Cocaine exerts its stimulatory effect by inhibiting the dopamine transporter (DAT). However, novel benztropine-and rimcazolebased inhibitors show reduced stimulant effects compared with cocaine, despite higher affinity and selectivity for DAT. To investigate possible mechanisms, we compared the subjective effects of different inhibitors with their molecular mode of interaction at the DAT. We determined how different inhibitors affected accessibility of the sulfhydryl-reactive reagent [2-(trimethylammonium)ethyl]-methanethiosulfonate to an inserted cysteine (I159C), which is accessible when the extracellular transporter gate is open but inaccessible when it is closed. The data indicated that cocaine analogs bind an open conformation, whereas benztropine and rimcazole analogs bind a closed conformation. Next, we investigated the changes in inhibition potency of [ 3 H]dopamine uptake of the compounds at a mutant DAT (Y335A) characterized by a global change in the conformational equilibrium. We observed a close relationship between the decrease in potencies of inhibitors at this mutant and cocaine-like responding in rats trained to discriminate cocaine from saline injections. Our data suggest that chemically different DAT inhibitors stabilize distinct transporter conformations and that this in turn affects the cocaine-like subjective effects of these compounds in vivo.

A specific role for the dopamine D3 receptor in behavior has yet to be elucidated. We now report that dopamine D2/D3 agonists elicit dose-dependent yawning behavior in rats, resulting in an inverted U-shaped dose-response curve. A series of experiments was directed toward the hypothesis that the induction of yawning is a D3 receptor-mediated effect, whereas the inhibition of the yawning observed at higher doses is due to competing D2 receptor activity. We compared several dopaminergic agonists with a range of in vitro D3 selectivity, including PD-128,907 [(5aR-trans)-5,5a,6,7,8, 9,9a,10-octahydro-6-propylpyrido[2,3-g]quinazolin-2-amine dihydrochloride], pramipexole (NЈ-propyl-4,5,6,7-tetrahydrobenzothiazole-2,6-diamine), 7-OH-DPAT [(Ϯ)-7-hydroxy-2-dipropylaminotetralin HBr], quinpirole [trans-(Ϫ)-(4aR)-4,4a,5,6,7,8, 8a,9-octahydro-5-propyl-1H-pyrazolo[3,4-g]quinoline HCl], bromocriptine [(ϩ)-2-bromo-12Ј-hydroxy-2Ј-(1-methylethyl)-5Ј-(2-methylpropyl) ergotaman-3Ј,6Ј-18-trione methanesulfonate], and apomorphine [(R)-(Ϫ)-5,6,6a,7-tetrahydro-6-methyl-4H-dibenzo-[de,g]quinoline-10,11-diol HCl] with respect to their ability to induce yawning in rats. A series of D2/D3 antagonists differing in selectivity for D3 over D2 receptors were evaluated for their ability to alter the effects of the dopamine agonists. The antagonists, and PG01037 (N-{4-[4-(2,3-dichlorophenyl)-piperazin-1-yl]-trans-but-2-enyl}-4-pyridine-2-yl-benzamide HCl) were used to determine effects on doseresponse curves for D2/D3 agonist-induced yawning. In addition, the potential contribution of cholinergic and/or serotonergic mechanisms to the yawning response was investigated using a series of pharmacological tools including scopolamine [(a,S)-a-(hydroxymethyl)benzeneacetic acid (1a,2b,4b,5a,7b)-9-methyl-3-oxa-9-azatricyclo[3.3.1.02,4]-non7-yl ester hydrobromide], mianserin (1,2,3,4,10,14b-hexahydro-2-methyldibenzo[c,f]pyrazino[1,2-a]azepine HCl), and the D3-preferring antagonists nafadotride, U99194, SB-277011A, and PG01037 to differentially modulate yawning induced by PD-128,907, physostigmine [(3aS)-cis-1,2,3,3a,8,8a-hexahydro-1,3a,8-trimethylpyrrolo[2,3-b]indol-5-ol methylcarbamate hemisulfate], and N- [3-(trifluoromethyl)phenyl]piperazine HCl. The results of these experiments provide convergent evidence that dopamine D2/D3 agonist-induced yawning is a D3 agonist-mediated behavior, with subsequent inhibition of yawning being driven by competing D2 agonist activity. Thus, dopamine agonist-induced yawning may represent an in vivo method for selectively identifying D3 and D2 receptormediated activities. Dopamine D3 receptors have received considerable interest since originally cloned (Sokoloff et al., 1990). The D3 receptor shares significant sequence homology with the dopamine D2 receptor but displays a much more restricted, limbic pattern of distribution compared with that of the D2 , trans-N-[4-[2-(6-cyano-1,2,3,4-tetrahydroisoquinolin-2-yl)

