There is certainly evidence that the polycyclic tryptamine psychedelic ibogaine acts in a similar manner at the serotonin transporter, stabilizing the inward cytoplasmic-facing state of the transporter (Jacobs behavioral reinforcing liabilityof a DAT inhibitor. Supplementary Material 01Click here to view.(129K, pdf) Acknowledgements This work was supported by NIH grant DA013261.. possessing the diphenylmethoxy moiety of benztropine and GBR12909 were dissimilar to cocaine-like compounds. In tests with specific isomers of cocaine and tropane analogues, compounds with 3 stereochemistry tended to exhibit benztropine-like binding, whereas those with 3 stereochemistry were more cocaine-like. Our results point to the importance of specific molecular featuresmost notably the presence of a diphenylmethoxy moietyin determining a compounds binding profile. This study furthers the concept of using DAT mutants to differentiate cocaine-like inhibitors from atypical inhibitors does not appear to dictate the reinforcing effects of a given stimulant compound, despite the clear case for DAT involvement in stimulant action. DAT inhibiting compounds may have dramatic, mild or even a complete lack of behaviorally rewarding effects. The disparate psychostimulant and reinforcing effects of various DAT inhibitors may be due to differential molecular interactions with the DAT. That T-3775440 hydrochloride is, inhibitors may interact with different binding sites on the DAT or induce distinct conformational changes in the DAT upon binding. These hypotheses are not mutually exclusive; one can envisionand might even anticipatethat two structurally unique ligands with at least partially divergent binding sites would differentially alter target protein conformation. Experimentally, this idea is supported by the finding that cocaine and benztropine differentially affect the vulnerability of extracellular-facing DAT cysteine residues towards reaction with impermeant sulfhydryl reducing reagents, indicating that these inhibitors trigger different conformational shifts upon binding (Reith molecular dynamics simulation to dock small-molecule inhibitors into a threaded DAT model, Beuming and colleagues recently mapped the binding site of CFT and cocaine to the DAT substrate pocket (Beuming, 2008). However, co-crystallization of LeuTAa with a bound transport inhibitorthe tricyclic antidepressant (TCA) desipraminerevealed a distal binding site located above the substrate-binding pocket, near the extracellular vestibule (Zhou (2001). [3H]CFT binding inhibition assays Transfected HEK293 cell suspensions were prepared according to the method outlined previously (Chen and the aqueous phase was extracted with dichloromethane (DCM; 215 mL). Combined organic phases were dried over Na2SO4 and concentrated and purified by column chromatography (DCM:EtOAc, 100:1) to give the product to yield D-247. It was purified by column chromatography (DCM:MeOH, 9:1) to give pure D-247 (175 mg, 55%). The free base was converted to the oxalate salt, mp 215 C 217C; 1H-NMR (CDCl3; 400 MHz): 2.28 (s, 3H), 2.44C2.55 (m, 8H), 2.69C2.70 (t, 2H), 3.58C3.61 (t, 2H), 5.37 (s, 1H), 7.21-7.35 (m, 10H). Anal. [(C20H26N2O?2(COOH)2] C, H, N. Synthesis of Mouse monoclonal antibody to MECT1 / Torc1 1-(2-(benzhydryloxy)ethyl)piperazine (D-248) Compound 5 (0.25 g, 1 mmol), piperazine (0.345 g, 4 mmol), anhyd. K2CO3 (0.276 g, 2.0 mmol) and potassium iodide (cat.) were refluxed in acetonitrile (20 mL) overnight. The solvent was removed to yield D-248. Purification by column chromatography (EtOAc:MeOH:triethylamine, 4:2:0.5) yielded pure D-248 (145 mg, 48%). The freebase was converted to the HCl salt, mp 114-116C; 1H-NMR (CDCl3, 400 MHz): 2.50 (brs, 4H), 2.63C2.66 (t, 2H), 2.86C 2.89 (t, 4H), 3.56C3.59 (t, 2H), 3.84C3.92 (brs, 1H), 5.35 (s, 1H), 7.20C7.33 (m, T-3775440 hydrochloride 10H). Anal. [(C19H24N2O?2HCl?H2O)] C, H, N. Synthesis of 3-(benzhydryloxy)-8-(3-phenylpropyl)-8-azabicyclo[3.2.1]-octane (D-249) Tropine (0.5 g, 3.54 mmol) was heated with stirring to 160C with benzhydryl chloride (0.7 mL, 3.89 mmol) for 1 hr. The resulting residue was allowed to cool and dissolved in DCM and then evaporated to obtain an oily residue, which was then stirred with ether to yield creamy colored solid, benztropine HCl (0.8 g, 65%). Freebase was liberated from this salt using 2 M Na2CO3 solution. The aqueous layer was extracted with EtOAc (210 mL). Combined organic layers were dried T-3775440 hydrochloride (Na2SO4) and the solvent evaporated, yielding pale yellow oil. Benztropine freebase (90.5 g, 1.626 mmol) was then dissolved in 1,2-dichloroethane (10 mL). Anhydrous Na2CO3 (0.69 g, 6.5 mmol) and ACE-Cl (0.71 mL, 6.5 mmol) were added and the mixture was refluxed for 3 hrs, filtered and the filtrate was dissolved in MeOH (10 mL) and allowed to stir overnight. The solvent was evaporated and the oily residue was dissolved in DCM and washed with 20% ammonia solution (220 mL). The T-3775440 hydrochloride organic layer was washed with water and dried. The solvent was to yield 7 as brown oil (0.29 g, 60%). Compound 7 (1 mmol), 3-bromo-1-phenylpropane (0.17 mL, 1.1 mmol), Na2CO3 (0.211 g, 2 mmol) and potassium iodide (cat.) were stirred in DMF (15 mL) at 60C overnight. The solvent was evaporated under reduced pressure; the residue was dissolved in DCM, washed with water, dried and evaporated 0.05 was used a threshold for significance in all statistical analyses. Results Apparent Affinity of DAT Inhibitors: SAR From Cocaine to Benztropine The or conformation..