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.

Adsorption of Antifungal Drugs Inside Pristine and Functionalized Fullerenes and Nanotubes: DFT Investigation.

BACKGROUND: Econazole, sulconazole and tioconazole usage as antifungal agents is limited due to poor pharmacokinetic properties. Pristine and hydroxylated structures of the C240 fullerene and single walled carbon nanotube (SWCNT) were proposed as transporters of these imidazoles potentially enhancing their pharmacokinetics. METHODS: To assess possibility of creation of the endohedral complexes of the azoles and carbon nanostructures, their adsorption and interaction energies were calculated with the hybrid exchange-correlation density functional B97-1 and 6-31(d,p) basis set. Interactions within the transporter - drug complexes were investigated with the Atoms in Molecules (AIM) Theory and Reduced Density Gradient (RDG). RESULTS AND CONCLUSIONS: The adsorption energies of the studied azoles depend on type and surface modification of the transporter. Hydroxylation of the fullerene and nanotube surface makes an opportunity for chemisorption of the investigated antifungal drugs. The pristine and hydroxylated nanotube complexes exhibit thermodynamic stability. The complexes of the fullerenes are thermodynamically unstable but its kinetic stability could be significant thus allowing for the such structures to exist. The energetic instability would enhance liberation of the encapsulated molecule from the complex. It is advantageous in the context of drug release.

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.

Novel 4-aryl-pyrido[1,2-c]pyrimidines with dual SSRI and 5-HT1A activity. Part 4.

This project describes the synthesis, pharmacological and pharmacodynamic tests on two series of novel derivatives of 2H-pyrido[1,2-c]pyrimidine with potential binary binding to 5-HT1A receptors and SSRI + serotonin transporters. The influence of piperidinyl-indole (8.1-8.7) and tetrahydropyridinyl-indole (8.8-8.32) residues and indole 5-position substituents (R3 = Br, Cl, F) present in the pharmacophore element of ligands on their binding to both molecular targets was tested. A considerable impact of piperidinyl-indole residue on binding to both targets was confirmed and compounds with a high binding affinity were identified: Ki 5-HT1A = 12.4 nM; Ki SERT = 15.6 nM 8.1; Ki 5-HT1A = 5.6 nM; Ki SERT = 20.7 nM 8.7, while the presence of a tetrahydropyridinyl-indole residue was found to reduce the affinity of ligands to 5-HT1AR. The presence of chlorine (R3) in this series resulted in a notable reduction in binding to both targets (5-HT1A and SERT). Selected compounds had their metabolic stability in a first-pass test (human liver microsomes, NADPH) determined in vitro, and R1 and R2 substituents present on the terminal residue of pyrido[1,2-c]pyrimidine were recognized as having an impact on stability.

A method for rapid screening of interactions of pharmacologically active compounds with albumin.

We determine the association constants for ligand-protein complex formation using the flow injection method. We carry out the measurements at high flow rates (F=1 mL min(-1)) of a carrier phase. Therefore, determination of the association constant takes only a few minutes. Injection of 1 nM of the ligand (10 µL of 1 µM concentration of the ligand solution) is sufficient for a single measurement. This method is tested and verified for a number of complexes of selected drugs (cefaclor, etodolac, sulindac) with albumin (BSA). We obtain K=4.45×10(3) M(-1) for cefaclor, K=1.00×10(5) M(-1) for etodolac and K=1.03×10(5) M(-1) for sulindac in agreement with the literature data. We also determine the association constants of 20 newly synthesized 3ß- and 3α-aminotropane derivatives with potential antipsychotic activity--ligands of 5-HT1A, 5-HT2A and D2 receptors with the albumin. Results of the studies reported here indicate that potential antipsychotic drugs bind weakly to the transporter protein (BSA) with K≈10(2)-10(3) M(-1). Our method allows measuring K in a wide range of values (10(2)-10(9) M(-1)). This range depends only on the solubility of the ligand and sensitivity of the detector.

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.

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--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.

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 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.

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.

Molecular properties impact on bioavailability of second generation triazoles antifungal agents.

The bioavailability of active compounds depends on their two main features: solubility and permeability. The experimental determination of these factors is rather cumbersome. The free enthalpies of salvation deltaG in water and chloroform, and the electrostatic potential surface around examined molecules were ab initio calculated by HF method for voriconazole, posaconazole and ravuconazole. These quantities are assumed to be the new determinants correctly describing both solubility and the affinity of biologically active compounds to lipophilic tendency to cross cellular membranes. The values of deltaG were compared to the theoretically and experimentally determined partition coefficients. The calculated values of deltaG and electrostatic potentials appeared to be consistent with these partition coefficients. It leads to conclusion that these theoretically derived parameters deltaG and electrostatic potential could be useful tools for fast and precise classification of chemical substances within the Biopharmaceutics Classification System (BCS).

Prediction of bioavailability of selected bisphosphonates using in silico methods towards categorization into a biopharmaceutical classification system.

The physicochemical properties relevant to biological activity of selected bisphosphonates such as clodronate disodium salt, etidronate disodium salt, pamidronate disodium salt, alendronate sodium salt, ibandronate sodium salt, risedronate sodium salt and zoledronate disodium salt were determined using in silico methods. The main aim of our research was to investigate and propose molecular determinants thataffect bioavailability of above mentioned compounds. These determinants are: stabilization energy (deltaE), free energy of solvation (deltaG(solv)), electrostatic potential, dipole moment, as well as partition and distribution coefficients estimated by the log P and log D values. Presented values indicate that selected bisphosphonates a recharacterized by high solubility and low permeability. The calculated parameters describing both solubility and permeability through biological membranes seem to be a good bioavailability indicators of bisphosphonates examined and can be a useful tool to include into Biopharmaceutical Classification System (BCS) development.

Endohedral complexes of fullerene C60 with small convalent molecules (H2O, NH3, H2, 2H2, 3H2, 4H2, O2, O3) in the context of potential drug transporter system.

The fullerene C60 has chemical properties which seem to predestinate it to be effective transporter of drugs in biological system. To prove this, the DFT/B3LYP (6-31G*) calculations were performed especially in order to determine the structures and energies of the inclusion complexes of C60 with small molecules. It was found that the small molecule is more compact when it is located in the centre of the C60 cage than as isolated molecule. The calculated inclusion energies in case of: H2O, NH3, O2, O3, H2, 2H2, 3H2, 4H2 are: 1.84, 3.81, 3.75, 21.07, 1.97, 20.10, 47.78 and 77.54 kcal/mol, respectively. The charge transfer and the influence of the complexed small molecules on the electrostatic potential distribution inside and outside of the C60 cavity are discussed.