Medicinal Chemistry - Volume 8, Issue 1, 2012

Medicinal Chemistry is now starting the eighth year of its existence. During the last seven years it has come to be recognised as a highly respectable journal that publishes cutting edge research articles in the field of medicinal chemistry and rational drug design. The journal owes its success and popularity to the very hard work of the Co-Editors, Associate Editors, Regional Editors and Editorial Advisory Board Members to whom I am grateful for their kind guidance and enthusiastic support. I am also very grateful to the hard working staff of Bentham Science Publishers including Mrs. Afsheen Shahab, Senior Production Editor, Mr. Akhtar Waheed and Mr. Asad Ali. I look forward to another good year for the journal with the publication of high quality publications from eminent experts.

The current issue focuses on current development for the production of anti-cancer molecules, principles and evaluation of factors associated with cancer risk, diagnostics and therapy, taking into consideration that cancer is a diverse disease with complex pathobiology. The papers for this hot topic issue have been received from the scientific conference which was held in Kragujevac, Serbia (March 16-19, 2011) entitled “PRECLINICAL TESTING OF ACTIVE SUBSTANCES AND CANCER RESEARCH” with an internationally recognized symposium on “ANTI-CANCER AGENTS, CARDIOTOXICITY AND NEUROTOXICITY”. The scientific themes of the conference have been: preclinical testing of active substances on cancer models, application of cell biology and molecular biology in testing of active substances, chemical synthesis of potentially active substances, biochemical isolation of bioactive substances, natural bioactive substances, toxicological aspects of active substances, microbial aspects of active substances, oxidative stress and cancer, anti-cancer agents, cardiotoxicity and neurotoxicity, and nutraceuticals and cancer. Scientific conference in figures - number of registered participants was 115, 31 researchers from abroad (11 from France, 9 from FYR Macedonia, 5 from Greece, 2 from Canada, 2 from Russia, 1 from Belarus, 1 from Slovakia), 2 review lectures were given, 44 oral presentations were made, 35 posters presentations were given, and 81 abstracts were printed in the Conference Abstract book (ISBN 978-86-7760-064-8). FP7 Info Day was organized at the scientific conference with participation of representatives of European Commission and Serbian government; 9 oral presentations were made during the FP7 Info Day together with presentations of two projects financed within FP7. The conference was organized under the auspices of EU 7th Framework Program funding scheme and with participation of Faculty of Science (Kragujevac, Serbia), Faculty of Medicine University of Kragujevac (Kragujevac, Serbia), Institute Curie (Paris, France), School of Medicine, Aristotle University (Thessaloniki, Greece), Faculty of Natural Sciences and Mathematics (Skopje, FYR Macedonia) and Serbian Physiological Society.

We have studied the kinetics of the complex formation of gold(III) complexes, [Au(en)Cl2]+ (dichlorido( ethylendiamine)aurate(III)-ion) [Au(dach)Cl2] (dichloride(1,2-diaminocyclohexane)aurate(III)-ion) and [Au(bipy)Cl2]+ (dichlorido(2,2'-bipyridyl)aurate(III)-ion) with guanosine5'-monophosphate (5'-GMP). It was shown that 5'-GMP have a high affinity for gold(III) complex, which may have important biological implications, since the interactions of Au(III) with DNA are thought to be responsible for the anti-tumor activity. The [Au(bipy)Cl2]+ complex is more reactive than [Au(en)Cl2]+ or [Au(dach)Cl2]+. The activation parameters for all studied reactions suggest an associative substitution mechanism. The cytotoxicity of gold(III) complexes was tested on A549 human lung carcinoma epithelial cell line and was evaluated by cytotoxic (MTT and LDH test) and apoptotic assays. The results showed that all tested gold(III) complexes displayed cytotoxic effect on A549 cells. Among the tested gold (III) complexes, AuBIPY showed the best cytotoxic effects.

