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Published in final edited form as: Bioorg Med Chem Lett. 2016 Apr 1;26(10):2422–2427. doi: 10.1016/j.bmcl.2016.03.119

Lobelane analogues containing 4-hydroxy and 4-(2-fluoroethoxy) aromatic substituents: Potent and selective inhibitors of [3H]dopamine uptake at the vesicular monoamine transporter-2

Shyam R Joolakanti a,#, Justin R Nickell b,#, Venumadhav Janganati a, Guangrong Zheng a, Linda P Dwoskin b, Peter A Crooks a,*
PMCID: PMC4977194  NIHMSID: NIHMS777369  PMID: 27080180

Abstract

A series of lobelane and GZ-793A analogues that incorporate aromatic 4-hydroxy and 4-(2-fluoroethoxy) substituents were synthesized and evaluated for inhibition of [3H]dopamine (DA) uptake at the vesicular monoamine transporter-2 (VMAT2) and the dopamine transporter (DAT), and [3H]serotonin uptake at the serotonin transporter (SERT). Most of these compounds exhibited potent inhibition of DA uptake at VMAT2 in the nanomolar range (Ki = 30–70 nM). The two most potent analogues, 7 and 14, both exhibited a Ki value of 31 nM for inhibition of VMAT2. The lobelane analogue 14, incorporating 4-(2-fluoroethoxy) and 4-hydroxy aromatic substituents, exhibited 96- and 335-fold greater selectivity for VMAT2 versus DAT and SERT, respectively, in comparison to lobelane. Thus, lobelane analogues bearing hydroxyl and fluoroethoxy moieties retain the high affinity for VMAT2 of the parent compound, while enhancing selectivity for VMAT2 versus the plasmalemma transporters.

Keywords: 4-Hydroxyphenyl lobelane analogues, Fluorinated lobelane analogues, Vesicular monoamine transporter-2, Dopamine uptake, Dopamine and Serotonin transporter

Graphical abstract

graphic file with name nihms777369f7.jpg

Methamphetamine (METH) abuse is a serious burden on the United States, with approximately 100,000 new METH users every year1. METH abuse constitutes significant health risks, such as long term neuronal damage, psychosis, paranoia, insomnia, anxiety, aggression, delusions, and hallucinations, and chronic use ultimately may lead to death2. Currently, there are no FDA-approved medications to treat METH addiction. METH users describe a sudden rush of pleasure lasting for several minutes to hours upon self-administration of the drug. These reinforcing properties of METH result from METH-induced prolonged release of dopamine (DA) into the extracellular space, where it interacts with postsynaptic DA receptors3. METH enters dopaminergic presynaptic terminals via passive diffusion through the plasmalemma membrane, and as a substrate for the plasmalemma DA transporter (DAT)4. Once inside the terminal, METH evokes the release of vesicular DA from synaptic vesicles into the cytosol through an interaction with the vesicular monoamine transporter-2 (VMAT2)4. Since METH also inhibits the activity of the mitochondrial enzyme, monoamine oxidase (MAO), the METH-evoked increase in cytosolic DA is not subjected to metabolism, and the formation of dihydroxyphenylacetic acid (DOPAC)5. The increase in cytosolic DA concentrations makes DA more readily available for release into the extracellular compartment via reversal of DAT. The resulting release of DA from the presynaptic terminal, and the subsequent enhanced stimulation of postsynaptic DA receptors results in the reinforcing effects and the high abuse liability associated with METH6.

