Identifying barbiturate binding sites in a nicotinic acetylcholine receptor with [3H]allyl m-trifluoromethyldiazirine mephobarbital, a photoreactive barbiturate.

At concentrations that produce anesthesia, many barbituric acid derivatives act as positive allosteric modulators of inhibitory GABAA receptors (GABAARs) and inhibitors of excitatory nicotinic acetylcholine receptors (nAChRs). Recent research on [(3)H]R-mTFD-MPAB ([(3)H]R-5-allyl-1-methyl-5-(m-trifluoromethyldiazirinylphenyl)barbituric acid), a photoreactive barbiturate that is a potent and stereoselective anesthetic and GABAAR potentiator, has identified a second class of intersubunit binding sites for general anesthetics in the α1ß3γ2 GABAAR transmembrane domain. We now characterize mTFD-MPAB interactions with the Torpedo (muscle-type) nAChR. For nAChRs expressed in Xenopus oocytes, S- and R-mTFD-MPAB inhibited ACh-induced currents with IC50 values of 5 and 10 µM, respectively. Racemic mTFD-MPAB enhanced the equilibrium binding of [(3)H]ACh to nAChR-rich membranes (EC50 = 9 µM) and inhibited binding of the ion channel blocker [(3)H]tenocyclidine in the nAChR desensitized and resting states with IC50 values of 2 and 170 µM, respectively. Photoaffinity labeling identified two binding sites for [(3)H]R-mTFD-MPAB in the nAChR transmembrane domain: 1) a site within the ion channel, identified by photolabeling in the nAChR desensitized state of amino acids within the M2 helices of each nAChR subunit; and 2) a site at the γ-α subunit interface, identified by photolabeling of γMet299 within the γM3 helix at similar efficiency in the resting and desensitized states. These results establish that mTFD-MPAB is a potent nAChR inhibitor that binds in the ion channel preferentially in the desensitized state and binds with lower affinity to a site at the γ-α subunit interface where etomidate analogs bind that act as positive and negative nAChR modulators.

How did phenobarbital's chemical structure affect the development of subsequent antiepileptic drugs (AEDs)?

Phenobarbital has been in clinical use as an antiepileptic drug (AED) since 1912. The initial clinical success of phenobarbital and other barbiturates affected the design of subsequent AEDs (e.g., phenytoin, primidone, ethosuximide), developed between 1938 and 1962, the chemical structures of which resemble that of phenobarbital. However, the empirical discovery of carbamazepine (1962) and the serendipitous discovery of valproic acid (1967) led to subsequent AEDs having chemical structures that are diverse and completely different from that of phenobarbital. Sixteen AEDs were introduced between 1990 and 2012. Most of these AEDs were developed empirically, using mechanism-unbiased anticonvulsant animal models. The empirical nature of the discovery of these AEDs, coupled with their multiple mechanisms of action, explains their diverse chemical structures. The antiepileptic market is therefore crowded. Future design of new AEDs must have a potential for treating nonepileptic central nervous system (CNS) disorders (e.g., bipolar disorder, neuropathic pain, migraine prophylaxis, or restless legs syndrome). The barbiturates were once used as sedative-hypnotic drugs, but have been largely replaced in this role by the much safer benzodiazepines. In contrast, phenobarbital is still used worldwide in epilepsy. Nevertheless, the development of nonsedating phenobarbital derivatives will answer a clinical unmet need and might make this old AED more attractive.

Allyl m-trifluoromethyldiazirine mephobarbital: an unusually potent enantioselective and photoreactive barbiturate general anesthetic.

We synthesized 5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (14), a trifluoromethyldiazirine-containing derivative of general anesthetic mephobarbital, separated the racemic mixture into enantiomers by chiral chromatography, and determined the configuration of the (+)-enantiomer as S by X-ray crystallography. Additionally, we obtained the (3)H-labeled ligand with high specific radioactivity. R-(-)-14 is an order of magnitude more potent than the most potent clinically used barbiturate, thiopental, and its general anesthetic EC(50) approaches those for propofol and etomidate, whereas S-(+)-14 is 10-fold less potent. Furthermore, at concentrations close to its anesthetic potency, R-(-)-14 both potentiated GABA-induced currents and increased the affinity for the agonist muscimol in human α1ß2/3γ2L GABA(A) receptors. Finally, R-(-)-14 was found to be an exceptionally efficient photolabeling reagent, incorporating into both α1 and ß3 subunits of human α1ß3 GABA(A) receptors. These results indicate R-(-)-14 is a functional general anesthetic that is well-suited for identifying barbiturate binding sites on Cys-loop receptors.

The influence of sulfur substituents on the molecular geometry and packing of thio derivatives of N-methylphenobarbital.

