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.

Characterization of lipophilicity and antiproliferative activity of E-2-arylmethylene-1-tetralones and their heteroanalogues.

A molecular library based on E-2-arylmethylene-1-tetralone has been designed and synthesized. A reversed phase high performance liquid chromatographic (RP-HPLC) method has been developed and applied to separate them and to characterize their lipophilicity. The chromatographic method applied here was suitable to separate the structural (ortho and para) isomers of compounds and was sensitive enough to differentiate their lipophilicities. The measured (k') and computer calculated (CLOGP) lipophilicity values has been compared. Good linear correlation has been found in the case of these structurally related molecules. In vitro biological assay has been performed with Methylene blue dying to investigate the antiproliferative potency of the compounds synthesized in this work. The measured (k') and calculated (CLOGP) lipophilicities of the compounds were compared with the antiproliferative activities and an optimum value of lipophilicity has been found for these compounds.

Comparison of measured and calculated lipophilicity of substituted aurones and related compounds.

A molecule library containing 55 aurone- and thioaurone-type structures has been designed and synthesised. Reversed phase high performance liquid chromatographic (RP-HPLC) method has been developed to separate these compounds and to characterise their lipophilicity by experimental method (k'). The experimental lipophilicity data have been compared with the computer calculated lipophilicity parameters (CLOGPs) of the same molecules. In general, good correlations between the measured and calculated lipophilicities have been found with the exception of structure isomers and compounds capable for hydrogen bonding. The chromatographic method was suitable to separate the structure (ortho and para) isomers of aurone and thioaurones and was sensitive enough to differentiate their lipophilicities. Our findings suggest the usefulness of the chromatographic method in fast characterisation of the lipophilicity of structurally closely related molecules.

Determination of the basicity of Mannich ketones by capillary electrophoresis.

Capillary electrophoretic (CE) method to characterise Mannich ketones (MKs) containing morpholine moiety was developed. Basicity (pKa,exp) of the MKs was characterised by measured data derived from the electrophoretic mobility values obtained in the CE separation. The MKs were found to be weaker bases than the parent morpholine molecule itself and the experimentally determined basicity values proved to be dependent on the chemical structure of the MKs. Since the basicity of the MKs has an influence on their reactivity and biological activity it seems to be useful to determine experimentally the pKa,exp values of the newly synthesised compounds to support rational drug design or screening of the molecule libraries.

Activation of caspase-3 protease during the process of ursolic acid and its derivative-induced apoptosis.

The apoptosis-inducing effect of the triterpene saponins, namely, ursolic acid and its natural derivative, methyl-ursolate beta-D-glucoside on A431 human epidermoid carcinoma cells was studied. The cells treated with 5-50 microg/ml of ursolic acid resulted in a dose- and time-dependent decrease in cell number, due to an increase of apoptotic cells as evidenced by MTT assay together with morphological changes. The highest dose (50 microg/ml) of ursolic acid resulted in approximately 90% inhibition in tumor cell growth after 96 hours of treatment and 60% of apoptosis after 48 hours. To the contrary, when the same treatment was carried out with methyl-ursolate beta-D-glucoside, after 96 hours of treatment the percentage of cell growth inhibition was found to be only 30% at the dose of 50 microg/ml and the value of apoptosis did not exceed 10%. Similarly to these results, ursolic acid effectively induced proteolytic activation of caspase-3 protease in a dose-dependent manner while its derivative showed only weak activity in this enzyme assay. The addition of DEVD-CHO prior to ursolic acid and methyl-ursolate beta-D-glucoside treatment effectively prevented the loss of triterpenes-induced viability. In summary, the triterpene saponins investigated contain an apoptotic-inducing activity in A431 cells and in the case of ursolic acid it is associated with proteolytic activation of caspase-3 and/or other similar caspases. Our results also indicated that methylation of COOH-28 together with the glycosylation of C3 of ursolic acid have a strong impact on its antitumor activity.

The somatostatin analogue peptide TT-232 induces apoptosis and chromosome breakage in cultured human lymphocytes.