Lobeline is the principal alkaloid of the Indian tobacco plant, Lobelia inflata. Lobeline inhibits DA uptake into synaptic vesicles via an interaction with the tetrabenazine (TBZ) binding site on VMAT2, but does not inhibit MAO activity8. Lobeline attenuates METH self-administration in a rodent model, but importantly, does not substitute for METH in the self-administration paradigm, indicating that it lacks abuse liability9. Lobeline also decreases METH-induced hyperactivity and behavioral sensitization in rats9. Also, lobeline decreases METH-evoked DA release from superfused rat striatal slices, while concurrently increasing extracellular DOPAC8c. Based on these preclinical findings, lobeline was considered a lead candidate as a treatment for METH abuse. Unfortunately, lobeline also acts as a nicotinic acetylcholine receptor antagonist10. Drugs which exhibit such neurochemical profiles have the potential to generate untoward side-effects in the clinical population. To overcome these limitations, several structure-activity relationship (SAR) studies were performed which involved modifying the chemical structure of lobeline, and assessing the influence of these structural alterations on selectivity of the novel compounds for VMAT2 versus nicotinic receptors and plasmalemma neurotransmitter transporters, DAT and the serotonin transporter (SERT)10.

Lobelane is a structurally modified derivative of lobeline (Fig-1), which exhibits 10- to 15-fold higher potency for inhibiting VMAT2 function compared to lobeline11. Importantly, structural defunctionalization of lobeline to afford lobelane markedly diminished nicotinic receptor affinity of lobelane and associated analogues. Lobelane also inhibits METH-evoked DA release from rat striatal slice preparations11. To improve upon the physicochemical properties of lobelane, we synthesized a lobelane analogue, GZ-793A, by structural modification of the N-methyl group in the molecule to an N-(2S)-1,2-dihydroxyl-propyl moiety (Fig. 1). This structural modification significantly improved affinity for VMAT2 and drug-likeness properties12. Notably, this compound blocked METH self-administration in rats, but had no effect on responding for food13.

Figure 1.

Figure 1

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Chemical structure of lobeline, lobelane, nor-lobelane, GZ-793A. Lobeline is the principal alkaloid of Lobelia inflata. Lobelane is a chemically defunctionalized, saturated derivative of lobeline. GZ-793A is a 4-methoxy analogue of nor-lobelane incorporating an N-(2S)-1,2-dihydroxypropyl substituent.

In the present study, we report on the synthesis of several new lobelane derivatives that incorporate novel functional groups onto the phenyl rings of lobelane and GZ-793A.

Moreover, several of the synthesized compounds were fluorine-containing analogues and are of potential value in positron emission tomography (PET) studies when prepared incorporating the 18F positron-emitting isotope (half-life ~ 120 minutes). The incorporation of fluorine into the structure of a drug molecule also influences lipophilic, electronic, and metabolic properties, all of which can critically influence both the pharmacodynamic and pharmacokinetic properties of the drug14.

The synthesis of the lobelane and GZ-793A derivatives utilized compound 3 as a starting point (Scheme 1). Compound 3 was synthesized by the reaction of 4-hydroxybenzaldehyde (1) with 2,6-lutidine (2) in acetic anhydride at reflux temperature. Intermediately, 3 was subjected to hydrogenation using Adams catalyst (PtO2) in acetic acid to yield a saturated piperidino derivative, which was further converted to hydrochloride salt 4 using HCl in diethyl ether. Compound 4 was reacted with NaBH3CN/HCHO to obtain N-methylated compound 5. Compounds 4 and 5 were de-acetylated in presence of K2CO3 in MeOH and followed by hydrochloride salt formation to afford the corresponding free hydroxyl compounds 6 and 7, respectively. Compound 4 was reacted with S-glycidol in ethanol, followed by de-acetylation in presence of K2CO3 in MeOH and formation of hydrochloride salt to obtain the N-(2S)-1,2-dihydroxylpropyl derivative 8 (Scheme 1).

Scheme 1.

Scheme 1

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Reagents and conditions: (a) Ac2O, reflux, 24 h, 48%; (b) 10% (w/v)PtO2/H2, AcOH, 12 h, 65%; (c) K2CO3/MeOH, r.t., 1 h 90–95% (d)HCHO/NaBH3CN, EtOH, 2 h 61%; (e) (S)-glycidol, EtOH, reflux, 2 h 57–60%; (f) 2 M HCl in ether.

