New arylpiperazine derivatives as antagonists of the human cloned 5-HT4 receptor isoforms

New derivatives of arylpiperazine 9 were designed from ML 10302, a potent 5 -HT4 receptor agonist in the gastrointestinal system. Compounds were synthe sized by condensation of a number of available arylpiperazines or heteroary lpiperazines with 2-bromoethyl 4-amino-5-chloro-2-methoxybenzoate. They wer e evaluated in binding assays on the recently cloned human 5-HT4(e) isoform stably expressed in C6 glial cells with [H-3]GR 113808 as the radioligand. The affinity values (K-i) depended upon the substituent on the aromatic ri ng. A chlorine atom produced a marked drop in activity (K-i > 100 nM), whil e a m-methoxy group gave a compound with nanomolar affinity (K-i = 3 nM). T he most potent compounds were the heterocyclic derivatives with pyrimidine, pyrazine, pyridazine, or pyridine moieties (compounds 9r, 9t, 9u, 9x, resp ectively). K-i values for 9a and 9r were determined for the 5-HT4(a), 5-HT4 (b), 5-HT4(c), and 5-HT4(d) receptor isoforms transiently expressed in COS cells. The results indicated that the compounds were not selective. They pr oduced an inhibition of the 5-HT-stimulated cyclic AMP synthesis in the C6 glial cells stably expressing the 5-HT4(e) receptor and shifted the 5-HT co ncentration-effect curve on adenylyl cyclase activity with pK(D) values of 7.44 and 8.47, respectively. In isolated human atrial myocytes, 9r antagoni zed the stimulatory effect of 5-HT on the L-type calcium current (I-Ca) wit h a K-D value of 0.7 nM.