Binding of beta-carbolines and related agents at serotonin (5-HT(2) and 5-HT(1A)), dopamine (D(2)) and benzodiazepine receptors.

A large series of beta-carbolines was examined for their ability to bind at [3H]agonist-labeled 5-HT(2A) serotonin receptors. Selected beta-carbolines were also examined at 5-HT(2C) serotonin receptors, 5-HT(1A) serotonin receptors, dopamine D(2) receptors, and benzodiazepine receptors. Indolealkylamines and phenylisopropylamines were also evaluated in some of these binding assays. The beta-carbolines were found to bind with modest affinity at 5-HT(2A) receptors, and affinity was highly dependent upon the presence of ring substituents and ring saturation. The beta-carbolines displayed little to no affinity for 5-HT(1A) serotonin receptors, dopamine D(2) receptors and, with the exception of beta-CCM, for benzodiazepine receptors. Examples of beta-carbolines, indolealkylamines (i.e. N,N-dimethyltryptamine analogs), and phenylisopropylamines have been previously shown to produce common stimulus effects in animals trained to discriminate the phenylisopropylamine hallucinogen DOM (i.e. 1-(2, 5-dimethoxy-4-methylphenyl)-2-aminopropane) from vehicle. Although the only common receptor population that might account for this action is 5-HT(2A), on the basis of a lack of enhanced affinity for agonist-labeled 5-HT(2A) receptors, as well as on their lack of agonist action in the PI hydrolysis assay, it is difficult to conclude that the beta-carbolines behave in a manner consistent with that of other classical hallucinogens.

Synthesis and in vitro and in vivo activity of (-)-(1R,5R,9R)- and (+)-(1S,5S,9S)-N-alkenyl-, -N-alkynyl-, and -N-cyanoalkyl-5, 9-dimethyl-2'-hydroxy-6,7-benzomorphan homologues.

Two of the synthesized (-)-(1R,5R,9R)-N-homologues (N-but-3-enyl- and N-but-3-ynyl-5,9-dimethyl-2'-hydroxy-6,7-benzomorphan (9, 13)) were found to be about 20 times more potent than morphine in the mouse tail-flick assay (ED(50) = 0.05 mg/kg), and (-)-(1R,5R, 9R)-N-but-2-ynyl-5,9-dimethyl-2'-hydroxy-6,7-benzomorphan ((-)-(1R, 5R,9R)-N-but-2-ynylnormetazocine, 12) was about as potent as the opioid antagonist N-allylnormetazocine (AD(50) in the tail-flick vs morphine assay = 0.3 mg/kg). All of the homologues examined had higher affinity for the kappa-opioid receptor than the mu-receptor except (-)-N-but-2-ynyl-normetazocine (12), which had a kappa/mu ratio = 7.8 and a delta/mu ratio = 118. The (-)-N-2-cyanoethyl (3), -allyl (8), and -but-3-ynyl (13) analogues had good affinity (<10 nM) for delta-opioid receptors. Two homologues in the (+)-(1S,5S,9S)-normetazocine series, N-pent-4-enyl (24) and N-hex-5-enyl (25), were high-affinity and selective sigma(1)-ligands (K(i) = 2 nM, sigma(2)/sigma(1) = 1250, and 1 nM, sigma(2)/sigma(1) = 750, respectively); in contrast, N-allylnormetazocine (22) had relatively poor affinity at sigma(1), and its sigma(1)/sigma(2) ratio was <100.

Rigid phencyclidine analogues. Binding to the phencyclidine and sigma 1 receptors.

Three phencyclidine (PCP) analogues possessing a highly rigid carbocyclic structure and an attached piperidine ring which is free to rotate were synthesized. Each analogue has a specific fixed orientation of the ammonium center of the piperidinium ring to the centrum of the phenyl ring. The binding affinities of the rigid analogues 1-piperidino-7,8-benzobicyclo[4.2.0]octene (14), 1-piperidinobenzobicyclo[2.2.1]heptene (16), and 1-piperidinobenzobicyclo[2.2.2]octene (13) for the PCP receptor ([3H]TCP) and th-receptor (NANM) were determined. The three analogues show low to no affinity for the PCP receptor but good affinity for the th-receptor and can be considered th-receptor selective ligands with PCP/th ratios of 13, 293, and 368, respectively. The binding affinities for the th-receptor are rationalized in terms of a model for the th-pharmacophore.

Zipeprol: preclinical assessment of abuse potential.

