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Published in final edited form as: Neuropharmacology. 2023 Dec 27;245:109827. doi: 10.1016/j.neuropharm.2023.109827

Structure-Activity Relationships for Locomotor Stimulant Effects and Monoamine Transporter Interactions of Substituted Amphetamines and Cathinones

Lauren R Fitzgerald a, Brenda M Gannon a, Donna Walther b, Antonio Landavazo c, Takato Hiranita d, Bruce E Blough c, Michael H Baumann b, William E Fantegrossi a
PMCID: PMC10842458  NIHMSID: NIHMS1956080  PMID: 38154512

Abstract

Substitutions to the phenethylamine structure give rise to numerous amphetamines and cathinones, contributing to an ever-growing number of abused novel psychoactive substances. Understanding how various substitutions affect the pharmacology of phenethylamines may help lawmakers and scientists predict the effects of newly emerging drugs. Here, we established structure-activity relationships for locomotor stimulant and monoamine transporter effects of 12 phenethylamines with combinations of para-chloro, β-keto, N-methyl, or N-ethyl additions. Automated photobeam analysis was used to evaluate effects of drugs on ambulatory activity in mice, whereas in vitro assays were used to determine activities at transporters for dopamine (DAT), norepinephrine (NET), and 5-HT (SERT) in rat brain synaptosomes. In mouse studies, all compounds stimulated locomotion, except for 4-chloro-N-ethylcathinone. Amphetamines were more potent stimulants than their β-keto counterparts, while para-chloro amphetamines tended to be more efficacious than unsubstituted amphetamines. Para-chloro compounds also produced lethality at doses on the ascending limbs of their locomotor dose-effect functions. The in vitro assays showed that all compounds inhibited uptake at DAT, NET, and SERT, with most compounds also acting as substrates (i.e., releasers) at these sites. Unsubstituted compounds displayed better potency at DAT and NET relative to SERT. Para-chloro substitution or increased N-alkyl chain length augmented relative potency at SERT, while combined para-chloro and N-ethyl substitutions reduced releasing effects at NET and DAT. These results demonstrate orderly SAR for locomotor stimulant effects, monoamine transporter activities, and lethality induced by phenethylamines. Importantly, 4-chloro compounds produce toxicity in mice that suggests serious risk to humans using these drugs in recreational contexts.

Keywords: Structure Activity Relationship, Amphetamine, Cathinone

1. INTRODUCTION

Despite attempts at early intervention and increased interdiction efforts, psychostimulant misuse and associated deaths are continuing to rise (Kariisa et al., 2019; Crime, 2020a). Illicit drug manufacturers skirt drug control laws by introducing minor chemical modifications to parent psychostimulant compounds, resulting in a host of novel psychoactive substances (NPS) (Warrick et al., 2012; Crime, 2020b). The proliferation of NPS that are structurally related to amphetamine-type psychostimulants poses major challenges to their timely identification, scientific evaluation, and legal regulation (Crime, 2020b). Phenethylamine compounds like amphetamine (AMP) and its analogues interact with dopamine transporters (DAT), norepinephrine transporters (NET), and serotonin transporters (SERT) in a manner dependent on their molecular size. In general, larger compounds function as non-transported monoamine uptake inhibitors, while smaller compounds inhibit uptake and also function as transportable substrates (i.e., releasers) which facilitate non-exocytotic monoamine release by reversing the normal direction of transporter flux (Rothman and Baumann, 2003). Structurally similar amphetamine analogues likely share these mechanisms of action, thereby providing viable alternatives to the illicit parent compounds (Giné et al., 2014; Marusich et al., 2016; Palamar et al., 2016). Indeed, substituted amphetamines and substituted cathinones are frequently detected in seized drug mixtures in abuse-ready preparations (Warrick et al., 2012; Seely et al., 2013; Oliver et al., 2019; Guirguis et al., 2017; Grifell et al., 2017). Para-chlorinated amphetamine analogues are of particular interest in this regard as these substitutions have been shown to increase serotonergic effects and concomitant toxicities (Fuller et al., 1988, 1965; Murnane et al., 2012).

