4-Aminophenoxyacetic acids as a novel class of reversible cathepsin K inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.12.053

We have designed and synthesized a novel series of 3-biphenylamino acid amides as cathepsin K inhibitors based on compound I. In these inhibitors, we have discovered 4-aminophenoxyacetic acids 43 and 47 with good IC₅₀ values, although lipophili

Full text of DOI:10.1016/j.bmcl.2005.12.053

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1502–1505
4-Aminophenoxyacetic acids as a novel class of reversible
cathepsin K inhibitors
Tsuyoshi Shinozuka,a,* Kousei Shimada,a Satoshi Matsui,a Takahiro Yamane,a
Mayumi Ama,a Takeshi Fukuda,a Motohiko Takib and Satoru Naitoa
aMedicinal Chemistry Research Laboratories, Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
bLead Discovery Research Laboratories, Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
Received 30 September 2005; revised 7 December 2005; accepted 12 December 2005
Available online 27 December 2005
Abstract—We have designed and synthesized a novel series of 3-biphenylamino acid amides as cathepsin K inhibitors based on com-
pound I. In these inhibitors, we have discovered 4-aminophenoxyacetic acids 43 and 47 with good IC50 values, although lipophilic
groups are favorable for the hydrophobic S10 pocket.
Ó 2005 Elsevier Ltd. All rights reserved.
Cathepsin K1 is a cysteine protease that is highly ex-
pressed in osteoclasts. The primary role of cathepsin K
is the degradation of type I collagen, one of the main con-
stituents of bone matrix. The inhibition of cathepsin K
has been expected to provide a therapeutic means for
the treatment of osteoporosis, osteoarthritis, rheumatoid
arthritis, and Paget’s disease. Derivatives of cyanamide,2
nitrile,3 ketone,4 aldehyde,5 and ketoamide6 are known as
cathepsin K inhibitors, which covalently interact with the
thiol group of the cysteine residue at the active site of the
enzyme. Besides those inhibitors, arylamine derivatives
were reported by a Novartis group7 and were shown to
be non-covalent and reversible inhibitors. Herein, we re-
port on arylamine-based inhibitors of cathepsin K whose
P3 unit is replaced with a biphenyl group. We also report
subsequent replacement of the methyl group in the P10
unit with an acetate group led to a novel class of reversible
cathepsin K inhibitors.
bonyl (Cbz) group8 revealed that the carbonyl oxygen
of the Cbz group did not interact with the enzyme.
Therefore, we converted the Cbz group of compound I
to a biphenyl group by tethering the carbonyl oxygen
to the benzylic carbon as shown in Figure 1 for obtain-
ing structurally novel inhibitors as such P3 sub-struc-
tures had not been reported when we started this
project.3
Compounds 11–35 were prepared from the correspond-
ing carboxylic acids and amines. As described in Scheme
1, aryloxycarboxylic acids 3 were prepared by etheration
of phenols 1 with a-bromo esters 2. The synthesis of N-
aryl amino acids 6 was accomplished by coupling aryl
P3
P2
P1
P1'
OMe
O
H
Our modification was started from Novartis compound
I7 which was chosen because non-covalent inhibitors
were generally thought to be more advantageous in
terms of toxicity compared to covalent inhibitors.
Scrutiny of the X-ray crystallographic data of cathepsin
K complexed with inhibitors bearing a benzyloxycar-
N
O
N
N
H
H
O
I
OMe
H
N
X
N
H
O
X = O or NH
Keywords: Cathepsin K inhibitors; Osteoporosis; 3-Biphenylamino
acid amides; 4-Aminophenoxyacetic acids.
*
Figure 1. Schematic representation of the assumed binding mode of
Novartis compound I and O- and NH-linked compound.
Corresponding author. Tel.: +81 3 3492 3131; fax: +81 3 5436
8563; e-mail: sinozu@sankyo.co.jp
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.12.053

