Pharmacological Evaluation of Tropane Analogues at the Serotonin Transporter

Abstract

Tropane alkaloids and their derivatives represent a diverse class of small molecules with a broad range of therapeutic applications. Many tropanes regulate synaptic levels of neuromodulators by interacting with monoamine transporters such as the dopamine (DAT) and serotonin (SERT) transporters. While DAT inhibition plays an important role in the addictive potential of tropanes like cocaine, recent evidence suggests that SERT modulation may oppose the effects of DAT inhibition. Moreover, SERT modulators such as 3,4-methylenedioxymethamphetamine (MDMA), ibogaine, and selective-serotonin reuptake inhibitors (SSRIs) have demonstrated potential as treatments for a broad range of conditions including depression, addiction, and post-traumatic stress disorder (PTSD). Here, we profiled a variety of structurally distinct subclasses of tropanes in SERT inhibition, efflux, and pharmacochaperone assays. We identified several compounds capable of potently modulating SERT in ways similar to fluoxetine, MDMA, or noribogaine. In particular, UCD0168 and UCD0820 emerged as potent SERT inhibitors that act as full and partial serotonin releasing agents (SRAs) in SERT-transfected HEK293T cells, respectively. Our work demonstrates that it is possible to use the tropane scaffold as a starting point for identifying both MDMA-like and noribogaine-like SERT modulators, and we provide several new tropane-containing hit structures for creating optimized therapeutics relying on SERT modulation.

Graphical Abstract

INTRODUCTION

The tropane alkaloids are a pharmacologically diverse class of natural products that contain a characteristic N-methyl-8-azabicyclo[3.2.1]octane core structure. These compounds are found across several plant families, including Erythroxylaceae, Solanaceae, and Convolvulaceae.¹ Given their diverse and potent pharmacological properties, many natural and semi-synthetic tropanes have been approved by the U.S. Food and Drug Administration (FDA) for therapeutic applications. Notable examples include atropine (mydriatic), cocaine (local anesthetic), ioflupane (diagnostic), maraviroc (HIV antiretroviral), retapamulin (antibiotic), scopolamine (antiemetic), tiotropium bromide (chronic obstructive pulmonary disease; COPD), and trospium (urinary antispasmodic). Beyond their approved medicinal uses, tropanes are perhaps best known for the highly addictive and potent euphorigenic properties of cocaine. As a result, a considerable amount of scientific research has been dedicated to understanding how cocaine and its analogues produce addiction as well as pharmacological strategies to counteract this effect.^2,3,4,5 It is now widely accepted that DAT inhibition is largely responsible for mediating the rewarding and psychostimulant effects of cocaine,^6,7,8 but far less is known about the role that SERT inhibition plays in these processes. However, it is important to mention that while DAT is believed to be primarily responsible for the euphorigenic effects of cocaine, selective DAT inhibitors based on benztropine have been shown to be behaviorally distinct in animal models of addiction and have led to potential therapeutics.^9,10,11

Recent evidence suggests that cocaine’s potent inhibition of SERT constrains its addictive potential. In fact, prevention of cocaine binding to SERT using SERT knockin mice facilitated compulsive drug-seeking behavior, and increasing cortical serotonin levels with the SSRI citalopram decreased compulsive self-stimulation of dopaminergic neurons in the ventral tegmental area (VTA).¹² It has also been recognized that cocaine use causes a depletion of 5-HT which leads to depressive-symptoms upon withdrawal.^13,14 An emerging class of therapeutics that targets both DAT and SERT systems, aligning with the dual deficiency model of psychostimulant dependence, have shown promise.^15,16 Both SERT-targeting tricyclic antidepressants (TCAs) and SSRIs have been used to treat addiction with co-occurring depression,¹⁷ though their efficacy is limited. While a meta-analysis of randomized clinical trials found that certain SSRIs (e.g., fluoxetine, citalopram, and sertraline) provided some benefit, SSRI monotherapy overall was not a particularly effective treatment option for substance use disorders.¹⁸

