Behavioral Effects of Four Novel Synthetic Cathinone Analogs in Rodents

Abstract

A new generation of novel cathinone compounds have been developed as stimulant substitutes to avoid drug control laws and detection of use by blood tests. Dipentylone, N-ethylhexedrone, 4-chloroethcathinone (4-CEC), and 4’-methyl-α-pyrrolidinohexiophenone (MPHP) were tested for in-vivo psychostimulant-like effects to assess their abuse liability. Locomotor activity was assessed in an open-field assay using Swiss-Webster mice to screen for locomotor stimulant effects and to identify behaviorally-active dose ranges, times of peak effect, and durations of action. Discriminative stimulus effects were assessed in separate groups of Sprague-Dawley rats trained to discriminate cocaine or methamphetamine from vehicle. Dipentylone, N-ethylhexedrone, 4-CEC and MPHP dose-dependently increased locomotor activity. Dipentylone, N-ethylhexedrone, and MPHP produced maximal stimulant effects similar to cocaine and methamphetamine. 4-CEC was less efficacious, producing peak stimulant effects of about 74% of that of methamphetamine. The compounds were less potent than methamphetamine and approximately equipotent with cocaine. The doses of cocaine, methamphetamine, dipentylone and 4-CEC that produced peak effects lasted 2 to 3 h, the peak dose of N-ethylhexedrone lasted 4 h, and the peak dose of MPHP lasted 6 h. All four compounds fully substituted for the discriminative stimulus effects of methamphetamine and cocaine, although full substitution by 4-CEC occurred at doses that substantially decreased response rate. Only 4-CEC fully substituted for MDMA. These data provide evidence that the novel cathinone compounds dipentylone, N-ethylhexedrone, 4-CEC and MPHP demonstrate potential for abuse as psychostimulants, given their ability to stimulate locomotor activity and their substitution for the discriminative stimulus effects of methamphetamine and cocaine.

1. Introduction

The ongoing emergence of new psychoactive substances (NPS) is a continuing public health problem ¹. Cathinone and cannabinoid analogs are the two most commonly used synthetic compounds ^1,2. Many of the NPS produce severe adverse effects in recreational users. Users of cathinones self-reported adverse effects such as anxiety, hallucinations, nervousness, paranoia, angina, myocardial infarction, tachycardia, and difficulty urinating ³. Published reports from poison control centers and clinical case studies found similar signs and symptoms, including extremely high blood pressure, confusion, psychotic-like and/or aggressive behaviors ^4,5. Life-threatening effects such as convulsions, myocardial infarction, arrhythmias, cardiac arrest, metabolic acidosis and prolonged rhabdomyolysis can be seen following a few doses of some of the cathinones ^6–9.

Four synthetic cathinones (4’-methyl-α-pyrrolidinohexiophenone (MPHP), N-ethylhexedrone (also known as α-ethylaminocaprophenone, N-ethylamino-hexanophenone, N-ethylnorhexedrone, Hexen and NEH), dipentylone (also known as N,N-dimethylpentylone BetaK-Dmbdp; Bk-dmbdp; DMBDP; Valerophenone), and 4-chloroethcathinone (4-CEC)) have been flagged by the US Drug Enforcement Administration (DEA) for evaluation due to increasing indications that these compounds may be public health risks. Chemical structures are shown in Figure 1. There is currently very little published data on these compounds, mostly reports of usage.

Figure 1.

Chemical structures of the synthetic cathinones tested in the present study.

MPHP is an older compound, first appearing on the illicit drug markets more than 10 years ago ^10,11. Its use can lead to toxic liver damage and rhabdomyolysis ¹². MPHP was tested in rats trained to discriminate 3,4-methylenedioxymethamphetamine (MDMA) from saline, but failed to fully substitute, producing a plateau of MDMA-like responding of around 50% up to doses that suppressed response rate ¹³. More recently, N-ethylhexedrone was found in sample of seized drugs ¹⁴. A case report of severe toxicity by a regular user of “Hexen” indicated signs and symptoms consistent with serotonin syndrome: hyperthermia, tachycardia, ocular clonus and nystagmus, and reduced consciousness ¹⁵. Dipentylone has been detected in wastewater in Europe for the first time, thereby flagging it as an emerging NPS ¹⁶. 4-CEC has also been found in samples of seized drug seizures in the past few years ^17–19, and has also been found in drug samples by a voluntary harm-reduction drug-checking service, although it is not common ²⁰. Two of the compounds (MPHP and N-ethylhexedrone) are under temporary C-I scheduling by the US DEA ²¹.

