In this project, an extensive range of benzodiazepine analogues have been synthesised via Schmidt reaction of specially prepared flavanone, 4-quinolone and l-thioflavanone precursors; nitrogen insertion being effected by use of trimethylsilyl azide in trifluoroacetic acid. In some cases, several of the benzodiazepine analogues have also been prepared by alternative cyclisation routes. A detailed kinetic-mechanistic study of the Schmidt reaction of flavanones has been carried out using 'H NMR spectroscopy to explain the observed regiochemistry of nitrogen insertion. The reaction rates, for the formation of both amide and tetrazolo derivatives have been found to be influenced by the electronic effects of the A- and B-ring substituents. A series of benzodiazepine analogues have been shown to undergo regioselective A-ring chlorination with t-butylhypochlorite; the products being characterised by 'H NMR, IR and mass spectroscopy. The mass spectrometric fragmentation patterns of series of 2-aryl-4-quinolones, and 2-aryl-l ,4-benzodiazepinones and their tetrazolo[l ,5-dl analogues have been elucidated using a combination of low-resolution, high-resolution and metastable-peak analyses. The binding affinities of various benzodiazepine analogues for rat brain benzodiazepine receptors have been evaluated using a radioreceptor assay technique. Structure-activity relationships were investigated to establish the effects of various A-, B- and Coring substituents on binding affinity. The conformational preferences of selected systems have been studied using a combination of multi-pulse 'H NMR spectroscopy, X-ray crystallography and computer modelling techniques with a view to establishing the influence of conformation on binding affinity.

An extensive range of oxygen and sulphur substituted benzodiazepine analogues has been synthesised via Baeyer-Villiger and Schmidt reactions of specially prepared flavanone and N-acetyl-4-quinolone precursors. Alternative, cyclisation routes have also been used to prepare some of these compounds. Ring-opening reactions of 1,5-benzodioxepinones have been investigated and a detailed kinetic-mechanistic study of the Baeyer-Villiger reaction of flavanones has been carried out using 1 H NMR spectroscopy to explain the observed regiochemistry of oxygen insertion. The electron-impact mass spectrometric fragmentation patterns of series of 4-aryl-l ,5-benzoxathiepinones, 3-aryl-4, I-benzoxathiepinones and 3-aryl-4,1-benzoxathiepines have been studied using a combination of low-resolution, highresolution and metastable-peak analyses. The 170 NMR spectroscopic properties of various oxygenated analogues have also been studied. The binding affinities of selected benzodiazepine analogues for rat brain benzodiazepine receptors have been evaluated using a radioreceptor assay technique; at certain concentrations, some of test compounds exhibited remarkable potentiation of diazepam binding, others the ability to displace diazepam from benzodiazepine receptors. A conformational analysis of the 7-membered ring systems has been undertaken, using lH NMR spectroscopic, computer modelling and x-ray crystallographic techniques, and certain conformational preferences have been identified.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

In this study, we investigated the effects of the peripheral benzodiazepine ligands, Ro5-4864 (putative antagonist) and PK 11195 (putative antagonist) on prolactin stimulated mitogenesis in Nb2 cells. Ro5-4864 and PK 11195 at 10⁻⁹ M maximally enhanced prolactin stimulated mitogenesis. At 10⁻⁶ M Ro5-4864 inhibited prolactin stimulated mitogenesis. Clonazepam, a ligand for the central benzodiazepine receptor had no effect on mitogenesis. Interaction studies were undertaken to determine if Ro5-4864 and PK 11195 act on the same site. The ability of each ligand to enhance the mitogenic action of prolactin was blocked by a 10⁻⁶ M concentration of the other ligand. Finally, simultaneous addition of 10⁻⁹ M of the ligands resulted in no additive effect over each ligand alone. These data show that peripheral benzodiazepine ligands modulate prolactin-stimulated mitogenesis and suggests they interact at the same binding site.

A new method is described for the quantitative analysis of urinary benzodiazepines by gas chromatography/mass spectrometry. Development work was aimed at satisfying federal requirements for methods used in forensic urine drug testing which have become the standard in the laboratory industry. Trimethylsilyl (TMS), tert-butyl-dimethylsilyl (TBDMS) and benzophenone derivatives were tested in the development of the new assay. TBDMS derivatives were found to be the most suitable for the analysis of six common benzodiazepine metabolites. Precision for all metabolites tested, as measured by the within run coefficient of variation, was less than 7% at 100 ng/ml (n = 15). Assay sensitivity varied with the specific analyte in the range of 5 to 10 ng/ml. Validation of the procedure included the reanalysis of benzodiazepine positive urine specimens obtained from a forensic drug testing laboratory and comparison of the results from the independent assays. These specimens were tested first by radioimmunoassay using a 100 ng/ml cutoff and then confirmed by GC/MS. Sensitivity was sufficient to confirm the presence of benzodiazepine metabolites in all specimens tested.

