Synthesis and evaluation of 4-substituted benzylamine derivatives as β-tryptase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.02.064

Since β-tryptase is considered a critical mediator of asthma, potent tryptase inhibitors may be useful as new agents for the treatment of asthma. We investigated 4-substituted benzylamine derivatives and obtained M58539 (15h) as a potent inhibitor of β-tryptase (IC₅₀ = 5.0 nM) with high selectivity against other serine proteases, low molecular weight, c log P value less than 5, lack of amidino and guanidino groups, and independence of Zn²⁺ ion.

Full text of DOI:10.1016/j.bmcl.2006.02.064

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2986–2990
Synthesis and evaluation of 4-substituted benzylamine derivatives
as b-tryptase inhibitors
Yutaka Miyazaki,* Yutaka Kato, Tadashi Manabe, Hiroyasu Shimada,
Masashi Mizuno, Takayuki Egusa, Munetaka Ohkouchi, Ikuya Shiromizu,
Tomokazu Matsusue and Ichiro Yamamoto
Pharmaceutical Research Center, Mochida Pharmaceutical Co., Ltd, 722 Uenohara, Jimba, Gotemba, Shizuoka 412-8524, Japan
Received 29 December 2005; revised 22 February 2006; accepted 23 February 2006
Available online 15 March 2006
Abstract—Since b-tryptase is considered a critical mediator of asthma, potent tryptase inhibitors may be useful as new agents for the
treatment of asthma. We investigated 4-substituted benzylamine derivatives and obtained M58539 (15h) as a potent inhibitor of b-
tryptase (IC50 = 5.0 nM) with high selectivity against other serine proteases, low molecular weight, clogP value less than 5, lack of
amidino and guanidino groups, and independence of Zn2+ ion.
Ó 2006 Elsevier Ltd. All rights reserved.
b-Tryptase, a serine protease, has been implicated in
allergic and inflammatory diseases like asthma.1–4 In
fact, APC-366, a tryptase inhibitor, was reported to be
effective in asthmatic patients.5 Other than APC-366,
several compounds, for example, BABIM6 and AMG-
126737,7,8 have been reported to be potent inhibitors
of b-tryptase. Most, however, contain strongly basic
groups such as amidino or guanidino groups. Further,
AMG-126737 has relatively high molecular weight
(>500). These properties are considered unfavorable
for oral administration. BABIM is known to require
Zn2+ ion for inhibition of b-tryptase (Fig. 1).
O
H
NH2
O
H
N
N
N
N
NH
N
OH O
HN
NH
HN
H2N
NH2
·HCl
HN NH2
BABIM
APC-366
MW : 440.5
MW : 332.4
O
O
H
H
H2N
N
N
S
NH2
NH
S
O
O
O
O
NH
Our interest therefore focused on development of new
inhibitors with the following properties.
AMG-126737
MW : 678.8
Figure 1. The chemical structure of APC-366, BABIM, and AMG-
126737.
(1) High selectivity against other serine proteases.
(2) Low molecular weight less than 500.
(3) Low clogP value less than 5.
groups and its clogP value of 1 is less than 5, although
its molecular weight is slightly above 500. It was there-
fore selected as a seed compound, and further investiga-
tion was carried out in order to obtain optimal
compounds.
(4) Lack of strongly basic groups such as amidino and
guanidino groups.
(5) Independence of metal ions such as Zn2+ ion.
Compound 1 (Fig. 2) was found by means of high-
throughput screening. It does not contain strongly basic
First, benzylamine derivatives containing a piperidino
function at the 4-position were synthesized, as shown
in Scheme 1. Condensation of 410 with 3 derived from
211 yielded seed compound (1). Reduction of the car-
bamoyl group in 1 with BH3–THF complex afforded
Keywords: b-Tryptase; Serine protease.
Corresponding author. Tel.: +81 550 89 7881; fax: +81 550 89
0945; e-mail: ymiya@mochida.co.jp
*
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.02.064