The fluorine-containing derivatives of lobelane and GZ-793A were synthesized from intermediate 3. Compound 3 was deacetylated in the presence of K2CO3 in MeOH to afford 9, which was O-alkylated utilizing ethylfluorotosylate and sodium hydroxide in DCM/MeOH (2:1) to yield a mixture of monofluoroethoxy derivative 10 and difluoroethoxy derivative 11 (Scheme 2). After careful separation of these two compounds by silica gel column chromatography, 10 and 11 were each hydrogenated using Adams catalyst (PtO2) in acetic acid to yield the corresponding saturated piperidino derivatives, which were further converted to their hydrochloride salts to obtain 12 and 13, respectively. Compounds 12 and 13 were then treated with NaBH3CN/HCHO to obtain the N-methylated derivatives 14 and 15. N-(2S)-1,2-Dihydroxypropyl derivatives 16 and 17 were obtained by reacting 12 and 13 with S-glycidol in ethanol (Scheme 2). The synthesized compounds were fully characterized by 1H NMR, 13C NMR and high resolution mass spectral analysis.

Scheme 2.

Scheme 2

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Reagents and conditions: (a) K2CO3/MeOH, r.t. 1 h; (b) F-CH2-CH2-OTs, NaOH, DCM/MeOH (2:1), 2 h 30–33% (c) 10% (w/v)PtO2/H2, AcOH, 12 h, 60–65%; (d) DCM/2M HCl in ether; (e) HCHO/NaBH3CN, EtOH, 2 h 58–61%; (f) (S)-glycidol, EtOH, reflux, 2 h 57–60%

Compounds 4–8 and 12–17 were evaluated initially for inhibition of VMAT2 function (Table 1 & 2; Figure 2). The majority of the compounds exhibited affinities for VMAT2 within a relatively restricted range of 30–70 nM. Incorporation of 4-acetoxy substituents into the phenyl rings of nor-lobelane afforded 4, which exhibited a Ki value of 0.22 µM in the VMAT2 assay. N-Methylation of 4 afforded 5, which had 3-fold improved inhibitory potency at VMAT2 (Ki = 0.071 µM). Hydrolysis of the 4-acetoxy groups in 4 and 5 gave the diphenolic analogues 7 and 6, respectively, with about 2-fold further improvement in inhibitory potency at VMAT2 (Ki = 0.031 µM and 0.046, respectively). These data indicate that introduction of aromatic hydroxyl substituents into the lobelane molecule retains the high inhibitory potency at VMAT2 exhibited by lobelane. Interestingly, this trend was also observed for compound 8, a diphenolic analogue of GZ-793A, which exhibited Ki of 0.039 µM in the VMAT2 assay (Table 1).

Table 1.

Inhibition of [3H]DA uptake at VMAT2 and DAT, and inhibition of [3H]5-HT uptake at SERT by precursor non-fluorinated lobelane analogues

Compound VMAT2
[3H]DA Uptake
(Ki; µM)		 DAT
[3H]DA Uptake
(Ki; µM)		 SERT
[3H]5-HT Uptake
(Ki; µM)
Lobelane 0.067 ± 0.0071 1.05 ± 0.031 3.6 ± 0.351
GZ-793A 0.029 ± 0.0081 1.44 ± 0.271 9.36 ± 2.741
graphic file with name nihms777369t1.jpg 0.22 ± 0.021a 1.47 ± 0.403 2.13 ± 0.466
graphic file with name nihms777369t2.jpg 0.071 ± 0.008 2.13 ± 0.87 11.6 ± 1.46
graphic file with name nihms777369t3.jpg 0.046 ± 0.005 1.35 ± 0.34 12.0 ± 2.00
graphic file with name nihms777369t4.jpg 0.031 ± 0.004 0.866 ± 0.14 1.03 ± 0.17
graphic file with name nihms777369t5.jpg 0.039 ± 0.003 1.43 ± 0.73 12.9 ± 2.98
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a

Data represent mean ± SEM (n = 3–4 rats/analogue)

1

Data taken from reference 15 (Horton et al., 2011)

Table 2.

