Browsing by discipline "Farmaseuttinen kemia"

11β-hydroxysteroid dehydrogenase/reductase (11β-HSD) enzymes 1 and 2 regulate the amount of cortisone and cortisol in human tissues. Since overexpression of 11β-HSD1 especially in the adipose tissue causes symptoms of metabolic syndrome, selective inhibition of 11β-HSD1 provides a way to treat this syndrome and type II diabetes. Inhibition of 11β-HSD2 causes cortisol-dependent mineralocorticoid activation, which leads to hypertensive side effects. There are several reported 11β-HSD1 inhibitors, for selective 11β-HSD2 inhibitition, only a few compounds have been developed. The difference between 11β-HSD1 and 2 ligand binding sites is unknown, which complicates the search of selective inhibitors to both of the enzymes. This study was done with two aims: (1) to identify the difference between the two isozymes, (2) to create pharmacophore models for selective 11β-HSD2 inhibitiors. These tasks were approached with computational methods: homology modeling, docking, ligand-based pharmacophore modeling and virtual screening. The homology model of 11β-HSD2 was constructed using SwissModeler and it showed satisfying superimposition both with is template 17β-HSD1 and 11β-HSD1. The difference between the enzymes could not be identified by visual inspections Therefore, seven compounds, of which six are 11β-HSD2 -selective, were docked both to 11β-HSD1 and 11β-HSD2 ligand binding sites using the program GOLD. The docking results revealed that the compounds orientate differently in the enzymes. To 11β-HSD1, the compounds were anchored similar than unselective compound carbenoxolone, whereas in 11β-HDS2, they adopted a flipped binding mode. The flipped binding mode in 11β-HDS2 enables hydrogen bonds to Ser310 and to Asn171, both residues that are only present in 11β-HSD2. Pharmacophore modeling and virtual screening were done using the program LigandScout3.0. The ligand-based pharmacophores were based on the six 11β-HSD2 selective compounds, which were also used for the docking studies. Both of the models consisted of six features (hydrogen bond acceptors, hydrogen bond donor and hydrophobic feature) besides the exclusion volumes. The most important features considering the 11β-HSD2 selectivity seem to be the hydrogen bond acceptor feature that could interact with the Ser310 and the hydrogen bond donor feature next to it. The interaction pair for this hydrogen bond donor feature was not observed in the homology model. However, a possibility of water molecule as an interaction pair was evaluated and it seems to be a possible solution to the problem. Since both of the models were able to find the selective 11β-HSD2 inhibitors and exclude the unselective ones from the test set database, they were employed for the screening of the database that consists of 2700 compounds stored at the University of Innsbruck. From the hits of these screenings ten compounds were selected and sent to biological testing. The results of the biological tests will decide how well the models represent the theory of the 11β-HSD2 selectivity.

The aim of this thesis was to synthesize abietic acid derivatives, and to determine their antimicrobial activity. The study was performed in two parts. The first part was carried out at VTT (Technical Research Centre of Finland) and the second part at the Faculty of Pharmacy, University of Helsinki, the Division of Pharmaceutical Chemistry and Technology. Abietic acid as well as its isomers and precursors are important antimicrobial substances among plant kingdom. They are produced in big scale by conifers, which extractives of pitch and wood they are. Abietic acid and its many derivatives have shown to have many bioactivities. Emerging antibiotic resistance of bacteria, viral diseases that are spreading and mutating throughout the world and serious diseases caused by protozoa, cause an ever-growing need to develop new active agents against these pathogens. Abietic acid derivatives are worthy candidates for research into new drug substances. At VTT a synthetic route with abietic acid chloride as intermediate was used (synthetic route 1). It did not work well in the synthesis of esters and yields were low. The synthetic route worked fairly well for amide synthesis. The purifications and analysis of the compounds, two esters and one amide of abietic acid, were left partially unfinished, and none of the products of these reactions were tested for bioactivity. At the Division of Pharmaceutical Chemistry and Technology eight derivatives of abietic acid, from abietic acid and four different amino acids, were obtained via synthetic route 2. Some of these were sent to tests for antibacterial activity. Six synthesized compounds were sent to tests for their bioactivity against bacteria. However, the results were not obtained in time for this Master's thesis, and therefore any conclusions of their structure-activity relationships could not be drawn.

