Biochemical and behavioral characterization of novel methylphenidate analogs.

As part of a project to develop treatment agents for cocaine abuse, (+/-)-threo-methylphenidate (TMP) and 11 analogs were characterized biochemically and behaviorally to assess their potential as anti-cocaine medications. The compounds contained aryl and/or nitrogen substitutions, and/or replacement of the ester function by an alcohol or ether. All of the analogs, except for the N-methyl-substituted compounds, showed increased inhibitory potency against (3)H-(-)-2-beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane 1,5-naphthalenedisulfonate ([(3)H]WIN 35,428) ([(3)H]WIN) binding to the dopamine transporter, compared with TMP. In general, parallel results were obtained for inhibition of [(3)H]dopamine ([(3)H]DA) uptake. Although compounds with N-substitutions were proportionally less potent at blocking DA uptake than WIN binding (compared with the unsubstituted compounds), one such compound that was 6-fold more potent against [(3)H]WIN binding than [(3)H]DA uptake did not attenuate inhibition by cocaine of synaptosomal [(3)H]DA transport. The compounds were significantly less potent in displacing [(3)H]citalopram binding from the serotonin transporter. In cocaine discrimination studies in rats, all but two of the analogs (both N-substituted) completely generalized with the cocaine stimulus. Robust positive correlations were observed between potency in the drug discrimination assay and activity at the dopamine transporter, but not the serotonin transporter. When tested for their ability to alter cocaine discrimination, four of the analogs (three of which had N-substitutions and shallow dose-response curves as cocaine substitutes) actually enhanced cocaine discrimination, often at combined doses of cocaine and test compound that were inactive when given separately. Taken together, the results suggest that TMP analogs may have potential as substitution therapies for the treatment of cocaine abuse.

Synthesis and pharmacology of site specific cocaine abuse treatment agents: a new synthetic methodology for methylphenidate analogs based on the Blaise reaction.

In order to make new analogs of the dopamine (DA) uptake inhibitor methylphenidate, a synthetic methodology based on the Blaise reaction was developed. The reaction between alpha-bromophenylacetic acid esters, zinc and alpha-cyano-omega-mesylates gave stable primary enamines. After reduction of the enamines with cyanoborohydride, the amines could be cyclized to methylphenidate analogs in which the amine ring size and aromatic ring were varied. These compounds were tested for inhibitory potency against [(3)H]WIN 35,428 binding to the cocaine recognition site and [(3)H]DA uptake using rat striatal tissue. When the heterocyclic ring size was varied, the six-membered ring of methylphenidate appeared to be the optimum ring size. When the aryl ring was varied the 4-trifluoromethylphenyl analog was less potent than methylphenidate, the beta-naphthyl congener was considerably more potent, whereas the alpha-naphthyl congener was less potent. Most of the compounds tested had ratios of uptake to binding inhibition (discrimination ratio) that were similar to cocaine and were therefore not lead compounds for the development of cocaine antagonists.

Synthesis and pharmacology of site-specific cocaine abuse treatment agents: 2-substituted-6-amino-5-phenylbicyclo[2.2.2]octanes.

