​The reaction of N′’(aryl)benzothiohydrazides with 2-chloro-6-((substituted)amino)-3-nitro-4-oxo-4H-thieno[2,3-b]thiopyran-5-carboxylate (13-15) under basic conditions (NEt3) in acetonitrile proceeds via thiophene ring-opening processes and yields, upon addition of iodomethane, the respective 1,3,4-thiadiazoline-6-sulfanylthiopyran-4(1H)-one hybrids. The new compounds were characterized by HRMS and NMR spectral data and confirmed by single-crystal X-ray crystallography. The cytotoxicity affinities for compounds 10-17 were evaluated in cross-correlations with their anti-inflammation and radical scavenging capacities. Compound 13 exhibited the highest cytotoxic properties, with IC50 values ranging from 160 nM in mammary T47D to less than 20.35 µM in colorectal CACO2 among 12 diverse cancer monolayers. Compound 17c significantly reduced lung and mammary cancer cell viability, with anti-tumorigenesis IC50 values of less than 10 µM. These new compounds have the potential to be further optimized into novel selective cytotoxic treatments.​