Separation of 1,4-benzodiazepines by micellar elektrokinetic capillary chromatography

In this work the applicability of micellar elektrokinetic capillary chromatography (MECC) for the determination of benzodiazepines (BZD) has been studied. The applied method was used for the simultaneous separation of 8 BZDs (alprazolam, bromazepam, chlordiazepoxide, diazepam, flunitrazepam, medazepam, oxazepam, nitrazepam), and also for the study of stability in acidic medium. A fast and reliable method has been developed; using a separation buffer composed of sodium tetraborate 25 mM (pH 9.5), SDS (50 mM) and methanol (at least 12%) as an organic modifier.     

Copper-psychoactive drug complexes: a voltammetric approach to complexation by 1,4-benzodiazepines

Copper complexation by the 1,4-benzodiazepines medazepam, diazepam, flurazepam, nitrazepam, and clonazepam was investigated using differential pulse polarography and cyclic voltammetry at a mercury electrode in 0.10 M KNO3 and pH 7.0 +/- 0.1. Because the 1,4-benzodiazepines are easily reduced at a mercury electrode through the two-electron reduction of the 4,5-azomethine functional group, copper reduction, as well as that of the ligands, was analyzed under varying experimental conditions. In most situations adsorption phenomena occurred and their influence on voltammetric signals had to be carefully analyzed. The voltammetric behavior was then interpreted in terms of complex formation. The results showed that all benzodiazepines can act as ligands toward copper(II) ions, forming 1:1 and 1:2 complexes with similar stabilities. The stoichiometric acidity constants of the benzodiazepines under study were also determined by potentiometric titration in water-ethanol medium and 0.10 M KNO3 and then extrapolated to 0% concentration of ethanol.     

Substitution and racemization of 3-hydroxy- and 3-alkoxy-1,4-benzodiazepines in acidic aqueous solutions

Oxazepam (OX), 3-O-methyloxazepam, 3-O-ethyloxazepam, temazepam (TMZ), 3-O-methyltemazepam, and 3-O-ethyltemazepam underwent acid-catalyzed nucleophilic substitution reaction (hydrolysis) in an acetonitrile–oxygen-18 water mixture to form either OX or TMZ in which the 3-hydroxyl group was either partially or fully labeled with an oxygen-18 atom. The dependence of the hydrolysis rates on solvent composition, temperature, ionic strength, and in deuterated solvent was studied by reversed-phase high-performance liquid chromatography (HPLC). The rates of racemization of enantiomeric compounds in acidic aqueous solutions were studied by both spectropolarimetry and chiral stationary phase HPLC. In acetonitrile: 2.5 M H₂SO₄ (4:1, v/v) at 50°C, enantiomers of OX and TMZ underwent racemization at rates ≥40-fold faster than the rates of hydrolysis. Enantiomeric 3-O-alkyl derivatives of OX and TMZ in acidic aqueous solutions did not themselves undergo racemization and it was their hydrolysis products (either OX or TMZ) that underwent racemization. © 1995 Wiley-Liss, Inc.     

Investigation into mechanisms mediating the inhibitory effect of 1,4-benzodiazepines on mast cells by gene expression profiling

AimsThis study aims to identify by a molecular genetic approach potential targets in mast cells at which 1,4-benzodiazepines may cause their inhibitory effect on mast cell activity.Main methodsGene expression analyses with microarray gene chip and/or quantitative PCR were performed using 1,4-benzodiazepine-treated human mast cell leukemia HMC-1.2 cells, promyelocytic leukemia HL-60 cells and human mast cells from healthy volunteers and patients with mast cell activation disease (MCAD). Pathway analysis was applied to search for enriched biological functions and canonical pathways within differentially regulated genes.Key findingsBoth neoplastic and normal human mast cells express several GABA_A receptor subunits at the mRNA level. In mast cells from MCAD patients expression of some GABA_A receptor subunits and expression of the translocator protein TSPO are increased compared with those from healthy controls. Expression of the protein tyrosine kinases Lyn, Fgr and Yes1 was increased in HMC-1.2 cells as compared with the ontogenetically related HL60 cells. Differences in gene regulation in HMC-1.2 cells after treatment with the 1,4-benzodiazepines clonazepam, flunitrazepam and 4-chlorodiazepam suggested that signaling and gene expression induced by clonazepam was similar to that of flunitrazepam but different from that of 4-chlorodiazepam. This conclusion is supported by the results of the pathway analysis.SignificanceA novel type of GABA_A receptors on mast cells appears to be involved in the inhibition of mast cell activity by 1,4-benzodiazepines. These receptors seem to be composed without γ subunits suggesting unique pharmacological properties. An action at Src-kinases, or at TSPO located in the plasma membrane may also be involved.     

