Amide Hydrolysis Research Articles

Absorption, metabolism, and excretion of oral [14C] radiolabeled donafenib: an open-label, phase I, single-dose study in humans.

The study aims to investigate the absorption, metabolism, and excretion of donafenib, a deuterated derivative of sorafenib, in healthy Chinese male volunteers. Six healthy Chinese male volunteers were administered a single oral dose of 300mg donafenib containing 120 µCi of [14C]-donafenib. The study involved collecting and analyzing plasma, urine, and feces samples to determine the recovery and distribution of total radioactivity, identify metabolites, and assess the metabolic pathways of donafenib. The mean recovery of total radioactivity was 97.31% of the administered dose. Six metabolites were identified, with the parent drug being the major radioactive component in plasma (67.52% of total radioactivity) and feces (83.17% of the dose). The N-oxidation metabolite (M2) was prominent in plasma. Donafenib was predominantly excreted via feces, indicating liver metabolism, with minimal renal excretion. The metabolic pathways of donafenib were similar to those of sorafenib, but the metabolite profiles differed significantly. Notably, the amide hydrolysis metabolite M6, present in sorafenib, was absent in donafenib. Donafenib is primarily metabolized in the liver and excreted through feces, with a metabolic profile that differs from sorafenib due to the deuterium isotope effect. These differences in metabolic characteristics may contribute to donafenib's improved safety and efficacy as a treatment for advanced hepatocellular carcinoma (HCC).

Detection and Identification of New Chlorphenesin Carbamate Metabolites in Human Urine and Metabolomic Characterization Using Liquid Chromatography-Q Exactive-HF-Orbitrap-Mass Spectrometry.

Chlorphenesin carbamate is a skeletal muscle relaxant, and one of its metabolites is 4-chlorophenoxyacetic acid (4-CPA), which is included on the Prohibited List of the World Anti-Doping Agency (WADA). The non-prohibited substance, chlorphenesin carbamate, as a potential source of 4-CPA, needs to be identified more strongly in doping control protocols. In this paper, the metabolism of chlorphenesin carbamate in human urine was studied. Ten volunteers were recruited to take chlorphenesin carbamate tablets orally, and urine samples were collected before and after being tested. The urine samples were detected by liquid chromatography-Q Exactive HF orbitrap mass spectrometry (LC-Q Exactive HF-MS) in full MS and full MS-ddMS2 scanning modes. Based on accurate molecular formulae determination and fragmentation pattern analysis by mass spectrometry, a total of 29 chlorphenesin carbamate metabolites were found, comprising 18 newly identified metabolites and 11 previously reported metabolites. There were five potential metabolic processes listed: hydroxylation, amide hydrolysis, C-oxidation, O-glucuronidation, sulfation, and their combinations. Chlorphenesin carbamate and the 29 metabolites were compared for their detection windows, and the findings indicated that the two recently reported metabolites, M9 and M10, had longer detection windows than the metabolites documented by the WADA. These metabolites are anticipated to be long-lasting biomarkers for the detection of chlorphenesin carbamate intake. By analyzing the results of metabolomic profiling, it was found that the metabolites with significant changes were mainly related to histidine metabolism and β-alanine metabolism pathways. This paper provides a comprehensive report on the metabolic profile of urinary chlorphenesin carbamate and reveals a point of view the changes in the metabolic in the human body, which is conducive to supporting the detection of chlorphenesin carbamate and meclofenoxate for doping control, as well as a better understanding of the mechanism of action of chlorphenesin carbamate as a drug.

Metabolic profiling of the synthetic cannabinoid APP-CHMINACA (PX-3) as studied by in vitro and in vivo models.

