Structural Modification of the Designer Stimulant α-Pyrrolidinovalerophenone (α-PVP) Influences Potency at Dopamine Transporters.

α-Pyrrolidinovalerophenone (α-PVP, 7) is an illegal synthetic stimulant that is being sold on the clandestine market as "flakka" and "gravel". The potent pharmacological effects of α-PVP are presumably mediated by inhibition of dopamine uptake at the dopamine transporter (DAT). However, little is known about how structural modification of α-PVP influences activity at DAT. Eleven analogs of α-PVP were synthesized and examined for their ability to inhibit uptake of [(3)H]dopamine and [(3)H]serotonin in rat brain synaptosomes. None of the analogs significantly inhibited [(3)H]serotonin uptake when tested at 10 µM at the serotonin transporter (SERT). All of the analogs behaved as DAT reuptake inhibitors, but potencies varied over a >1500-fold range. Potency was primarily associated with the nature of the α-substituent, with the more bulky substituents imparting the highest potency. Expansion of the pyrrolidine ring to a piperidine reduced potency up to 10-fold, whereas conformational constraint in the form of an aminotetralone resulted in the least potent compound. Our study provides the first systematic and comparative structure-activity investigation on the ability of α-PVP analogs to act as inhibitors of DAT.

Stereoselective Actions of Methylenedioxypyrovalerone (MDPV) To Inhibit Dopamine and Norepinephrine Transporters and Facilitate Intracranial Self-Stimulation in Rats.

The designer stimulant methylenedioxypyrovalerone (MDPV) is a potent reuptake inhibitor at transporters for dopamine (DAT) and norepinephrine (NET) that produces a constellation of abuse-related behavioral effects. MDPV possesses a chiral center, and the abused formulation of the drug is a racemic mixture, but no data are available on the pharmacology of its isomers. Here, the individual optical isomers of MDPV were prepared and examined with respect to their neurochemical actions on neurotransmitter reuptake and behavioral effects in an assay of intracranial self-stimulation (ICSS) in rats. In assays of DAT uptake inhibition, S(+)MDPV (EC50 = 2.13 nM) was more potent than either (±)MDPV (EC50 = 4.85 nM) or R(-)MDPV (EC50 = 382.80 nM); the three drugs were less potent at NET uptake inhibition, with the same rank order of potency. Neither racemic MDPV nor its optical isomers inhibited the reuptake of serotonin at concentrations up to 10 µM. S(+)MDPV produced an abuse-related and dose-dependent facilitation of ICSS, and the potency of S(+)MDPV (significant facilitation at doses ≥ 0.1 mg/kg) was greater than that of the racemate (significant facilitation at doses ≥ 0.32 mg/kg). R(-)MDPV failed to alter ICSS at doses up to 100 times greater than the lowest effective dose of S(+)MDPV. The results indicate that abuse-related neurochemical and behavioral effects of racemic MDPV reside primarily with its S(+) isomer.

Quantitative structure-activity relationship analysis of the pharmacology of para-substituted methcathinone analogues.

BACKGROUND AND PURPOSE: Methcathinone (MCAT) is a potent monoamine releaser and parent compound to emerging drugs of abuse including mephedrone (4-CH3 MCAT), the para-methyl analogue of MCAT. This study examined quantitative structure-activity relationships (QSAR) for MCAT and six para-substituted MCAT analogues on (a) in vitro potency to promote monoamine release via dopamine and serotonin transporters (DAT and SERT, respectively), and (b) in vivo modulation of intracranial self-stimulation (ICSS), a behavioural procedure used to evaluate abuse potential. Neurochemical and behavioural effects were correlated with steric (Es ), electronic (σp ) and lipophilic (πp ) parameters of the para substituents. EXPERIMENTAL APPROACH: For neurochemical studies, drug effects on monoamine release through DAT and SERT were evaluated in rat brain synaptosomes. For behavioural studies, drug effects were tested in male Sprague-Dawley rats implanted with electrodes targeting the medial forebrain bundle and trained to lever-press for electrical brain stimulation. KEY RESULTS: MCAT and all six para-substituted analogues increased monoamine release via DAT and SERT and dose- and time-dependently modulated ICSS. In vitro selectivity for DAT versus SERT correlated with in vivo efficacy to produce abuse-related ICSS facilitation. In addition, the Es values of the para substituents correlated with both selectivity for DAT versus SERT and magnitude of ICSS facilitation. CONCLUSIONS AND IMPLICATIONS: Selectivity for DAT versus SERT in vitro is a key determinant of abuse-related ICSS facilitation by these MCAT analogues, and steric aspects of the para substituent of the MCAT scaffold (indicated by Es ) are key determinants of this selectivity.