Inhibition of [3H]DA uptake at VMAT2 and DAT, and inhibition of [3H]5-HT uptake at SERT by fluorinated lobelane analogues

Compound VMAT2
[3H]DA Uptake
(Ki; µM)		 DAT
[3H]DA Uptake
(Ki; µM)		 SERT
[3H]5-HT Uptake
(Ki; µM)
Lobelane 0.067 ± 0.0071 1.05 ± 0.031 3.6 ± 0.351
GZ-793A 0.029 ± 0.0081 1.44 ± 0.271 9.36 ± 2.741
graphic file with name nihms777369t6.jpg 0.032 ± 0.003a 1.66 ± 0.34 1.05 ± 0.29
graphic file with name nihms777369t7.jpg 0.040 ± 0.002 1.67 ± 0.30 0.640 ± 0.034
graphic file with name nihms777369t8.jpg 0.031 ± 0.003 2.97 ± 0.46 10.4 ± 0.54
graphic file with name nihms777369t9.jpg 0.14 ± 0.011 3.43 ± 0.73 7.43 ± 1.84
graphic file with name nihms777369t10.jpg 0.063 ± 0.003 5.47 ± 0.92 1.43 ± 0.23
graphic file with name nihms777369t11.jpg 0.120 ± 0.003 3.69 ± 0.72 33.0 ± 2.92
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a

Data represent mean ± SEM (n = 3–4 rats/analogue)

1

Data taken from reference 15 (Horton et al., 2011)

Figure 2. Lobelane and GZ-793A analogues inhibit [3H]DA uptake at VMAT2 in rat striatal vesicles.

Figure 2

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Concentration-response curves for inhibition of VMAT2. Order of presentation for the analogues within the figure legend is from high to low affinity for VMAT2. Control (CON) represents [3H]DA uptake in the absence of compound. Data are mean (± SEM) specific [3H]DA uptake expressed as a percentage of control (24.8 ± 1.06 pmol/min/mg, control n = 42 rats; n = 3–4 rats/compound)

Evaluation of nor-lobelane analogues 12 and 13, the mono-4-(2-fluoroethoxy) and di-4-(2-fluoroethoxy) derivatives of 7, indicated that replacing the phenolic groups in these compounds with 2-fluoroethoxy groups had little effect on inhibition of DA uptake at VMAT2 (Ki’s = 0.032 and 0.040, respectively) in relation to analogue 7. N-Methylation of 12 afforded compound 14, one of the most potent inhibitors (Ki = 0.031 µM) of vesicular DA uptake in the series. However, while the di-4-(2-fluoroethoxy) analogue 13 afforded a Ki value of 0.040 µM, which is not different from that of lobelane or GZ-793A, insertion of either a N-methyl group (compound 15, Ki = 0.14 µM) or a N-(2S)-1,2-dihydroxypropyl group (compound 17, Ki = 0.120 µM) in place of the piperidino hydrogen atom in 13 reduces potency at VMAT2 by 3 to 3.5-fold. It is important to note that compound 16, a mono-4-(2-fluoroethoxy) analogue of GZ-793A, exhibited a Ki value of 0.063 µM in the VMAT2 assay, and represents a lead fluorine-containing analogue with improved water solubility over lobelane.

Potency of VMAT2 inhibition alone is insufficient to determine whether the discovery of these novel lobelane analogues have clinical potential as treatments for METH abuse. Accordingly, we also examined the ability of these compounds to inhibit the plasmalemma transporters, DAT and SERT (Tables 1 and 2; Figures 3 and 4). Generally, all of the analogues in this study exhibited affinities for DAT comparable to those for both lobelane (Ki = 1.05 µM) and GZ-793A (Ki = 1.44 µM)15. Likewise, a preponderance of the compounds inhibited SERT with affinities not different from those for lobelane (Ki = 3.6 µM) and GZ-793A (Ki = 9.36 µM)15. Notable exceptions to these generalizations are compound 13 which has 6-fold greater affinity for SERT than lobelane, compound 16 which has a 7-fold greater affinity for SERT than GZ-793A, and compound 17 which exhibits a 4-fold decrease in affinity for SERT when compared to GZ-793A. Thus, compound 17 exhibits 30- and 275-fold greater selectivity for VMAT2 versus DAT and SERT, respectively. While incorporation of 2-fluoroethoxy moieties into the structural scaffold of GZ-793A does not alter affinity for VMAT2 in comparison to both lobelane (Ki = 67 nM) and GZ-793A (Ki = 29 nM), the greater selectivity for VMAT2 exhibited by these compounds suggests a low likelihood of abuse liability15.

