Solution Structures of the Prototypical 18 kDa Translocator Protein Ligand, PK 11195, Elucidated with <sup>1</sup>H/<sup>13</sup>C NMR Spectroscopy and Quantum Chemistry

Eighteen kilodalton translocator protein (TSPO) is an important target for drug discovery and for clinical molecular imaging of brain and peripheral inflammatory processes. PK 11195 [<b>1a</b>; 1-(2-chlorophenyl)-<i>N</i>-methyl-(1-methylpropyl)-3-isoquinoline carboxamide] is the major prototypical high-affinity ligand for TSPO. Elucidation of the solution structure of <b>1a</b> is of interest for understanding small-molecule ligand interactions with the lipophilic binding site of TSPO. Dynamic <sup>1</sup>H/<sup>13</sup>C NMR spectroscopy of <b>1a</b> revealed four quite stable but interconverting rotamers, due to amide bond and 2-chlorophenyl group rotation. These rotamers have been neglected in previous descriptions of the structure of <b>1a</b> and of the binding of <b>1a</b> to TSPO. Here, we used quantum chemistry at the level of B3LYP/6-311+G­(2d,p) to calculate <sup>13</sup>C and <sup>1</sup>H chemical shifts for the rotamers of <b>1a</b> and for the very weak TSPO ligand, <i>N</i>-<i>desmethyl-</i>PK 11195 (<b>1b</b>). These data, plus experimental NMR data, were then used to characterize the structures of rotamers of <b>1a</b> and <b>1b</b> in organic solution. Energy barriers for both the amide bond and 2′-chlorophenyl group rotation of <b>1a</b> were determined from dynamic <sup>1</sup>H NMR to be similar (ca.17 to 18 kcal/mol), and they compared well with those calculated at the level of B3LYP/6-31G*. Furthermore, the computed barrier for <i>Z</i> to <i>E</i> rotation is considerably lower in <b>1a</b> (18.7 kcal/mol) than in <b>1b</b> (25.4 kcal/mol). NMR (NOE) unequivocally demonstrated that the <i>E</i> rotamer of <b>1a</b> is the more stable in solution by about 0.4 kcal/mol. These detailed structural findings will aid future TSPO ligand design and support the notion that TSPO prefers to bind ligands as amide <i>E</i>-rotamers.