Screening of drugs of abuse has to combine sensitivity, selectivity and repeatability. The conventional screening methods include immunoassay screening followed by a more sensitive confirmation method. The aim of the study was to develop a simple, yet sensitive sample preparation method for screening of benzodiazepines and amphetamine derivatives in urine samples with silicon micropillar array electrospray ionization chip (µPESI) coupled to mass spectrometric analysis. Another aim was to evaluate the suitability of µPESI in biological sample analysis. Ideally, the developed method would provide an alternative to immunoassay screening method in forensic urine analysis. The sample preparation methods were separately optimized for benzodiazepines and amphetamine derivatives. Methods used included solid- phase extraction with Oasis HLB cartridge and C18-phase containing ZipTip®-pipette tip, liquid-liquid extraction, and dilution and filtering without prior extraction. Optimization focused, however, on ZipTip®-extraction. The compounds were spiked in blank urine to their cut-off levels, 200 ng/ml for benzodiazepines and 300 ng/ml for amphetamine derivatives. For benzodiazepines, every extraction phase was optimized. The sample pH was adjusted to 5, the ZipTip® phase was conditioned with acetonitrile and washed with a mixture of water (pH 5) and acetonitrile (10 % v/v) and the sample was eluted with a mixture of acetonitrile, formic acid and water (95:1:4 v/v/v). For amphetamine derivatives, pH values of sample and solvents were optimized. The sample pH was adjusted to 10, the ZipTip® phase was conditioned with a mixture of water and ammoniumbicarbonate (pH 10, 1:1 v/v), washed with a mixture of water and acetonitrile (1:5 v/v) and the sample was eluted with methanol. The optimized methods were tested with authentic urine samples obtained from Yhtyneet Medix Laboratories and compared to the results of quantitative GC/MS analysis. Benzodiazepine samples were hydrolyzed prior to extraction to improve recovery. All samples were measured with Q-TOF Micro apparatus and hydrolyzed benzodiazepine samples additionally with microTOF apparatus in Yhtyneet Medix Laboratories. Based on the results the developed method needs more optimization to function properly. The main problems were lack of reproducibility and poor sample ionization. Manual sample preparation and adding to the chip sample introduction spot increased variation. Authentic benzodiazepine samples gave false negative and authentic amphetamine derivative samples false positive results. False negatives may be due to the lack of sensitivity and false positives due to the contamination of sample cone, chips or solvents.

The aims of this work were (1) to compare the three dimensional structures of different S- adenosylmethionine (SAM)-dependent methyltrasferases and (2) to screen in silico a commercial library for potential methyltransferase inhibitors. In this work we decided to focus on DNA methyltransferase-like enzyme (DNMT2) and catechol-O-methyltransferase(COMT). There were two different parts in my work. The first part was to analyze the 3Dstructures of DNMT2 and COMT in relation with their amino acid sequences. The structures of DNMT2 and COMT were compared together by means of superimposition with Sybyl 8. The ligand binding properties were studied by manual and automatic docking of known inhibitors in order to understand the binding specificity of these methyltransferases. The softwares I used for docking were Autodock 4.2 and Gold 4.0. The sequence alignments and superimposition of the known crystal structures showed that the structures of DNMT2 and COMT share a similar fold. Furthermore the main similarities between the structures of these enzymes are in the co-enzyme binding sites. The only significant difference in the binding sites is the place of one tyrosine residue, which causes a slight change in the conformation of the bound co-enzyme. Unlike co- enzyme binding sites, the substrate binding sites of DNMT2 and COMT are different. There is indeed a bound magnesium ion in the substrate binding site of COMT but not in the substrate binding site of DNMT2. Because the substrate binding sites are more different than the co-enzyme binding sites, we decided to screen the potential active ligands only at the substrate binding sites. The second part of the work was virtual screening. I used a subset of 20.000 molecules of ChemBridge DIVER Set that can be purchased commercially. The softwares I used for library preparation were CONCORD and Balloon, from which Balloon created more reasonable 3D structures for the docking. I did two parallel screenings to the crystal structure of COMT (PDB code 3BWM) with docking program GOLD 4.0, which is the only program that can take account metal coordination. To DNMT2 I did two sets of screenings, one with GOLD 4.0 and another with Autodock 4.2. I used known COMT inhibitors as control in the COMT run and known DNA methyltransferase inhibitors as control in DNMT2 run. Before docking to the three dimensional structure of DNMT2, one loop near the substrate binding site had to be modeled. I used Swiss-Modeler and Modeller softwares for that. Docking to COMT was successful according to the rank of the known COMT inhibitors compared to the subset of the FIMM library that was screened. I created the hitlist of 60 compounds based on the scores of these compounds, pharmacophore search and visual examination. 30 of these compounds were purchased and are currently being tested. The results of the DNMT2 run were not as reliable as the results of COMT run mentioned before, since the DNMT2 run was unable to retrieve known inhibitors better than random. The reason for that can be the quality of the model of the missing loop or the chosen controls. Furthermore only one of the ten small molecules that we used as controls is proved to be DNMT2 inhibitor, the others are DNMT1 and DNMT3 inhibitors and while the binding sites of DNMT1, DNMT2 and DNMT3 are very similar, they are, however, not completely identical.

