Phenyl ring-substituted lobelane analogs: inhibition of [³H]dopamine uptake at the vesicular monoamine transporter-2

Abstract

Lobeline attenuates the behavioral effects of methamphetamine via inhibition of the vesicular monoamine transporter (VMAT2). To increase selectivity for VMAT2, chemically defunctionalized lobeline analogs, including lobelane, were designed to eliminate nicotinic acetylcholine receptor affinity. The current study evaluated the ability of lobelane analogs to inhibit [³H]dihydrotetrabenazine (DTBZ) binding to VMAT2 and [³H]dopamine (DA) uptake into isolated synaptic vesicles and determined the mechanism of inhibition. Introduction of aromatic substituents in lobelane maintained analog affinity for the [³H]DTBZ binding site on VMAT2 and inhibitory potency in the [³H]DA uptake assay assessing VMAT2 function. The most potent (K(i) = 13-16 nM) analogs in the series included para-methoxyphenyl nor-lobelane (GZ-252B), para-methoxyphenyl lobelane (GZ-252C), and 2,4-dichlorphenyl lobelane (GZ-260C). Affinity of the analogs for the [³H]DTBZ binding site did not correlate with inhibitory potency in the [³H]DA uptake assay. It is noteworthy that the N-benzylindole-, biphenyl-, and indole-bearing meso-analogs 2,6-bis[2-(1-benzyl-1H-indole-3-yl)ethyl]-1-methylpiperidine hemifumarate (AV-1-292C), 2,6-bis(2-(biphenyl-4-yl)ethyl)piperidine hydrochloride (GZ-272B), and 2,6-bis[2-(1H-indole-3-yl)ethyl]-1-methylpiperidine monofumarate (AV-1-294), respectively] inhibited VMAT2 function (K(i) = 73, 127, and 2130 nM, respectively), yet had little to no affinity for the [³H]DTBZ binding site. These results suggest that the analogs interact at an alternate site to DTBZ on VMAT2. Kinetic analyses of [³H]DA uptake revealed a competitive mechanism for 2,6-bis(2-(4-methoxyphenyl)ethyl)piperidine hydrochloride (GZ-252B), 2,6-bis(2-(4-methoxyphenyl)ethyl)-1-methylpiperidine hydrochloride (GZ-252C), 2,6-bis(2-(2,4-dichlorophenyl)ethyl)piperidine hydrochloride (GZ-260C), and GZ-272B. Similar to methamphetamine, these analogs released [³H]DA from the vesicles, but with higher potency. In contrast to methamphetamine, these analogs had higher potency (>100-fold) at VMAT2 than DAT, predicting low abuse liability. Thus, modification of the lobelane molecule affords potent, selective inhibitors of VMAT2 function and reveals two distinct pharmacological targets on VMAT2.

Fig. 1.

Chemical structures of standards. TBZ is a benzoquinolizine compound that depletes vesicular neurotransmitter content by reversibly inhibiting VMAT2. Ro4-1284 is also a compound belonging to the benzoquinolizine family that influences storage of catecholamines in a similar manner. Lobeline is a lipophilic, nonpyridino alkaloid present in L. inflata. Lobelane is a defunctionalized, saturated meso-analog of lobeline.

Fig. 2.

Chemical structures of lobelane and derivative analogs bearing phenyl ring substituents. For clarity of presentation, compounds are grouped according to structural similarity of phenyl ring substituents: 1) compounds with an extended distance between the N-methyl and piperidine ring and analogs containing phenyl and methyl substituents; 2) compounds bearing moieties that contain oxygen; and 3) analogs bearing highly electronegative atoms/groups on the phenyl rings. GZ-246B, 2,6-bis[2-phenylethyl]piperidine hydrochloride; GZ-282, 2,6-bis(2-phenylethyl)-1-ethylpiperidine hydrochloride; GZ-289, 2,6-bis(2-phenylethyl)-1-n-propylpiperidine hydrochloride; GZ-253B, 2,6-bis(2-(4-methylphenyl)ethyl)piperidine hydrochloride; GZ-253C, 2,6-bis(2-(4-methylphenyl)ethyl)-1-methylpiperidine hydrochloride; GZ-273B, 2,6-bis(2-(2-methoxyphenyl)ethyl)piperidine hydrochloride; GZ-273C, 2,6-bis(2-(2-methoxyphenyl)ethyl)-1-methylpiperidine hydrochloride; GZ-270B, 2,6-bis(2-(4-hydroxyphenyl)ethyl)piperidine hydrochloride; GZ-250B, 2,6-bis(2-(3,4-methylenedioxyphenyl)ethyl)piperidine hydrochloride; GZ-250C, 2,6-bis(2-(3,4-methylenedioxyphenyl)ethyl)-1-methylpiperidine hydrochloride; GZ-261B, 2,6-bis(2-(3-methoxyphenyl)ethyl)piperidine hydrochloride; GZ-261C, 2,6-bis(2-(3-methoxyphenyl)ethyl)-1-methylpiperidine hydrochloride; GZ-266B, 2,6-bis(2-(4-acetoxyphenyl)ethyl)piperidine hydrochloride; GZ-274B, 2,6-bis(2-(2-fluorophenyl)ethyl)piperidine hydrochloride; GZ-274C, 2,6-bis(2-(2-fluorophenyl)ethyl)-1-methylpiperidine hydrochloride; GZ-260B, 2,6-bis(2-(2,4-dichlorophenyl)ethyl)piperidine hydrochloride; GZ-275B, 2,6-bis(2-(3-fluorophenyl)ethyl)piperidine hydrochloride; GZ-275C, 2,6-bis(2-(3-fluorophenyl)ethyl)-1-methylpiperidine hydrochloride; GZ-254B, 2,6-bis(2-(4-fluorophenyl)ethyl)piperidine hydrochloride; GZ-254C, 2,6-bis(2-(4-fluorophenyl)ethyl)-1-methylpiperidine hydrochloride; GZ-264B, 2,6-bis(2-(3-trifluoromethylphenyl)ethyl)piperidine hydrochloride; GZ-264C, 2,6-bis(2-(3-trifluoromethylphenyl)ethyl)-1-methylpiperidine hydrochloride.

