2-1.36 (m, 1H). Calcd exact mass:
451.2042. MS (electrospray): MH+ 451.2 (100%).
Synthesis of 39f. Isonipecotamide (10.0 g, 78.0 mmol) was
placed in a 250 mL Parr hydrogenation bottle and dissolved
in 70 mL MeOH. Aqueous formaldehyde (37%) (17.5 mL (235
mmol) was added. The solution was stirred for 3 h. Raney
nickel (2.25 g of a 50% w/w slurry) in water was added. The
mixture was hydrogenated on a Parr apparatus at 60 psi H2
30 (1.57 g, 6.03 mmol) was dissolved in 15 mL anhydrous
THF. A solution of 21 (1.47 g, 6.03 mmol) was added in
anhydrous THF (15 mL). The reaction was heated at 70 °C
for 6 h, cooled to room temperature, filtered, and concentrated.

7532 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Palmer et al.
The residue was purified by flash chromatography on silica
gel using 9:1 CH2Cl2:MeOH eluent, giving 1.70 g (70%) of Boc-
4-[4-(4-carboxyphenyl)-thiazol-2-yloxy]-piperidine as a cream
colored solid. This material (982 mg, 2.43 mmol) and 16 (673
mg, 2.43 mmol) were dissolved in DMF (10 mL). DIPEA (1.7
mL, 9.8 mmol) was added. The solution was stirred for 5 min.
HATU (1.02 g, 2.69 mmol) was added, and the mixture was
stirred for 4 h. The solvents were removed under reduced
pressure. The residue was dissolved in EtOAc (150 mL),
washed with 1 × 50 mL 10% aqueous citric acid, 1 × 50 mL
water, 1 × 50 mL 10% aqueous Na2CO3, 1 × 50 mL water,
and 1 × 50 mL brine, dried over anhydrous MgSO4, filtered,
and concentrated. The residue was purified by flash chroma-
tography on silica gel using 1:1 CH2Cl2:EtOAc eluent, to obtain
1.08 g (78%) of Boc-4-(4-{4-[1-(cyanomethylcarbamoyl)-cyclo-
hexylcarbamoyl]-phenyl}-thiazol-2-yloxy)-piperidine.
General Synthesis Procedure E, Illustrated for Prepa-
ration of 39n. 4-Methylpiperazinyl thiourea (34, 15.0 g, 95
mmol) was added to THF (500 mL) containing 21 (22.0 g, 95.0
mmol). The mixture was refluxed for 3 h. The product, 22 (Y
) N-methylpiperazinyl) was filtered, washed with acetone and
ether, and dried. The yield was 22.0 g, 60%. The product was
characterized by LC-MS (FAB LC-MS. 304.2 (M + H+)).
To a stirred solution of thiocarbonyldiimidazole (23, 17.8 g,
100 mmol) in THF (300 mL) at room temperature was added
N-methyl piperazine (23, R ) Me, 10.0 g, 100 mmol). The
reaction mixture was stirred at room temperature for 1 h and
then at 55 °C for 1 h. The mixture was cooled to room
temperature, and approximately 20 mL of THF was removed
under vacuum. Ammonia in ethanol (200 mL of a 2.0 M
solution) was added. The mixture was stirred overnight. The
reaction was then heated at 55 C for 2 h and cooled to room
temperature. The product, 22 Y ) N-methyl piperazine, was
filtered, washed with ether, and dried. Yield 15.0 g (90%). 1H
NMR (DMSO-d6, ppm): δ 7.5 (br. s. 2 H), 3.68 (m, 4 H), 2.23
(m, 4 H), 2.13 (s, 3 H).
To a solution of 274 mg (0.482 mmol) of this material in
THF (1.0 mL) at 0 °C was added MsOH (94 µL, 1.4 mmol).
