Four additional compounds (5c-5f, Figure ) were prioritized based on the physicochemical marker properties. Compound 5c combines fragments of ethyl modification to N10 of 2a and the tail ethyl ester of 4a. Due mainly to the tail ester, this modification improves the inhibition for both TbPTR1 (IC50 1 nM) and LmPTR1 (IC50 0.1 μM). The activity against TbDHFR is similar to that of the N10-modified parent 2a, whereas LmDHFR and hDHFR inhibition are again influenced by the tail modification (IC50 LmDHFR 5c: 0.2 μM, 4a: 0.1 μM; hDHFR 5c: 13 μM, 4a: 12 μM). Compound 5d merges the ethyl N10 fragment of 2a with the unsubstituted benzene of 4d. In TbPTR1, this boosts the nanomolar IC50 of 4d to the subnanomolar range, while the activity towards LmPTR1 remains similar to 4d. This profile can be related to the N10 ethyl, which seems disfavored in LmPTR1 as judged by the modest inhibition of the parent 2a (IC50 13.3 μM).