A series of 2-substituted-6-amino-5-phenylbicyclo[2.2.2]octanes was synthesized and tested for inhibitor potency in [(3)H]WIN 35,428 (WIN) binding at the dopamine (DA) transporter and [(3)H]DA uptake assays. To demonstrate transporter selectivity for the compounds, inhibitor potency was also tested using [(3)H]nisoxetine and [(3)H]paroxetine binding assays for the norepinephrine (NE) and serotonin (5-HT) transporters, respectively. Synthesis was accomplished by bisannulation of the enamine derived from phenylacetaldehyde and dimethylamine with 2-cyclohexenone to give a mixture of endo- and exo-trans-6-amino-5-phenylbicyclo[2.2. 2]octan-2-ones. The separated ketones were reduced to the four diastereomeric alcohols which were converted to acetyl and benzoyl esters. The ketones, alcohols, and acetyl esters generally have low affinity for the three transporters and do not effectively inhibit the uptake of [(3)H]DA. In all cases, the benzoates show significantly greater inhibition of WIN binding compared to the corresponding ketones, alcohols, or acetate esters. One compound, (1R/S,4R/S)-6R/S-(N,N-dimethylamino)-5R/S-phenylbicyclo[2.2. 2]oct-2S/R-yl benzoate, is almost as potent as cocaine in binding to the DA transporter (IC(50) = 270 nM versus 159 nM for cocaine). The C-2 epimer, (1R/S,4R/S)-6R/S-(N, N-dimethylamino)-5R/S-phenylbicyclo[2.2.2]oct-2R/S-yl benzoate, was selective and potent in binding to the 5-HT transporter (IC(50) = 53 nM versus 1050 nM for cocaine) as compared to the DA transporter (IC(50) = 358 nM). A preliminary molecular modeling study of the benzoyl esters indicates that their relative potencies in the WIN binding assay are not correlated to the nitrogen to benzoate phenyl (centroid) distance or to the deviation of the nitrogen from the plane defined by the benzoate ring.

Synthesis and pharmacology of site-specific cocaine abuse treatment agents: 2-(aminomethyl)-3-phenylbicyclo[2.2.2]- and -[2.2.1]alkane dopamine uptake inhibitors.

As part of a program to develop site-specific medications for cocaine abuse, a series of 2-(aminomethyl)-3-phenylbicyclo[2.2.2]- and -[2.2.1]alkane derivatives was synthesized and tested for inhibitory potency in [3H]WIN 35,428 binding and [3H]dopamine uptake assays using rat striatal tissue. Selected compounds were tested for their ability to substitute for cocaine in rat drug discrimination tests. Synthesis was accomplished by a series of Diels-Alder reactions, using cis- and trans-cinnamic acid derivatives (nitrile, acid, acid chloride) with cyclohexadiene and cyclopentadiene. Standard manipulations produced the aminomethyl side chain. Many of the compounds bound with high affinity (median IC50 = 223 nM) to the cocaine binding site as marked by [3H]WIN 35,428. Potency in the binding assay was strongly enhanced by chlorine atoms in the 3- and/or 4-position on the aromatic ring and was little affected by corresponding methoxy groups. In the [2.2.2] series there was little difference in potency between cis and trans compounds or between N, N-dimethylamines and primary amines. In the [2.2.1] series the trans exo compounds tended to be least potent against binding, whereas the cis exo compounds were the most potent (4-Cl cis exo: IC50 = 7.7 nM, 27-fold more potent than 4-Cl trans-exo). Although the potencies of the bicyclic derivatives in the binding and uptake assays were highly correlated, some of the compounds were 5-7-fold less potent at inhibiting [3H]dopamine uptake than [3H]WIN 35,428 binding (for comparison, cocaine has a lower discrimination ratio (DR) of 2.5). The DR values were higher for almost all primary amines and for the trans-[2.2.2] series as compared to the cis-[2.2.2]. Most of the compounds had Hill coefficients approaching unity, except for the [2. 2.2] 3,4-dichloro derivatives, which all had nH values of about 2.0. Two of the compounds were shown to fully substitute for cocaine in drug discrimination tests in rats, and one had a very long duration of action.

Effects of methylphenidate analogues on phenethylamine substrates for the striatal dopamine transporter: potential as amphetamine antagonists?