Somatostatin receptors are supposed to be important in the regulation of apoptosis. In this study, we measured apoptosis occurring spontaneously, or induced by the synthetic somatostatin analogue, the peptide TT-232. We examined isolated human peripheral blood lymphocytes (PBL) from 32 nurses exposed bedside to cytostatic drugs, 12 chronic lymphoid leukaemia (CLL) patients prior to treatment, and 19 unexposed, healthy donors without anamnestic occupational exposure to genotoxic agents. Cells were stimulated by phytohaemagglutinin-P (PHA) and cultured for 69 h with or without 15 microg/ml TT-232, respectively. Cell kinetic parameters and apoptosis were determined by flow cytometry after staining with FITC-labeled anti-BrdU and propidium iodide (PI) and the results on spontaneous and peptide-induced apoptosis were compared with the obtained chromosome aberration frequencies (CA). The peptide TT-232 unexpectedly induced chromosome breakage in addition to apoptosis. The mean spontaneous apoptotic fractions were 6.65+/-0.89%, 6.46+/-0. 53%, and 3.07+/-0.57%, and the mean CA yields in the samples without TT-232 were 1.74+/-0.46%, 2.44+/-0.40%, and 4.50+/-1.05%, for healthy subjects, nurses, and CLL patients, respectively. A total of 15 microg/ml TT-232 treatment in healthy subjects increased the mean CA frequency (10.38+/-1.57%), as well as the apoptotic cell fraction (2.63+/-0.45 times higher than the corresponding untreated sample). In TT-232-treated PBLs of nurses, CA remained unchanged and the mean apoptotic cell fraction showed only a slight increase (1.24+/-0.11 times higher than the untreated). Among CLL patients, TT-232 treatment significantly increased both CA (up to 17.83+/-4.04%) and the ratio of apoptotic cells (21.78+/-11.00 times higher than the untreated). These results demonstrated significant differences in apoptosis sensitivity in controls, nurses and CLL donors, after 15 microg/ml TT-232 treatment. Data also indicate that the induced CA yields in CLL donors with high CA are in correlation with TT-232-induced apoptosis.

Cytostatic, cytotoxic and protein tyrosine kinase inhibitory activity of ursolic acid in A431 human tumor cells.

The effect of the tritepene, ursolic acid, on the proliferation of A431 human epidermoid carcinoma cells was studied. According to our investigations, ursolic acid is a potent inhibitor of A431 cell growth. Ursolic acid markedly reduced A431 cell growth in a time- and dose-dependent manner. We found a good correlation between the results of direct cell counting and the MTT test. During long periods of drug exposure, ursolic acid exhibited both cytotoxic and cytostatic activity. The effect was partially reversible on drug removal. The greatest cytotoxicity was observed both in the trypan blue test and in the MTT test at 50 mM. Investigations on tyrosine kinase inhibition were performed by biochemical and cellular assays on A431 cells. Ursolic acid inhibited tyrosine kinase activity of A431 cells in biochemical assay in a dose-dependent manner with an IC50 of 24 mM. In cellular assay, when A431 cells were pretreated with ursolic acid for 24, 48 and 168 hours at various concentrations (5, 10, 20, 30 and 50 mM), lower values of IC50 were measured: 6.8 microM for 24 hours, 5.2 mM for 48 hours and 1.4 mM for 168 hours. The results suggest that ursolic acid exerts an antiproliferative effect through the inhibition of tyrosine kinase enzymes.

Novel lipoamino acid- and liposaccharide-based system for peptide delivery: application for oral administration of tumor-selective somatostatin analogues.

Lipoamino acid and liposaccharide conjugates of somatostatin analogue TT-232 were synthesized to modify the physicochemical properties of the parent peptide. The relative position, the number, and the nature of the lipid and/or saccharide moieties were varied. Experiments in vitro clearly showed that many compounds modified at the N- and/or C-terminus with lipid or sugar moieties retained the biological activity of the parent compound. An interesting construct was synthesized containing lipid and sugar units at opposite ends of the somatostatin analogue, so that the entire molecule could be considered as an amphipathic surfactant.

[Preparation and characterization of potential antineoplastic agents]. / Potenciális daganatellenes hatású vegyületek elóállítása és vizsgálata.

