Fullerene Derivatives of Nucleoside HIV Reverse Transcriptase Inhibitors-In Silico Activity Prediction.

Here we present new derivatives of nucleoside reverse transcriptase inhibitors with a C20 fullerene. The computational chemistry methods used in this study evaluate affinity of designed compounds towards the HIV-1 reverse transcriptase (RT) binding site and select the most active ones. The best of the designed compounds have superior or similar affinity to RT active site in comparison to most active test compounds, including drugs used in anti-HIV therapy.

HPLC METHOD FOR SEPARATING ENANTIOMERS OF IMIDAZOLE DERIVATIVES - ANTIFUNGAL COMPOUNDS.

The aim of this study was to test separation possibility of enantiomers of nine active substances belonging to imidazole derivatives: bifonazole, butoconazole, econazole, enilconazole, fenticonazole, isoconazole, miconazole, sertaconazole and tioconazole. The study was performed using HPLC method and the CHI- RALCEL OJ column (10 gm; 250 x 4.6 mm), the mobile phase flow rate of 0.8 mL/min and detection at 220 rim. Mobile phases containing hexane and the following modifiers: alcohols (2-propanol, ethanol, methanol) and diethylamine were tested. At first isocratic elution was used but some enantiomers eluted after a long retention time and their peaks were asymmetrical and too wide. Therefore, a gradient elution was developed allow- ing to obtain satisfactory retention times and other parameters of enentioseparation of the compounds.

QUANTITATIVE DETERMINATION OF RESIDUAL MONOMETHYLAMINE CONTENT IN NEBIVOLOL HYDROCHLORIDE BY HPIC WITH SUPPRESSED CONDUCTIVITY DETECTION.

The aim of this paper was to develop a simple analytical method which could be used to determine a synthesis-derived amount of monomethylamine (MMA) residue present in nebivolol hydrochloride. High-performance ion chromatography (HPIC) method with suppressed conductivity detection was used for this purpose. The HPIC analysis was performed with IonPac CS 14 column (250 x 4 mm) containing a macroporous weak cation-exchange stationary phase eluted with 10 mM methanesulfonic acid (MSA). Validation of the method confirmed its selectivity by achieving a satisfactory separation of alkyl- and alkanolamines and metal cations. The method also showed a sufficient precision (RSD < 5.0%) and accuracy (recovery 90-103%). The calibration plot was linear in the range 0.03-2.4 µg/mL of MMA (r² = 0.9997). The calculated limit of quan- tification LOQ was 0.03 µg/mL. Amount of the MMA contained in nebivolol hydrochloride was determined by direct reading from the calibration curve and by the multiple standard additions method. Both methods showed satisfactory precision (RSD < 10.0%) and they can be used to determine the monomethylamine content in the studied active substance.

Novel renin inhibitors containing derivatives of N-alkylleucyl-ß-hydroxy-γ-amino acids.

