Miklós Idei

Capillary Electrophoretic Analysis of Somatostatin Analog Peptides. Effect of Organic Solvents as Buffer Modifiers

Abstract Capillary electrophoretic (CE) method utilizing triethylammonium phosphate/organic modifier solvents as CE buffer was developed for analysis of new proprietary Somatostatin analog peptides synthetized in our laboratory. Acetonitrile, methanol, ethanol and i-propanol were applied as organic modifiers. Applicability of the systems in the analysis of Somatostatin analog peptides was evaluated and optimum conditions of the separation were determined. The effects of the organic solvents on the migration times of the S-analog peptides, elution order and selectivity in the CE systems were investigated. The migration time, elution order and selectivity can be influenced modifying the composition of the electrophoretic buffer with organic solvents. Applying different organic solvents as modifiers the elution order of the peptides can be changed in different ways. The effect of the alcohols on the CE migration time of the peptides is quite opposite to that of the acetonitrile. The degree of the effect of t...

Comparison of high-performance liquid chromatography and capillary electrophoresis in the analysis of somatostatin analogue peptides

HPLC and CE methods were developed for analysis of somatostatin analogue (S-analogue) peptides utilizing triethylammonium phosphate-organic solvent modifier solvents as the CE buffer and HPLC eluent. Acetonitrile, methanol, ethanol and 2-propanol were applied as organic modifiers. The applicability of HPLC and CE systems was evaluated and compared. Optimum conditions for the separation were determined for both methods. Retention (migration) time, elution order and selectivity can be influenced by modifying the composition of the eluent (buffer) with organic solvents not only in HPLC but also in CE. Although the HPLC system reacted to changes in the organic solvent concentration in a much more sensitive way than the CE system did (from the point of view of retention time), CE proved to be a more suitable method for separating the peptides investigated. Baseline separation could be achieved within 6-9 min by CE, a result which was impossible to achieve with HPLC working in the isocratic mode. In CE the effect of the alcohols on migration times proved to be opposite to that of acetonitrile. Whereas ACN decreased, the alcohols increased the migration times in a concentration-dependent way. The results suggest that CE can be applied very advantageously in peptide analysis. Its performance regarding selectivity, resolution, theoretical plate number, duration and cost is comparable or sometimes superior to that of HPLC.

New antitumor leads from a peptidomimetic library

A parallel combinatorial library of over 1600 compounds has been designed and synthesized for the development of new potential peptidomimetic protein tyrosine kinase (PTK) inhibitor leads. These peptidomimetic molecules are aimed at intervening with the substrate binding site of the pp60c−src enzyme. The new structures were based on kown PTK inhibtors with at least two variously substituted aromatic moieties attached by spacer groups of different length and flexibility. Eleven bis-aryl-type inhibitory compounds were found in the range of 18–100 μM IC50 concentrations from combinations of 12 different substituents. Molecular modeling of the active compounds showed a characteristic distance of 12–14 Å between the farthest sp2 carbon atoms of the two aromatic rings. Conformational analysis of several peptide substrates recently found for pp60c−src PTK showed that the energyminimized conformers had the same distance between the two aromatic moieties. Several compounds in the library not only showed remarkable PTK inhibitory activity but also a significant apoptosis-inducing effect on HT-29 human colon tumor cells.

EVALUATION OF HYDROPHOBICITY AND ANTITUMOR ACTIVITY OF A MOLECULE LIBRARY OF MANNICH KETONES OF CYCLOALKANONES

A series of Mannich ketones of cycloalkanones were synthesized to study the relative importance of structure and specific substitutions in relation to their hydrophobicity and antitumor activity. Substitutions were carried out with morpholinyl, pirrolidinyl, piperidyl, and tetrahydro-isoquinolyl groups in various position on four different base structures. Hydrophobicity of Mannich ketones was characterized by chromatography data (log k′) and by software calculated parameters (c log P). Cell proliferation inhibitory activity of cycloalkanones was evaluated by MTT and apoptosis assays on A431 human adenocarcinoma cells. Our results suggest that the higher the hydrophobicity values (log k′ and c log P) the higher the antitumor- and apoptotic activity of Mannich ketones. Determination of hydrophobicity by measuring the log k′ or by calculating the c log P values of the compounds may help to predict their biological activities.

