The gas chromatography mass spectrometry of the major metabolites of flurazepam

Clatworthy, A.J.; Jones, L. V.; Whitehouse, Martin J.

doi:10.1002/bms.1200040411

Cited by 20 publications

(7 citation statements)

References 7 publications

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“…GABA A Rs are the target of a range of insomnia medicines, including flurazepam and ZOL. To investigate the molecular effects of insomnia treatments on n α 1 GABA A Rs, we examined the interactions with either DID, the major metabolite of flurazepam 44 (K i 16.9 ± 1.7 nM), or ZOL (K i 22.9 ± 2.7 nM) and n α 1 GABA A Rs ( Figure 3a and 3b ). Both compounds engage the receptor ECD at the α1 *+ /γ2 - interface, which is spatially equivalent to the GABA pockets, each sandwiched at a β + /α - interface.…”

Section: Mainmentioning

confidence: 99%

Regulated assembly and neurosteroid modulation constrain GABA_Areceptor pharmacologyin vivo

Sun

Zhu

Clark

et al. 2023

Preprint

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Type A GABA receptors (GABAARs) are the principal inhibitory receptors in the brain and the target of a wide range of clinical agents, including anesthetics, sedatives, hypnotics, and antidepressants. However, our understanding of GABAAR pharmacology has been hindered by the vast number of pentameric assemblies that can be derived from a total 19 different subunits and the lack of structural knowledge of clinically relevant receptors. Here, we isolate native murine GABAAR assemblies containing the widely expressed α1 subunit, and elucidate their structures in complex with drugs used to treat insomnia (zolpidem and flurazepam) and postpartum depression (the neurosteroid allopregnanolone). Using cryo-EM analysis and single-molecule photobleaching experiments, we uncover only three structural populations in the brain: the canonical α1β2γ2 receptor containing two α1 subunits and two unanticipated assemblies containing one α1 and either an α2, α3 or α5 subunit. Both of the noncanonical assemblies feature a more compact arrangement between the transmembrane and extracellular domains. Interestingly, allopregnanolone is bound at the transmembrane α/β subunit interface, even when not added to the sample, revealing an important role for endogenous neurosteroids in modulating native GABAARs. Together with structurally engaged lipids, neurosteroids produce global conformational changes throughout the receptor that modify both the pore diameter and binding environments for GABA and insomnia medications. Together, our data reveal that GABAAR assembly is a strictly regulated process that yields a small number of structurally distinct complexes, defining a structural landscape from which subtype-specific drugs can be developed.

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Section: Mainmentioning

confidence: 99%

Regulated assembly and neurosteroid modulation constrain GABA_Areceptor pharmacologyin vivo

Sun

Zhu

Clark

et al. 2023

Preprint

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“…A number of mass spectra of 1,4-benzodiazepine derivatives together with EI-induced fragmentation mechanisms are well know from the literature [13][14][15][16][17][18][19][20][21][22] but no derivates with functionalized methylene group in position 2, which exhibit new features in their mass spectrometric fragmentation, have been reported.…”

Section: Metabolites Resulting From Major Biotransformation Pathwaysmentioning

confidence: 99%

“…Of structural significance are the typical benzophenone fragments [C 6H4CICO]+ (m/e 139, 5) and [M-C 6H4CI]+ (4) both with subsequent loss of CO to 5.1 and 4.1 [13,17,18,22,23,24]. Consequently both metabolites are benzophenones with the structures given in scheme 3.…”

Section: Metabolites M 5 and Mmentioning

confidence: 99%

“…16 and 17) show identity with that of the reference compounds KC 3757 and KC 3756, respectively. The molecular ions of both compounds are decomposing according to fragmentation mechanisms which are discussed in scheme 5 and which are in principle known from published results of similar compounds [13][14][15][16][17][18][19][20][21][22] …”

Section: Metabolites M 10 and M 13mentioning

confidence: 99%

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Metabolism and pharmacokinetics of metaclazepam (Talis®), Part III: Determination of the chemical structure of metabolites in dogs, rabbits and men

