Locomotor activity

Figure 2 shows the time course data for the test compounds. Because of the large amount of data, only doses which produced peak depressant or stimulant effects are shown, along with vehicle data for comparison. Treatment with 2C-C resulted in time- and dose-dependent depression of locomotor activity following 30 and 100 mg/kg (F (5,41) = 32.94, p < 0.001); these effects occurred within 10 min following injection and lasted 30 to 120 min. Convulsions were observed in 2/8 mice, and tremors in 6/8 mice at 30 min following 100 mg/kg 2C-C. Lethality occurred in 1/8 mice within 120 min following 100 mg/kg. Treatment with 2C-I resulted in time- and dose-dependent depression of locomotor activity following 3–30 mg/kg (F (5,42) = 20.90, p < 0.001); depressant effects occurred within 10 min following injection and lasted 30–60 min (Fig. 1). Treatment with 2C-T-2 resulted in time- and dose-dependent depression of locomotor activity following 3 and 10 mg/kg (F (5,42) = 19.88, p < 0.001); depressant effects occurred within 20 min following injection and lasted 30–50 min. Time- and dose-dependent depression of locomotor activity following 3 and 10 mg/kg DOC occurred within 10 min following injection (F (5,42) = 44.58, p < 0.001) and lasted 30–80 min.

Fig. 2 Average horizontal activity counts/10 min (ambulation counts) as a function of time (0–8 h) and dose of test compound. Data for the vehicle and the dose which produced peak depressant effects are shown in each panel. 2C-D and DOC also produced stimulant effects, and the data for the dose which produced peak stimulant effects are also shown. N = 8 for each treatment Full size image

Treatment with 2C-D resulted in both stimulation and depression of locomotor activity. Depressant effects of 10 and 30 mg/kg 2C-D (F (4,35) = 11.62, p < 0.001) occurred within 10 min following injection and lasted 20–40 min. Stimulant effects of 3 mg/kg 2C-D (F (4,35) = 1.46, p = 0.236) occurred within 50 min following injection and lasted 180 min. Lethality occurred in 8/8 mice within 30 min following 100 mg/kg 2C-D. 2C-E stimulated locomotor activity at low doses and depressed locomotor activity at higher doses. The depressant effects of 10 and 30 mg/kg 2C-E (F (5,42) = 23.56, p < 0.001) occurred within 10 min following injection and lasted 50–70 min. Stimulant effects of 0.3 and 1 mg/kg 2C-E (F (7,56) = 2.71, p = 0.017) occurred within 50 min after injection and lasted 70–100 min.

Drug discrimination

Table 1 shows the summary data for the drug discrimination studies. 2C-C fully substituted for the discriminative stimulus effects of DOM (ED 50 = 0.95 ± 0.09 mg/kg) and MDMA (ED 50 = 1.48 ± 0.15 mg/kg). 2C-C (5 mg/kg) produced a maximum of 75 % drug-appropriate responding in both DMT- and LSD-trained rats, whereas 2C-C (10 mg/kg) produced minimal drug-appropriate responding in METH-trained rats. 2C-C decreased response rates following 2.5 mg/kg in MDMA-trained rats (F (4,20) = 6.18, p = 0.002), 10 mg/kg in DMT-trained rats (F (5,25) = 6.41, p = 0.001) and LSD-trained rats (F (6,30) = 5.51, p = 0.001), and 25 mg/kg in METH-trained rats (F (4,8) = 5.98, p = 0.016). The adverse effects were observed following 25 mg/kg 2C-C, including reddening of the extremities (3/3 rats) and salivation (1/3 rats), and this dose was not tested in DMT- or LSD-trained rats.

Table 1 Maximum percent drug-appropriate responding (DAR) of each test compound in each of the training groups and effects on rate of responding at that dose. Full size table