Comparative Characterization of Experimental and Calculated Lipophilicity and Anti-Tumour Activity of Isochromanone Derivatives

Compound lipophilicity connected to ADME(T)(a) has great importance in drug development and it has to be evaluated by the generally used drug developmental process. In addition to the importance of lipophilicity in ADMET, recently it has been reported that lipophilicity of small molecules correlates with their antiproliferative activity because of certain specific hydrophobic and lipophilic interactions. Due to the complexity of ADME(T) parameters an efficient and fast method is needed to characterize the many promising candidate lead molecules as a preselection in order not to be rejected from the latter phase of drug development. In the present paper we provide an overview of the importance of lipophilicity of drug candidates for biological action and for ADME(T) and describe a novel approach for drug-likeness characterization of a molecular library using correlation study between lipophilicity and biological activity. Lipophilicity and molecular characteristics have been measured, predicted and optimized for a diverse library from which the best members have been selected to describe their biological, chemical and drug-likeness properties. Molecules were selected from the family of alpha,beta-unsaturated ketones and thorough HPLC characterization for lipophilicity and morphological, antiproliferative and flow cytometric studies were carried out on them. Based on the results 17 member isochromanone library including E and Z geometric isomers were selected for further characterization. In this focused library linear correlation has been found between the calculated and measured lipophilicity and significant parabolic correlation was found between the antiproliferative effect and lipophilicity. Using our efficient and fast method, from a diverse library, we identified an outstandingly effective inhibitor of A431 tumour cell growth via a PARP(a) cleavage dependent apoptosis. In summary the optimized HPLC analyses of lipophilicity combined with the cell-culture assay, introduced above, resulted in the determination of an optimal lipophilicity range. This optimized lipophilicity range should be used in designing novel antiproliferative compounds.

A NOVEL NORMALIZED RETENTION FACTOR IN MICELLAR ELECTROKINETIC CHROMATOGRAPHY

Characteristic properties of the expression k″ = (tm—to)/(tmc—to) and its applicability in micellar electrokinetic capillary chromatography (MEKC) were compared to the previous expression, k′ = (tm—to)/to(1‐tm/tmc), introduced by Terabe. It was proved with theoretical calculations (curve shape analysis) that the properties of function k″ are in full accordance with the properties of the MEKC system and k″ could be applied advantageously to characterize hydrophobicity of the analytes. This conclusion is now supported by experimental data obtained with homolog series of alkylbenzenes and alkylphenones as well as with hydrophobic protected peptides. Migration times, k′, k″ values, and software‐calculated hydrophobicity data are summarized and analyzed in the present study. Since k″ is a normalized parameter, good relationships between the migration time, the software‐calculated hydrophobicity, and the k″ values were obtained. Differences in hydrophobicity of the analytes could be estimated in a more realistic way with the aid of function k″ than by using function k′. Hydrophobicity data estimated on the basis of the k″ values proved to be in good accordance with the expectations based on the migration times and on the chemical structures of the compounds investigated.

Characterisation of mixed lithium dodecyl sulphate/lithium perfluorooctanesulphonate pseudo-stationary phases in MEKC.

