Chagas’ disease is an infection caused by the protozoan Trypanosoma cruzi that represents a major public health threat in Latin America. The development of new drugs against T. cruzi is still required since the only two drugs (nifurtimox and benznidazole) currently used cause severe side effects. Previously we developed 4-carbohydrazide derivatives of 1H-pyrazolo[3,4-b]pyridine as antichagasic agents, which the hit compound was the N’-4-hydroxybenzylidene-carbohydrazide derivative. In order to verify the influence of the substituent position and the carbohydrazide moiety replacement for 1,3,4-oxadiazoline moiety, herein we described the synthesis and in vitro evaluation of trypanocidal activity and cytotoxicity of eleven new 1,6-diphenyl-4-(substituted)-1H-pyrazolo[3,4-b]pyridine derivatives. New N’-hydroxybenzylidene-carbohydrazide derivatives were synthesized by reaction of 4-carbohydrazide-1,6-diphenyl-3-methyl-1H-pyrazolo[3,4-b]pyridine with the corresponding substituted benzaldehyde (2-OH; 3-OH; 3,4-OH; 3-OH-4-OCH₃). The 1,3,4-oxadiazoline derivatives were synthetized from the corresponding hydroxybenzylidene-carbohydrazide compounds by treatment with refluxing acetic anhydride. The new derivatives containing the 1,3,4-oxadiazoline moiety have the hydroxyl group replaced by the acetyl group as a protective group for the hydroxyl function. Therefore, an additional deprotection step was carried out in order to obtain two novel hydroxylated 1,3,4-oxadiazoline derivatives from 3-OAc, 4-OAc derivatives. All the new 1,6-diphenyl-3-methyl-4-(substituted)-1H-pyrazolo[3,4-b]pyridine derivatives were obtained with yields ranging from 70 to 95% and had their structures elucidated by spectroscopic methods. These compounds were evaluated in vitro against intracellular amastigote form of T. cruzi, using the benznidazole drug as the positive control and had their cytotoxicity profiles determined on LLCMK₂ mammalian cells. All derivatives tested have shown some trypanocidal activity. The N’-2-hydroxybenzylidene-carbohydrazide and 2-(N’-acetyl-1,3,4-oxadiazolin-2-yl)-phenyl acetate derivatives showed the most significant antichagasic activity (IC₅₀= 0.85 and 1.11 μM, respectively) in comparison to benznidazole drug (IC₅₀= 3.98 μM) in addition to low cytotoxicity. These results suggest that the 2-substituted position of phenyl group connected to the carbohydrazide or oxadiazoline moiety play an important role in antichagasic activity of 1,6-diphenyl-4-(substituted)-1H-pyrazolo[3,4-b]pyridines compounds. Furthermore, our results indicate bioisosteric replacement of carbohydrazide moiety by 1,3,4-oxadiazoline ring.