Identification of 6-substituted 4-arylsulfonyl-1,4-diazepane-2,5-diones as a novel scaffold for human chymase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.03.038

A novel series of 6-substituted 4-sulfonyl-1,4-diazepane-2,5-diones were designed, synthesized and evaluated as human chymase inhibitors. Structure-activity relationship studies led to the identification of a potent inhibitor, (6S)-6-(5-chloro-2-methoxybe

Full text of DOI:10.1016/j.bmcl.2007.03.038

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3431–3434
Identification of 6-substituted 4-arylsulfonyl-1,4-diazepane-
2,5-diones as a novel scaffold for human chymase inhibitors
a,
a
a
Taisaku Tanaka,a,* Tsuyoshi Muto, Hiroshi Maruoka, Seiichi Imajo,
*
Harukazu Fukami,b Yoshiaki Tomimori,a Yoshiaki Fukudaa and Takashi Nakatsukaa
aBiomedical Research Laboratories, Daiichi Asubio Pharma Co., Ltd, 1-1-1 Wakayamadai Shimamoto-cho, Mishima-gun,
Osaka 618-8513, Japan
bFoods Function and Health Science Laboratory, Department of Bioscience and Biotechnology, Faculty of Bioenvironmental Science,
Kyoto-gakuen University, 1-1 Nanjootani, Sogabe-cho, Kameoka, Kyoto 621-8555, Japan
Received 28 November 2006; revised 8 March 2007; accepted 13 March 2007
Available online 16 March 2007
Abstract—A novel series of 6-substituted 4-sulfonyl-1,4-diazepane-2,5-diones were designed, synthesized and evaluated as human
chymase inhibitors. Structure–activity relationship studies led to the identification of a potent inhibitor, (6S)-6-(5-chloro-2-meth-
oxybenzyl)-4-[(4-chlorophenyl)sulfonyl]-1,4-diazepane-2,5-dione, with an IC50 of 0.027 lM.
Ó 2007 Elsevier Ltd. All rights reserved.
Chymase (EC 3.4.21.39) is a chymotrypsin-type serine
protease localized in the secretory granules of mast cells,
and is known as a major non-ACE, angiotensin-II (Ang-
II)-generating enzyme in human tissue.1 In addition to
Ang-II formation, chymase mediates the production of
endothelin-1 (1–31)2 and modification of apolipopro-
teins,3 and several reports have suggested that these
functions of chymase are involved in cardiovascular dis-
eases such as atherosclerosis,4 restenosis,5 and heart fail-
ure.6 On the other hand, chymase has also been shown
to participate in the production of collagen,7 activation
of metalloproteases,8 and processing of cytokines such
as interleukin-1b,9 transforming growth factor-b1,10
and stem cell factor.11 In addition to such activities,
the localization of chymase in mast cells has prompted
the study of its role in allergy and fibrosis. It has been
demonstrated that chymase is involved in the progres-
sion of dermatitis12 and chronic inflammation following
cardiac6,13 and pulmonary fibrosis.14 Thus, inhibition of
chymase is expected to provide therapeutic means for
the treatment of cardiovascular diseases, allergic inflam-
mation, and fibrotic disorders.15
In a previous paper, we have reported on novel human
chymase inhibitors possessing a 3-phenylsulfonylquinaz-
oline-2,4-dione scaffold.16 Although several compounds
identified in that report are effective in some pathologi-
cal animal models,15 these compounds are not suffi-
ciently efficacious for use as therapeutic agents. Thus,
we continued to explore a new class of human chymase
inhibitors, based on the insights from docking studies of
these quinazoline derivatives with human chymase. The
docking structure of the active site of human chymase
with compound 116 revealed that the inner part of the
S1 pocket is not completely filled with the aromatic part
of the quinazoline moiety of compound 1, and it seems
that the hydrophobic interaction of 1 at the S1 pocket is
not fully efficient. On the basis of these findings, we
designed benzyl-substituted monocyclic lactams to
increase the flexibility of the aromatic part of the
inhibitors, in order to sufficiently interact with the S1
pocket, and identified a novel scaffold, 6-substituted
4-arylsulfonyl-1,4-diazepane-2,5-diones (2), for human
chymase inhibitors (Fig. 1). In this paper, we describe
the synthesis and the pharmacological evaluation of this
new class of human chymase inhibitors.
