Metabolism of MMB022 and identification of dihydrodiol formation in vitro using synthesised standards.

MMB022 (methyl 3-methyl-2-[1-(pent-4-en-1-yl)-1H-indole-3-carboxamido]butanoate) is a new synthetic cannabinoid with an alkene at the pentenyl side chain, a rare functional group for synthetic cannabinoids. Metabolite identification is an important step for detection of synthetic cannabinoids in humans, since they are generally extensively metabolised. The aims of the study were to tentatively identify in vitro phase I metabolites, to confirm major metabolites using synthesised metabolites, to examine metabolic pathways thoroughly, to study metabolic stability and to suggest metabolites appropriate for urine screening. MMB022 and its synthesised metabolites were incubated with human liver microsomes (HLM) and the supernatants were analysed by liquid chromatography-quadrupole time-of-flight mass spectrometry. Sixteen metabolites were identified, which were generated via dehydrogenation, dihydrodiol formation, ester hydrolysis, hydroxylation, and combinations thereof. A major biotransformation of the alkene at the pentenyl side chain was confirmed to be dihydrodiol formation. The major metabolites were ester hydrolysis (M15) and dihydrodiol (M8) metabolites, whereas the metabolite derived from the combination of ester hydrolysis and dihydrodiol (M5) was the 4th most abundant metabolite. The metabolic pathways were investigated using synthesised metabolites and revealed that M5 is an end product of the pathways, indicating that it might become a more abundant metabolite in vivo depending on the rate of metabolism in humans. The major pathway of MMB022 to M5 was determined to be via M8 formation. Intrinsic clearance of MMB022 was determined to be 296 mL/min/kg and t1/2 was 2.1 min indicating a low metabolic stability. M15, M8 and potentially M5 are suggested as suitable urinary targets.


| INTRODUCTION
The emergence and prevalence of new psychoactive substances (NPS) is still a continuing problem in Europe. 1 identified in authentic biological samples, 7,8 and has become increasingly prevalent in infused papers at Scottish prisons since June 2019. 9 Due to the structural similarity, MMB022 might become one of the next prevalent cannabinoids.
A carbon-carbon double bond on the pentyl side chain is a rare modification among synthetic cannabinoids. It has been reported as a minor component of products with other NPS, analogs, including N-(4-pentenyl)-JWH-122, 10 N-(4-pentenyl)-XLR-11 (also known as N-(4-pentenyl)-UR-144), 11 and N-(4-pentenyl)-AKB-48. 12 However, the first cannabinoids seen as the main components were JWH-022 2 and MDMB-4en-PINACA 9 and not until the latter became prevalent in 2019 did they receive much attention. Therefore, information on the metabolism of such a double bond is limited. So far, MDMB-4en-PINACA is the only synthetic cannabinoid with an alkene ( Figure 1) whose metabolism has been studied. 7 7 Although dihydrodiol formation from an alkene is a common biotransformation in metabolism, e.g., ezlopitant alkene (CJ-12,458) 13 and α-methylstyrene, 14 this assumption is inconclusive given that an alkene can be metabolized to an alcohol, 13 and thus further hydroxylation could lead to isomers of the dihydrodiol structure.
Therefore, unambiguous identification of the dihydrodiol formation is necessary to confirm the major biotransformation of the alkene in the pentenyl side chain of synthetic cannabinoids.
Consequently, we hypothesize that a major metabolic pathway of a terminal alkene of MMB022 is dihydrodiol formation. Therefore, the aims of the study were to tentatively identify metabolites of MMB022 using human liver microsomes (HLM) and to confirm the

| Metabolic stability
MMB022 was incubated with HLM as described above for metabolite identification with the following changes: the drug concentration was 1 μmol/L; the drug was added last to initiate the reaction; the incubation duration was 0, 2, 4, 6, 8, and 10 min; incubation was performed in triplicate; and the injection volume was 1 μL.
where 21 mL/min/kg was used for human hepatic blood flow (Q H ). 15

| Synthesized reference material analysis
Solutions of synthesized reference material (MMB022 metabolites: dihydrodiol, epoxide, ester hydrolysis, ester hydrolysis with dihydrodiol, ester hydrolysis with epoxide) were made in methanol to the concentration of 100 ng/mL, except for the metabolite with ester hydrolysis with epoxide metabolite to 10 μg/mL, respectively. One μL of each solution was injected into LC-QTOF-MS system.

| LC-QTOF-MS analysis
An Agilent 1290 Infinity Table 1. Product ion spectra of MMB022 and metabolites are shown in Figure 2 and Supplemental 2.
The detected biotransformations included dehydrogenation, dihydrodiol formation, ester hydrolysis, hydroxylation, and combinations thereof. The top two most abundant metabolites by peak area were ester hydrolysis (M15) and dihydrodiol (M8), while the metabolite in combination of ester hydrolysis and dihydrodiol (M5) ranked fourth (Table 1). It should be noted that although the peak abundance When the synthesized metabolites were incubated with HLM, all but M5 underwent further metabolism (

| Metabolic pathways
Metabolism of synthetic cannabinoids has been studied frequently during the last decade. However, an alkene functional group on the terminal carbons of a pentyl side chain is an uncommon structure for synthetic cannabinoids and, to date, MDMB-4en-PINACA is the only synthetic cannabinoid, for which metabolism of such an alkene group has been studied. 7 In the present study, we have demonstrated that similarly to MDMB-4en-PINACA, the double bond of MMB022 was metabolized to a dihydrodiol (M8), whose structure was confirmed by a synthesized reference standard. Likewise, M5 was confirmed to contain a dihydrodiol structure, in addition to ester hydrolysis.
According to the literature, dihydrodiol formation from metabolism of an alkene is via epoxidation catalyzed by CYP enzymes, followed by hydration of the epoxide either spontaneously or facilitated by epoxide hydrolase. 13,14 In the present study, the potential intermediate epoxide metabolites (IM1 and IM2) were not directly detected as metabolites. However, the incubation of the epoxide (IM2) resulted in the dihydrodiol metabolite (M8) and M1-M5, while the incubation of M8 also resulted in M1-M5 (Table 2) produced from the incubation of M8 was much greater than that from M15 ( Table 2), indicating that the pathway from M8 to M5 was the major pathway to M5 (Figure 4). 10-minute metabolic stability incubation, a considerably smaller peak area was observed for M5 in comparison with M8 and M15 ( Figure 6).

| Metabolic stability
This result seems to indicate that the metabolic transformation of M8 and M15 to M5 is a relatively slow process.
Since none of the metabolites retain the parent drug skeletal structure, their presence does not confirm the consumption of MMB022.
However, to our knowledge, these metabolites are not yet reported for other synthetic cannabinoids and thus are deemed suitable for screening purposes. The detection of both M15 and M8 should give more confidence regarding the specificity, as these metabolites together show the full picture of the parent drug skeletal structure.
Finally, the present study was conducted using HLM. Although these two metabolites can be expected to be abundant in human urine samples, authentic human urine samples need to be examined to corroborate the findings of the present study.

| CONCLUSION
MMB022 was metabolized by HLM via dehydrogenation, dihydrodiol formation, ester hydrolysis, and hydroxylation. As was hypothesized, it was confirmed, using synthesized reference standards, that the ter- University.

CONFLICT OF INTEREST
The authors declare that they have no conflict of interest.