Ashish P. Vartak

The vesicular monoamine transporter-2: An important pharmacological target for the discovery of novel therapeutics to treat methamphetamine abuse

Methamphetamine abuse escalates, but no approved therapeutics are available to treat addicted individuals. Methamphetamine increases extracellular dopamine in reward-relevant pathways by interacting at vesicular monoamine transporter-2 (VMAT2) to inhibit dopamine uptake and promote dopamine release from synaptic vesicles, increasing cytosolic dopamine available for reverse transport by the dopamine transporter (DAT). VMAT2 is the target of our iterative drug discovery efforts to identify pharmacotherapeutics for methamphetamine addiction. Lobeline, the major alkaloid in Lobelia inflata, potently inhibited VMAT2, methamphetamine-evoked striatal dopamine release, and methamphetamine self-administration in rats but exhibited high affinity for nicotinic acetylcholine receptors (nAChRs). Defunctionalized, unsaturated lobeline analog, meso-transdiene (MTD), exhibited lobeline-like in vitro pharmacology, lacked nAChR affinity, but exhibited high affinity for DAT, suggesting potential abuse liability. The 2,4-dicholorophenyl MTD analog, UKMH-106, exhibited selectivity for VMAT2 over DAT, inhibited methamphetamine-evoked dopamine release, but required a difficult synthetic approach. Lobelane, a saturated, defunctionalized lobeline analog, inhibited the neurochemical and behavioral effects of methamphetamine; tolerance developed to the lobelane-induced decrease in methamphetamine self-administration. Improved drug-likeness was afforded by the incorporation of a chiral N-1,2-dihydroxypropyl moiety into lobelane to afford GZ-793A, which inhibited the neurochemical and behavioral effects of methamphetamine, without tolerance. From a series of 2,5-disubstituted pyrrolidine analogs, AV-2-192 emerged as a lead, exhibiting high affinity for VMAT2 and inhibiting methamphetamine-evoked dopamine release. Current results support the hypothesis that potent, selective VMAT2 inhibitors provide the requisite preclinical behavioral profile for evaluation as pharmacotherapeutics for methamphetamine abuse and emphasize selectivity for VMAT2 relative to DAT as a criterion for reducing abuse liability of the therapeutic.

The Novel Pyrrolidine nor-Lobelane Analog UKCP-110 (cis-2,5-di-(2-Phenethyl)-pyrrolidine Hydrochloride) Inhibits VMAT2 Function, Methamphetamine-evoked Dopamine Release,and Methamphetamine Self-administration in Rats

Both lobeline and lobelane attenuate methamphetamine self-administration in rats by decreasing methamphetamine-induced dopamine release via interaction with vesicular monoamine transporter-2 (VMAT2). A novel derivative of nor-lobelane, cis-2,5-di-(2-phenethyl)-pyrrolidine hydrochloride (UKCP-110), and its trans-isomers, (2R,5R)-trans-di-(2-phenethyl)-pyrrolidine hydrochloride (UKCP-111) and (2S,5S)-trans-di-(2-phenethyl)-pyrrolidine hydrochloride (UKCP-112), were evaluated for inhibition of [3H]dihydrotetrabenazine binding and [3H]dopamine uptake by using a rat synaptic vesicle preparation to assess VMAT2 interaction. Compounds were evaluated for inhibition of [3H]nicotine and [3H]methyllycaconitine binding to assess interaction with the major nicotinic receptor subtypes. In addition, compounds were evaluated for inhibition of methamphetamine-evoked endogenous dopamine release by using striatal slices. The most promising compound, UKCP-110, was evaluated for its ability to decrease methamphetamine self-administration and methamphetamine discriminative stimulus cues and for its effect on food-maintained operant responding. UKCP-110, UKCP-111, and UKCP-112 inhibited [3H]dihydrotetrabenazine binding (Ki = 2.66 ± 0.37, 1.05 ± 0.10, and 3.80 ± 0.31 μM, respectively) and had high potency inhibiting [3H]dopamine uptake (Ki = 0.028 ± 0.001, 0.046 ± 0.008, 0.043 ± 0.004 μM, respectively), but lacked affinity at nicotinic receptors. Although the trans-isomers did not alter methamphetamine-evoked dopamine release, UKCP-110 inhibited (IC50 = 1.8 ± 0.2 μM; Imax = 67.18 ± 6.11 μM) methamphetamine-evoked dopamine release. At high concentrations, UKCP-110 also increased extracellular dihydroxyphenylacetic acid. It is noteworthy that UKCP-110 decreased the number of methamphetamine self-infusions, while having no effect on food-reinforced behavior or the methamphetamine stimulus cue. Thus, UKCP-110 represents a new lead in the development of novel pharmacotherapies for the treatment of methamphetamine abuse.