Steric parameters, molecular modeling and hydropathic interaction analysis of the pharmacology of para-substituted methcathinone analogues.

BACKGROUND AND PURPOSE: There is growing concern over the abuse of certain psychostimulant methcathinone (MCAT) analogues. This study extends an initial quantitative structure-activity relationship (QSAR) investigation that demonstrated important steric considerations of seven 4- (or para-)substituted analogues of MCAT. Specifically, the steric character (Taft's steric ES ) of the 4-position substituent affected in vitro potency to induce monoamine release via dopamine and 5-HT transporters (DAT and SERT) and in vivo modulation of intracranial self-stimulation (ICSS). Here, we have assessed the effects of other steric properties of the 4-position substituents. EXPERIMENTAL APPROACH: Definitive steric parameters that more explicitly focus on the volume, width and length of the MCAT 4-position substituents were assessed. In addition, homology models of human DAT and human SERT based upon the crystallized Drosophila DAT were constructed and docking studies were performed, followed by hydropathic interaction (HINT) analysis of the docking results. KEY RESULTS: The potency of seven MCAT analogues at DAT was negatively correlated with the volume and maximal width of their 4-position substituents, whereas potency at SERT increased as substituent volume and length increased. SERT/DAT selectivity, as well as abuse-related drug effects in the ICSS procedure, also correlated with the same parameters. Docking solutions offered a means of visualizing these findings. CONCLUSIONS AND IMPLICATIONS: These results suggest that steric aspects of the 4-position substituents of MCAT analogues are key determinants of their action and selectivity, and that the hydrophobic nature of these substituents is involved in their potency at SERT.

Abuse-related and abuse-limiting effects of methcathinone and the synthetic "bath salts" cathinone analogs methylenedioxypyrovalerone (MDPV), methylone and mephedrone on intracranial self-stimulation in rats.

RATIONALE: Abuse of synthetic cathinones, popularized as "bath salts," has increased dramatically in the USA since their debut in 2010. Preclinical behavioral studies may clarify determinants of the abuse-related effects produced by these compounds. OBJECTIVES: This study examined behavioral effects of (±)-methcathinone, (±)-3,4-methylenedioxypyrovalerone (MDPV), (±)-3,4-methylenedioxymethcathinone (methylone), and (±)-4-methylmethcathinone (mephedrone) in rats using intracranial self-stimulation (ICSS). METHODS: Male Sprague-Dawley rats (n = 18) with electrodes targeting the medial forebrain bundle responded for multiple frequencies of brain stimulation and were tested in two phases. First, dose-effect curves for methcathinone (0.1-1.0 mg/kg), MDPV (0.32-3.2 mg/kg), methylone (1.0-10 mg/kg), and mephedrone (1.0-10 mg/kg) were determined. Second, time courses were determined for effects produced by the highest dose of each compound. RESULTS: Methcathinone produced dose- and time-dependent facilitation of ICSS. MDPV, methylone, and mephedrone produced dose- and time-dependent increases in low rates of ICSS maintained by low brain stimulation frequencies, but also produced abuse-limiting depression of high ICSS rates maintained by high brain stimulation frequencies. Efficacies to facilitate ICSS were methcathinone ≥ MDPV ≥ methylone > mephedrone. Methcathinone was the most potent compound, and MDPV was the longest acting compound. CONCLUSIONS: All compounds facilitated ICSS at some doses and pretreatment times, which is consistent with abuse liability for each of these compounds. However, efficacies of compounds to facilitate ICSS varied, with methcathinone displaying the highest efficacy and mephedrone displaying the lowest efficacy to facilitate ICSS.

