Mineralocorticoid receptor (MR) blockade has come into focus as a promising approach for the treatment of cardiovascular diseases such as hypertension and congestive heart failure. In order to identify a novel class of nonsteroidal MR antagonists that exhibit significant potency and good selectivity over other steroidal hormone receptors, we designed a novel series of benzoxazin-3-one derivatives and synthesized them from 6-(7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazin-6-yl)-2H-1,4-benzoxazin-3(4H)-one (1a), high-throughput screening (HTS) hit compound. Our design was based on a crystal structure of an MR/compound complex and a docking model. In the course of lead generation from 1a, a 1,2-diaryl framework was characterized as a key structure with high binding affinity. On the basis of scaffold hopping and optimization studies, benzoxazin-3-one derivatives possessing 1-phenyl-3-trifluoromethylpyrazol-5-yl moiety at the 6-position were identified as a novel series of potent and selective MR antagonists. Among these compounds, 6-[1-(4-fluoro-2-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-5-yl]-2H-1,4-benzoxazin-3(4H)-one (14n) showed highly potent activity and good selectivity and also exhibited a significant antihypertensive effect in deoxycorticosterone acetate-salt hypertensive rats. On the basis of these results, compound 14n was progressed for further pharmacological evaluation.
6‐Aminohexanoic‐acid‐oligomer hydrolase of Flavobacterium sp. KI72 was purified to homogeneity by column chromatography three times, and by preparative polyacrylamide gel electrophoresis twice. The purified enzyme had the following characteristics.
The molecular weight was estimated to be 84000 by Sephadex G‐200 molecular‐sieve chromatography. The enzyme consisted of two homologous subunits of 42000, judged from sodium dodecylsulfate/polyacrylamide gel electrophoresis.
The optimum pH for activity was between 8 and 9, the optimum temperature was 40° C for a 1‐h reaction. The Michaelis‐Menten constants and turnover numbers for the 6‐aminohexanoic acid dimer and trimer were 5.9 mM and 2.4 s−1, and 6.2 mM and 2.0 s−1 respectively.
The enzyme was inhibited by 0.37 mM diisopropylfluorophosphate and by 0.013 mM p‐chloromercuribenzoate.
The enzyme was active on 6‐aminohexanoic acid oligomers from dimer to hexamer and icosamer but not on hectamer, and the activity decreased with the increase of the polymerization number of the ougomer. The oligomers were hydrolyzed so as to remove the 6‐aminohexanoic acid residue successively from the amino terminus. The enzyme could not hydrolyze other linear amides, cyclic amides, dipeptides, tripeptides or casein.
6‐Aminohexanoic‐acid‐oligomer hydrolase was classified as a new member of the linear amidases (EC 3.5.1.‐).
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