The mixture was allowed to stand at room-temperature
overnight. The mother liquor was decanted. The precipitate
was triturated three times with 2:1 Et2O:THF. The residue
was purified by reverse phase HPLC using a 20 f 65% MeCN/
H2O + 0.05% TFA gradient elution to obtain 42 mg (15%) of
22, Y ) N-methyl piperazine (20 g, 50.80 mmol), was taken
up in DMF (350.0 mL), and 16 (15.0 g, 50.8 mmol) was added,
followed by triethylamine (27.6 mL, 200 mmol) and HATU
(20.0 g, 50.8 mmol). The solution was stirred overnight at room
temperature. DMF was removed under vacuum. Saturated
sodium bicarbonate (200 mL) was added, and the mixture was
stirred for 30 min. Water (100 mL) and ether (100 mL) were
added, and the mixture was stirred for an additional 15 min.
The solution was filtered. The solid was washed with water
twice and dried. The solid was then taken up in 10% methanol
in dichloromethane and dried over anhydrous magnesium
sulfate. (This is done to remove any traces of water, which
interferes with the recrystallization procedure.) The solvent
was removed in vacuo. This was then redissoved in hot 10%
methanol in dichloromethane. The solution was allowed to cool
to room temperature, and ether was added till the solution
became turbid. This was then stirred for 30 min, and the
product was filtered, washed with ether, and dried. The
product was characterized by LC-MS and NMR. Yield (20 g,
1
39g as a white solid. H NMR (300 MHz, DMSO-d6): δ 8.58
(br s, 1H), 8.24 (t, J ) 6 Hz, 1H), 8.04 (s, 1H), 7.94 (s, 4H),
7.67 (s, 1H), 5.29 (m, 1H), 4.06 (d, J ) 6 Hz, 2H), 3.08-3.35
(m, 4H), 1.92-2.33 (m, 6H), 1.68-1.84 (m, 2H), 1.45-1.60 (m,
5H), 1.2-1.36 (m, 1H). Calcd exact mass: 467.1991. MS
(electrospray): MH+ 468.2 (100%).
General Synthesis Procedure D, Illustrated for Prepa-
ration of 39j. (4-Methyl-piperazin-1-yl)-thiourea was synthe-
sized following the procedure according to ref 17. A suspension
of this compound (6.6 g, 0.038 mol) and 4-(2-bromo-acetyl)-
benzoic acid (9.2 g, 0.038 mol) in ethanol (300 mL) was refluxed
for 2 h. The reaction mixture was cooled at 4 °C for about 1 h,
and the yellow solid that formed was filtered and washed with
ethyl ether. The product, 4-[2-(4-methylpiperazin-1-ylamino)-
thiazol-4-yl]-benzoic acid hydrobromide, was obtained as a
yellow solid (12.8 g, 84%, LC/MS: M + 1: 319.0).
1
80%). H NMR (DMSO-d6, ppm): δ 8.13 (m, 1 H), 8.03 (m, 4
To a suspension of the hydrobromide salt (2.5 g, 0.0062 mol)
in methanol (20 mL) was bubbled HCl gas for 15 min. The
solution was left stirring overnight at room temperature. The
solid that crashed out from the solution was filtered and
washed with ethyl ether. Methyl 4-[2-(4-methylpiperazin-1-
ylamino)thiazol-4-yl]-benzoate dihydrochloride was obtained
as an yellow solid (2.5 g, 100%, LC/MS, M + 1: 332.6.
H), 7.51 (s, 1 H), 4.89-4.13 (m, 5 H), 3.21-3.67 (m, 6 H), 2.81
(s, 3 H), 2.11-2.23 (m, 2 H), 1.55-1.72 (m, 6 H). 13C NMR
(DMSO-d6, ppm): δ 21.27, 27.59, 31.67, 38.56, 45.71, 47.91,-
53.69,59.27, 104.31, 117.77,125.07, 128.16, 133.34, 137.10,
149.78, 165.96, 170.49, 174.92. MS (ES): 466.4 (M + H+).
Analysis (C28H34N4O3): (C, H, N)
39b. Synthesized via general procedure E: 1H NMR
(DMSO-d6) (270 MHz): δ 8.22 (1H, t, J ) 5.4 Hz), 8.00 (1H,
s), 7.96 (4H, s), 7.44 (1H, s), 5.15 (1H, m), 4.05 (2H, d, J ) 5.7
Hz), 3.31 (6H, s), 3.04 (2H, s), 2.89 (2H, s), 2.30 (3H, s), 2.11
(4H, m), 1.76 (2H, m), 1.53 (5H, m), 1.26 (1H, m). LC/MS: M
+ 1: 481.6.