Pharmacophore identification for sigma-1 (sigma1) receptor binding: application of the "deconstruction-reconstruction-elaboration" approach.

At least two different types of sigma (sigma1 and sigma2) receptors have been identified. A structural feature common to high-affinity (Ki <10 nM) sigma1 ligands is: C-N(R)-X-Ph; both C and Ph are associated with regions of bulk tolerance. Numerous other ligands bind, but typically do so with lower affinity.

Binding of beta-carbolines at 5-HT(2) serotonin receptors.

A series of ring-substituted (i.e., methoxy and bromo) 3,4-dihydro- and 1,2,3,4-tetrahydro-beta-carbolines was examined at 5-HT(2A) and 5-HT(2C) serotonin receptors. Whereas most of the methoxy-substituted derivatives typically displayed affinities similar to their unsubstituted parents, certain (particularly 8-substituted) bromo derivatives displayed enhanced affinity. A binding profile was obtained for selected beta-carbolines.

Behavioral characterization of 2-O-desmethyl and 5-O-desmethyl metabolites of the phenylethylamine hallucinogen DOM.

The present investigation was undertaken to test the hypothesis that known metabolites of the phenylethylamine hallucinogen 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM) are pharmacologically active. This hypothesis was tested by evaluating the ability of racemic DOM metabolites 2-O-desmethyl DOM (2-DM-DOM) and 5-O-desmethyl DOM (5-DM-DOM) to substitute for the stimulus properties of (+)lysergic acid diethylamide (LSD). The data indicate that both metabolites are active in LSD-trained subjects and are significantly inhibited by the selective 5-HT(2A) receptor antagonist M100907. Full generalization of LSD to both 2-DM-DOM and 5-DM-DOM occurred, and 5-DM-DOM was slightly more potent than 2-DM-DOM. Similarly, 5-DM-DOM had a slightly higher affinity than 2-DM-DOM for both 5-HT(2A) and 5-HT(2C) receptors. Additionally, it was of interest to determine if the formation of active metabolite(s) resulted in a temporal delay associated with maximal stimulus effects of DOM. We postulated that if metabolite formation resulted in the aforementioned delay, direct administration of the metabolites might result in maximally stable stimulus effects at an earlier pretreatment time. This hypothesis was tested by evaluating (1) the time point at which DOM produces the greatest degree of LSD-appropriate responding, (2) the involvement of 5-HT(2A) receptor in the stimulus effects of DOM at various pretreatment times by administration of M100907 and (3) the ability of 2-DM-DOM and 5-DM-DOM to substitute for the stimulus properties of LSD using either 15- or 75-min pretreatment time. The data indicate that (a) the DOM stimulus produces the greatest degree of LSD-appropriate responding at the 75-min time point in comparison with earlier pretreatment times and (b) the stimulus effects of DOM are differentially antagonized by M100907 and this effect is a function of DOM pretreatment time prior to testing. Both 2-DM-DOM and 5-DM-DOM were found to be most active, at all doses tested, using a 75-min versus a 15-min pretreatment time. The present data do not permit unequivocal acceptance or rejection of the hypothesis that active metabolites of (-)-DOM provide a full explanation of the observed discrepancy between brain levels of (-)-DOM and maximal stimulus effects.

Binding of nicotine and homoazanicotine analogues at neuronal nicotinic acetylcholinergic (nACh) receptors.

A total of 20 substituted analogues of nicotine (1a) and homoazanicotine (3a) were examined in order to determine whether or not they might bind in a similar manner at alpha4beta2 nicotinic acetylcholinergic (nACh) receptors. It was found that parallel structural changes in the two series resulted in parallel shifts in affinity. Evidence suggests that the two series are binding in a comparable fashion.