Taken into consideration limited data about effects of palladium on cardiovascular system, the aim of our study was to compare toxicity of inorganic and organic palladium compounds on the isolated rat heart. The hearts (total number n=30, 6 for each experimental group) excised from Wistar albino rats, male sex, age 8 weeks, and body mass 180-200 g, were retrogradely perfused according to the Langendorff technique at constant perfusion pressure (70 cm H2O). After the insertion of sensor in the left ventricle, the parameters of heart function: maximum rate of left ventricular pressure development (dP/dt max), systolic left ventricular pressure (SLVP), diastolic left ventricular pressure (DLVP), mean blood pressure (MBP) and heart rate (HR)), were continuously registered. The experiments were performed during control conditions, and in the presence of perfusion with incresing concentration of the following: (triethanolamine (TEA), triethanolamine acetate (TEAA), palladium(II)chloride (PdCl2), and trans-dichlorobis(triethanolamine-N)palladium(II) complex (trans-[PdCl2(TEA)2])) started every 30 minutes (30, 60, 90, 120 minute). dP/dt max was not affected significantly by either TEAA, TEA, PdCl2 or Pd complex. SLVP was, also, not affected significantly by either TEAA, TEA, PdCl2, or Pd complex. DLVP was significantly decreased by both TEAA and PdCl2, while TEA and Pd complex did not show significant effect. MBP was significantly decreased only by PdCl2, while TEAA, TEA and Pd complex did not show significant effect. HR was significantly decreased by all compounds- PdCl2, TEAA, TEA and Pd complex. In our study, inorganic palladium compound (PdCl2) induced clear depression of the isolated rat heart contractility, manifested as drop in diastolic and mean blood pressure , and as decrease of the heart rate. On the other hand, it seems that palladium, when bound in an organic compound (linked to TEA in Pd complex), does not contribute significantly to cardio-toxicity in our experimental conditions.

Effects of infrared (IR) radiation generated by a low-power Co2-laser on the membrane of cultured dissociated nociceptive neurons of newborn rat spinal ganglia were investigated using the whole-cell patch-clamp method. Lowpower IR radiation diminished the voltage sensitivity of activation gating machinery of slow sodium channels (Nav1.8). Ouabain known to block both transducer and pumping functions of Na+,K+-ATPase eliminated IR irradiation effects. The molecular mechanism of interaction of Co2-laser radiation with sensory membrane was proposed. The primary event of this interaction is the process of energy absorption by ATP molecules. The transfer of vibrational energy from Na+,K+- ATPase-bound and vibrationally excited ATP molecules to Na+,K+-ATPase activates this enzyme and converts it into a signal transducer. This effect leads to a decrease in the voltage sensitivity of Nav1.8 channels. The effect of IR-radiation was elucidated by the combined application of a very sensitive patch-clamp method and an optical facility with a controlled Co2-laser. As a result, the mechanism of interaction of non-thermal low-power IR radiation with the nociceptive neuron membrane is suggested.

Neuroepithelial tumor cells were cultured in vitro. The biopsy material was taken from 93 children at removal of the brain tumors during neurosurgical operations. The individual features of the cells sensitivity of primary cultures in respect to protocol-approved chemotherapy drugs and changes in the Interleukin-6 (Il-6) level in the culture medium after the application of chemotherapy were established. The initial level of Il-6 exceeded 600.0 pg/ml in the cultural medium with histologically verified pilomyxoid astrocytoma cells, and ranged from 100.0 to 200.0 pg/ml in the medium at cultivation of ganglioneuroblastoma and pilocytic astrocytoma. A decrease in the Il-6 level in the medium culture of primary tumors cells was observed after the application of chemotherapeutic agents on the cells of pilomyxoid astrocytoma, astrocytomas, and pilocytic desmoplastic/nodular medulloblastoma. The production of Il-6 increased after application of cytostatic drugs on the cells of oligoastrocytomas. A decrease in Il-6 level after application of Cisplatin and Methotrexate and a 5-10 fold increase in the level of Il-6 after application of Etoposide, Carboplatin, Cytarabine, and Gemcitabine were registered in the medium with ganglioneuroblastoma. To improve the cytotoxic action of chemotherapeutic agents, the combined application of cytostatics with heterocyclic compounds was carried out. A computer modeling of ligand-protein complexes of carbamide using the Dock 6.4 and USF Chimera program packages was performed with molecular mechanics method. Special attention was drawn to the ability of several isoxazole heterocycles and isothiazolyl to inhibit the tyrosine kinase. It was proved in vitro that the joint application of chemotherapeutic agents and heterocyclic compounds could reduce the concentration of the cytostatic factor by 10 or more times, having maintained the maximum cytotoxic effect. It was assumed that the target amplification of cytotoxic action of chemotherapeutic agents had prospects for reducing toxic side effects of chemotherapy in vivo, which would be carried out only after the preclinical studies.