Figure 3. Lobelane and GZ-793A analogues inhibit [3H]DA uptake at DAT in rat striatal synaptosomes.

Figure 3

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Concentration-response curves for inhibition of DAT. Order of presentation for the analogues within the figure legend is from high to low affinity for DAT. Control (CON) represents [3H]DA uptake in the absence of compound. Data are mean (± SEM) specific [3H]DA uptake expressed as a percentage of control (19.5 ± 1.27 pmol/min/mg, control n = 43 rats; n = 3–4 rats/compound).

Figure 4. Lobelane and GZ-793A analogues inhibit [3H]5-HT uptake at SERT in rat striatal synaptosomes.

Figure 4

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Concentration-response curves for inhibition of SERT. Order of presentation for the analogues within the figure legend is from high to low affinity for SERT. Control (CON) represents [3H]5-HT uptake in the absence of compound. Data are mean (± SEM) specific [3H]5-HT uptake expressed as a percentage of control (4.46 ± 0.234 pmol/min/mg, control n = 44 rats; n = 4 rats/compound).

Similar to lobelane and GZ-793A, all the compounds evaluated in this study exhibited higher affinities for VMAT2 than DAT and SERT. Most compounds showed 15 to 40-fold greater selectivity for VMAT2 versus the plasmalemma transporters. The combination of 4-(2-fluoroethoxy) and 4-hydroxy aromatic substitution present in compounds 14 and 16 afforded the greatest selectivity for VMAT2 versus DAT, 96 and 87-fold respectively. Lobeline analogue 14 was one of the most potent VMAT inhibitors in the series. Likewise, incorporation of 4-acetoxy and 4-hydroxy aromatic substituents into the phenyl rings of lobelane resulted in improvement in selectivity for VMAT2 versus SERT; compounds 5, 6 and 8 exhibited selectivity ratios of 163, 260 and 331-fold, respectively. Overall, compound 14 demonstrated 96 and 335-fold selectivity for VMAT2 versus DAT and SERT, respectively, and was the most selective analogue of the series.

In summary, a series of lobelane and GZ-793A analogues that incorporate aromatic 4-hydroxy and aromatic 4-(2-fluoroethoxy) substituents were synthesized and evaluated for their ability to inhibit [3H]DA uptake at VMAT2 and DAT, and [3H]5-HT uptake at SERT. Incorporation of these moieties into the structure of lobelane generally maintained inhibitory potency at VMAT2 relative to the parent compound. The potent VMAT2 inhibitor 14, which incorporates 4-(2-fluoroethoxy) and 4-hydroxy aromatic substituents, exhibited the greatest selectivity for VMAT2 versus DAT and SERT. These results also indicate that fluorinated lobelane and GZ-793A analogues retain the high inhibitory potency and selectivity of lobelane and GZ-793A for VMAT2, and as such constitute promising candidates for the visualization of VMAT2 via PET scanning in the clinical population.

Supplementary Material

NIHMS777369-supplement.docx (105.9KB, docx)

Acknowledgments

This research was supported by NIH U01 DA13519, TR000117 and NIH/COBRA P20 GM109005 grants, and an Arkansas Research Alliance (ARA) Scholar award.

