Diss. (sammanfattning) Uppsala : Uppsala universitet, 2007. / Härtill 8 uppsatser.

This thesis describes the structure elucidation of drug metabolites in biological samples by the use of high performance liquid chromatography (HPLC) atmospheric pressure ionization (API) tandem mass spectrometry (MS/MS). Due to their different advantages, various mass analyzers have been used in the different experiments. The metabolism of clemastine, flutamide, and meloxicam were studied in vitro and/or in vivo in different species such as humans, dogs, and horses. Accurate mass measurements with the quadrupole-time of flight mass spectrometer and MSn data supplied by the ion trap instrument were useful in the structural investigation of the product ions of the drugs and their metabolites. Different scan modes of the triple quadrupole mass spectrometer resulted in great flexibility, selectivity, and sensitivity in the qualitative and semi-quantitative studies. Additionally, hydrogen/deuterium exchange and experiments with atmospheric pressure chemical ionization were conducted, and the fungus Cunninghamella elegans was utilized to produce amounts of drug metabolites sufficient for structural investigation. Six isomers of oxidized clemastine were detected and characterized in C. elegans incubations and their retention times and mass spectral data were compared to the metabolites detected in urine samples. Two of the metabolites were concluded to be diastereomeric N-oxides. In urine from horses treated with meloxicam, the peak of 5'-hydroxymethylmeloxicam resulted in much higher intensity than the parent drug or the other metabolites, and it was detectable for at least 14 days after the last dose in some of the horses. That is useful information in the development of analytical methods for the detection of prohibited use of meloxicam. A mercapturic acid conjugate of hydroxyflutamide was detected in urine from cancer patients, which indicated that a reactive metabolite was formed. This metabolite could be responsible for the adverse events reported for flutamide. The results from the four papers included in the thesis clearly demonstrate the usefulness and the flexibility of the HPLC-API-MS/MS technique.

Tuberculosis (TB) is a major infectious disease, killing about 2 million people annually throughout the world. Today's TB treatment is a lengthy procedure involving a combination of antibiotics. No new TB drug has been introduced onto the market in the past 40 years, and the emergence of multi- and extensively drug-resistant TB calls for new drugs. Finding new drug targets is important and one such target is the Mycobacterium tuberculosis enzyme glutamine synthetase (GS), which catalyses the formation of glutamine from glutamic acid. In this work, novel GS inhibitors and new Pd(0)-catalyzed methods have been developed. A microwave-enhanced Pd(0)-catalyzed α-arylation reaction was developed using water as solvent, and a phenylglycine scaffold was identified using structure-based design. A series of α-arylated phenylglycine derivates was produced at moderate to good yields. Some of these were biologically evaluated against GS. A novel scaffold, 3-amino-imidazo[1,2-a]pyridine, was identified by high-throughput screening directed towards GS. This type of compound could be easily produced via a Ugi-type, microwave-promoted multi-component reaction in 20 min. The scaffold was investigated by changing one substituent at a time, and in an experimental design where 8 factors were varied in the same design. Several potent inhibitors were identified; amongst them the most potent inhibitor to date (IC50 = 0.38 µM). Two discrete structure-activity relationships were established, and one of the inhibitors was co-crystallized. The first general aminocarbonylation of aryl chlorides and the first aminocarbonylation of alkenyl phosphates were developed. Alkenyl chlorides, bromides and triflates were investigated in the same transformation utilizing Mo(CO)6 as a solid carbon monoxide source. Two different Pd(0)-based catalytic systems were developed. A wide variety of aryl chlorides and amines could be transformed into the corresponding amides with good yields. The alkenyl substrates produced low to good yields.

Tuberculosis kills approximately 1.7 million people each year around the world making it one of the most lethal infectious diseases. This thesis concerns the development of two computational tools that can support the early stages of drug discovery, and their use in an anti-tubercular drug discovery program. One of the tools developed is a statistical molecular design (SMD) approach that generates information-rich libraries biased towards a lead structure. The other metod is a post-filtering technique to increase the success of virtual screening, has also been developed. Both methods have been validated using literature data. Ribonucleotide reductase (RNR) has been identified as a potential anti-tubercular target, and our focus has been to develop small-molecule inhibitors of this target. The enzyme consists of two subunits (a large R1 and a small R2 subunit) that have to associate in order to generate a bioactive complex. It had previously been shown that a heptapeptide corresponding to the small R2 subunits C-terminal inhibited the enzyme. In order to investigate the requirements for inhibitory effect of the peptide a library was designed using the developed SMD approach. The designed library was synthesized and evaluated for biological activity and an OPLS-DA model was derived to understand which positions were most important for activity. In order to identify small-molecule inhibitors of RNR a combined shape- and structure-based virtual screen was performed, employing ROCS, GlideXP and the developed post-filtering technique. Starting from a library of 1.5 million compounds 24 was acquired and evaluated for enzymatic activity. The best compounds were almost as potent as the starting peptide, but considerably more drug-like.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2010. / Härtill 4 uppsatser.

