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.

The main focus of this thesis is on the effects caused by α-helical peptides on liposome structure, the impact of cholesterol on the peptide-membrane interactions, and the effect of aggregate curvature on the peptide affinity. Results of the studies show that the membrane destabilizing effect of the cationic α-helical peptide melittin is modulated by cholesterol. Melittin induces leakage from pure phospholipid liposomes in a manner that is compatible with the presence of small pores. In the case of cholesterol-supplemented liposomes leakage coincides, however, with major structural transformations and rupture of the liposomes. Cholesterol decreases the affinity of melittin for phospholipid bilayers, but once the peptide has adsorbed to the membrane the presence of cholesterol does not offer any resistance against melittin-induced membrane destabilization. Our investigations indicate that cholesterol affects the alamethicin-lipid bilayer interactions in a similar way. PEG-stabilized bilayer disks are formed upon addition of polyethylene glycol (PEG)-lipids to lipid mixtures with high bending rigidity. The partial segregation of components within the bilayer disk, suggested by theoretical calculations and experimental observations, was verified by small angle neutron scattering. By means of a novel competitive binding assay it was shown that the three α-helical peptides melittin, alamethicin, and magainin have high affinity for the curved rim of PEG-stabilized bilayer disks. The bilayer disks have structural, and other properties, that make them interesting for the formulation of peptides and membrane-associated proteins. For stability reasons dry formulations are often preferred. The PEG-stabilized bilayer disks were shown to retain their structure in rehydrated samples that had been freeze- or spray-dried in the presence of lactose.

Chicken antibodies, commonly referred to as IgY, have several properties that make them suitable for oral treatment of infections and there is essentially no risk for development of resistance. The overall aims of this thesis were to investigate Anti-Pseudomonas IgY as prophylaxis against infections with Pseudomonas aeruginosa for cystic fibrosis (CF) patients and to characterize the antibody treatment. We found that Anti-Pseudomonas IgY has affinity for P. aeruginosa flagellin, the major component of the flagellum. This is important since the flagellum is required for host invasion and establishment of infection. Flagellin induces inflammation. The main cause of morbidity and mortality among CF patients is chronic colonization of the airways with P. aeruginosa. We have studied prophylactic treatment of 17 Swedish CF patients with Anti-Pseudomonas IgY for up to twelve years. The results were compared with a control group of 23 Danish CF patients. Patients treated with IgY had 2.3 P. aeruginosa positive cultures/100 treatment months vs. 7.0 cultures/100 treatment months in the control group (p=0.028), and the time from inclusion to the first recolonization was significantly longer in the IgY-treated group (p=0.012). Lung function was preserved and patients treated with IgY had good nutritional status at the end of the study. Furthermore, other bacteria have not emerged instead of P. aeruginosa. Freeze-drying of IgY and the content of IgY preparations for oral use was investigated. Besides IgY, 26 egg yolk proteins were identified. Some of the proteins are known to have antimicrobial and immunostimulatory effects, and could have a positive additive effect to IgY treatment. Cholesterol levels were low. Conclusion: Anti-Pseudomonas IgY is a promising complement in the prevention of Pseudomonas aeruginosa infections in CF patients, partly explained by the fact that IgY binds to flagellin. / Felatkigt ISBN ändrat: Tidigare 978-91-554-7477-5 nu 978-91-554-7478-2

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.

This thesis describes the synthesis of molecules designed for inhibition of two aspartic proteases, viral HIV-1 PR and human BACE-1. It also reports on the structure activity relationships of the targeted enzyme inhibitors. It is estimated that currently 33 million people are infected with HIV, the causative agent of AIDS. The virus targets T-lymphocytes and macrophages of the human immune system. The HIV-1 PR plays an important role in the viral replication, and by inhibiting the enzyme the disease progression can be slowed down or even halted. Herein is reported the design and synthesis of a series of HIV-1 PR inhibitors with novel P2 substituents of which several inhibit the enzyme in the nanomolar range. The aim of the second work was to further develop the inhibitors by the introduction of fluorine. Several attempts were performed to fluorinate different P2-substituents. Alzheimer’s disease (AD) is neurodegenerative, progressive and fatal disorder of the brain. It is associated with accumulation of plaques and tangles that cause impairment and functional decline of brain tissue which result in loss of memory and cognition. The plaques are mainly constituted of amyloid-β peptides that are generated in two steps from the amyloid precursor protein (APP). The cleavage sequence is initiated by the aspartic protease BACE-1, which makes the enzyme a key target in the effort of finding a therapy that aim to slow down the progression of AD. Herein are enclosed the development of two series of potent BACE-1 inhibitors. In the first work a synthetic strategy was developed to truncate a previously reported hydroxyethylene core structure in order to generate more drug-like inhibitors. This generated a series of truncated inhibitors where two amide bonds have been replaced with an ether - or alternatively a secondary amine linkage. A number of these inhibitors show potency against BACE-1. In the second part of the work the aim was investigate the effect of alterations in the P1 position. Five scaffolds with new P1 substituents were designed, synthesized and coupled with two different P2-P3 substituents. This resulted in a series of potent inhibitors that inhibit BACE-1 in the nanomolar range. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: Submitted. Paper 2: Submitted. Paper 3: Manuscript.

