Novel tropane-based irreversible ligands for the dopamine transporter

3 alpha-(Diphenylmethoxy)tropane (benztropine) and its analogues are tropan e ring-containing dopamine uptake inhibitors that display binding and behav ioral profiles that are distinct from cocaine. We previously prepared a ben ztropine-based photoaffinity label [I-125]-(N-[4-(4'-azido-3'-iodophenyl)bu tyl]-3 alpha-[bis(4'-fluorophenyl)methoxy]tropane, [I-125] 1, that covalent ly attached to the 1-2 transmembrane spanning region of the dopamine transp orter (DAT). This was in contrast to the 4-7 transmembrane spanning region labeled by a cocaine-based photoaffinity label, [I-125] 2 (RTI 82). To char acterize further these different binding domains, photoaffinity ligands tha t had the 4'-azido-3'-iodophenyl substituent extended from the same positio n on the tropane ring were desirable. Thus, identification of the optimal a lkyl linker between this substituent and the tropane nitrogen in the benztr opine series was investigated to ultimately prepare the identical N-substit uted analogue of 2. In this pursuit, the N-[4-(4'-azido-3'-iodophenyl)propy l] analogue of 3 alpha-[bis(4'-fluorophenyl)methoxy]tropane (9a) was synthe sized as well as two isothiocyanate analogues that do not require photoacti vation (10a,b) for irreversible binding. The synthesis of these target comp ounds was achieved using a modification of the strategy developed for 1. Ev aluation of these compounds for displacing [H-3]WIN 35 428 binding at DAT i n rat caudate putamen revealed that the 4'-azido-3'-iodophenylbutyl substit uent, found in 1, provided optimal binding affinity and was chosen to repla ce the N-CH3 group on 2. Both the 4'-azido-3'-iodophenyl- and the 4'-isothi ocyanatophenylbutyl analogues of 2 (25 and 26, respectively) were synthesiz ed. Both products bound to DAT with comparable potency (IC50 = 30 nM) to RT I 82 (2). In addition, compound 26 demonstrated wash-resistant displacement of [H-3]WIN 35 428 in HEK 293 cells stably transfected with hDAT. These li gands will provide important tools for further characterizing the binding d omains for tropane-based dopamine uptake inhibitors at the DAT.