Zipeprol was evaluated in a number of in vitro and in vivo assays predictive of stimulant, depressant, or opioid abuse potential. Zipeprol had affinity for mu and kappa opioid binding sites as well as sigma binding sites. However, it failed to exert opioid-like agonist actions in rodents, and did not attenuate withdrawal signs in morphine- or pentobarbital-dependent rats. Zipeprol did not substitute for either amphetamine or pentobarbital in drug discrimination assays in rhesus monkeys. On the other hand, it suppressed morphine withdrawal signs in rhesus monkeys in two assays, and it acted as a quadazocine-sensitive reinforcer in monkeys trained to self-inject alfentanil. Zipeprol also acted as a reinforcer in monkeys trained to self-inject methohexital. In a dose range of 10-18 mg/kg, zipeprol induced convulsions in monkeys. Zipeprol appears to have abuse potential and a novel spectrum of action involving both opioid and non-opioid effects.

(E)-8-benzylidene derivatives of 2-methyl-5-(3-hydroxyphenyl)morphans: highly selective ligands for the sigma 2 receptor subtype.

The determination of the structure and function of the sigma receptor subtypes and their physiological role(s) has been impeded by the unavailability of selective ligands. We have developed a new class of sigma subtype selective receptor ligands that are (E)-8-benzylidene derivatives of the synthetic opioid (+/-)-, (+)-, and (-)-2-methyl-5-(3-hydroxyphenyl)morphan-7-one (1). The derivatives can be prepared by reaction of 1, (+)-1, and (-)-1 with the appropriate benzaldehyde under Claisen-Schmidt conditions. Incorporation of substituted (E)-8-benzylidene moieties onto the 7-keto precursor of (+)-2-methyl-5-(3-hydroxyphenyl)morphan, (+)-1, produces compounds (-)-2 through (-)-7 (5.8-32.0 nM, sigma 1), which have between a 25- and 131-fold increase in affinity for the sigma 1 receptor subtype relative to the keto precursor (+)-1 (Ki = 762 nM, sigma 1). Compound (-)-2 is the most selective of this group (16-fold) for the sigma 1 subtype versus sigma 2. Substitution of an (E)-8-benzylidene moiety onto the 7-keto precursor of (-)-2-methyl-5-(3-hydroxyphenyl)morphan, (-)-1, produces compounds (+)-2-(+)-9 (6.4-52.6 nM, sigma 2), which have at least a 475-3906-fold increase in affinity for the sigma 2 receptor subtype relative to the keto precursor (-)-1 (Ki = 25 x 10(3) nM). This enhancement of sigma 2 receptor affinity is accompanied by substantial selectivity of all of these dextrorotatory products for the sigma 2 relative to the sigma 1 subtype (32-238-fold), and thus, they are among the most sigma 2 selective compounds currently known. Furthermore, the sigma 1 subtype is highly enantioselective for the levorotatory isomers, (-)-2-(-)-7 (41-1034-fold), whereas the sigma 2 subtype is only somewhat enantioselective for the dextrorotatory isomers, (+)-2-(+)-7 (2.6-9.3-fold). All of these derivatives retain substantial affinity for the mu opioid receptor. Despite the high affinity of the dextrorotatory derivatives for the mu opioid receptor, the high affinity and selectivity for sigma 2 over sigma 1 sites will surely prove beneficial as tools for the delineation of the function and physiological role of sigma 2 receptors.

A marked change of receptor affinity of the 2-methyl-5-(3-hydroxyphenyl)morphans upon attachment of an (E)-8-benzylidene moiety: synthesis and evaluation of a new class of sigma receptor ligands.