This has led to modern efforts to identify SERT-modulators with more profound therapeutic properties. Perhaps the best known SERT-modulators to emerge from these efforts are the entactogen MDMA and the alkaloid ibogaine. Both preclinical and clinical studies have suggested that MDMA may be useful for treating post-traumatic stress disorder (PTSD) and perhaps certain substance use disorders.^{19,20,21,22,23} Through a variety of mechanisms, including acting as a substrate for SERT, MDMA facilitates serotonin release into the extracellular space.¹⁹ Serotonin release is widely believed to play a key role in the prosocial effects of MDMA in mice²⁴ and its therapeutic effects in humans.²⁵ Similarly, the suspected antiaddictive properties of ibogaine and its active metabolite noribogaine^26,27,28 have been hypothesized to be due to their unique abilities to stabilize an inward-open conformation of the SERT.^29,30

The pharmacochaperoning effects of ibogaine and noribogaine, which reinstate SERT transporter maturation, surface localization, and functional activity, have also been associated with their conformation stabilizing effects.^31,32,33,34 In contrast, several canonical (outward-open) substrates and inhibitors have failed to replicate this phenomenon.^35,36,37,38 Given that several tropane-containing compounds have mimicked the effects of SERT inhibitors/modulators in previous studies, we reasoned that this scaffold could prove to be a fruitful starting point for medicinal chemistry efforts to identify novel SERT modulators. Fortunately, we recently reported an efficient synthetic strategy towards tropanes enabling late-stage functionalization of the tropane core structure.³⁹ We used this strategy to access a variety of tropane analogues with various substitution patterns at N8, C6/C7, and C3 (Figure 1).⁴⁰ Here, we evaluated these compounds in assays relevant to SERT inhibition, efflux, and pharmacochaperoning (Figure 2), and we identified UCD0168 and UCD0820 as promising potent full and partial SRAs, respectively.

Figure 1. Structures of tropane analogues from various chemical classes.

Compounds are grouped into structural classes based on either analogy to well-known compounds or shared structural features. Compound numbers represent experimental compound codes from the UC Davis Institute for Psychedelics and Neurotherapeutics proprietary compound library.

Figure 2. Assays for evaluating SERT modulation.

Schematics depict three distinct assays using HEK293T cells heterologously expressing SERT. (A) Inhibition assays measure the amount of [³H]5-HT uptake in the presence of a SERT inhibitor. (B) Efflux assays measure the amount of pre-loaded [³H]5-HT released following compound stimulation. (C) Pharmacochaperone assays measure the amount of [³H]5-HT uptake following stimulation with a pharmacochaperone. The boxes on the left show zoomed-in views of the box found on the cell surface (right). Tx = treatment

RESULTS AND DISCUSSION

Effects of Tropane Analogues on SERT Inhibition

Using cocaine (10 μM) as a positive control, we assessed the abilities of various tropane analogues (10 μM) to inhibit SERT transport of radiolabeled serotonin (5-HT) (Figure 2A). A broad range of tropane analogues demonstrated meaningful levels of SERT inhibition at 10 μM including scopolamine, benztropine, and many novel analogues that had remained synthetically inaccessible until recently⁴⁰ (Figure 3A). While the activity of benztropine as a SERT inhibitor has been reported previously,⁴¹ to the best of our knowledge, no such activity has been reported for scopolamine. In general, compounds bearing N-methyl substituents performed better than those with larger groups appended to N8 including those with phenethyl and benzyl moieties (i.e., N8-substituted and tropinone-like, respectively).

Figure 3. Assays for evaluating SERT modulation.

