With the structure of 9 in hand, further optimization attempts focused towards engaging an attractive hydrophobic groove identified between His149 and Thr93 (named HT channel) (Figure ). It was hypothesized that this new channel could be readily accessible by further modifications of the 4fluorobenzylamino group, particularly meta-substitutions. We therefore next synthesised compounds 11, 12, 13, 14 by introducing methyl, ethyl or allyl group at position 3 of the benzylamine to form favourable hydrophobic interactions with the HT channel. Indeed, ligand 13, bearing an allyl group, yielded the highest binding affinity (ITC KD = 380 nM, ΔH = -7.1 kcal/mol, Table and Supplementary Figure ). To reveal the ligand binding modes, we solved crystal structures of SBC soaked with 11, 12 and 13. Surprisingly, upon inspection of the structures we realized that the electron density for the allyl group of 13 was largely missing in the bound ligand. We postulated that this observation could be a result of steric clash forcing an unnatural binding mode, potentially resulting from pre-existing crystal contact constraints of apo SBC crystals preventing the soaked ligand to adopt its preferred binding mode.