2C-D was tested at two time points, 15 and 70 min, which corresponded with peak depressant and peak stimulant locomotor activity effects, respectively. At 15 min after administration, 2C-D fully substituted for the discriminative stimulus effects of DMT (ED 50 = 3.14 ± 0.15 mg/kg), DOM (ED 50 = 0.77 ± 0.10 mg/kg), and LSD (ED 50 = 0.71 ± 0.12 mg/kg). 2C-D (2.5 mg/kg) produced 61 % drug-appropriate responding in MDMA-trained rats, and 10 mg/kg produced 15 % METH-appropriate responding (Table 1). The response rate was decreased following 1 and 2.5 mg/kg in DOM-trained rats (F (4,20) = 6.23, p = 0.002), 5 mg/kg in MDMA-trained rats (F (5,25) = 9.04, p < 0.001), 10 mg/kg in DMT-trained rats (F (4,20) = 5.13, p = 0.005), and 5 and 10 mg/kg in METH-trained rats (F (4,20) = 6.86, p = 0.001). In the METH-trained rats, 2/6 rats exhibited reddening of the extremities following 10 mg/kg 2C-D, and 4/6 rats failed to complete the first fixed ratio. At 70 min after administration, 2C-D fully substituted for the discriminative stimulus effects of DMT (ED 50 = 2.99 ± 0.13 mg/kg) and LSD (ED 50 = 3.04 ± 0.13 mg/kg). 2C-D (10 mg/kg) produced partial substitution of drug-appropriate responding in DOM-trained rats and MDMA-trained rats, and 25 mg/kg 2C-D partially substituted in (+)-METH-trained rats. The response rate was decreased following 10 mg/kg in DOM-trained rats (F (4,20) = 4.34, p = 0.011), 25 mg/kg in MDMA-trained rats (F (5,25) = 4.18, p = 0.007), and 25 and 50 mg/kg in (+)-METH-trained rats (F (6,12) = 5.49, p = 0.006). Two of the three rats receiving 50 mg/kg 2C-D exhibited salivation and failed to complete the first fixed ratio.

2C-E fully substituted for the discriminative stimulus effects of DMT (ED 50 = 0.95 ± 0.20 mg/kg), DOM (ED 50 = 0.84 ± 0.08 mg/kg), LSD (ED 50 = 0.62 ± 0.10 mg/kg), and MDMA (ED 50 = 2.48 ± 0.10 mg/kg) but produced minimal METH-appropriate responding. The response rate was decreased following 2.5, 5, and 10 mg/kg in MDMA-trained rats (F (9,45) = 10.25, p < 0.001), and 5 and 25 mg/kg in METH-trained rats (F (10,50) = 4.01, p < 0.001). Loss of muscle tone was observed at 10 mg/kg in MDMA-trained rats, and 25 mg/kg 2C-E completely suppressed responding.

2C-I fully substituted for the discriminative stimulus effects of DMT (ED 50 = 0.68 ± 0.11 mg/kg) and LSD (ED 50 = 1.66 mg/kg ± 0.12). In MDMA-trained rats, 2.5–10 mg/kg 2C-I produced a maximal 65 % drug-appropriate responding, and 1 mg/kg 2C-I produced only 38 % drug-appropriate responding in METH-trained rats (Table 1). The response rate was decreased with doses of 2.5–10 mg/kg 2C-I in MDMA-trained rats (F (4,20) = 3.66, p = 0.022) and with 1 and 5 mg/kg in METH-trained rats (F (4,20) = 5.08, p = 0.005).

2C-T-2 produced 73 % drug-appropriate responding following 2.5 mg/kg in DMT-trained rats. A 10 mg/kg dose of 2C-T-2 elicited hind limb paralysis, salivation, and loss of muscle tone, and was not tested further. 2C-T-2 failed to substitute for LSD, MDMA, or (+)-METH. 2C-T-2 substantially decreased response rates following 2.5 and 5 mg/kg in rats trained to DMT (F (5,25) = 6.66, p < 0.001), LSD (F (4,20) = 4.60, p = 0.008), MDMA (F (3,15) = 3.46, p = 0.043), and (+)-METH (F (3,15) = 14.75, p < 0.001). In each case, 5/6 rats failed to complete the first fixed ratio at the highest dose tested. Decreased muscle tone was observed in 3/6 rats following 5 mg/kg 2C-T-2 in LSD-trained rats.