Effects of infrared (IR) radiation generated by a low-power Co2-laser on sensory neurons of chick embryos were investigated by organotypic culture method. Low-power IR radiation firstly results in marked neurite suppressing action, probably induced by activation of Na+,K+-ATPase signal-transducing function. A further increase in energy of radiation leads to stimulation of neurite growth. We suggest that this effect is triggered by activation of Na+,K+-ATPase pumping function. Involvement of Na+,K+-ATPase in the control of the transduction process was proved by results obtained after application of ouabain at very low concentrations. Physiological significance of low-power IR radiation and effects of ouabain at nanomolar level was investigated in behavioral experiments (formalin test). It is shown that inflammatory pain induced by injection of formalin is relieved both due to ouabain action and after IR irradiation.

Objectives: Glass ionomer cements (GICs) are commonly used as restorative materials. Responses to GICs differ among cell types and it is therefore of importance to thoroughly investigate the influence of these restorative materials on pulp stem cells that are potential source for dental tissue regeneration. Eight biomaterials were tested: Fuji I, Fuji II, Fuji VIII, Fuji IX, Fuji Plus, Fuji Triage, Vitrebond and Composit. We compared their cytotoxic activity on human dental pulp stem cells (DPSC) and correlated this activity with the content of Fluoride, Aluminium and Strontium ions in their eluates. Methods: Elution samples of biomaterials were prepared in sterile tissue culture medium and the medium was tested for toxicity by an assay of cell survival/proliferation (MTT test) and apoptosis (Annexin V FITC Detection Kit). Concentrations of Fluoride, Aluminium and Strontium ions were tested by appropriate methods in the same eluates. Results: Cell survival ranged between 79.62% (Fuji Triage) to 1.5% (Fuji Plus) and most dead DPSCs were in the stage of late apoptosis. Fluoride release correlated with cytotoxicity of GICs, while Aluminium and Strontium ions, present in significant amount in eluates of tested GICs did not. Significance: Fuji Plus, Vitrebond and Fuji VIII, which released fluoride in higher quantities than other GICs, were highly toxic to human DPSCs. Opposite, low levels of released fluoride correlated to low cytotoxic effect of Composit, Fuji I and Fuji Triage.

The antioxidant, antibacterial and antiproliferative activities, total phenolic content and concentrations of flavonoids of A. flavum extracts were determined. The total phenolic content was determined with Folin-Ciocalteu reagent and it ranged between 42.29 to 80.92 mg GA/g. The concentration of flavonoids in various extracts of A. flavum was determined using spectrophotometric method with aluminum chloride and obtained results varied from 64.07 to 95.71 mg RU/g. The antioxidant activity was monitored spectrophotometrically and expressed in terms of IC50 (μg/ml), and its values ranged from 64.34 to 243.34 μg/ml. The highest phenolic content and capacity to neutralize DPPH radicals were found in acetone extract. Antibacterial efficacy was defined by determining minimum inhibitory and minimum bactericidal concentrations using microdilution method. Significant antibacterial activity, especially for ethyl acetate extract, was observed. The best activity was showed against G+ bacteria, Staphylococcus aureus ATCC 25923 and Bacillus subtilis, while Escherichia coli was one of the least sensitive bacteria. Antiproliferative activity of the methanolic extract on HCT- 116 cell line was determined by MTT assay. Results showed that A. flavum has good antiproliferative activity with IC50 values of 28.29 for 24 h and 35.09 for 72 h. Based on these results, A. flavum is a potential source of phenols as natural antioxidant, antibacterial and anticancer substance of high value. Phenolic content of extracts depend on the solvents used for extraction.