To a solution this material (3.0 g, 0.0074) in dry DMF (15
mL) at 0 °C, a 60% suspension of NaH in oil (900 mg, 0.023
mol) was added and the reaction mixture stirred for 15 min.
Then, methyl iodide (0.500 mL, 0.00814 mol) was added and
the mixture stirred overnight at room temperature. The
reaction mixture was diluted with ethyl acetate and washed
with water and brine. After drying over sodium sulfate, the
solution was concentrated to dryness to obtain a yellow solid
(2.0 g, LC/MS, M + 1: 346.2). The solid was dissolved in MeOH
(100 mL) and a solution of NaOH (924 mg, 0.023 mol) in water
(20 mL) was added. The reaction mixture was stirred overnight
at room temperature. The methanol was removed under
vacuum, and the pH of the aqueous solutions was acidified to
pH 5 by adding 1 N HCl. The mixture was extracted several
times with ethyl acetate, and the combined organic layers were
washed with brine and dried over sodium sulfate. After
evaporation of the solvent, the title compound was obtained
as a yellow solid (0.820 mg, 33% overall yield). 1H NMR
(DMSO-d6): d 7.93 (4H, m), 7.44 (1H, s), 3.40 (4H, m), 3.27
(3H, s), 3.21 (4H, m), 3.16 (3H, s). This material was coupled
with 16 as described previously to obtain the title compound.
1H NMR (DMSO-d6) (270 MHz): δ 8.18 (1H, t, J ) 5.4 Hz),
7.95 (1H, s), 7.88 (5H, s), 4.03 (2H, d, J ) 5.4 Hz), 3.10 (3H,
s), 2.82 (4H, m), 2.19 (3H, s), 2.10 (4H, m), 1.74 (2H, m), 1.52
(5H, m), 1.25 (1H, m). Analysis: C, 60.58%; H, 6.71%, N,
1978%. Found: C, 59.11%; H, 7.02%; N, 19.08%. LC/MS: M
+ 1: 496.5.
39e. Synthesized via general procedure E: 1H NMR
(DMSO-d6) (270 MHz): δ 8.22 (1H, t, J ) 5.5 Hz), 8.00 (1H,
s), 7.95 (2H, d, J ) 8.4 Hz), 7.90 (2H, d, J ) 8.4 Hz), 7.39 (1H,
s), 4.05 (2H, d), 3.75 (1H, m), 3.50 (4H, m), 2.30 (3H, s), 2.11
(4H, m), 1.77 (2H, m), 1.53 (5H, m), 1.27 (1H). MS (ES): M +
H+: 453.2.
39i. Synthesized via general procedure E: 1H NMR
(DMSO-d6) (270 MHz): δ 8.22 (1H, t), 8.00 (1H, s), 7.91 (2H,
s), 7.90 (2H, s), 7.46 (1H, s), 4.05 (2H, d), 3.83 (1H, m), 3.46
(5H, m), 3.15 (2H, t), 2.09 (4H, t), 1.75 (2H, m), 1.53 (6H, m),
1.23 (3H, s), 1.21 (3H, s). MS: M + H+: 508.8.
39k. Synthesized via general procedure E: 1H NMR
(DMSO-d6) (270 MHz): δ 8.22 (1H, t), 8.00 (1H, s), 7.91 (2H,
s), 7.90 (2H, s), 7.43 (1H, s), 4.06 (2H, t,m), 2.74(1H, m), 2.65
(3H, m), 2.49 (1h, m), 2.09 (5H, m), 1.69 (5H, m), 1.53 (8H,
m), 1.23 (3H, s), 1.21 (3H, s). MS: M + H+: 523.639n. MS: M
+ H+: 503.4.