Racemic compounds 2 were synthesized as shown in
Scheme 1. The Baylis–Hillman reaction of substituted
benzaldehydes 3 with ethyl acrylate followed by acetyla-
tion gave adducts 4. SN20-type substitution of the acet-
oxy groups of 4 by sodium azide, and subsequent
reduction of the introduced azide groups, led to amines
Keywords: Chymase; Inhibitor; 4-Arylsulfonyl-1,4-diazepane-2,5-
dione.
*
Corresponding authors. Tel.: +81 75 962 8481; fax: +81 75 962
6448; e-mail addresses: tanaka.taisasku.fu@asubio.co.jp; muto.
tsuyoshi.ba@asubio.co.jp
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.03.038

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T. Tanaka et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3431–3434
Figure 1. Design of 1,4-diazepane-2,5-dione scaffold.
5, which were converted to b-amino acids 617 by stan-
dard methods. Condensation of 6 with p-chlorobenzen-
sulfonamide gave acylsulfonamides 7, which were
transformed to bromoacetamides 8 or their sodium
salts. Ring closure of 8 to a 7-membered ring by intra-
molecular alkylation, followed by catalytic hydrogena-
tion, afforded compounds 2.18 Compounds 2c, 2k, and
2l were prepared by reduction of precursor compounds
2a, 2m, and 2n, respectively. Compounds 2q and 2r were
synthesized from 9q, which was obtained by a similar
procedure to that described above. Scheme 2 shows
the synthesis of the optically active compounds (S)-
and (R)-2m. Optically active b-amino acids (S)- and
(R)-14 were synthesized19a from compounds (S)- and
(R)-13, which were obtained by asymmetric hydrogena-
tion of itaconic acid 12 using (S,S)- or (R,R)-phenyl-
Scheme 2. Reagents and conditions: (a) H2, [RhCl(cod)2], (R,R)- or
(S,S)-Ph-CAPP, Et3N, MeOH, 40 °C, 3 h; (b) PhI(OH)OTs, MeCN,
reflux, 1 h; (c) (Boc)2O, aq NaOH, THF, rt, 2 h; (d) aq KOH, MeOH,
rt, 3 h; (e) EDCI HCl, DMAP, p-chlorobenzensulfonamide, CH2Cl2,
rt, 1 h; (f) 1 M HCl/AcOH, rt, 30 min; (g) bromoacetyl chloride, aq
NaOH, CH2Cl2, 0 °C, 30 min (isolated as sodium salt); (h) DMF, rt,
72 h.
CAPP,19b,9c respectively, as a chiral ligand. Using the
same procedure described in Scheme 1, (S)- and (R)-14
were converted to optically active (R)-2m and (S)-2m,
respectively.
