Systematic study on the TD-DFT calculated electronic circular dichroism spectra of chiral aromatic nitro compounds: A comparison of B3LYP and CAM-B3LYP.

B3LYP is one of the most widely used functional for the prediction of electronic circular dichroism spectra, however if the studied molecule contains aromatic nitro group computations may fail to produce reliable results. A test set of molecules of known stereochemistry were synthesized to study this phenomenon in detail. Spectra were computed by B3LYP and CAM-B3LYP functionals with 6-311++G(2d,2p) basis set. It was found that the range separated CAM-B3LYP gives better predictions than B3LYP for all test molecules. Fragment population analysis revealed that the nitro groups form highly localized molecule orbitals but the exact composition depends on the functional. CAM-B3LYP allows sufficient spatial overlap between the nitro group and distant parts of the molecule, which is necessary for the accurate description of excited states especially for charge transfer states. This phenomenon and the synthesized test molecules can be used to benchmark theoretical methods as well as to help the development of new functionals intended for spectroscopical studies.

Structure elucidation of a process-related impurity of dapoxetine.

Unknown by-product associated with the synthesis of dapoxetine was isolated. The structure elucidation of this new compound using accurate mass data and NMR spectroscopy is presented herein. The unambiguous resonance assignment concluded to the formation of a tricyclic compound 4-phenyl-2H,3H,4H-naphtho[1,2-b]pyran, a new impurity of dapoxetine which has never been reported previously. A proposed mechanism for the formation of the new carbon-carbon bond is discussed. For the separation of dapoxetine and the process-related impurities, a gradient HPLC method was developed.

Synthetic and quantum chemical study on the regioselective addition of amines to methyl maleamate.

Synthetic and theoretical studies were performed to gain insight into the regioselectivity in the mechanism of aspartyl-isoaspartyl formation, modeled by additions of ammonia and primary amines to methyl maleamate. Reactions between maleamate and aliphatic, araliphatic amines or O-methyl acetimidate lead to the formation of N-substituted isoasparaginates. The size of the amine and the activating effect of the amide and ester group on the double bond are the determining factors of the site of addition. The formation of both isomers was observed only in the case of ammonia addition. The regioselectivity was predicted on the basis of the charge distribution for low-energy methyl maleamate conformers, calculated at the B3LYP/6-311++G(2df,2pd)//B3LYP/6-31+G(d) level, both in gas phase and in methanol. The methyl isoasparaginate over methyl asparaginate product ratio was computed based on the free energy Boltzmann distribution of their conformers. The calculated 2 : 1 ratio is in agreement with the experimental regioselectivity of the addition of nitrogen nucleophiles.

Species-specific lipophilicity of thyroid hormones and their precursors in view of their membrane transport properties.

A total of 30 species-specific partition coefficients of three thyroid hormones (thyroxine, liothyronine, reverse liothyronine) and their two biological precursors (monoiodotyrosine, diiodotyrosine) are presented. The molecules were studied using combined methods of microspeciation and lipophilicity. Microspeciation was carried out by (1)H NMR-pH and UV-pH titration techniques on the title compounds and their auxiliary derivatives of reduced complexity. Partition of some of the individual microspecies was mimicked by model compounds of the closest possible similarity, then correction factors were determined and introduced. Our data show that the iodinated aromatic ring system is the definitive structural element that fundamentally determines the lipophilicity of thyroid hormones, whereas the protonation state of the aliphatic part plays a role of secondary importance. On the other hand, the lipophilicity of the precursors is highly influenced by the protonation state due to the relative lack of overwhelmingly lipophilic moieties. The different logp values of the positional isomers liothyronine and reverse liothyronine represent the importance of steric and electronic factors in lipophilicity. Our investigations provided clear indication that overall partition, the best membrane transport - predicting physico-chemical parameter depends collectively on the site-specific basicity and species-specific partition coefficient. At physiological pH these biomolecules are strongly amphipathic due to the lipophilic aromatic rings and hydrophilic amino acid side chains which can well be the reason why thyroid hormones cannot cross membranes by passive diffusion and they are constituents of biological membranes. The lipophilicity profile of thyroid hormones and their precursors are calculated and depicted in terms of species-specific lipophilicities over the entire pH range.

