Browsing by discipline "Pharmaceutical Chemistry"

Cytochrome P450 (CYP) -enzymes are one of the most important enzymes in the metabolism of xenobiotics. Because many xenobiotics are metabolized with each other by the same CYP-enzymes, it is possible that metabolic interactions will take place. These interactions can be the inhibition or induction of the metabolism of another xenobiotic. The interaction can be harmful e.g. when it causes an accumulation of a toxic metabolite or when it inhibits the metabolism of an active drug substance. The aim of this study was to develop a quantitative method for determining metabolic interactions between drugs and environmental chemicals in human liver microsome (HLM) incubations. HLMs contain high concentrations of CYP-enzymes, enabling the use of CYP-model reactions for observing interactions. The model reactions chosen for this study were O-deethylation of phenacetin (CYP1A2), 7-hydroxylation of coumarin (CYP2A6), 4'-hydroxylation of diclofenac (CYP2C9), 1'-hydroxylation of bufuralol (CYP2D6) and 6β-hydroxylation of testosterone (CYP3A4). Michaelis-Menten constants (Km) and maximal enzymatic activities (Vmax) were determined for each model reaction. The suitability of the model reactions for inhibition studies was assessed with specific inhibitors. The quantitative method was developed for an ultra-high performance liquid chormatograph (UPLC) and for a quadrupole time of flight mass spectrometer (QTOF). Samples were ionized with electrospray ionization (ESI) using positive mode. Device parameters were the same for all the metabolites. The analytical method validation was partly performed according to ICH (International Conference on Harmonisation) guidelines. A sufficient linearity (R2>0,99) and specificity was achieved for the quantitative method. The achieved limits of quantitation (LOQ) were low enough (1-120 nM) for quantitation of the small concentrations of the metabolites formed in the inhibition assays. The measurement reproducibility and the reproducibility and accuracy of the method did not fulfill the acceptance criteria for all the metabolites. Improvement of the results should be tried by e.g. exploring different device parameters. 1'-hydroxydiclofenac was found likely to degrade in the matrix solution because of the acidic conditions, making the reliability of the results poor for this metabolite. The Km value obtained for coumarin differed markedly from literature values, which can be due to a too long incubation time. Therefore, incubation conditions should be optimized for this model reaction in coming studies. The Km values obtained for the model reactions of CYP1A2, CYP2D6 and CYP3A4 were similar to those found in literature. Also the IC50 values were quite well within the range of values reported in literature for the inhibitors of the above mentioned model reactions. The effects of four different polymers, F68, F127, Tetronic 1307 and polyvinyl alcohol (PVA) on the enzyme activities were also studied, at a concentration of 1 mg/ml. In principal, at this concentration the polymers did not cause significant changes in the enzyme activities, although inhibition of the CYP2C9 could have been significant. However, the reliability of CYP2C9 model reaction was found to be poor with the used method. In the future this developed method should be further validated, and the incubation conditions for the model reaction of CYP2A6 should be optimized. After this, the IC50 values for the polymers could be studied to get more reliable information about their potential CYP-inhibition properties.

Neurosteroids are steroids which are active in the central nervous system. They have many biological and physiological functions in human body. Fluctuations of the neurosteroid concentrations are related to many diseases such as depression, schizophrenia and epilepsy. Neurosteroid levels are measured to understand their role in brain function and human behavior. The aim of the work was to develop a gas chromatographic-atmospheric pressure fotoionization-tandem mass spectrometric (GC-APPI-MS/MS) method for analyzing 19 neurosteroids and their metabolites in urine. Neurosteroids are excreted in urine mainly as conjugates, so they have to be hydrolyzed before analysis. Sample purification is done by liquid-liquid extraction and the analytes are subsequently derivatized to enhance their volatility. Because widely used β-glucuronidase/arylsulfatase-enzyme from Helix pomatia oxidases 3β-hydroxy-5-ene and 3β-hydroxy-5α-reduced steroids, we decided to use β-glucuronidase from Escherichia coli and acid hydrolysis instead of H. pomatia. The quantification of the total neurosteroid concentration in urine was challenging because β-glucuronidase enzyme from E. coli did not hydrolyze glucuronides completely and acid hydrolysis deconjugated also glucuronides in addition to sulfate conjugates. In addition the internal standard d4-allopregnanolone was noticed to be impure and degrade during acid hydrolysis. The limits of detection were reasonably low for the method (2 pg/ml-1 ng/ml). The retention times of the analyte peaks were very repeatable (RSD 0,06-0,11%) and the repeatability of the method was acceptable for all compounds (RSD < 27%). Urine samples from two males and two females were analyzed with the preliminary validated method. We could determine estimated concentrations for dehydroepiandrosterone, dihydrotestosterone, androstenedione, testosterone, estrone, β-estradiol, estriol, 5α-tetrahydrodeoxocorticosterone, cortisone, corticosterone and hydrocortisone. The developed method did not meet all the aims of this work. The method needs further validation and more exact investigation about the effect of the selected hydrolysis method on intact steroids. Also the internal standard should be changed to some other compound, preferably a non-deuterated one.