Determination of 1,4- and 1,5-benzodiazepines in urine using a computerized gas chromatographic—mass spectrometric technique

A method for the determination of benzodiazepines and their main metabolites in urine after acid hydrolysis is described. The extract is analyzed by computerized gas chromatography—mass spectrometry. An on-line computer allows rapid detection using mass fragmentography with the masses m/e 211, 230, 241, 244, 249, 262, 276, and 285. The mass fragmentogram and the underlying mass spectra of the hydrolysis products (benzophenones and analogues) are documented.     

C5-piperazinyl-1,4-benzodiazepines, water-soluble, orally bioa vailable CCKB/gastrin receptor antagonists

A novel series of potent, water-soluble benzodiazepine based CCK_B/gastrin antagonists has been prepared which incorporate an N-methylpiperazine group at the C5 position of the benzodiazepine ring system. The N1-n-propyl analogue (7b) is a high affinity, selective and potent receptor antagonist in vitro, with good bioavailability and excellent oral absorption providing high plasma levels in vivo.     

Chromatographic separation of 2,3-benzodiazepines

A review of chromatographic methods for the determination of 2,3-benzodiazepines (2,3-BZs) is presented. The determinations are performed to investigate the presence of potential impurities in drug substances and to study their pharmacokinetic profile in biological samples, either in animals or in humans. Several methods dealt with a pretreatment of samples, i.e., liquid–liquid extraction by using a variety of solvents, solid-phase extraction, direct injection of specimens into the chromatographic apparatus. Different chromatographic techniques have been used. High-performance liquid chromatography allows optimal sensitivity and specificity by using ultraviolet or diode array detection methods. Gas chromatography-mass spectrometry and gas chromatography with nitrogen-phosphorous or electron-capture detectors have been also reported. Suitable methods for the separation of enantiomers of 2,3-BZs have been described. Thin-layer chromatography has been shown to be capable to isolate analytes from biological samples as urine or faeces. The reported chromatographic techniques are currently applied to define the metabolic pathways of 2,3-BZs in experimental and clinical studies.     

Benzodiazepine compounds as inhibitors of the src protein tyrosine kinase: screening of a combinatorial library of 1,4-benzodiazepines.

We screened 1680 spatially separated compounds of a diverse combinatorial library of 1,4-benzodiazepines for their ability to inhibit the kinase activity of protein tyrosine kinases Src, Yes, Abl, Lck, Csk, and fibroblast growth factor receptor. This screening yielded novel ligands for the protein tyrosine kinase Src. In the 1, 4-benzodiazepine-2-one scaffold, the preferred substituent at position R(1) was 4-hydroxyphenylmethyl or a 3-indolemethyl derived from a tyrosine or tyrptophan used in building the benzodiazepine, while the substituent at R(2), introduced by alkylating agents, was preferably aromatic in nature. The preferred ring structure introduced on the bicyclic ring of the scaffold by acid chlorides was a p-hydroxy phenyl group. The lead compound, designated as N-L-Yaa, has a L-4-hydroxyphenylmethyl ring at R(1) and a biphenylmethyl substituent at R(2). The compound has an IC(50) of 73 microM against Src, 2- to 6-fold lower than against other protein tyrosine kinases and >10-fold lower than against other nucleotide-utilizing enzymes. The mechanism of binding of N-L-Yaa to Src is mixed against the peptidic substrate with a K(i) of 35 microM and noncompetitive against ATP-Mg with a K(i) of 17 microM. Multiple inhibition analysis of the lead compound in the presence of other competitive inhibitors demonstrated that the binding of the lead compound is nonexclusive to the other competitive inhibitor. The inhibitor was found to be nontoxic to the AFB-13-human fibroblasts cells and inhibited the colony formation of HT-29 colon adenocarcinoma cells that are dependent on Src activity.     

Up-regulation of L-type high voltage-gated calcium channel subunits by sustained exposure to 1,4- and 1,5-benzodiazepines in cerebrocortical neurons

The aim of this study is to examine how sustained exposure to two 1,4-benzodiazepines (BZDs) with different action period, diazepam and brotizolam, and a 1,5-BZD, clobazam, affects L-type high voltage-gated calcium channel (HVCC) functions and its mechanisms using primary cultures of mouse cerebral cortical neurons. The sustained exposure to these three BZDs increased [⁴⁵Ca²⁺] influx, which was due to the enhanced [⁴⁵Ca²⁺] entry through L-type HVCCs but not through of Cav2.1 and Cav2.2. Increase in [³H]diltiazem binding after the exposure to these three BZDs was due to the increase in the binding sites of [³H]diltiazem. Western blot analysis showed increase of Cav1.2 and Cav1.3 in association with the increased expression of α2/δ1 subunit. Similar changes in [³H]diltiazem binding and L-type HVCC subunit expression were found in the cerebral cortex from mouse with BZD physical dependence. These results indicate that BZDs examined here have the potential to increase L-type HVCC functions mediated via the enhanced expression of not only Cav1.2 and Cav1.3 but also α2/δ1 subunit after their sustained exposure, which may participate in the development of physical dependence by these BZDs.     