(A) Single concentration inhibition assays were conducted at 10 μM with values represented as percentages normalized to the vehicle (0.1% DMSO = 0%) and positive (100 μM cocaine = 100%) controls. (B) Single concentration efflux assays were conducted at 10 μM with values represented as percentages normalized to the vehicle (0.1% DMSO = 0%) and positive (100 μM p-chloroamphetamine (PCA) = 100%) controls. (C) Single concentration inhibition assays were conducted at 20 μM with values represented as percentages normalized to the vehicle (0.2% DMSO = 0%) and positive (20 μM noribogaine = 100%) controls. Values for cocaine (10 μM) and (±)-MDMA (10 μM) for inhibition and efflux, respectively, were taken from a previous report of ours.⁴⁵ Given that noribogaine was used as a the plate control for the pharmacochaperoning assays (i.e., it was set to 100%), no error bars are shown. Data represent means ± standard error of mean (SEM). The dotted line indicates 50% activity for a particular assay. Colors indicate compound classes shown in Figure 1 with gray bars indicating control compounds.

Given that cocaine (10 μM) produced ~50% inhibition, we used this level of inhibition as our cutoff for identifying hits for further medicinal chemistry optimization. Of the tropane analogues tested, UCD0184 stood out as the only compound that reached this threshold, producing near full inhibition of SERT at 10 μM. This finding supports a previous study that found UCD0184 to be a highly potent and selective inhibitor of SERT with approximately 10- and 100-fold selectivity over DAT and NET, respectively.⁴² This selectivity has been attributed to the orientation of the N8 lone pair, with equatorial and axial orientations of the N8 methyl group favoring DAT and SERT, respectively.⁴³ Computational calculations and nuclear magnetic resonance (NMR) studies revealed that UCD0184 preferentially adopts the axial conformation.^42,44

Effects of Tropane Analogues on SERT Efflux

In sharp contrast to SERT inhibition, nearly all tropane analogues that we profiled exhibited negligible activity in SERT efflux assays (Figure 3B). This was quite surprising given that our group and others have found that cocaine, a prototypical tropane-containing compound, can induce [³H]5-HT efflux in HEK293T and C6 glioma cells heterologously expressing SERT.^45,46 Interestingly, tropacocaine lacks the C2 methyl ester substituent present in cocaine and has no effect on [³H]5-HT efflux. In fact, none of the analogues that we profiled contained a C2 methyl ester, suggesting that this substituent plays a critical role in cocaine’s [³H]5-HT efflux activity. This agrees with previous efforts demonstrating that tropacocaine has reduced activity relative to cocaine in both serotonin uptake inhibition and SERT binding affinity assays.⁴⁷

The only compound we tested that induced any appreciable 5-HT efflux was UCD0184. This was intriguing given that it was the only 3-aryl-tropidine profiled and all other C3-substituted tropane analogues possessed tetrahedral geometry at C3. Though not often appreciated, this dual efflux-inhibition activity emphasizes the rich and complex pharmacology of SERT modulation. Several other ligands, including MDMA, have demonstrated similar SERT-mediated efflux (Figure 3B).

Effects of Tropane Analogues on SERT Pharmacochaperoning

Pharmacochaperoning is a phenomenon where small molecules help correct the misfolding of mutant proteins by binding to them in the endoplasmic reticulum and/or Golgi apparatus, stabilizing correctly folded conformations, and facilitating their export to the cell surface.³⁴ While SERT inhibition and efflux assays are routinely used to assess the pharmacological properties of SERT ligands, pharmacochaperoning assays are employed much more infrequently. Thus, there is a limited amount of information available on SERT pharmacochaperones, with ibogaine and noribogaine being some of the few known SERT pharmacochaperones.^35,36,37,48 A recent unpublished preprint identified the point-mutant variant SERT-N²¹⁷S in some patients with psychiatric disorders and reported that it exhibited impaired cell surface trafficking. Notably, treatment with noribogaine appeared to rescue this trafficking defect in vitro.⁴⁹ Iboga alkaloids possess a characteristic isoquinuclidine core,⁵⁰ and given that this bicycle is a constitutional isomer of the tropane core, we reasoned that tropane analogues might possess SERT pharmacochaperoning properties like ibogaine/noribogaine.