Synthetic cathinone (CATH) analogues have been studied less intensively than substituted amphetamines, but acute and chronic toxicities associated with their misuse have been reported as a cause for concern (Crime, 2021; Štefková et al., 2017; Angoa-Pérez et al., 2017). Chemically, CATH is the β-keto derivative of AMPH, and all cathinone analogues bear a β-keto moiety. It would be beneficial for scientists and legislators alike to determine whether established structure-activity relationships (SAR) for substituted amphetamines can also be applied to their cathinone counterparts, because small structural changes to drug molecules often result in large alterations in pharmacological effects (Rothman and Baumann, 2003; Baumann et al., 2018). Though it might be predicted that SAR for amphetamines and cathinones would be similar, this notion has not been rigorously evaluated using a large sample of compounds forming a rational SAR series. To this end, these experiments utilized in vivo and in vitro methods to systematically study 12 structurally-related compounds (Figure 1) to establish the effects of para-chloro ring-substitution, addition of a β-keto group, and addition of an N-methyl or N-ethyl group on the phenethylamine scaffold. The simplest compound in this series is AMP, a prototypical psychostimulant in humans and laboratory animals (Cho and Segal, 1994; Rasmussen, 2015). The N-methyl derivative of AMP is methamphetamine (METH), and the N-ethyl derivative is ethamphetamine (EA). METH is twice as potent as AMP in vivo, while EA is half as potent as AMP (Cho and Segal, 1994; van der et al., 1962). Para-chloro ring-substitution of AMP, METH, and EA yields 4-chloroamphetamine (4CA), 4-chloromethamphetamine (4CMA), and 4-chloroethamphetamine (4CEA), respectively. It has been widely documented that these chloro substituted AMP derivatives are more serotonergic than their unchlorinated analogues, which are typically more dopaminergic (Cho and Segal, 1994; Johnson et al., 1990; Fuller, 1992; Fuller et al., 1965; Murnane et al., 2012).

Figure 1: Structures.

Figure 1:

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Array of 12 compounds where the structures become increasingly complex, differing from one another by either a para-chlorine substitution, β-keto substitution, or N-methyl or N-ethyl substitution. The top row includes amphetamine (AMP), methamphetamine (METH), and ethamphetamine (EA). The second row depicts the para-chlorinated compounds of row 1, including 4-Cl amphetamine (4CA), 4-Cl methamphetamine (4CMA), and 4-Cl ethamphetamine (4CEA), respectively. Similarly, the third row contains cathinone (CATH), methcathinone (MC), and ethcathinone (EC), and the fourth row contains 4-Cl cathinone (4CC), 4-Cl methcathinone (4CMC), and 4-Cl ethcathinone (4CEC). Within each row, columns 2 and 3 contain the N-methyl and N-ethyl substituted analogue of column 1, respectively. The third and fourth rows depict the β-ketone containing analogues of the first and second rows, respectively.

CATH is a naturally occurring psychostimulant found in the fresh leaves of the khat shrub Catha edulis (Feng et al., 2017; Simmons et al., 2018) and has effects similar to those of AMP (Cho and Segal, 1994; Glennon, 1986; Schechter et al., 1984). N-methyl substitution of CATH produces the β-keto analogue of METH, methcathinone (MC). Similar to METH, MC has psychostimulant-like effects in animals and humans (Goldstone, 1993), and its chronic use is associated with the development of a Parkinsonian syndrome (Stepens et al., 2008; Sikk and Taba, 2015). N-ethyl substitution of CATH yields the β-keto derivative of EA, ethcathinone (EC), a compound that does not appear to be widely misused as of yet. Para-chloro ring-substitution of CATH, MC, and EC produces 4-chlorocathinone (4CC), 4-chloromethcathinone (4CMC), and 4-chloroethcathinone (4CEC), respectively. Chloro-containing cathinones have become prevalent constituents of confiscated drug products, with 4CMC among the five most frequently seized cathinones in Europe in 2016 (Wojcieszak et al., 2020). Determining whether or not established SAR for substituted amphetamines extends to emerging cathinones will allow researchers to better understand the abuse liability and potential toxic effects of stimulant NPS (Gannon et al., 2018a, 2018b), and enable scientists to predict effects of novel substituted cathinones, based on well-established effects of substituted amphetamines with the same structural modifications. Such information will allow scientists and lawmakers to prioritize experimental interest in compounds with purported health risks, and may speed up the regulation of such compounds.

2. MATERIALS AND METHODS

2.1. Animals

Male NIH Swiss mice (Charles River Laboratories, Wilmington MA, USA) were used for locomotor experiments. Mice weighing 25–30 g at delivery were group-housed three per cage at the University of Arkansas for Medical Sciences (UAMS) animal facility (Little Rock, AR, USA). Mice were maintained on a 12-hour light/dark cycle at 22 ± 2°C and 45–50% humidity, with food and water available ad libitum. All locomotor test conditions used groups of eight drug-naïve mice unless otherwise noted. Mouse experimental procedures were approved by the UAMS Institutional Animal Care and Use Committee (IACUC). Male Sprague-Dawley rats (Envigo, Frederick MD, USA) were used for in vitro transporter assays. Rats weighing 250–300 g at delivery were group-housed three per cage at the National Institute on Drug Abuse (NIDA) Intramural Research Program (IRP) animal facility (Baltimore, MD, USA). Rats were maintained on a 12-h light/dark cycle at 22 ± 2°C and 45–50% humidity, with food and water available ad libitum. Rat experimental procedures were approved by NIDA IRP IACUC. All animal facilities were accredited by the American Association for Laboratory Animal Care, and procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council of the National Academies 2011).