There are dozens of bromotyrosine alkaloids extracted from the marine sponge Pseudoceratina purpurea. Bromotyrosines have for example antibacterial, antiviral and antitumor activity in vitro and therefore bromotyrosines are potential drug lead molecules. By far, bromotyrosines have been extracted from different marine sponges of the Verongida order for the use in biological activity research. Collecting marine sponges by scuba diving is neither fast nor ecological and therefore finding an effective synthesis strategy is vital so that the research could continue. One new bromotyrosine, purpurealidin I, was found from the marine sponge Pseudoceratina purpurea in the four year (2010-2014) Marex-project. The aim of this thesis was to synthesize the compound purpurealidin I and its derivatives. Synthesized compounds were tested against hepatitis C and chikungunya viruses. It is important to find new potent drug molecules, because approximately 350 000-500 000 people die from hepatitis C and there is no curative medication for the chikungunya. Purpurealidin I is synthesized from tyrosine and tyramine parts, which will be put together to form an amide bond in the final step of the synthesis. The synthesis of purpurealidin I was not completed during the Master's thesis. However there were two purpurealidin I derivatives and four purpurealidin I tyramine part derivatives that were successfully synthesized. One of the compounds is purpurealidin E, which can be extracted from the sponge Pseudoceratina purpurea. The t-Boc derivative of purpurealidin E was examined against hepatitis C and chikungunya and the compound showed moderate activity against hepatitis C virus, but it wasn't active against chikungunya virus. The original plan to synthesize purpurealidin I is possible, although some reactions and purification of crude products need to be optimized in order to get better yields. For the future research derivatives of the t-Boc derivative of purpurealidin E should be synthesized and studied against hepatitis C virus.

River blindness (onchocerciasis) is a human helminth disease that is caused by Onchocerca volvulus filaria. It is endemic in tropical areas in Africa and Latin America. About 37 million people are infected. River blindness manifests in cutaneous and eye symptoms that are caused by the youngest forms (microfilariae). Against river blindness there has been mass drug treatments with ivermectin which has an effect on microfilariae. There is a need for a drug that kills adults or sterilizes females. A vaccine would be even better. Antibiotics are a new treatment because O. volvulus has Wolbachia bacterium as an obligate symbiont. Doxycycline kills at least 60 per cent of adults and sterilizes females. But the course lasts many weeks. A promising compound is emodepside that has a new mechanism of action for an anthelmintic drug. Numerous compounds has been examined to get drugs for filarial diseases. Some of them inhibit enzymes with which filariae evade human immune defence. Others disturb moulting that takes place four times. A good target for a drug is essential for the parasite but absent from mammals. Betulin is a triterpene that is abundant in birch bark. Betulin and many of its derivatives are pharmacologically active compounds that are examined particularly as cancer and HIV drugs. The research group of medicinal chemistry in University of Helsinki has synthesized and examined many derivatives. Some of them are promising for example against Leishmania protozoans, Chlamydia pneumoniae bacterium and alphaviruses. That is why those compounds should be tested against other causes of diseases like filariae. Both Wolbachia and C. pneumoniae have the same lipid biosynthesis pathway that is essential for both. Heterocycloadducts between betulin and nitrogen heterocycle were synthesized whose alcohols were oxidized to carbonyls. In both Leishmania donovani and L. braziliensis examinations the most effective compound was heterocycloadduct of formylbetulin. Although the compounds have not been examined against filariae, in future it would be worth an effort to synthesize derivatives that has nitrogen instead of carbonyls because the compound effective on C. pneumoniae is dioxime.

The aim of this study was to synthesize antimicrobial and anti-biofilm agents using abietic (AA) and dehydroabietic acids (DHAA). Bacterial biofilms are formed when bacteria cells cluster together within a self-produced extracellular matrix. This lifestyle makes bacteria highly resilient to different environmental stresses and conventional antibiotics when compared to single-cell bacteria. Currently, there are no approved anti-biofilm agents as drugs and only a few number of compounds can selectively target biofilms and eradicate them at low concentrations. Potent drugs targeting them are needed. AA and DHAA are abietane-type diterpenoids found in the resin of conifer trees. Antibacterial effects of resin acids have been widely studied, specifically against methicillin-resistant Staphylococcus aureus strain (MRSA). Through the combination of DHAA with different amino acids, Manner et al. (2015) discovered a new class of hybrid compounds that target both planktonic and biofilm bacteria in Staphylococcus aureus. The study group also discovered two of the most potent abietane-type anti-biofilm agents reported so far in literature. This thesis followed the work of the research group by designing and synthesizing additional AA and DHAA derivatives to target bacterial biofilms. Rings A, B and C of the diterpenoid core were modified and 24 derivatives were successfully synthesized. Amino acids were attached to the compounds either before or after ring modification. Standard structural elucidation techniques were used to confirm the structure of the synthesized compounds.