A parallel combinatorial library of over sixteen hundred compounds has been designed and synthesized for the development of new potential peptidomimetic protein tyrosine kinase (PTK) inhibitor leads that is aimed for intervening with the substrate binding site of the pp60c-src enzyme. The new structures were based on known PTK inhibitors having 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 micromolar IC50 concentrations from combinations of twelve different substituents. Molecular modeling of the active compounds showed a characteristic distance of 13-14 A between the farthest sp2 carbon atoms of the two aromatic rings. Conformational analysis of several peptide substrates recently found for pp60c-src PTK [5,6,7] showed that the energy minimized conformers had the same distance between 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.

A novel normalized retention factor in micellar electrokinetic chromatography.

Characteristic properties of the expression k'' = (t(m)-t(o))/(t(mc)-t(o)) and its applicability in micellar electrokinetic capillary chromatography (MEKC) were compared to the previous expression, k' = (t(m)-t(o))/t(o)(1-t(m)/t(mc)), 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.

Analysis of polyanionic macromolecular carrier poly-(N-vinylpyrrolidone-co-maleic acid) and its bioconjugats by capillary electrophoresis.

Anionic carrier poly(N-vinylpyrrolidone-co-maleic acid) and its conjugates, prepared with coupling of 2-cyano-3-hydroxy-5-amino-2-pentenoyc(4-trifluoromethyl anilide) or (6', 7'-dimethyl-l'-quinoxalinyl)-4-(2' amino) acetanilide to the carrier, were analyzed by capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) in the following buffers: 0.25 N triethylammonium phosphate (TEAP); sodium dodecyl sulfate (SDS; 25-150 mM) in TEAP (pH 2.25-6.30); 0.1 N Na-borate buffer (pH range 7-11) and SDS (25-150 mM) in Na-borate buffer (pH range 7-11). The presence of strong carboxyl groups (dissociated even at pH 2.25) on the polymer chain was proved by the CZE method. It was also proved by potentiometric titration that carboxyls with a wide range of acidity were on the polymer chain. CZE was able to differentiate among the analytes possessing carboxyl groups of different acidic strengths at pH 2.25. These components were not distinguished by CZE at high pH values (11.0). Interaction between the analyte and SDS affected the separation at this pH, and hence good resolution was obtained by MEKC. Informative separations were achieved both for the carrier and the conjugates in TEAP buffer at pH 2.25 by the CZE method. Optimal separation was achieved in borate buffer containing 75 mM SDS at pH 11.0 for the carrier and at pH 7.7 for the conjugates in MEKC.

Synthesis of gonadotropin-releasing hormone III analogs. Structure-antitumor activity relationships.

Following the observation that the activity of gonadotropin-releasing hormone III (GnRH-III) in the suppression of growth of MDA-MB-231 and MCF-7 breast cancer cells surpasses that of GnRH and other analogs thereof, analogs of GnRH-III were synthesized to investigate the structural basis for the improved antitumor activity. Compounds synthesized include analogs with changes in the central sequence in which GnRH-III differs from GnRH and in the C- and N-terminal regions. The results indicate that a salt bridge between Asp6 and Lys8 stabilizes the active conformation of GnRH-III and show the importance of the Trp7. Replacement of the C-terminal Gly-NH2 with D-Ala-NH2 was not well tolerated, but replacement with ethylamide was. Replacement of pGlu1 with Ac-D-Trp appears to have a significantly deleterious effect on a unique conformation of GnRH-III which is responsible for its binding to the receptors on cancer cell lines and the resultant antitumor activity.

Synthesis, conformation, biodistribution, and hormone-related in vitro antitumor activity of a gonadotropin-releasing hormone antagonist-branched polypeptide conjugate.