The binding of [³H]flunitrazepam (FLU) to membranes prepared from mammalian brain, retina and kidney was investigated by means of conventional filtration assay techniques. In the mouse brain a study of the ontogeny of [³H]FLU binding was conducted. Specific [³H]FLU binding was present early in development and there was a rapid increase in receptor density (Bmax) during the first 2 weeks of neonatal life. This increase could be described by the function, a·eᵏˣ, where a = 1.8 pmol/brain, k = 0.23 weeks⁻¹ and x = time in weeks (r = 0.98). By 3-4 weeks of age, adult levels of [³H]FLU binding were reached (∼115 fmol/mg tissue). Notable changes in the equilibrium dissociation constant (K(d)) during development were not observed. γ-Aminobutyric acid (GABA) has previously been shown to increase the affinity of [³H]FLU binding in the adult rat brain; in the present studies this effect was shown to be present throughout the development of the mouse brain. Kinetic analyses of the GABA enhancement of [³H]FLU binding indicated that the change in K(d) was due to a decrease in the rate constant of dissociation (k₋₁). [³H]FLU binding has been shown to occur in the mammalian retina and it has all the characteristics of cerebral [³H]FLU binding. Monosodium glutamate (MSG) is toxic to retinal neurons and it was used to ascertain the putative cellular localization of retinal BZD binding sites. Nine weeks following neonatal MSG administration, histologic evidence showed the virtual absence of ganglion cells and a marked reduction in the number of inner nuclear neurons in MSG retinae. A corresponding 73 percent decrease in GABA content and a 77 percent decrease in the Bmax of [³H]FLU were found in the retinae from MSG treated rats as compared to controls. There were no significant changes in [³H]FLU binding in the cerebella, cerebral cortices and hypothalami from MSG treated rats. The binding of [³H]FLU was characterized for the rat kidney. Binding was specific, saturable and of moderately high affinity (Bmax, ∼320 fmol/mg tissue; K(d), ∼11 nM). Drug specificity studies with renal membranes showed that inhibition of [³H]FLU binding by various BZD's did not correlate either with their pharmacologic potency as anxiolytic agents or with their potency as inhibitors of [³H]FLU binding in the brain. An intrarenal distribution of specific [³H]FLU binding was found in the bovine kidney; specific binding was greatest in the outer cortex and virtually absent in the medulla, the minor calyx and the renal artery. In rats made hypertensive by simultaneous deoxycorticosterone acetate and NaCl administration, there was a significant 35-43 percent increase in the Bmax of renal [³H]FLU binding. Binding in the cerebral cortex of these animals was unchanged. The inhibition of [³H]FLU binding by a triazolopyridazine (CL 218,872) was studied in membranes prepared from bovine retina, rat cerebral cortex, cerebellum and kidney. The affinity of CL 218,872 for the inhibition of [³H]FLU binding was greatest in the cerebellum, followed by the retina, cerebral cortex and kidney (60, 150, 200, and 1,800 nM, respectively). The slope factors (Hill coefficients) were ∼1 for the kidney, ∼0.9 for the cerebellum and ∼0.7 for the cerebral cortex and retina. A nonlinear least squares regression analysis of the data from the cerebral cortex, retina and cerebellum gave an excellent fit for a model containing 2 binding sites. In washed membrane preparations from the cerebral cortex, cerebellum and retina, Kᵢ values for CL 218,872 were significantly decreased an average of 60 percent in the presence of 100 μM GABA. (+)Bicuculline could antagonize this effect of GABA. GABA had no effect upon the Kᵢ of CL 218,872 in renal membranes.

Thesis (M.S.) in Nursing--University of Maine, 2004. / Includes vita. Includes bibliographical references (leaves 59-79).