T. Shinozuka et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1502–1505
1503
R2 R3
CO2Me
OBn
R2 R3
a
R1
R1
+
+
BocHN
BocHN
Br
OH
O
CO2H
OH
N
Y
a
1
4
2
3
36
37
38: Y = Ns
39: Y = H
b
OBn
R2 R3
R2 R3
CO2H
b
R1
R1
H2N
CO2H
N
NsHN
Br
H
OR5
5
6
H
N
c
N
H
N
Scheme 1. Synthesis of carboxylic acids. Reagents and conditions: (a)
KOH, tert-BuOH, reflux; (b) CuI, K2CO3, DMA, 90 °C.
H
O
40: R5 = Bn
41: R5 = H
42: R5 = CO2t-Bu
43: R5 = CO2H
d
e
f
bromides 4 with L-leucine or 1-aminocyclohexanecarb-
oxylic acid (5) using a copper catalyst.9
g
The preparation of compounds 11–35 is shown in
Scheme 2. Mitsunobu reaction10 of N-Boc aminoetha-
nols 711 with 2-nitrobenzenesulfonamide 8 afforded N-
Boc ethylenediamines 9, which were deprotected with
PhSH to provide ethylenediamine derivatives 10.12 Sub-
sequent coupling reaction of 10 with carboxylic acid 3 or
6 furnished compounds 11–35.
41
44: R5 = CH2CN
h
N
N
45: R5
=
N
N
H
Scheme 3. Synthesis of compounds 42, 43, and 45. Reagents and
conditions: (a) DEAD, PPh3, THF; (b) PhSH, K2CO3, MeCN, 82% (2
steps); (c) TFA, CH2Cl2, then 6, WSCÆHCl, HOBt, DMF, 99%; (d) H2,
Pd/C, EtOH, 87%; (e) tert-butyl bromoacetate, NaH, DMF/THF,
81%; (f) HCl, 1,4-dioxane, 74%; (g) NaH, bromoacetonitrile, DMF/
THF, 59%; (h) NaN3, NH4Cl, DMF, 100 °C, 69%.
The preparation of compounds 42, 43, and 45 is shown
in Scheme 3. Mitsunobu reaction of N-Boc aminoetha-
nol 3611 with 2-nitrobenzenesulfonamide 37 and subse-
quent deprotection provided 39.12 The coupling of 39
with carboxylic acid 6, followed by cleavage of benzyl
ether (H2, Pd/C, EtOH), provided phenol 41. Alkylation
of 41 with tert-butyl bromoacetate afforded ester 42.
Saponification of ester 42 furnished carboxylic acid 43.
Tetrazole 45 was prepared from nitrile 44 under the usu-
al reaction conditions. Ester 46 and carboxylic acid 47
were prepared in the same manner.
Table 1. IC50 values of human cathepsin K inhibitors 11–28
OMe
R2 R3
R1
H
N
X
N
H
O
R1
R2
R3
IC50 (lM)
a
Compound
X
I
0.09
3
11b
12b
13b
14b
15b
16b
17
O
H
i-Bu
i-Bu
H
H
H
H
H
H
O
2-Ph
3-Ph
4-Ph
1.2
Purified procathepsin K is efficiently activated at lower
pH, such as pH 4.0.13 In addition, the activity of mature
cathepsin K at pH 3.8 was around 40% when compared
at pH 6.0 using Cbz-Phe-Arg-AMC as a substrate (data
not shown). Therefore, we examined the inhibitory
activity of O-linked compounds against recombinant
human cathepsin K at pH 3.8 as shown in Table 1. Iso-
butyl and cyclohexyl groups were employed as the P2
unit, and isobutyl derivatives (compounds 11–16) were
prepared as racemic mixtures. Non-substituted phenoxy
derivative 11 exhibited an IC50 value in the micromolar
O
i-Bu
i-Bu
0.090
0.10
0.069
0.60
3
O
O
3-PhO
4-PhO
H
i-Bu
i-Bu
O
O
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
i-Bu
18
19
O
3-Ph
4-Ph
4-Et
0.32
0.19
0.13
0.20
0.099
11
O
20
21
O
O
4-i-Pr
4-c-hex
H
22
23
O
NH
NH
NH
NH
NH
NH
24
25
2-Ph
3-Ph
4-Ph
3-Ph
3-c-hex
>20
0.083
0.10
0.040
0.060
26
27
OMe
H
H
BocHN
BocHN
OH
N
R
28
i-Bu
a
R4
R4
a Inhibition of recombinant human cathepsin K activity in a fluores-
cence assay using 8 lM Cbz-Phe-Arg-AMC as a substrate in 200 mM
NaOAc, 10 mM DTT, 120 mM NaCl, and buffer (pH 3.8). The IC50
values represent the average of at least n = 2.
7
9: R=Ns
10: R=H
b
OMe
NsHN
b Racemic compound.
OMe
R2 R3
R1
H
8
N
c
X
N
H
O
R4
range, whereas compound I indicated good inhibition
(IC50 = 0.09 lM).14 In isobutyl derivatives, substitution
of a phenyl group to a phenoxy group in compound
11 at the 3- or 4-position increased the inhibitory
activity by 30 times (compounds 11, 13, and 14), while
11-35 (X=O or NH)
Scheme 2. Synthesis of compounds 11–35. Reagents: (a) DEAD, PPh3,
THF; (b) PhSH, K2CO3, MeCN; (c) TFA, CH2Cl2, then 3 or 6,
WSCÆHCl, HOBt, DMF.

