Chagas disease, caused by Trypanosoma cruzi , affects millions of people worldwide and remains inadequately treated because the currently available drugs, benznidazole (Bzn) and nifurtimox (Nfx), show variable efficacy and significant toxicity across different disease stages. Given that purine nucleoside analogues have demonstrated antitrypanosomal activity and may target essential parasite proteins such as cruzipain ( Tc CZP) and bromodomain factor 2 ( Tc BDF2), this study aimed to determine whether a set of 20 purine derivatives possessed antiparasitic properties in relation to these targets using ex vivo , in vitro and in silico approaches. The results indicated that, in a screening assay against circulating trypomastigotes from two Mexican strains of T. cruzi (NINOA and INC-5), most of these purines outperformed the reference drugs Nfx and Bzn, according to the half-maximal lethal concentration (LC ₅₀ ) values (which ranged from 6.28 to 144 µM for the NINOA strain and from 18.42 to 166.87 µM for the INC-5 strain), with the most active purines being the most active purines 9e , 9i , 9j , 9m , 9n , and 9t . Structure-activity relationship (SAR) analyses explained the influence of some fragments in the purine scaffold related to the trypanocidal effect. Subsequently, selected purines were tested against intracellular Tulahuen-2 amastigotes, where four demonstrated activities at sub-10 µM concentrations, and 9m achieved approximately 1.5-fold greater potency than Bzn. Due to the low inhibitory activity of the purines tested against Tc CZP (the best compound, 9j , had an IC₅₀ of 87.1 µM), this target was ruled out as the source of the trypanocidal activity. However, when the compounds were evaluated on Tc BDF2, the results showed that 9i and 9t were the two best compounds with binding capacities to Tc BDF2 (K _d = 13.5 and 19.6 µM, respectively), which was more closely aligned with trypanocidal activity. In addition, 9i and 9t did not bind to Tc BDF3, indicating selectivity among these targets. Molecular docking and 200 ns molecular dynamics simulations supported the stable binding of purine 9i to the Tc BDF2 bromodomain pocket. In addition, purines 9i , 9j , 9m , and 9t were predicted to have good pharmacokinetic profiles for oral administration of the drug. Overall, trisubstituted purines emerged as promising antichagasic leads, with Tc BDF2 engagement appearing more consistent with the observed trypanocidal activity, thereby guiding future optimisation towards a balance of potency, selectivity, and cytotoxicity.