Methylphenidate (MPD) was found to inhibit competitively the striatal dopamine transporter (DAT) and bind at sites on the DAT in common with both cocaine (a non-substrate site ligand) and amphetamine (a substrate site ligand). Some methylphenidate analogues modified on the aromatic ring and/or at the nitrogen were tested to determine whether the profile of inhibition could be altered. None was found to stimulate the release of dopamine in the time frame (< or = 60 s) of the experiments conducted, and each of the analogues tested was found to noncompetitively inhibit the transport of dopamine. It was found that halogenating the aromatic ring with chlorine (threo-3,4-dichloromethylphenidate hydrochloride; compound 1) increased the affinity of MPD to inhibit the transport of dopamine. A derivative of MPD with simultaneous, single methyl group substitutions on the phenyl ring and at the nitrogen (threo-N-methyl-4-methylphenidate hydrochloride; compound 2) bound at a site in common with MPD. A benzyl group positioned at the nitrogen (threo-N-benzylmethylphenidate hydrochloride; compound 3) imparted properties to the inhibitor in which binding at substrate and non-substrate sites could be distinguished. This analogue bound at a mutually interacting site with that of methylphenidate and had a K(int) value of 4.29 microM. Furthermore, the N-substituted analogues (compounds 2 and 3), although clearly inhibitors of dopamine transport, were found to attenuate dramatically the inhibition of dopamine transport by amphetamine, suggesting that the development of an antagonist for substrate analogue drugs of abuse may be possible.

Fourphit, an acylating phencyclidine derivative, attenuates cocaine-induced hyperactivity in rats.

Fourphit (4-isothiocyanato-1-[1-phenylcyclohexyl]piperidine), an acylating phencyclidine derivative that irreversibly inhibits stimulant binding to the dopamine transporter in vitro (Schweri et al., J. Pharmacol. Exp. Ther. 261:936-942, 1992), was tested in rats for its ability to block the increased locomotor activity caused by cocaine. Administration of Fourphit (20 mg/kg, i.v.) significantly reduced the hyperactivity caused by challenge with either 15 or 40 mg/kg (-)cocaine x HCl (i.p.) 24 h later. It also increased the amount of thigmotaxis and decreased the rearing frequency of rats given the higher dose of cocaine. Only negligible effects on behavior were found upon acute administration of the compound by itself, or in response to a saline challenge 24 h later. Activity during the dark cycle immediately following Fourphit administration, however, was moderately depressed. Contrary to the results predicted from its activity in vitro, Fourphit did not inhibit the ex vivo binding of [3H]methylphenidate to stimulant receptors in the striatal tissue of treated rats. These results show that Fourphit can antagonize some behavioral actions of cocaine, but these effects are not likely due to covalent modification of the site on the dopamine transporter recognized by cocaine.

Synthesis and pharmacology of potential cocaine antagonists. 2. Structure-activity relationship studies of aromatic ring-substituted methylphenidate analogs.

As part of a program to develop medications which can block the binding of cocaine to the dopamine transporter, yet spare dopamine uptake, a series of aromatic ring-substituted methylphenidate derivatives was synthesized and tested for inhibitory potency in [3H]WIN 35,428 binding and [3H]dopamine uptake assays using rat striatal tissue. Synthesis was accomplished by alkylation of 2-bromopyridine with anions derived from various substituted phenylacetonitriles. In most cases, erythro compounds were markedly less potent than the corresponding (+/-)-threo-methylphenidate (TMP; Ritalin) derivatives. The ortho-substituted compounds were much less potent than the corresponding meta- and/or para-substituted derivatives. The most potent compound against [3H]WIN 35,428 binding, m-bromo-TMP, was 20-fold more potent than the parent compound, whereas the most potent compound against [3H]dopamine uptake, m,p-dichloro-TMP, was 32-fold more potent. Threo derivatives with m- or p-halo substituents were more potent than TMP, while electron-donating substituents caused little change or small loss of potency. All of the derivatives had Hill coefficients approaching unity, except m,p-dichloro-TMP, which had an nH of 2.0. Although the potency of the (+/-)-methylphenidate derivatives in the two assays was highly correlated (R2 = 0.986), the compounds m-chloro-,m-methyl-, and p-iodo-TMP were 4-5-fold more potent at inhibiting [3H]-WIN 35,428 binding than [3H]dopamine uptake (cocaine has a ratio of 2.3). These and other compounds may be promising candidates for further testing as potential partial agonists or antagonists of cocaine.

Mercuric chloride and p-chloromercuriphenylsulfonate exert a biphasic effect on the binding of the stimulant [3H]methylphenidate to the dopamine transporter.