Since permanently high levels of GnRH analogues are necessary to exert direct and/or indirect antitumor effect on mammary tumors, much emphasis was put on the development of retarded-release devices (e.g. microcapsules) for GnRH derivatives. Alternatively, these compounds can be covalently coupled to high-molecular mass carrier molecules for the design of bioconjugates acting as (a) prodrugs producing prolonged release or (b) macromolecular therapeutics. In order to evaluate the feasibility of this approach, a prototype construct has been prepared with a potent GnRH antagonist Ac(D-Trp1,3, D-Cpa2, D-Lys6, D-Ala10)-GnRH (MI-1544). As a carrier, a representative of a new generation of synthetic, biodegradable branched poly[Lys(Xi-DL-Alam)] (XAK) type polypeptides with poly(L-lysine) backbone has been used in which X is an acetylated derivative of glutamic acid (AcEAK). This polyanionic polypeptide with free gamma-carboxyl groups was conjugated to MI-1544, which has only a single amino group at position 6. In this paper, we describe (i) the synthesis and structure (primary structure, conformation) properties of the MI-1544-AcEAK conjugate with a 33% degree of substitution, (ii) the effect of the covalent attachment of MI-1544 to AcEAK on its blood clearance and tissue distribution, and (iii) the hormone-related indirect (ovulation inhibitory) or direct (antiproliferative) antitumor activity of the conjugate studied by in vitro assays. Data obtained with 111In- and 125I-labeled conjugates have demonstrated that in fact the body/blood survival of MI-1544 was prolonged by 1.5-3 times. The direct in vitro antitumor effect of MI-1544 was maintained or even enhanced in the MI-1544-AcEAK conjugate. Furthermore, we have shown that this conjugate was able to antagonize the effect of GnRH in vitro or to act as free MI-1544 both in short- and long-term inhibition of ovulation even after single subcutaneous injection. These data suggest that it is feasible to use a biodegradable polymeric polypeptide for development of a macromolecular therapeutic with GnRH antagonists.

A tumor-selective somatostatin analog (TT-232) with strong in vitro and in vivo antitumor activity.

We report a series of new in vitro and in vivo data proving the selective antitumor activity of our somatostatin structural derivative, TT-232. In vitro, it inhibited the proliferation of 20 different human tumor cell lines in the range of 50-95% and induced a very strong apoptosis. In vivo TT-232 was effective on transplanted animal tumors (Colon 26, B16 melanoma, and S180 sarcoma) and on human tumor xenografts. Treatment of MDA-MB-231 human breast cancer xenografted in mice with low submaximal doses of TT-232 [0.25 and 0.5 mg/kg of body weight (b.w.)] caused an average 80% decrease in the tumor volume resulting in 30% tumor-free animals surviving for longer than 200 days. Treatment of prostate tumor (PC-3) xenografted animals with 20 mg/kg of b.w. of TT-232 for 3 weeks resulted in 60% decrease in tumor volume and 100% survival even after 60 days, while 80% of nontreated animals perished. We have demonstrated that TT-232 did not bind to the membrane preparation of rat pituitary and cortex and had no antisecretory activity. TT-232 was not toxic at a dose of 120 mg/kg of b.w. in mice. Long-term incubation (24 h) of tumor cells with TT-232 caused significant inhibition of tyrosine kinases in good correlation with the apoptosis-inducing effect. The level of p53 or KU86 did not change following TT-232 treatment, suggesting a p53-independent apoptotic effect. Preincubation of human breast cancer cells (MDA-MB-453) with TT-232 for 2 h decreased the growth factor receptor autophosphorylation. All of these data suggest that TT-232 is a promising and selective antitumor agent.

Analysis of macromolecular branched chain polypeptides by capillary electrophoresis and micellar electrokinetic chromatography.

Amphoteric poly(Lys-[Glu1.0-DL-Ala4.1]), (EAK) and anionic poly(Lys-Ac-Glu0.98-DL-Ala3.98]), (AcEAK) branched chain polypeptides were analyzed by capillary electrophoresis (CE) and micellar elektrokinetic chromatography (MEKC) in the following buffers. A1: 0.25 N triethyl ammonium phosphate (TEAP) buffer (pH 2.25); A2: 100 mM sodium dodecyl sulfate (SDS) in buffer A1; B1: Na-borate buffer (pH 7.7); B2: 100 mM SDS in buffer B1; C1: Na-borate buffer (pH 11.0); C2: 100 mM SDS in buffer C1. Both EAK and AcEAK could be separated by a CE mechanism at pH 2.25 and by an MEKC mechanism at pH 11.0. Optimum results were achieved with CE in buffer A1 and with MEKC in buffer C2.