The metabolic pathways of APP-CHMINACA were characterized to select the markers of intake for implementation into analytical assays used by the clinical and forensic communities. We have combined the evidences obtained by both in vitro experiments and administration studies on mice. APP-CHMINACA was incubated with either human or mouse liver microsomes. Urine and blood samples were collected at different time points from mice after injection of a 3mg/kg dose of the test compound. Samples were analyzed using liquid chromatography-tandem mass spectrometry. The in vitro studies allowed to isolate eight different metabolic reactions, formed by two metabolic routes, with no differences between human and mouse liver microsomes. The main biotransformation route involved the hydrolysis of the distal amide group and the subsequent hydroxylation on the cyclohexyl-methyl ring. The second route involved multiple hydroxylation of the parent compound, followed by reduction to generate minor metabolites. In blood samples, the most abundant substances identified were APP-CHMINACA unchanged and the metabolites formed by the hydrolysis of the distal amide together with its hydroxylated products. In urine samples, four metabolites formed following the hydroxylation of the distal amide hydrolysis metabolite were detected as the most abundant and long-term metabolites. The outcomes of our study showed that the most suitable markers to detect the intake of APP-CHMINACA in blood and urine samples in the framework of toxicological, clinical and forensic investigations were the metabolite formed by the hydrolysis of the distal amide and its hydroxylated products.

One‐Pot Synthesis of 2‐Substituted‐4,5‐dimethyloxazoles from Amides and Acetylene Gas in the KOH/MeOH/DMSO Catalytic Triad

AbstractPharmaceutically prospective 2‐substituted‐4,5‐dimethyloxazoles are synthesized in 16–58% yield via cascade assembly of one molecule of primary amide with two molecules of acetylene gas in the KOH/MeOH/DMSO catalytic triad (90 °C, 2 h). This self‐organization of oxazoles demonstrates unexpected successful competition with the alkaline hydrolysis of amides. The mechanism involving amide N‐vinylation, enamine‐imine isomerization, imine ethynylation and intramolecular O‐vinylation cascade sequence has been deduced and supported by quantum‐chemical calculations using the B2PLYP−D3/6‐311+G**//B3LYP/6‐31+G*+IEF PCM (B3LYP/6‐31+G*) approach.

Transglutaminase 2-mediated histone monoaminylation and its role in cancer.

Transglutaminase 2 (TGM2) has been known as a well-characterized factor regulating the progression of multiple types of cancer, due to its multifunctional activities and the ubiquitous signaling pathways it is involved in. As a member of the transglutaminase family, TGM2 catalyzes protein post-translational modifications (PTMs), including monoaminylation, amide hydrolysis, cross-linking, etc., through the transamidation of variant glutamine-containing protein substrates. Recent discoveries revealed histone as an important category of TGM2 substrates, thus identifying histone monoaminylation as an emerging epigenetic mark, which is highly enriched in cancer cells and possesses significant regulatory functions of gene transcription. In this review, we will summarize recent advances in TGM2-mediated histone monoaminylation as well as its role in cancer and discuss the key research methodologies to better understand this unique epigenetic mark, thereby shedding light on the therapeutic potential of TGM2 as a druggable target in cancer treatment.

Deciphering iopamidol transformation during the joint disinfection of monochloramine and peroxymonosulfate: A base-catalyzed mutual activation

The joint disinfection of monochloramine (MCA) and peroxymonosulfate (PMS) can combine the advantages of both sides; and the iodine-containing micropollutants are widely present in drinking water. For the first time, the conversion of iopamidol (IPM) was investigated in the MCA/PMS system. For IPM degradation, the observed pseudo-first-order rate constant (kobs) was 0.0076 min−1, and the apparent rate constant (kapp) was 6.14 × 10−7 μM−2.16 min−1. The reaction orders for MCA (m) and PMS (n) were fitted to be 1.14 and 1.02, respectively. The mutual activation of MCA and PMS was a base-catalyzed reaction, which was highly exothermic and thermodynamically favored with a free energy of − 78.74 kcal/mol. It is believed that Cl• and 1O2 contributed to IPM degradation. The kobs value was increased by an alkaline pH and the presence of Cl−, while reduced by the existence of NOM, HCO3− and NH4+. The N in MCA was converted to NH4+-N, NO2−-N and NO3−-N, and 47.0 % of I in IPM was converted to IO3−. In the MCA/PMS system, IPM transformation was mainly via amide hydrolysis, deiodination and substitution reaction by reactive species. The study provides an insight into the fate of iodine-containing micropollutants during the joint disinfection of MCA and PMS.

