In the search for promising anti-proliferative agents that might be helpful in the treatment of cancer effectively, several compounds in a series (4b–j) comprising 1,4-bis (5-substituted -2-thiono-2H-tetrahydro-1,3,5-thiadiazin-3-yl) butane derivatives have been isolated. The aimed two privileged thiadiazinane pharmacophores were symmetrically assembled in one molecular frame via 1,4-diaminobutane; the endogenous compound produced by the breakdown of some amino acids that’s known as putrescine. The thiadiazinane rings bearing variable substituents at N-5 as well. The structure of the new derivatives, which were obtained by domino-reactions in water are confirmed by NMR and ESI-MS spectra. Data of 1H-NMR and 13C-NMR. NMR-spectra revealed symmetrical structural features. The anti-proliferative activity was evaluated against five different human cancer cell lines. Compounds 4b, 4d, 4e, and 4j (IC50 range 0.11–0.24 μM), were found potent against Hep3B (hepatocellular carcinoma). Compounds 4d and 4e are also potent (IC50 = 0.42 and 0.41 μM) against U-87 MG (Brain (glioblastoma astrocytoma)). Moreover, 4d provided (IC50 = 0.53 μM) against HepG2 (hepatocellular carcinoma), (A549 (lung carcinoma), and HT-29 (human colorectal adenocarcinoma), as well as normal cell line (fibroblast F180). All the derivatives 4b, 4d, 4e, and 4j are not only more potent, but also relatively safer than doxorubicin in the cell-lines mentioned. The anti-proliferative profile indicates that these compounds are good leads as anti-cancer agents and merit further studies to optimize their structure, detect their bio-targets and in vivo activity.