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  • Analysis of Table shows that compound b in which the

    2019-11-19

    Analysis of Table 1 shows that compound 7b, in which the biphenyl scaffold does not bear any substituent, displays its inhibitory potency in the micromolar range (IC50 = 11 μM). If the amide bridge is moved from position 1 to 3 of the biphenyl system, product 7c is yielded, whose potency is about 4 times lower. Adding a CF3 group (which is present in A771726) to 7b, at either position 4′ (compound 7d) or position 3′ (compound 7e) of the biphenyl moiety, again decreases the potency, approximately by one quarter and by one half, respectively. Conversely, considerable modulation in the inhibitory activity occurs when fluoro substitution is performed on the biphenyl system. Fluorine is a lipophilic substituent of small size, endowed with high electronegativity; its presence may affect the binding affinity of a compound to its target protein in many ways [25], [26]. The introduction of two or four fluorine atoms on the phenyl ring adjacent to the oxadiazole enhances the inhibitory potency of compounds 7f and 7g about 12-fold and 166-fold that of the parent structure. In both 7f and 7g, the introduction of a CF3 group at position 4 or 3 of the second phenyl ring (products 7h, 7i, 7l, 7m) decreases the inhibitory potency which, nevertheless, remains higher than that of 7b. By contrast, the presence of a trifluoromethoxy group at position 3′ (7n) affords the most active compound of the series, whose potency is comparable to that of BQN. In an attempt to rationalize the activity profile across the series, a molecular modeling study was carried out. A conformational search using an implicit solvent model was accomplished for each of the newly synthesized compounds; this was followed by refinement of the geometry of local minima through a quantum-mechanical (QM) method. Subsequently, flexible docking of the compounds was performed in three different crystallographic structures recovered from the Protein Data Bank (see Experimental). The complexes of the rat enzyme with BQN (PDB ID 1UUO), of the human enzyme with a BQN analog (PDB ID 1D3G), and finally of human DHODH with a biphenylaminocarbonyl derivative of cyclopentenecarboxylic GK921 (PDB ID 2BXV), were chosen. The rat isoform was taken into account to explore whether small inter-species differences may play a role, while the two human complexes were selected in order to ascertain whether differences in the conformation of Arg136 and Gln47 side chains might affect the binding mode, as observed by Baumgartner and co-workers [23]. Our docking protocol was able to accurately reproduce the BQN experimental pose in 1UUO and 1D3G (RMSD 0.52 and 0.40 Å, respectively). Moving to derivatives 7a–n, no major differences were observed among the poses obtained on 1UUO and 1D3G; in both cases the compounds were found to bind in a BQN-like fashion, namely with the deprotonated hydroxyfurazan moiety facing Arg136, thus effectively mimicking the carboxyl group of BQN and related compounds (Fig. 4a). While on 1D3G and 1UUO the hydroxyfurazan was in no case found to interact with Tyr356 in a leflunomide-like fashion, a significant fraction of such poses was found when 2BXV was used as the docking target (Fig. 4b). In spite of the good qualitative agreement, the scoring function implemented in AutoDock (see Experimental) was not able to reproduce the experimentally observed differences in inhibitory potency across compounds 7a–n. However, when the binding pose of the furazan derivatives in 1D3G was compared with the QM-refined global minimum obtained from the implicit solvent conformational search, we found excellent agreement for the derivatives bearing fluorine atoms on the biphenyl system, especially with regard to the dihedral angle between the benzene ring and the amide group (107.1° in the docking pose, 113.6° in the global minimum in solution for 7n, Fig. 5a). Conversely, for compounds lacking aromatic fluorine atoms, in the putative bioactive conformation the two moieties are tilted by about 100° (106.1° for 7d, Fig. 5b), while in solution the benzene ring lies in the same plane as the amide bond. Since such a conformation would not fit into the ubiquinone site due to steric hindrance, the non-fluorinated compounds necessarily suffer a conformational strain penalty to assume a pose compatible with the constraints imposed by the geometry of the enzyme cavity. The same considerations also apply to the leflunomide-like poses found on 2BXV, with similar dihedral angles between the benzene ring and the amide group, the latter lying on the opposite side of the benzene ring compared to brequinar-like poses. This suggests that aromatic fluorine atoms not only optimize interactions with the hydrophobic amino acids lining the active site (in particular, Ala59 and leucines 46, 58, 359), but also stabilize the putative bioactive pose, thus enhancing inhibition. For compound 7n, the trifluoromethoxy group protrudes out of the distal benzene ring plane, establishing additional contacts with Tyr38 (Fig. 4), which probably further contribute to making it the most active compound of the series.