New chemotypes for cathepsin K inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.03.036

Cyano pyrimidine acetylene and cyano pyrimidine t-amine, which belong to a new chemical class, were prepared and tested for inhibitory activities against cathepsin K and the highly homologous cathepsins L and S. The use of novel chemotypes in the developm

Full text of DOI:10.1016/j.bmcl.2008.03.036

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2599–2603
New chemotypes for cathepsin K inhibitors
Naoki Teno,* Osamu Irie, Takahiro Miyake, Keigo Gohda, Miyuki Horiuchi,à
Sachiyo Tada,§ Kazuhiko Nonomura, Motohiko Kometani,
Genji Iwasaki and Claudia Betschart–
Novartis Institutes for BioMedical Research, Ohkubo 8, Tsukuba, Ibaraki 300-2611, Japan
Received 5 February 2008; revised 10 March 2008; accepted 13 March 2008
Available online 16 March 2008
Abstract—Cyano pyrimidine acetylene and cyano pyrimidine t-amine, which belong to a new chemical class, were prepared and
tested for inhibitory activities against cathepsin K and the highly homologous cathepsins L and S. The use of novel chemotypes
in the development of cathepsin K inhibitors has been demonstrated by derivatives of compounds 1 and 8.
Ó 2008 Elsevier Ltd. All rights reserved.
Osteoporosis is a debilitating disease that is caused by
an imbalance between bone matrix resorption and bone
remodeling. Cathepsin K, which is selectively and highly
expressed in osteoclasts, is a lysosomal cysteine protease
of the papain superfamily that has high homology to
cathepsins S and L.1,2 Studies using cathepsin K anti-
sense3 and cathepsin K deficient mice4 have shown that
the proteinase is primarily involved in osteoclastic bone
resorption. Cathepsin K inhibitors therefore are re-
garded as a potential therapy for the treatment of bone
loss, such as osteoporosis.5 One significant consider-
ation in the design of cathepsin K inhibitors is selectivity
for its highly homologous lysosomal cysteine proteases,
cathepsins L and S. This has been shown to be possible
in many inhibitors having new scaffolds that have been
described in previous publications.6–9
itors with P2 (R2 group in Fig. 1) and P3 (R1 group in
Fig. 1) moieties that make a significant contribution to
S3
S3
N
N
HN
N
HN
N
N
N
S2
S2
Purine
nitrile
R1
N
Cyano pyrimidine Cyano pyrimidine
amide
t-amine
O
As depicted in Figure 1, we have reported novel scaf-
folds, A,6 B,8 and C9 for non-peptidic cathepsin K inhib-
R1
N
N
N
R1
N
N
H
N
N
N
HN
N
N
N
R2
R2
HN
R2
Keywords: Cathepsin K; Non-peptidic inhibitors; New chemotype;
Cyano pryrimidine acetylene; Cyano pyrimidine t-amine.
N
A
B
E
*
Corresponding author. Tel.: +81 29 865 2360; fax: +81 29 865
2308; e-mail: naoki.teno@novartis.com
Cyano pyrimidine
Pyrrolopyrimidine
Present address: Computer-Aided Molecular Modeling Research
Center Kansai, 1-3-12, Honjyocho, Higashinada-ku, Kobe, Hyogo
658-0012, Japan.
acetylene
R1
N
R1
N
à
§
Present address: YASUTOMI & Associates, MT-2 BLDG, 5-36,
Miyahara 3-chome, Yodogawa-ku, Osaka 532-0003, Japan.
Present address: Central Research Laboratories, Sysmex Co., 4-4-4,
Takatsuka-dai, Nishi-ku, Kobe, Hyogo 651-2271, Japan.