<p>Capillary electroseparation methods<b> </b>are exciting new techniques with very broad application areas and vast potential in pharmaceutical and biomedical analysis.</p><p> To improve the limit of detection (LOD) capillary zone electrophoresis (CZE) has been combined with isotachophoretic (ITP) preconcentration in a single capillary. Using the ITP-CZE combination the LOD can be improved at least 100-fold. Laser-induced fluorescence (LIF) detection is more sensitive and more selective than the most common detection technique, UV, and the intensity and focusing capability of LIF fits well with the small dimensions in CZE. The total sensitivity enhancement attained for a new acetylcholinesterase inhibitor, NXX-066, by using ITP-CZE-LIF was more than 5500-fold compared to CZE-UV.</p><p> Capillary electrochromatography (CEC) combines the high separation efficiency of CZE with the vast possibilities to improve selectivity of HPLC. We have examined different ways to solve the problem of extensively tailing peaks and studied the influence of the mobile phase composition on the electrochromatographic performance for a number of tricyclic antidepressants and related quaternary ammonium compounds. (1) Adding aliphatic amines to the mobile phase in reversed phase CEC. The effect on the chromatographic performance was coupled to the hydrophobicity of the additive and the amine of our choice was dimethyloctylamine. (2) Silica-based cation exchangers with different pore sizes. The large-pore materials promoted pore flow, but this had no positive influence on the performance. The small-pore (highest surface area) particles gave the best selectivity. (3) Designing special continuous beds. As the bed is covalently attached to the capillary wall, problems related to retaining frits are avoided. The stationary phase most suitable for our analytes had a molar ratio of 1:80 between the functional ligands, vinyl sulphonic acid and isopropyl groups, respectively. The LOD was lowered 26000-fold by dissolving the sample in a low-conducting medium.</p>

<p>Capillary electrophoresis-mass spectrometry has been used successfully for the analysis of a wide range of analytes such as chiral local anaesthetics, sulphonated reactive dyes and endogenous neurotransmitters and neuropeptides.</p><p>The partial filling technique was used in CE-MS for chiral separation of bupivacaine and ropivacaine using the non-volatile selector β-cyclodextrin. By only partially filling the capillary with selector and using capillaries coated with polyacrylamide to suppress the electroosmotic flow, introduction of the selector into the mass spectrometer was avoided. An impurity of 0.25% of the R-enantiomer of ropivacaine in the S-form could be detected.</p><p>The partial filling technique was developed further using CE employing two different selectors in separate plugs in the capillary. This enhanced the separation efficiency and offered greater flexibility in controlling the separation.</p><p>By using transient-isotachophoresis (tITP)-CE-MS it was possible to concentrate and detect classical neurotransmitters and neuropeptides with masses ranging from 104 Da to 1642 Da. γ -Aminopropyltriethoxysilane coated capillaries were used to minimize adsorption of the peptides onto to capillary surface. Endogenous dopamine, glutamate, γ-aminobutyric acid (GABA), acetylcholine, methionine-enkephalin and substance P 1-7 were detected in the striatum of marmoset monkey.</p><p>Sulphonated dyes obtained from single textile fibres were analysed using CE-MS. Capillary electrophoresis was found to be a good way of removing the excess amounts of glucose present in the sample that would otherwise interfere with the electrospray ionisation. </p><p>Automatic function switching, originally developed for use together with liquid chromatography, was found to be a great method for acquiring MS/MS data when doing infusion experiments saving both time and sample without decreasing the quality of the MS/MS data. It was also found to be a more time efficient way than using the precursor ion scanning mode on the Q-TOF to obtain precursor ion data.</p>

