Rational design of potent and selective NH-linked aryl/heteroaryl cathepsin K inhibitors

Article abstract of DOI:10.1016/j.bmcl.2004.05.087

Prior reports from our laboratories have identified the nonpeptidic inhibitor 2 as a potent and selective Cathepsin K (Cat K) inhibitor. Modelling studies suggested that the introduction of a NH linker between the P3 aryl and P2 leucinamide moieties would allow the formation of a H-bond with the Gly66 residue of Cat K, hopefully increasing potency. Aniline 4 was thus synthesized and showed improved potency over its predecessor 2. Further modelling concluded that a 2-substituted five membered ring could more adequately place the P3 moiety of 4 into the S3 pocket of Cat K. The synthesis of the 2-substituted thiophene 5 confirmed this hypothesis by displaying a slight increase in potency against Cat K (>10-fold increase in potency vs 2) and a good selectivity profile against Cathepsins B, L, and S. This rationally designed inhibitor 5 also displayed increased potency in a functional bone resorption assay (10nM) versus 2 (95nM).

Full text of DOI:10.1016/j.bmcl.2004.05.087

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4291–4295
Rational design of potent and selective NH-linked aryl/heteroaryl
cathepsin K inhibitors
a,
a,
a
a
*
*
€
Joel Robichaud, Christopher Bayly, Renata Oballa, Peppi Prasit,
Christophe Mellon,a Jean-Pierre Falgueyret,b M. David Percival,b
Gregg Wesolowskic and Sevgi B. Rodanc
aDepartment of Medicinal Chemistry, Merck Frosst Centre for Therapeutic Research, 16711 TransCanada Highway,
Kirkland, QC, Canada H9H 3L1
bDepartment of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, 16711 TransCanada Highway,
Kirkland, QC, Canada H9H 3L1
cDepartment of Bone Biology and Osteoporosis, Merck Research Laboratories, West Point, PA, USA
Received 28 April 2004; revised 28 May 2004; accepted 28 May 2004
Available online 19 June 2004
Abstract—Prior reports from our laboratories have identified the nonpeptidic inhibitor 2 as a potent and selective Cathepsin K (Cat
K) inhibitor. Modelling studies suggested that the introduction of a NH linker between the P3 aryl and P2 leucinamide moieties
would allow the formation of a H-bond with the Gly66 residue of Cat K, hopefully increasing potency. Aniline 4 was thus syn-
thesized and showed improved potency over its predecessor 2. Further modelling concluded that a 2-substituted five membered ring
could more adequately place the P3 moiety of 4 into the S3 pocket of Cat K. The synthesis of the 2-substituted thiophene 5
confirmed this hypothesis by displaying a slight increase in potency against Cat K (>10-fold increase in potency vs 2) and a good
selectivity profile against Cathepsins B, L, and S. This rationally designed inhibitor 5 also displayed increased potency in a func-
tional bone resorption assay (10 nM) versus 2 (95 nM).
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Cathepsin K (Cat K) is a cysteine protease that is highly
expressed by osteoclasts and has been shown to be a key
enzyme involved in bone resorption.1 Secreted in the
extracellular acidic lacunae at the interface of the
osteoclast and bone tissue, the enzyme’s primarily role
consists of type I collagen degradation, one of the main
Extensive research in the field of bone biology in recent
years has yielded new discoveries that have further en-
hanced our appreciation of the intricate mechanisms
governing bone turnover. These processes are mediated
by highly specialized cells named osteoblasts and oste-
oclasts that are responsible for bone tissue formation
and resorption, respectively. Much of this research
interest was fueled by the hope that the understanding of
these processes would identify potential targets for
therapeutic intervention for degenerative diseases such
as osteoporosis.
P3
P2
P1
X
n
H
H
N
N
N
O
H
H
O
N
O
N
N
H
N
O
1
N
N
4, X = CH,
n = 2
R
5, X = S,
n = 1
2 R = piperazine
3 R = H
N
H
Figure 1. Amide bond replacement of dipeptide 1 affords biaryls 2 and
3, which are further modified to the NH-linked biaryl 4 and hetero-
biaryl 5 to reintroduce a favorable H-bond interaction with the Cat K
enzyme.