Present address: Novartis Institutes for BioMedical Research, CH-
4002 Basel, Switzerland.
N
R2
N
HN
R2
N
N
N
C
D
–
Figure 1. Structure-based design of diverse pyrimidine-based scaffold.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.03.036

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N. Teno et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2599–2603
the interaction with S2 and S3 subsites. The derivatives
based on the scaffolds of purine nitrile A6 are potent
inhibitors for cathepsin K but they exhibit only moder-
ate specificity toward the highly homologous cathepsins
L and S. Starting from the purine structure, cyano
pyrimidine amide B8 has been explored as a series of
cathepsin K inhibitors. The novel scaffold C9 derived
from the pyrimidine derivatives contributed to the dis-
covery of new and specific cathepsin K inhibitors. In this
communication, we report two additional new chemo-
types, cyano pryrimidine acetylene D and cyano pyrim-
idine t-amine E (Fig. 1).
respectively). The acetylene inhibitor 1 displayed an
inhibitory activity for cathepsin K at single digit nM
range with 100-fold selectivity against cathepsins L
and S (Table 1). As indicated in Table 2, the parent com-
pound 8 for scaffold E represented moderate inhibition
for cathepsin K with selectivity against the highly
homologous cathepsins L and S. Therefore, both the
structures 1 and 8 were selected as a starting point for
further optimization. We expected to achieve a further
increase in the potency by extending the P3 moiety into
the S3 subsite in cathepsin K, which is a crucial binding
pocket comprising Asp61 and Tyr67. Crystal structures
of cathepsins L11 and S12 indicate that the correspond-
ing residues to the two amino acids in cathepsin K are
Glu63 and Leu69 in cathepsin L, and Lys64 and
Phe70 in cathepsin S. Since the comparison of the S3
subsites among human cathepsins K, L, and S revealed
significant variations, higher selectivity over related pro-
teases, cathepsins L and S, was anticipated.
In general, when designing selective inhibitors for cys-
teine proteases, it is crucial to create ideas to occupy
the S2 and S3 subsites by appropriate parts extending
from the core structure of the inhibitor. Of the various
types of the P2 substituents investigated, a neopentyl
as the P2 moiety of scaffolds B and C proved to be the
most favorable for the S2 subsite.8,9 Encouraged by this
observation, the neopentyl moiety was chosen as the
first candidate for the P2 moiety for the new scaffolds.
The modeling suggested that the P3 moiety can reach
to the S3 subsite if an appropriate rigid spacer could lin-
early extend from the 5-position of the pyrimidine to-
ward the S3 subsite. The compound 1 with acetylenes
as rigid linkers was designed as the first derivative for
scaffold D (Fig. 2). To further pursue the structural
diversity of the scaffold for cathepsin K inhibitor, we fo-
cused on the amino group at the 4-position of the pyrim-
idine. For scaffold E, the compound 8 was designed to
elucidate whether the appropriate residue at the 4-posi-
tion of the pyrimidine pointed toward the S3 subsite.
Unlike the peptidic inhibitors with rigorous hydrogen
bonds between the peptide sequence and amino acids
of the enzyme,10 non-peptidic inhibitors 1 and 8 may
have a flexible P2/S2 and P3/S3 interaction (molecular
parts colored by beige and pink in Figures 3 and 4,
Figure 3. Compound 1 (beige) docked into the active site of human
cathepsin K.
N
R1
N
N
N
N
R1
HN
N
N
8 = R1: H
10 = R1:
9 = R1: I
1 = R1:
3 = R1:
2 = R1:
4 = R1:
N
N
N
N
N
N
N
O
N
O
Cl
N
N
O
S N
O
Cl
5 = R1:
= R1:
6
N
N
N
N
N
R1
Cl
7 = R1:
O
S N
O
N
O
12 = R1:
14 = R1:
11 = R1:
13 = R1:
15 = R1:
N
N
N
N
N
N
N
N
N
N
Figure 2. Structures of cathepsin K inhibitors with scaffold D or E.

