Pharmacological effect of (±)-trans-3-methylfentanyl and (±)-cis-3-methylfentanyl

(±)-trans-3-methylfentanyl and (±)-cis-3-methylfentanyl are stereoisomeric analogues of 3-methylfentanyl. Although both exhibit high potency as µ-opioid receptor (MOR) agonists, their pharmacological profiles, such as analgesic potency, differ markedly. Current experimental data remain limited primarily to analgesia and MOR binding affinity.

To comprehensively evaluate and compare the acute toxicity and the potency of abuse liability of (±)-trans-3-methylfentanyl and (±)-cis-3-methylfentanyl.

Acute toxicity was assessed using the up-and-down procedure in mice. The potency of abuse liability was evaluated through four complementary paradigms: conditioned place preference (CPP), drug self-administration (SA), drug discrimination, and naloxone-precipitated withdrawal. In addition, dopamine D2 receptor (D2R) binding affinity was quantified in vitro using surface plasmon resonance (SPR), complementing in vivo behavioral findings.

The median lethal dose (LD₅₀) of (±)-trans-3-methylfentanyl and (±)-cis-3-methylfentanyl was identical and was 101 mg/kg (s.c.). The minimum doses to induce CPP were 30 µg/kg for (±)-trans-3-methylfentanyl and 3 µg/kg for (±)-cis-3-methylfentanyl, indicating a 10-fold higher potency of (±)-cis-3-methylfentanyl in inducing reward-associated learning. Conversely, in SA experiments, the peak dose to induce SA was 0.05 µg/kg/infusion for (±)-trans-3-methylfentanyl and 0.25 µg/kg/infusion for (±)-cis-3-methylfentanyl, demonstrating that the trans-isomer exhibits fivefold greater reinforcing potency. Drug discrimination experiment revealed that the discriminative stimulus potency of (±)-trans-3-methylfentany (ED₅₀ = 0.29 µg/kg) was 1.4 times lower than that of (±)-cis-3-methylfentany (ED₅₀ = 0.20 µg/kg). Repeated administration of (±)-trans-3-methylfentanyl and (±)-cis-3-methylfentanyl produced naloxone-precipitated withdrawal symptoms, confirming physical dependence liability. Notably, (±)-trans-3-methylfentanyl showed higher D2R binding affinity than (±)-cis-3-methylfentanyl in the SPR experiment.

This study provides the first integrated assessment of acute toxicity and multifaceted abuse liability across key behavioral and molecular endpoints for these two stereoisomers. The divergent structure-activity relationships observed, particularly the inverse potency patterns in CPP versus SA and differential D2R engagement, highlight the critical influence of stereochemistry on neuropharmacological outcomes. These findings advance mechanistic understanding of fentanyl analogue action and inform future risk assessment and structure-based design of opioid therapeutics.