Here, we demonstrate the application of desorption atmospheric pressure photoionization (DAPPI) as a screening method at the Criminal Laboratory of the Finnish National Bureau of Investigation for samples confiscated by the Finnish criminal police. DAPPI is a fast mass spectrometric technique to analysis compounds directly from the sample surface in ambient atmosphere. In DAPPI, the sample is thermally desorbed from the sample surface using hot solvent vapor, after which the analytes are ionised in the gas-phase by photon-initiated gas-phase reactions. DAPPI was applied to the direct analysis of confiscated drugs, anabolic steroids and explosives of various matrices without any sample preparation. Confiscated drug samples included e.g. tablets, powders, herbal mixtures, herbal products [Catha edulis (khat) leaves, opium, Cannabis sativa, Psilocybe mushrooms] and ampules and tablets containing anabolic steroids. Powders were sprinkled on a 2-sided tape on a microscope slide, after which the excess powder was shaken away from the tape surface. Liquid samples were analysed from a kitchen paper, after application of 1 Äl of oil from ampules. Other samples were analysed by simply placing them on the DAPPI sampling stage and by directing the solvent plume on the sample surface. DAPPI proved to be a fast and specific analysis technique to this type of forensic analysis. DAPPI does not require any sample preparation, which therefore is well suited for fast forensic analysis, especially for plant samples and oily anabolic steroids, which are considered very challenging with conventional methods. Contamination of the mass spectrometer could be avoided by adjustment of the distance of the sample from the mass spectrometer inlet. Memory effects or contamination of the MS instrument were not observed even after several weeks of DAPPI measurements. DAPPI was also used for trace detection of the explosives trinitrotoluene (TNT), nitroglycol (NK), nitroglycerine (NG), penitrit (PETN), cyclonite (RDX), octogen (HMX) and picric acid. These organic explosives are nitrated compounds, which are divided based on their chemical structure into nitroaromatics (TNT and picric acid), nitroamines (RDX and HMX) and nitrate esters (PETN, NG and NK). Explosive dilutions were analysed with DAPPI from a polymer surface [poly(methyl methacrylate), PMMA] after application and drying of 1 Äl of sample. Also forensic analysis of post-blast residues from different matrices were done. DAPPI was effective in the ionisation of nitroamines and nitrate esters as their adducts with anions such as nitrate, acetate, formate and acetate. TNT used to form negative molecular ions through electron capture and picric acid formed deprotonated molecules through proton transfer. A DAPPI-MS method was developed for all explosives but the identification of the very low concentration explosive traces from wild variety of matrices proved to be difficult.