Investigation of the affinities of two new beta-carbolines for rat brain imidazoline(2) receptors.

The beta-carbolines are possible endogenous ligands and modulators of the imidazoline binding sites (I-BSs). Relatively little is known about this class of compound and its interaction with the I-BS. Presented here are the binding data for two aryl ring substituted dihydro-beta-carbolines at the imidazoline(2) binding site (I(2)-BS) and alpha(2)-adrenoceptor: BG-326 (5-bromo-4,9-dihydro-3H-b-carboline) and BG-350 (5-methoxy-4,9-dihydro-3H-b-carboline). Both compounds show good affinity and selectivity for the I(2)-BS.

Homoazanicotine: a structure-affinity study for nicotinic acetylcholine (nACh) receptor binding.

We have recently identified 3-[(1-methyl)-4,5-dihydro-1H-imidazol-2-yl)methyl]pyridine (homoazanicotine, 8) as a novel nicotinic acetylcholinergic (nACh) receptor ligand. In the present investigation, after we determined that 8 binds selectively at nicotinic (K(i) = 7.8 nM) vs muscarinic (K(i) > 10,000 nM) acetylcholinergic receptors, we examined its structure-affinity relationships for nACh receptor binding. The features investigated included the influence of (i) the composition of connector that separates the two rings, (ii) the N-methyl group, (iii) the ring opening of the imidazoline ring, (iv) the pyridine nitrogen atom, and (v) the aromatization of the imidazoline ring on nACh receptor affinity. As with nicotine, the parent structure seems optimal and most structural changes reduce nACh receptor affinity. Also, as with nicotine analogues, alteration of the spacer group influences affinity in a manner that is somewhat different than that seen with the parent structure.

Discriminative stimulus properties of alpha-ethyltryptamine optical isomers.

alpha-Ethyltryptamine (alpha-ET) possesses central stimulant and hallucinogenic activity. Also, in tests of stimulus generalization using rats trained to discriminate the controlled substance analog (i.e., designer drug) N-methyl-1-(3,4-methylenedioxyphenyl)-2-aminopropane (MDMA) from vehicle, alpha-ET substituted for MDMA. These previous studies employed racemic alpha-ET. Because psychoactive phenylalkylamines with abuse potential can produce one or more of three distinct stimulus effects (i.e., amphetamine-, DOM- and/or PMMA-like effects) in animals trained to discriminate either the stimulant (+)amphetamine, the hallucinogen 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM), or N-methyl-1-(4-methoxyphenyl)-2-aminopropane (PMMA) from vehicle, and because these effects can be stereoselective, the individual optical isomers of alpha-ET were examined in groups of animals trained to discriminate (+)amphetamine, DOM, PMMA and MDMA from saline vehicle. (-)alpha-ET (ED(50)=7.8 mg/kg), but not (+)alpha-ET (maximum of 53% drug-appropriate responding), substituted for (+)amphetamine, whereas (+)alpha-ET (ED(50)=2.7 mg/kg), but not (-)alpha-ET (maximum of 33% drug-appropriate responding), substituted for DOM. Both optical isomers of alpha-ET substituted for PMMA and MDMA with ED(50) values of 1.6 and 1.4 mg/kg (PMMA-trained animals) and 1.3 and 2.0 mg/kg (MDMA-trained animals) for (-)alpha-ET and (+)alpha-ET, respectively. The results of this investigation suggest that both optical isomers of alpha-ET are capable of producing an MDMA/PMMA-like effect at nearly comparable doses, and that the stimulant or amphetamine-like nature of alpha-ET resides primarily with its (-)isomer whereas hallucinogenic or DOM-like character resides primarily with the (+)enantiomer.

beta-carboline binding to imidazoline receptors.

