(S)-N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-butyl-1H-indazole-3carboxamide (ADB-BUTINACA) is an emerging synthetic cannabinoid that was first identified in Europe in 2019 and entered Singapore's drug scene in January 2020. Due to the unavailable toxicological and metabolic data, there is a need to establish urinary metabolite biomarkers for detection of ADB-BUTINACA consumption and elucidate its biotransformation pathways for rationalizing its toxicological implications.

We characterized the metabolites of ADB-BUTINACA in human liver microsomes using liquid chromatography Orbitrap mass spectrometry analysis. Enzyme-specific inhibitors and recombinant enzymes were adopted for the reaction phenotyping of ADB-BUTINACA. We further used recombinant enzymes to generate a pool of key metabolites in situ and determined their metabolic stability. By coupling in vitro metabolism and authentic urine analyses, a panel of urinary metabolite biomarkers of ADB-BUTINACA was curated.

Fifteen metabolites of ADB-BUTINACA were identified with key biotransformations being hydroxylation, N-debutylation, dihydrodiol formation, and oxidative deamination. Reaction phenotyping established that ADB-BUTINACA was rapidly eliminated via CYP2C19-, CYP3A4-, and CYP3A5-mediated metabolism. Three major monohydroxylated metabolites (M6, M12, and M14) were generated in situ, which demonstrated greater metabolic stability compared to ADB-BUTINACA. Coupling metabolite profiling with urinary analysis, we identified four urinary biomarker metabolites of ADB-BUTINACA: 3 hydroxylated metabolites (M6, M11, and M14) and 1 oxidative deaminated metabolite (M15).

Our data support a panel of four urinary metabolite biomarkers for diagnosing the consumption of ADB-BUTINACA.

Since the first detection of synthetic cannabinoids (SCs) in commercial products such as Spice in 2008, more than 208 new SCs have appeared in the drug market (1). Designed to mimic (−)-trans-Δ9-tetrahydrocannabinol (Δ9-THC) found in cannabis, SCs exert their psychoactive effects by binding to cannabinoid receptor 1 and thereby inducing euphoria (2, 3). To mitigate abuse, several countries have scheduled SCs as illicit drugs (4). Most SCs are extensively metabolized by cytochrome P450 enzymes (CYP450), carboxylesterases (CES), and UDP-glucuronosyltransferases (UGT), resulting in the parent SC being virtually undetectable in urine (5–7). Consequently, there is a need to identify the metabolites of SCs as optimal urinary biomarkers for their forensic management.

(S)-N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-butyl-1H-indazole-3-carboxamide (ADB-BUTINACA) (Supplemental Fig. 1, A) is a new SC that has been increasingly detected in the illicit market (1, 8). Since 2020, ADB-BUTINACA has been listed in various legislative acts of controlled substances worldwide, including Singapore (9–11). There are currently 3 commercially available reference metabolites of ADB-BUTINACA: the N-4 hydroxybutyl metabolite, N-butanoic acid metabolite, and oxidative deaminated metabolite (Supplemental Fig. 1, B–D). These 3 metabolites were predicted according to the published metabolism of the structurally similar N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-pentylindazole-3-carboxamide (ADB-PINACA) (12). Based on in-house observation, these metabolites were absent or detected at low concentrations in authentic urine samples of ADB-BUTINACA abusers. Hence, it is important to comprehensively elucidate the metabolism of ADB-BUTINACA to establish its urinary biomarkers and understand its toxicological implications.

An ideal scenario is to confirm the observed metabolites of SCs in human urine using commercially available reference metabolites. However, this is often impossible for novel SC where such reference metabolites are typically unavailable. In the context of ADB-BUTINACA, one viable alternative is to generate a pool of key metabolites in situ for determining their metabolic properties. Here we aimed to investigate the hepatic metabolism of ADB-BUTINACA systematically and establish its (1) major Phase I metabolites, (2) hepatic clearance, (3) key metabolic pathways and enzymes, (4) metabolic stability of key metabolites, and (5) optimal urinary biomarkers.

ADB-BUTINACA, its N-4 hydroxybutyl, N-butanoic acid, oxidative deaminated metabolites and internal standard (IS) D5-AM2201 N-(4-hydroxypentyl) metabolite [(1-(5-fluoro-4-hydroxypentyl)-1H-indol-3-yl-2,4,5,6,7-d5)(naphthalene-1-yl)methanone] were kindly provided by the Health Sciences Authority, Singapore. UltraPool human liver microsomes (HLM), Supersomes human CYP450 and CES recombinant enzymes (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5, CES1b, and CES2), Gentest NADPH Regenerating System, and Gentest UGT Reaction Mix were purchased from Corning. CYP450-specific chemical inhibitors [α-naphthoflavone (CYP1A2), quercetin (CYP2C8), sulfaphenazole (CYP2C9), S-(+)-N-3-benzyl-nirvanol (CYP2C19), ticlopidine (CYP2C19 and CYP2D6), quinidine (CYP2D6), ketoconazole (CYP3A4 and CYP3A5)] were purchased from Sigma-Aldrich. High-performance chromatography-grade acetonitrile (ACN) was obtained from Merck. All other commercially available chemicals were of analytical grade.

