Novel 2,4-disulfonylsubstituted 5-aminothiazoles were synthesized and their anticancer activity was assessed at a high dose (10 μM) against NCI 60 cancer cell lines. Compounds 24 and 25 showed the antiproliferative activity with mean growth inhibition about 66.0%. Replacing 4-hydroxypiperidine 24 with the more hydrophilic N-methyl piperazine 25 increased the number of sensitive cell lines while replacing these hydrophilic groups with lipophilic ones abolished the anticancer activity. The COMPARE analysis showed that the tested compounds had a moderate positive correlation with alkylating agents (CCNU and methyl CCNU) and with a purine nucleotide biosynthesis inhibitor analog (L-cysteine). The results indicate that the above mechanisms of antitumor action of standard compounds are not the main ones for the tested compounds due to the lack of a high correlation. The results of this study allow us to consider compounds 24 and 25 as a basis for their further functionalization to obtain more active compounds.