Scheme 1. Reagents and conditions: (a) ethyl acrylate, DABCO, La(OTf)3, N(CH2CH2OH)3, rt, 24–48 h or ethyl acrylate, DABCO, MeOH (3
equiv), rt, 24–48 h; (b) AcCl, Et3N, CH2Cl2, 0 °C, 1 h; (c) NaN3, DMSO, rt, 1 h; (d) PPh3, H2O, THF, rt, 18 h; (e) aq NaOH, EtOH, rt, 1 h; (f)
(Boc)2O, aq NaOH, THF, 0 °C, 1 h or (CF3CO)2O, pyridine, THF, rt, 1 h; (g) EDCI HCl, DMAP, p-chlorobenzensulfonamide, CH2Cl2, rt, 1 h; (h)
i—1 M HCl/AcOH, rt, 1 h, ii— bromoacetyl chloride, Et3N, CH2Cl2, H2O, 0 °C, 30 min; (i) i—aq NaOH, MeOH, rt, 1 h, ii—bromoacetyl chloride,
aq NaOH, CH2Cl2, 0 °C, 30 min; (j) NaH, DMF, 60 °C, 18 h (in the case of X = H) or DMF, 60 °C, 18 h (in the case of X = Na); (k) H2, Pt(sulfided)–
C, THF, rt, 18 h; (l) H2, Pd–C, THF, rt, 14 h; (m) H2, PtO2, AcOH, rt, 8 h; (n) TFA, CH2Cl2, 0 °C, 1 h; (o) BrCH2COOtBu, NaI, NaHCO3, DMF, rt,
20 h; (p) MeNH2 HCl, Et3N, 2-chloro-1,3-dimethylimidazolinium chloride, CH2Cl2, rt, 2 h.

T. Tanaka et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3431–3434
3433
Table 2. Inhibitory activity of compounds 2m–2x against recombinant
human chymase
The compounds described above were tested for their
in vitro inhibitory activity against recombinant human
chymase20 using Suc-Ala-Ala-Pro-Phe-MCA as a sub-
strate.21 We initially examined the substituents on the
6-position of the 1,4-diazepane-2,5-dione core, which
would interact with the S1 pocket (Table 1). Compound
2b possessing a benzylidene substituent was less potent
than the corresponding benzyl derivative 2a. Similar
activity trends were observed in the other benzylidene ser-
ies corresponding to 2d–2m (data not shown), suggesting
that the benzene ring in the fixed benzylidene substituent
is not located in the proper position in the S1 pocket. The
more bulky cyclohexylmethyl (2c) and 2-naphthylmethyl
(2d), or more hydrophilic pyridylmethyl groups (2e and
2f), did not show high inhibitory activity. Introduction
of a chlorine atom to the benzene ring of 2a retained or
slightly increased activity (2g–2i), and the 20-Cl substitu-
ent (2i) was the most preferable. Other substituents at
the 20-position such as fluoro (2j) and alkoxy (2k and 2l)
also conferred high activity. The combination of 20-meth-
oxy and 50-chloro substituents (2m) synergistically en-
hanced the activity, so that the compound was 10- or
fivefold more potent than 2h or 2k, respectively. Next,
we investigated the effect of the substituents at the 20-
and 50-positions of 2m (Table 2). Elongation of the meth-
oxy moiety of 2m decreased the activity (2n–2p), whereas
addition of a carboxyl (2q) or carboxamide (2r) group re-
sulted in the same level of activity as 2m. The fact that 2q
and 2r having bulky and hydrophilic substituents at the
20-position showed high inhibitory activity indicated that
substituents at the 20-position were not located inside the
S1 pocket, which is space-restricted and hydrophobic,
and the carboxyl or methylcarbamoyl group was situated
Compounda
R1
R2
IC50 (lM)
b
2m
2n
2o
2p
2q
2r
Me
Et
50-Cl
0.034
0.083
0.22
0.28
0.039
0.042
0.026
0.027
1.1
50-Cl
nPr
50-Cl
nBu
CH2COOH
CH2CONHMe
50-Cl
50-Cl
50-Cl
2s
Me
Me
Me
Me
Me
Me
50-F
2t
50-CN
50-OH
50-OMe
40,50-Cl2
30-Cl, 50-F
2u
2v
2w
2x
7.4
0.023
4.2
a Compounds 2m–2x are racemates at the 6-position.
b For details of the assay conditions, see Ref. 21.
at a hydrophilic region and formed a favorable interac-
tion, such as hydrogen bonding, with another enzyme site
(e.g., S2 or S3). The lower activity of compounds 2n–2p,
which have longer, hydrophobic alkyl chains at the 20-alk-
oxy group, supports this idea. Replacement of the chlo-
rine atom at the 50-position with a fluoro (2s) or cyano
(2t) group did not alter the potency, but replacement with
a more hydrophilic hydroxy (2u) or methoxy (2v) group
decreased the activity remarkably. These results support
the idea that substituents at the 50-position lie in the inner
part of the hydrophobic S1 pocket. Introduction of an
additional chlorine atom at the 40-position did not affect
the activity (2m vs 2w), but introduction at the 30-position
did reduce the activity (2s vs 2x), probably due to restric-
tion of the space around the 30-position in the S1 pocket.