Comparative analysis of somatostatin analog peptides by capillary electrophoresis and micellar elektrokinetic chromatography.

Capillary electrophoresis (CE) and micellar electrokinetic chromatography (MEKC) methods, utilizing uncoated silica capillary and triethyl ammonium phosphate or sodium borate buffers in the pH range of 2.25-11.0, containing sodium dodecyl sulfate (SDS) (0-100 mM) for analysis of somatostatin-analog peptides were developed. The method presented here was compared with the reversed-phase high performance liquid chromatographic (RP-HPLC) and CE methods developed for analysis of peptides. The peptides investigated in this work can be separated by CE on the basis of their electrophoretic mobility in aqueous buffer of low pH value (pH 2.25) or by MEKC on the basis of their hydrophobicity in SDS containing buffer of high pH value (pH 11.0). Optimal MEKC separation of the investigated peptides has been achieved at pH 11.0 in an Na-borate buffer containing 100 mM SDS. CE at pH 2.25 proved insensitive to the hydrophobicity of the peptides investigated. By contrast, results obtained with MEKC at pH 11.0 proved to be anologous to those obtained by RP-HPLC, with highly hydrophobic peptides-migrating slower than peptides without hydrophobic moieties.

Calculation of modified capacity factor in micellar electrokinetic chromatography.

In addition to the expression k' = (tm-t(o))/t(o) (1-tm/tmc), we propose the expression k" = (tm-t(o))/(tmc-t(o)) to calculate the capacity factor in micellar electrokinetic chromatography (MEKC), where tm, t(o), and tmc are the migration time of the analyte, the flow marker, and the micelles, respectively. The k' and k" values that were obtained from simulated data as well as from MEKC analysis of different peptides (in 100 mM sodium dodecyl sulfate/0.1 N sodium borate buffer at pH 11.0) were calculated and compared. The k" value is equal to zero for an analyte remaining in the aqueous phase whereas it is equal to one for an analyte always staying in the micellar phase. By applying k" a finite capacity factor can be obtained for an analyte, indicating its partition between the two moving phases (aqueous and micellar) even in those cases when tm equals tmc. The slope of the curve k" as a function of tm is constant through the whole migration window and therefore peak compression does not occur when applying k" to calculate the capacity factor. A given difference in k" corresponds the same difference in migration times and this value does not depend on the position within the migration window. Since k" is a normalized parameter it is easy to evaluate the significance of a given difference in capacity factor or to estimate the relative position of an analyte with a given capacity factor in the migration window by applying k". Therefore, k" seems to be an adequate parameter to calculate the capacity factor in MEKC and, similar to K', it also refers to the hydrophobicity of the analyte.

Investigation of the chemical stability of a new growth hormone-releasing hormone (GHRH) analogue by HPLC.

The stability of a new active growth hormone-releasing hormone analogue (D-Ala2,Nle27,(gamma-amino-butyric acid)30-GHRH(1-30)-NH2) was investigated during storage at different temperatures in aqueous solution. Samples stored for various periods of time were analysed by HPLC. It is concluded that in aqueous solution D-Ala2, Nle27,(gamma-amino-butyric acid)30-growth hormone-releasing hormone (1-30)-NH2 is stable: at least for 36 days at 4 degrees C; for 28 days at 25 degrees C; and for 10 days at 37 degrees C.

Arginine supply for nitric oxide synthesis and arginase is mainly exogenous in elicited murine and rat macrophages.

L-arginine, the precursor of nitric oxide(NO) is provided mainly by extracellular sources in casein-elicited murine and rat peritoneal macrophages. Free extracellular L-arginine(Arg), esters, peptides and proteins containing Arg are the best sources in accordance with the fact that proteolytic activity is high in peritoneal macrophages. The recycling of Arg from citrulline(Cit) was observed but at a low rate. This situation is different from that in endothelial cells where half of Arg is recycled from citrulline. No significant anaplerotic reaction from glutamic acid(Glu) can be demonstrated in peritoneal macrophages.

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.