In the next step, we aimed at investigating hit series 21 in more detail. The goal was to explore different heterocycles instead of the pyrazole ring of 21 and the introduction of functional groups at the hydrophobic tail to establish interactions with the catalytic His residues (Table ). Since such compounds were not commercially available at the time, they were synthesised in-house. This was achieved by coupling of heteroaryl carboxylic acids with either isopropyl amine to give amides 116-118, or with amino alkyl esters to give esters 119-121 (Scheme 1). Hydrolysis or aminolysis of the esters prepared gave carboxylic acid analogues 122-125 and primary amides 126 and 127, respectively. The full synthesis protocols are given in the supporting information. For three ligands, only the heterocycle was varied compared to the hit compound (116-118, Table and Table ). For the pyrroles 116 and 117 no binding could be detected using either BLI or X-ray crystallography. In contrast, the crystal structure in complex with the imidazole analogue 118 could be determined (Figure ) and BLI gave a KD of 480 µM which translates to a ligand efficiency (LE) of 0.39 kcal/mol/heavy atom. This compound adopts the same binding mode as the parent hit 21 (Figure and Figure ). The lack of binding of the investigated pyrrole suggests that the N1 nitrogen atoms in the heterocycles of 21 and 118 act as hydrogen bond acceptors. Next, we investigated compounds that had a functional group attached to the hydrophobic linker. The two catalytic histidines of FabF are known to bind carbonyl containing compounds such as the carboxylic acid groups found in platensimycin and platencin (Figure ) or in several of our fragment hits (20, Figure ; 56, Figure ; 113, Figure ), thioester groups found in thiolactomycin, and amide groups found in cerulenin (Figure ). Accordingly, we sought to attach similar functional groups to our fragment screening hit 21. At the same time, we varied the length of the carbon linker atoms to probe the ideal chain length (compounds 119-127 in Table ).