ADB-BUTINACA was incubated in a mixture containing 86 μL of 100 mM potassium phosphate buffer (pH 7.4), 5 μL of HLM, 5 μL of NADPH solution A, and 1 μL of solution B to a total incubation volume of 100 μL and final substrate concentration at 10 μM (Supplemental Table 1). Another experiment was similarly performed replacing NADPH solutions with buffer to examine the production of metabolites via non-CYP450 pathway. For each experiment, the mixture was prewarmed at 37 °C and mixed at 120 rpm for 5 min in a shaking incubator. The reaction was initiated with the addition of the substrate. At 0, 1, and 2 h, 80 μL of the reaction mixture was transferred to an equal volume of ice-cold ACN. The quenched samples were centrifuged at 2775 g, 4 °C for 30 min and the supernatant was subjected to liquid chromatography Orbitrap mass spectrometry analysis (Supplemental Methods 1.1). The metabolites were putatively identified based on mass accuracy (94% total chromatographic peak area (Fig. 2). Biotransformation, m/z of parent ion, mass error, elemental composition, retention time, chromatographic peak area at 1 h and 2 h, and estimated percentage of metabolite at 2 h are summarized for metabolites M1 to M15 in Table 1. MS/MS spectra and fragmentation patterns are shown in Fig. 1 for the most abundant 5 metabolites (M4, M6, M8, M12, and M14), M11 and M15, and in Supplemental Figs. 2 and 3 for the other minor metabolites. Hydroxylation was the dominant Phase I reaction (M5, M6, M9, M11, M12, and M14), with sequential reactions producing ketone (M8, M10), dihydroxylated (M1, M3, and M7) and dehydrogenated (M13) metabolites (Fig. 2). Other reactions included N-debutylation (M4), dihydrodiol formation (M2), and oxidative deamination (M15).

(A) Combined extracted ion chromatogram and (B) proposed metabolic pathways of ADB-BUTINACA to 15 major metabolites post–2-h incubation with NADPH in human liver microsomes. Bold arrow indicates major pathway. Enzymes contributing to the formation of top 3 monohydroxylated metabolites (M6, M12, and M14) are shown.

M5, M6, M9, M11, M12, and M14 are monohydroxylated metabolites as indicated by +15.9949 Da mass shift from parent drug. Based on MS/MS spectra, these metabolites were further classified into 3 categories. For M5, M6, and M9, the mass shift was observed in all product ions, except m/z 145.0397 and m/z 163.0499, suggesting a hydroxylation at the N-butyl chain. M5 was identified as N-(4-hydroxybutyl) metabolite by comparison with retention time and MS/MS spectrum of the reference metabolite. For M11 and M14, the same mass difference was observed in all product ions, including m/z 161.0344 and 179.0450 (m/z 145.0398 and 163.0499 +O, respectively), suggesting a hydroxylation at the indazole moiety. For M12, its MS/MS spectrum presented the same product ions as ADB-BUTINACA except for m/z 330.1807 and 302.1860, which were associated with respective losses of amine and carboxamide, indicating that hydroxylation occurred at the tert-butyl chain. Notably, the product ion at m/z 302.1860 displayed a low signal intensity in favor of m/z 272.1766, which was produced by a sequential loss of hydroxymethyl (-CHOH). This phenomenon was similarly observed for ADB-PINACA (12) and ADB-FUBINACA (19).

M1, M3, and M7 were distinct dihydroxylated metabolites despite a common +31.9903 Da mass shift. Details of their fragmentation patterns are further described in the Supplemental Results.

Oxidation occurred for M6 and M9, resulting in ketone formation of M8 and M10 (+O −2H). The mass shifts of parent and product ions from ADB-BUTINACA, apart from ions at m/z 145.0401 and 163.0504, indicated ketone formation at the N-butyl chain. M13 presented the same mass shift (+13.9794 Da) for parent ion but was only observed at product ion of m/z 327.1826 (amine loss, m/z 313.1863, +O −2H), indicating hydroxylation and dehydrogenation at the tert-butyl chain. This reaction was likely an imine formation rather than ketone formation due to the stability of imine (12, 19).

M4 is an N-debutylated metabolite as indicated by the mass loss of 56.0631 Da (−C4H8). Major fragments were generated by losses of carboxamide (m/z 230.1294) and dimethylbutanamide (m/z 163.0502). This reaction is commonly observed for SCs with an N-aliphatic chain (12, 20–22) and for ADB-FUBINACA with a methylenefluorophenyl moiety (19).

M2 with a 34.0058 Da increase (+O +H2O) suggested a dihydrodiol formation. Details of the fragmentation patterns are further described in the Supplemental Results. This biotransformation is common among SCs with an indazole core (23, 24).

M15 is an oxidative deaminated metabolite that yielded −0.9851 Da shift (−NH2 +OH) of parent ion but same product ions as ADB-BUTINACA. This was the only prominent metabolite detected in HLM incubation without NADPH. This biotransformation is common among SCs containing terminal ester and amide, and CES is the typical enzyme catalyzing this reaction (13, 25).

In the presence of NADPH, t1/2 of ADB-BUTINACA was 14.74 min (95% CI 14.28 to 15.24 min). Its elimination rate constant k was 0.047 min−1, which was scaled to yield in vitro CLint, uninhibited of 0.094 mL/min/mg, CLint, liver of 6664 mL/min and CLH, parent of 715 mL/min with hepatic extraction ratio of 0.5. No observable metabolism of ADB-BUTINACA was seen in the absence of NADPH or in negative control (Supplemental Fig. 4), confirming that the metabolism of ADB-BUTINACA was mainly mediated by CYP450.

CYP450 selective inhibitors ticlopidine, S-(+)-N-3-benzyl-nirvanol and ketoconazole demonstrated substantial inhibition of metabolic activity, with twice the amount of ADB-BUTINACA remaining compared to control after 30 min incubation (P values