Fig. 3.

Lobelane analogs containing bulky aromatic moieties. Compounds GZ-272B, AV-1-292C, and AV-1-294, which, respectively contain bulky biphenyl, N-benzylindole, and indole moieties, are shown.

Fig. 4.

Lobelane and phenyl-substituted lobelane analogs inhibit [³H]DTBZ binding to striatal vesicle membranes. Top, inhibition of [³H]DTBZ binding by VMAT2 standards and lobelane analogs whose structures share an extended distance between the N-methyl and the piperidine ring and contain phenyl, N-benzylindole, indole, and methyl substituents. Middle, inhibition of [³H]DTBZ binding by analogs bearing moieties that contain oxygen. Bottom, concentration-response curves for analogs bearing highly electronegative atoms or groups on the phenyl rings. Insets, the order of presentation for the analogs is from high to low affinity at the [³H]DTBZ binding site. Control represents [³H]DTBZ binding in the absence of compound. Data are mean (± S.E.M.) specific [³H]DTBZ binding as a percentage of control (1431 ± 97.24 fmol/mg; n = 3–4 rats/compound).

Fig. 5.

Lobelane and its phenyl ring-substituted analogs inhibit [³H]DA uptake in rat striatal synaptic vesicle preparations. Top, data are representative of inhibition of [³H]DA uptake by the VMAT2 standards, analogs with an extended distance between the N-methyl and the piperidine ring, and analogs containing phenyl, N-benzylindole, indole, and methyl substituents. Middle, inhibition of [³H]DA uptake by analogs bearing moieties that contain oxygen. Bottom, concentration response curves of analogs bearing highly electronegative atoms or groups on the phenyl rings. Insets, the order of presentation for the analogs is from high to low affinity for the [³H]DA uptake site. Control represents [³H]DA uptake in the absence of compound. Data are mean (± S.E.M.) specific [³H]DA uptake as a percentage of control (50.7 ± 2.59 pmol/min/mg; n = 3–5 rats/compound).

Fig. 6.

Lack of correlation between phenyl ring-substituted lobelane analog inhibition of [³H]DTBZ binding and [³H]DA uptake. Data presented are K_i values obtained from concentration-response curves for analog-induced inhibition of [³H]DTBZ binding and [³H]DA uptake (Figs. 4 and 5, respectively). Spearman analysis of these data indicates a lack of correlation (Spearman r = 0.22; p = 0.22) between the ability of the compounds of this series to inhibit [³H]DTBZ binding to VMAT2 and inhibit [³H]DA uptake at VMAT2.

Fig. 7.

Kinetic analysis of the inhibition of [³H]DA uptake by lobeline, lobelane, TBZ, GZ-252B, GZ-252C, GZ-272B, and GZ-260C. The mechanism of [³H]DA uptake inhibition for VMAT2 standards was determined for analogs exhibiting the highest potency inhibiting [³H]DA uptake at VMAT2 and the analog having no affinity for the [³H]DTBZ binding site, but which had moderate affinity inhibiting [³H]DA uptake at VMAT2. Concentration-response curves for VMAT2 standards are at the top, and those for the analogs are at the bottom. Concentrations of all compounds used for the kinetic analysis were the respective K_i concentrations from the inhibition curves illustrated in Fig. 5. The concentrations of inhibitor are included in parentheses adjacent to the compound name. V_max and K_m values (± S.E.M.) are presented in Table 2 (n = 6–8 rats/compound).

Fig. 8.

Lobelane analog-evoked [³H]DA release from isolated synaptic vesicles. Data represent the ability of VMAT2 standards and the most potent compounds within this series in the [³H]DA uptake inhibition assay to evoke DA release from synaptic vesicles. Inset, the order of presentation of EC₅₀ values for the analogs and standards is from high to low potency for the ability to release [³H]DA. Control represents [³H]DA release in the absence of compound. Data are mean (± S.E.M.) specific [³H]DA release as a percentage of the control (n = 3–4 rats/compound).