New thiazolo[2,3-a] pyrimidinedione derivatives were synthesized by two different chemical methods.One method included the addition of a mixture of glacial acetic acid ,acetic anhydride (2 : 1), chloroacetic acid and  anhydrous sodium acetate to mercapto-thieno[2,3-d]pyrimidinone derivatives 5aor 5b under reflux to give thieno[2,3-d]thiazolo[3,2-a]pyrimidinedione derivatives 6a or 6b which react with selected aldehydes 7,8, 9a-c,10,11at a next step to give our targeted products. The other method is the direct reaction of the solutions of  5a or 5b  in the presence of the previous reagents except  chloroacetic acid   under reflux  with  series of aldehydes yielding the new derivatives7a, 7b, 8a, 8b, 9d, 9e, 9f, 9g, 9h,9i, 10a,10b,11a and11b.  The chemical structure of these compounds was confirmed by various spectroscopic analysis. In vitro cytotoxic activity was investigated for all compounds against HCT-116, HepG2, and MCF-7 cancer cell lines. Two compounds were potent against all cell lines, 8a with IC50 4.7, 5.6, and 6.2 μM and9hwith IC50 5.4, 7.8, and 5.6 against HCT-116, HepG2, and MCF-7 respectively. Molecular docking against inosine monophosphate dehydrogenase2 showed that compound 8a had the top ranked free energy of binding ΔG -8.68 (kcal/mol) and RMSD 1.2 Å.