The (E)-8-benzylidene and (E)-8-(3,4-dichlorobenzylidene), 7-ketone derivatives, 5 and 6, of the synthetic opiate 2-methyl-5-(3-hydroxyphenyl)morphan [5-(3-hydroxyphenyl)-2-methyl-2-azabicyclo[3.3.1]nonane, 1], were synthesized from the 7-ketone derivatives 2 or 4 via the Claisen-Schmidt reaction. The corresponding enantiomers of 5 and 6 were obtained in > 99% optical purity from the optical isomers of 4, resolved with the O,O'-dibenzoyltartaric acids. The absolute configurations of the enantiomers of 4 were determined by conversion, via Clemmensen reduction, to the enantiomers of 1, the configurations of which are known. The determination of the regioisomer and configurational isomer of 5, with respect to the introduced benzylidene group, was determined from a single-crystal X-ray analysis. 1H NMR data was used to confirm that 6 possessed the same configuration as 5. Radioreceptor binding studies in rat and guinea pig brain preparations revealed that (-)-(1S,5S)-5 displayed an 11-fold decrease in affinity for the opioid mu receptor and an increase in affinity for sigma receptors of 81-fold (low nanomolar affinity) relative to the ketone precursor (+)-(1S,5S)-4. An analogous, albeit less dramatic, trend was seen with compound (-)-(1S,5S)-6. Compounds (-)-(1S,5S)-5 and (-)-(1S,5S)-6 are distinct from the typical sigma-opiates in that they have very low affinity for either PCP sites or muscarinic receptors. The high affinity and selectivity of these novel sigma receptor ligands suggests that they will be valuable for the elucidation of the functional roles of sigma receptors.

Antipodal alpha-N-(methyl through decyl)-N-normetazocines (5,9 alpha-dimethyl-2'-hydroxy-6,7-benzomorphans): in vitro and in vivo properties.

The enantiomeric (-)- and (+)-N-(methyl through decyl) normetazocines (5,9 alpha-dimethyl-2'-hydroxy-6,7-benzomorphans) were synthesized and their in vitro and in vivo activities determined. Increasingly bulky enantiomeric N-alkyl homologs were prepared until their interaction with the sigma 1 receptor decreased and their insolubility became a hindrance to their evaluation in vivo and/or in vitro. The (-)-methyl, -pentyl, -hexyl, and -heptyl homologs were essentially as potent as, or more potent than, morphine in the tail-flick, phenylquinone, and hot-plate assays for antinociceptive activity; the (-)-propyl homolog had narcotic antagonist activity between that of nalorphine and naloxone in the tail-flick vs morphine assay, and it also displayed antagonist properties in the single-dose suppression assay in the rhesus monkey. The antinociceptively potent (-)-heptyl homolog did not substitute for morphine in monkeys but did show morphine-like properties in a primary physical-dependence study in continuously infused rats. All five potent compounds showed high affinity for the mu opioid receptor from both rat and monkey preparations and the kappa opioid receptor (< 0.05 microM), and all except the (-)-methyl homolog interacted reasonably well at the delta receptor (K(i) < 0.1 microM). The (-)-propyl compound was equipotent (K(i) 1.5-2.0 nM) at mu and kappa receptors. The pattern of interaction of the (-)-enantiomeric homologs with mu receptors from rat and monkey preparations was similar, but not identical. The enantioselectivity of the homologs for mu receptors was greater in the rat than in the monkey preparation for all but the N-H and butyl compounds, and the enantioselectivity of the lower homologs (methyl through butyl) for the mu (monkey) receptor was greater than for the kappa or delta receptors. However, bulkier homologs (hexyl through decyl) displayed higher enantioselectivity at kappa or delta receptors than at the mu (monkey) receptor. The (+)-butyl through (+)-octyl homologs were essentially equipotent with, or more potent than, (+)-pentazocine at the sigma receptor. Only the (+)-H and (+)-methyl homologs had high affinity (< 0.05 microM) at PCP binding sites.

Novel 1-phenylcycloalkanecarboxylic acid derivatives are potent and selective sigma 1 ligands.

Carbetapentane (1, 2-[2-(diethylamino)ethoxy]ethyl 1-phenyl-1-cyclopentanecarboxylate) binds with high affinity to sigma sites and is a potent antitussive, anticonvulsant, and spasmolytic agent. However, carbetapentane interacts at muscarinic binding sites as well, and it is not clear whether either of these receptor systems is involved in the mechanism(s) of action(s) of this drug. In an attempt to determine whether these psychoactivities can be attributed to interaction at sigma sites, a series of carbetapentane analogs were prepared. Phenyl ring substitution; contraction, expansion, and replacement with a methyl group of the cyclopentyl ring; replacement of the carboxylate function with an amide, methyl ether, and methylamine; and replacement of the N,N-diethyl substituent with a morpholino or piperidino moiety were investigated. All of these novel analogs were evaluated for binding to sigma 1 and sigma 2 sites, and comparison of binding at muscarinic m1 and m2 and PCP (1-(1-phenylcyclohexyl)piperidine) receptors was performed. All of the compounds were selective for sigma 1 over sigma 2 sites, with the three most selective analogs being compounds 34 (65-fold), 35 (78-fold), and 39 (51-fold). None of the compounds were active at PCP sites, and chemical modification including (1) replacing the ester function, (2) replacing the cyclopentyl ring with a smaller ring system (cyclopropyl) or a methyl group, and (3) replacing the diethylamino moiety with a morpholino group resulted in > 220-fold selectivity over muscarinic receptor binding. Therefore, several of these novel compounds are potent, sigma 1-selective ligands which can now be investigated as potential antitussive, anticonvulsant, and antiischemic agents. These studies may reveal whether sigma 1 sites play a role in the pharmacological actions of these drugs.

