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We have investigated a series of phenolic diaryl amino piperidine delta opioid receptor agonists, establishing the importance of the phenol functional group and substitution on the piperdine nitrogen for delta agonist activity and selectivity versus the mu and kappa opioid receptors. This study uncovered compounds with improved agonist potency and selectivity compared to the standard, non-peptidic delta agonist SNC-80. In vivo anti-nociceptive activity of analog 8e in two rodent models is discussed, demonstrating the potential of delta agonists to provide a novel mechanism for pain relief.

Nonpeptide delta opioid agonists are analgesics with a potentially improved side-effect and abuse liability profile, compared to classical opioids. Andrews analysis of the NIH nonpeptide lead SNC-80 suggested the removal of substituents not predicted to contribute to binding. This approach led to a simplified lead, N, N-diethyl-4-[phenyl(1-piperazinyl)methyl]benzamide (1), which retained potent binding affinity and selectivity to the human delta receptor (IC(50) = 11 nM, mu/delta = 740, kappa/delta > 900) and potency as a full agonist (EC(50) = 36 nM) but had a markedly reduced molecular weight, only one chiral center, and increased in vitro metabolic stability. From this lead, the key pharmacophore groups for delta receptor affinity and activation were more clearly defined by SAR and mutagenesis studies. Further structural modifications on the basis of 1 confirmed the importance of the N, N-diethylbenzamide group and the piperazine lower basic nitrogen for delta binding, in agreement with mutagenesis data. A number of piperazine N-alkyl substituents were tolerated. In contrast, modifications of the phenyl group led to the discovery of a series of diarylmethylpiperazines exemplified by N, N-diethyl-4-[1-piperazinyl(8-quinolinyl)methyl]benzamide (56) which had an improved in vitro binding profile (IC(50) = 0.5 nM, mu/delta = 1239, EC(50) = 3.6 nM) and increased in vitro metabolic stability compared to SNC-80.

The design, synthesis, and pharmacological evaluation of a novel class of delta opioid receptor agonists, N, N-diethyl-4-(phenylpiperidin-4-ylidenemethyl)benzamide (6a) and its analogues, are described. These compounds, formally derived from SNC-80 (2) by replacing the piperazine ring with a piperidine ring containing an exocyclic carbon carbon double bond, were found to bind with high affinity and exhibit excellent selectivity for the delta opioid receptor as full agonists. 6a, the simplest structure in the class, exhibited an IC(50) = 0.87 nM for the delta opioid receptors and extremely high selectivity over the mu receptors (mu/delta = 4370) and the kappa receptors (kappa/delta = 8590). Rat liver microsome studies on a selected number of compounds show these olefinic piperidine compounds (6) to be considerably more stable than SNC-80. This novel series of compounds appear to interact with delta opioid receptors in a similar way to SNC-80 since they demonstrate similar SAR. Two general approaches have been established for the synthesis of these compounds, based on dehydration of benzhydryl alcohols (7) and Suzuki coupling reactions of vinyl bromide (8), and are herewith reported.

The novel substituted benzamide eticlopride, (S)-(-)-5-chloro-3-ethyl-N-[(1-ethyl-2-pyrrolidinyl)methyl]-6-methoxy salicylamide hydrochloride (A38503; FLB 131), was radiolabelled to high specific activity and used for in vivo receptor binding studies in the rat brain. Intravenous injections of [3H]eticlopride resulted in a rapid accumulation of radioactivity in several brain regions: striatum greater than olfactory tubercle greater than septum greater than substantia nigra greater than frontal cortex greater than cerebellum. Approximately 95% of the radioactivity recovered from the striatum was in the form of authentic eticlopride, as determined by thin-layer chromatography. Two hours after injection, the ratio between the amount of radioactivity present in the striatum and in the cerebellum was approximately 10:1. The in vivo binding of [3H]eticlopride was saturable in all dopamine-rich areas, with a very low proportion of non-specific binding. The specific in vivo binding of [3H]eticlopride was blocked by several dopamine antagonists, including haloperidol, (+)-butaclamol, spiperone, d,l-sulpiride and remoxipride. The dopamine agonist N-n-propylnorapomorphine, but not apomorphine, was found to be a potent blocker of in vivo [3H]eticlopride binding. Serotonin and noradrenaline receptor antagonists did not prevent the in vivo binding of [3H]eticlopride. Autoradiographic analysis of the in vivo [3H]eticlopride binding showed a high density of binding sites in the striatum, nucleus accumbens and the olfactory tubercle. Moderate binding was found in the hippocampal formation and in the entorhinal area, but little or no binding was detected in other cortical regions. [3H]Eticlopride binding in all these areas was blocked by pretreatment with (+)-butaclamol. Taken together, these findings show that the substituted benzamide compound [3H]eticlopride passes readily into the brain and binds with high specificity to dopamine or neuroleptic receptors in dopamine-rich brain areas. Thus, eticlopride may be a useful tool in studies of dopamine D-2 receptors in vivo.

