Synthesis and pharmacological evaluation of neurosteroid photoaffinity ligands.

Neuroactive steroids are potent positive allosteric modulators of GABA receptors (GABAR), but the locations of their GABAR binding sites remain poorly defined. To discover these sites, we synthesized two photoreactive analogs of alphaxalone, an anesthetic neurosteroid targeting GABAR, 11β-(4-azido-2,3,5,6-tetrafluorobenzoyloxy)allopregnanolone, (F4N3Bzoxy-AP) and 11-aziallopregnanolone (11-AziAP). Both photoprobes acted with equal or higher potency than alphaxalone as general anesthetics and potentiators of GABAR responses, left-shifting the GABA concentration - response curve for human α1β3γ2 GABARs expressed in Xenopus oocytes, and enhancing [H]muscimol binding to α1β3γ2 GABARs expressed in HEK293 cells.