Thioacetamide (TAA) exerts hepatotoxic, neurotoxic and carcinogenic effects. The aim of our study was to investigate the effects of TAA on lipid peroxidation and catalase activity in various rat brain regions. Male Wistar rats were divided into following groups: 1. control, saline-treated; 2. thioacetamide-treated groups, TAA300 (300 mg/kg), TAA600 (600 mg/kg) and TAA900 (900 mg/kg). Daily dose of TAA (300 mg/kg) was administered intraperitoneally once (TAA300), twice (TAA600) and three times (TAA900) in consecutive days. Brain samples were collected 24 h after the last dose of TAA and malondialdehyde (MDA) level and catalase activity were determined in cortex, brainstem and hippocampus. MDA level was significantly increased while catalase activity was significantly lower in all brain regions in TAA900 group in comparison with control group. In TAA600 MDA level was increased in the brainstem and cortex when compared to control (p<0.01). The same dose of TAA 600 mg/kg induced a significant decline in catalase activity in the brainstem and cortex and an increase in its activity in the hippocampus when compared to control (p<0.01). In TAA300 an increase in MDA level was evident only in the brainstem. Catalase activity was significantly higher in the cortex and hippocampus in TAA300 group in comparison with control (p<0.01). Based on these results, it may be concluded that various rat brain regions have different sensitivity to TAA-induced lipid peroxidation with hippocampus being less sensitive than cerebral cortex and brainstem.

Nitric oxide (NO), one of the gaseous neurotransmitters, is produced in reaction catalyzed by family of NO synthases (NOS). The involvement of neuronal NOS (nNOS) in seizures induced by homocysteine thiolactone has not been studied. Therefore, the aim of this study was to determine the effects of 7-nitroindazole, a selective nNOS inhibitor, on behavioral manifestations of homocysteine - induced seizures in adult rats. Adult male Wistar albino rats were treated with 7-nitroindazole (25, 50 and 75 mg/kg, i.p.) 30 min before injection of subconvulsive dose of homocysteine (D,L homocysteine thiolactone 5.5 mmol/kg, i.p.). Convulsive behavior was assessed during 90 min upon homocysteine administration by the following parameters: seizure incidence, duration of latency, number of seizure episodes per rat and their severity. Severity of seizures was evaluated using a descriptive scale graded from 0 to 4. It was shown that 7-nitroindazole increased seizure incidence, shortened latency time to first seizure, increased number of seizure episodes per rat and increased severity of seizures induced by homocysteine in rats. 7- nitroindazole in dose of 25 mg/kg led to a statistically significant increase in the seizure incidence and number of seizure episodes per rat, while doses of 50 and 75 mg/kg significantly increased severity of homocysteineinduced seizures. It could be concluded that inhibition of nNOS by 7-nitroindazole potentiates seizures induced by homocysteine in rats….

