Metabolism of MMB022 and identification of dihydrodiol formation in vitro using synthesized standards

Abstract

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 the detection of synthetic cannabinoids in humans, since they are generally extensively metabolized. The aims of the study were to tentatively identify in vitro phase I metabolites, to confirm major metabolites using synthesized metabolites, to examine metabolic pathways thoroughly, to study metabolic stability and to suggest metabolites appropriate for urine screening. MMB022 and its synthesized metabolites were incubated with human liver microsomes (HLM) and the supernatants were analyzed 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 fourth most abundant metabolite. The metabolic pathways were investigated using synthesized 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 t 1/2 was 2.1 min, indicating a low metabolic stability. M15, M8, and potentially M5 are suggested as suitable urinary targets.

New synthetic cannabinoids carrying a cyclobutyl methyl side chain: Human Phase I metabolism and data on human cannabinoid receptor 1 binding and activation of Cumyl‐CBMICA and Cumyl‐CBMINACA

“…The presence of epoxide in the samples seems not likely but cannot be ruled out without the use of a reference standard, because the epoxidized structure is isomeric to monohydroxylated metabolites. A recently published metabolism study of MMB‐4en‐PICA (MMB‐022) by Watanabe et al has shown that a theoretical epoxide intermediate in the context of dihydrodiol biotransformation of the 4‐pentenyl tail group was not observed in pHLM incubations 16 …”

“…15 The presence of epoxide in the samples seems not likely, but cannot be ruled out without the use of a reference standard, since the epoxidized structure is isomeric to monohydroxylated incubations. 16 For the unambiguous identification of a Cumyl-CBMICA uptake the metabolites M24…”

The metabolism of the synthetic cannabinoids ADB‐BUTINACA and ADB‐4en‐PINACA and their detection in forensic toxicology casework and infused papers seized in prisons

“…Two other relatively abundant metabolites featuring mono‐hydroxylation on the indazole ring are reported in this study, one of which is a phase 1 metabolite (B16, ranked third in abundance, as estimated by peak area) and the other a phase II glucuronidated metabolite (B5, ranked sixth in abundance, as estimated by peak area). The suggested metabolite structures are based on evaluation on LC‐QTOF‐MS data and previous studies of the biotransformation of similar SCRAs 33,34 . Comparison of retention times of the metabolites observed in the HHeps incubation samples with those obtained from the analysis of high purity synthesized reference standards under the same conditions or NMR is required for unequivocal structural confirmation.…”

“…The T A B L E 1 ADB-BUTINACA metabolites with biotransformations, chemical formulas, retention times, exact masses of the protonated molecules, mass errors from all samples, peak areas, and rankings of metabolites based on average peak area in 5 h human hepatocyte incubations and comparison with previously reported metabolites following incubation of ADB-BUTINACA with human liver microsomes 32 suggested metabolite structures are based on evaluation on LC-QTOF-MS data and previous studies of the biotransformation of similar SCRAs. 33,34 Comparison of retention times of the metabolites observed in the HHeps incubation samples with those obtained from the analysis of high purity synthesized reference standards under the same conditions or NMR is required for unequivocal structural confirmation.…”

An insight into the metabolism of New Psychoactive Substances : Structural elucidation of urinary metabolites of synthetic cannabinoids and fentanyl analogues using synthesized reference standards

“…134, Exclusive use of human liver microsomes has been used to study the metabolism of, MMB022, 3,5-AB-CHMFUPPYCA, ADB-FUBINACA, 5F-ADB, CUMYL-PINACA, CUMYL-4CN-B7AICA, CUMYL-4CN-BINACA, 5F-CUMYL-PINACA, AKB-48, STS-135, MAM-2201 and XLR-11 among others. 136,[164][165][166][167][168][169][170][171] The following synthetic cannabinoids have been studied using only hepatocytes, BB-22, 5C-AKB48, EG-018, PB-22, 5F-PB-22 SDB-006, AKB-48 and XLR-11 among others. [172][173][174][175][176][177][178] Several metabolism studies of synthetic cannabinoids in urine have been carried out, including of ADB-FUBINACA, AM-694, AM-2201, JWH-007, JWH-019, JWH-203, JWH-307, MAM-2201, UR-144, XLR-11, APINAC, BB-22, EG-018, EG-2201, MDMB-CHMCZCA, MDMB-FUBINACA and 5Cl-THJ-018.…”