Further studies of the structure-activity relationships of 1-[1-(2-benzo[b]thienyl)cyclohexyl]piperidine. Synthesis and evaluation of 1-(2-benzo[b]thienyl)-N,N-dialkylcyclohexylamines at dopamine uptake and phencyclidine binding sites.

We previously reported (J. Med. Chem. 1993, 36, 1188-1193) that changes to the ring size of the piperidine and cyclohexyl rings of the high-affinity and selective dopamine (DA)-uptake inhibitor 1-[1-(2-benzo[b]thienyl)cyclohexyl]piperidine (BTCP, 2) caused different, and in some cases opposite, changes in affinity for sites on the DA transporter labeled by [3H]BTCP and [3H]-cocaine. These results suggested that the radioligands label different sites on the transporter. In the present study, we extend the structure-activity relationships (SAR) of BTCP by studying the binding characteristics of a series of N,N-disubstituted 1-(2-benzo[b]thienyl)cyclohexylamines 7-32 at the DA transporter. Cyclohexyl was selected as opposed to other ring sizes since it corresponds to BTCP. The binding results indicate that a considerable degree of structural variation is permitted for the N-substituents, while still retaining nanomolar affinity for sites on the transporter (studied in rat forebrain homogenates). As observed in our earlier study, the differential effects of structural change on binding to sites on the DA transporter labeled by these radioligands suggests that they are different and distinct binding sites. In general, and up to a point, increasing the size and lipophilicity of the N substituents resulted in improvements in binding but appeared to have less predictable effects on DA-uptake inhibition (as measured in rat brain synaptosomes). The binding of these compounds to sites labeled by [3H]BTCP appeared to correlate best with IC50 for DA-uptake inhibition. To our surprise, the monoalkyl N-substituted BTCP derivatives displayed the highest affinity for the DA transporter of all the compounds in this series. For example, the N-(cyclopropylmethyl) derivative 14 displayed IC50's = 23 nM ([3H]cocaine) and 1 nM ([3H]-BTCP), and the N-butyl derivative 10 showed IC50's = 60 nM ([3H]cocaine) and 0.3 nM ([3H]-BTCP). BTCP exhibited IC50's of 39 nM ([3H]cocaine) and 5 nM ([3H]BTCP) in this assay. The observation that N,N-dibutyl derivative 31 exhibited low ratios of IC50 [3H]cocaine/IC50 DA reuptake and IC50 [3H]BTCP/IC50 DA reuptake suggests that it may be a potential candidate for cocaine antagonism studies. The effect of additional amino, amide, and aromatic groups on the N-substituents was examined, and the results are discussed. The failure of all of the compounds in this series to bind phenycyclidine receptors coupled with their high affinity and range of selectivities at the DA transporter identifies many of them as useful tools for probing the mode of action of BTCP at this site.

Synthesis and binding properties of MK-801 isothiocyanates; (+)-3-isothiocyanato-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten- 5,10-imine hydrochloride: a new, potent and selective electrophilic affinity ligand for the NMDA receptor-coupled phencyclidine binding site.