To assess the potential of compounds to serve as pharmacochaperones for SERT, we relied on the overexpression of a SERT mutant (SERT-PG^601,602AA) that is prone to misfolding and exhibits reduced surface expression and activity.35 After treating cells for 24 h to promote plasma membrane expression of functional SERT, radiolabeled 5-HT was added to the cultures and uptake was assessed (Figure 3C). Of the 30 tropane-containing compounds that we screened, 27% exhibited pharmacochaperoning effects greater than 30%, with compounds being identified from nearly every structural class (Figure 3C). As expected, the compound with the greatest structural similarity to ibogaine, UCD0002, was relatively active in this assay. However, it’s efficacy was only 49% that of noribogaine. The best pharmacochaperone identified was UCD0812, a scopolamine-like compound with an efficacy of 72% compared to noribogaine. Unlike scopolamine, UCD0812 lacks affinity for muscarinic receptors,⁴⁰ and thus, could represent a SERT pharmacochaperone with fewer side-effects.

Despite the fact that many tropane analogues possessed some level of pharmacochaperoning activity at SERT, no structural class emerged as being clearly more efficacious than the others. Thus, it was unclear which scaffold to prioritize for medicinal chemistry optimization. In stark contrast, UCD0184 was one of the few tropane analogues that did not act as a SERT pharmacochaperone, but it was clearly superior to all other compounds in assays of SERT inhibition and efflux. As a result, we decided to perform additional structure-activity relationship (SAR) studies to better understand why UCD0184 exhibited such high efficacy in SERT inhibition and efflux assays.

SAR Studies of UCD0184 Analogues in SERT Inhibition and Efflux Assays

We synthesized several analogues of UCD0184 by treating tropinone with various substituted indoles under acidic conditions (Figure 4A). We also accessed UCD0891 and UCD0813 through organolithium addition to the carbonyl of tropinone followed by acid-mediated dehydration (Figure 4B). Next, we performed concentration-response studies to determine EC₅₀ and E_max values (reported as Span) for SERT inhibition (Figure 5) and efflux (Figure 6). Trigonal geometry at C3 as well as the presence of an indole substituent at that position were critical for achieving high activity, as the phenyl substituted analogues UCD0891 and UCD0813 exhibited drastically reduced potency and efficacy compared to UCD0184. Comparing UCD0184 to UCD0168 revealed that indole N-methylation has minimal to no impact on potency or efficacy in either inhibition or efflux assays. However, SERT activity could be effectively modulated by changing the position and identity of the indole substituent.

Figure 4. Synthesis of UCD0184 analogues.

(A) Five analogues were synthesized directly from tropinone following treatment with acid and various substituted indoles. (B) Addition of an organolithium nucleophile to tropinone yielded UCD0891, which was subsequently converted to UCD0813 via acid-catalyzed dehydration.

Figure 5. Concentration-response studies evaluating UCD0184 analogues in SERT inhibition assays.

Concentration-response curves for UCD0184 analogues in SERT inhibition assays were normalized to vehicle (0.1% DMSO; 0%) and positive (100 μM cocaine; 100%) controls. Data are presented as mean ± standard error of mean (SEM). The curve for UCD0184 (Span = 105%, EC₅₀ = 1.10 μm) is shown on each graph for reference. COCA = cocaine; FLX = fluoxetine.

Figure 6. Concentration-response studies evaluating UCD0184 analogues in SERT efflux assays.

Concentration-response curves for UCD0184 analogues in SERT efflux assays were normalized to vehicle (0.1% DMSO; 0%) and positive (100 μM PCA; 100%) controls. Data are presented as mean ± standard error of mean (SEM). The curve for UCD0184 (Span = 75.1%, EC₅₀ = 582 nm) is shown on each graph for reference. Error bars for UCD0891 extend beyond the range depicted. Complete confidence intervals could not be calculated for UCD0820 and UCD0813, and thus, the span and EC₅₀ values reported for these compounds should be interpreted with caution.