Pyrrolidine Analogues of Lobelane: Relationship of Affinity for the Dihydrotetrabenazine Binding Site with Function of the Vesicular Monoamine Transporter 2 (VMAT2)†

Ring size reduction of the central piperidine ring of lobelane yielded pyrrolidine analogues that showed marked inconsistencies in their ability to bind to the dihydrotetrabenazine (DTBZ) binding site on the vesicular monoamine transporter-2 (VMAT2) and their ability to inhibit VMAT2 function. The structure-activity relationships indicate that structural modification within the pyrrolidine series resulted in analogues that interact with two different sites, i.e., the DTBZ binding site and an alternative site on VMAT2 to inhibit transporter function.

Design, Synthesis and Interaction at the Vesicular Monoamine Transporter-2 of Lobeline Analogs: Potential Pharmacotherapies for the Treatment of Psychostimulant Abuse

The vesicular monoamine transporter-2 (VMAT2) is considered as a new target for the development of novel therapeutics to treat psychostimulant abuse. Current information on the structure, function and role of VMAT2 in psychostimulant abuse are presented. Lobeline, the major alkaloidal constituent of Lobelia inflata, interacts with nicotinic receptors and with VMAT2. Numerous studies have shown that lobeline inhibits both the neurochemical and behavioral effects of amphetamine in rodents, and behavioral studies demonstrate that lobeline has potential as a pharmacotherapy for psychostimulant abuse. Systematic structural modification of the lobeline molecule is described with the aim of improving selectivity and affinity for VMAT2 over neuronal nicotinic acetylcholine receptors and other neurotransmitter transporters. This has led to the discovery of more potent and selective ligands for VMAT2. In addition, a computational neural network analysis of the affinity of these lobeline analogs for VMAT2 has been carried out, which provides computational models that have predictive value in the rational design of VMAT2 ligands and is also useful in identifying drug candidates from virtual libraries for subsequent synthesis and evaluation.

Quinlobelane: a water-soluble lobelane analogue and inhibitor of VMAT2.

Replacing the phenyl groups in the structure of the VMAT2 inhibitor, lobelane with either pyridyl, quinolyl or indolyl groups affords novel analogues with improved water solubility. The synthetic methodologies reported herein also underscore the paucity of hydrogenation methods that offer selectivity in the synthesis of the different classes of heteroaromatic lobelane analogues. The quinolyl group was the only replacement for the phenyl group in lobelane that retained VMAT2 inhibition.

3-O-Phosphate ester conjugates of 17-β-O-{1-[2-carboxy-(2-hydroxy-4-methoxy-3-carboxamido)anilido]ethyl}1,3,5(10)-estratriene as novel bone-targeting agents

A series of 3-O-phosphorylated analogs (4-10) of a novel bone-targeting estradiol analog (3) were synthesized after a thorough study of the reaction of 3 with a selection of phosphoryl chlorides under a variety of reaction conditions. Evaluation of these novel phosphate analogs for affinity for hydroxyapatite revealed that they bind with equal or higher affinity when compared to the bone tissue accumulator, tetracycline.

Improved and Scalable Synthetic Route to the Synthon 17-β-(2-Carboxyethyl)-1,3,5(10)-estratriene: An Important Intermediate in the Synthesis of Bone-Targeting Estrogens

An improved, highly scalable methodology for the multigram-scale preparation of an important synthon, 17-β-(2-carboxyethyl)-1,3,5(10)-estratriene, is described. Previous approaches have failed to provide useful quantities of the analytically pure product because of facile retro-Michael breakdown of the β-alkoxy carbonyl precursors during workup and isolation operations. The synthetic approach described herein has been designed specifically to sidestep this problematic breakdown process. This new scalable method of preparation overcomes a major hurdle in the exploration of structure–activity relationships centered around novel estradiol derivatives with bone-targeting properties and also provides a scalable process for subsequent developmental work.

Effects of a lobelane analog on [11C]DTBZ binding to the VMAT2

Introduction: The in vivo PET imaging of the vesicular monoamine transporter type 2 (VMAT2) has become a valuable means for assessing losses of monoaminergic terminal density in animal and human studies. Both carbon-11 ([C]dihydrotetrabenazine, DTBZ) and fluorine-18 ([F]fluoropropyl-dihydrotetrabenazine, AV-133) radioligands are now in routine use. Recently, analogs of lobelane were synthesized that exhibited in vitro inhibition of vesicular transport of dopamine but low affinities for the [H]DTBZ binding site on the VMAT2 [1]. These compounds may be useful as pharmacological tools to study the VMAT2, and might provide a window to a second class of radioligands suitable for in vivo imaging of these transporters. We report here initial in vivo studies of one such compound, 1-methyl-cis-2,5-dibenzyl-pyrrolidine (1), that has a high potency for inhibition of dopamine transport into vesicles (19 nM) and low in vitro binding affinity for the DTBZ site (2.46 μM).