A series of beta-carbolines were prepared and their affinities for imidazoline (I(1) and I(2)) sites evaluated. Selected compounds were also examined at alpha(2)-adrenoceptors. Some of the beta-carbolines were found to bind with high affinity to I(2)-sites and this affinity was dependent on both the planarity of the molecule and the presence of the aryl ring substituents. Good I(1)-affinity was observed with two of the compounds but none of the tested compounds bound to alpha(2)-adrenoceptors. The hallucinogenic properties of beta-carbolines have been linked to activity at 5-HT receptors, in particular 5-HT(2), however, it is apparent from this study that many of these compounds display substantially higher affinity for the imidazoline sites. This finding, and those showing modulation of some behavioural effects of morphine by I(2)-ligands, suggests that imidazoline sites may be interesting new targets in drug abuse research.

1-[2-methoxy-5-(3-phenylpropyl)]-2-aminopropane unexpectedly shows 5-HT(2A) serotonin receptor affinity and antagonist character.

Certain phenylethylamines, such as 1-(4-bromo-2,5-dimethoxyphenyl)-2-aminopropane (DOB; 1a), are high-affinity 5-HT(2) agonists. Previous structure-affinity studies have concluded that both the 2,5-dimethoxy substitution pattern and the nature of substituents at the 4-position are important determinants of high affinity. We recently demonstrated that replacement of the bromo group of DOB with a 3-(phenyl)propyl substituent results in retention of affinity and that, counter to established structure-affinity relationships, the 2,5-dimethoxy substitution pattern is no longer a requirement for the binding. The present investigation extends these findings by examining a series of analogues, 3, lacking a 5-methoxy group. It was additionally found that shifting the phenylalkyl substituent from the 4- to the 5-position (e.g., 4i) also results in retention of affinity. For example, 1-(2-methoxy-5-(3-phenylpropyl)-2-aminopropane (6; the alpha-methyl derivative of 4i) binds at 5-HT(2A) receptors with high affinity (K(i) = 13 nM) and possesses 5-HT(2A) antagonist character. Thus, not only is the 2,5-dimethoxy substitution pattern not a requirement for the binding of certain phenylethylamines at 5-HT(2A) receptors, the presence of a 4-position substituent (previously thought to serve as a modulator of affinity of DOB-like agents) is also not required. Striking differences in the 5-HT(2A) binding requirements of the present compounds as compared to DOB-like agents suggest multiple substituent-dependent modes of binding.

The binding of arylguanidines at 5-HT(3) serotonin receptors: a structure-affinity investigation.

The 5-HT(3) receptor binding affinities of nine pairs of aryl-substituted arylguanidines and arylbiguanides were examined and the results suggest the likelihood that both classes of agents utilize common receptor binding features. The effects of structural modification were also examined using CoMFA. 1-(3,4,5-Trichlorophenyl)guanidine (5-HT(3) K(i)=0.7 nM) was identified as a very high-affinity arylguanidine. The structures of the high-affinity arylguanidines are inconsistent with current 5-HT(3) pharmacophore models.

PMMA-stimulus generalization to the optical isomers of MBDB and 3,4-DMA.

Psychoactive phenylisopropylamines can produce one or more of several different stimulus effects in animals. These effects are typified by the hallucinogen 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM), the central stimulant amphetamine, and by N-methyl-1-(4-methoxyphenyl)-2-aminopropane (PMMA), an agent whose actions are not yet well understood. The optical isomers of two phenylisopropylamines known to lack DOM and amphetamine-stimulus character, that is N-methyl-1-(3,4-methylenedioxyphenyl)-2-aminobutane (MBDB) and 1-(3,4-dimethoxyphenyl)-2-aminopropane (3,4-DMA), were examined in rats trained to discriminate 1.25 mg/kg of PMMA from vehicle. The PMMA stimulus (ED(50)=0.4 mg/kg) generalized to all four agents: S(+)-MBDB (ED(50)=0.8 mg/kg), R(-)-MBDB (ED(50)=2.0 mg/kg), S(+)-3,4-DMA (ED(50)=2.6 mg/kg) and R(-)-3,4-DMA (ED(50)=3.9 mg/kg). The results show that these agents produce stimulus effects similar to those produced by PMMA. Both isomers of MBDB have been previously demonstrated to substitute for N-methyl-1-(3,4-methylenedioxyphenyl)-2-aminopropane (MDMA) in rats trained to discriminate MDMA from vehicle, but MBDB-trained animals failed to recognize DOM or amphetamine. Similar results were obtained with the 3,4-DMA optical isomers in the present investigation using rats trained to discriminate MDMA, DOM or (+)-amphetamine from vehicle; both isomers of 3,4-DMA substituted for an MDMA stimulus, but not for a DOM or amphetamine stimulus. Taken together, the evidence suggests that PMMA, S(+)-MBDB, R(-)-MBDB, S(+)-3,4-DMA, R(-)-3,4-DMA, and S(+)-MDMA can produce common stimulus effects in rats. The present findings also better define the PMMA stimulus and the structural requirements necessary to produce this type of stimulus effect.

