J. Med. Chem. 1986,29, 1663-1668
in position two, may perturb the geometry of the ethylamine fragment and realign the orientation of the Leu5side chain differently from 1-c once the phenolic and phenyl rings bind to their respective sites affording a conformer with enhanced I*-receptorpreference. In accordance with this hypothesis is the observation that the D-Leuj diastereomer of 1-d has considerably reduced selectivity (by a factor of 4 based on the ICboratio between the MVD and GPI assays).16 Conclusion In the series of 7cu-(l-hydroxy-l-methylphenalkyl)oripavines maximal analgesic activity has been reported to be associated with a 19-phenylpropyl chain as in PEO. The fact that analgesic potency is drastically reduced by shortening or lengthening the phenylalkyl chain implicates an additional well-defined binding site. We found that the conformation reported for the cyclic opioid peptide Hm - c y c l o[-~-N~-Om-Gly-Phe-Leu-] provides the possibility of topographical congruency between the CZlaromatic ring in PEO and that of the phenylalanyl side chain of the peptide. Accordingly, the two may interact with a common lipophilic receptor site. Low-energy peptide conformers were obtained by a bimolecular energy minimization procedure with PEO, and it was found that the ethylamine fragment of the tyramine moiety in the former assumes a different conformation than its counterpart in the latter such that the basic nitrogens occupy distinct spatial loci approximately 1.1A apart. The variation in receptor selectivity among the members of the homologous series shown in Figure 1 may arise from subtle differences in
1663
backbone conformation which governs the direction of the leucine side chain and the geometrical orientation of the ethylamine fragment once the phenolic and phenylalanyl rings are bound to the receptor. Previous conformational comparisons of flexible opioid peptides with rigid opiates resulted in intermediate- to high-energy conformers for those peptides having maximum resemblence to rigid alkaloids.36 Since the rigidity of the cyclic opioid derivative 1-c precludes any major backbone rearrangement, we found that an alternate approach is to anchor the minimal common element (the phenolic group) and attach the basic nitrogens and the secondary aromatic rings of the respective molecules by “spring constants” followed by the relaxation. Since the procedure balances mutual spatial orientations of pharmacophoric elements, it may be regarded analogous to an induced fit.
Acknowledgment. We are indebted to the financial assistance of the Natural Sciences and Engineering Research Council of Canada (operating Grant A-1529) to J.D. while at the University of Sherbrooke and to A.M. (operating Grant A-0329). We also acknowledge Isabelle Lepage for typing the manuscript. Registry No. IC, 83159-95-9; 2, 14521-98-3. (36) Loew, G. H.; Burt, S.K. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 7. (37) Hudson, D.; Sharpe, R.; Szelke, M. Znt. J. Peptide Protein Res. 1980, 15, 122.
In Vitro Labeling of Serotonin-S2Receptors: Synthesis and Binding Characteristics of [3H]-7-Aminoketanserin Walter Wouters,*+Cornelus G. M. Janssen,* Jacky Van Dun,t Jos B. A. Thijssen,* and Pierre M. Laduront Department of Biochemical Pharmacology and Department of Drug Metabolism & Pharmacokinetics, Janssen Pharmaceutica, B-2340 Beerse, Belgium. Received November 25, 1985 [3H]-7-Aminoketanserin (7-amino-3-[2- [4-(2-tritio-4-fluoro6enzoyl)-l-piperidinyl]ethyl]-2,4(lN,3H)-quinazolinedione), an amino derivative of the selective serotonin-Sz antagonist hetanserin, was synthesized and tested for in vitro labeling of serotonin-S2 receptors. The compound showed a very high affinity for both membrane-bound and detergentsolubilized serotonin-Sz receptors with K Dvalues of 0.35 and 2.03 nM, respectively. At nanomolar concentrations, binding to serotonin-SI sites was totally absent. Serotonin-SPreceptor binding was characterized by a slow dissociation and a very low nonspecific binding. In rat frontal cortex preparations, binding could be displaced by nanomolar concentrations of different serotonin antagonists and micromolar concentrations of serotonin agonists. Compounds with other pharmacological profdes were poorly or not active. Introduction of an amino function in this new radioligand led to a decreased lipophilicity. Therefore, besides being a valuable radioligand for routine binding studies, [3H]-7-aminoketanserin will probably be a good ligand for labeling serotonin-S2receptors on intact cells.
