(N)-Methanocarba-adenosines as monoamine transporter allosteric inhibitors: Extended N6 groups for bitopic stabilization

A human dopamine transporter (DAT) cryo-EM structure was recently reported, as stabilized by an allosteric inhibitor, i.e. rigid nucleoside MRS7292 1, a tropane orthosteric inhibitor and Zn²⁺. We have synthesized multiple North (N)-methanocarba-adenosine analogues of 1, with N⁶, C2 and 4' modifications to examine their effects on DAT radioligand binding (either enhancement or inhibition) and at the norepinephrine (NET) and serotonin (SERT) transporters (generally inhibition). Small N⁶ groups provided the most pronounced DAT enhancement (≥500 % at 10 μM for N⁶-methyl 2 and N⁶-cyclopropyl 8 5'-ethyl esters, and N⁶-methyl-4'-cyanomethyl 44 analogues). Various N⁶-(ω-phenyl-alkyl) groups caused binding inhibition and compensated for the removal of stabilizing interactions shown in the 1-DAT structure, e.g. an N7 H-bond, by accessing a previously uncharacterized DAT distal binding region. The optimal chain length was five methylenes for bitopic N⁶-(ω-phenyl-alkyl) derivatives, e.g. 16 and 31, having favored 5'-ethyl ester (but not 4'-cyanomethyl) and 2-(arylethynyl) groups. Surprisingly, the previously noted reduced DAT activity with a 2-iodo group, could be compensated by N⁶-(ω-phenyl-alkyl) groups. N⁶-(6-Phenylhexyl)-2-iodo compounds, 53 (5'-hydroxy, K_i 0.89 μM, pan-inhibitor) and 54 (4'-cyanomethyl, K_i 0.40 μM), inhibited DAT binding. Chiral, branched N⁶ groups displayed binding stereoselectivity. Key nucleosides were docked to outward-facing hDAT cryo-EM structures. Molecular dynamics simulation predicted π-π interactions of the N⁶-(ω-phenyl-alkyl) substituent and aromatic side chains of F208^EL2 and H375^EL4 (aromatic conserved at NET) to define a preferred binding mode for the extended chain. Thus, we characterized the transporter SAR of this series, either enhancement or inhibition of orthosteric radioligand binding, and transport inhibition, and with DAT structural predictions.