2.2. Drugs

(+)-S-amphetamine sulfate (AMP) and (±)-R/S-4-chloroamphetamine HCl (4CA) were purchased from Millipore-Sigma (St. Louis, MO, USA). (+)-S-methamphetamine HCl (METH), (+)-S-ethamphetamine HCl (EA), (±)-R/S-cathinone HCl (CATH), and (±)-R/S-methcathinone HCl (MC) were supplied by the NIDA Drug Supply Program (NDSP, Rockville, MD, USA). (±)-R/S-4-chloro-methamphetamine HCl (4CMA), (±)-R/S-4-chloroethamphetamine HCl (4CEA), (±)-R/S-ethcathinone HCl (EC), (±)-R/S-4-chlorocathinone HCl (4CC), and (±)-R/S-4-chloromethcathinone HCl (4CMC) were synthesized at RTI International Center for Drug Discovery by one of us (BEB). (±)-R/S-4-chloro-ethcathinone HCl (4CEC) was received from the United States Drug Enforcement Administration (DEA) Special Research and Testing Laboratory. For the mouse locomotor experiments, compounds were dissolved in 0.9% physiological saline and stored in glass vials at 3–5°C until the time of use. Mice received intraperitoneal (IP) drug injections in a volume of 0.1 mL/10g. For the in vitro assays, compounds were dissolved in 100% dimethylsulfoxide (DMSO) at a concentration of 1 mM and stored at −80°C until the day of assay.

2.3. Experiment 1. Effects of structure on drug induced horizontal locomotor activity.

Mice were randomly assigned to receive a single IP injection of one of the test drugs (Figure 1) or saline. Approximately 30 min after mice were moved from the vivarium to the experimental testing room, they were removed from their home cage, weighed, and administered a single IP injection of the assigned dose of the assigned test compound. Immediately after drug administration, mice were placed into the center of a clear acrylic chamber (43.20 × 43.20 × 29.80 cm; Med Associates Inc., Fairfax, VT, USA) equipped with photobeam arrays and housed within closed, lighted, noise-masking cabinets. Activity was recorded for 6 h post injection as infrared beam breaks and converted to distance traveled using Activity Monitor 7 Software (Med Associates Inc.).

2.4. Experiment 2. Effect of structure on transporter uptake and release in rat brain synaptosomes.

Uptake inhibition and release assays were conducted using crude synaptosomes prepared from rat brain tissue as previously described (Gannon et al., 2018a; Partilla et al., 2016; Solis et al., 2017; Rothman et al., 2012). Briefly, rats were decapitated after CO2 narcosis, brains were rapidly extracted, and synaptosomes were prepared in 10% sucrose. Synaptosomes from rat caudate tissue were used for DAT assays, whereas synaptosomes from rat forebrain minus caudate tissue were used for NET and SERT assays.

For uptake inhibition assays, freshly prepared rat brain synaptosomes were added to tubes containing Krebs-phosphate buffer (KPB), selective uptake inhibitors, the specific test drug, and the appropriate [3H]neurotransmitter. Specifically, 50 nM GBR 12935 was added to NET and SERT assays to block DAT, whereas 100 nM nomifensine was added to SERT assays to block NET. Five nM [3H]dopamine, 10 nM [3H]norepinephrine, or 5 nM [3H]5-HT was used as the radiolabeled transmitter for DAT, NET, or SERT uptake assays, respectively.

For the release assays, freshly prepared synaptosomes were incubated with selective uptake inhibitors and [3H]substrate for 30 min, to achieve steady state. Specifically, 100 nM desipramine was added to DAT release assays to block NET, 100 nM citalopram was added to DAT and NET assays to block SERT, and 1 μM reserpine was added to all assays to prevent trapping of substrates in synaptic vesicles. Nine nM [3H] MPP+ was used as the radiolabeled substrate for DAT and NET release assays, whereas 5 nM [3H]5-HT was used as the radiolabeled substrate for SERT release assays. After the incubation step, the preloaded synaptosomes were added to tubes containing the KPB, the appropriate blockers, and the specified test drug. The uptake inhibition and release assays were terminated by vacuum filtration and retained radioactivity was quantified by liquid scintillation counting as previously described (Baumann et al., 2013).