Clozapine derived 2,3-dihydro-1H-1,4- and 1,5-benzodiazepines with D4 receptor selectivity: synthesis and biological testing

Novel 4-arylpiperazin-1-yl-substituted 2,3-dihydro-1H-1,4- and 1H-1,5-benzodiazepines and their aza-analogues were synthesized as debenzoclozapine derivatives for evaluation as potential D4-ligands. While Ki values of some of the title compounds came within the range of clozapine, they showed an impressively greater selectivity over other dopamine receptor subtypes, especially D2. For the most promising compounds, intrinsic activity and binding properties to serotonin 5-HT1A and 5-HT2 were also determined.     

A classic compilation

Kurtén, B. 1988: On Evolution and Fossil Mammals . XVII + 301 pp. Columbia University Press. ISBN 0–231–05868–3. Price: USD40.00 (bound).     

Design and discovery of 1,3-benzodiazepines as novel dopamine antagonists

A series of novel 1,3-benzodiazapine based D1 antagonists was designed according to the understanding of pharmacophore models derived from SCH 23390 (1b), a potent and selective D1 antagonist. The new design features an achiral cyclic-amidine that maintains desired basicity. Solid phase synthesis was developed for SAR development of the novel dopamine antagonists.     

Enantiomers of 2-[(Acylamino)ethyl]-1,4-benzodiazepines, potent ligands of kappa-opioid receptor: chiral chromatographic resolution, configurational assignment and biological activity

Compounds 2a and 3a-e are racemic 2-[(acylamino)ethyl]-1,4-benzodiazepines, tifluadom analogs, with high affinity and selectivity towards the kappa-opioid receptor. We describe the enantiomeric separation of all compounds through liquid chromatography with chiral stationary phases, as well as the resolution of the enantiomers of the most interesting compounds, 2a and 3a, by the semipreparative column Chiralpak AD. The configuration of the resolved enantiomers was investigated: the comparative study of CD and (1)H NMR spectra shows that compounds (-)-2a and (-)-3a have the same absolute configuration of (+)-(S)-tifluadom. A study on the stereoselective interaction with opiate receptors is reported.     

New 7,8-ethylenedioxy-2,3-benzodiazepines as noncompetitive AMPA receptor antagonists

A series of 1-aryl-3,5-dihydro-7,8-ethylenedioxy-4H-2,3-benzodiazepin-4-ones 2a-f, were synthesized and screened as anticonvulsant agents in DBA/2 mice against sound-induced seizures. The new compounds display anticonvulsant properties although the ED(50) values are higher than those of prototypes 1-aryl-3,5-dihydro-7,8-methylenedioxy-4H-2,3-benzodiazepin-4-ones (1) and GYKI 52466, well-known noncompetitive AMPA receptor antagonists. Functional tests were performed to evaluate the antagonistic activity at the AMPA and kainate receptors.     

Simple and sensitive high-performance liquid chromatographic method for the determination of 1,5-benzodiazepine clobazam and its active metabolite N-desmethylclobazam in human serum and urine with application to 1,4-benzodiazepines analysis

A HPLC–UV determination of clobazam and N-desmethylclobazam in human serum and urine is presented. After simple liquid–liquid extraction with dichloromethane the compounds and an internal standard diazepam were separated on a Supelcosil LC-8-DB column at ambient temperature under isocratic conditions using the mobile phase: CH₃CN–water–0.5 M KH₂PO₄–H₃PO₄ (440:540:20:0.4, v/v and 360:580:60:0.4, v/v for serum and urine, respectively). The detection was performed at 228 nm with limits of quantification of 2 ng/ml for serum and 1 ng/ml for urine. Relative standard deviations for intra- and inter-assay precision were found below 8% for both compounds for all the tested concentrations. The described procedure may be easily adapted for several 1,4-benzodiazepines.     

Synthesis of 1,4-anhydro-D-fructose and 1,4-anhydro-D-tagatose

1,4-Anhydro-D-fructose and 1,4-anhydro-D-tagatose were prepared from 1,2-O-isopropylidene-D-glucofuranose via the common intermediate 3,5,6-tri-O-benzyl-D-glucitol. The title compounds may be interesting anti-oxidants and feature activities akin to their natural pyranoid counterpart, 1,5-anhydro-D-fructose.