Dopamine D3 receptor subtypes have been hypothesized to play a pivotal role in modulating the reinforcing and drug-seeking effects induced by cocaine. However, definitive pharmacological investigations have been hampered by the lack of highly D3 receptor selective compounds that can be used in vivo. To address this problem, the potent and D3-receptor-selective antagonist NGB 2904 (1, 9H-fluorene-2-carboxylic acid {4-[(2,3-dichlorophenyl)-piperazin-1-yl]-butyl}-amide, Ki (hD3) = 2.0 nM, Ki (hD2L) = 112 nM, D2/D3 selectivity ratio of 56) was chosen as a lead structure for chemical modification in an attempt to reduce its high lipophilicity (c log D = 6.94) while optimizing D3 receptor binding affinity and D2/D3 selectivity. A series of >30 novel analogues were synthesized, and their binding affinities were evaluated in competition binding assays in HEK 293 cells transfected with either D2(L), D3, or D4 human dopamine receptors using the high affinity, selective D2-like receptor antagonist (125)I-IABN. Structural diversity in the aryl amide end of the molecule was found to have a major influence on (sub)nanomolar D3 receptor affinity and D2/D3 selectivity, which was optimized using a more rigid trans-butenyl linker between the aryl amide and the piperazine. Several analogues demonstrated superior D3 receptor binding affinities and selectivities as compared to the parent ligand. Compound 29 (N-{4-[4-(2,3-dichlorophenyl)-piperazin-1-yl]-trans-but-2-enyl}-4-pyridine-2-yl-benzamide) displayed the most promising pharmacological profile (Ki (hD3) = 0.7 nM, Ki (hD2L) = 93.3 nM, D2/D3 selectivity ratio of 133). In addition, this ligand inhibited quinpirole stimulation of mitogenesis at human dopamine D3 receptors transfected into Chinese hamster ovary (CHO) cells, with an EC50 value of 3.0 nM. Compound 29 was a nearly 5 times more potent antagonist at the D3 receptor than 1 (EC50 = 14.4 nM). Moreover, a decrease in c log D value of approximately 2 orders of magnitude was determined for this novel D3-receptor-preferring ligand, compared to 1. In summary, chemical modification of 1 has resulted in compounds with high affinity and selectivity for D3 receptors. The most promising candidate, compound 29, is currently being evaluated in animal models of cocaine abuse and will provide an important tool with which to elucidate the role of D3 receptors in drug reinforcement in vivo.

Dopamine D3 receptor antagonists and partial agonists have been shown to modulate drug-seeking effects induced by cocaine and other abused substances. Compound 6 [PG01037, (N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)-trans-but-2-enyl)-4-pyridine-2-ylbenzamide)] and related analogues are currently being evaluated in animal models of drug addiction. In these studies, a discrepancy between in vitro binding affinity, in vivo occupancy, and behavioral potency has been observed. The purpose of this study was to examine (1) modifications of the 2-pyridylphenyl moiety of 6 and (2) hydroxyl, acetyl, and cyclopropyl substitutions on the butylamide linking chain systematically coupled with 2-fluorenylamide or 2-pyridylphenylamide and 2-methoxy- or 2,3-dichloro-substituted phenylpiperazines to measure the impact on binding affinity, D2/D3 selectivity, lipophilicity, and function. In general, these modifications were well tolerated at the human dopamine D3 (hD3) receptor (Ki = 1-5 nM) as measured in competition binding assays. Several analogues showed >100-fold selectivity for dopamine D3 over D2 and D4 receptors. In addition, while all the derivatives with an olefinic linker were antagonists, in quinpirole-stimulated mitogenesis at hD3 receptors, several of the hydroxybutyl-linked analogues (16, 17, 21) showed partial agonist activity. Finally, several structural modifications reduced lipophilicities while retaining the desired binding profile.