2.5. Statistical Analysis

GraphPad Prism (Prism version 8 or 9, La Jolla, CA, USA) and Systat software (SigmaPlot/SigmaStat suite version 11, Inpixon, Palo Alto, CA, USA) were used for statistical analyses. For the mouse studies, horizontal locomotor activity data are presented as mean ± standard error of the mean (SEM), expressed as cumulative distance traveled (m) for 6 h post injection. These data were subjected to one-way analysis of variance (ANOVA) across doses for each drug, and all pairwise comparisons were made using Tukey’s HSD test. For the in vitro experiments, dose-response data for test compounds in DAT, NET, and SERT uptake and release assays are depicted as mean ± standard deviation (SD). Concentration-response data were fit using nonlinear regression, from which IC50, EC50, and Emax values were derived, with 95% confidence intervals.

3. RESULTS

3.1. Experiment 1. Effect of structure on drug induced horizontal locomotor activity.

Figure 2 presents dose-effect data for drug-induced horizontal locomotor activity over a 6 h period following injection, plotted as a function of dose. All drugs produced significant main effects on locomotor activity as compared to the saline control (AMP: F=17.71, p<0.0001; METH: F=10.13, p<0.0001; EA: F=24.85, p<0.0001; 4CA: F=20.12, p<0.0001; 4CMA: F=6.807, p=0.0004; 4CEA: F=41.03, p<0.0001; CATH: F= 5.038, p=0.0026; MC: F=11.08; p<0.0001; EC: F=37.08, p=0.0264; 4CC: F=2.969, p=0.0327; 4CMC: F=7.624, p=0.0002; 4CEC: F=8.416, p<0.0001). Tukey’s HSD test showed that all drugs except 4CEC significantly and dose-dependently increased activity as compared to saline (Figure 2). AMP, METH, EA, and MC dose-effect curves contained descending limbs due to the induction of motor stereotypy at the largest doses tested, indicated by biphasic time-activity curves at these doses. For all remaining compounds except 4CEA and 4CEC, descending limbs were not obtained due to observed lethal effects. A one-way ANOVA of Emax values (Table 1) indicated a significant main effect of drug (F=9.136, p<0.0001). The rank-order of locomotor stimulatory effectiveness was 4CMA > 4CEA > EA > 4CMC ≈ 4CA > EC ≈ MC > AMP ≈ METH > CATH ≈ 4CC > 4CEC (see Table 1). Tukey’s HSD test confirmed that many of these Emax values were significantly different among various compounds, such that 4CMA was significantly more effective than AMP (q=7.280, p=0.0002), METH (q=7.526, p=0.0001), 4CA (q=5.163, p=0.0327), CATH (q=9.385, p<0.0001), MC (q=6.876, p=0.0006), EC (q=6.485, p=0.0017), 4CC (q=9.898, p<0.0001), 4CMC (q=5.148, p=0.0337), and 4CEC (q=11.09, p<0.0001); EA was significantly more effective than CATH (q=5.221, p=0.0291), 4CC (q=5.734, p=0.0099) and 4CEC (q=6.928, p=0.0006); 4CA was significantly more effective than 4CEC (q=5.929, p=0.0064); 4CEA was significantly more effective than CATH (q=6.759, p=0.0009), 4CC (q=7.272, p=0.0002) and 4CEC (q=8.466, p<0.0001); and 4CMC was significantly more effective than 4CEC (q=5.944, p=0.0062 ).

Figure 2: Ambulatory Distance.

Figure 2:

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Average locomotor activity over 6 h ± SEM elicited by IP administration of saline (open circles) or the given dose of the specified drug (filled squares). Abscissa: ‘Saline’ represents saline control data and numbers refer to dose of the respective drug expressed as mg/kg on a log scale. Each point represents a separate group of animals; n = 8. Ordinate: mean distance traveled in meters recorded over 6 hours post injection. Asterisks indicate a significant difference (p<0.05) between dose administered and saline using a one-way ANOVA. Octothorpes indicate a significant difference (p<0.05) between dose administered and next lowest dose administered.

Table 1:

Total distance (in meters) traveled for 6 hours following treatment with study compounds expressed as Emax ± SEM; IC50 and 95% confidence intervals for monoamine uptake inhibition at DAT, NET, and SERT in nM; EC50 and 95% confidence intervals for substrate release at DAT, NET, and SERT in nM.