Serotonin-S2receptors have been identified in the central nervous system as well as in the periphery. These receptor sites were shown to mediate the antagonism of various serotonergic effects, measured both in vivo and in vitro, such as behavioral excitation in rodents induced by serotonin mimetic agents (“the serotonin syndrome”), serotonin-induced co&actions i n isolated aiteries, and serotonin-induced shape changes and aggregation of blood p1atelets.l Several radioligands have been used for the in vitro biochemical characterization of serotonin-S2 receptors, including [3H]spiperone,2[3H]LSD,3[1251]LSD,4[3H]miDepartment of Biochemical Pharmacology. 1Department of Drug Metabolism & Pharmacokinetics. 0022-2623/86/1829-1663$01.50/0
anserin,j and [3H]metergoline.6 However, most of these ligands were either not selective for serotonin-S2sites or showed a too high nonspecific binding. Recently [3H]ketanserin was introduced as a high-affinity, selective (1) \-I
Leysen, J. E.; de Chaffoy de Courcelles, D.; De Clerck, F.; Niemegeers, C. J. E.; Van-Nueten, J. M. Neuropharmacology
1984,23, 1493. (2) Leysen, J. E.; Niemegeers, C. J. E.; Tollenaere, J. P.; Laduron, P. M. Nature (London) 1978,272, 168. (3) Peroutka, S.J.; Snyder, S. H. Mol. Pharmacol. 1979,16,687. (4) Hartig, P. R.; Kadan, M. J.; Evans, M. J.; Krohn, A. M. Eur. J. Pharmacol. 1983, 89, 321. (5) Peroutka, S. J.; Snyder, S. H. J. Pharmacol. Exp. Ther. 1981,
216, 142. (6) Hamon, M.; Mallat, M.; Herbet, A.; Nelson, D. L.; Audinot, M.; Pichat, L.; Glowinski, J. J. Neurochem. 1981, 36, 613.
0 1986 American Chemical Society
Wouters et al.
1664 Journal of Medicinal Chemistry, 1986, Vol. 29, No. 9
HCI conc.
0
methyl isobutyl k e l o n c
VI h
VI I h .
Figure 1. Synthesis of [3H]-7-aminoketanserin. serotonin-S2receptor ligand that did not bind to serotonin& sites (a serotonin binding site labeled by nanomolar concentrations of [3H]serotonin).7 By use of [3H]ketanserin, membrane-bound as well as detergent-solubilized serotonin-S2 receptors could be specifically labeled.8,9 However, in both cases there was a relatively high amount of nonspecific binding. In the present paper we describe the synthesis and binding characteristics of a derivative of ketanserin namely [3H]-7-aminoketanserin. This new radioligand combines the selectivity of ketanserin with a very low nonspecific binding.
Results Synthesis of [3H]-7-Aminoketanserin(VII). In order to obtain specifically ortho-labeled VI1 (Figure l),to avoid unnecessary loss of radioactivity and to acquire a high specific activity, the synthetic strategy toward tritiated aminoketanserin required the introduction of the tritium label via dehalogenation in the latest possible stage. Friedel-Craft acylation of 3-fluorobromobenzene with IO ' afforded an isomeric mixture of I1 and I11 that was separated by HPLC. Deacetylation of I1 and coupling of the deprotected piperidine derivative IV with Vll furnished bromoaminoketanserin VI, which was reductively dehalogenated (tritium; Pd 10% on charcoal) to title compound VII. Some physicochemical properties of 7-aminoketanserin are listed in Table 11. Leysen, J. E.; Awouters, F.; Kennis, L.; Laduron, P. M.; Vandenberk, J.; Janssen, P. A. J. Life Sci. 1981, 28, 1015. Leysen, J. E.; Niemegeers, C. J. E.; Van Nueten, J. M.; Laduron, P. M. Mol. Pharmacol. 1982,21, 301. Wouters. W.: Van Dun, J.: Levsen, J. E.; Laduron, P. M. Eur. J . Pharmacal. 1985, 115, 1. Duncan. R. L.. Jr.: Helslev. G. C.: Welstead. W. J.. Jr.: Da Vanzo, J. P.; FunderburklW. H.;'Lunsford, C. D. J . Med. Chem. 1970, 13, 1. Unlabeled VI1 was obtained in a 35% yield by coupling V (this reference) with 4-(4-fluorobenzoyl)piperidine (this reference). Satisfactory elemental combustion analyses were obtained (C, H *0.3%). The compound was TLC and HPLC identical to tritiated VII: U S . Patent Application 678 422, 1985.
Table I. Inhibition by 7-Aminoketanserin of Radioligand Binding in Nine Binding Models" binding -log Ki,b KiJKi model [3H]ligand IC,,,M nM (serotonin-S,) serotonin-S, ketanserin 9.25 0.17 1 serotonin-S, serotonin 5.25 4416 25976 histamine-H, pyrilamine 8.65 1.22 7.2 7.82 5.49 32.3 a,-adrenergic WB-4101 dopamine-D2 haloperidol 6.38 164 965 qadrenergic clonidine