Novel Bifunctional Amidase Catalyzing the Degradation of Propanil and Aryloxyphenoxypropionate Herbicides in Rhodococcus sp. C-1.

Propanil residues can contaminate habitats where microbial degradation is predominant. In this study, an efficient propanil-degrading strain C-1 was isolated from paddy and identified as Rhodococcus sp. It can completely degrade 10 μg/L-150 mg/L propanil within 0.33-10 h via the hydrolysis of the amide bond, forming 3,4-dichloroaniline. A novel bifunctional amidase, PamC, was identified in strain C-1. PamC can catalyze the hydrolysis of the amide bond of propanil to produce 3,4-dichloroaniline as well as the hydrolysis of the ester bonds of aryloxyphenoxypropionate herbicides (APPHs, clodinafop-propargyl, cyhalofop-butyl, fenoxaprop-p-ethyl, fluazifop-p-butyl, haloxyfop-p-methyl, and quizalofop-p-ethyl) to form aryloxyphenoxypropionic acids. Molecular docking and site-directed mutagenesis confirmed that the catalytic triad Lys82-Ser157-Ser181 was the active center for PamC to hydrolyze propanil and cyhalofop-butyl. This study presents a novel bifunctional amidase with capabilities for both amide and ester bond hydrolysis and enhances our understanding of the molecular mechanisms underlying the degradation of propanil and APPHs.

Comparative study of N-vinyl pyrrolidone and cyclic ketene acetal copolymer degradation under alkaline, enzymatic, or wastewater conditions

The (bio)degradability of cyclic ketene acetal (CKA)-containing polymers is dependent on the polymer structure and degradation conditions. Copolymers of N-vinyl pyrrolidone (NVP), with different CKA comonomers and architectures, were subjected to (bio)degradation under alkaline, enzymatic and wastewater conditions. Grafting CKA-co-NVP moieties onto a poly(ethylene oxide) (PEO) backbone promoted the enzymatic hydrolysis of CKA esters and improved the overall polymer biodegradability. Limited biodegradation was observed for poly(CKA-co-NVP) in the presence of wastewater sludge during an industrial biodegradability test under OECD 301F guidelines. Under less stringent test conditions poly(CKA-co-NVP) achieves small, yet non-negligible biodegradation. Structural analysis of the degradation products shed light into the degradation mechanisms, revealing CKA ester hydrolysis in all cases, while in presence of wastewater sludge, additional CKA end-group oxidation was clearly observed. However, effective amide hydrolysis or pyrrolidone unit biodegradation was not detected, with pyrrolidone-containing segments persisting through the biodegradation test.

In vitro metabolism study of ADB-P-5Br-INACA and ADB-4en-P-5Br-INACA using human hepatocytes, liver microsomes, and in-house synthesized references.

Synthetic cannabinoids (SCs) remain a major public health concern, as they continuously are linked to severe intoxications and drug-related deaths worldwide. As new SCs continue to emerge on the illicit drug market, an understanding of SC metabolism is needed to identify formed metabolites that may serve as biomarkers in forensic toxicology screening and for understanding the pharmacokinetics of the drugs. In this work, the metabolism of ADB-4en-P-5Br-INACA and ADB-P-5Br-INACA ((S)-N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-5-bromo-1-(pent-4-en-1-yl)-1H-indazole-3-carboxamide, (S)-N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-5-bromo-1-pentyl-1H-indazole-3-carboxamide respectively) were investigated using human hepatocytes in vitro and in-house synthesized references. Both SCs were incubated with pooled human hepatocytes over 3h, with the aim to identify unique and abundant metabolites using liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). In total nine metabolites were identified for ADB-4en-P-5Br-INACA and 10 metabolites for ADB-P-5Br-INACA. The observed biotransformations included dihydrodiol formation, terminal amide hydrolysis, hydroxylation, dehydrogenation, carbonyl formation, glucuronidation, and combinations thereof. The major metabolites were confirmed by in-house synthesized references. Recommended biomarkers for ADB-P-5Br-INACA and ADB-4en-P-5Br-INACA are the terminal hydroxy and dihydrodiol metabolite respectively.