Drug Distance (m) Uptake Inhibition, IC50 (95% CI) in nM Monoamine Release, EC50 (95% CI) in nM
Emax ± SEM DAT NET SERT DAT NET SERT
AMP 499.51 ± 32.90 321.4 (249.5, 414.0) 240.7 (202.3, 286.4) 11468.3 (9036.5, 14554.6) 13.0 (10.5, 16.1) 22.4 (14.5, 34.8) 4008.7 (2877.4, 5584.7)
METH 479.91 ± 55.33 161.4 (133.7, 195.0) 227.0 (195.0, 264.2) 3850.3 (2636.3, 5623.4) 11.4 (8.1, 16.1) 12.8 (8.1,20.3) 695.0 (532.1,907.8)
EA 747.59 ± 77.76 379.3 (335.0, 429.5) 369.4 (331.1,412.1) 1605.1 (1250.3, 2060.6) 44.1 (30.7, 63.2) 28.1 (15.6, 50.7) 333.0 (238.8, 464.5)
4CA 668.06 ± 92.84 756.0 (612.4, 933.3) 289.1 (251.8, 331.9) 714.5 (511.7, 997.7) 42.2 (27.1,65.6) 26.2 (14.0, 48.8) 28.3 (23.6, 33.9)
4CMA 1079.03 ± 176.07 803.5 (676.1,955.0) 434.5 (339.6, 555.9) 151.9 (119.7, 192.8) 54.7 (35.2, 85.1) 36.5 (22.8, 58.5) 29.9 (21.3, 42.1)
4CEA 870.01 ± 78.48 488.7 (394.5, 605.3) 608.8 (477.5, 776.2) 573.5 (456.0, 721.1) 238.0 (117.2, 483.1) 162.6 (87.3, 302.7) 33.8 (23.2, 49.2)
CATH 331.98 ± 21.77 776.2 (644.2, 935.4) 396.3 (328.9, 477.5) 22361.5 (13677.3, 36559.5) 34.8 (25.3, 47.9) 25.6 (14.6, 45.1) 7594.5 (4688.1, 12302.7)
MC 531.69 ± 43.77 445.7 (370.7, 535.8) 364.8 (285.1,466.7) 20965.2 (14223.3, 30903.0) 23.6 (18.7, 29.9) 26.1 (14.9, 45.7) 4576.1 (3169.6, 6606.9)
EC 562.82 ± 43.32 839.5 (665.3, 1059.3) 847.2 (734.5, 977.2) 9440.6 (7568.3, 11776.1) 267.6 (152.1,471.0) 88.3 (50.0, 156.0) 1923.1 (1482.5, 2494.6)
4CC 291.12 ± 64.37 4466.8 (3681.3, 5420.0) 1135.0 (984.0, 1309.2) 632.4 (521.2, 767.4) 221.8 (155.6, 316.2) 85.1 (45.9, 157.8) 128.4 (92.0, 179.1)
4CMC 669.26 ± 131.31 841.4 (679.2, 1042.3) 511.1 (420.7, 620.9) 533.3 (450.8, 631.0) 46.4 (28.6, 75.2) 90.9 (64.6, 127.9) 124.0 (86.9, 177.0)
4CEC 196.09 ± 23.83 1574.0 (1230.3, 2013.7) 3054.9 (2600.2, 3589.2) 579.4 (465.6, 721.1) 353.6 (45.7, 2735.3) 5194.0 (2606.2, 10351.4) 152.6 (108.1,215.3)
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3.3. Experiment 2. Effect of structure on transporter uptake and release in rat brain synaptosomes.

Figure 3A shows concentration-response curves for drug-induced inhibition of uptake at DAT (white circles), NET (gray squares), and SERT (black triangles). Data were fit with a nonlinear regression for DAT, NET, and SERT individually. All compounds exhibited fully efficacious concentration-dependent inhibition of monoamine uptake, and potencies for DAT and NET were generally similar to one another for each individual compound. For AMP and CATH, potency at SERT was poor, but SERT activity was systematically improved by lengthening the amine substituent to methyl and to ethyl. Addition of a 4-chloro ring-substitution improved SERT potency, regardless of the amine substituent length. Calculated IC50 values and their associated 95% confidence intervals for each transporter are listed in Table 1.

Figure 3: In Vitro Assays.

Figure 3:

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Panel A illustrates the effects of the test drugs on inhibition of [3H]neurotransmitter uptake by DAT (white circles), NET (gray squares), or SERT (black triangles) in rat brain tissue. Abscissa: molar drug concentration. Ordinate: percent of maximal uptake expressed as mean ± SD for n=3 experiments performed in triplicate. Panel B depicts the effects of the test drugs on release of [3H]MPP+ at DAT (white circles) and NET (gray squares), or [3H]5-HT at SERT (black triangles) in rat rain tissue. Abscissa: molar drug concentration. Ordinate: percent of maximal release expressed as mean ± SD for n=3 experiments performed in triplicate.

Figure 3B presents concentration-effect curves for drug-induced release at DAT (white circles), NET (gray squares), and SERT (black triangles). Data were fit using a nonlinear regression model for DAT, NET, and SERT individually. Results here largely recapitulate the uptake inhibition data, with most compounds exhibiting fully efficacious concentration-dependent stimulation of monoamine release. Activities at DAT and NET were generally similar to one another for each individual compound, with the exception of the two 4-chloro-ethyl compounds (4CEA and 4CEC) which exhibited reduced efficacy to release dopamine and reduced potency to release norepinephrine. For AMP and CATH, the potency for release of [3H]5-HT via SERT was poor, but was systematically improved by lengthening the amine substituent to methyl and to ethyl. Addition of a 4-chloro ring-substitution improved SERT release potency, regardless of the amine substituent length. Calculated EC50 values and their associated 95% confidence intervals for each transporter are listed in Table 1.