Pharmacological mechanisms of the four test compounds have been tested in one study ²². According to this study, N-ethylhexedrone was a reuptake inhibitor at dopamine (DAT), norepinephrine (NET) and serotonin transporters (SERT), and had a 100-fold DAT/SERT ratio and a 10-fold DAT/NET ratio. MPHP was a reuptake inhibitor somewhat more selective for DAT, having a 160-fold DAT/SERT ratio and a 10-fold DAT/NET ratio. Dipentylone was equipotent at inhibiting DAT and NET, and was about 10-fold more selective for DAT and NET over SERT. In contrast, 4-CEC was to some degree SERT selective, being 3-fold more potent at SERT than DAT and was equipotent at DAT and NET. It was reported that 4-CEC also produced serotonin release, but not dopamine or norepinephrine release. However, in this study the other cathinone compounds were not tested for their ability to release monoamines.

The purpose of the present study was to provide behavioral data of the abuse potential of these 4 cathinone compounds. Locomotor activity was tested in mice to determine whether the test compounds produce locomotor stimulant effects similar to those of cocaine and methamphetamine. The locomotor assay is also useful for quickly identifying the active dose range and time course, as it correlates highly with potencies in the rat drug-discrimination assay. Drug discrimination is a useful animal model of the subjective effects that correlates well with human use ²³. Rats were trained to discriminate methamphetamine, cocaine, or 3,4-methylenedioxymethamphetamine (MDMA) from saline, and the four test compounds were tested for substitution for the discriminative stimulus effects of methamphetamine, cocaine and MDMA.

2. Materials and Methods

2.1. Subjects

Male Swiss–Webster mice (n=280) were obtained from Envigo (Indianapolis, IN) at approximately 8 weeks of age and tested at approximately 10 weeks of age. Mice were group housed (4 per cage) on a 12:12-h light/dark cycle and were allowed free access to food and water.

Male Sprague-Dawley rats (n=66) were obtained from Envigo. All rats were housed individually and were maintained on a 12:12 light/dark cycle (lights on at 7:00 AM). Body weights were maintained at 320–350 g by limiting food to 15 g/day which included the food received during operant sessions. Water was readily available. All housing and procedures were in accordance with Guidelines for the Care and Use of Laboratory Animals ²⁴ and were approved by the University of North Texas Health Science Center Animal Care and Use Committee.

2.2. Locomotor Activity

The study was conducted using 40 Digiscan (model RXYZCM, Omnitech Electronics, Columbus, OH) locomotor activity testing chambers (40.5 X 40.5 X 30.5 cm) housed within sound-attenuating chambers in sets of two. A panel of infrared beams (16 beams) and corresponding photodetectors were located in the horizontal direction along the sides of each activity chamber. A 7.5-W incandescent light above each chamber provided dim illumination and fans provided an 80-dB ambient noise level within the chamber.

Separate groups of 8 mice were injected with either vehicle (0.9% saline), or a dose of methamphetamine (0.25, 0.5, 1, 2, 4 mg/kg), cocaine (5, 10, 20, 40 mg/kg), dipentylone (2.5, 5, 10, 25, 50 mg/kg), N-ethylhexedrone (2.5, 5, 10, 25, 50 mg/kg), 4-CEC (2.5, 5, 10, 25, 50 mg/kg), or MPHP (2.5, 5, 10, 25, 50 mg/kg) immediately prior to locomotor activity testing. Separate vehicle controls were tested for each test compound. In all studies, horizontal activity (interruption of photocell beams) was measured for 8 hours within 10-min periods, in order to establish a time-course of locomotor effects, beginning at 0800 h (1 h after lights on). Studies typically began with 1 mg/kg, after which higher and/or lower doses were tested from no effect, defined as not statistically different from vehicle, to full effect, defined as less than 50% of vehicle control for depressants. Stimulants typically produce an inverted-U shaped dose-effect curve for the locomotor stimulant effects. Increasing doses are tested until a peak has been reached and at least one dose on the descending limb of the dose-effect curve has been tested.