The room-temperature crystal structures of four new thio derivatives of N-methylphenobarbital [systematic name: 5-ethyl-1-methyl-5-phenylpyrimidine-2,4,6(1H,3H,5H)-trione], C(13)H(14)N(2)O(3), are compared with the structure of the parent compound. The sulfur substituents in N-methyl-2-thiophenobarbital [5-ethyl-1-methyl-5-phenyl-2-thioxo-1,2-dihydropyrimidine-4,6(3H,5H)-dione], C(13)H(14)N(2)O(2)S, N-methyl-4-thiophenobarbital [5-ethyl-1-methyl-5-phenyl-4-thioxo-3,4-dihydropyrimidine-2,6(1H,5H)-dione], C(13)H(14)N(2)O(2)S, and N-methyl-2,4,6-trithiophenobarbital [5-ethyl-1-methyl-5-phenylpyrimidine-2,4,6(1H,3H,5H)-trithione], C(13)H(14)N(2)S(3), preserve the heterocyclic ring puckering observed for N-methylphenobarbital (a half-chair conformation), whereas in N-methyl-2,4-dithiophenobarbital [5-ethyl-1-methyl-5-phenyl-2,4-dithioxo-1,2,3,4-tetrahydropyrimidine-6(5H)-one], C(13)H(14)N(2)OS(2), significant flattening of the ring was detected. The number and positions of the sulfur substituents influence the packing and hydrogen-bonding patterns of the derivatives. In the cases of the 2-thio, 4-thio and 2,4,6-trithio derivatives, there is a preference for the formation of a ring motif of the R(2)(2)(8) type, which is also a characteristic of N-methylphenobarbital, whereas a C(6) chain forms in the 2,4-dithio derivative. The preferences for hydrogen-bond formation, which follow the sequence of acceptor position 4 > 2 > 6, confirm the differences in the nucleophilic properties of the C atoms of the heterocyclic ring and are consistent with the course of N-methylphenobarbital thionation reactions.

Monolithic silica columns with chemically bonded beta-cyclodextrin as a stationary phase for enantiomer separations of chiral pharmaceuticals.

An enantioselective silica rod type chiral stationary phase (CSP) is presented; a novel combination of the well known enantiomer separation properties of beta-cyclodextrin and the unique properties concerning the flow behavior of silica monoliths. Two different synthesis routes are described, and it was found that the in situ modification of a plain silica rod column turned out to be the best. The chromatographic behaviour of the beta-cyclodextrin silica rod was studied and compared with a very similar commercially available beta-cyclodextrin bonded particulate material (ChiraDex). Even if the amount of beta-cyclodextrin bound to the silica rod was only about half of the amount of beta-cyclodextrin bound to ChiraDex) particles, good resolutions were achieved for a set of chiral test components like Chromakalin, Prominal, Oxazepam, Methadone and some other drugs. By taking advantage of the unique features of the silica rods relating to their flat H/u (Van Deemter) curves, fast enantiomer separations could be demonstrated.

Determination of two ternary mixtures containing phenobarbitone by second derivative of the ratio spectrum-zero-crossing and HPLC methods.

A spectrophotometric method is developed for the determination of ternary mixtures with overlapping spectra. The method is based on the use of the second derivative of the ratio spectrum with a zero-crossing technique. The ratio spectrum was obtained by dividing the absorption spectrum of the mixture by that of one of the components. The concentration of the other components are then determined from their respective calibration graphs treated similarly. The method is accurate, non-destructive and do not require resolutions of equations. The method has been applied for the resolution of two ternary mixtures, namely, phenobarbitone, methylphenobarbitone and phenytoin (1), and phenobarbitone, papaverine HCl and piperazine acefyllinate (2). Also, a HPLC method was developed for determination of phenobarbitone, papaverine and HCL and piperazine acefyllinate. The HPLC method depends upon using ODS column with a mobile phase consisting of acetonitrile-5 mM aqueous heptane sulfonic acid sodium salt (50:50, v/v) and adjusted to apparent pH 4 using acetic acid. Quantitation was achieved with UV detection at 220 nm based on peak area. The proposed methods were applied for the determination of the two ternary combinations in synthetic mixtures and in commercial pharmaceutical products. The results obtained were precise and accurate.

Prediction of adsorption from multicomponent solutions by activated carbon using single-solute parameters. Part II--Proposed equation.

Prediction of multicomponent adsorption is still one of the most challenging problems in the adsorption field. Many models have been proposed and employed to obtain multicomponent isotherms from single-component equilibrium data. However, most of these models were based on either unrealistic assumptions or on empirical equations with no apparent definition. The purpose of this investigation was to develop a multicomponent adsorption model based on a thermodynamically consistent equation, and to validate that model using experimental data. Three barbiturates--phenobarbital, mephobarbital, and primidone--were combined to form a ternary system. The adsorption of these barbiturates from simulated intestinal fluid (without pancreatin) by activated carbon was studied using the rotating bottle method. The concentrations, both before and after the attainment of equilibrium, were determined with a high-performance liquid chromatography system employing a reversed-phase column. The proposed equation and the competitive Langmuir-like equation were both fit to the data. A very good correlation was obtained between the experimental data and the calculated data using the proposed equation. The results obtained from the original competitive Langmuir-like model were less satisfactory. These results suggest that the proposed equation can successfully predict the trisolute isotherms of the barbituric acid derivatives employed in this study.