39l. Synthesized via general procedure E: 1H NMR
(DMSO-d6) (270 MHz): δ 8.22 (1H, t), 8.00 (1H, s), 7.91 (2H,
s), 7.90 (2H, s), 7.43 (1H, s), 4.04 (2H, d), 3.87 (2H, m), 3.45
(4H, m), 2.62 (5H, t), 2.49 (2H, m), 2.09 (2H, m), 1.69 (4H, m),
1.53 (8H, m). MS: M + H+: 537.6.

Tri-Ring P3 Benzamide-Containing Aminonitriles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7533
39n. Synthesized via general procedure E. 1H NMR
(DMSO-d6) (270 MHz): δ 8.19 (1H, t, J ) 5.4 Hz), 7.97 (1H,
s), 7.92 (2H, d, J ) 8.8 Hz), 7.88 (2H, d, J ) 8.8 Hz), 7.42 (1H,
s), 4.04 (2H, d, J ) 5.7 Hz), 3.46 (6H, m), 3.30 (3H, s), 2.55
(4H, m), 2.10 (2H, m), 1.74 (2H, m), 1.52 (5H, m), 1.25 (3H,
m). LC/MS: M + 1: 511.6.
Acknowledgment. The authors thank Joanne Fa-
nucchi for help with preparation of this manuscript and
Drs. Jochen Knolle and Ann Welton for helpful discus-
sions.
Supporting Information Available: Elemental analysis
data for compounds 1-3, 5, 11-14, 18b,c,e-i,n,o, 19j, and
39n. This material is available free of charge via the Internet
at http://pubs.acs.org.
39p. Synthesized via general procedure E: 1H NMR
(DMSO-d6) (270 MHz): δ 8.20 (1H, t), 8.00 (1H, s), 7.90 (4H,
s), 7.39 (1H, s), 4.01 (4H, d, m), 3.02 (3H, t), 2.09 (2H, m), 1.74
(5H, m), 1.49 (17H, m). MS: M + H+: 535.4.
General Procedure F, Illustrated for 39q. A 1:1 mixture
of tert-butylpiperazine13a (23, R ) t-Bu) and 2,4-dibromothia-
zole (35) in DMF (in the presence of 2 equiv of triethylamine)
was heated for 3 h, cooled, and purified by flash chromatog-
raphy (10% MeOH/CH2Cl2) to give 36 (R ) t-Bu) in 50% yield.
This material was coupled with 37 using the palladium-
assisted cross-coupling procedure as described for the biphe-
nylamide derivatives 19a-m, above.
Enzyme Assays. All enzymes used in these studies, with
the exception of human cathepsin B, were produced by Celera
Genomics. Cathepsin B was from human liver and was
purchased from Athens Research and Technology (Athens,
GA). The substrates used in these studies were purchased from
the following vendors: Z-Phe-Arg-AMC, Boc-Leu-Lys-Arg-
AMC, and Z-Val-Val-Arg-AMC were from Bachem (Torrance,
CA) and Z-Leu-Arg-AMC was from Calbiochem-Novabiochem
(San Diego, CA). Bovine serum albumin was purchased from
the Sigma Chemical Company (St. Louis, MO). Routine buffer
components and all other chemicals used in these experiments
were of the highest available quality.
Methods. Enzyme inhibition studies were performed under
several sets of conditions, and each set was tailored to provide
the optimal activity of the given cathepsin being assayed. Each
cathepsin was incubated with the inhibitor, present at variable
concentrations, under the conditions specified below. Enzyme
and inhibitor were incubated together for 30 min at room
temperature (21 to 24 °C) in 96-well U-bottom, microtiter
plates (Falcon, from Becton Dickinson, Franklin Lakes, NJ).
After the preincubation phase, reactions were initiated with
the addition of the 7-amino-4-methylcoumarin (AMC) sub-
strate specified below. The hydrolysis of these substrates yields
7-amino-4-methylcoumarin which was monitored fluorometri-
cally (excitation at 355 nm and emission at 460 nm) using an
FMAX 96-well plate reader from Molecular Devices (Sunny-
vale, CA) interfaced with a Macintosh computer. Under these
experimental conditions, 1 µM of product produces a fluores-
cence signal of approximately 125 units. The velocity of the
enzyme-catalyzed reaction was obtained from the linear por-
tion of the progress curves (usually the first 5 min after
addition of substrate).
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