2.3. Discrimination Procedures

Standard behavior-testing chambers (Coulbourn Instruments, Allentown, PA, Model E10–10) were connected to IBM-PC compatible computers via LVB interfaces (Med Associates, East Fairfield, VT). Response levers were positioned to the left and right of the food hopper. A house light was centered over the hopper close to the ceiling and was illuminated only when the levers were active. The computers were programmed in Med-PC for Windows, version IV (Med Associates) for the operation of the chambers and collection of data.

Using a two-lever choice methodology, a pool of rats was trained to discriminate either methamphetamine (1 mg/kg), cocaine (10 mg/kg), or MDMA (1.5 mg/kg) from saline. Rats received an injection of either saline or drug and were subsequently placed in the behavior-testing chambers, where food (45 mg food pellets; Bio-Serve, Frenchtown, NJ) was available as a reinforcer for every ten responses on a designated injection-appropriate lever. The pretreatment time was 10 min for cocaine and methamphetamine, and 15 min for MDMA. Each training session lasted a maximum of 10 min, and the rats could earn up to a maximum of 20 food pellets. The rats received approximately 60 of these sessions before they were used in tests for substitution of the experimental compounds. Rats were used in testing once they had achieved 9 of 10 sessions at 85% injection-appropriate responding for both the first reinforcer and total session. The training sessions occurred on separate days in a double alternating fashion (drug-drug-saline-saline-drug; etc.) until the training phase was complete, after which substitution tests were introduced into the training schedule such that at least one saline and one drug session occurred between each test (drug-saline-test-saline-drug-test-drug; etc.). The substitution tests occurred only if the rats had achieved 85% injection-appropriate responding on the two prior training sessions.

Dipentylone (2.5, 5, 10, 25 mg/kg), N-ethylhexedrone (0.25, 0.5, 1, 2.5 mg/kg), 4-CEC (0.25, 0.5, 1, 2.5, 5, 10 mg/kg) and MPHP (0.5, 1, 2.5, 5, 10, 25 mg/kg) were tested for substitution in methamphetamine-trained rats. Dipentylone (1, 2.5, 5, 10, 25 mg/kg), N-ethylhexedrone (0.5, 1, 2.5, 5, 10, 25 mg/kg), 4-CEC (0.5, 1, 2.5, 5, 10 mg/kg) and MPHP (2.5, 5, 10, 25 mg/kg) were tested for substitution in cocaine-trained rats. Dipentylone (2.5, 5, 10, 25, 50 mg/kg), N-ethylhexedrone (0.5, 1, 2.5, 5, 10, 25 mg/kg), 4-CEC (0.1, 0.25, 0.5, 1 mg/kg) and MPHP (1, 2.5, 5, 10, 25 mg/kg) were tested for substitution in MDMA-trained rats.

Test sessions lasted for a maximum of 20 min. In contrast with training sessions, both levers were active, such that 10 consecutive responses on either lever led to reinforcement. Data were collected until all 20 reinforcers were obtained, or for a maximum of 20 min. Each cathinone test compound was tested in groups of six rats. A repeated-measures design was used, such that each rat was tested at all doses of a given drug, including vehicle and training-drug controls. The dose effect of each compound was tested from no effect to full effect or rate suppression (<20% of vehicle control) or adverse effects. Starting doses and pretreatment times were inferred from the locomotor activity testing. For dose-effect experiments, intraperitoneal (i.p.) injections (1 ml/kg) of vehicle or test compound were administered with a pretreatment of 15 min for each compound, except 4-CEC, which had a pretreatment time of 30 min. Rats that failed to complete the first fixed ratio were excluded from the analysis of drug-appropriate responding, but were used for analysis of response rate.