The substituted benzamide drug raclopride, [((-)-(S)-3,5-dichloro-N-((1-ethyl-2-pyrrolidinyl) methyl)-6-methoxy-salicylamide tartrate; FLA 870(-); A40664] was shown to be a potent and selective antagonist of dopamine D-2 receptors by its high affinity for striatal 3H-spiperone binding sites and low potency to block dopamine stimulated adenylate cyclase in vitro. In vitro studies showed that 3H-raclopride binds with a high affinity (KD = 1.2 nM) and a low proportion of non-specific binding to rat striatal homogenates. The binding of 3H-raclopride is saturable (Bmax = 23.5 pmoles/g wet wt) and reversible (dissociation half-time = 30 min) with a regional distribution of the specifically bound drug showing the following rank-order: striatum greater than nucleus accumbens greater than olfactory tubercle greater than septum greater than hypothalamus greater than hippocampus greater than frontal cortex. After in vivo administration, 3H-raclopride accumulates preferentially in dopamine rich brain areas with approximately 10 times higher levels in the striatum than in the cerebellum, when examined 30 min after injection. The in vivo binding of 3H-raclopride was saturable, reversible and showed a low component of non-specific binding. More than 90% of the drug reached the brain in a non-metabolized form as judged by thin-layer chromatography. Pharmacological analysis of 3H-raclopride binding showed that it could be displaced by dopamine agonists and antagonists but not by serotoninergic or noradrenergic drugs. Taken together, the results suggest that 3H-raclopride labels dopamine D-2 receptors with high specificity in the rat brain both in vitro and in vivo, and thus, that it should be a useful tool in studies of central dopamine D-2 receptors.

1. The use of raclopride, a new compound of the salicylamide series, as a ligand for the labelling of dopamine-D2 receptors in vitro and in vivo is described. 2. 3H-Raclopride has a high affinity for the dopamine-D2 receptors (Kd = 1 nM in rat striatum) with much less affinity for any other receptor. 3. 3H-Raclopride enters the brain easily and has therefore also been used in in vivo binding and autoradiography. The nonspecific binding is very low both in vitro and in vivo. 4. Raclopride has been labelled with 11C, and is used as a marker for dopamine-D2 receptors in the living human brain using positron emission tomography.

This paper describes the synthesis of 5-[(3-bromo-2,6-dimethoxybenzamido)-methyl]-5-hydroxy-2-pyrrolidon e (3) and its 1-ethyl analogue 2, two urinary metabolites of the dopamine D-2 antagonist remoxipride [1, (S)-3-bromo-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2, 6-dimethoxybenzamide]. Two synthetic schemes leading to a common intermediate, 5-benzamido-4-oxopentanoic acid 4, were developed. This key intermediate permits conversion into either metabolite. Reaction of 4 with isobutyl chloroformate furnished a mixed carbonic anhydride, which upon treatment with ethylamine or ammonia gave the 4-oxopentanamides 5 and 6, respectively. Ring-closure afforded the corresponding 5-hydroxy-2-pyrrolidones 2 and 3.

The substituted benzamide compound eticlopride, (S)-(-)-5-chloro-3-ethyl-N-[(1-ethyl-2-pyrrolidinyl) methyl]-6-methoxysalicylamide hydrochloride (FLB 131), has been shown to selectively block dopamine-D2 binding sites in the rat brain. The compound was tritium-labelled to high specific radioactivity and was used for in vitro receptor binding studies. [3H]Eticlopride was found to bind specifically to rat brain homogenates with the highest binding in the striatum and lowest in the hippocampus. The binding was saturable with a high number of binding sites (49.5 pmol/g) and with very high affinity (0.17 nM). As with other benzamides, the binding of [3H]eticlopride was highly sodium-dependent. Lesioning of the striatal neurons with ibotenic acid reduced the binding by 50% while lesioning of the nigrostriatal pathways with 6-hydroxydopamine was without effect on the observed binding. The binding of [3H]eticlopride was inhibited potently by neuroleptic drugs, while compounds known not to interact with the dopamine-D2 binding sites were inactive. It is concluded that this new dopamine-D2 antagonist may be a useful tool for the study of dopamine-D2 binding sites due to its high affinity and good selectivity.

The binding of 3H-alaproclate, a selective 5-hydroxytryptamine uptake inhibitor, to membranes prepared from the rat cerebral cortex was investigated by a filtration technique. It was found that 3H-alaproclate bound with high affinity to three or four different sites and to one low affinity site. The binding to two of these sites was displaceable by 1 microM proadifen (SKF 525A), an inhibitor of drug metabolism. From iterative nonlinear regression analysis the KD-values of these sites were calculated to about 1 and 28 nM and the Bmax values 1.5 and 19 pmol/g wet tissue, respectively. The high affinity binding that was not displaceable by proadifen but by 10 microM alaproclate had KD-values of 1 nM and 6 nM and Bmax-values of 0.4 and 2 pmol/g wet tissue. The low affinity binding that was not displaceable by proadifen had a KD-value of about 200 nM and a Bmax-value of about 90 pmol/g tissue. The possible relationship between the proadifen sensitive high affinity binding of 3H-alaproclate and the brain cytochrome P-450 is discussed.