• A novel bis(3-amino-1 H -pyrazolo[3,4- b ]pyridine) is prepared in a good yield. • A regioselective synthesis of new series of piperazine-linked bis(pyrimidines) hybrids is performed. • The new structures were elucidated by elemental and spectral analyses. • DFT and Fukui indices calculations are used to provide additional evidence for the proposed structures. • MM2, and MMFF94 minimization are used to investigate the plausible formation mechanism of the bis(pyrimidines) . Bis(enaminone), linked to benzofuran units via piperazine spacer, was prepared and used as a key intermediate in this study. It reacted for 8 h at reflux with two equivalents of 2-cyanothioacetamide in dioxane in the presence of TEA to give the corresponding bis(pyridine-2(1 H )-thione) derivative. The previous compound was reacted with two equivalents of iodomethane in methanolic sodium methoxide solution at reflux for 1 h to yield the corresponding bis(2-methylthiopyridine) derivative. Boiling of the prior compound with hydrazine hydrate in pyridine at reflux for 12 h gave the precursor bis(3-amino-1 H -pyrazolo[3,4- b ]pyridine) derivative in a good yield. Next, a regioselective protocol was conducted for the synthesis of a diverse series of the target bis(pyrido[2′,3′:3,4]pyrazolo[1,5- a ]pyrimidine) derivatives in good to excellent yields. The protocol involved the reaction of bis(3-amino-1 H -pyrazolo[3,4- b ]pyridine) with two equivalents of prop-2-en-1-ones in DMF in the presence of two equivalents of barium hydroxide at reflux for 5–7 h. To gain a better understanding of the regioselectivity observed in these reactions, DFT calculations were performed using B3LYP method and 6-31+G(d,p) basis set. The favored mechanism and the experimental regioselectivity of this reaction were explained by calculations of activation energy, natural atomic charge, Fukui indexes derived from density functional theory, MM2, and MMFF94 calculations. The current study demonstrated that using the previous computational tools, the regioselectivity of the applied reaction protocol could be correctly predicted.