2.4. Drugs

(+)-Methamphetamine hydrochloride, (−)-cocaine HCl, (±)-3,4-methylenedioxymethamphetamine HCl, dipentylone HCl (1-(1,3-benzodioxol-5-yl)-2-(dimethylamino)-1-pentanone), N-ethylhexedrone HCl (2-(ethylamino)-1-phenyl-1-hexanone), 4-chlorethcathinone HCl (1-(4-chlorophenyl)-2-(ethylamino)-1-propanone), and 4’-methyl-α-pyrrolidinohexiophenone (2-(pyrrolidin-1-yl)-1-(p-tolyl)hexan-1-one) were all supplied by the National Institute on Drug Abuse Drug Supply Program. Optically active cathinones were provided as racemates. The N-ethylhexedrone saline solutions were acidic (pH approximately 5.7) at concentrations used to deliver the 10 to 25 mg/kg doses. These solutions were buffered to approximately pH 7 using 1 M NaOH prior to injection.

2.5. Data Analysis

Locomotor activity data were expressed as the mean number of photocell counts in the horizontal plane (ambulation counts) during each 10-min period of testing. A 30-min period, beginning when maximal stimulation of locomotor activity first appeared at the lowest effective dose, was used for analysis of dose-response data. A two-way repeated measures analysis of variance (Dose X Ten-minute periods) was conducted on horizontal activity counts/10 min interval. A one-way analysis of variance (Dose) was conducted on horizontal activity counts for the 30-min period of maximal effect (defined as the earliest time period in which a peak effect was observed), and planned comparisons were conducted for each dose against saline control using single degree-of-freedom F tests. A one-way ANOVA (Drug Treatment) was conducted on peak ambulation adjusted for control (peak effects = locomotor activity drug − locomotor activity vehicle) for the five test compounds. The cut-off for statistical significance was set at p<0.05.

Drug discrimination data are expressed as the mean percentage of drug-appropriate responses occurring in each test period. Graphs for percent drug-appropriate responding and response rate were plotted as a function of dose of test compound (log scale). Percent drug-appropriate responding was shown only if at least 3 rats completed the first fixed ratio. Full substitution was defined as ≥80% drug-appropriate responding and not statistically different from the training drug. Rates of responding were expressed as a function of the number of responses made divided by the total session time (sec). Response rate data were analyzed by one-way repeated measures analysis of variance. Effects of individual doses were compared to the vehicle control value using a priori contrasts.

The potencies (ED₅₀ and 95%-confidence intervals) of the test compounds in both assays were calculated by fitting straight lines to the linear portion of the dose-response data for each compound by means of OriginGraph (OriginLab Corporation, Northampton, MA). A one-way ANOVA was conducted to compare locomotor potencies and peak effects. A two-way ANOVA was conducted on the ED₅₀ values for cocaine and methamphetamine drug discrimination.

2.6. Results

2.6.1. Locomotor Activity

Time course data are shown in Figure 2. For clarity, only the effects of the peak dose of each compound are shown. Dose-effect curves were constructed from the 30-min period of peak effect and are shown in Figure 3. Potencies and peak effects for each test compound and the methamphetamine and cocaine controls are shown in Table 1. One-way ANOVA of potencies showed an overall significant effect [F(5,47)=21.95, p<0.001]. Methamphetamine was at least 10-fold more potent than cocaine and the test compounds. Cocaine and the test compounds were equipotent. One-way ANOVA of peak locomotor activity showed an overall significant effect [F(5,138)=11.161, p<0.001]. 4-CEC was significantly less efficacious than the other compounds.

Figure 2.

Locomotor activity time course of MPHP, N-ethylhexedrone, dipentylone and 4-CEC compared to that of methamphetamine and cocaine. Average horizontal activity counts/10 min as a function of time (10 min bins) for the peak dose of each compound. Data are from independent groups of 8 mice per dose. Asterisks indicate doses significantly different from vehicle during the time of peak effect (p<0.05).

Figure 3.

Locomotor activity dose effect of MPHP, N-ethylhexedrone, dipentylone and 4-CEC compared to that of methamphetamine and cocaine. Average horizontal activity counts/10 min in the period of peak effect (methamphetamine, 4-CEC: 20–50 min, cocaine, N-ethylhexedrone, MPHP: 0–30 min) as a function of dose. Data are from independent groups of 8 mice per dose. Asterisks indicate doses significantly different from vehicle during the time of peak effect (p<0.05). Error bars show standard error of the mean.