In search for new drugs lowering arterial blood pressure, which could be applied in anti-hypertensive therapy, research concerning agents blocking of renin-angiotensin-aldosteron system has been conducted. Despite many years of research conducted at many research centers around the world, aliskiren is the only one renin inhibitor, which is used up to now. Four novel potential renin inhibitors, having structure based on the peptide fragment 8-13 of human angiotensinogen, a natural substrate for renin, were designed and synthesized. All these inhibitors contain unnatural moieties that are derivatives of N-methylleucyl-ß-hydroxy-γ-amino acids at the P2-P1' position: 4-[N-(N-methylleucyl)-amino]-3-hydroxy-7-(3-nitroguanidino)-heptanoic acid (AHGHA), 4-[N-(N-methylleucyl)-amino]-3-hydroxy-5-phenyl-pentanoic acid (AHPPA) or 4-[N-(N-methylleucyl)-amino]-8-benzyloxycarbonylamino-3-hydroxyoctanoic acid (AAHOA). The previously listed synthetic ß-hydroxy-γ-amino acids constitute pseudodipeptidic units that correspond to the P1-P1' position of the inhibitor molecule. An unnatural amino acid, 4-methoxyphenylalanin (Phe(4-OMe)), was introduced at the P3 position of the obtained compounds. Three of these compounds contain isoamylamide of 6-aminohexanoic acid (ε-Ahx-Iaa) at the P2'-P3' position. The proposed modifications of the selected human angiotensinogen fragment are intended to increase bioactivity, bioavailability, and stability of the inhibitor molecule in body fluids and tissues. The inhibitor Boc-Phe(4-OMe)-MeLeu-AHGHA-OEt was obtained in the form of an ethyl ester. The hydrophobicity coefficient, expressed as log P varied between 3.95 and 8.17. In vitro renin inhibitory activity of all obtained compounds was contained within the range 10(-6)-10(-9) M. The compound Boc-Phe(4-OMe)-MeLeu-AHPPA-Ahx-Iaa proved to be the most active (IC50 = 1.05 × 10(-9) M). The compounds Boc-Phe(4-OMe)-MeLeu-AHGHA-Ahx-Iaa and Boc-Phe(4-OMe)-MeLeu-AHPPA-Ahx-Iaa are resistant to chymotrypsin.

NEW RENIN-INHIBITORS--STABILITY AND ACTIVITY DETERMINATION. PART III.

A series of new four potential renin inhibitors containing pseudodipeptides were synthesized. Stability for all compounds (1-4) in homogenates of liver, kidney, lung and in serum, gastric, intestinal juice and in the presence of α-chymotrypsin was determined. Compound 1 was unstable, compounds 2, 3 were stable, compound 4 was partly unstable, (liver and kidney homogenates, (α-chymotrypsin solution). Inhibitory activity of the compounds was measured in vitro by HPLC determination of lowering concentration of substrate (angiotensinogen) in the presence of renin and the potential renin inhibitor (compounds 1-4). Compound 1, 2, 3 and 4 showed inhibitory activity (1.7 x 10(-6), 9.6 x 10(-7), 1.05 x 10(-9) and 1.31 x 10(-7)M, respectively).

IDENTIFICATION AND DETERMINATION OF RUPATADINE AND FEXOFENADINE BY DENSITOMETRIC METHOD.

Simple, precise and accurate densitometric methods were developed for the determination of two antihistamine drugs. rupatadine and fexofenadine. Silica gel 60 F254 HPTLC plates were used as stationary phase, while mixtures of acetonitrile - water - 25% ammonia (90 : 10 : 1, v/v/v) and acetonitrile - methanol -acetate buffer at pH 5.5 (3 : 2 : 5, v/v/v) were used as mobile phases for rupatadine and fexofenadine, respectively. The detection of rupatadine and fexofenadine was conducted out at 256 and 210 nm, respectively. The limit of detection and the limit of quantification for rupatadine were found to be 0.3 and 0.1 µg/spot, respectively, and for fexofenadine, 5 and 2 µg/spot, respectively.

DEVELOPMENT OF CHROMATOGRAPHIC METHOD FOR DETERMINATION OF DRUGS REDUCING CHOLESTEROL LEVEL--STATINS AND EZETIMIBE.

The presented developed HPLC method and GC method may be used to separate and determine all analyzed 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) and ezetimibe using a single columns and a uniform methodology. In order to perform qualitative and quantitative tests of statins and ezetimibe the Symmetry C18 column 250 mm x 4.6 mm, 5 µm, the mobile phase: acetonitrile:water (70:30, v/v), adjusted to pH = 2.5 and a spectrophotometric detector for the HPLC method were used. For GC method column HP-1; 30 m x 0.25 mm x 0.25 µm and FID detector were selected. All results and statistical data obtained indicate good method sensitivity and precision. The RSD values are appropriate for both newly developed methods.

IMPACT OF STRESS FACTORS ON OPTICAL ISOMERISM OF BENAZEPRIL HYDROCHLORIDE.