Benzodiazepines are the most commonly prescribed group of psychotherapeutic pharmaceuticals on a global scale and have been on the market since the 1960s. Benzodiazepines remain in the aqueous effluent from sewage treatment plants and have been found in natural aquatic environments. The aim of this study was to investigate if there is benzodiazepines in natural sediment from Lake Ekoln situated downstream River Fyris in Uppsala, where previous studies have detected high concentrations in the water. The study tested following hypotheses: (1) benzodiazepines are accumulating in sediments; and (2) breakdown of benzodiazepines is slow in sediment resulting in them being preserved in sediments that are several years/decades old. An extraction method for sediment was developed followed by liquid chromatography-tandem mass spectrometry for analysis of oxazepam, alprazolam, clonazepam, flunitrazepam, diazepam and prazepam. All investigated benzodiazepines occurred in the sediment of Lake Ekoln; hence, in line with hypothesis 1, benzodiazepines are accumulating in natural sediments. Further, all benzodiazepines were found in the upper 10 cm of the sediment and oxazepam, clonazepam and diazepam was found at depth below 20 cm, corresponding to an age of more than 20 years indicating that benzodiazepines resists degradation processes in sediment and are persistent for decades as predicted by hypothesis 2. To my knowledge this is the first study demonstrating that benzodiazepines are accumulated and preserved in natural sediments.

The purpose of this study was to determine the effects of triazolam, a short-acting benzodiazepine, on nocturnal arousals, body movements, and quality of sleep in healthy, postmenopausal females. A double-blind control by constancy cross-over design was employed. Twelve subjects were randomly assigned to the sequence paradigm placebo, drug, placebo, drug or to the sequence paradigm drug, placebo, drug, placebo. Each subject slept for six nights in a sleep laboratory that was specifically designed to decrease the artificiality of the laboratory setting. EEG, EOG, and EMG measurements were recorded on a polysomnograph. Body movements were recorded on videotape and measured by radar and ultrasound instruments. Sleep quality was measured using both Likert and visual analogue scales. In examining arousal activity during sleep period time, the findings demonstrated a significant decrease in wakes after sleep onset (WASO), sleep stage one episodes, and sleep stage shifts when triazolam was compared to a placebo reference. There was no significant effect on K-complex activity associated with movement. In examining body movements during the sleep period time, the findings demonstrated a significant decrease in major body movements when triazolam was compared to a placebo reference. Minor body movements were increased, but not at significant levels. In examining the subject's perceived quality of sleep, their satisfaction with sleep was significantly increased on nights following triazolam administration when compared with nights following placebo administration.

Rigid analogues of the tricyclic antidepressant imipramine and the phenothiazine tranquilizer promazine were designed and their syntheses were attempted. Conformational rigidity was expected to reduce the side effects of these drugs by limiting their binding to multiple receptors. Ortho-directed metalation followed by acylation provided synthetic intermediates for the formation of the desired pentacyclic congeners. The known dilithiation of phenothiazine and iminodibenzyl and n-butyllithium, followed by acylation with dimethylformamide, gave carboxaldehydes at the 1 and 4 positions respectively. Ortho-lithiated nicotinamides were acylated by these aldehydes exclusively at the 4 position to provide the key intermediate alcohol amides. Difficulties in amide hydrolysis are discussed. Catalytic hydrogenation over palladium-on-carbon in refluxing acetic acid yielded carboxylic acids, apparently via the gamma-lactones formed in situ. The lactones could not be isolated easily due to instability to oxidation. Pentacyclic lactams were formed by dehydration, and borane was used to reduce the carbonyl function. Only the iminodibenzyl lactam was reduced, and problems encountered in subsequent pyridine ring reduction are discussed. Cis and trans ring fusion isomers were identified by ¹³C nmr. Attempted one-pot synthesis of this pentacycle and a regioisomer by double acylation of 4,5-dilithioiminodibenzyl with 2,3-pyridinedicarboxylic anhydride, and 3,4-pyridinedicarboxylic anhydride failed. Mechanistic considerations are discussed regarding regiochemistry and reactivity of the nitrogen and carbon anions involved. Ortho-lithiation of 3-bromopyridine to form 3-pyridyne in the presence of the preformed N-lithioiminodibenzyl-4-carboxaldehyde was unsuccessful in providing a pentacyclic benzonaphthyridinobenzazepine. The resulting 2- and 4-lithiated 3-bromopyridines were trapped by the aldehyde instead. Both hydroxymethylbromopyridines were identified by their proton coupling patterns in the pyridine ring. These compounds are discussed as potential precursors to pentacyclic benzazepinopyridobenzazepines. Several other attempts at forming benzonaphthyridinobenzazepines and naphthyridinophenothiazines were unsuccessful. Intermediates were obtained by carbon acylation of the dilithiated iminodibenzyl and phenothiazine with arecoline esters, arecaidine, and pyridine-3-carboxaldehyde. Dibenzylic alcohol reduction is discussed, as is its labile oxidation. None of the resulting pyridylmethyl heterocycles could be cyclized.