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.
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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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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
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(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.