Three new site-directed irreversible (wash-resistant) ligands for the high-affinity phencyclidine (PCP) binding site associated with the N-methyl-D-aspartate (NMDA) receptor were synthesized and their binding characteristics were studied. (+)-3- And (+)-2-isothiocyanato-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cycl ohepten-5,10 - imine hydrochloride ((+)-8a,b.HCl) were prepared in four steps from the corresponding nitro derivatives (+)-4a,b, which were obtained by nitration of (+)-3 (MK-801). In the same way the optical antipode (-)-8a.HCl was synthesized from (-)-3. At a concentration of 100 nM, the 3-isothiocyanate derivative (+)-8a irreversibly labeled approximately 50% of the (+)-[3H]-3 binding sites, compared to 20 microM needed for its optical antipode (-)-8a and the 2-isothiocyanate (+)-8b. The apparent Ki values for reversible inhibition of (+)-[3H]-3 binding by (+)- and (-)-8a and (+)-8b were 37,838, and 843 nM, respectively. In contrast, metaphit (1b) and etoxadrol m-isothiocyanate (2b), two previously reported irreversible ligands for the PCP binding site, label about 50% of the (+)-[3H]-3 binding sites at 100 microM and 250 nM, respectively, with apparent Ki values for reversible inhibition of 535 and 94 nM. Compound (+)-8a is also a selective affinity ligand, displaying little or no irreversible in vitro affinity at 100 microM for opioid, benzodiazepine, muscarinic, and dopamine receptors. At a 25 microM concentration, (+)-8a caused an irreversible 52% reduction of binding to sigma 1-receptors. Compound (+)-8a is the most potent known electrophilic affinity label for the PCP binding site. Its potency and selectivity should enable it to be a valuable tool for the elucidation of the structure and function of the NMDA receptor-associated PCP binding site in the mammalian central nervous system.

Synthesis and biological evaluation of 1-[1-(2-benzo[b]thienyl)cyclohexyl]piperidine homologues at dopamine-uptake and phencyclidine- and sigma-binding sites.

Piperidine and cyclohexyl ring homologues of the high-affinity dopamine (DA) uptake inhibitor 1-[1-(2-benzo[b]thienyl)cyclohexyl]piperidine (BTCP, 3) were each prepared in four steps from the appropriate cycloalkanones. These compounds were tested for their ability to displace [3H]BTCP and [3H]cocaine and to inhibit [3H]DA uptake in rat striatal homogenates. The ratios IC50([3H]cocaine)/IC50([3H]BTCP) ranged from 62 for BTCP to 1.5 for 1-[2-(benzo[b]thienyl)-cyclopentylamine (17); cocaine gave a ratio of 0.6. This indicates that BTCP is the most selective of all the compounds tested for sites labeled by [3H]BTCP whereas cocaine is most selective for sites labeled by [3H]cocaine. The wide differences in the relative abilities of these compounds to displace [3H]BTCP and [3H]cocaine suggests that these two radioligands are labeling different sites on the transporter. In general, the compounds structurally related to BTCP exhibited greater selectivity for sites labeled by [3H]BTCP. However, several of the BTCP-related derivatives showed greater (compared with BTCP and cocaine) ability to displace [3H]cocaine. Most notably, 1-[1-(2-benzo[b]thienyl)cyclohexyl]pyrrolidine (7) exhibited a 3.4-fold greater affinity for these sites compared with BTCP and a 9-fold greater affinity at these sites than cocaine. Most of the BTCP homologues displayed greater ability to inhibit [3H]DA uptake in rat forebrain synaptosomes than cocaine. BTCP and 7 were the most potent of all the compounds tested in terms of their ability to inhibit uptake of [3H]DA. IC50 ratios for [3H]cocaine binding/[3H]DA uptake ranged from 0.47 for 1-[1-(2-benzo[b]thienyl)cyclopentyl]homopiperidine (11) to 8.8 for 1-(2-benzo[b]thienyl)cyclohexylamine (4). The importance of this ratio remains unclear in terms of identification of potential cocaine antagonists. As for BTCP, all of the compounds tested showed Ki values > 10,000 nM for displacement of [3H]TCP from rat brain homogenates. These compounds were able to displace the highly selective sigma receptor probe [3H]-(+)-pentazocine from guinea pig brain homogenates with Ki values ranging from 125 to 9170 nM. The significance of their sigma-binding activity in light of their dopaminergic properties is unclear. The diverse binding properties of these compounds at the DA-uptake site and their spectrum of inhibitory activities for [3H]DA uptake identifies them as a useful base for the development of subtype selective probes at this site. These compounds will allow further study of the structure and function of the "cocaine" receptor as well as the development of potential cocaine antagonists.

Synthesis, configuration, and activity of isomeric 2-phenyl-2-(N-piperidinyl)bicyclo[3.1.0]hexanes at phencyclidine and sigma binding sites.