DOC was tested at two time points, 15 and 60 min, which corresponded with the peak depressant and peak stimulant locomotor activity effects, respectively. At 15 min following administration, DOC fully substituted for the discriminative stimulus effects of DOM (ED 50 = 0.13 ± 0.16 mg/kg) and LSD (ED 50 = 0.39 ± 0.33 mg/kg). DOC produced 65 % DMT-appropriate responding following 1 mg/kg, and less than 50 % drug-appropriate responding in MDMA-trained and METH-trained rats. The response rate was decreased following 2.5 mg/kg DOC in rats trained to LSD (F (7,35) = 3.86, p = 0.003), MDMA (F (5,25) = 3.81, p = 0.011), and METH (F (5,25) = 3.86, p = 0.010). With 2.5 mg/kg DOC, substantial rate suppression was observed, such that 4/6 or 5/6 rats tested in each case failed to respond, and decreased muscle tone was observed in 12/24 rats. At 60 min, DOC fully substituted for the discriminative stimulus effects of DMT (ED 50 = 0.61 ± 0.19 mg/kg), DOM (ED 50 = 0.26 ± 0.19 mg/kg), and LSD (ED 50 = 0.23 ± 0.10 mg/kg). In MDMA-trained rats, DOC produced 60 % drug-appropriate responding following 1 mg/kg, and no drug-appropriate responding at any dose in METH-trained rats. Doses of 1 mg/kg and higher decreased the response rates in rats trained to DMT (F (5,25) = 3.66, p = 0.013), LSD (F (5,25) = 2.96, p = 0.031), MDMA (F (6,30) = 3.96, p = 0.005), and METH (F (6,30) = 4.07, p = 0.004). Substantial rate suppression and failure to complete the first fixed ratio were observed following 2.5 mg/kg DOC in MDMA-trained rats (4/6 rats) and 5 mg/kg in METH-trained rats (5/6 rats).

In vitro pharmacology: interaction with serotonin receptors

In HEK-h5-HT 1A cells, the phenethylamines were tested for their affinities for the [3H]8-OH-DPAT binding site and their effect on 5-HT 1A function (Table 2). The agonist [3H]8-OH-DPAT binds to the 5-HT 1A high affinity state, and the agonist activation of the receptor results in increased binding of [35S]GTPγS to the Gαi/o subunit of G proteins and reflects receptor function. All six compounds had lower affinities (high nanomolar to low micromolar) for the [3H]8-OH-DPAT binding site than serotonin and LSD (p values <0.001, one-way ANOVA followed by Tukey's multiple comparison test). 2C-C, 2C-I, and 2C-T-2 had higher affinities than DOM, DOC, MDMA, and METH (p values <0.05). 2C-E had higher affinity than MDMA and METH (p values <0.05); and DOC had lower affinity than DMT (p < 0.001). In the [35S]GTPγS functional assay, serotonin and LSD had very high potency, higher than all other compounds (p values <0.001). 2C-I and 2C-T-2 had similar low micromolar potencies, and 2C-T-2 was more potent than MDMA and METH (p values <0.001). The four other phenethylamines had minimal efficacy (<25 %). 2C-I and 2C-T-2 had similar efficacies to serotonin and LSD, and 2C-I had higher efficacy than DOM, MDMA, and METH (p < 0.05).

Table 2 Pharmacology of 2C-C, 2C-D, 2C-E, 2C-I, 2C-T-2, and DOC at 5-HT 1A , 5-HT 2A , and 5-HT 2C receptors: effects on binding and function Full size table

In HEK-h5-HT 2A cells, the phenethylamines were tested for their affinity for the [125I]DOI binding site and effects on the 5-HT 2A signaling pathways. Agonist binding to 5-HT 2A receptors can activate both phospholipase A2 (increasing the AA release from the plasma membranes) and phospholipase C (increasing the inositol phosphate cascade (IP-1 assay)). The binding affinities of the drugs were significantly different (p < 0.0001, one-way ANOVA, Table 2). LSD had higher affinity than all other tested compounds (p values <0.001). The phenethylamines had high affinities, and their K i values did not differ from each other or from serotonin or DOM (p > 0.05), and were higher than DMT, MDMA, and METH (p values <0.001).

In the 5-HT 2A [3H]AA release assay, 2C-C, 2C-D, 2C-E, 2C-T-2, DOC, LSD, and 5-HT were agonists with similar efficacies (p = 0.26, one-way ANOVA, Table 2), while potencies differed significantly (p < 0.0001, one-way ANOVA). LSD and serotonin had potencies in the low nanomolar range, with LSD having higher potency (p < 0.01). 2C-T-2, 2C-E, and DOC were very potent with EC 50 values that did not differ from serotonin and LSD. 2C-T-2, 2C-C, 2C-E, DOC, LSD, and serotonin were more potent than 2C-D and DMT (p values <0.05 to 0.001). In contrast, 2C-I minimally stimulated the release (Fig. 3a), and no EC 50 was determined. This finding was unexpected, since 2C-I substituted for the discriminative effects of LSD and DMT, which stimulated [3H]AA release (Table 2 and Fig. 3a). 2C-I fully inhibited serotonin-stimulated [3H]AA release with low potency, similar to ketanserin (p > 0.05, two-tailed t test, Fig. 3b). To confirm that the same compound was used in behavioral and biochemical assays, an aliquot of 2C-I from the behavioral assays was tested and confirmed 2C-I antagonism of serotonin-mediated [3H]AA release.