Lipophilicity and methylene selectivity of mixed pseudo‐stationary phases (PSPs) (containing lithium dodecyl sulphate (LDS) and lithium perfluorooctanesulphonate (LiPFOS) in different molar ratios) applied in MEKC have been investigated. Micellar proportion (tprop,mic, a quantity expressing that how much time is spent by the analyte in the micellar phase related to its whole migration time), CLOGP50 value (showing the value of hydrophobicity of a molecule spending exactly 50% of its migration time in the PSP) and methylene selectivity have been determined for different LDS/LiPFOS mixed phases. Values of the above‐mentioned parameters have been determined for analytes with different chemical structures (alkylbenzene and alkylphenone homologous series, alcohols). Good linear correlation was obtained between either the micellar proportion, CLOGP50, or methylene selectivity and the phase composition for the mixed phases. Lipophilicity and methylene selectivity of the mixed LDS/LiPFOS PSPs can be calculated and can continuously be changed by mixing the two single phases (LDS and LiPFOS) in the appropriate (and calculable) portion.

Optimization of Important Early ADME(T) Parameters of NADPH Oxidase-4 Inhibitor Molecules

Through their reactive oxygen species (ROS) producing function, NADPH oxidase (NOX) enzymes have been linked to several oxidative stress related diseases. In our recently published paper [1] we have already shown the NOX4 inhibitory effect of diverse, molecule sub-libraries and their biological importance. We also presented our work connected to potential anti-tumour molecules and the relationship between their biological activity and physico-chemical properties [2]. As an extension of these studies further physico-chemical and biological investigation has been carried out on a molecule group included NOX4 inhibitory chromanone compounds. Here we describe the optimization of early ADME(T) parameters determining lipophilicity, phospholipophilicity and permeability linked to structure-activity relationship. We prove that optimal lipo- and phospholipophilicty can be also determined in case of NOX4 inhibitors and a comparison will be made between the chemically similar isochromanone and chromanone molecular libraries. It will be also shown how to predict the effect of different substituents on permeability, lipo- and phospholipophilicity and also the biological differences between anti-tumour molecules and NOX4 inhibitors according to their penetration ability.

Lipophilicity of Substituted Aurones and Related Compounds Measured on Immobilized Artificial Membrane (IAM) and Conventional C8 (MOS) Columns

Abstract A high performance liquid chromatographic method utilizing an immobilized artificial membrane (IAM HPLC) column has been developed to separate the members of a library containing 38 aurone and thioaurone type structures and to characterise their lipophilicity. The experimental lipophilicity data (k′IAM) have been compared with the previously determined ones C8(MOS) column (k′MOS) and with their calculated lipophilicity parameters (CLOGP). In general, good correlations between the measured and calculated lipophilicities have been found both for the IAM and MOS column. The IAM column showed higher efficiency in separation of isomeric aurones or thioaurones than the MOS one and, consequently, it showed higher potential in differentiation of their lipophilicities. Our findings proved the usefulness of the HPLC method in fast characterisation of the lipophilicity of drug candidates closely related in structure.

CALCULATION OF PHASE RESIDENCE TIMES IN MICELLAR ELECTROKINETIC CHROMATOGRAPHY

Method to calculate micellar phase residence time [tmic = tmc k‴, time spent by the analyte in the micellar phase] and aqueous phase residence time [taq, = t0 (1 − k‴), time spent by the analyte in the aqueous phase] was developed, where tm, to and tmc are the migration time of the analyte, the flow marker and the micelles, respectively. It was proved that migration time of the analyte is the sum of the phase residence times [tm = tmic + taq = t0 (1 − k‴) + k‴ tmc]. Two physico-chemical parameters characterizing hydrophobicity of the analyte, namely the ratio of the moles of the analyte in the micellar (nmc) and aqueous (naq) phase (nmc/naq = tmic/taq) as well as distribution coefficient (K) have also been determined from the phase residence times. Good correlation was found between the micellar phase residence times and computer calculated hydrophobicity values (S) in the case of hydroxy- and aldehyde benzene derivatives, benzene- and alkyl phenone homolog series and in the case of seven hydrophobic protected peptides. Comparison of hydrophobicity values and phase residence times showed that the higher the hydrophobicity of the analyte the longer the time spent in the micellar (hydrophobic) phase. The micellar phase residence time determinable experimentally in MEKC reflects the hydrophobicity of the molecule investigated.