As shown in Table 3, the representative compound 2m
exhibited high selectivity against other serine proteases,
Table 1. Inhibitory activity of compounds 2a–2m against recombinant
human chymase
Compounda
X
A
IC50 (lM)
b
Table 3. IC50 valuesa of inhibition of human chymase and other serine
proteases for compound 2m
Chymostatin
1
—
—
—
—
0.043
0.018
0.42
1.5
a
Enzyme
IC50 (lM)
2a
2b
2c
2d
2e
2f
–CH2–
–CH@c
–CH2–
–CH2–
–CH2–
–CH2–
–CH2–
–CH2–
–CH2–
–CH2–
–CH2–
–CH2–
–CH2–
Ph
Ph
Human chymase
Bovine a-chymotrypsin
Human cathepsin G
Bovine trypsin
0.034
0.3
2.9
Cyclohexyl
2-Naphthyl
3-Pyridyl
4-Pyridyl
4-Cl–Ph
3-Cl–Ph
2-Cl–Ph
2-F–Ph
2-OMe–Ph
2-OEt–Ph
2-OMe-5-Cl–Ph
>10
>10
>10
>10
0.46
0.31
0.11
0.15
0.14
0.18
0.034
>10
>10
Human elastase
a For details of the assay conditions, see Refs. 21,22.
2g
2h
2i
2j
Table 4. Inhibitory activity of compounds (S)-2m and (R)-2m against
recombinant human chymase
2k
2l
a
Compound
IC50 (lM)
2m
a Compounds 2a, 2c–2m are racemates at the 6-position.
b For details of the assay conditions, see Ref. 21.
c (E)-Isomer.
(S)-2m
(R)-2m
0.027
>10
a For details of the assay conditions, see Ref. 21.

3434
T. Tanaka et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3431–3434
13. Kanemitsu, H.; Takai, S.; Tsuneyoshi, H.; Nishina, T.;
Yoshikawa, K.; Miyazaki, M.; Ikeda, T.; Komeda, M.
Hypertens. Res. 2006, 29, 57.
14. (a) Tomimori, Y.; Muto, T.; Saito, K.; Tanaka, T.;
Maruoka, H.; Sumida, M.; Fukami, H.; Fukuda, Y. Eur.
J. Pharmacol. 2003, 478, 179; (b) Sakaguchi, M.; Takai, S.;
Jin, D.; Okamoto, Y.; Muramatsu, M.; Kim, S.; Miyazaki,
M. Eur. J. Pharmacol. 2004, 493, 173.
15. (a) Takai, S.; Jin, D.; Muramatsu, M.; Okamoto, Y.;
Miyazaki, M. Eur. J. Pharmacol. 2004, 501, 1; (b) Muto,
T.; Fukami, H. IDrugs 2002, 5, 1141.
16. Fukami, H.; Imajo, S.; Ito, A.; Kakutani, S.; Shibata, S.;
Sumida, M.; Tanaka, T.; Niwata, S.; Saitoh, M.; Kiso, Y.;
Miyazaki, M.; Okunishi, H.; Urata, H.; Arakawa, K.
Drug Des. Discov. 2000, 17, 69.
including chymotrypsin and chymotrypsin-type protease
cathepsin G. Finally, we examined the effect of chirality at
the 6-position of the 1,4-diazepane core, and found that
only (S)-2m exhibited inhibitory activity (IC50,
0.027 lM) (Table 4).