Y. Miyazaki et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2986–2990
2987
prepared from the protected 4-aminomethylbenzalde-
hyde (22)16 via 23, as shown in Scheme 2.
H2N
O
N S
N
NH
O
O
R
X
Finally, a benzylamine derivative (29) with an ether type
side chain instead of the secondary amine group in 15a
was synthesized. Compound 27, prepared by DIBAL
reduction of 26,18 was alkylated by bromoacetonitrile
in the presence of a phase-transfer catalyst, and subse-
quent reduction of the cyano group by NaBH4/CoCl2
afforded the amine (28). The amine (28) was easily con-
verted into the corresponding sulfonamide, which was
deprotected to the ether (29). The spectral data of the
products shown in Schemes 1–3 are not contradicted
by the proposed structures.
IC50 : 21.1μM9
MW : 506.7
clogP : 2.43
1
Figure 2. The chemical structure of compound 1. See above-mentioned
reference for further information.
5. 4-(40-Hydroxymethyl)piperidinobenzonitrile (6)12 was
smoothly converted to the amino-aldehyde (7) in the
usual manner. Reductive amination of 7 with 1-sulfonyl-
piperazine (8)13 and with monosulfonyl-ethylenediamine
(10)14 followed by deprotection yielded compounds 9
and 11, respectively.
Optimization was performed from the middle region (X)
of 1. The results of modification of the region are listed
in Table 1. Activity was about 4-fold stronger than for 1,
when reduction of the amide moiety (5) or replacement
of the piperidine ring of the sulfonamide region with a
piperazine ring (9) was performed.
Second, compounds containing an open chain structure
in the central part of the molecule were synthesized as
illustrated in Scheme 2. Thus, N-protected 4-iodo-ben-
zylamine (12) was allowed to react with allyl alcohol
in the presence of Pd(OAc)2 to afford aldehyde (13).
Reductive amination of 13 with 10 yielded 14, which
was deprotected to obtain 15a. Similarly, compounds
15b–h were prepared from 13 by sulfonylation with aryl-
sulfonyl chlorides (19b–h) and deprotection via 18.19 In
addition, N-methyl derivative (16) of 15a was prepared
from 14 by means of Leukart type reductive methyla-
tion. Compound 21 was synthesized from 4-cyanophe-
nylacetic acid (20)15 by condensation with 10 and
subsequent conversion of an amide carbonyl group to
a methylene group. Further, compound (25) containing
a carbonyl group on the ethylenediamine moiety was
In order to give more flexibility to the substrates, 11,
which featured replacement of the piperidine ring in X
region with an ethylenediamine, was examined. Com-
pound 11 was 13-fold more potent than 1. In order to
make X region even more flexible, 15a, which featured
replacement of two piperidine rings with open chains,
was examined and was significantly more potent (ca.
300-fold) than the seed compound 1.
Based on 15a, the middle region was optimized further.
The focus of this optimization was the length of this re-
gion, the addition of substituent at the amine, and the
need for the amine. Compound 21, the length of which
was shortened by one methylene, was 11-fold less potent
than 15a. N-methylation of the amine (16) reduced
activity 1.5-fold. The amide (25) was also significantly
BocHN
e, f
a - d
N
NH2
1
NC
N
OH
4
3
2
H2N
N
NH
a
N SO2
5
H2N
g, f
NC
N
N
OH
N
8
6
N
O2S
9
a - c
H2N
g, f
BocHN
N
N
CHO
HN
10
NH
O2S
7
11
H2N
O2
N S
HO
O
O2
HN N S
NH
O2S
4
8
10
Scheme 1. Synthesis of 4-piperidinobenzylamine derivatives. Reagents: (a) BH3-THF; (b) Boc2O; (c) (COCl)2, DMSO, Et3N; (d) AcONH4,
NaBH3CN; (e) WSC–HCl; (f) HCl; (g) NaBH(OAc)3, AcOH.

2988
Y. Miyazaki et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2986–2990
O2
S
H2N
BocHN
N
N
I
H
16
12
13
a
b, c
(HCHO)n
O2
OH
BocHN
BocHN
b
H
N
S
H
N
10
H
O
14
c
O2
S
H2N
b, d, e
H
17
N
N
H
15a
f, c
O2
S
H2N
H
Boc
N
BocHN
N
N
R
RSO2Cl
H
NH2
19b-h
15b-h
18
In this case, R are shown in Table 2.
g, h
10
NC
H2N
O
H
N
N
S
OH
H
O2
20
21
BocHN
i - k
l, c
24
BocHN
H
NH2
O
22
23
H2N
O2
S
H
N
N
H
O
25
MeO
N
O2
N
N
H2N
+
HO
S
NHCOCF3
N
O
N
H
O
-
MeO
reagent A
Cl
17
2417
Scheme 2. Synthesis of benzylamine derivatives. Substituted with open chain function at 4-position. Reagents: (a) Pd(OAc)2; (b) NaBH(OAc)3,
AcOH; (c) HCl; (d) Boc2O; (e) K2CO3; (f) Et3N; (g) WSC–HCl; (h) BH3-THF; (i) (EtO)2P(O)CH2CN, NaH; (j) NaBH4; (k) H2, PtO2; (l) reagent A.
See above-mentioned reference for further information.
BocHN
H2N
b, c
a
BocHN
BocHN
OH
CO2Et
26
27
O2
S
d, e
O
N
O
NH2
H
28
29
Scheme 3. Synthesis of derivative (29) containing an ethereal linkage on side chain. Reagents: (a) DIBAL; (b) BrCH2CN, NaOH, Bu4NHSO4;
(c) NaBH4, CoCl2; (d) 2-naphthalenesulfonyl chloride, Et3N; (e) HCl.
less potent than compound 15a. Compound 29, which
featured replacement of the nitrogen atom by an oxygen
atom, was 80-fold less potent than compound 15a.
was 15-fold more potent than 15a. These results suggest-
ed that the shape of 1-chloronaphthalene fits the binding
site exactly. Compound 15h was 4220-fold more potent
than seed compound 1.
Thus, compound 15a was evaluated as the starting point
for further optimization.
The most potent compound 15h, M58539, was tested
for selectivity against trypsin, factor Xa, plasmin,
thrombin, and elastase. Among them, M58539 dis-
played exceedingly high selectivity against tryptase as
shown in Table 3.
Modification of the naphthalene ring moiety (R) was
performed. The results of this modification are shown
in Table 2. The naphthalene in 15a was replaced by
other aromatic ring systems, that is, benzene (15b), in-
dan (15c), benzo[b]thiophene (15d, e), 1,2,3,4-tetrahy-
droiso-quinoline (15f), or benzothiazole (15g). Bicyclic
rings seemed superior to monocyclic rings, and 15h20
Finally, the influence of Zn2+ ion on the inhibitory effect
of M58539 was tested. BABIM exhibited very low inhib-
itory activity (1/670) in the absence of Zn2+ ion, while