Keywords: Cathepsin K inhibitors; Bone resorption; Modelling; Biaryl
piperazine.
* Corresponding authors. Tel.: +1-514-4283665; fax: +1-514-4284900;
e-mail: joel_robichaud@merck.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.05.087

4292
J. Robichaud et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4291–4295
constituents of bone matrix. It has been suggested that
the inhibition of Cat K could slow bone resorption and
it appears that Cat K represents a promising therapeutic
target for the treatment of osteoporosis.2
2. Results
As previously reported,3 work in our laboratories to
achieve these goals focused on reducing the peptidic
character of 14 by replacing its P2-P3 amide bond with a
phenyl to afford biaryl 3 (Fig. 1). The resulting inhibitor
3 retained potency while displaying an improved selec-
tivity profile. Further SAR around the biaryl core of 3
led to the discovery of the potent and selective Cat K
inhibitor 2 (humanized rabbit IC50 ¼ 11 nM).
However, this phenyl replacement removes the H-bond
donated by the P2-P3 amide of 1 to the backbone oxy-
gen of Gly66 in the enzyme (Fig. 2), instead leaving this
enzyme oxygen unpaired.5 This presumably incurs a
desolvation penalty to binding. The goal of this publi-
cation is to outline our efforts to re-engineer our inhib-
itors with this favorable H-bonding interaction, thus
leading us to inhibitors such as 4 and 5.6
Figure 3. The average structure resulting from molecular dynamics of
a simple aniline linker (desbromo-6) in Cat K. The H-bond between
the inhibitor P2-P3 NH and the backbone oxygen of Gly66 (visible
under the active site surface) is shown as a dashed yellow line.
Hydrogens depict ortho- and meta-substitution positions of the aniline;
neither is optimal.
Modelling studies8 suggested that the insertion of a NH
H-bond donor between P2 and the biaryl core could
rectify this problem. Molecular dynamics results on
fully-solvated covalently-bound inhibitor/enzyme com-
plex showed that the aniline NH would introduce a
correctly-placed nonbasic H-bond donor while retaining
the nonpeptidic P2-P3 linker of this series (Fig. 3). A
good overall H-bonding configuration and a good
overall fit of the inhibitor was maintained. The P3
piperazine analog to 3 would still lie in P3, although
with a different orientation (data not shown).
An aminoaryl linker would substantially change the
orientation of the P3-fragment compared to the simple
phenyl linker so the meta-substitution of the biaryl core
would have to be re-examined. The aniline molecular
dynamics result suggested that neither ortho nor meta-
substitution appeared optimal: without allowing the
aniline phenyl to shift, ortho-substitution would steri-
cally clash with S3 while meta-substitution would be
directed slightly out of the S3 subsite into solvent.
Allowing for some degree of movement of the ani-
line phenyl, both ortho- and meta-substitutions on
the phenyl (depicted by the hydrogen substitutions on
the phenyl in Fig. 3) could be substituted to access P3.
Also, to evaluate whether the effect on potency of
inserting an NH linker was attributable to H-bonding or
simply a geometric consequence of the added atom,
CH2, O, and S linkers were also tested. In addition to
removing the H-bond, these replacements also examine
the effects of modifying the coplanirity of the phenyl
with the X–CH bond from P2: O will favor a coplanar
structure (as with the aniline), CH2 will favor perpen-
dicular, whereas S will favor neither.
The biological results obtained (Table 1) from the var-
iously linked aryl bromides were in accordance with the
modelling hypothesis. The NH linker showed markedly
increased potency compared to any other linkers, irre-
spective of the substitution on the phenyl ring. The most
potent compounds proved to be the ortho- and meta-
substituted NH-linked aryl bromides 6 and 7 which
displayed potencies of 54 and 72 nM against Cat K,
respectively.
Figure 2. The crystal structure of the active site of Cat K with bound
peptidomimetic inhibitor having a P2-P3 amide similar to 1.7 The H-
bond between the P2-P3 amide NH and the backbone oxygen of Gly66
(visible underneath the active site surface) is shown as a dashed yellow
line. This irreversible vinylsulfone inhibitor covalently binds to the
Cys25 (not shown), the P1-P3 portion adopting a peptide-like binding
orientation.