Glucose and cell swelling induce insulin secretion by alternative signaling pathways. Swelling-induced secretion is in most systems independent of calcium and various mediators of glucose stimulation. Comparison of two insulinoma tumor cell lines revealed surprising difference; INS-1E cells in contrast to INS-1 cells and isolated rat pancreatic islets do not respond to hypotonicity in the presence of calcium. To delineate the role of cholesterol the effect of its extraction or addition on the insulin secretion in response to glucose and cell swelling was compared. INS-1E cells have significantly higher cholesterol content than INS-1 cells (58.5 ± 2.9 and 46.3 ± 2.5 mg chol/mg prot respectively). After cholesterol desorption by 1.0, 5.0 and 10.0 mM of carboxymethyl-β-cyclodextrin, methyl-β-cyclodextrin, or 2-hydroxypropyl-β- cyclodextrin the response to hypotonicity in INS-1E cells emerged. On the contrary, supplementation of INS-1 cells with cholesterol inhibited their response to cell swelling. Cyclodextrin pretreatment inhibited glucose-induced insulin secretion from INS-1 cells while INS-1E cells were more resistant to their effect. Conclusion: Cellular cholesterol content substantially affects secretory process; both high and low levels could be inhibitory. Absence of swelling-induced insulin secretion in INS-1E cells despite adequate response to glucose is related to their high cholesterol content. Optimal cholesterol concentration is different for either type of stimulation; swelling-induced mechanism is more sensitive to higher cholesterol content. The difference is likely to reflect involvement of sequential type exocytosis after cell swelling. Sensitivity of secretory processes suggests that either hypercholesterolemia or excessive effort to decrease plasma cholesterol in patients could have adverse effect on insulin secretion.

Thin-Layer Chromatography is a technique widely used for qualitative analysis of organic compounds, isolation of the individual compounds from multicomponent mixtures, quantitative analysis, and preparative-scale isolation. In many cases, it outperforms the other chromatographic techniques. Firstly, there is a multitude of the chromatographic systems that can be applied in TLC. Many kinds of TLC and high performance TLC (HPTLC) precoated plates are commercially available such as: inorganic adsorbent layers (silica or silica gel and alumina), organic layers (polyamide, cellulose), polar bonded stationary phases (diol, cyanopropyl, and aminopropyl), nonpolar bonded stationary phases (RP2, RP8, RP18). Sorbents applied in TLC have the different surface characteristics and, hence, different physicochemical properties. Moreover, there is a wide choice of mobile phases that can be used to separate mixture components; these belong to various selectivity groups and, thus, have different properties. In TLC, UV absorption of the mobile phase solvents does not play a significant negative role in detection and quantification of the analytes, because the mobile phase is evaporated from the plate prior to the detection. Only high viscosity of a solvent can be viewed as a sole property limiting its choice as a mobile phase component. These plate and mobile phase characteristics allow a choice from among an unparalleled abundance of TLC systems that offer a broad spectrum of separation selectivities, which is particularly important when complex mixtures of the natural origin have to be separated. Another advantage of TLC is that each plate is used only once, thereby allowing simpler sample preparation methods compared to other chromatographic techniques. Highly sorbed materials in analyzed samples can be left behind in a layer, and do not interfere in the analysis of subsequent samples. Multiple samples can be analyzed at the same time on a single TLC or HPTLC plate, reducing the time and solvent volume used per sample; the processing of standards and samples on the same plate leads to advantages in the accuracy and precision of quantification by densitometry. TLC enables usage of numerous special development techniques. Most separations are carried out by a capillary flow development with a single mobile phase (isocratic) in the ascending or horizontal configuration. There are also special modes of developing a chromatogram: gradient elution with stepwise mobile phase variations and techniques consisted on multiple development of the plate. Multiple development consists of repeated development of the chromatogram on the same or on increased distances with the same or various eluents with drying the plate between the individual development runs. It results in narrowing of the spots or zones and improved resolution. Moreover, the circular and anticircular development methods can also be applied. The use of techniques with forced-flow of mobile phase (overpressure planar chromatography or electro-planar chromatography) has been also reported. TLC is also the easiest technique with which to perform multidimensional (i.e., two-dimensional) separations. A single sample is applied in the corner of a plate, and the layer is developed in the first direction with mobile phase 1. The mobile phase is evaporated, and the plate is then developed with mobile phase 2 at a right angle (perpendicular or orthogonal direction); mobile phase 2 has different selectivity characteristics compared to mobile phase 1. In this way, complete separation can be achieved of very complex mixtures (e.g., of the components of a plant extract) over the entire layer surface. Particularly valuable separation results can be achieved when using various mobile phase systems to benefit from different separation mechanisms. For example, with cellulose one can apply nonaqueous mobile phase to achieve the adsorption mechanism of retention and aqueous mobile phase to achieve the partition mechanism. In a similar way, with the polar chemically bonded stationary phases one can use nonaqueous mobile phases to achieve the adsorption mechanism of retention and the aqueous mobile phases to achieve the reversed-phase mechanism. Shifting from the adsorption to the partition mode causes marked differences in the separation selectivity....