Table 1. Potencies of the test compounds in producing methamphetamine- or cocaine-like discriminative stimulus effects or increasing locomotor activity.

Potency data are shown as average ED₅₀ values (mg/kg) ± the standard error of the mean. Peak effect data are shown as beam breaks/10 min bin.

Compound	Locomotor Activity Peak Effect	Locomotor Activity Potency	Methamphetamine Discrimination Potency	Cocaine Discrimination Potency
Methamphetamine	5948±256	0.41 ± 0.05	0.26±0.11	–
Cocaine	5640±277	5.03 ± 0.06	–	3.69 ± 0.08
MPHP	6384±232	6.62 ± 0.08	2.95 ± 0.16	11.80 ± 0.10
N-Ethylhexedrone	5837±357	6.54 ± 0.10	1.44 ± 0.07	3.42 ± 0.12
Dipentylone	6123±303	5.29 ± 0.09	8.70 ± 0.12	8.27 ± 0.12
4-CEC	4398±193	5.69 ± 0.08	1.33 ± 0.14	1.49 ± 0.14

(+)-Methamphetamine produced time- and dose-dependent stimulation of locomotor activity (Figure 2). Two-way ANOVA conducted on horizontal activity counts/10 min bin indicated a significant effect for Dose [F(5,42)=7.1, p<.001], a significant effect of 10-Minute Periods [F(47,1974)=78.1, p<.001], and a significant interaction of Periods and Dose [F(235,1974)=5.0, p<.001]. One-way ANOVA conducted on the period of maximal stimulant effect (20–50 min) indicated a significant effect [F(5,42)=12.9, p<.001] as seen in Figure 3. Increases in locomotor activity were seen following 0.5, 1 and 2 mg/kg, with peak effects observed following 1 mg/kg. Peak effects occurred within 20 min and lasted 180 min (Figure 2). A higher dose of 4 mg/kg returned locomotor activity to baseline levels.

Similarly, ANOVA of cocaine effects on locomotor activity indicated a significant effect of Dose [F(5,42)=4.1, p=.004], 10-Minute Periods [F(47,1974)=77.1, p<.001], and the interaction of Periods and Dose [F(235,1974)=3.3, p<.001]. Stimulant effects occurred within 10 minutes following injection and lasted 110 minutes. One-way ANOVA conducted on the period of maximal stimulant effect (0–30 min) indicated a significant effect [F(5,42)=14.3, p<.001]. Increases in locomotor activity were seen following 5, 10, 20 and 40 mg/kg, with peak effects observed following 20 mg/kg.

MPHP produced time- and dose-dependent stimulation of locomotor activity. There was a significant effect for Dose [F(5,42)=23.6, p<.001], 10-Minute Periods [F(47,1974)=56.81, p<.001], and the interaction of Periods and Dose [F(235,1974)=4.6, p<.001]. One-way ANOVA conducted on the period of maximal stimulant effect (20–50 min) indicated a significant effect [F(5,42)=5.82, p<.001]. Increases in locomotor activity were seen following 5, 10 and 25 mg/kg, with peak effects observed following 25 mg/kg. Peak effects occurred within 10 min and lasted 240 min. A higher dose of 50 mg/kg returned locomotor activity to baseline levels.

ANOVA of N-ethylhexedrone effects on locomotor activity indicated a significant effect of Dose [F(5,42)=8.2, p<.001], 10-Minute Periods [F(47,1974)=81.0, p<.001], and the interaction of Periods and Dose [F(235,1974)=4.7, p<.001]. Stimulant effects occurred within 10 minutes following injection and lasted 240 minutes. One-way ANOVA conducted on the period of maximal stimulant effect (0–30 min) indicated a significant effect [F(5,42)=4.7, p=.002]. Increases in locomotor activity were seen following 10 and 25 mg/kg, with peak effects observed following 25 mg/kg. A higher dose of 50 mg/kg decreased locomotor activity from peak levels, but not to baseline levels.