N. Teno et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2599–2603
2601
to the poorly selective inhibitor 5 against cathepsins L
and S. The sulfonamides 6 and 7 caused no substantial
change in a potency for cathepsin K and the selectivity
against cathepsins L and S. Although there is a possibility
on the optimization of the P3 moiety to improve the selec-
tivity against untargeted cathepsins, it turned out that 1,
6, and 7 are selective cathepsin K inhibitors.
The derivatives derived from 8 as representatives of scaf-
fold E are shown in Table 2. As indicated above and in
Figure 4, the structure of 8 implies that the para-substitu-
ents in the benzyl group of 8 contributed to the increase in
inhibitory activity against cathepsin K while having selec-
tivity against other cathepsins. Indeed, even synthetic
intermediate 9 showed an about 3.5-fold-increase in the
inhibitory activity for the target enzyme. In addition,
10–15 had substantial changes (8- to 18-fold-increase) in
the target enzyme inhibition compared to 8. As depicted
in Figure 4, compounds 10 (yellow) and 15 (orange) occu-
pied the critical subsites in the active site of cathepsin K.
Note here that all of the inhibitors listed in Table 2 had
no affinity to the highly homologous cathepsins L and
S. From the examples prepared in this series, it is evident
that the P3 moiety extended from scaffold E points toward
the S3 subsite of cathepsin K.
Figure 4. Compound 8 (pink), 10 (yellow), and 15 (orange) docked
into the active site of human cathepsin K.
Table 1. Inhibition of human cathepsins K, L, and S by compounds 1–
7
a
IC50 (nM)
Compound
Cat K
Cat L
Cat S
1
2
3
4
5
6
7
7.1
10
980
>1000
150
760
920
840
460
68
The synthesis of inhibitors discussed here is illustrated in
1
and 2. The cyclization13,14 occurred
3.2
8.6
7.2
7.1
3.7
Schemes
1000
190
predominantly to yield 16 (scaffold C) under the condi-
tions, Pd(PPh3)2Cl2, CuI, Et3N, DMF, 80 °C.9 More-
over, we found that a change of the solvent to THF or
dioxane and/or reaction temperature (rt to 60 °C) in this
reaction led to non-cyclized product 1 (scaffold D). Fur-
ther investigation on the reaction condition using differ-
ent acetylenes as a starting material was carried out in
order to obtain non-cyclized derivatives as major prod-
ucts. The synthesis of 2–5 is outlined in Scheme 1, a cou-
pling of 17, and 19a and 19b (prepared from 18a and 18b
and bromo-ethyne) carried out by using Pd(PhCN)2Cl2
and P(t-Bu)3 as catalyst and ligand15 gave 2 and 3,
respectively. The preparation of 4 and 5 was performed
by using Pd(PPh3)2Cl2, CuI, Et3N, DMF, 60 °C. Sulfo-
nyl chloride derivatives 20 and 21 were coupled with
2216 to afford sulfonamide 6 and 7, respectively.
>1000
>1000
>1000
800
a Inhibition of recombinant human cathepsins K, L, and S in a fluo-
rescence assay.6
Table 2. Inhibition of human cathepsins K, L, and S by compounds 8–
15
a
IC50 (nM)
Compound
Cat K
Cat L
Cat S
8
9
69
19
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
10
11
12
13
14
15
4.8
3.8
6.4
7.3
3.6
8.6
The 2-chloropyrimidin derivative 23 was prepared from
2,4-dichloropyrimidine and 2,2-dimethyl-propylamine,
which was coupled with commercially available benzyl
bromide derivatives to yield 24a–c (Scheme 2). Amina-
tion of 24c with 1H-[1,2,4]triazole in DMF gave 24d.
The intermediates, 24a, 24b, and 24d were reacted with
NaCN in DMSO-H2O to yield 8, 9, and 10, respectively.