Mercuric chloride was found to have a biphasic effect on the binding of the radiolabeled stimulant [3H]methylphenidate to membranes from a crude synaptosomal preparation of rat striatal tissue. Binding was enhanced at low concentrations of HgCl2, reaching a maximum of 62% above control values at 2.5 microM HgCl2. It was inhibited in a dose-dependent manner at concentrations greater than 5 microM HgCl2, with an IC50 of 7.2 microM. The increase in binding observed at the low concentrations of HgCl2 was shown by Scatchard analysis to be due to an increase in the affinity of [3H]methylphenidate for its binding site on the dopamine transporter, while a decrease in both affinity and Bmax accompanied the reduction of [3H]methylphenidate binding observed at the higher concentrations of the inorganic mercury compound. The sodium salt of the organic mercurial p-chloromercuriphenylsulfonic acid also caused an increase in [3H]methylphenidate binding (159% above controls at 2,000 microM), followed by an immediate decrease in binding at higher concentrations of the reagent. Because both of these mercury-containing compounds have a high propensity for interacting with sulfhydryl groups, these data suggest that the cysteine residues in the dopamine transporter molecule may play an important role in the regulation of stimulant binding to the uptake complex.

Can stimulant binding and dopamine transport be differentiated? Studies with GBR 12783 derivatives.

Both 3- and 4-substituted GBR 12783 derivatives were synthesized in an effort to create site-directed cocaine antagonists. The potencies of these compounds to inhibit stimulant ([3H]WIN 35,428) binding and synaptosomal [3H]dopamine uptake were determined and compared with a large number of compounds assayed under identical experimental conditions. Three groups of compounds were identified which affected stimulant binding and dopamine transport to varying degrees. The 3-substituted GBR 12783 derivatives resembled the known non-amphetamine stimulant agents, in that they were approximately equipotent in inhibiting [3H]WIN 35,428 binding and [3H]dopamine transport. The 4-substituted GBR 12783 derivatives formed a second group that was approximately seven times more potent in the inhibition of [3H]dopamine uptake compared to [3H]WIN 35,428 binding. The third group, approximately one hundred-fold more potent at inhibiting [3H]dopamine uptake than stimulant binding, consisted mainly of substrates for the dopamine transporter. Although these GBR derivatives did not meet the criteria for potential cocaine antagonists, the results demonstrate that slight modifications of the molecular structure of a stimulant drug can differentially affect binding and transport. This finding holds great promise for the eventual development of a true pharmacological antagonist of cocaine.

Synthesis and pharmacology of irreversible affinity labels as potential cocaine antagonists: aryl 1,4-dialkylpiperazines related to GBR-12783.

As part of a program aimed at designing irreversible antagonists of the stimulant and reinforcing properties of cocaine, derivatives of GBR-12783 containing electrophilic substituents were synthesized. GBR-12783, a potent and selective inhibitor of both stimulant binding and dopamine transport, was modified to incorporate either isothiocyanate or maleimido groups at the meta- or para-positions in one phenyl ring of the geminal diphenyl portion of the molecule. The effect of these compounds, as well as their respective amino- or nitro-substituted precursors, on stimulant binding to rat striatal tissue was studied using the [3H]methylphenidate radioreceptor assay. Under the assay conditions used, the compounds were found to have IC50s (nM) ranging from 11.9 (m-nitro) to 1677 (p-maleimido); the parent compound, GBR-12783, had an IC50 of 12.0. Using a washout technique (repeated washing with 100 mM KCl) which completely removed the tightly bound, but reversible GBR-12783, both the m- and p-isothiocyanate compounds were found to irreversibly inhibit binding of [3H]methylphenidate to the stimulant recognition site. The m-maleimido derivative also irreversibly inhibited binding, albeit with lower efficacy than was observed with the isothiocyanate compounds. Neither the p-maleimido, nor the amino or nitro intermediates, were capable of irreversible inhibition.