4. DISCUSSION

The increasing prevalence of NPS abuse highlights the need to determine their pharmacological effects and associated toxicities quickly and efficiently. Determining whether or not established SAR for substituted amphetamines can be applied to analogous substituted cathinones can help scientists and regulators prioritize which compounds to study in order to speed up the process of research and evidence-based legislation. This study assessed locomotor stimulant effects in vivo and monoamine transporter interactions in vitro for 6 substituted amphetamines and their 6 substituted cathinone analogues. In particular, we assessed the relationships between pharmacological endpoints and structural modifications of β-keto addition, para-chloro ring-substitution, N-methylation and N-ethylation. This study utilized the simplest approach to SAR determination, investigating a series of compounds distinguished by single moiety substitutions (Glennon and Dukat, 2017). Previous studies have examined similar endpoints and compounds, however no previous studies have specifically assessed these 12 compounds with their systematic single moiety substitutions in an attempt to investigate SAR in this rigorous manner. We have thus included results from compounds previously studied by ourselves and others to provide a complete data set for this series of drugs using the same experimental conditions within the same laboratories.

4.1. Addition of a β-keto moiety:

Previous studies have profiled pharmacological effects of amphetamines and cathinones, with the majority comparing one or two cathinones to a singular amphetamine analogue – typically METH or MDMA (Štefková et al., 2017; Baumann et al., 2013; Soares et al., 2019; Kiyatkin and Ren, 2017; Fantegrossi et al., 2013). For most prior studies, comparisons among drugs involved multiple moiety substitutions, thus only limited information is available concerning the impact of the β-ketone substitution which transforms the amphetamine to its cathinone analogue. The locomotor data we present here demonstrate that, with the exception of METH vs MC, the addition of a β-keto group decreases in vivo efficacy for producing locomotor effects in mice. Comparing the lowest dose of each drug which yielded a significant locomotor stimulant effect suggests that the β-keto analogues also exhibit decreased in vivo potency. Specifically, AMP, METH, and EA all elicited significant stimulant effects at doses where CATH, MC, and EC failed to increase motor activity above saline control levels. The same is true for 4CA vs 4CC and for 4CEA vs 4CEC, but not for 4CMA vs 4CMC. Few previous studies have directly compared amphetamines to their β-keto analogues, but at least two early publications with CATH demonstrate reduced motor stimulation, as compared to AMPH, in rats (Kalix, 1980) and in mice (Zelger et al., 1980). Our data are thus in general agreement with these previous data.

Our in vitro experiments demonstrate that the addition of a β-keto group generally worsens potency for monoamine uptake inhibition and release at DAT, NET, and SERT. With the exception of 4CMA vs 4CMC, addition of a β-keto group decreases potency to inhibit uptake through DAT and NET, with IC50 values typically increasing at least 2-fold. The effects of β-keto addition on potency to inhibit uptake at SERT were less robust than observed at the catecholamine transporters, but for unchlorinated compounds, potency at SERT was already negligible. With the exception of 4CA vs 4CC, potency for uptake inhibition at SERT was always either lower or unchanged as compared to the amphetamine parent drug. The β-keto analogues also displayed decreased potency to stimulate release at all three transporters as compared to their amphetamine counterparts. The reduced effect of β-keto analogues was especially pronounced for release at DAT, where IC50 values were typically increased by at least 2-fold, likely explaining the decreased psychostimulant potency and efficacy in our locomotor studies.

4.2. Para-chloro ring substitution:

Previous studies have highlighted relatively weak locomotor effects associated with para ring-substituted cathinones, including 4CEC, 4CMC, mephedrone, flephedrone, 4-methylethcathinone, and 4-methyl-α-pyrrolidinopropiophenone (Gatch et al., 2019; Gatch et al., 2015a; Gatch et al., 2015b; Gatch et al., 2021; Gatch et al., 2013). Presently, all six unchlorinated compounds (AMP, METH, EA, CATH, MC, and EC) elicited robust dose-dependent increases in locomotor activity in mice. Furthermore, comparing the locomotor effects of the 4-Cl cathinone derivatives (4CC, 4CMC, 4CEC) to their unsubstituted cathinone analogues (CATH, MC, EC respectively), we found that there was no obvious trend regarding locomotor potency or effectiveness. Importantly, our dose-effect curve for locomotor effects of 4CMC is similar to that reported previously in rats, with a dose of 1 mg/kg producing saline-like activity but higher doses producing dose-dependent stimulant effects (Chojnacki et al., 2023). Among the amphetamines, para-chloro substitution (AMP, METH, and EA to 4CA, 4CMA, and 4CEA, respectively) systematically increased locomotor efficacy (see Emax values in Table 1). Additionally, the 4-chloro compounds induced lethal effects at doses which did not elicit motor stereotypy and appeared on the ascending-limb for locomotor activity, resulting in monotonic dose-effect functions for these substances. These lethal effects are at least partially related to 5-HT release (see below.)