One-pot four-step direct synthesis of 2,5-furandicarboxylic acid from 2,5-diformylfuran under oxygen-free conditions

2,5-Furanedicarboxylic acid (FDCA) is a valuable compound with the potential as a renewable and green alternative to terephthalic acid in polyester production. To address the issues of flammability, explosiveness, and product separation challenges associated with the current aerobic oxidation of 5-hydroxymethylfurfural (5-HMF) to FDCA route, herein, for the first time, a one-pot four-step direct reaction pathway for the synthesis of FDCA from 2,5-diformylfuran (DFF) and hydroxylamine salt is proposed. Under optimized conditions of 120 ℃ for 8 h, [HSO3-b-mim]·CF3SO3 catalyst and p-xylene-water dual liquid phase solvent, 100 % DFF conversion and 91.0 % FDCA separation yield were obtained. Notably, [HSO3-b-mim]·CF3SO3 exhibited excellent stability, maintaining its activity after being recycled more than five times. Through online experimentation and density functional theory (DFT) calculations, a four-step reaction mechanism involving aldehyde oximation, oxime dehydration, nitrile hydrolysis, and amide hydrolysis was proposed. This reaction pathway offers significant industrial application value due to its high FDCA yield, simple product separation, easy recovery and recycling of ionic liquid, and elimination of the need for oxygen.

Temperature and pH-dependent stability of fentanyl analogs: Degradation pathways and potential biomarkers.

The collection, storage, and transport of samples prior to and during analysis is of utmost importance, especially for highly potent analogs that may not be present in high concentrations and are susceptible to pH or thermally mediated degradation. An accelerated stability study was performed on 17 fentanyl analogs (fentalogs) over a wide range of pH (2-10) and temperature (20-60°C) conditions over 24 h. Dilute aqueous systems were used to investigate temperature and pH-dependent kinetics using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Liquid chromatography-quadrupole/time-of-flight-mass spectrometry (LC-Q/TOF-MS) was used for structural elucidation of degradants. With the exception of remifentanil, all fentalogs evaluated were stable at pH 6 or lower. Fentalogs were generally unstable in strongly alkaline environments and at elevated temperatures. Remifentanil was the least stable drug and N-dealkylated fentalogs were the most stable. Fentanyl degraded to acetylfentanyl, norfentanyl, fentanyl N-oxide, and 1-phenethylpyridinium salt (1-PEP). A total of 26 unique breakdown products were observed for 15 of the fentanyl derivatives studied. Common degradation pathways involved N-dealkylation, oxidation of the piperidine nitrogen, and β-elimination of N-phenylpropanamide followed by oxidation/dehydration of the piperidine ring. Ester and amide hydrolysis, demethylation at the propanamide, and O-demethylation were observed for selected fentalogs only. The potential for analyte loss should be considered during the pre-analytical phase (i.e., shipping and transport) where environmental conditions may not be controlled, as well as during the analysis itself.

Leveraging High-Resolution Ion Mobility-Mass Spectrometry for Cyclic Peptide Soft Spot Identification.