Seas are one of the most biodiverse and species-rich areas on the planet. Many of the underwater species are yet to be found and identified. The marine based drug discovery and the clinical pipeline of marine compounds have increased lately. Thus, there is a strong believe that the marine-derived compounds will provide new pharmaceutical lead compounds. Marine sponges are one of the most studied marine species. Sponges can be found in shallow and deep waters all over the world. Pseudoceratina purpurea is a Verondiga order sponge that is known to be a source of bromotyramines. Bromotyramines are tyramine derivatives that have represented biological activity including cytotoxity, antivirality and antimicrobial effects. Purpurealidin E is a bromotyramine that has been identified from Pseudoceratina purpurea. Purpurealidin E hasn't showed remarkable biological activity by itself, but it can be used as starting point for synthesis of novel bromotyramine derivatives. By forming an amide bond between carboxylic acid and primary amine of purpurealidin E, new bromotyramines can be synthesized. In this master's thesis, purpurealidin E was successfully synthesized. Total amount of 11 novel bromotyramine derivatives were synthesized by amide coupling. Three of the new bromotyramine derivatives and purpurealidin E were purified and their biological activity against hepatitis C virus (HCV) was evaluated. Purpurealidin E did not show any antiviral activity, but all the three compounds showed potential biological activity against HCV. This work can be considered to a continuum to the now ended MAREX project (Exploring Marine Resources for Bioactive Compounds: From Discovery to Sustainable Production and Industrial Applications).

Melanoma is the most severe case of skin cancer and there is no curative treatment if it has progressed. Despite the recent advances in drug therapy tens of thousands of patients die of melanoma annually. There is still need for new antimelanoma drugs for which marine compounds are a potential source. Halogens are common elements in drug molecules as they enhance their molecular properties. So far most of the halogenated drugs contain fluorine and/or chlorine but the role of bromine and iodine is probably growing in the future due to halogen bonding. Bromotyrosines are originally isolated from Verongiida-order sponges but whether they are truly of bacterial origin is under controversy. All bromotyrosine compounds consist of brominated tyrosine and/or tyramine residues or their derivatives. Purpurealidin I is one of the newest bromotyrosine derivatives extracted from Pseudoceratina purpurea and it has shown activity against melanoma. In this study eight new purpurealidin I derivatives were synthesized following a successful route previously designed. All synthesized derivatives contained the original N-oxime structure which's stereochemistry was determined to be E by X-ray crystallography. Cytotoxicity against A375 melanoma cells was determined for seven compounds synthesized here and for 15 compounds synthesized previously. All seven compounds and one previously synthesized purpurealidin I analog were active with CC50 values between 4,7 and 22,1 µM. The previously synthesized bromotyrosine derivative intermediates and aerophobin-1 analogs were not active. The selectivity of the active compounds was calculated by determining their CC50 value against Hs27 fibroblast cells. None of the compounds showed remarkable selectivity the most selective 2-pyridin containing derivative having four times better selectivity against melanoma. The tyrosine part and N-oxime seem to be important parts to preserve while the tyramine part can be modified more freely and maintain the activity. Still more derivatives need to be synthesized and tested to confirm these observations. More data is also needed considering the selectivity of the compounds.

The characteristics of macrolides are discussed in general level in the theoretical part of this Master's thesis. The discussion is focused on the properties of two macrolides in molecular level and their tendency to form tautomeric forms highlighting the structural similarities and differences of these macrolides, which will affect both the mechanisms of action and the metabolism. Attention is also paid to biosynthesis and manufacturing process keeping focus on downstream process, especially the impurities, which arise from the macrolide biosynthesis. Also the principles of argentation chromatography are discussed. In the experimental part of Master's thesis a purification method for one macrolide was developed using argentation chromatography. Conventional chromatographic purifications cannot separate the macrolide from its impurities. The purity of the macrolide after argentation chromatography was 98.6%. Also a new crystallization method was developed, which produces anhydrous form of the macrolide instead of traditional monohydrate form. A method for analysing the macrolide using HPLC was developed. The method was validated according to ICH guidelines. The tautomeric forms and the impurities of the macrolide were analysed using LC/MS. One of these impurities was isolated and analysed with NMR thus confirming its identity. An analysed NMR spectrum of this impurity has not been published according to our best knowledge. A previously unknown impurity was identified based on MS analysis and retention time.