N-(3-Iodophenyl)trozamicol (IPHT) and Related Inhibitors of Vesicular Acetylcholine Transport: Synthesis and Preliminary Biological Characterization A. B. Khare, 1 R. B. Langason, 1 S. M. Parsons, 2 R. H. Mach 3 and S. M. N. Efange 1 1 DEPARTMENTS OF RADIOLOGY, MEDICINAL CHEMISTRY, AND NEUROSURGERY, UNIVERSITY OF MINNESOTA, MINNEAPOLIS, MINNESOTA, USA; 2 DEPARTMENT OF CHEMISTRY AND THE GRADUATE PROGRAM IN BIOCHEMISTRY AND MOLECULAR BIOLOGY, UNIVERSITY OF CALIFORNIA, SANTA BARBARA, CALIFORNIA, USA; AND 3 DEPARTMENTS OF RADIOLOGY AND PHYSIOLOGY AND PHARMACOLOGY, WAKE FOREST UNIVERSITY SCHOOL OF MEDICINE, WINSTON-SALEM, NORTH CAROLINA, USA ABSTRACT. Four isomeric N-(halophenyl)trozamicol analogues (6a– d) were synthesized and evaluated as potential vesicular acetylcholine transporter (VAChT) ligands. Of the four compounds, N-(3-bromophenyl) trozamicol (6b) and N-(3-iodophenyl)trozamicol (6d) displayed the highest affinity for the VAChT in vitro, whereas the para-substituted compound 6c showed the lowest affinity for this transporter. Tissue distribution studies of N-(3-[ 125 I]iodophenyl)trozamicol ([ 125 I]6d, [ 125 I]IPHT) suggest that the central distribution of the latter is consistent with cholinergic innervation. However, only moderate target-to-background ratios were obtained, suggesting little improvement over the N-(halobenzyl)trozamicols described previously. NUCL MED BIOL 26;6:609 – 617, 1999. © 1999 Elsevier Science Inc. All rights reserved. KEY WORDS. Vesicular acetylcholine transport, Vesamicol, Radioligands INTRODUCTION The deanol analogue trans-2-(4-phenylpiperidino)cyclohexanol (1, vesamicol, AH5183) causes respiratory distress, paralysis, and death in laboratory animals (5). Investigation of the mechanism of action of this compound has led to the identification (3) and subsequent cloning of a vesicular acetylcholine transporter (VAChT) (1, 2, 4, 19, 28, 34, 40, 52, 53). This membrane protein transports newly synthesized acetylcholine (ACh) from the cytosol into the cholin- ergic synaptic vesicle, the primary vehicle for the impulse-mediated quantal release of ACh (reviewed in refs. 31, 35, 36). The VAChT is localized exclusively in cholinergic neurons (20, 22, 42– 45, 55). Consequently, radiolabeled VAChT ligands may be used to study cholinergic function with single photon emission computed tomog- raphy (SPECT) or positron emission tomography (PET). Although vesamicol, the prototypical VAChT ligand, displays moderately high affinity for the VAChT (37), this compound also binds to -adrenoceptors (30, 54) and sigma receptors (16). The marginal selectivity of this ligand has sparked efforts to develop a new class of selective high-affinity VAChT ligands (10 –12, 17, 24 –27, 32, 33, 38, 39, 46 – 48). Previously (12), we reported that the vesamicol analogue 4-azavesamicol (trozamicol, 2) was a poor ligand for the VAChT; however, N-alkylation of trozamicol (to yield derivatives such as 3 and 4) dramatically increases affinity for this transporter (Fig. 1). In the present investigation, part of our continuing effort to develop high-affinity VAChT ligands and to map the vesamicol binding site of the VAChT, the synthesis and affinities of N-phenyltrozamicol and some of its analogues are reported. Also provided is a preliminary account of the tissue distribution of one analogue, N-(3-[ 125 I]iodophenyl)trozamicol ([ 125 I]IPHT). Although trozamicol displays little or no affinity for the VAChT, the ease of functionalization of this molecule (by derivatization at the nitrogen atom) provides ready access to a pool of compounds that can be used to probe the vesamicol binding site of the VAChT. Previously (12), we identified the N-(halobenzyl)trozamicols as a class of high-affinity VAChT ligands. meta-Iodobenzyltrozamicol (MIBT) and 4-fluorobenzyltrozamicol (FBT), two compounds from this class, have been studied extensively in rodents and primates as potential radioligands for mapping cholinergic innervation in vivo (6, 9, 13–15, 18, 29, 49). Although these compounds have provided valuable insights into various aspects of cholinergic function, their ability to provide specific information regarding the topography of the vesamicol binding site of the VAChT (such as the ligand conformation required for optimum binding) is limited by the flexibility of the benzyl fragment. In addition, both MIBT and FBT display undesirable levels of nonspecific binding in the cortex and cerebellum in vivo, thus limiting their utility as imaging agents. In our continuing effort to map the vesamicol binding site of the VAChT and to develop even better radiotracers for studying the cholinergic system in vivo, we therefore developed the N-phenyl- trozamicols as a class of conformationally restricted VAChT li- gands. The target compounds were expected to display affinity for VAChT because 5 exhibits moderate affinity for this transporter (38). EXPERIMENTAL Materials Synthetic intermediates were purchased from Aldrich Chemical (Milwaukee, WI), and were used as received. Tetrahydrofuran Address correspondence to: S. Mbua Ngale Efange, Ph.D., Department of Radiology (Mayo Box 292), University of Minnesota Academic Health Center, 420 Delaware St. SE, Minneapolis, MN 55455, USA; e-mail: efang001@maroon.tc.umn.edu Received 4 October 1998. Accepted 10 February 1999. Nuclear Medicine & Biology, Vol. 26, pp. 609 – 617, 1999 ISSN 0969-8051/99/$–see front matter Copyright © 1999 Elsevier Science Inc. All rights reserved. PII S0969-8051(99)00013-X