The surrounding of the compound tail features several hydrophobic residues, particularly in the two T. brucei targets (Figure ). Directional interactions with the tail moiety may have limited benefit for the binding affinity in PTR1, since the flexibility of the solvent-exposed tail likely has an entropic contribution. Hydrophobic interactions are geometrically less restrained than, for instance, hydrogen bonds. Therefore, anticipating less pronounced entropic penalties on binding in our designed derivatives, we replaced the methyl ester in the tail of 1b by the more flexible ethyl and propyl in 4a and 4b, respectively. Two aspects may result in selectivity benefits from this approach: The tail region is enclosed by more hydrophobic moieties in parasite DHFR than in the hDHFR off-target (Figure ) and residues surrounding the tail have previously been demonstrated to show differing conformational variability in the crystal structures when comparing parasitic targets with the off-target. To combine exploitation of the differing pattern of hydrophobic residues in the tail environment of targets and off-targets with improved enclosure in PTR1 (Figure ). Additional hydrophobic residues in the target pocket entrance regions, like Pro99 of TbPTR1 (Figure ), can be targeted with an altered geometry in combination with methoxylations: 4f and 4g combine a one-carbon spacer between N10 and PABA and amide-linked methoxylated tail portions. In addition, an etheryl linkage to a non-substituted (4h), methoxylated (4i) or trimethoxylated (4j) benzyl group was explored. Compounds 4f-4j were collectively designed to interact with the different hydrophobic, aromatic and positively charged surrounding residues found around the tail region in the various targets (Figure ).