A series of new analogues of N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl] 2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-carboxamide (1; GR127935) as potent and selective 5-HT(1B/1D) antagonists were synthesized and evaluated pharmacologically. Their receptor binding profiles were comparable to that of 1. The 1,3,4-oxadiazole isomer 2 and the 4'-aminocarbonyl and 4'-amidinyl analogues (9 and 10) of 1 had higher affinities at the rat 5-HT(1B) receptor (IC(50) = 0.93, 1. 3, and 0.5 nM, respectively) and calf 5-HT(1D) receptor (IC(50) = 37, 10, and 3 nM, respectively) than did 1 (1.6 and 52 nM for rat 5-HT(1B) and calf 5-HT(1D) receptors, respectively). In the functional in vitro testing of 5-HT(1B/1D) antagonistic properties, 2, 9, 10, 11b (O-demethylated derivative of 2), 13a (O-methylsulfonyl analogue of 2), and 16 (which differs from 2 with a sulfonamide linker) showed more pronounced effects in the K(+)-induced 5-HT release in the cortex of guinea pig than did 1 and 3 (SB224289). Compounds 2, 9, and 10 were equally potent as 1 in rabbit saphenous vein model (pA(2) > 9). A biochemical study of 2 with in vivo microdialysis in the rat brain showed that it is capable of augmenting citalopram (a selective serotonin reuptake inhibitor, SSRI) induced 5-HT release in rat ventral hippocampus, while preventing the decrease in acetylcholine release elicited by citalopram administration. The molecular structure of 2 was determined by single-crystal X-ray analysis. The log P and log D values of these compounds were calculated. This study contributes to the SAR study of N-piperazinylphenyl biphenylcarboxamides as selective and potent 5-HT(1B/1D) antagonists.