The novel semirigid derivatives (+)-cis-1-[2-phenyl-2-bicyclo[3.1.0]hexyl]piperidine [(+)-8], its enantiomer (-)-8, and (+-)-trans-1-[2-phenyl-2-bicyclo[3.1.0]hexyl]piperidine [(+/-)-9] were synthesized as probes to investigate the mode of interaction of phencyclidine (PCP) with its binding site on the N-methyl-D-aspartate receptor complex. Each target compound was obtained in five steps starting from cyclopent-2-enone. (+)- and (-)-8 were obtained in greater than 98% optical purity through three recrystallizations from ethanol of the (S)-(+)- and (R)-(-)-mandelate salts of intermediate (+-)-cis-2-phenyl-2-bicyclo[3.1.0]hexylamine ([(+/-)-16]. Crystallization of the (R)-(-)-mandelate salt afforded (1R,2R,5S)-(-)-16, whereas the (S)-(+)-mandelate salt afforded (1S,2S,5R)-(+)-16; the absolute configuration was determined by single-crystal X-ray analysis of (-)-16.(R)-(-)-mandelate. Single-crystal X-ray analysis of (+/-)-9-picrate confirmed its trans configuration and provided conformational data. (+)- and (-)-8 and (+/-)-9 were examined for their ability to interact with PCP and sigma binding sites in vitro using [3H]TCP and [3H]pentazocine as radioligands. The binding was compared with that of PCP and contrasted with the rigid symmetrical phencyclidine derivatives cis- and trans-1-[3-phenyl-3-bicyclo[3.1.0]hexyl]piperidines (6 and 7). The results of the study indicated that the conformations of PCP represented by 6-9 are not optimal for potent interaction at either of these sites. Affinities ranged from 582 nM [(+/-)-9] to 29,000 nM [(+)-8] at PCP binding sites and from 1130 nM [(-)-8] to 16,300 nM (7) at sigma sites. In this assay, PCP exhibited affinities of 64.5 nM at PCP and 1090 nM at sigma sites. Qualitative correlation between the sigma and PCP binding data suggests some similarities between these binding sites. An axial phenyl and equatorial piperidine ring with the nitrogen lone pair of electrons antiperiplanar to the phenyl ring has been postulated as the receptor-active conformation of PCP-like ligands at the PCP binding site. Comparison of the binding data of 7-9 with that of the previously described methylcyclohexyl-PCP derivatives allowed its rationalization in terms of this model. It is likely that the lowered affinity in this bicyclo[3.1.0]hexane series is a consequence of nonoptimal geometry (pseudoequatorial phenyl or pseudoboat) for binding as opposed to the presence of steric bulk which proved deleterious in the methylcyclohexyl-PCP derivatives.

Fourphit: a selective probe for the methylphenidate binding site on the dopamine transporter.

Fourphit, a phencyclidine derivative containing an isothiocyanate substitution at the 4-position of the piperidine ring, inhibits the binding of the radiolabeled psychomotor stimulant, [3H]methylphenidate, to sites on the dopamine transport complex in membranes prepared from the crude synaptosomal fraction of rat striatal tissue with an IC50 of 7.1 microM. The inhibition caused by Fourphit is irreversible and is associated with a decrease in the Bmax, but not the KD, of [3H]methylphenidate binding. Pretreatment with saturating concentrations of unlabeled methylphenidate effected a modest (but statistically significant) protection of the stimulant binding site from inactivation by Fourphit, indicating that the acylating phencyclidine derivative may act directly at this site. Preincubation with Fourphit rather than vehicle did not alter the dissociation rate of [3H]methylphenidate when measured in the presence of excess amfonelic acid, nor was any difference detected in the off-rate of [3H]methylphenidate when excess Fourphit was substituted for excess unlabeled methylphenidate as the displacing agent. This lack of effect on the dissociation kinetics of [3H]methylphenidate provides further evidence that Fourphit does not act allosterically at the methylphenidate binding site. Unlike Metaphit (an isomer of Fourphit containing the isothiocyanate moiety at the meta position of the aromatic ring), Fourphit can discriminate between the methylphenidate binding site and the phencyclidine binding site associated with the N-methyl-D-aspartate receptor: Metaphit irreversibly inactivates both binding sites, whereas Fourphit binds reversibly to the phencyclidine binding site. The data suggest that Fourphit may be useful as a relatively selective affinity label for the site on the dopamine transport complex recognized by methylphenidate and other psychomotor stimulants.

Analogues of the dioxolanes dexoxadrol and etoxadrol as potential phencyclidine-like agents. Synthesis and structure-activity relationships.

