The 5-HT3 receptor, a pentameric ligand-gated ion channel (pLGIC), is an important therapeutic target. During a recent fragment screen, 6-chloro-N-methyl-2-(4-methyl-1,4-diazepan-1-yl)quinazolin-4-amine (1) was identified as a 5-HT3R hit fragment. Here we describe the synthesis and structure–activity relationships (SAR) of a series of (iso)quinoline and quinazoline compounds that were synthesized and screened for 5-HT3R affinity using a [3H]granisetron displacement assay. These studies resulted in the discovery of several high affinity ligands of which compound 22 showed the highest affinity (pKi > 10) for the 5-HT3 receptor. The observed SAR is in agreement with established pharmacophore models for 5-HT3 ligands and is used for ligand–receptor binding mode prediction using homology modeling and in silico docking approaches.