In conclusion, we have described here the design,
synthesis, and evaluation of 6-substituted 1,4-diaze-
pane-2,5-diones as novel and potent human chymase
inhibitors. From insights obtained from the docking of
compound 1 with the active site of human chymase,
we designed 6-benzyl-substituted 4-sulfonyl-1,4-diaze-
pane-2,4-diones to interact sufficiently with the S1 pocket.
Modification of the benzyl moiety of a prototype such as
2a led to the development of a potent inhibitor such as
(S)-(2m), which exhibited an IC50 of 0.027 lM.
17. Compounds 6o and 6p for the synthesis of 2o and 2p were
synthesized from 6m via intermediate 15 by a five-step
sequence of reactions, as shown below.
Acknowledgment
We are grateful to Dr. T. Nishihara for his helpful
advice and valuable discussion during this study.
References and notes
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18. In the synthesis of compound 2u (R1, 2-OMe, 5-OH), the
final deprotection reaction (TFA, CH2Cl2) was needed to
remove the methoxymethyl group which protected the
phenolic hydroxy group during the reaction sequence.
19. (a) Takeuchi, Y. Jpn. Kokai Tokkyo Koho, JP
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20. The expression and purification of recombinant human
chymase are described in Ref. 11b.
21. The human chymase assay was performed as follows:
recombinant human chymase was preincubated with the
test compounds for 10 min in 50 mM Tris/HCl buffer (pH
7.5) containing 1 M NaCl and 0.01% Triton X-100. The
enzyme reaction was initiated with substrate Suc-Ala-Ala-
Pro-Phe-MCA (Peptide Institute), supplied at 100 lM,
and was stopped with 30% acetic acid after 10 min
incubation. The intensity of the fluorescence of the
AMC produced by chymase was measured (extinction
380 nm, emission 460 nm), and the IC50 value was
calculated from the inhibition of AMC formation at each
concentration of the tested compound.
22. The inhibitory effects on the enzymatic activity of serine
proteases were evaluated by a similar procedure as in Ref.
21. The enzymes and substrates used for the assays were as
follows: Suc-Ala-Ala-Pro-Phe-MCA (Peptide Institute)
for bovine pancreatic a-chymotrypsin (Sigma) and human
neutrophil cathepsin G (Calbiochem); Boc-Gln-Ala-Arg-
MCA (Peptide Institute) for bovine pancreatic trypsin
9. Mizutani, H.; Schechter, N.; Lazarus, G.; Black, R. A.;
Kupper, T. S. J. Exp. Med. 1991, 174, 821.
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Keski-Oja, J. J. Biol. Chem. 1995, 270, 4689; (b) Tomi-
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Sumida, M.; Fukami, H.; Fukuda, Y. Eur. J. Pharmacol.
2003, 478, 179.
11. (a) Longley, B. J.; Tyrrell, L.; Ma, Y.; Willams, D. A.;
Halaban, R.; Langley, K.; Lu, H. S.; Schechter, N. M.
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mori, Y.; Muto, T.; Fukami, H.; Saito, K.; Horikawa, C.;
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Yamashiro, K.; Sumida, M.; Kakutani, S.; Fukuda, Y.
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Saito, K.; Miura, K.; Wada, A.; Tsudzuki, M.; Fukuda, Y.
Int. Arch. Allergy Immunol. 2002, 128, 229.
(Nacalai
Tesque);
MeOSuc-Ala-Ala-Pro-Val-pNA
(Bachem) for human neutrophil elastase (Calbiochem).
The assay buffer was as follows: 0.1 M Tris/HCl (pH 7.5)
containing 1 M KCl and 0.01% Triton X-100 for a-
chymotrypsin; 50 mM Tris/HCl buffer (pH 7.5) containing
1 M NaCl and 0.01% Triton X-100 for cathepsin G and
elastase; 50 mM Tris/HCl buffer (pH 7.5) containing
20 mM CaCl2 for trypsin.