Thyroxine lipophilicity is dominated by its zwitterionic microspecies.

Species-specific partition coefficients were determined for a triprotic molecule for the first time. Thyroxine, the vitally important thyroid hormone which exists in solution in the forms of eight microspecies due to its phenolate, amino and carboxylate basic sites, was studied by combined methods of microspeciation and lipophilicity. Partition of the individual microspecies was mimicked by model compounds of the closest possible similarity, then correction factors were determined and introduced. The non-charged microspecies is only 2.40 times as lipophilic as its zwitterionic protonation isomer, showing that for thyroxine the iodinated aromatic rings are the structural elements that determine the lipophilicity of this molecule, and the protonation state of the other substituents plays only a minor role. The overwhelming dominance of the zwitterionic form, however, ensures that its contribution to the overall lipophilicity exceeds 14,500 times that of the non-charged one. This fact is so far the sharpest counter-example of the widespread belief that passive diffusion into lipophilic media is predominated by the non-charged species. The lipophilicity profile of thyroxine is expressed, calculated and depicted in terms of species-specific lipophilicities over the entire pH range.

Triprotic site-specific acid-base equilibria and related properties of fluoroquinolone antibacterials.

The complete macro- and microequilibrium analyses of six fluoroquinolone drugs - ciprofloxacin, enrofloxacin, norfloxacin, pefloxacin, ofloxacin and moxifloxacin - are presented. Previous controversial literature data are straightened up, the protonation centers are unambiguously identified, and the protonation macro- and microconstant values are reported. The macroconstants were determined by (1)H NMR-pH titrations while the microconstants were determined by a multi-modal spectroscopic-deductive methodology, in which methyl ester derivatives were synthesized and their NMR-pH titration data contributed to the evaluation of all the microconstants. The full (1)H, (13)C and (15)N NMR assignments, NMR-pH profiles, macro- and microprotonation schemes and species-specific diagrams are included. Our studies show that the fluoroquinolones have three protonation centers: the carboxylate group, the N-1' and N-4' piperazine nitrogens and concentration of the uncharged microspecies is way below the values published earlier. The results could be well interpreted in terms of structural properties. The protonation macro- and microconstant values allow the pre-planned method development in techniques such as capillary zone electrophoresis and also, the interpretation of fluoroquinolone mechanism of biological action, including the pharmacokinetic properties, and antibacterial activities that are all heavily influenced by the states of protonation.

Lipophilicity of zwitterions and related species: a new insight.

The experimental determination of microscopic partition coefficients for protonation isomers is elaborated for the first time, and applied for niflumic acid, an ampholytic, mainly zwitterionic drug for pains in joints and muscles. The acid-base microequilibria of niflumic acid are also characterized by NMR-pH and deductive methods using auxiliary compounds of reduced complexity. The results show that 16 times as many zwitterionic than non-charged microspecies exist in aqueous solution. Partition of the individual microspecies was mimicked by model compounds of the closest possible similarity, then correction factors were also determined and introduced. Thus the long-awaited intrinsic partition coefficients of the non-charged vs. zwitterionic species could be calculated. The non-charged microspecies is 390 times as lipophilic as its zwitterionic protonation isomer. The microscopic partition coefficients are also in line with the experimentally determined distribution coefficients. These results make evident that contribution of the zwitterionic microspecies to the overall lipophilicity is not negligible, especially at the isoelectric pH region of the compound.

Synthesis and characterization of long-chain tartaric acid diamides as novel ceramide-like compounds.

Ceramides play a crucial role in the barrier function of the skin as well as in transmembrane signaling. In this study long aliphatic chain tartaric acid diamides able to replace ceramides in an in vitro model of the stratum corneum lipid matrix due to their similar physico-chemical properties were synthesized from diacetoxysuccinic anhydride in four steps. Their pro-apoptotic effect on fibroblast cells was also investigated.

A PAMPA study of the permeability-enhancing effect of new ceramide analogues.

