Pharmacological activity and structural analysis of a benzamide (TKS159) and its optical isomers in an in vitro study and in an in vivo study in mice.

We previously conducted an in vitro study of 4-amino-5-chloro-2-methoxy-N-(1-ethyl-2-hydroxymethyl-4-pyrrolidinyl)benzamide (2S,4S)-(1, TKS159) and its three optical isomers (2S,4R)-(2), (2R,4S)-(3) and (2R,4R)-(4) with respect to their binding ability to the 5-HT(4) receptors, as well as an in vivo study on their gastric emptying-accelerating ability in rats. Consequently, we reported that steric configuration at positions 2 and 4 of the pyrrolidine ring is important in determining their pharmacological activity. The optical isomer (2R,4S)-(3) exhibited the most potent binding ability. However, the compound (2S,4S)-(1, TKS159) exhibited the most potent gastric emptying-accelerating ability in rats. A difference was thus found between binding ability and gastric emptying-accelerating ability in rats. Therefore, we conducted an in vitro study of TKS159 (1) and its three optical isomers to examine their agonistic activity on the 5-HT(4) receptors, as well as an in vivo study in mice to examine their gastric emptying-accelerating ability. Consequently, a tendency for correlation was found between the activity and the ability. TKS159 (1) exhibited the most potent pharmacological activity, well reflecting the results from the previous in vivo study in rats. Furthermore, the present in vitro and in vivo studies reverified the importance of steric configuration at positions 2 and 4 of the pyrrolidine ring. In addition, we also made an X-ray crystallographic analysis of the optical isomer (2R,4S)-(3), which has the S-configuration at position 4 similar to TKS159 (1), and discussed molecular structures in conjunction with the previously reported results from the X-ray crystallographic analysis of TKS159 (1).

Synthesis and pharmacological activity of 4-amino-5-chloro-2-methoxy-N-[(2S,4S)-1-ethyl-2-hydroxymethyl-4- pyrrolidinyl]benzamide (TKS159) and its optical isomers.

Of 4-amino-5-chloro-2-methoxy-N-(1-ethyl-2-hydroxymethyl-4- pyrrolidinyl)benzamide, four optical isomers, (2S,4S)-1 (TKS159), (2S,4R)-25, (2R,4S)-26 and (2R,4R)-27, were prepared from optically active 4-amino-1-ethyl-2-hydroxymethylpyrrolidine di-p-toluenesulfonate [(2S,4S)-14, (2S,4R)-17, (2R,4S)-20 and (2R,4R)-23, respectively]. The requisites, (2S,4S)-14, (2S,4R)-17, (2R,4S)-20 and (2R,4R)-23, were prepared from a commercially available trans-4-hydroxy-L-proline. The absolute configurations of (2S,4S)-1 (TKS159), (2S,4R)-25, (2R,4S)-26 and (2R,4R)-27 were spectroscopically determined. Of the benzamide derivatives, four optical isomers, (2S,4S)-1, (2S,4R)-25, (2R,4S)-26 and (2R,4R)-27, showed a relatively potent affinity for 5-hydroxytryptamine 4 (5-HT4) receptors in a radioligand binding assay ([3H]GR113808). The activities of 25-27 were less effective than that of 1 for the gastric emptying of a phenol red semisolid meal in rats. All this suggests that the most potent of the isomers was 4-amino-5-chloro-2-methoxy-N-[(2S,4S)-1-ethyl-2- hydroxymethyl-4-pyrrolidinyl]benzamide (1).