Next, we tested analogues with electron-donating (methoxy) and electron-withdrawing (fluoro) substituents at the 5-position of the indole. Introduction of a methoxy group (UCD0809) resulted in a 3.5-fold decrease in SERT inhibition potency compared to UCD0184, but it completely abolished SERT efflux. In stark contrast, introduction of a 5-fluoro substituent (UCD0093) drastically increased SERT inhibition potency with minimal effect on SERT efflux. Transposing the fluoro substituent from the 5- to the 6-position of the indole improved SERT inhibition potency further to 62.4 nM, making UCD0820 ~6-fold more potent than fluoxetine. Additionally, UCD0820 exhibited extremely potent partial SERT-mediated efflux of serotonin (Figure 6 and 7). Our group has observed this phenomenon previously with noribogaine and 10-fluoroibogamine,⁴⁵ while others have observed partial serotonin release using PAL-1045.^51,52 Relatively few partial SRAs have been reported, and the exact mechanism by which they mediate this effect remains unclear.^53,54 Regardless, partial SRAs like noribogaine and UCD0820 may offer advantages over full releasers due to their reduced potential for serotonergic side effects resulting from high initial serotonin signaling and/or subsequent serotonin depletion.^55,56 However, it should be noted that we performed our efflux assays under stationary conditions rather than relying on a continuous flow superfusion strategy employed by some labs.⁵⁷ A limitation of stationary efflux assays is the increased potential for inhibitors to appear as releasing agents by preventing the re-uptake of leaked [³H]5-HT. While this is a possibility that should be considered when interpreting our results, the drastic differences in potencies between inhibition and efflux assays for UCD0809 and UCD0820 suggest that their effects on efflux are distinct from re-uptake inhibition.

Figure 7. UCD0184 and UCD0820 are full and partial SRAs, respectively.

(A) A bias plot depicting % SERT efflux and % SERT inhibition at 8 concentrations (1 pM – 10 μM). The dotted line indicates the theoretical profile for a perfectly balanced compound. While UCD0184 is relatively balanced, UCD0820 acts as a full inhibitor but only partial SRA. (B) Ratios of spans from inhibition and efflux assays. The dotted line indicates a ratio of 1.

Comprehensive Binding Profiles for Select Tropidine Analogues

Given that UCD0184, UCD0168, UCD0093, and UCD0820 were the most potent SERT inhibitors that we identified, we decided to use radioligand binding assays to thoroughly assess their selectivity profiles across a range of central nervous system targets (Figure 8). As expected, all four compounds demonstrated substantial binding to SERT at 10 μM. Like many other tropane-containing setron drugs (e.g., tropisetron, zatosetron, etc.), all of the tropidine analogues that we evaluated exhibited high affinity for 5-HT3 receptors. They were also universally potent ligands for sigma 1 and sigma 2 receptors.

Figure 8. Comprehensive binding profiles for UCD0184, UCD0168, UCD0093, and UCD0820.

The effects of tropidine analogues (10 μM) on a wide range of targets were assessed by the National Institute of Mental Health Psychoactive Drug Screening Program (NIMH PDSP).⁵⁸ Data represent % inhibition of the binding of a radioactively labeled ligand specific for each target. Assays were conducted as N = 1 in quadruplicate with the averaged results being presented. GABAA refers to the benzodiazepine binding site from rat brain.