A series of dioxolane analogues based on dexoxadrol ((4S,6S)-2,2-diphenyl-4-(2-piperidyl)-1,3-dioxolane) and etoxadrol ((2S,4S,6S)-2-ethyl-2-phenyl-4-(2-piperidyl)-1,3-dioxolane) were prepared and tested for their ability to displace [3H]TCP (1-[1-(2-thienyl)cyclohexyl]piperidine) from PCP (1-(1-phenylcyclohexyl)piperidine) binding sites in rat brain tissue homogenates. Qualitative structure-activity relationships within this series were explored through modifications of the three major structural units of dexoxadrol, the piperidine, 1,3-dioxolane, and aromatic rings of the molecule. N-Alkyl derivatives of dexoxadrol were found to be inactive, as were those analogues where the dioxolane ring was modified. Phenyl-substituted etoxadrol analogues were compared to similarly substituted PCP analogues and distinct differences were found in their structure-activity relationships suggesting that the aromatic rings in these two drug classes interact differently with the PCP binding sites. The replacement of the phenyl ring in etoxadrol by either a 2- or 3-thienyl ring led to compounds with affinity comparable to etoxadrol, and the replacement of the ethyl moiety on etoxadrol's dioxolane ring with propyl (7) or isopropyl (8) led to compounds which were more potent than etoxadrol or PCP. The most potent compound was (2S,4S,6S)-2-ethyl- 2-(1-chlorophenyl)-4-(2-piperidyl)-1,3-dioxolane (11), where a chlorine moiety was placed in the ortho position in the aromatic ring of etoxadrol. Its potency was comparable with TCP in vitro.

Synthesis, stereochemistry, and biological activity of the 1-(1-phenyl-2-methylcyclohexyl)piperidines and the 1-(1-phenyl-4-methylcyclohexyl)piperidines. Absolute configuration of the potent trans-(-)-1-(1-phenyl-2-methylcyclohexyl)piperidine.

The (-)- and (+)-isomers of the cis- and trans-Ph/Me 1-(1-phenyl-2-methylcyclohexyl)piperidines have been synthesized and the achiral cis- and trans-Ph/Me 1-(1-phenyl-4-methylcyclohexyl)piperidines were prepared, and their in vitro [displacement of [3H]TCP (1-[1-(2-thienylcyclohexyl)]piperidine) from the PCP (1-(1-phenylcyclohexyl)piperidine) binding site] and in vivo (rotarod assay) activities determined. The 1-(1-phenyl-2-methylcyclohexyl)piperidine isomers were resolved by classical crystallization procedures, through the diastereomeric salts obtained with d- and l-10-camphorsulfonic acid. The relative stereochemistry of the cis- and trans-Ph/Me 1-(1-phenyl-2-methylcyclohexyl)piperidines and the achiral cis- and trans-Ph/Me 1-(1-phenyl-4-methylcyclohexyl)piperidines was established by using 13C and 1H NMR. Both (-)-trans-1-(1-phenyl-2-methylcyclohexyl)piperidine ((-)-2) and (+)-trans-1-(1-phenyl-2-methylcyclohexyl)piperidine ((+)-2) were examined by single-crystal X-ray analysis, and the absolute configuration of (-)-2 was determined to be 1S,2R. The (-)-2 was found to be about five times more potent than PCP in vitro and twice as potent in vivo. It is the most potent of all of the simple methyl-substituted cyclohexyl PCP isomers and is among the most potent PCP-like compounds which have been synthesized. It was nine times more potent in vitro and four times more potent in vivo than (+)-2. The racemic cis-1-(1-phenyl-2-methylcyclohexyl)piperidine (3), and its enantiomers ((+)-3 and (-)-3), were essentially inactive in vitro and in vivo. The cis-Ph/Me 1-(1-phenyl-4-methylcyclohexyl)piperidine (18) was more potent than trans-Ph/Me 1-(1-phenyl-4-methylcyclohexyl)piperidine (17), but considerably less potent than (-)-2. The enantioselectivity observed at the PCP binding site for (-)-2 could indicate that this site can discriminate between enantiotopic edges of the achiral PCP (choosing the pro-1-S edge), as does the mu-opioid receptor in the prodine series of opioids. Benzimidoyl or benzoyl group replacement of the phenyl ring in the 1-(1-phenyl-2-methylcyclohexyl)piperidine series gave compounds which showed little in vitro and in vivo activity.

Specificity of phencyclidine-like drugs and benzomorphan opiates for two high affinity phencyclidine binding sites in guinea pig brain.