Cyclic peptides are an important class of molecules that gained significant attention in the field of drug discovery due to their unique pharmacological characteristics and enhanced proteolytic stability. Yet, gastrointestinal degradation remains a major hurdle in the discovery of orally bioavailable cyclic peptides. Soft spot identification (SSID) of the regions in the cyclic peptide sequence susceptible to amide hydrolysis by proteases is used in the discovery stage to guide medicinal chemistry design. SSID can be an arduous task, traditionally performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), often resulting in complex and time-consuming manual analysis, particularly when isomeric linear peptide metabolites chromatographically coelute. Here, we present an alternative orthogonal approach that entails a high-resolution ion mobility (HRIM) system based on Structures for Lossless Ion Manipulation (SLIM) technology interfaced with quadrupole time-of-flight (QTOF) mass spectrometry to address some of the challenges associated with SSID. Two strategies were used to resolve linear isomeric peptide metabolites: labeled and label-free, both utilizing the HRIM platform. The label-free strategy leverages negative polarity to ionize the isomers which achieves better separation of the gas phase ions in the ion mobility (IM) dimension as compared to positive polarity, which is a more conventional approach when studying proteins and peptides. The second approach uses an isotope-labeled dimethyl tag on the terminal amine group, acting as a "shift reagent" to influence the mobility of isomers in the positive mode. This method resulted in baseline separation for the isomers of interest and produced unique product ions in the fragmentation spectra for unambiguous soft spot identification. Both label-free and labeled strategies demonstrated the ability to solve the challenges associated with SSID for cyclic peptides.

Insights on the degradation mechanism of neotame using UV/periodate: Roles of reactive species, kinetics, and pathways

As a new artificial sweetener (ASs), the jeopardize of neotame (NEO) to aquatic environment is attracting concern due to its widespread usage and difficulty in removing from wastewater. UV/periodate (UV/PI) is being considered as an effective process for the degradation of micropollutants in wastewater. However, there are few studies of the roles of reactive species (RS) and the effects of complex water matrices on the degradation of NEO via UV/PI. The degradation of NEO in aqueous media by UV/PI was investigated herein. NEO was effectively removed (>97 %) by UV/PI within 4 min. The degradation of NEO was mainly attributed to •OH (75.7 %), IO3• (23.0 %) and 1O2 (1.3 %) with the observed second-order rate constants of 7.71 × 109, 1.56 × 108 and 2.94 × 105 M−1 s−1, respectively. The contribution of IO3• and 1O2 to NEO degradation was greater at acidic and high PI concentrations, but significantly decreased in the presence of natural organic matter. The presence of anions in the water did not obviously impact NEO degradation. During the degradation of NEO, •OH attacked NEO through hydroxylation reaction, while IO3• and 1O2 predominantly participated in decarboxylation and amide hydrolysis reactions. Toxicity evaluation showed that the UV/PI treatment of NEO enhanced the toxicity to Vibrio fischeri within 5 min, and the toxicity reduced with the prolongation of treatment time. The study provides meaningful information on the roles of IO3• and 1O2 for NEO degradation by UV/PI.

Diketopyrrolopyrrole based molecular semiconductors with intrinsic conductivity

Intrinsically conducting diketopyrrolopyrrole (DPP) compounds functionalised with pendant quaternary ammonium groups have been synthesised. The hydroxide forms were found to be the product of base hydrolysis of the DPP amide during synthesis, likely made possible by restricted resonance. This gave mixed chemical compositions of solutions and conductive films formed by drop-casting. Chemical decomposition of the hydrolysed compounds was also observed at temperatures above 85 °C. Conductivities of approximately 3 × 10−4 S m−1 were observed in processed films of DPP ammonium hydroxides. It was also found that trifluoroacetic acid (TFA) DPP precursors gave clear electron paramagnetic resonance (EPR) signals indicative of doping and exhibited conductivities one order of magnitude larger than the hydroxides. This opens the possibility of using ammonium TFAs as moieties for doping organic semiconductors.

The Power of Molecular Dynamics Simulations and Their Applications to Discover Cysteine Protease Inhibitors.