ANOVA of 4-CEC effects on locomotor activity failed to indicate a significant effect of Dose [F(5,42)=2.3, p=.059], but did indicate significant effects of 10-Minute Periods [F(47,1974)=57.2, p<.001], and the interaction of Periods and Dose [F(235,1974)=2.5, p<.001]. Stimulant effects occurred within 10 minutes following injection and lasted 120 minutes. One-way ANOVA conducted on the period of maximal stimulant effect (20–50 min) indicated a significant effect [F(5,42)=12.32, p<.001]. Increases in locomotor activity were seen following 10 and 25 mg/kg, with peak effects observed following 25 mg/kg. A higher dose of 50 mg/kg returned locomotor activity to baseline levels.

Dipentylone produced time- and dose-dependent stimulation of locomotor activity. There was a significant effect for Dose [F(5,42)=12.769, p<.001], 10-Minute Periods [F(47,1974)=80.166, p<.001], and the interaction of Periods and Dose [F(235,1974)=6.297, p<.001]. One-way ANOVA conducted on the period of maximal stimulant effect (20–50 min) indicated a significant effect [F(5,42)=12.32, p<.001]. Increases in locomotor activity were seen following 10 and 25 mg/kg, with peak effects observed following 25 mg/kg. Peak effects occurred within 10 min and lasted 360 min. A higher dose of 50 mg/kg returned locomotor activity to baseline levels.

2.6.2. Drug Discrimination

All four compounds fully substituted for the discriminative stimulus effects of both methamphetamine and cocaine (Figure 4). ED₅₀ values are shown in Table 1. There was a significant effect of training drug [F(1,40)=17225, p<.001], test compound [F(3,40)=30692, p<.001], and the interaction [F(3,40)=11240, p<.001]. MPHP, N-ethylhexedrone, and 4-CEC were equipotent at producing methamphetamine-like discriminative stimulus effects. Dipentylone was 3- to 6-fold less potent than the other compounds. Similar potencies were observed in the cocaine-trained rats, with the notable exception being MPHP, which was 4-fold less potent in the cocaine-trained rats than in the methamphetamine-trained rats.

Figure 4.

Substitution for the discriminative stimulus effects of methamphetamine (Meth, left panels), cocaine (center panels), and MDMA (right panels): Top panels show percentage of total responses made on the drug-appropriate lever. Bottom panels show rate of responding in responses per second (r/s). n=6 unless otherwise shown, except dimethylone in methamphetamine-trained rats n=9. Vehicle and training drug controls are pooled averages. Ctrl indicates vehicle and training drug control values. * indicates response rate different from vehicle control (p < 0.05). Error bars show standard error of the mean.

N-Ethylhexedrone failed to alter rate of responding at the doses tested. In the methamphetamine-trained rats, dipentylone increased response rate following 5 and 10 mg/kg, with the maximum effect (152% of vehicle control) following 10 mg/kg, and decreased to 52% of vehicle control following 25 mg/kg such that 1 of 6 rats failed to complete the first reinforcer [F(4,20)=13.94, p<.001]. Dipentylone failed to alter rate of responding in the cocaine-trained rats. MPHP increased response rate to 137% of vehicle control following 10 mg/kg in the methamphetamine-trained rats [F(5,25)=2.87, p=.035], and increased response rate to 135% of vehicle control following 25 mg/kg in the cocaine-trained rats [F(4,20)=2.98, p=.044]. 4-CEC decreased response rate to 44% of vehicle control following 10 mg/kg in the methamphetamine-trained rats [F(6,30)=4.71, p=.002] and decreased response rate to 30% of vehicle control in the cocaine-trained rats [F(5,25)=4.56, p=.004] such that two of six rats failed to complete the first reinforcer.

In contrast, only 4-CEC fully substituted in MDMA-trained rats (ED₅₀=0.43±0.04 mg/kg). 4-CEC produced no effect on response rate. N-Ethylhexedrone produced a plateau of 50–70% drug-appropriate responding from 1 to 25 mg/kg. Response rate was decreased following 25 mg/kg of N-ethylhexedrone such that only 3 of 6 rats earned a reinforcer [F(6,30)=8.92, p<.001]. MPHP produced a plateau of 39–67% drug-appropriate responding from 2.5 to 10 mg/kg. Response rate was decreased following 25 mg/kg of MPHP such that only 2 of 4 rats tested earned a reinforcer [F(5,15)=11.29, p<.001]. Lethality was observed in 1/5 rats following 25 mg/kg of MPHP. Dipentylone produced a maximum of 60% MDMA-appropriate responding following 25 mg/kg. Response rate was decreased following 50 mg/kg of dipentylone such that only 2 of 6 rats earned a reinforcer [F(5,25)=7.45, p<.001].