The investigations demonstrated clearly a unique function and role of endogenous formaldehyde (HCHO) and ozone (O3) in the antibiotic effect of diverse molecules having different chemical structure. Elimination of HCHO and/or O3 from the layer chromatographic spots resulted in a decrease in the antimicrobial activity. On the basis of detection and measure of endogenous HCHO and O3 BioArena enables to both direct isolation and biological evaluation of new bioactive compounds.

Components of 50% aqueous ethanol chamomile (Matricaria recutica L.) flower extract, previously found antibacterial in a TLC-bioautographic study, were separated and isolated by the use of on-line overpressured layer chromatography (OPLC). This system consisted of an OPLC 50 BS system, an on-line coupled flow-through UV detector, and a manual fraction collector. The collected fractions were investigated by GC-MS analysis and by TLC re-chromatography with subsequent visualization, performed after use of the vanillin-sulphuric acid reagent, or under UV illumination, or applying bioautographic detection. The main compounds of the collected 11 fractions were identified by GC-MS. The results showed that the antibacterial effect of 50% aqueous ethanol extract of chamomile is ascribable to cis-, trans-spiroethers, and the coumarins like herniarin and umbelliferone.

Seventeen various extraction procedures based on precipitation of proteins in milk samples spiked with flumequine were tested. Several criteria were taken into account, when choosing the most effective. The supernatants were analyzed by thin-layer chromatography - direct bioautography (TLC-DB) and high performance liquid chromatography (HPLC-UV). The results obtained from both methods indicate as the best the same deproteinization procedure. The addition of acetonitrile to milk in 1:1 volume proportions gave the highest concentration of flumequine in supernatant and prompt coagulation of proteins in milk samples.

This paper summarizes the application of thin-layer chromatography coupled with biodetection for the search of substances characterized with properties relevant to the treatment of neurodegenerative diseases. The main focus is on the screening of plant extracts for the presence of acetycholinesterase inhibitors as well as direct antioxidants, taking into account the cholinergic hypothesis of Alzheimer's disease and oxidative stress theory of neurodegeneration. Practical aspects of performing TLC-bioassay tests are discussed and typical problems are addressed. Some practical hints are also given referring to the variety of compound classes that may be analyzed by means of these methods.

A series of vegetal extracts have been chemically altered by ethanolysis. The effect of the reaction on the inhibition of the enzyme β-glucosidase properties of the mixtures was studied using thin layer chromatography (TLC) with biodetection. Glucosidase inhibitory activity guided fractionation of one of the produced chemically engineered extracts led to the isolation of apigenin and ethyl p-cumarate. Both compounds were generated during the chemical modification step.

Two dimensional thin layer chromatography on DIOL polar bonded stationary phase was performed to optimize the separation of some anioxidant phenolic compounds from Eupatorium cannabinum extracts. Ethyl-methyl ketone mixed with n-heptane and ethyl acetate mixed with n-heptane were used as non-aqueous mobile phases in normal phase separations (1st direction of development in 2D-HPTLC mode) and methanol mixed with water was used as a mobile phase in reversed phase (2nd direction of development in 2D-HPTLC mode). The plates were sprayed by use of Merck TLC sprayer using 2-(diphenylboryoxy)-ethylamine and PEG4000 (Merck, Darmstadt, Germany) or DPPH and photographed in Camag Cabinet UV lamp at 254 nm and 365 nm by use of Fuji 8 mpx camera. Satisfactory separations of antioxidant phenolic compounds in Eupatorium cannabinum extracts were obtained by use of optimized 2D-HPTLC systems.