Since their first occurrence in 2008, a rapid increase of new psychoactive substances (NPS) on the recreational drug market has been observed. One class of NPS are synthetic opioids, which poses a health risk due to the high affinity and high potency of most of these compounds for the opioid receptors. Four opioid receptor subtypes are known, the mü-opioid (MOR), the delta-opioid (DOR), the kappa-opioid (KOR) and the nociceptin receptor, which are G protein-coupled receptors (GCPRs). MOR and DOR agonists often express mood-enhancing and euphoric properties. However, opioid overdoses can lead to respiratory depression and result in death. Nevertheless, for many NPS, including new synthetic opioids, data on toxicological and toxicokinetic properties are scarce.

The aim of the present study was to investigate eight structurally closely related U-opioids (U-47700, isopropyl U-47700, U-49900, U-47931E, N-methyl U-47931E, U-48520, U-51754, and U-48800) for their potential of MOR and KOR activation, due to the relative lack of pharmacological data for the U-opioids.

The [35S]-GTPγS assay, was used to study receptor activation and is based on the exchange of GDP with radiolabelled GTP. This exchange is one of the first events of the GPCR-signal cascade.

For each receptor two respective and well-studied compounds were chosen as reference compounds: hydromorphone and fentanyl for MOR, and U-69593 and U-50488 for KOR. Owing to their close structural similarity of the investigated U-compounds, structure activity relationships have also been derived.

The most prevalent compound of this set, U-47700, was also the most potent compound of the test set at MOR, with an EC50 value of 111nM. However, the two MOR reference compounds hydromorphone and fentanyl had lower EC50 values of 6.75nM and 24.9nM, respectively, hence, being 16 and 4 times more potent than U-47700. At KOR, U-51754 was the most potent substance out of the test set, with an EC50 value of 120nM. Nevertheless, the two U-opioids U-69593 and U-50488, which have been used as KOR reference compounds, were 4 and 5 times more potent than U-51754 at KOR, with EC50 of 29.3nM and 24.8nM, respectively.

The studied compounds can be categorized into the U-47700 and U-50488 group. It has been previously reported that the compounds of the U-47700 group show higher receptor binding affinities for MOR and compounds of the U-50488 group for KOR, hence the presence or absence of the methylene group has an influence on the receptor binding affinity at MOR and KOR. In general, our results on the receptor activation of MOR and KOR are in good alignment with reported receptor binding affinities, confirming that the U-47700 group has a higher affinity and activity at MOR and the U-50488 group at KOR.

The following structural features were advantageous for activating MOR: two chlorine substituents in 3,4-position at the aromatic ring, the absence of the methylene group between the amide group and the aromatic ring, a methyl group at the amide nitrogen, and/or a dimethylamine residue at the amine nitrogen of the cyclohexane ring. Further, the following structural features were beneficial for KOR activation: a methylene group between the amide group and the aromatic ring, a pyrrolidine residue at the amine nitrogen of the cyclohexane ring, a methyl group at the amide nitrogen, and/or chlorine substitution in 3,4-position at the aromatic ring.

While synthetic opioids are often considered more potent than conventional opioids, there are some occurring on the NPS market, which are less potent. Hence, performing these studies is important to identify the effect of structural alterations on the receptor activity and in understanding the potential risk to the public health.