and for cytotoxicity on L6 cells derived from rat skeletal myoblasts (ATCC CRL-1458) (Table ). Inhibition of the growth of T. cruzi amastigotes by the compounds was evaluated by determining the inhibitor concentration required for inhibiting the growth of the parasite by 50% (IC50 values). Meanwhile, the synthesized 3-benzylmenadione derivatives and heterocyclic analogues were also tested for cytotoxicity (CC50 values) using rat L6 cells. In the assays. The standard drug against Chagas disease, benznidazole was also tested as reference displaying an IC50 value of 2.25 µM. Compound 4a was modified by depletion, addition, or substitution of chemical functions on several key positions of its scaffold. Therefore, a first investigation on the importance of the methylene group between the 4'-tert-butylbenzylic ring Bn2 and the piperazine was conducted to reveal the same central motif present in posaconazole (blue). When removed, the compound 21e lost all of its anti-T. cruzi activity. In a similar manner, the menadione core was deleted (26) showing a loss of activity as well. Next, the piperazine moiety was evaluated. The previously synthesized monoamines 27a-b (Figure ) showed to have no inhibition effect on T. cruzi. Moreover, when the ring size of the diazacycle was increased, the IC50 value also slightly increased compared to 4a. Whereas the diazepane moiety (15f) appeared to be nonselective, its spirocyclic analogues 15aa-ab, despite its lower activity, presented a greater selectivity. Spirocyclic derivatives can be very interesting because of their ability to modify certain physico-chemical and pharmacokinetics properties such as an improved solubility or even an increased metabolic stability compared to their single ring analog. The 6-fluoro (4b)