There is a major need in drug discovery for quick, precise, and cost-effective high-throughput screening (HTS) systems in the early stages of drug research. The Parallel Artificial Membrane Permeability Assay (PAMPA) aims at predicting the passive membrane properties of drugs. Since 1998, model membranes have been developed to predict gastro-intestinal absorption or transport through the blood-brain barrier. This paper presents recent results in a project aiming to improve the prediction of transdermal penetration. Using the PAMPA system, we investigated the effect of four newly synthetized ceramide analogues (certramides) on the permeability of three model compounds (ciprofloxacin, nifedipine, and verapamil). The certramides differ in the length of one alkyl chain, while the other alkyl chain and the head group remained the same. A relationship between the membrane concentration of certramides (from 0 to 100%) and the permeability of compounds was found, and the results of different certramides were compared. The strongest effect on permeability was caused by the ceramide analogue CTR(C12-C16). The reproducibility of the experiments and the impact of presence or absence of organic solvents (dodecane and CHCl3) in the membrane were also investigated.

Triprotic acid-base microequilibria and pharmacokinetic sequelae of cetirizine.

(1)H NMR-pH titrations of cetirizine, the widely used antihistamine and four related compounds were carried out and the related 11 macroscopic protonation constants were determined. The interactivity parameter between the two piperazine amine groups was obtained from two symmetric piperazine derivatives. Combining these two types of datasets, all the 12 microconstants and derived tautomeric constants of cetirizine were calculated. Upon this basis, the conflicting literature data of cetirizine microspeciation were clarified, and the pharmacokinetic absorption-distribution properties could be interpreted. The pH-dependent distribution of the microspecies is provided.

Proton speciation and microspeciation of serotonin and 5-hydroxytryptophan.

Protonation equilibria of the neurotransmitter serotonin and its precursor 5-hydroxytryptophan (5-HTP) are characterized at the macroscopic and microscopic levels. 1H-NMR-pH and UV-pH titrations were carried out to determine the macroconstants. Microconstants were obtained by appropriate combination of interactivity parameters and model compounds, allowing the calculation of all the twelve microconstants, the eight microspecies concentrations, and three site-interactivity parameters of 5-HTP, for which no microconstants have been reported earlier. NMR-pH Profiles, macro- and microscopic protonation schemes, and species-specific distribution diagrams are presented. The physicochemical data obtained can help to understand the steric and electronic features governing the selectivity of binding and to design new therapeutic agents.

Enantiomer separation of imidazo-quinazoline-dione derivatives on quinine carbamate-based chiral stationary phase in normal phase mode.

The normal phase mode liquid chromatographic enantiomer separation capability of a quinine tert-butyl-carbamate-type chiral stationary phase (CSP) has been investigated for a set of polar [1,5-b]-quinazoline-1,5-dione derivatives. This class of chiral heterocycles is currently under development as potential alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and/or N-methyl-D-aspartic acid (NMDA) receptor antagonists. The effect of the nature and concentration of polar modifier, i.e., ethanol and isopropanol, in n-hexane-based mobile phases, as well as the substituent pattern of the phenyl ring attached to the quinazolone framework on retention factor, enantioselectivity, and resolution was investigated. The Soczewinski competitive adsorption model was used to describe the relationship between the retention and the binary mobile phase compositions. According to this model, linear plots of the logarithms of retention factor versus molar fractions of the polar modifiers were obtained over a wide concentration range (X(B) between 0.15 and 0.35). Addition of equimolar ethanol yields higher resolution than isopropanol, R(S) values ranging between 1.54 and 2.75, whereas the latter allows to achieve moderately increased enatioselectivity. The resolution was further improved by using a ternary mixture of n-hexane:methanol:isopropanol/85:5:10 (v/v). The most pronounced selectivity factor alpha and resolution R(S) values were obtained for the para-hydroxy substituted compound, indicating that chiral recognition is sensitive to steric and stereoelectronic factors. In the course of optimization, the temperature-dependence on the chiral separation was also investigated. It turned out that the enantiomer separation is predominantly enthalpically driven in normal phase mode.

Site-specific protonation microequilibria of penicillin and cephalosporin beta-lactam core molecules.