Prediction of adsorption from multicomponent solutions by activated carbon using single-solute parameters.

The adsorption of 3 barbiturates--phenobarbital, mephobarbital, and primidone--from simulated intestinal fluid (SIF), without pancreatin, by activated carbon was studied using the rotating bottle method. The concentrations of each drug remaining in solution at equilibrium were determined with the aid of a high-performance liquid chromatography (HPLC) system employing a reversed-phase column. The competitive Langmuir-like model, the modified competitive Langmuir-like model, and the LeVan-Vermeulen model were each fit to the data. Excellent agreement was obtained between the experimental and predicted data using the modified competitive Langmuir-like model and the LeVan-Vermeulen model. The agreement obtained from the original competitive Langmuir-like model was less satisfactory. These observations are not surprising because the competitive Langmuir-like model assumes that the capacities of the adsorbates are equal, while the other 2 models take into account the differences in the capacities of the components. The results of these studies indicate that the adsorbates employed are competing for the same binding sites on the activated carbon surface. The results also demonstrate that it is possible to accurately predict multicomponent adsorption isotherms using only single-solute isotherm parameters. Such prediction is likely to be useful for improving in vivo/in vitro correlations.

Chiral separations of enantiomeric pharmaceuticals by capillary electrophoresis using sulphobutyl ether beta-cyclodextrin as isomer selector.

Capillary electrophoresis has developed into an extremely useful technique for the separation of optical isomers. High efficiencies and the availability of many types of isomer selectors allowing rapid and inexpensive methods development make capillary electrophoresis (CE) an attractive alternative to gas chromatography (GC) and high-pressure liquid chromatography (HPLC) for the determination of chiral purity. In this research the separation of the enantiomers of some chiral pharmaceuticals was investigated using anionic sulphobutyl ether-beta-cyclodextrins as isomer selectors. These chiral selectors have a large countercurrent mobility, making them inherently advantageous as selectors as compared to neutral cyclodextrins. The effects of pH, buffer composition and selector concentration on the chiral separation of these compounds was investigated. All of the compounds studied were successfully resolved by the sulphobutyl ether beta-cyclodextrins (SBE-beta-CDs) typically with run times of less than 20 min using low concentrations of the SBE selector (1-5 mM).

Synthesis of N-beta-D-glucopyranosyluronate derivatives of barbital, phenobarbital, metharbital, and mephobarbital.

The synthesis and characterization of barbital, phenobarbital, metharbital, and mephobarbital glucuronides is reported. The condensation of per(trimethylsilyl)-barbital and -phenobarbital with methyl 1,2,3,4-tetra-O-acetyl-beta-D-glucopyranuronate in the presence of trimethylsilyl trifluoromethanesulfonate gave moderate yields of the N1-(beta-D-glucopyranosyluronate) barbiturate derivatives. The diastereomers of the phenobarbital derivatives were resolved by use of C18 reversed-phase HPLC. The homologous N3-methyl barbiturate N1-glucuronates were prepared by reaction of the barbital and phenobarbital N1-glucuronate derivatives with diazomethane. The absolute configuration of the phenobarbital N1-beta-D-glucopyranuronate epimers was determined by oxidative removal of the glycon from the mephobarbital N1-beta-D-glucopyranuronate epimers to give the optical isomers of mephobarbital. The spectroscopic data for this series of compounds will facilitate the characterization of N-glycosylated imide xenobiotics that may be detected as mammalian metabolites in biodisposition studies.

Molecular modelling of the inclusion complexes between beta-cyclodextrin and (R)/(S)-methylphenobarbitone and its application to HPLC.

Molecular modelling of beta-cyclodextrin and optimisation of its potential energy suggests that a favoured conformation is that distorted from a symmetrical torus. The inclusion of water molecules into the torus cavity simulates the increased stability in an aqueous solvent. Complexes of beta-cyclodextrin with (R)- and (S)-enantiomers of methylphenobarbitone have been modelled and energetically optimised by the application of molecular mechanics. The simulations suggests that the guest molecules adopt an orientation in which the phenyl ring is projected into the torus cavity, with in each case the plane of the ring parallel to a longer axis of the distorted torus and slightly displaced from the axis through the torus cavity. It is suggested that the asymmetry in the macrocyclic ring contributes to chiral recognition as a result of additional discriminatory binding to the barbiturate ring residue of each enantiomer, which occupy different 3D geometries. The enantiomers form complexes of different minimum potential energies. The resulting difference in complex stability can be related to the behaviour of beta-cyclodextrin, as a mobile phase additive in reverse-phase HPLC to effect chiral separation of rac-methylphenobarbitone during chromatography.