Y. Miyazaki et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2986–2990
2989
Table 1. SAR for X region modification
120.0
Compound
X
IC50 (lM)
100.0
80.0
60.0
40.0
20.0
0.0
N
NH
O
M58539
1
5
21.1
5.1
5.6
1.6
N
N
BABIM
without Zn2+
BABIM
N
N
NH
150mM Zn / M58539
150mM Zn / BABIM
1mM EDTA / M58539
1mM EDTA / BABIM
N
9
N
N
11
15a
21
16
25
29
HN
NH
-20.0
0.1
1
10
100
1000
10000 100000
0.073
HN
Log concentration (nM)
NH
NH
HN
Figure 3. The influence of zinc ion on inhibitory effect.
0.81
0.12
1.3
N
M58539 exhibited high inhibitory activity independent
of Zn2+ ion (Fig. 3).
Me
NH
NH
NH
O
HN
O
As described in this paper, through modification of
4-substituted benzylamine derivatives, we obtained a
very active compound, M58539, as a potent inhibitor of
b-tryptase (IC50 = 5.0 nM) with the desired properties of
high selectivity against other serine proteases (>20,000),
low molecular weight (432.0), low clogP value (4.04),
lack of amidino and guanidino groups, and indepen-
dence of Zn2+ ion.
5.7
Table 2. SAR for naphthalene ring moiety modification
Compound
X
IC50 (lM)
15a
0.073
References and notes
15b
0.76
0.20
0.13
0.21
0.19
0.27
0.005
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15c
15d
S
S
15e
15f
NH
N
15g
S
Me
15h M58539
Cl
Table 3. Selectivity of M58539 and BABIM against serine proteases
Compound
M58539
Enzyme
IC50 (lM)
Selectivity
Tryptase
Trypsin
FXa
0.005
>100
>100
>20,000
>20,000
>20,000
>20,000
>20,000
Plasmin
Thrombin
Elastase
>100
>100
>100
BABIM
Tryptase
Trypsin
FXa
0.051
4.4
4.5
86
88
Plasmin
Thrombin
Elastase
26
31
510
608
>100
>1960

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d, J = 8 Hz), 7.13 (2H, d, J = 8 Hz), 4.79 (1H, s), 4.28 (2H,
d, J = 5 Hz), 3.33–3.10 (4H, m), 2.80 (2H, d, J = 7 Hz),
2.58 (2H, d, J = 8 Hz), 1.93–1.75 (2H, m), 1.46 (9H, s),
1.44 (9H, s).
1
20. Spectroscopic data of 15h: H NMR (DMSO-d6) d: 8.92
(2H, s), 8.61 (1H, d, J = 2 Hz), 8.35–8.15 (3H, m), 8.15–
8.05 (1H, m), 8.12 (1H, d, J = 9 Hz), 7.90 (1H, dd, J = 2,
9 Hz), 7.40 (2H, d, J = 8 Hz), 7.26 (2H, d, J = 8 Hz), 4.05–
3.90 (2H, m), 3.15–2.80 (6H, m), 2.87 (3H, s), 2.64 (2H, t,
J = 8 Hz), 1.95–1.80 (2H, m).