Acid-base chemistry of 6-aminopenicillanic acid (6-APA) and 7-aminocephalosporanic acid (7-ACA), the fundamental units of the two classical antibiotic families is characterised at the macroscopic and microscopic levels. (1)H NMR-pH and pH-potentiometric titrations were combined to monitor the previously unreported extent of the site-specific protonation of 6-APA and 7-ACA. Microscopic protonation constants were derived either from direct multiple fittings of NMR-pH titration curves of adjacent carbon-bound (1)H nuclei, using in situ pH-indicator molecules, or from newly synthesised methyl ester derivatives. The results indicate that even the amino protonation of both structures occurs well below pH 5, the major protonation route includes the zwitterionic forms, and protonation at the carboxylate site brings about a three-fold diminish in the amino basicity and vice versa.

Complete resolution of the microscopic protonation equilibria of N-methyl-D-aspartic acid and related compounds.

Protonation equilibria of N-methyl-D-aspartate (NMDA, a specific glutamate receptor agonist) and its derivatives are characterized at the macroscopic and microscopic levels. (1)H NMR-pH and pH-potentiometric titrations were carried out to determine the macroconstants. Microconstants were obtained by appropriate combination of acidity and NMR parameters of the parent compound and its three synthetic derivatives. These derivatives were close models of the NMDA minor microspecies, allowing the calculation of all the 12 microconstants, the 8 microspecies concentrations and 3 site interactivity parameters. Reliability of the microconstants was assessed by three independent test methods. It was found that protonation of the secondary amino site decreases the beta- and alpha-carboxylate basicities almost exactly by one and two orders of magnitude, respectively, whereas protonation of one of the carboxylates lessens the basicity of the other one by a factor of 3. NMR-pH profiles, macro- and microscopic protonation schemes and species-specific distribution diagrams are presented.

Liquid chromatographic enantiomer separations of novel quinazolone derivatives on quinine carbamate based chiral stationary phases using hydro-organic mobile phases.

Quinine carbamate-type weak chiral anion-exchange selectors (SOs) and the respective chiral stationary phases (CSPs) have been used for the direct liquid chromatographic enantiomer separation of a wide range of chiral acids. In the present work, we demonstrate that these CSPs can also be extended to chiral discrimination of a set of neutral polar potential NMDA (N-methyl-D-aspartic acid) and/or AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) antagonist imidazo-quinazoline-dione derivatives (selectands, SAs) using acetonitrile and methanol containing hydro-organic and buffered mobile phases. The influence of mobile phase composition, column temperature and structure variation of the SAs and SOs on retention and enantioselectivity was systematically investigated to gain insight into the overall chiral recognition mechanism. As was expected for the reversed-phase mode, acetonitrile has a stronger eluotropic effect compared to methanol. Except for two analytes, the acetonitrile containing mobile phases provided baseline resolution (R(S)) of the enantiomers with R(S) values ranging between 1.68 and 2.76. Using methanol as the organic modifier enhanced the enantioselectivity. The enthalpic and entropic terms for the SO-SA association were calculated from the linear van't Hoff plots. Data reveal that the enantiomer separations are predominantly enthalpically driven.

[Enantioseparation of quinazolone derivatives using an AGP-based liquid chromatography stationary phase]. / Az AGP-alapú folyadék-kromatográfiás állófázis alkalmazása kinazolon származékok enantiomerjeinek elválasztásában.

An AGP-based chiral stationary phase has been applied successfully for enantioseparation of 4(3H)-quinazolone derivatives. Chiral-AGP is proved to be an excellent selector, as optimized chromatographic conditions allow with one exception baseline resolution for the enantiomers of the potential cholecystokinin antagonist compounds (alpha = 1.19-1.85). Retention and enantioselectivity could be modified to a large extent by varying the eluent pH and adding organic solvents with different types i.e. acetonitrile and 2-propanol to the buffered mobile phase. It was established that by increasing the eluent pH from 6.0 to 7.0 the retention factors of of the model compounds bearing no protonable groups are increased in the presence of 7 v/v % (1.33 M) acetonitrile. However further increasing the acetonitrile content up to 10 v/v % or addition of 2-propanol in equimolar concentration (1.33 M) no similar changes could be detected with the same modification in the eluent pH. These observations are explained by changes in the sorption properties of the selector determined simultaneously by the type and concentration of organic modifier and also the eluent pH. The experimental data give further insight into the chromatographic mechanism on a Chiral-AGP column.