Recently, the presence of two high affinity binding sites for phencyclidine were described in guinea pig brain, with one site coupled to the glutamate excitatory amino acid receptor, specifically activated by N-methyl-D-aspartate (NMDA) (site 1) and the other site associated with the dopamine (DA) reuptake carrier (site 2). Phencyclidine and its analogs, as well as the benzomorphan opiates, are known to interact with binding sites for phencyclidine. In this study, the equilibrium dissociation constants (Kd) of these compounds for the two binding sites for phencyclidine were determined. Phencyclidine and 1-[1-(2-thienyl)cyclohexyl]piperidine (TCP), an analog of PCP, were essentially non-selective between the two sites and also were the two drugs of the group observed to have the highest affinity for site 2. (+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cycloheptene-5,10-imine [(+)MK801] was the most selective agent for site 1, while none of the drugs tested showed selectivity for site 2. In humans, phencyclidine produces psychotomimetic effects, while (+)MK801 has been reported to produce minimal, if any, psychotomimetic effects, at doses sufficient to reduce seizures. These clinical observations, in conjunction with the present biochemical binding data, suggest that (+)MK801 may serve as a "marker" for site 1 and that the psychotomimetic effects of phencyclidine might be mediated by site 2.

Synthesis, phencyclidine-like pharmacology, and antiischemic potential of meta-substituted 1-(1-phenylcyclohexyl)-1,2,3,6-tetrahydropyridines.

A series of 1-[1-arylcyclohexyl]-1,2,3,6-tetrahydropyridines were prepared by the reaction between 1-(1-cyanocyclohexyl)-1,2,3,6-tetrahydropyridine (1) and an appropriately substituted Grignard reagent. The resulting compounds were tested for their phencyclidine binding site affinities. Selected compounds were then tested for their ability to produce ketamine appropriate responding in monkeys and/or to show neuroprotective effects in a baby rat hypoxia/ischemia model. While it was found that binding site affinity correlated well with discriminative stimulus effects, it was found to be a poor indicator of neuroprotective efficacy within this series.

Synthesis and anticonvulsant activity of 1-phenylcyclohexylamine analogues.

Thirty-eight analogues of 1-phenylcyclohexylamine (PCA), a phencyclidine (PCP) derivative, were examined for their activities in the mouse maximal electroshock (MES) seizure test and in a motor-toxicity assay. In addition, we determined the binding affinities of the compounds for PCP acceptor sites in rat brain membranes labeled with [3H]-1-[1-(2-thienyl)cyclohexyl]piperidine. Many of the analogues were protective against MES seizures (ED50s of 5-41 mg/kg, ip) and all of these compounds caused motor toxicity. The potencies in the motor toxicity and MES seizure tests showed a moderate correlation with the affinities for PCP sites. Several analogues exhibited a greater separation of potencies in the motor toxicity and MES seizure tests than did the parent compound PCA. These were obtained by (i) 3-methylation of the cyclohexyl ring trans to the phenyl ring, (ii) methoxylation at the ortho position on the phenyl ring, and (iii) contraction of the cyclohexane ring to form the corresponding cyclopentane.

Synthesis and structure-activity relationship of C5-substituted analogues of (+-)-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine [(+-)-desmethyl-MK801]: ligands for the NMDA receptor-coupled phencyclidine binding site.

A series of eight C5-substituted analogues of (+-)-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (1) have been prepared by the directed lithiation-alkylation (and acylation) of its (+-)-N-tert-butylformamidinyl derivative 2 followed by formamidine solvolysis. An additional 10 analogues were prepared by elaboration of the C5-ethyl ester derivative. Analogues possessing large (e.g. propyl and larger) lipophilic substituents displace [3H]-1-(1-thienylcyclohexyl)piperidine [( 3H]TCP) from the high-affinity phencyclidine (PCP) binding site in rat brain homogenates only at high concentrations (Ki greater than 1000 nM); however, the presence of a polar amino functionality (e.g. 2-aminoethyl) offsets this effect (Ki = 20 nM). Thus, the boundary condition for lipophilic substituents larger than ethyl appears to be polar in nature. Interaction of the 11 relatively small (MR less than 14) C5-substituted analogues of 1 with the high-affinity PCP binding site associated with the N-methyl-D-aspartate (NMDA) receptor is best described by the equation log (1/Ki) = -5.83F + 0.64 pi + 7.41 (r = 0.90).