A large family of enzymes with the function of hydrolyzing peptide bonds, called peptidases or cysteine proteases (CPs), are divided into three categories according to the peptide chain involved. CPs catalyze the hydrolysis of amide, ester, thiol ester, and thioester peptide bonds. They can be divided into several groups, such as papain-like (CA), viral chymotrypsin-like CPs (CB), papainlike endopeptidases of RNA viruses (CC), legumain-type caspases (CD), and showing active residues of His, Glu/Asp, Gln, Cys (CE). The catalytic mechanism of CPs is the essential cysteine residue present in the active site. These mechanisms are often studied through computational methods that provide new information about the catalytic mechanism and identify inhibitors. The role of computational methods during drug design and development stages is increasing. Methods in Computer-Aided Drug Design (CADD) accelerate the discovery process, increase the chances of selecting more promising molecules for experimental studies, and can identify critical mechanisms involved in the pathophysiology and molecular pathways of action. Molecular dynamics (MD) simulations are essential in any drug discovery program due to their high capacity for simulating a physiological environment capable of unveiling significant inhibition mechanisms of new compounds against target proteins, especially CPs. Here, a brief approach will be shown on MD simulations and how the studies were applied to identify inhibitors or critical information against cysteine protease from several microorganisms, such as Trypanosoma cruzi (cruzain), Trypanosoma brucei (rhodesain), Plasmodium spp. (falcipain), and SARS-CoV-2 (Mpro). We hope the readers will gain new insights and use our study as a guide for potential compound identifications using MD simulations.

Rapid and Definitive Identification of Cyclic Peptide Soft Spots by Isotope-Labeled Reductive Dimethylation and Mass Spectrometry Fragmentation.

Cyclic peptides are an emerging therapeutic modality over the past few decades. To identify drug candidates with sufficient proteolytic stability for oral administration, it is critical to pinpoint the amide bond hydrolysis sites, or soft spots, to better understand their metabolism and provide guidance on further structure optimization. However, the unambiguous characterization of cyclic peptide soft spots remains a significant challenge during early stage discovery studies, as amide bond hydrolysis forms a linearized isobaric sequence with the addition of a water molecule, regardless of the amide hydrolysis location. In this study, an innovative strategy was developed to enable the rapid and definitive identification of cyclic peptide soft spots by isotope-labeled reductive dimethylation and mass spectrometry fragmentation. The dimethylated immonium ion with enhanced MS signal at a distinctive m/z in MS/MS fragmentation spectra reveals the N-terminal amino acid on a linearized peptide sequence definitively and, thus, significantly simplifies the soft spot identification workflow. This approach has been evaluated to demonstrate the potential of isotope-labeled dimethylation to be a powerful analytical tool in cyclic peptide drug discovery and development.

Metabolism of ADB-FUBIATA in zebrafish and pooled human liver microsomes investigated by liquid chromatography-high-resolution mass spectrometry.

ADB-FUBIATA is one of the most recently identified new psychoactive substance (NPS) of synthetic cannabinoids. The co-use of in vitro (human liver microsomes) and in vivo (zebrafish) models offers abundant metabolites and may give a deep insight into the metabolism of NPS. In vivo and in vitro metabolic studies of new synthetic cannabinoid ADB-FUBIATA were carried out using zebrafish and pooled human liver microsome models. Metabilites were structurally characterized by liquid chromatography-high-resolution mass spectrometry. In total, 18 metabolites were discovered and identified in the pooled human liver microsomes and zebrafish, including seventeen phase I metabolites and one phase II metabolite. The main metabolic pathways of ADB-FUBIATA were hydroxylation, dehydrogenation, N-dealkylation, amide hydrolysis, glucuronidation, and combination thereof. Hydroxylated metabolites can be recommended as metabolic markers for ADB-FUBIATA because of the structural characteristics and high intensity. These metabolism characteristics of ADB-FUBIATA were useful for its further forensic or clinical related investigations.

In vivo and in vitro metabolite profiling of nirmatrelvir using LC-Q-ToF-MS/MS along with the in silico approaches for prediction of metabolites and their toxicity.