The intermediate 25 was prepared by Sonogashira cou-
pling of 9 and prop-2-yn-1-ol in the presence of
Pd(PPh3)2Cl2 and CuI as catalysts. The hydroxyl group
of 25 was converted to chloride by methanesulfonyl
chloride to give 26. Various amines or heteroaromatic
derivatives were coupled with 26 to afford 11–15.
a Inhibition of recombinant human cathepsins K, L, and S in a fluo-
rescence assay.6
The initial design of the P3 moiety (R1 in Fig. 1) in scaf-
fold D or E aims to have electric and hydrophobic affin-
ity with Asp61 and Tyr67, respectively, in the S3 subsite
of cathepsin K. The designed derivatives having scaffold
D or E are illustrated in Figure 2.
The IC50 values of derivatives of 1 with scaffold D are
listed in Table 1. Acetylene inhibitors 2–5 having a hetero-
cyclic or a heteroaromatic group as the P3 moiety inhib-
ited cathepsin K equipotently to 1 but their selectivity
against cathepsin L and S showed a declining trend. In
particular, introduction of the imidazole derivative led
In summary, the new inhibitors reported here are an
important first step in tackling chemical modification.
The chemical modification of 1 and 8 with the scaffolds
D and E was focused on the P3 moiety which contrib-

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N. Teno et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2599–2603
N
N
N
N
N
Br
a
b
N
N
16 (27%)
HN
N
+
N
N
N
1 (25%)
d
17
2 (65%), 3 (46%)
4 (72%), 5 (65%)
c
R
R-H
Br
+
e
18a, 18b,
18c, 18d
19a (88%), 19b (91%),
19c (quant.), 19d (quant.)
Cl
N
Cl
N
O
18d, 19d: R =
N
18c, 19c: R =
18a, 19a: R =
18b, 19b: R =
N
N
N
O
S
O
HN
R-Cl
+
N
20: R=
Cl
O
f
6 (31%), 7 (51%)
HN
22
N
O
S
O
N
21: R=
20, 21
Scheme 1. Reagents and conditions: (a) Pd(PPh3)2Cl2, CuI, Et3N, DMF, 80 °C, 2 h; (b) Pd(PPh3)2Cl2, CuI, Et3N, THF, 60 °C; (c) DMF, 60 °C, 5–
7 h; (d) 17, Pd(PhCN)2Cl2, CuI, P(t-Bu)3, HN(i-Pr)2, Dioxane, rt, 12 h; (e) 17, Pd(PPh3)2Cl 2, CuI, Et3N, DMF, 60 °C, 0.5 h; (f) Et3N, CH2Cl2, 0 °C,
stirred at rt for 12 h.
N
R
a
b
NH2
N
N
N
N
Cl
+
+
Br
HN
N
Cl
Cl
N
Cl
R
23
R=H, (quant.): 24a
R=I, (quant): 24b
R=-CH Br, (94 %): 24c
2
c
R=
(85 %): 24d,
N
d
N
N
8 (75 %), 9 (quant), 10 (32%)
N
N
e
f
N
N
N
N
+
HO
g
9
N
N
HO
Cl
26
25
11 (52%), 12 (75%), 13 (88%), 14 (90%), 15 (64%)
Scheme 2. Reagents and conditions: (a) K2CO3, THF, 80 °C, 8 h, 58%; (b) NaH, DMF, rt, 18 h; (c) DMF, rt, 18 h; (d) NaCN, DABCO, DMSO-
H2O, 75 °C, 24 h; (e) Pd(PPh3)2Cl2, CuI, EtN(i-Pr)2, DMF, rt, 1 h, rt, 82%; (f) methansulfonyl chloride, EtN(i-Pr)2, CH2Cl2, rt, 15 h; (g) various
amines or heteroaromatic rings (2.0 equiv), DMF, rt, 15 h.
uted to the selectivity against other cathepsins. The
capability of the novel chemotypes in the development
of cathepsin K inhibitors has been demonstrated by
the derivatives represented by 1 and 8, and appropriate
moieties extending from each scaffold engaged with key
pockets in the active site of cathepsin K.
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