The CH2 and O linkers are the most similar to NH in
terms of size and bond lengths, differing mainly in their
H-bonding capability, CH2 being neutral and O being a

J. Robichaud et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4291–4295
Table 1. Inhibition of Cat K with NH, CH2, O, and S-linked inhibitors
4293
H
N
N
X
Br
O
Linker X
ortho-Isomer
Cat K IC50 (nM)a
meta-Isomer
Cat K IC50 (nM)a
para-Isomer
Cat K IC50 (nM)a
NHb
CH2
O
6
9
54
2352
1147
433
7
10
13
16
72
3394
1475
684
8
11
14
17
208
2349
2999
675
12
15
S
a IC50 values represent an average of at least n ¼ 2, variation between obtained values were within 2-fold. Humanized rabbit Cat K was used (see
Ref. 3a for conditions used).
b Aniline compounds (6, 7, and 8) are chiral and of S-configuration.
hydrogen bond acceptor. The >20-fold improvement of
the NH compared to these two linkers affirms the
importance of the H-bond to Gly66. The only other
series registering potencies below 1 lM was the S-linked
aryl bromides.
of 3 and 2 nM, respectively, against Cat K. In contrast,
the para-substituted phenyl piperazine 19 displayed a
potency of 27 nM. While the selectivity profile of these
two NH-linked biaryl piperazines 4 and 18 against
Cathepsins B, L, and S was lacking in comparison to the
biaryl piperazine 2, the potency of the ortho-isomer 4 in
the functional bone resorption assay9 was improved
(IC50 ¼ 9 nM, compared to 95 nM for 2). The meta-
isomer 18 on the other hand showed comparatively less
potency in this functional assay (IC50 ¼ 61 nM).
Aware that the extent of substitution in P3 may have a
dramatic influence on potency and selectivity, we deci-
ded to introduce our previously optimized phenyl
piperazine P3 moiety3 onto our three promising NH-
linked aryl bromides (6, 7, and 8). Closely tracking the
SAR of the aryl bromide series, the most potent inhib-
itors proved to be the ortho- and meta-substituted
biphenyl piperazines 4 and 18 (Table 2) with potencies
Re-examination of the modelling studies in light of these
experimental results suggested that the similar potencies
of ortho- and meta-substitutions represented extremes of
Table 2. Inhibition of Cat K, B, L, and S by other N-linked inhibitors
H
N
N
R
O
R
Compd
IC50 (nM)
Cat Lb
Bone res.c
Cat Ka
Cat Bb
3950
Cat Sb
2010
HN
N
2
11d
3725
95
ortho
meta
para
4
18
19
3
2
1812
93
2101
108
158
24
9
61
37
HN
Br
N
27
26
115
34
HN
S
20
43
158
458
897
71
HN
S
HN
N
5
1d
123
352
102
10
HN
a IC50 values represent an average of at least n ¼ 2, variation between obtained values were within 2-fold. Humanized rabbit Cat K was used (see
Ref. 3a for conditions used).
b Human enzyme was used for these assays (see Ref. 3a for conditions used).
c See Ref. 9 for assay conditions.
d Compound 2 is 3 nM against human Cat K, whereas compound 2 is 0.2 nM against human Cat K.

4294
J. Robichaud et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4291–4295
4. Chemistry
The synthesis of the NH-linked biaryls (Scheme 1) be-
gan with the copper-catalyzed cross-coupling reaction of
the required dibromobenzene (ortho, meta, and para)
with L
-leucine in DMF under basic conditions.10 This
sequence afforded the desired aniline-acids 21, 22, and
23 in good yields, which were coupled with amino-
acetonitrile hydrochloride to afford the corresponding
isomeric amides 6, 7, and 8. Suzuki cross-coupling of
these aryl bromides with boronic acid 243 afforded the
desired Boc-protected piperazine biaryls 25, 26, and 27,
which were carefully deprotected3 using methanesulf-
onic acid in dry THF to yield the three positional iso-
mers 4, 18, and 19.