Benazepril hydrochloride contains two stereogenic centers, but is currently available as single enantiomer (S,S configuration) for the treatment of hypertension. Its enantiomer (R,R configuration) and the diastereoisomeric pair (R,S and S,R) can be regarded as impurities. Stereochemical stability of S,S isomer of benazepril hydrochloride and its potential susceptibility to conversion in the.active substance and in Lisonid tablets were examinated. The separation with the use of the TLC method with the following system: chromatographic plates Chiralplate and a mobile phase: methanol - acetonitrile - 1 mM copper(II) acetate (4 : 2 : 4, v/v/v) with saturation of glacial acetic acid for 1 h and the HPLC method system: Chiral AGP column (150 x 4.0 man x 5 µm) and a mobile phase: phosphate buffer pH = 6.0 - methanol (80 : 20, v/v) were obtained. Active substance - benazepril hydrochloride and Lisonid tablets 20 mg were subjected to the impact of different stress factors. Samples were examined after 1 and 6 weeks. It was found that none of the applied stress factors caused the transformation of the S,S enantiomer of benazepril hydrochloride in the substance and tablets to other identified stereoisomers - only the compound decomposition has occurred.

SYNTHESIS AND STUDY OF HALOGENATED BENZYLAMIDES OF SOME ISOCYCLIC AND HETEROCYCLIC ACIDS AS POTENTIAL ANTICONVULSANTS.

A series of potential anticonvulsants have been synthesized. There are eight fluorobenzylamides and three chlorobenzylamides of isocyclic or heterocyclic acids. Two not halogenated benzylamides were also synthesized to compare the effect of halogenation. The aim of the research performed was to evaluate whether halogenation of the mother structure is able to improve its anticonvulsant activity. The compounds were tested in Anticonvulsant Screening Project (ASP) of Antiepileptic Drug Development Program (ADDP) of NIH. Compound 1 showed MES ED50 = 80.32 mg/kg, PI = 3.16. Compound 7 showed CKM ED50 = 56.72 mg/kg. Compound 8 showed MES ED50 = 34.23 mg/kg and scPTZ ED50 > 300 mg/kg, PI = 8.53.Compound 13 showed 6Hz ED50 = 78.96, PI = 3.37. The results indicate that fluorination does not improve activity, whereas chlorination in our experiment even reduces it.

New validated HPLC methodology for the determination of (-)-trans-paroxetine and its enantiomer in pharmaceutical formulations with use of ovomucoid chiral stationary phase.

A new chromatographic method for the enantioseparation and the determination of (-)-trans-paroxetine and (+)-trans-paroxetine has been developed with the aid of amylose ovomucoid-based chiral stationary phase. The method is faster and five times more sensitive than procedures recommended previously: limit of detection and limit of quantification are 5 and 16 ng/mL, respectively [modified (Ferretti et al. in J Chromatogr B 710:157-164, 1998): 20 and 60 ng/mL]. It was carefully validated and applied for the determination of (-)-trans-paroxetine and (+)-trans-paroxetine in Parogen (Mc Dermott Laboratories Ltd.) and Xetanor (Actavis) coated tablets.

Mass spectrometry and molecular modeling studies on the inclusion complexes between alendronate and ß-cyclodextrin.

Complexation of alendronate sodium (AlnNa) with ß-cyclodextrin (ß-CD) was studied by means of ESI-mass spectrometry. The experimental results show that stable 1:1 inclusion complexes between selected bisphosphonates and ß-CD were formed. In addition, complexes with different stoichiometry were observed. DFT/B3LYP calculations were performed to elucidate the different inclusion behavior between alendronate and ß-CD. Molecular modeling showed that the inclusion complex of Aln-ß-CD where the two phosphonate groups bound to the central carbon atom of bisphosphonate were inserted into the cavity of ß-CD from its "top" side was thermodynamically more favorable than when they were inserted from its "bottom" side; the complexation energy was -74.05 versus -60.85 kcal/mol. The calculations indicated that the formation of conventional hydrogen bonds was the main factor for non-covalent ß-CD:Aln complex formation and stabilization in the gas phase.