Nirmatrelvir (NRV), a 3C-like protease or Mpro inhibitor of SARS-CoV-2, is used for the treatment of COVID-19 in adult and paediatric patients. The present study was accomplished to investigate the comprehensive metabolic fate of NRV using in vitro and in vivo models. The in vitro models used for the study were microsomes (human liver microsomes, rat liver microsomes, mouse liver microsomes) and S9 fractions (human liver S9 fractions and rat liver S9 fractions) with the appropriate cofactors, whereas Sprague-Dawley rats were used as the in vivo models. Nirmatrelvir was administered orally to Sprague-Dawley rats, which was followed by the collection of urine, faeces and blood at pre-determined time intervals. Protein precipitation was used as the sample preparation method for all the samples. The samples were then analysed by liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (LC-Q-ToF-MS/MS) using an Acquity BEH C18 column with 0.1% formic acid and acetonitrile as the mobile phase. Four metabolites were found to be novel, which were formed via amide hydrolysis, oxidation and hydroxylation. Furthermore, an in silico analysis was performed using Meteor Nexus software to predict the probable metabolic changes of NRV. The toxicity and mutagenicity of NRV and its metabolites were also determined using DEREK Nexus and SARAH Nexus.

ADB-HEXINACA-a novel synthetic cannabinoid with a hexyl substituent: phase I metabolism in authentic urine samples, a case report and prevalence on the German market.

Synthetic cannabinoid receptor agonists (SCRAs) are one of the largest groups of new psychoactive substances (NPS). Yet, another novel analog started spreading on the NPS market around 2021. Soon after, the substance could be analytically characterized in herbal material as ADB-HEXINACA, an SCRA containing a hexyl-substituted tail on the indazole core. Here, we present suitable urinary markers to prove the consumption of this analog, a case report of acute polydrug intoxication and data on its prevalence in Germany. Anticipated phase I metabolites were detected in 12 authentic urine samples that were collected for abstinence control and analyzed by ultra-performance liquid chromatography coupled to a time-of-flight mass spectrometer (UPLC-qToF-MS). The results of in vivo samples were confirmed by analysis of in vitro incubates with pooled human liver microsomes (pHLMs). Forensic samples were used to assess the prevalence of ADB-HEXINACA. Thirty-two phase I metabolites were detected in the authentic urine samples. The main metabolites resulted from amide hydrolysis in combination with either monohydroxylation or ketone formation at the hexyl moiety (M15 and M26), the monitoring of which is recommended as a proof of consumption. ADB-HEXINACA was detected in 3.5% of SCRA positive urine samples collected for abstinence control in Freiburg up to December 2022 and in 5.5% of the SCRA positive blood/serum samples. The hexyl substituent of ADB-HEXINACA allows for the detection of specific urinary biomarkers suggested as analytical targets to confirm its prior intake. ADB-HEXINACA had a rather low prevalence in Germany, alternating months of higher prevalence with periods of total absence.

Room Temperature Amide Hydrolysis Enabled by TiO2 (001) Surface

AbstractAmides are crucial components of biomolecules and are extensively used in polymer, pharmaceutical, and agrochemical production. Their direct hydrolysis offers great potential for exploring protein structures and producing valuable carboxylic acids in biological and industrial applications. Nevertheless, activating the resonance‐stabilized C−N bond in amides poses a formidable challenge. Extensive research over the past decades has reported various transition metal‐based complexes and solid catalysts that catalyze this reaction. These catalysts possess Lewis acid (LA) sites and exhibit enhanced activity when further combined with Brönsted acid (BA) sites. In this study, we present the first demonstration of amide hydrolysis on TiO2, a rock‐forming material, offering valuable insights into its surface activity. By using acetamide as the model compound, we observed that the thermodynamically stable (101) surface of TiO2 remained inert up to 95 °C. Surprisingly, the high‐energy (001) surface of TiO2 activated amide hydrolysis at a temperature as low as 25 °C. Contrary to previous reports, the fluorine‐modified (001) surface with additional BA sites required temperatures above 70 °C likely due to hydrogen bond stabilization by nearby fluorine atoms. These findings provide guidance for the development of cost‐effective catalysts with improved activity.