For any given amphetamine or cathinone lacking ring substitution, inhibition of uptake through DAT and NET were similar to each other, and inhibition of uptake at SERT was much less potent compared to the IC50 values observed at the catecholamine transporters. Previous literature indicates that para-halogenation of substituted cathinones yields compounds with reduced affinity for DAT and increased affinity for SERT, inverting the typical DAT/SERT selectivity ratio observed among abused psychostimulants (Chojnacki et al., 2023; Maier et al., 2021). Presently, we observed that the addition of a 4-chloro substituent significantly improved potency to inhibit SERT uptake to the extent that the chlorinated compounds became either equipotent or more selective for SERT over DAT. Importantly, this trend applies to both substituted amphetamines and substituted cathinones. Although consistent across all drugs, the range for this increase in potency was quite large, with the smallest effect being an approximately 3-fold improvement in potency when EA was chlorinated to 4CEA, and the largest measurable effect being an approximately 25-fold improvement in potency when METH was chlorinated to 4CMA. In the case of AMP, CATH and MC, the magnitude of this chlorination effect could not be calculated because the potency of the unsubstituted drug for SERT was so poor.

Regarding monoamine release, it is well documented that para-chloro ring substitution of psychostimulant-like compounds results in more potent 5-HT releasing derivatives (Cho and Segal, 1994; Chojnacki et al., 2023; Miczek et al., 1975; Murnane et al., 2012; Fuller et al., 1965). For cathinone compounds in particular, the effect of para ring substitution is directly related to molecular size of the substituent, where larger functional groups engender greater potency at SERT release relative to DAT release (Bonano et al., 2015; Negus and Banks, 2017). At the mechanistic level, the increased SERT selectivity for larger para ring substituents is facilitated by a larger orthosteric binding pocket in SERT as compared to DAT (Sakloth et al., 2015). Similar to inhibition of uptake, the present experiments showed that potencies to stimulate catecholamine release were similar to each other for each amphetamine or cathinone lacking ring substitution, while potency to induce release through the SERT was always less potent compared to the catecholamine transporters. Thus, unchlorinated compounds were more selective for DAT and NET than for SERT. Addition of a 4-chloro moiety always increased potency to stimulate release through SERT, regardless of side chain length.

4.3. N-substitution:

Although the number of amphetamine analogues with different amine substituents is relatively low in recreational drug markets (Cho and Segal, 1994), N-methyl and N-ethyl substitutions are sometimes found. Pharmacological activity of amphetamine-type drugs is decreased substantially if the N-alkyl chain is lengthened beyond ethyl, as previous studies show that N-propylamphetamine and N-butylamphetamine are ~4-fold and ~6-fold less potent than amphetamine in rats (Woolverton et al., 1980). Among the amphetamines and the cathinones in our present studies, longer N-alkyl chains were associated with greater locomotor stimulant effects, reflected in larger Emax values. Lengthening the side chain to methyl and then to ethyl tended to systematically increase Emax values for locomotor effects among the amphetamines and the cathinones, although variability was large and, in some cases, reliable assessments of Emax values could not be accomplished due to induction of lethality at doses on the ascending limbs of locomotor dose-effect curves.

Potency to inhibit catecholamine uptake was not systematically affected by N-alkyl chain length, but SERT potency was systematically improved when unsubstituted compounds were modified through N-methylation and N-ethylation, and this was recapitulated for the transporter release assays. All compounds were fully efficacious at releasing all three monoamines, except for 4CEA and 4CEC, which did not fully induce release through the DAT at any tested concentration. For 4CEA and 4CEC, release through SERT was both more potent and more efficacious than release through the catecholamine transporters, highlighting the pronounced serotonergic effects produced by the “double hit” of longer N-alkyl side chains and 4-chloro ring-substitution of these two molecules. The unique transporter pharmacology observed here for 4CEA and 4CEC has been described previously as “hybrid” transporter activity, characterized by substrate-type releasing activity at SERT but uptake inhibition at DAT (Saha et al., 2015). Recently, Chojnacki and colleagues (Chojnacki et al., 2023) also demonstrated analogous hybrid transporter activity for 4CEC.