Physicochemical properties of lomefloxacin, levofloxacin, and moxifloxacin relevant to the biopharmaceutics classification system.

The aim of this investigation was to identify the impact of physicochemical properties of three fluoroquinolones (second, third, and fourth generation) on bioavailability in relation to the Biopharmaceutics Classification System (BCS) by in silico and in vitro methods. These properties were estimated by analyzing the electrostatic potential pattern and values of the free energy of solvation as well as the distribution coefficients and true partition coefficients of the studied compounds. This study was based on theoretical quantum-chemical methods and the in vitro shake-flask technique with two immiscible phases (n-octanol and phosphate buffer) as well as the experimental potentiometric method to estimate protonation macro- and micro-constants. Properties identified in the in vitro and in silico studies were similar and indicated high lipophilic properties of the studied molecules as well as their good solubility in a polar medium. It appears that both the theoretical methods and simple in vitro studies are useful tools for predicting the bioavailability of medicinal substances based on the BCS principles.

HPLC method for identification and quantification of three active substances in a dermatological preparation--Viosept ointment.

The study was aimed at developing a HPLC method to identify and quantify domiphen bromide, tripelennamine hydrochloride and clioquinol in Viosept ointment. The tested substances were successfully separated using Inertsil ODS-3 (250 x 4.6 mm, 5 µm) as a stationary phase and a gradient elution. Detection at 310 nm wavelength was applied for tripelennamine hydrochloride and clioquinol, and at 215 nm wavelength for domiphen bromide. Methods of extraction of the tested substances were developed: domiphen bromide and clioquinol were extracted with acetone from heated solutions, and tripelennamine hydrochloride was extracted in a hexane-water system. Validation procedure confirmed the method to be sufficiently selective, precise and accurate. Correlation coefficients of calibration curves pointed out that they were linear within the examined concentration range.

New renin inhibitors--stability and activity determination. Part I.

A series of new six potential renin inhibitors containing pseudodipeptides were synthesized. Stability for all compounds (1-6) in homogenates of liver, kidney, lung and in serum, gastric, intestinal juice and in the presence of alpha-chymotrypsin was determined. Compound 5 was unstable, compound 6 was stable, other compounds were partly unstable, compound 2 was stable except kidney homogenate and compound 4 was stable except liver homogenate. Inhibitory activity of the compounds was measured in vitro by HPLC determination of lowering concentration of substrate (angiotensinogen) in the presence of renin and the potential renin inhibitor (compounds 1-6). Compound 2, 4 and 6 showed inhibitory activity (1.4 x 10(-6), 5.2 x 10(-6), 1.5 x 10(-7) M, respectively). Other compounds (1, 3, 5) showed no inhibitory activity up to 10(-5) M.

New renin inhibitors containing phenylalanylhistidyl-gamma-amino acid derivatives in P3 - P1' position.

Five potential inhibitors of renin have been designed and obtained. In the molecule position P3 - P1', crucial for indicating inhibitory activity, all contain phenylalanylhistidylaminoalcanoyl group, ready for interaction with the hydrophobic pocket S3 - S1 of renin molecule. The aminoalcanoyl fragment consists of pseudo-dipeptidic units derivative of gamma-amino acids: of 4-amino-3-hydroxybutanoic acid (AHBA) [26], 4-amino-5-(4-ethoxyphenyl)-3-hydroxypentanoic acid (AEPHPA) [13], 4-amino-5-cyclohexyl-3-hydroxypentanoic acid (ACHPA) (1) and 4-amino-3-hydroxynonanoic acid (AHNA) [21]. At the P3 - P2 position of obtained compounds an unnatural fragment, derivative of Phe-His dipeptide, was placed ande isoamyl amid of 6-amino-hexanoic acid was attached at the end of the molecule (epsilonAhx-Iaa). The preliminary in vitro tests indicated that all compounds were inactive. However, they provided valuable information on P3 - P2 fragment possible structure modification able to produce a resonable renin activity inhibition. All synthesized inhibitors were chymotrypsin-resistant.