3. Discussion

MPHP, N-ethylhexedrone, dipentylone and 4-CEC are synthetic cathinone compounds that are of increasing concern in illicit markets in Europe and the US. The current study assessed the locomotor stimulant and methamphetamine- and cocaine-like discriminative stimulus effects of these four compounds. All four compounds produced psychostimulant-like increases in locomotor activity. All were similar in potency to cocaine, but 4-CEC was less efficacious than the other synthetic cathinones and cocaine. None of the compounds were as potent as methamphetamine, which typically is about 10-fold more potent than cocaine in producing locomotor stimulant effects (see Table 1). MPHP, N-ethylhexedrone, dipentylone and 4-CEC did vary in duration of action. The peak dose of cocaine lasted 2 h, the peak doses of methamphetamine, dipentylone and 4-CEC lasted 3 h, the peak dose of N-ethylhexedrone lasted 4 h, and the peak dose of MPHP lasted 6 h.

These findings are similar to our earlier work on cathinones in the open-field locomotor activity. Other studies have also assessed locomotor activity of cathinones ^25–29, but differences in procedures such as chambers (size, open-field vs. home cage), species, strain, measurement (photo beams vs. camera), and adaptation to the chamber make it difficult to compare data between laboratories. Our laboratory does not adapt the subjects to the chamber before testing, which allows detection of early-onset depressant effects such as that observed in MDMA, flephedrone, 4-fluoroamphetamine, MDAI ^30–33. The use of mice in the locomotor activity assay predicts very well the potency of psychostimulants in the methamphetamine and cocaine drug discrimination in our previous studies.

Along with 4-CEC, a number of other cathinones produce weak locomotor stimulant effects in the open-field locomotor activity test, including mephedrone (4-methylmethcathinone, 4-MMC), clephedrone (4-chloro-methcathinone, 4-CMC), flephedrone (4-fluoromethcathinone, 4-FMC), 4-methylethcathinone (4-MEC), and 4’-methyl-alpha- pyrrolidinopropiophenone (4-mePPP) ^31,33–35. An obvious commonality among all these compounds is that they are substituted at the 4-position on the phenyl ring. In support of the hypothesis that a 4-position substitution is important, the non-cathinone, 4-fluoroamphetamine was also a weak locomotor stimulant ³⁰, but 3-FMC, the 3-substituted analog of 4-FMC was a strong locomotor stimulant. This agrees with findings that compounds substituted at the 4-position had far more pronounced serotonergic effects both in vitro ³⁶ and in vivo ³⁷.

An exception to this trend is MPHP in the present study, which has a methyl substitution at the 4-position. However, all the low efficacy compounds have a single carbon appended to the alpha position, whereas MPHP has a 4-carbon side chain. Earlier work has reported that the 4-position phenyl substitutions increase SERT activity ^36,37 and MDMA-like discriminative stimulus effects ¹³, whereas lengthening the alpha side-chain increases DAT selectivity ³⁸ and decreases MDMA-like discriminative stimulus effects ¹³.