Borchers¹,

Achtert²,

Hausleiter³

et al. 1984

European Journal of Drug Metabolism and Pharmacokinetics

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The metabolism of 7-bromo-1-methyl-2-methoxymethyl-5-(2'-chlorophenyl)-2, 3-dihydro-1H-1,4-benzodiazepine (metaclazepam, Talis) in animals and men is described. Based upon mass spectrometry fifteen metabolites could be identified. Qualitative and quantitative differences in the biotransformation products of metaclazepam in comparison with the well known metabolites of other drugs in the 1,4-benzodiazepine class could be demonstrated. Metabolites with a benzodiazepine-2-one structure representing the most characteristic feature of other 1,4-benzodiazepines and their metabolites, were found in trace amounts only. The major metabolic pathways of metaclazepam led via stepwise demethylation of the O-methyl and/or the N-methyl group to O-demethyl-metaclazepam (M 2), N-demethyl-metaclazepam (M 7) and bis-demethyl-metaclazepam (M 6). Further aromatic hydroxylation yielded the metabolite M 1. Two metabolites with amino-benzophenone structure (M 5, M 8) which are in general known to result from other 1,4-benzodiazepines could be detected. Additionally a 3-oxo-benzodiazepine (M 4) was found. Minor biotransformation pathways led to a chlorophenyl-bromo-benzodiazepine (M 9) by loss of the side chain from bis-demethyl-metaclazepam and N-demethyl-metaclazepam. By further oxidation and degradation the 2-oxo-benzodiazepine M 10 and the dihydro-quinazoline M 12 were formed. The respective N-methylated metabolites M 13 and M 16 were possibly generated by the same pathway. Still open is the formation of M 15, a 1-methyl-3-hydroxy-4-(2'-chlorophenyl)-6-bromo-1,2-dihydroquinoline and M 11, a 2-methyl-4-(2'-chlorophenyl)-6-bromo-quinazoline. The substitution of bromine by a hydroxyl group during the formation of M 14 can be explained by a NIH-shift mechanism. Quantitative investigations show that the methoxymethyl side chain in the benzodiazepine ring system of metaclazepam acts as an effective barrier with respect to the metabolic attack at position two. We assume that this barrier only can be overcome by complete side chain degradation. This multi-step reaction can hardly compete with more favourable and faster conjugation and elimination processes.

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“…1). Hydrolysis of the conjugates can also be achieved by glucuronidases, but typically takes more than 5 h [8]. Thus, the acid-hydrolysis method is especially useful for urine samples, because the drugs are excreted to urine largely in a hydroxylated or conjugated form [2].…”

Section: Discussionmentioning

confidence: 99%

Positive and negative ion mass spectrometry of benzophenones, the acid-hydrolysis products of benzodiazepines

et al. 1987

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Positive electron impact (EI), positive chemical ionization (CI), and negative CI mass spectra of 14 benzophenones are presented. In the positive EI mode, intense molecular peaks appeared for most compounds; some other peaks due to splitting at both sides of the carbonyl group also appeared. In the positive CI mode, [M + 1]+ quasi-molecular ions together with [M + C2H5]+ peaks were observed for all compounds; some fragment peaks were common to those in the positive EI mode. In the negative CI mode, the spectra were much simpler than those in the positive EI or CI mode. In the 1 Torr negative CI mode, some spectra showed only single molecular anions; in the 0.01 Torr negative CI mode, halogen or nitro peaks appeared in addition to the molecular anions. An extraction procedure for benzophenones from human urine and plasma after heating in strong acid, and their separation by gas chromatography (GC) are also presented to serve for their actual identification by GC/mass spectrometry.

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The gas chromatography mass spectrometry of the major metabolites of flurazepam

Cited by 20 publications

References 7 publications

Regulated assembly and neurosteroid modulation constrain GABA_Areceptor pharmacologyin vivo

Regulated assembly and neurosteroid modulation constrain GABA_Areceptor pharmacologyin vivo

Metabolism and pharmacokinetics of metaclazepam (Talis®), Part III: Determination of the chemical structure of metabolites in dogs, rabbits and men

Positive and negative ion mass spectrometry of benzophenones, the acid-hydrolysis products of benzodiazepines

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The gas chromatography mass spectrometry of the major metabolites of flurazepam

Cited by 20 publications

References 7 publications

Regulated assembly and neurosteroid modulation constrain GABAAreceptor pharmacologyin vivo

Regulated assembly and neurosteroid modulation constrain GABAAreceptor pharmacologyin vivo

Metabolism and pharmacokinetics of metaclazepam (Talis®), Part III: Determination of the chemical structure of metabolites in dogs, rabbits and men

Positive and negative ion mass spectrometry of benzophenones, the acid-hydrolysis products of benzodiazepines

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Regulated assembly and neurosteroid modulation constrain GABA_Areceptor pharmacologyin vivo

Regulated assembly and neurosteroid modulation constrain GABA_Areceptor pharmacologyin vivo