In addition to their 4 primary targets (i.e., SERT, 5-HT3, sigma 1, and sigma 2), UCD0184, UCD0093, and UCD0820 were potent ligands for DAT, NET, H1, 5-HT2B, and adrenergic receptors. Despite only a single carbon shift of the small fluorine substituent, clear differences were evident between UCD0093 and UCD0820. The latter compound was the only tropidine tested that exhibited high affinity for the 5-HT2A receptor, but for the most part, its selectivity profile mirrored that of UCD0184. In stark contrast, UCD0168 appeared to be much more selective, indicating that methylation of the indole N1 position prevents binding to a range of targets. In fact, treatment with UCD0168 only led to >75% inhibition at 10 μM for 4 out of the 43 targets assessed.

Taken together, our studies reveal that 3-tropidine-substituted indoles are potent SERT modulators that can be easily accessed from tropinone. Moreover, additional indole substitution can be used to tune selectivity profiles and SERT activity. By fluorinating the 6-position, we were able to convert UCD0184, an MDMA-like full SRA, into UCD0820, a noribogaine-like partial SRA. Given that MDMA-like and noribogaine-like SERT modulators offer distinct advantages for treating diseases like PTSD and SUD, our work establishes the tropane scaffold as an excellent starting point for identifying both types of SERT modulators.

METHODS

Data Analysis and Statistics.

Statistical analyses were performed using GraphPad Prism (version 10.0.3). Data are represented as mean ± SEM, unless noted otherwise. Concentration-response curves were calculated using the log(agonist) vs. response (three parameters) setting.

Drugs.

All drugs used in these studies were commercially available or synthesized in house (see supporting information or a prior report⁴⁰). The NIDA Drug Supply Program provided (−)-cocaine hydrochloride and noribogaine. Fluoxetine hydrochloride (Millipore Sigma, F132-10MG), (±)-p-chloroamphetamine hydrochloride (Millipore Sigma, C9635-1G), scopolamine hydrobromide trihydrate (Acros, AC161750010), and benztropine mesylate (Millipore Sigma, SML0847-500MG) were purchased from commercial sources. All other tropanes used in these studies were freebases.

SERT Inhibition Assays.

Cells (HEK293T) were grown in Dulbecco’s Modified Eagle Media (DMEM) supplemented with 10% fetal bovine serum (FBS). Plastic Costar 96-well plates were seeded at a density of 100,000 cells/well 24 h prior to the experiment and concurrently transfected using Lipofectamine^™ 3000 Transfection Reagent according to the manufacturer’s protocol. Cells were transfected with 0.1 μg of hSERT-N1-pEYFP (Addgene #70105) per well before returning the plate to the 37 °C, 5% CO₂ atmosphere incubator overnight. Wells were washed (1 × 200 μL) with 1x Hank’s Balanced Salt Solution (HBSS) supplemented with 2 mM MgCl₂, 2 mM CaCl₂, and 5 mM HEPES and replenished with 100 μL of supplemented HBSS. The plate was placed in a 37 °C water bath for the remainder of the experiment and allowed to incubate for 30 min prior to beginning the experiment. The assay was initiated by adding a 100 μL mixture of 5-[1,2-³H(N)]-hydroxytryptamine creatinine sulfate ([ 3 H]5-HT; #NET498001MC, Revvity) and respective drug onto a plate at final concentrations of 20 nM [³H]5-HT and 10 μM – 1 nM (0.1% DMSO). After 10 min, the uptake was terminated by aspiration, and wells were washed with supplemented HBSS (3 × 200 μL). Cells were then lysed for 30 min by the addition of 30 μL of 10 mM NaOH. Next, 120 μL of OptiPhase HiSafe (#1200.437; Revvity) was added to the cell lysates. Counts per minute (CPM) were quantified after an additional 30 min using a MicroBeta2 (Revvity) microplate liquid scintillation counter.

SERT Efflux Assays.