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.

The enantiomeric specificity of the antihypertensive activity of 1-(phenylthio)-2-aminopropane, a synthetic substrate analogue for dopamine beta-monooxygenase.

We have found that (R,S)-1-(phenylthio)-aminopropane (4a), a synthetic alternate substrate for the terminal enzyme of norepinephrine biosynthesis, dopamine beta-monooxygenase (DBM), is both an indirect sympathomimetic and a potent antihypertensive agent in spontaneously hypertensive rats. We demonstrate herein that there is a distinct enantiospecific difference in the activities of (R)-1-(phenylthio)-2-aminopropane (4b) and (S)-1-(phenylthio)-2-aminopropane (4c). We find that 4c, the more potent DBM substrate analogue, exhibits both the indirect sympathomimetic activity and the antihypertensive activity previously observed for the racemate and inhibits the active transport of catecholamines at the nerve terminal. In contrast, 4b, which is less potent as a DBM substrate or as an inhibitor of catecholamine uptake, does not exhibit an indirect sympathomimetic effect and is not an effective antihypertensive agent. These results suggest that the greater selectivity of the S enantiomer for both the catecholamine reuptake transporter and the target enzyme DBM accounts for its greater potency as an indirect-acting sympathomimetic agent as well as its activity as an antihypertensive agent. These results are also consistent with the hypothesized mechanism of action of this class of sulfur-containing DBM substrate analogues.

N-ethylmaleimide irreversibly inhibits the binding of [3H]threo-(+-)-methylphenidate to the stimulant recognition site.

N-Ethylmaleimide, a nonspecific protein modifier which reacts selectively with the sulfhydryl group of cysteinyl residues under controlled conditions, irreversibly inhibited the binding of [3H]threo-(+/-)-methylphenidate to a subset of stimulant binding sites in striatal tissue membranes from the rat in vitro. The inhibition was marked by a decrease in the Bmax of binding of the radiolabelled stimulant drug, while the KD remained unchanged. Pretreatment with excess unlabelled methylphenidate afforded complete protection from inactivation of the binding site by N-ethylmaleimide. Uptake of [3H]dopamine into striatal synaptosomes was likewise reduced after treatment with N-ethylmaleimide; pretreatment with large concentrations of methylphenidate provided partial protection from inactivation of transport. These findings suggest that the stimulant recognition site on the dopamine transport complex contains one or more cysteinyl residues.

Metaphit inhibits dopamine transport and binding of [3H]methylphenidate, a proposed marker for the dopamine transport complex.

Metaphit, an acylating derivative of phencyclidine, was shown to interact with components of the dopamine nerve terminal in rat striatal tissue. This compound, previously demonstrated to be an irreversible inhibitor at the phencyclidine receptor, was shown in these experiments to irreversibly inhibit synaptosomal [3H]dopamine uptake. It also inhibited binding of [3H]methylphenidate to its recognition site, which is thought to be a subunit of the dopamine transporter. Although the inhibition was due primarily to a reduction in the binding and transport capacity of the systems studied, increases in the apparent KD of [3H]methylphenidate and the Km of [3H]dopamine were also observed. Differences in the behavior of Metaphit and phencyclidine in these dopaminergic systems compared to their effects on the NMDA receptor-linked phencyclidine receptor suggest that Metaphit may be interacting with two distinct molecular sites in the rat striatum.

Metaphit irreversibly inhibits [3H]threo-(+/-)-methylphenidate binding to rat striatal tissue.

Metaphit (1-[1-(3-isothiocyanatophenyl)cyclohexyl]-piperidine), a derivative of phencyclidine that contains an isothiocyanate group on the meta position of the aromatic ring, resembles its parent compound (phencyclidine) in its ability to inhibit the binding of the stimulant drug [3H]threo-(+/-)-methylphenidate to crude synaptosomal membranes from rat striatal tissue (IC50 = 1.4 and 6.2 microM for phencyclidine and Metaphit, respectively). Unlike phencyclidine, however, Metaphit appears to inhibit binding of the radiolabeled stimulant in an irreversible manner, as the degree of inhibition of binding of the stimulant does not diminish when the Metaphit-treated tissue is subjected to repeated washings before determination of the binding of [3H]threo-(+/-)-methylphenidate. This finding suggests that Metaphit may be a useful tool in the study of the molecular basis of stimulant action.