All four of the synthetic cathinones tested in the current study (MPHP, N-ethylhexedrone, dipentylone and 4-CEC) produced discriminative stimulus effects similar to those of methamphetamine and cocaine, although 4-CEC only substituted for methamphetamine and cocaine at doses that decreased rates of responding. This suggests that 4-CEC has fairly weak psychostimulant-like discriminative stimulus effects, and may be used less on the street as a substitute for cocaine or methamphetamine, particularly since doses of 4-CEC high enough to produce stimulant effects may also produce unpleasant adverse effects. Dipentylone also substituted for methamphetamine at a dose that suppressed response rate, which suggests that street use may also be associated with unwanted adverse effects. These findings extend earlier work reporting that most illicit synthetic cathinones fully substituted for the discriminative stimulus effects of cocaine and methamphetamine in our lab ^{31, 33–35, 39} and others ^{25, 4–44}. A notable exception is TH-PVP, which is unusual in that it is selective for SERT 10-fold over DAT and NET ²², poorly soluble, a locomotor depressant, and produced only weak substitution (<50% at 100 mg/kg and rate suppression) in methamphetamine- and cocaine-trained rats ³³. In addition, alpha-PVT, 3,4-MD-PBP and dimethylone produced only partial substitution in cocaine trained rats ^{33, 39} although another study reported full substitution with alpha-PVT using a lower training dose of cocaine ⁴⁰.

In contrast, only 4-CEC fully substituted in MDMA-trained rats with no effect on response rate. 4-CEC was 3.5-fold more potent in MDMA-trained rats than in the methamphetamine-trained rats. The remaining test compounds failed to fully substitute for the discriminative stimulus effects of MDMA, producing maximal MDMA-appropriate responding ranging between 60 and 70% up to doses that suppressed responding. Most notably, N-ethylhexedrone produced a 5-dose plateau ranging between 50 to 70% MDMA-appropriate responding. Many cathinones fully substitute for the discriminative stimulus effects of MDMA; however, several do not ^45–48. Recently, it has been reported that chemical structure plays a role, such that compounds with no substitutions on the phenyl ring, pyrrolidinyl cathinones and compounds with long alpha side chains produce less MDMA-appropriate responding ^{13, 49}, which is in agreement with other studies indicating that the alpha side chain length ³⁸ and 4-substitutions ^{37, 49} predict activity of cathinones.

The potencies of MPHP, N-ethylhexedrone and 4-CEC tested in methamphetamine-trained rats ranged between those of cocaine and methamphetamine (0.3 to 4 mg/kg), which is very typical for the cathinones tested in our laboratory ^{31, 33–35, 39}. However, the potency of dipentylone was low in both the methamphetamine- and cocaine-trained rats, and MPHP was almost 4-fold less potent in the cocaine-trained rats than in the methamphetamine-trained rats. Other low potency cathinones we have tested include 4-MEC, 4’-MePPP, alpha-PVT, dimethylone and dibutylone. 4’-MePPP also substituted for methamphetamine with low potency, and 4-MEC did not substitute at all in one study ⁴². Another study reported lower ED₅₀ values for alpha-PVT in both the cocaine and methamphetamine drug discrimination, but used a lower training dose of both compounds, which may account for the difference ⁴⁰. It is not clear what contributes to the lower potencies and/or efficacies of 4-MEC, 4’-MePPP, alpha-PVT, dimethylone, dibutylone, and dipentylone, or why MPHP should have lower potency in the cocaine-trained rats than in the methamphetamine-trained rats. All of the test compounds inhibit monoamine transport; MPHP, N-ethylhexedrone and dipentylone were selective for DAT/SERT ²². 4-CEC was more selective for SERT than DAT, but was a weak locomotor stimulant and fully substituted for methamphetamine and cocaine. However, there are no consistent structural differences among these compounds, nor are the relative binding affinities for DAT, SERT and NET predictive.

In conclusion, dipentylone, N-ethylhexedrone, and MPHP likely have abuse potential as substitutes for psychostimulants such as methamphetamine and cocaine, given the similar discriminative stimulus effects. Confirmation of these findings in studies of reward/reinforcement such as self-administration or place conditioning will be necessary. Previous reports of toxic liver damage and rhabdomyolysis in human users and the lethality in one rat may indicate that MPHP has significant additional public health risk. The compounds in the present study produced long-acting locomotor stimulant effects (3–5 h) which may lead to increased likelihood of adverse effects, especially when combined with other substances. 4-CEC produced psychostimulant effects only at doses that suppressed rates of responding. However, it is potently MDMA-like, so it may be an acceptable alternative to club drugs. Its slow onset may also diminish its abuse liability, but tests of rewarding and reinforcing effects need to be directly tested.

Data Availability Statement

The data that support the findings of this study are available from NIDA ATDP.