Natural products, which are enzymatically biosynthesized, have a broad range of biological activities. In particular, many flavonoids are known to contribute to human health with low toxicity. We previously reported that novel benzo[b]thiophenyl (BT) flavones with a 10π-electron BT ring B replacing the usual 6π-electron phenyl ring showed potent antiproliferative activity against human tumor cell lines. Interestingly, the activity profiles against cell cycle progression of the BT-flavones totally changed depending on the combination of substituents at the C-3 and C-5 positions. This finding encouraged an extension of these studies on the impact of BT to related flavonoids, such as chalcones, isoflavones, and aurones. Accordingly, 10 isoflavones, 29 chalcones, and four aurones were synthesized and evaluated for antiproliferative activity against five human tumor cell lines including a multi-drug-resistant cell line. Among these compounds, BT-isoflavone 7, BT-chalcones 48, 52, 57, 66, and 77, and BT-aurone 80 displayed significant antiproliferative effects against all tested tumor cell lines. The structure-antiproliferative activity relationships clearly demonstrated the importance of BT instead of phenyl as ring B for the isoflavone and chalcones, but not the aurones. Flow cytometry and immunocytochemical studies demonstrated that the active BT-flavonoids led to cell cycle arrest at the prometaphase by induction of multipolar spindle formation. The present studies should contribute greatly to the synthesis and functional analysis of biologically active flavonoid derivatives for chemical space expansion.