Cells (HEK293T) were grown in Dulbecco’s Modified Eagle Media (DMEM) supplemented with 10% fetal bovine serum (FBS). Plastic Costar 96-well plates were seeded at a density of 100,000 cells/well 24 h prior to the experiment and concurrently transfected using Lipofectamine^™ 3000 Transfection Reagent according to the manufacturer’s protocol. Cells were transfected with 0.1 μg of hSERT-N1-pEYFP (Addgene #70105) per well before returning the plate to the 37 °C, 5% CO₂ atmosphere incubator overnight. Wells were washed (1 × 200 μL) with 1x Hank’s Balanced Salt Solution (HBSS) supplemented with 2 mM MgCl₂, 2 mM CaCl₂, and 5 mM HEPES and replenished with 100 μL of supplemented HBSS. The plate was placed in a 37 °C water bath for the remainder of the experiment and allowed to incubate for 30 min prior to beginning the experiment. Next, 100 μL of HBSS containing 80 nM 5-[1,2-³H(N)]-hydroxytryptamine creatinine sulfate ([³H]5-HT; #NET498001MC, Revvity) was added to the wells for a final concentration of 40 nM, and the cells were incubated for 20 min to allow uptake into SERT-expressing cells. Uptake was terminated by aspiration of the wells followed by washing with supplemented HBSS (2 × 200 μL) and replenished with 180 μL of supplemented HBSS. Efflux was then initiated by adding 20 μL of drug (1 mM – 1 pM, 1% DMSO) solution in supplemented HBSS for 20 min at a final concentration of 10 μM – 1 nM (0.1% DMSO). Following the incubation, wells were washed with supplemented HBSS (3 × 200 μL). Cells were then lysed for 30 min by the addition of 30 μL of 10 mM NaOH. Next, 120 μL OptiPhase HiSafe (#1200.437; Revvity) was added to the cell lysates. Counts per minute (CPM) were quantified after an additional 30 min using a MicroBeta2 (Revvity) microplate liquid scintillation counter.

SERT Pharmacochaperoning Assays.

Cells (HEK293T) were grown in Dulbecco’s Modified Eagle Media (DMEM) supplemented with 10% fetal bovine serum (FBS). Plastic Costar 96-well plates were seeded at a density of 100,000 cells/well 24 h prior to the experiment and concurrently transfected using Lipofectamine^™ 3000 Transfection Reagent according to the manufacturer’s protocol. Cells were transfected with 0.1 μg of SERT-PG^601,602AA plasmid (kindly gifted by Michael Freissmuth) per well before returning the plate to the 37 °C, 5% CO₂ atmosphere incubator overnight. The next day, wells were washed once with 200 μL of a 1x Hank’s Balanced Salt Solution (HBSS) supplemented with 2 mM MgCl₂, 2 mM CaCl₂, and 5 mM HEPES before being exchanged with fresh media containing the respective drugs at final concentrations of 20 μM (0.2% DMSO). The plate was then returned to the 37 °C, 5% CO₂ atmosphere incubator for 24 h. The following day, the wells were washed with 200 μL of supplemented HBSS at intervals of 10 min for four times total. The wells were replenished with 100 μL of supplemented HBSS before adding 100 μL of HBSS containing 80 nM 5-[1,2-³H(N)]-hydroxytryptamine creatinine sulfate ([³H]5-HT; #NET498001MC, Revvity) at a final concentration of 40 nM. The SERT-expressing cells were allowed to uptake radiolabeled 5-HT for 10 min. Uptake was terminated by aspiration of the wells followed by washing with supplemented HBSS (2 × 200 μL). Cells were then lysed for 30 min by the addition of 30 μL of 10 mM NaOH. Next, 120 μL of OptiPhase HiSafe (#1200.437; Revvity) was added to the cell lysates. Counts per minute (CPM) were quantified after an additional 30 min using a MicroBeta2 (Revvity) microplate liquid scintillation counter.

Supplementary Material

SUPPORTING INFORMATION

Procedures for synthesizing tropidine analogues (PDF)

Characterization data for tropidine analogues (PDF)

¹H NMR for tropidine analogues (PDF)

¹³C NMR for tropidine analogues (PDF)