[The multidrug resistance modulators heterocondensed quinazolones]. / Multidrug rezisztencia gátló heterokondenzált kinazolonok szintézise.

Exploration for new MDR-modulators utilizing pyrazino[2,1-b]quinazolones as scaffolds disclosed after systematic synthetic investigation highly hydrophobic N-substituted derivatives as a readily accessible active tricyclic compounds. A versatile synthesis of 2-substituted-1,2,3,4-tetrahydro-6H-pyrazino[2,1-b]quinazoline-3,6-diones is presented starting from 2,3-substituted quinazolines. The new compounds have been characterized by elemental analyses, 1H nmr and in some cases by 13C ruler, and X-ray investigations.

[Synthesis of dicarboxylic acid monoamides]. / Amidcsoport szelektív kialakítása dikarbonsavakban.

The pharmaceutical and biological importance of the amide moiety is briefly surveyed. Relationships between the electron density and chemical reactivity of the amide site are shortly described. Synthetic methods for the selective formation of monoamino-dicarboxylic acid alpha and beta monoamides are summarised. The three major selective synthesis routes for the preparation of monoamides are introduced. The first class of synthetic methods consists of non selective formation of a- and b-esters, followed by their separation on the basis of different solubilities. Amidation of these esters results in the alpha- or beta-amides respectively. The second class of the reactions utilises the orientating capacity of the amino (ammonium) site, producing first various cyclic anhydrides, lactames, lactones, which are then decomposed in hydrolytic, aminolytic, etc. reactions, resulting selectively in the alpha- or beta-amides or esters. Reactants in the third class of the reactions are dicarboxylic acids with carbon-carbon pi-bonds, and ammonia (or alkyl-amine) which form the appropriate compound in addition reactions. Reactivities and selectivities are interpreted in terms of inductive effects, acidity differences and electronic effects of the various protecting groups. Some important analytical properties of monoamino-dicarboxylic acids and their monoamides are compiled.

[EFfect of quinazolone-alkyl-carboxylic acid derivatives on the transmembrane Ca2+ ion flux mediated by AMPA receptors]. / Kinazolon-alkil-karbonsav származékok hatása az alpha-amino-3-hidroxi-5-metil-4-izoxazol propionsav (AMPA) receptor által szabályozott transzmembrán Ca2+ ion fluxusra.

The excitatory neurotransmitter, Glu, plays a crucial role in many sensory and motor functions as well as in brain development, learning and memory and it is also involved in the pathogenesis of a number of neurological disorders, including epilepsy, Alzheimer's and Parkinson's diseases. Therefore, the study of Glu receptors (GluRs) is of therapeutical importance. We showed here by fluorescence monitoring of transmembrane Ca2+ ion fluxes in response to (S)-alpha-amino-3-hidroxi-5-metil-4-izoxazol propionic acid ((S)-AMPA) on the time scale of 0.00004-10 s that Ca2+ ion influx proceeds through faster and slower desensitizing receptors. Pharmacological isolation of the slower and faster desensitizing AMPA receptor was possible by fluorescence monitoring of Ca2+ ion translocation in response to (S)-AMPA in the presence and absence of various 2-methyl-4-oxo-3H-quinazoline-3-alkyl-carboxilic acid derivatives (Qxs): the acetic acid Q1 inhibits the slower desensitizing receptor response specifically, while the acetyl-piperidine Q5 is a more potent inhibitor of the faster desensitizing receptor response. In addition, spontaneous interictal activity, as induced by high [K+] conditions in hippocampal slices, was reduced significantly by Q5, suggesting a possible anticonvulsant property of Q5. Substitutions of Qxs into the GluR2 S1S2 binding core were consistent with their effect by causing variable degree of S1S2 bridging interaction as one of the main determinants of AMPA receptor agonist activity. The exploitation of differences between similar receptors will be important in the development and use of drugs with high pharmacological specificity.