Imidazoline-modified benzylimidazolines as h5-HT(1D/1B) serotonergic ligands.

Sumatriptan, a h5-HT1D and h5-HT1B receptor agonist used clinically as a migraine-abortive, produces certain side effects thought to result from its affinity for h5-HT1B receptors. The present investigation extends our work with benzylimidazolines as novel non-tryptamine h5-HT(1D/1B) ligands. The effect of N-methylation, N-benzylation, ring-aromatization, and variation of the imidazoline ring on affinity both at h5-HT1D and h5-HT1B receptors was examined. Several compounds were identified with good affinity and enhanced (i.e., > 100-fold) h5-HT1D versus hS-HT1B selectivity.

Influence of chain length and N-alkylation on the selective serotonin receptor ligand 9-(aminomethyl)-9,10-dihydroanthracene.

Comparison of the serotonin 5-HT2A receptor affinities of chain lengthened and N-alkylated analogues of the novel ligand 9-aminomethyl-9,10-dihydroanthracene (AMDA) and a structurally similar prototypical tricyclic amine imipramine suggests that the two agents bind to the receptor in different fashions. The demonstration that AMDA is highly selective for serotonin receptors (5-HT2A, K = 20nM; 5-HT2C, Ki=43nM) versus the dopamine D2 receptor (Ki>10,000nM), as well as the serotonin and norepinephrine transporters (Ki>10,000nM) further suggests that AMDA and the nonselective ligand imipramine interact with these target macromolecules in different ways.

Exploring the relationship between binding modes of 9-(aminomethyl)-9,10-dihydroanthracene and cyproheptadine analogues at the 5-HT2A serotonin receptor.

Comparison of the serotonin 5-HT2A receptor affinities of a parallel series of structural analogues of the novel ligand 9-aminomethyl-9,10-dihydroanthracene (AMDA) and a structurally similar prototypical tricyclic amine cyproheptadine suggests that the two agents bind to the receptor in different fashions. Examination of ligand-receptor model complexes supports the experimental data and suggests a potential origin for the differences in binding modes.

Chain-lengthened and imidazoline analogues of nicotine.

Analogues of nicotine (1) and azanicotine (3) were prepared with an additional methylene group inserted between the two rings (i.e., homonicotine and homoazanicotine; 6 and 5, respectively). Although 6 (Ki = 3110 nM) and 3 (Ki = 206 nM) bind at nACh receptors with > or = 100-fold lower affinity than nicotine (Ki = 2.1 nM), 5 displays high affinity (Ki = 7.8 nM). Like nicotine (ED50 = 12 microg/mouse), both 3 and 5 (ED50 = 21 and 19 microg/mouse, respectively) produced antinociceptive activity in the tail-flick assay following intrathecal administration. The antinociceptive actions of 3 and 5, unlike those of nicotine, were not antagonized by mecamylamine. Compounds 3 and 5 might represent novel analgesic agents that act via a non-nicotinic mechanism, or via a nicotinic mechanism that is distinct from that mediating the antinociceptive actions of nicotine.