Footnotes

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  • 16.Vesicular [3H]DA Uptake Assay. Inhibition of [3H]DA uptake was conducted as previously described,8b utilizing a striatal synaptic vesicle preparation. Briefly, rat striata were homogenized with 10 up and down strokes of a Teflon pestle homogenizer (clearance ~ 0.003”) in 14 ml of 0.32 M sucrose solution. Homogenates were centrifuged (2000g for 10 min at 4 °C), and the resulting supernatants were centrifuged again (10,000g for 30 min at 4 °C). Pellets were resuspended in 2 ml of 0.32 M sucrose solution and subjected to osmotic shock by adding 7 ml of ice-cold water to the preparation, followed by the immediate restoration of osmolality by adding 900 µL of 0.25 M HEPES buffer and 900 µL of 1.0 M potassium tartrate solution. Samples were centrifuged (20,000g for 20 min at 4 °C), and the resulting supernatants were centrifuged again (55,000g for 1 h at 4 °C), followed by addition of 100 µL of 10 mM MgSO4, 100 µL of 0.25 M HEPES and 100 µL of 1.0 M potassium tartrate solution prior to the final centrifugation (100,000g for 45 min at 4 °C). Final pellets were resuspended in 2.4 mL of assay buffer (25 mM HEPES, 100 mM potassium tartrate, 50 µM EGTA, 100 µM EDTA, 1.7 mM ascorbic acid, 2 mM ATP-Mg2+, pH 7.4). Aliquots of the vesicular suspension (100 µL) were added to tubes containing assay buffer, various concentrations of analogue inhibitor (0.1 nM–10 mM) and 0.1 µM [3H]DA in a final volume of 500 µL. Nonspecific uptake was determined in the presence of Ro4-1284 (10 µM). Reactions were terminated by filtration, and radioactivity retained by the filters was determined by liquid scintillation spectrometry (Liquid scintillation analyzer; PerkinElmer Life and Analytical Sciences, Boston, MA). Synaptosomal [3H]DA and [3H]5-HT Uptake Assays. Analogue-induced inhibition of [3H]DA and [3H]5-HT uptake into rat striatal synaptosomes, was determined using modifications of a previously described method.15 Striata were homogenized in 20 ml of ice-cold 0.32 M sucrose solution containing 5 mM NaHCO3, pH 7.4, with 16 up-and-down strokes of a Teflon pestle homogenizer (clearance ~0.005 inch). Homogenates were centrifuged at 2000g for 10 min at 4°C, and resulting supernatants were centrifuged at 20,000g for 17 min at 4°C. Pellets were re-suspended in 1.5 ml of Krebs' buffer, containing 125 mM NaCl, 5 mM KCl, 1.5 mM MgSO4, 1.25 mM CaCl2, 1.5 mM KH2PO4, 10 mM α-d-glucose, 25 mM HEPES, and 0.1 mM EDTA, with 0.1 mM pargyline and 0.1 mM ascorbic acid, saturated with 95% O2/5% CO2, pH 7.4. Synaptosomal suspensions (20 µg of protein/50 µl) were added to duplicate tubes containing 50 µl of analogue (7–9 concentrations; 0.1 nM-1 mM, final concentration) and 350 µl of buffer and incubated at 34°C for 5 min in a total volume of 450 µl. Samples were placed on ice, and 50 µl of [3H]DA or [3H]5-HT (10 nM, final concentration) was added to each tube for a final volume of 500 µl. Reactions proceeded for 10 min at 34°C and were terminated by the addition of 3 ml of ice-cold Krebs' buffer. Nonspecific [3H]DA and [3H]5-HT uptake were determined in the presence of 10 µM GBR 12909 and 10 µM fluoxetine, respectively. Samples were rapidly filtered through Whatman (Clifton, NJ) GF/B filters using a cell harvester (MP-43RS; Brandel Inc., Gaithersburg, MD). Filters were washed three times with 4 ml of ice-cold Krebs' buffer containing catechol (1 mM). Complete counting cocktail was added to the filters, and radioactivity was determined by liquid scintillation spectrometry (B1600 TR scintillation counter; PerkinElmer Life and Analytical Sciences

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Supplementary Materials

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