The most important part in bioanalysis is the sample cleanup process which is usually the most laborious and time consuming part of the analysis and very susceptible to errors. A functional bioanalysis has to be quick, easily automated, sensitive, selective and stable. It also needs to be suitable for high throughput analysis. Desorption atmospheric pressure photoionization (DAPPI) is a novel direct desorption/ionization technique for mass spectrometry that enables direct analysis of solids from surfaces or liquid samples from a suitable sample plate often without any sample preparation. The suitability of DAPPI-MS for biological samples was investigated by measuring the limits of detection for selected opioids and benzodiazepines and screening them from authentic urine samples. Limits of detection were measured for standard solutions and spiked urine. Opioids and benzodiazepines were analyzed from post mortem urine samples with an optimized DAPPI-MS method. Post mortem urine samples were analyzed with and without sample preparation. Sample preparation improved the sensitivity of the method remarkably. About 50 % of the analytes were detected without sample preparation and almost 100 % after sample cleanup. It is however difficult to estimate the suitability of DAPPI-MS as a screening method because not all analyte concentrations of the urine samples were known. Therefore we cannot be certain weither the results obtained without sample preparation are caused by the suppression of the urine matrix or if the concentrations of the analytes are below the limits of detection. The reliability of the method can further be improved by investigating the metabolites of the analytes and improving the system towards automation. On grounds of this research DAPPI-MS should be used cautiously as a screening method for urine samples without sample preparation and with only high enough analyte concentrations. DAPPI-MS shows promise as a screening method for opioids and benzodiazepines from urine when the sample cleanup is used before the analysis.

Protein phosphorylation is an important mechanism in cell signaling. Normally, one third of all proteins in a living organism is phosphorylated every moment. Abnormal amount of phosphorylated proteins is associated with many diseases. Proteins can also be oxidized in a living organism by reactive oxygen species. Usually oxidized proteins are degraded quickly in cells. Increased oxidation and decreased capability to degrade oxidized proteins can cause accumulation of these species in tissues. This has also been associated with several diseases and aging. Titanium dioxide is a widely used catalyst in photocatalytic reactions. When titanium dioxide absorbs UV radiation, hydroxyl radicals and superoxide anions are formed in aqueous solution. These radicals can then react with other compounds and degrade them. The most important factors affecting titanium dioxide photocatalysis are concentration and type of titanium dioxide catalyst, initial concentration of the substrate and pH. The purpose of this work was to investigate the effect of phosphorylation on degradation and oxidation of peptides in titanium dioxide photocatalysis. Since titanium dioxide columns have been used to enrich phosphopeptides, the adsorbtion of phosphorylated and unphoshorylated peptides on titanium dioxide is known to be different. Possibly this has also an effect on reaction products in photocatalysis. Peptides investigated were a non-phosphorylated, a monophosphorylated and a triply phosphorylated insulin receptor peptide. Samples were analyzed by ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS). First, the effect of pH on the degradation of the peptides was studied. Acidic conditions decreased the degradation of the non-phosphorylated peptide. Degradation of the phosphorylated peptides was significant in all pH conditions studied. In alkaline conditions all of the investigated peptides were degraded efficiently so these conditions were chosen for further experiments. Degradation of all three peptides was very fast in titanium dioxide photocatalysis. Peptides were degraded significantly already after 0.5 minutes UV exposure. The non-phosphorylated and monophosphorylated peptide produced mainly once (M+O) and doubly (M+2O) oxidized products. Oxidation products of the triply phosphorylated peptide were not detected. Oxidation sites could not be identified since the products were formed in such a small concentrations. Reproducibility was also poor in this study. Further studies are needed to find out, if phosphorylation has effect on oxidation of peptides. In the future, the reaction conditions and the analytical method need to be optimized.