[3H]Threo-(+/-)-methylphenidate binding to 3,4-dihydroxyphenylethylamine uptake sites in corpus striatum: correlation with the stimulant properties of ritalinic acid esters.

Saturable and stereoselective binding sites for [3H]threo-(+/-)-methylphenidate were characterized in rat brain membranes. The highest density of [3H]threo-(+/-)-methylphenidate binding sites was found in the synaptosomal fraction of corpus striatum. Scatchard analysis revealed a single class of noninteracting binding sites with an apparent dissociation constant (KD) of 235 nM and a maximum number of binding sites (Bmax) of 13.4 pmol/mg protein. Saturable, high-affinity binding of [3H]threo-(+/-)-methylphenidate to striatal synaptosomal membranes was dependent on the presence of sodium ions. A good correlation (r = 0.88; p less than 0.001) was observed between the potencies of various psychotropic drugs in displacing [3H]threo-(+/-)-methylphenidate from these sites and their potencies as inhibitors of [3H]3,4-dihydroxyphenylethylamine ( [3H]dopamine) uptake into striatal synaptosomes. A good correlation (r = 0.85; p less than 0.001) was also observed between the potencies of a series of ritalinic acid esters in inhibiting [3H]threo-(+/-)-methylphenidate binding to striatal synaptosomal membranes and their potencies as motor stimulants in mice. These observations suggest that the binding sites for [3H]threo-(+/-)-methylphenidate described here are associated with a dopamine uptake or transport complex, and that these sites may mediate the motor stimulant properties of ritalinic acid esters such as methylphenidate.

The effects of surgical and chemical lesions on striatal [3H]threo-(+/-)-methylphenidate binding: correlation with [3H]dopamine uptake.

The specific binding of [3H]threo-(+/-)-methylphenidate to membranes prepared from rat striatum was significantly reduced following either surgical lesions of the medial forebrain bundle or intracerebroventricular administration of 6-hydroxydopamine. The decrease in the density of [3H]threo-(+/-)-methylphenidate binding sites in striatum following chemical or surgical denervation was highly correlated with the decrease in [3H]dopamine uptake. In contrast, intracerebroventricular administration of 5,7-dihydroxytryptamine, AF64A, or chronic parenteral administration of reserpine did not alter either the number of apparent affinity of [3H]threo-(+/-)-methylphenidate binding sites. These data suggest that the specific binding sites for [3H]-threo-(+/-)-methylphenidate in striatum are localized to dopaminergic nerve terminals, and may be associated with the dopamine transport complex.

Strain differences in susceptibility to the convulsant actions of 3-carbomethoxy-beta-carboline.

NIH mice were found to be approximately three-fold more sensitive than NIH General Purpose mice to the convulsant actions of 3-carbomethoxy-beta-carboline. The convulsant action of 3-carbomethoxy-beta-carboline has been previously demonstrated to be mediated via an interaction with C.N.S. benzodiazepine receptors. The characteristics of the benzodiazepine receptor from the two strains appeared to be identical with respect to both binding affinity and capacity for [3H]3-carbomethoxy-beta-carboline and [3H]diazepam, as well as the relative decreases in apparent receptor affinity for [3H] 3-carbomethoxy-beta-carboline in the presence of 10 microM gamma-aminobutyric acid. Although the rate of degradation of 3-carbomethoxy-beta-carboline in plasma was similar in the two strains, a marked difference in brain levels of the drug (or an active metabolite) was observed after in vivo administration. These results suggest that pharmacokinetic, rather than pharmacodynamic factors are primarily responsible for the observed strain differences in sensitivity to 3-carbomethoxy-beta-carboline.