Radioiodinated Azide and Isothiocyanate Derivatives of Cocaine for Irreversible Labeling of Dopamine Transporters: Synthesis and Covalent Binding Studies John R. Lever, †, * Mu-Fa Zou, ‡ M. Laura Parnas, § Romain A. Duval, † Sara E. Wirtz, | Joseph B. Justice, | Roxanne A. Vaughan, § and Amy Hauck Newman ‡ Departments of Radiology, and Medical Pharmacology and Physiology, University of MissourisColumbia, Columbia, Missouri 65212 and Research Service, Harry S. Truman Veterans Administration Medical Center, Columbia, Missouri 65201, Medicinal Chemistry Section, National Institute on Drug Abuse, Intramural Research Program, Baltimore, Maryland 21224, Department of Biochemistry and Molecular Biology, University of North Dakota, Grand Forks, North Dakota 58203, and Department of Chemistry, Emory University, Atlanta, Georgia 30322. Received November 19, 2004; Revised Manuscript Received February 15, 2005 Two novel N-substituted-3-phenyltropane alkaloids have been labeled with iodine-125 for use as irreversible probes of dopamine transporter (DAT) binding sites. One contains an iodoaryl azide moiety for photolabeling, while the other bears an iodoaryl isothiocyanate for direct conjugation. Both radioligands were prepared in a one-flask procedure by electrophilic radioiodination of the corre- sponding aniline under no-carrier-added conditions, followed either by diazotization and treatment with sodium azide, or by addition of thiophosgene under basic conditions. Specifically, (-)-N-[4-(3- [ 125 I]iodo-4-azidophenyl)butyl]-2-carbomethoxy-3-(4-chlorophenyl)tropane ([ 125 I]MFZ-2-24) and (-)- N-[4-(3-[ 125 I]iodo-4-isothiocyanophenyl)butyl]-2-carbomethoxy-3-(4-chlorophenyl)tropane ([ 125 I]MFZ 3-37) were synthesized. Isolation by reversed-phase HPLC and solid-phase extraction gave good average yields of [ 125 I]MFZ-2-24 (67%, n ) 5) and [ 125 I]MFZ-3-37 (45%, n ) 3) with high radiochemical purities (96-99%) and specific radioactivities (>2000 mCi/µmol). The utility of the radioligands was demonstrated by their covalent linkage to rat striatal membranes, and immunoprecipitation of a single radiolabeled band at 80 kDa corresponding to the full-length DAT. INTRODUCTION The dopamine transporter (DAT) 1 is an 80 kDa phos- phoprotein, thought to have 12 transmembrane domains (TMs), that is regulated by protein kinases, controls synaptic dopamine concentrations, and plays a pivotal role in locomotion (1, 2). Inhibition of dopamine uptake also is recognized as a critical determinant of the reinforcing properties and abuse liability of cocaine and other stimulants (3, 4). Thus, the identification of thera- peutic DAT inhibitors that block the actions of stimulant drugs of abuse with minimal disruption of normal dopaminergic function is a topic of current interest (5, 6). In fact, dopamine, cocaine, amphetamine, and other structural classes of DAT ligands bind to recognition sites that seem to be at least partially discrete based upon functional, chimera, and mutagenesis studies. Accordingly, we have been developing irreversible ligands to assess and differentiate the features of DAT binding domains at the molecular level. These ligands include several radioiodinated photoaffinity probes: [ 125 I]R- TI-82 (7, 8), a tropane alkaloid congener of cocaine; [ 125 I]- DEEP (7,9) and [ 125 I]AD-96-129 (10) which are based upon GBR12,909; and [ 125 I]GA-2-34 (11), a benztropine with key pharmacophores drawn from both of the other structural classes (Figure 1). Studies to date confirm that distinct regions of the DAT primary sequence are labeled covalently depending upon ligand framework. In brief, [ 125 I]RTI-82 becomes incorporated specifically in TMs 4-6, [ 125 I]DEEP and [ 125 I]GA-2-34 preferentially attach to TMs 1-2, and [ 125 I]AD-96-129 labels TMs 1-2 and 4-6 to about the same extent (12, 13). The data for [ 125 I]AD- 96-129 suggests that TMs 1-2 and 4-6 should be close together in the three-dimensional structure of the DAT and, by comparison to the results from [ 125 I]DEEP, demonstrates the sensitivity of DAT binding to modest structural modifications (13). Interestingly, [ 125 I]GA-2-34 and [ 125 I]RTI-82 attach covalently to different TMs even though they both have a tropane ring system (12). This finding is consistent with work showing that benztropines exhibit potent DAT binding but do not display a behavioral profile like that * Address correspondence to John R. Lever, Ph.D., Depart- ment of Radiology, M292 Medical Sciences Bldg., University of MissourisColumbia, One Hospital Dr., Columbia, MO 65212. Phone: 573-814-6000 ext. 3686. FAX: 573-814-6551. E-mail: leverj@health.missouri.edu. † University of MissourisColumbia and Harry S. Truman Veterans Administration Medical Center. ‡ National Institute on Drug Abuse. § University of North Dakota. | Emory University. 1 Abbreviations: AD-96-129, 4-[2-(diphenylmethoxy)ethyl]-1- [(4-azido-3-iodophenyl)methyl]piperidine; DAT, dopamine trans- porter; DEEP, 1-[2-(diphenylmethoxy)ethyl]-4-[2-(4-azido-3- iodophenyl)ethyl]piperazine; DTT, dithiothreitol; GA-2-34, N-[n-butyl-4-(4-azido-3-iodophenyl)]-4,4-difluoro-3-(diphenyl- methoxy)tropane; GBR 12 ,909, 1-{2-[bis(4-fluorophenyl)methoxy]- ethyl}-4-(3-phenylpropyl)piperazine; HEK, human embryonic kidney; IP, immunoprecipitated; MFZ-2-24, (-)-N-[4-(3-iodo-4- azidophenyl)butyl]-2-carbomethoxy-3-(4-chlorophenyl)tro- pane; MFZ 3-37, (-)-N-[4-(3-iodo-4-isothiocyanophenyl)butyl]- 2-carbomethoxy-3-(4-chlorophenyl)tropane; RTI-82, 3-(4- chlorophenyl)tropane-2-carboxylic acid, 4-azido-3-iodophenylethyl ester; TM, transmembrane domain; WIN 35, 428, (-)-2-car- bomethoxy-3-(4-fluorophenyl)tropane. 644 Bioconjugate Chem. 2005, 16, 644-649 10.1021/bc0497214 CCC: $30.25 © 2005 American Chemical Society Published on Web 04/15/2005 Downloaded by COUPERIN CONSORTIUM FRANCE on July 9, 2009 Published on April 15, 2005 on http://pubs.acs.org | doi: 10.1021/bc0497214