Fig. 3 [3H]AA release from HEK-5-HT 2A cells. The experiments were conducted as described in the “Materials and methods” section. Data presented are means ± SEM. a Agonist assay. Basal activity is subtracted, and the data are normalized to the maximal stimulation by serotonin on each experimental day. n = 3–5 except n = 2 for 2C-I. b Antagonist assay. Nonspecific release, measured in the presence of 30 μM ketanserin, is subtracted from all data, and the data are normalized to the maximal release stimulated by serotonin. n = 3–4 Full size image

In the 5-HT 2A IP-1 functional assay, all the compounds tested were agonists (except METH with no measurable efficacy), with significantly different potencies (p < 0.001, one-way ANOVA, Table 2, Fig. 4a). LSD had higher potency (p values <0.001), while MDMA had lower potency (p values <0.001) than all other compounds. 2C-I and 2-C-T-2 were the most potent phenethylamines. 2C-I had higher potency than serotonin, 2C-D, and 2C-E (p values <0.05 to 0.001). 2C-T-2 had higher potency than serotonin, DOM, 2C-D, and 2C-E (p values <0.05 to 0.001). 2C-C, 2C-D, 2C-E, DOC, DOM, and serotonin had similar potencies. Efficacies differed significantly (p < 0.0001, one-way ANOVA). 2C-E had higher efficacy than LSD, 2C-I, and 2C-T-2 (p values <0.05). 2C-C, 2C-D, 2C-I, 2C-T-2, DOC, serotonin, LSD, and DOM had similar efficacies. DMT and MDMA had similar efficacy that was lower than all the other compounds (p values <0.01).

Fig. 4 Stimulation of IP-1 formation in HEK-5-HT 2A and HEK-5-HT 2C cells. The experiments were conducted as described in the “Materials and methods” section. a HEK-5-HT 2A cells. All the compounds are full or partial agonists. The average maximal stimulation by serotonin was 565 ± 46 nM IP1. n = 3–8. b HEK-5-HT 2C cells. All the compounds are full agonists. The average maximal stimulation by serotonin was 1,390 ± 180 nM. n = 4–7 Full size image

In HEK-h5-HT 2C cells, the phenethylamines were tested for their affinities for the [125I]DOI binding site and effects on 5-HT 2C -mediated IP-1 turnover. In the [125I]DOI binding assay, there were significant differences in affinities (p < 0.0001, one-way ANOVA). 2C-C, 2C-D, 2C-E, 2C-I, 2C-T-2, DOC, serotonin, and LSD had similar, low nanomolar affinities (Table 2). DOM had lower affinity than serotonin and LSD (p < 0.01) but was similar to the phenethylamines. DMT had similar affinity to DOM. MDMA and METH had similar affinities which were lower than all the other compounds (p < 0.001).

All compounds were 5-HT 2C agonists, activating the phospholipase C-inositol phosphate cascade, with significant differences in potencies (p < 0.001, one-way ANOVA, Table 2, Fig. 4b). The highest potency drugs were LSD, serotonin, 2C-I, 2C-T-2, and DOM. 2C-I was more potent than 2C-D, DMT, MDMA, and METH (p values <0.01). 2C-T-2 was more potent than DMT, MDMA, and METH (p values <0.001). 2C-C, 2C-D, 2C-E, and DOC had similar low- to mid-nanomolar potencies and were less potent than serotonin (p values <0.05) and LSD (except DOC). DMT, MDMA, and METH had very low potencies. Efficacies did not differ (p = 0.28, one-way ANOVA).

In vitro pharmacology: interaction with hDAT, hSERT, and hNET, and dopamine receptors

In the transporter assays, the phenethylamines had no measurable or very low affinity for hDAT and hNET in the binding assays, and very low potency (at least micromolar) in the [3H]dopamine and [3H]norepinephrine uptake assays (Table 3). Only 2C-I had measurable affinity (high nanomolar) for hSERT and very low potency in the [3H]serotonin uptake assay. In the release assays, the compounds had no releasing efficacy, while METH elicited robust release in all the cell lines. The phenethylamines also had no measurable affinity for the dopamine D1, D2, and D3 receptors (data not shown). Thus, the substituted phenethylamines have minimal interaction with hDAT, hNET, and dopamine receptors, and very low potency at hSERT.