1504
T. Shinozuka et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1502–1505
substitution at the 2-position (compound 12) brought a
little improvement in activity. The cyclohexyl derivatives
18 and 19 were less potent than the corresponding isobu-
tyl derivatives 13 and 14. 3-Phenoxy derivative 15
showed good inhibitory activity (IC50 = 0.069 lM),
whereas 4-phenoxy derivative 16 showed 10 times re-
duced activity compared to 15. Alkyl derivatives 20–22
retained good activity, and cyclohexyl derivative 22
was 2-fold more potent than 4-biphenyl ether 19.
Next the substituent on the benzene ring in the P10 unit
of ethyl derivatives 31 and 35 was examined. The result
is summarized in Table 3. Incorporation of a tetrazole
group in the P10 unit led to moderate inhibitor 45, which
was only 3-fold less potent than 35. This result encour-
aged us to synthesize carbonyl derivatives such as esters
and carboxylic acids, although it has been shown that
lipophilic groups are favorable for the hydrophobic
S10 pocket.7b tert-Butyl ester 42 showed a 2-fold increase
in the inhibitory activity compared to tetrazole 45. Inter-
estingly, the IC50 value of carboxylic acid 43 was
4.5 nM, which was more potent than ester 42 and similar
to that of methoxy derivative 35. 3-Biphenylaminocyclo-
hexyl analogues of the above derivatives were also pre-
pared and evaluated for inhibitory activity. Compound
47, a cyclohexane analogue of 43, possessed good inhib-
itory activity with an IC50 of 4.8 nM. The reason for the
good inhibitory activities of these carboxylic acid deriv-
atives was obscure.
We then examined the inhibitory activity of NH-linked
compounds as also summarized in Table 1. Biphenyl-
amine derivatives 25 and 26 possessed increased activity
compared to the corresponding biphenyl ethers 18 and
19. The improvement in activity could be ascribed to a
hydrogen bond between the amino group and the en-
zyme as mentioned in the literature.3 In the arylamine
series, 3-biphenylamine derivative 25 displayed better
activity than 4-biphenylamine derivative 26, and the 3-
biphenyl-L-leucyl group was found to be a better P2 sub-
stituent than the 3-biphenylaminocyclohexylcarbonyl
group (Compounds 25 and 27). The terminal phenyl
group of 27 was replaced with a cyclohexyl group; how-
ever, no enhancement in activity was attained. This is
unlike the same displacement in aryl ether 19, which
resulted in more potent 22.
In summary, a 3-biphenylamino group was found to be
an efficient P3 sub-structure for arylamine-based inhibi-
tors of cathepsin K. Addition of an ethyl group in the
(S)-configuration to the ethylenediamine moiety and a
carboxyl group in the P10 unit resulted in compounds
43 and 47 with a good IC50 value against cathepsin K.
We expect the addition of the acidic group brings differ-
ent features in enzyme selectivities and/or ADME prop-
erties from other cathepsin K inhibitors. These results
will be reported in due course.
It was expected that the introduction of an alkyl group
extending into the S1 pocket would enhance the inhibi-
tory activity, so derivatives with an alkyl side chain in
the ethylenediamine moiety of compounds 25 and 27
were synthesized and the inhibitory activities were deter-
mined, as depicted in Table 2. First, both stereoisomers
of methyl derivatives, 29 and 30, were prepared, and (S)-
isomer 29 was found to be more potent. Therefore,
derivatives of various alkyl side chains with the (S)-con-
figuration were examined, and the maximum activity
was observed for the ethyl group (compound 31). Simi-
larly, good activity was displayed by the isobutyl deriv-
ative substituted with an ethyl group (compound 35).
Table 3. IC50 values of human cathepsin K inhibitors 21 and 25–30
OR3
R1 R2
H
N
N
H
N
H
O
a
IC50 (nM)
Compound
R1
R2
R3
31
–(CH2)5–
Me
2.7
3.8
11
35
i-Bu
H
H
H
Me
Table 2. IC50 values of human cathepsin K inhibitors 25, 27, and
29–35
N
N
O
O
O
O
N
45
i-Bu
N
H
OMe
R1 R2
H
N
N
H
N
H
R3 R4
42
i-Bu
6.4
4.8
4.5
4.8
O
O
O
a
IC50 (nM)
Compound
R1
R2
R3
R4
46
–(CH2)5–
i-Bu
25
29
30
31
32
33
27
34
35
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
i-Bu
H
H
Me
H
H
H
H
H
H
H
83
10
Me
H
82
43b
47b
H
Et
2.7
3.9
5.6
40
OH
n-Pr
n-Bu
H
H
H
H
–(CH2)5–
i-Bu
i-Bu
Me
Et
10
OH
3.8
a Inhibition of recombinant human cathepsin K activity in a fluores-
cence assay using 8 lM Cbz-Phe-Arg-AMC as a substrate in 200 mM
NaOAc, 10 mM DTT, 120 mM NaCl, and buffer (pH 3.8). The IC50
values represent the average of at least n = 2.
a Inhibition of recombinant human cathepsin K activity in a fluores-
cence assay using 8 lM Cbz-Phe-Arg-AMC as substrate in 200 mM
NaOAc, 10 mM DTT, 120 mM NaCl, and buffer (pH 3.8). The IC50
values represent the average of at least n = 2.
b 2 HCl salt.

T. Shinozuka et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1502–1505
1505
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