New renin inhibitors--stability and activity determination. Part II.

A series of new six pseudodipeptic potential renin inhibitors were synthesized. Enzymatic stability for all compounds (1-6) in homogenates (liver, kidney, lung) and body fluids (serum, gastric, intestinal juice) and alpha-chymotrypsin was determined. Compounds 4 was stable, compound 5 was unstable and compounds 1, 2, 3, 6 were partly unstable. Inhibitory activity of the compounds was measured in vitro by HPLC determination of lowering concentration of substrate (angiotensinogen) in the presence of renin and the potential renin inhibitor (compounds 1-6). Compound 4, 5, 6 showed inhibitory activity (0.9 x 10(-6), 1.3 x 10(-8), 2.2 x 10(-6) M, respectively). Other compounds showed no inhibitory activity up to 10(-5) M.

Stability of new anticonvulsant derivatives of picolinic, nicotinic, cyclocarboxylic acids in body fluids and tissues.

The stability of new compounds with established anticonvulsant activity: picolinic acid 4-pyridyl-methylamide (Pic-4-PMA), cyclopentanecarboxylic acid benzylamide (Cpc-BZA), cycloheptanecarboxylic acid benzylamide (Chc-BZA), picolinic acid 2-fluoro-3-trifluoromethylbenzylamide (Pic-2F-3TFM-BZA), 2-chloronicotinic acid benzylamide (2-Cl-Na-BZA), 6-chloronicotinic acid benzylamide (6-Cl-Na-BZA) and 6-trifluoromethylnicotinic acid benzylamide (6-TFM-Na-BZA) in homogenates of body organs and in body fluids was determined after incubation. It was found that three compounds were stable against enzymes present in body fluids and organs and two were found to decompose in liver and kidney homogenates and two decomposed only in liver homogenate.

Synthesis of imperatorin analogs and their evaluation as acetylcholinesterase and butyrylcholinesterase inhibitors.

In this study, we synthesized several imperatorin analogs using imperatorin and xanthotoxin as substrates. The anti-cholinesterase activities of all compounds were evaluated in in vitro experiments according to the modified Ellman's method. For each synthesized compound, IC50 values for both enzymes were established. Galantamine hydrobromide was used as a positive control in the enzymatic experiments. All active compounds showed selectivity toward butyrylcholinesterase (BuChE) rather than acetylcholinesterase. The most active ones were 8-(3-methylbutoxy)-psoralen and 8-hexoxypsoralen with IC50 values for BuChE of around 16.5 and 16.4 µM, respectively. The results of our study may be considered as the beginning of a search for potential anti-Alzheimer's disease drugs based on the structure of natural furocoumarins.

Model structure-activity relationship studies of potential tropane 5HT1A, 5HT2A, and D2 receptor ligands.

The two-stages studies of structure-activity relationship for model ligands of 5HT1A, 5HT2A, and D2 receptors were performed. On the first stage, the pharmacophores of two potential ligands of known in vitro binding to 5HT1A, 5HT2A, D2 receptors and model pharmacophore of strongly interacting D2 receptor ligands were found and their parameters were related to affinity data. The analyzed parameters were hydrophobic, hydrophilic, aromatic, donor and acceptor of proton centers. The geometry of spatial distribution of these properties was also investigated in comparative analysis. The studied, model compounds were two 3ß-acylamine derivatives of tropane. The second stage includes docking of studied compounds to D2 receptor model and the comparison of its quality with in vivo binding data. The obtained results are consistent with in vitro binding data and applied procedure accurate estimates the affinity of potential ligands to D2 receptors.

Identification and determination of selected histamine antagonists by densitometric method.

The conditions for identification were determined for four histamine antagonists: clemastine fumarate, loratadine, cetirizine dihydrochloride and desloratadine by TLC (thin-layer chromatography) method. The selected chromatographic conditions were used to develop a densitometric method for the content determination of the histamine antagonists in medicinal products and substances. The statistical data showed adequate accuracy and precision of the developed methods.