<p>The aim of this work was to explore the structural and functional diversity of polypeptides that are found in plants. Expanding knowledge of simililarities between plant use of these compound and animal use promises exceptional opportunities for finding, from plant research, new structures with biomedical and biotechnological potential.</p><p>A fractionation protocol was developed and applied to many plant species, providing fractions enriched in polypeptides, amenable to chemical and biological evaluation. From one species, the common field pansy (<i>Viola arvensis</i>), a 29-amino-acid residue polypeptide was isolated, named varv A, which revealed a remarkable macrocyclic structure (i.e., N- and C-termini are joined) stabilised by three knotted disulfides<i>. </i></p><p>Varv A, together with an increasing number of homologous peptides, form the currently known peptide family of cyclotides. Their stable structure makes them an attractive scaffold for protein engineering. In addition, they display a wide range of biological activities (e.g., antimicrobial, cytotoxic, and insecticidal). As a part of this work, the cytotoxic effects of varv A and two other isolated cyclotides were evaluated in a human cell-line panel: all were active in the low µM range. Most likely, these effects involve pore formation through cell membranes.</p><p>Cyclotides were found to be common in the plant family<i> Violaceae; </i>with eleven cyclotides isolated and sequenced from V. arvensis, V. cotyledon, and<i> Hybanthus parviflorus. </i>For six members of the genus <i>Viola</i>, cyclotide expression profiles were examined by liquid chromatography-mass spectrometry (LC-MS): all expressed notably complex mixtures, with single species containing more than 50 cyclotides. These profiles reflect the evolution of the genus<i>.</i></p><p>To assess these mixtures, a rational strategy for MS based amino acid sequencing of cyclotides was developed, circumventing inherent structural problems, such as low content of positively charged amino acids and the macrocyclic structure. This was achieved by aminoethylation of cysteines, which, following tryptic digestion, produced fragments of size and charge amenable to MS analysis. This method was also modified and used for mapping of disulfide bonds<i>. </i></p><p>Methods for isolation and characterisation developed in this work may prove useful not only for further studies on macrocyclic polypeptides from plants, but also for other plant peptides and disulfide-rich peptides from animals.</p>

<p>Aspartyl proteases can generally be inhibited by peptide mimics containing an uncleavable peptide bond isostere at the proposed cleavage site. One such peptide bond isostere is the hydroxyethylamine moiety, which in this thesis has successfully been incorporated in potential inhibitors of the HIV-1-protease as well as the malarial proteases plasmepsin I and II.</p><p>The human immunodeficiency virus (HIV) has during the last 20 years given rise to a new fast-spread epidemic. The virus protease is one of the foremost targets for drug intervention. In an attempt to improve an earlier design, a P1'-anthranilic acid was exchanged for all four isomers of 2-aminocyclopentanecarboxylic acid, which were synthesized from racemic starting materials, the <i>trans</i> isomers via a novel synthetic route. None of the isomers enhanced potency as compared to the anthranilic acid.</p><p>Because of increasing development of resistance, the pharmaceutical intervention with malaria is becoming rapidly more difficult. A prominent new target for drug research is the hemoglobin degradation pathway. Two of the many proteases involved in this pathway are plasmepsin I and II. Two series of peptide mimics with the hydroxyethylamine were prepared and tested against these enzymes as well as against the similar human protease cathepsin D.</p><p>In the first series the central nitrogen of the target compounds is a secondary amine, derived from natural and unnatural amino acids, the side-chain of which was to bind in the S1'-site of the proteases. It was found that <i>para</i>-aryl substituted phenylalanines resulted in the most active inhibitors. While the P1- and P2-side-chains were kept constant at benzyl and isopropyl respectively, the P3 capping carboxylic acid was varied with a set of diverse carboxylic acids. It was found that many of the carboxylic acids were acceptable.</p><p>A selection of compounds was tested for inhibition of parasite growth in infected human erythrocytes and found to be active.</p><p>In the target compounds of the second series the P1'-side-chain was moved from the α-carbon of the initial amino acids to the adjacent nitrogen, thus rendering this a tertiary amine. The SAR of these compounds suggests that this side-chain cannot be larger than benzyl, which is in sharp contrast to the first series, where both isomers of phenylalanine (i.e. a benzyl group on the α-carbon) render inactive compounds.</p><p>Most of the compounds show a good degree of selectivity for the plasmepsins over cathepsin D, even though a few good inhibitors of the human enzyme could be identified also.</p>

<p>Carbon-carbon bond formation reactions are among the most important processes in chemistry, as they represent key steps in the synthesis of more complex molecules from simple precursors. This thesis describes mainly the development of novel regioselective applications of the mild and versatile palladium-catalyzed carbon-carbon coupling method, commonly known as the Heck reaction. In addition, this thesis will focus on environmentally friendly developments of the Heck reaction.</p><p>Novel ligand-controlled internal Heck vinylations of vinyl ethers and enamides to form branched electron-rich dienes were performed with high regioselectivity. The vinylation of 2-hydroxyethyl vinyl ether permits a chemoselective transformation of a vinylic triflate or bromide into a blocked α,β-unsaturated methyl ketone. Furthermore, a simple separation of the palladium catalyst was achieved with new fluorous-tagged bidentate ligands in combination with fluorous solid phase extraction. The reaction times could be reduced up to 1000 times with controlled microwave heating in the palladium-catalyzed reactions with, in the majority of cases, retained, high selectivity. </p><p>The development of a “green” regioselective arylation and vinylation method relying on an aqueous DMF-potassium carbonate system and excluding the toxic thallium salt has been accomplished. Ionic liquids as the versatile and environmentally friendly class of solvents have been used in rapid phosphine-free terminal Heck arylations with controlled microwave heating. Recycling of the catalytic medium was achieved after a simple product purification.</p>