4.4. Stereochemistry:

A limitation of the current experiments is the inconsistent use of single enantiomers for some substances and racemic mixtures for others. For all assays, S-enantiomers of AMP, METH, and EA were used, while the remaining compounds were tested as racemic mixtures. Thus, comparisons assessing the impact of N-alkyl chain length on amphetamine pharmacology are not affected by this discrepancy, but the remaining comparisons (i.e., β-ketone and para-chloro ring substitution) are confounded by potential stereochemical differences among compounds. Consistency in stereochemistry is clearly needed to draw rigorous conclusions regarding SAR, however if the goal is to evaluate the abuse liability of emerging NPS compounds, then using the most commonly available form of the specific substances might be preferable. To this end, the use of S-METH in the present studies seems more appropriate than using the racemic mixture because the majority of seized METH samples are the pure S-enantiomer (Wang et al., 2015). However, seized AMP is generally racemic (Losacker et al., 2022), so our use of S-AMP – though consistent with S-METH and S-EA – is perhaps not optimal. For NPS like the present cathinones, determination of enantiomer ratios in the products available on the illicit market is largely unknown, but enantioselective monitoring by various governments is becoming commonplace (Losacker et al., 2022). As various precursor substances are regulated and monitored by authorities, illicit synthesis of these substances may be altered to avoid the use of watchlist reagents, resulting in a shift in the stereochemical composition of the resulting products. Hence, analysis of enantio-separated psychostimulant mixtures is being optimized (Almeida et al., 2022), and future studies may benefit from using the more representative forms of these substances as they are determined, and as they may change over time.

4.5. Conclusions:

Established SAR is useful when forming hypotheses about the pharmacology of NPS, and the present studies indicate that not all SAR trends established for amphetamine analogues hold for cathinone derivatives. For example, para-chloro ring substitution of the amphetamines increased locomotor activity, but this was not the case for the cathinones. While the cathinones were weaker locomotor stimulants compared to their respective amphetamine analogues, there is evidence that reinforcing effects of cathinones can be greater than those of traditional psychostimulants (Gannon et al., 2018a, 2018b, 2017; Partilla et al., 2016; Solis et al., 2017; Rothman et al., 2012; Baumann et al., 2013; Glennon and Dukat, 2017; Fantegrossi et al., 2013; Gatch et al., 2021). These differences highlight the fact that SAR is endpoint-specific, even when the endpoints in questions are relatively similar in their relation to abuse liability and mediation by central dopaminergic systems. On the other hand, similar SAR among the amphetamines and cathinones was determined regarding the effects of para-chloro substitution, where all compounds studied exhibited improved potency at SERT for uptake and release, as compared to their unsubstituted analogues. Confirmation and extension of these findings in studies utilizing abuse-related endpoints including reinforcing effectiveness would be particularly useful to aid in emergency scheduling of emerging NPS. Finally, given that substituted amphetamines and cathinones are continuously being developed, further in vivo and in vitro studies expanding on the structural modifications studied here in an attempt to establish more thorough SAR should be considered.

Highlights.

  • Structure activity relationships are endpoint specific

  • Not all structure activity relationships shown for amphetamines hold for cathinones

  • 4-chloro compounds produce toxicity in mice that suggests serious risk to humans

Acknowledgements:

The authors thank the UAMS Department of Laboratory Animal Medicine for expert husbandry and veterinary services. This work was presented, in part, in LRF’s doctoral dissertation defense as follows: Russell LN (2022) In Vivo Structure-Activity Relationships of Substituted Amphetamines and Substituted Cathinones. Doctoral dissertation, University of Arkansas for Medical Sciences, Little Rock, AR.

Funding:

This work was supported, in part, by a contract with the US Department of Justice, Drug Enforcement Administration [HHSF223201610079C (WEF)] and by the National Institutes of Health [T32 DA022981 (LRF)]. The work of the Designer Drug Research Unit (DDRU) was generously supported by the NIDA IRP [Z1A DA000523 (MHB)]. Compounds made at RTI were supported by a grant from NIDA (DA012970). None of the funding sources participated in study design, in the collection, analysis, or interpretation of data, in the writing of the report, or in the decision to submit the article for publication.

Footnotes

Competing Interests:

Dr. Fantegrossi declares on behalf of all other authors that this research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest, and none of the authors have any competing interests relevant to these studies to declare. The findings and conclusions in this article are those of the authors and do not necessarily represent the views of the Drug Enforcement Administration, the Department of Justice, the National Institutes of Health, or any other office of the U.S. government.

Declaration of interests

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

William Fantegrossi reports financial support and equipment, drugs, or supplies were provided by US Department of Justice. Lauren Fitzgerald reports financial support was provided by National Institutes of Health. Michael Baumann reports financial support was provided by National Institute on Drug Abuse Intramural Research Program. Bruce Blough reports financial support was provided by National Institute on Drug Abuse.

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