The synthesis of the C-linked biaryls (Scheme 2) began
with the commercially available 3-(2-bromophenyl)
propionic acid, which was deprotonated with LDA and
alkylated with 1-iodo-2-methylpropane to yield the de-
sired carboxylic acid 28, which was then converted to
the desired amidoacetonitrile 9 according to the usual
PyBOP coupling reaction (the same route was used for
the meta- and para-isomers, all compounds were pre-
pared as racemic mixtures).
Figure 4. The average structure resulting from molecular dynamics of
5 in Cat K. The hydrogen bond between the inhibitor P2-P3 amine NH
and the backbone oxygen of Gly66 (visible underneath the active site
surface) is shown as a dashed yellow line.
allowed angles of P3 substitution, and that an inter-
mediate angle might be better. Since the angle between
1,2-substitution on a five-membered heteroaromatic ring
(around 72°) falls in between that of ortho (60°) and meta
(120°) substitution on a phenyl ring, replacing the aniline
phenyl with a five-membered heteroaryl moiety (i.e. thio-
phene) might yield improved potency by allowing the P3
phenyl piperazine to sit better in S3 (see Fig. 4).
The synthetic route utilized for the preparation of the O
and S-linked biaryls was analogous for both families of
compounds. The commercial 2-bromophenol and 2-
bromothiophenol (Scheme 2) were deprotonated with
NaH and alkylated with methyl 2-bromo-4-methyl-
B(OH)2
To test this hypothesis both the thiophene bromide 20
and the corresponding phenyl piperazine 5 were pre-
pared (Table 2). While the simple bromo-substituted
inhibitor 20 (43 nM against Cat K) showed no signifi-
cant improvement over the ortho-substituted bromo-
aniline 6 (54 nM), the fully elaborated phenyl piperazine
derivative 5 displayed a slightly improved potency of
1 nM against humanized rabbit Cat K (0.2 nM against
human Cat K) and a good selectivity profile against
Cathepsins B, L, and S (123-fold, 352-fold, and 102-
fold, respectively). Importantly, this potent inhibitor 5
also displayed very good potency (IC50 ¼ 10 nM) in the
functional bone resorption assay.
H
N
a
c
+
X
N
H
N
H
Br
Br
Br
O
N
O
24
N
b
d
R = H
X = OH X = NHCH2CN
ortho: 21
meta: 22
para: 23
R = Boc
ortho: 25
meta: 26
para: 27
N
N
R
ortho: 6
meta: 7
para: 8
ortho: 4
meta: 18
para: 19
RN
Scheme 1. Synthetic route to NH-linked derivatives. Reagents and
conditions: (a) )-leucine, K2CO3, CuI (cat), DMF, D; (b)
HClÆH2NCH2CN, PyBOP, Et3N, DMF; (c) aq Na2CO3, PdCl2(dppf),
DMF, 85–90 °C; (d) MeSO3 H, THF.
(
L
R
3. Conclusion
b
OH
H
N
N
X
Br
a
O
In summary, reintroducing a H-bond donating NH
linker in our nonpeptidic inhibitors led to anilines 4 and
18, which showed improved potencies over a previously
disclosed biaryl piperazine inhibitor 2. Further refine-
ment of the geometrical biaryl scaffold of 4 and 18 led to
the rational design of thiophene 5 which displayed good
selectivity against Cathepsins B, L, and S and a 10-fold
improvement in potency against Cat K in comparison
to 2. Inhibitor 5 also showed very good potency
(IC50 ¼ 10 nM) in the functional bone resorption assay.
Further SAR in this series is ongoing and will be dis-
closed in due course.
R = H
Br
O
X = C (9), O (12), S (15)
R = CH2CH(CH3)2 (28)
d, b
c
OMe X = O (29)
XH
X
X = S (30)
Br
Br
O
Scheme 2. General synthetic route to the CH2, O, and S-linked ana-
logs. Reagents and conditions: (a) LDA, THF, 0 °C, then 1-iodo-2-
methylpropane, rt; (b) HClÆH2NCH2CN, PyBOP, Et3N, DMF; (c)
NaH (60%), THF, then methyl 2-bromo-4-methylpentanoate; (d)
LiOH, MeOH.