The aim of the stydy was to evaluate how different chemical derivatization methods are suitable for characterization of regional isomers of different glucuronide conjugates. Glucuronidation is one of the phase II metabolic reactions where more water soluble and often inactive substances are produced. Different functional groups may be subjected to glucuronidation. It is important to determine the exact position of glucuronidation, as the isomers may possess different toxicological or pharmacological properties. For example morphine-6-glucuronide is pharmacologically more active than morphine itself. The glucuronide conjugates are commonly detected by liquid chromatography tandem mass spectrometry (LC-MS/MS) and/or nuclear magnetic resonance (NMR). MS/MSspectra of native molecule and glucuronidated molecule are usually similar because of an initial loss of 176 Da, i.e. monodehydrated glucuronic acid. This fact often makes it impossible to determine the site of glucuronidation. Samples of NMR-analysis requires larger amounts of sample materials than MS-analysis. Many of those derivatization reagents tested in this study were not reacting as they were supposed to react according to literature. O-phthalaldehyde (OPA) and 9-fluorenylmethyl chloroformate (FMOC) were forming derivatives as expected and those reagents are very suitable for glucuronide conjugates studies. At the end of the studies the site of the glucuronidation of dopamine- and serotonineglucuronides were evaluated by derivatization with OPA and FMOC. Derivatization with OPA and FMOC successfully gave information about the region of the glucuronide acid in dopamine- and serotoninemolecules. The assumptions supposed to be correct according to NMR-studies presented in literature.

Voltage-gated sodium channels play an essential role in the function of the nervous system as they are responsible for producing action potentials. Abnormal activity of sodium channels is in connection to several diseases such as epilepsy and chronic pain. Voltage-gated sodium channel blockers which are selective towards a specific isoform could provide more efficient and better tolerated drugs to treat these diseases when compared to the drugs used today. Clathrodin is an alkaloid isolated from Caribbean sea sponge Agelas clathrodes. Bioactivity studies have shown that clathrodin changes the function of voltage-gated sodium channels. The aim of this study was to synthesize two kinds of structure analogs of clathrodin and study their structure-activity relationship towards different isoforms of voltage-gated sodium channels. The study is part of the MAREX project (Exploring Marine Resources for Bioactive Compounds: From Discovery to Sustainable Production and Industrial Applications) funded by the European Union. Intention of the project is to find new bioactive compounds in marine organisms. A four-step route was developed for synthesizing 2-aminobenzothiazole analogs. A three-step route was developed as well but the last step seemed to be problematic for some of the compounds. The three-step route provided new compounds as intermediates and some of them were sent to tests for activity. Synthesis of 1H-pyrrole-2-carboxamide analogs of clathrodin failed. 4,5-dihydrooxazole was recognized as a problem as it was formed as a result of a cyclization reaction when bromination was tried on the intermediate. The formed structure was used in synthesizing 2-(1H-pyrrol-2-yl)-4,5-dihydrooxazole analogs of clathrodin. These reactions failed to give any final products which could have been tested for activity. Eight synthesized compounds were sent to tests for activity. Results were received from two of them and they showed no activity towards the voltage-gated sodium channels in 10 µM concentrations. Discussion about structure-activity relationship is not possible based on two compounds only.

Drug-drug interactions occur when a drug or a drug metabolite modifies the activity of a drug metabolizing enzyme. As a result the concentration of active drug can be too low to be effective or too high and possibly toxic. This is an increasing problem in drug therapy where polypharmacy is rather common today. Therefore, in drug discovery and development significant efforts have been made in order to predict such interactions in advance and avoid them, or at least minimize them. This study is focused on medetomidine, a drug metabolized by UDP-glucuronosyltransferases (UGT). The aim of the study was to find inhibitors for medetomidine glucuronidation. Also the mechanism of possible inhibition was of interest. It is already common to test interactions of a given enzyme substrate with other enzymes of the same family either in phase I or phase II of drug metabolism in humans. It is less common, however, to examine such interactions between enzymes of two different families. In the present study it is tested if the compounds which are possible inhibitors of cytochrome P450 monooxygenase (CYP) also inhibit UGTs. Inhibition of glucuronidation was studied with HPLC method previously developed for medetomidine glucuronidation. First glucuronidation of medetomidine was studied without inhibitor compounds. After that the impact of three possible inhibitors on medetomidine glucuronidation was studied and results were compared with the initial results. Three compounds were found to inhibit glucuronidation of medetomidine. Also an interesting change in UGT's enzyme kinetics after the binding of inhibitor was discovered. It is interesting that same compounds could inhibit both CYPs and UGTs. The results revealed that if a CYP and a UGT could bind for the same compound, it is also likely that structural analogues of that compound will interact with both enzymes. In drug discovery and development it is important to take into account both CYP-enzymes and less studied UGTs, and their possible interactions.