J. Robichaud et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4291–4295
4295
pentanoate11 to afford the desired ether 29 and thioether
30. The resulting esters were converted to the O and S-
linked nitrile inhibitors 12 and 15 as described in Scheme
2 (the same route was used for the meta- and para-iso-
mers, all compounds were synthesized as racemic mix-
tures).
R.; O’Shea, P.; Prasit, P.; Robichaud, J.; Tillyer, R.;
Wang, X. J. Org. Chem. 2003, 68, 2633.
4. Suzue, S.; Irikura, T. Chem. Pharm. Bull. 1968, 16, 1417.
5. DesJarlais, R. L.; Yamashita, D. S.; Oh, H.-J.; Uzinskas,
I. N.; Erhard, K. F.; Allen, A. C.; Haltiwanger, R. C.;
Zhao, B.; Smith, W. W.; Abdel-Meguid, S. A.; D’Alessio,
K.; Janson, C. A.; McQueney, M. S.; Tomaszek, T. A.;
Levy, M. A.; Veber, D. F. J. Am. Chem. Soc. 1998, 120,
9114.
6. For a similar approach, see: Greenspan, P. D.; Clark, K.
L.; Cowen, S. D.; McQuire, L. W.; Tommasi, R. A.;
Farley, D. L.; Quadros, E.; Coppa, D. E.; Du, Z.; Fang,
Z.; Zhou, H.; Doughty, J.; Toscano, K. T.; Wigg, A. M.;
Zhou, S. Bioorg. Med. Chem. Lett. 2003, 13, 4121.
7. McGrath, M. E.; Klaus, J. L.; Barnes, M. G.; Bromme, D.
Nat. Struct. Biol. 1997, 4, 105, PDB entry 1MEM.
8. Case, D. A.; Pearlman, D. A.; Caldwell, J. W.; Cheatham,
T. E., III; Ross, W. S.; Simmerling, C. L.; Darden, T. A.;
Merz, K. M.; Stanton, R. V.; Cheng, A. L.; Vincent, J. J.;
Crowley, M.; Tsui, V.; Radmer, R. J.; Duan, Y.; Pitera, J.;
Massova, I.; Seibel, G. L.; Singh, U. C.; Weiner, P. K.;
Kollman, P. A. (1999), AMBER 6, University of California,
San Francisco.
Access to the NH-linked thiophenes 20 and 5 was
accomplished starting from commercially available 3,4-
dibromothiophene and using the same synthetic route
described in Scheme 1.
References and notes
1. Motyckova, G.; Fisher, D. E. Curr. Mol. Med. 2002, 2,
407.
2. For recent reviews on this subject see: (a) Stroup, G. B.;
Lark, M. W.; Veber, D. F.; Bhattacharyya, A.; Blake, S.;
Dare, L. C.; Erhard, K. F.; Hoffman, S. J.; James, I. E.;
Marquis, R. W.; Ru, Y.; Vasko-Moser, J. A.; Smith, B. R.;
Tomaszek, T.; Gowen, M. J. Bone Miner. Res. 2001, 16,
1739; (b) Yamashita, D. S.; Dodds, R. A. Curr. Pharm.
Des. 2001, 6, 1.
3. (a) Robichaud, J.; Oballa, R.; Prasit, P.; Falgueyret, J.-P.;
Percival, M. D.; Wesolowski, G.; Rodan, S. B.; Kimmel,
D.; Johnson, C.; Bryant, C.; Venkatraman, S.; Setti, E.;
Mendoca, R.; Palmer, J. T. J. Med. Chem. 2003, 46, 3709;
(b) Chen, C.-Y.; Dagneau, P.; Grabowski, E. J. J.; Oballa,
9. Falgueret, J. P.; Oballa, R. M.; Okamoto, O.; Wesolowski,
G.; Aubin, Y.; Rydzewski, R. M.; Prasit, P.; Riendeau,
D.; Rodan, S. B.; Percival, M. D. J. Med. Chem. 2001, 44,
94.
10. Ma, D.; Zhang, Y.; Yao, J.; Wu, S.; Tao, F. J. Am. Chem.
Soc. 1998, 120, 12459.
11. Saito, K.; Harada, K. Bull. Chem. Soc. Jpn. 1989, 62,
2562.