This thesis focuses on the use of molecularly imprinted polymers as selective sorbents for solid-phase extraction (MISPE). The MISPE methods developed were mainly intended for use with biological samples, such as human urine and blood plasma. These body fluids are complex samples, which often need an effective clean-up step before analysis to reduce the levels of possible interfering substances from the matrix, especially if the analytes are present in trace amounts. Solid-phase extraction (SPE) is a well-established and routinely used method for clean-up and preconcentration of samples from diverse matrices. However, conventional SPE sorbents often lack selectivity, leading to co-extraction of interferences, which negatively affects the following detection method. One of the advantages of MISPE is the built-in selectivity for a target analyte, or class of structurally related analytes, enabling the efficient clean-up that is often required for biological samples. The built-in selectivity of MISPE originates from the preparation of a highly crosslinked copolymer network in the presence of an imprint molecule, i.e. the template. Subsequent removal of this template molecule leads to the creation of defined recognition sites, complementary to the shape and functionality of the template. In this work, molecularly imprinted polymers were synthesized for the first time for several types of target analytes, including diphosphate esters (Papers I-III) and a protein adduct (Paper IV) and evaluated as sorbents for solid-phase extraction. A MISPE method for extracting local anaesthetic drugs from human plasma was also evaluated (Paper V). The development of appropriate methods for using the prepared polymers to extract target analytes directly from body fluids, and the elucidation of factors that influence their performance, were major foci of all the work underlying this thesis. These are not straightforward tasks, since the recognition mechanism of the material is often based on polar interactions, which are not favoured in aqueous environments. In such cases, non-selective adsorption of the analyte(s) to the polymer surface often occurs. In order to use the MIP sorbent most effectively it is important to suppress this non-selective adsorption, without disrupting the selective adsorption of the target analyte(s) to the imprints. Generally in these studies, this strong analyte-polymer surface interaction could be repressed, and selective adsorption enhanced, by carefully optimising the conditions for washing the sorbent, in terms of organic solvent volumes, solvent polarity and the addition of an ionic modifier. The sample matrix, mainly urine, was found to strongly decrease the capacity of the MIP. Hence, this effect was further investigated. It was found that the presence of NaCl in the sample negatively affected the recovery and repeatability of the method. Furthermore, these parameters could be improved by adjusting the sample pH. It was important to control the pH of the sample, in order both to achieve selective extraction and to increase the extraction recoveries. The selectivity of MISPE for the extraction of diphosphate esters from human urine was demonstrated by comparing its performance with that of a conventional SPE sorbent, a mixed-mode-anion exchanger (MAX). Due to its efficient clean-up, MISPE generated extracts that yielded less complex ion chromatograms in subsequent LC/ESI-MS analysis than extracts from the MAX cartridge. Due to its efficient clean-up, MISPE generated extracts that yielded less complex ion chromatograms in subsequent LC/ESI-MS analysis than extracts from the MAX cartridge. Signal suppression from the interfering co-eluting compounds was detected when the MAX extracts were analysed, which was not the case for the MISPE extracts. These findings show the importance of efficient and selective sample preparation, even if a selective detector is used. Development of LC/ESI-MS methods was also an extensive component of this work (Papers I-IV). Different chromatographic conditions have been evaluated for the optimal separation and detection of the investigated compounds. Use of ion-pairing agents and suitable HPLC columns (Hypercarb and C18 Aquasil) for the acidic, polar analytes, was found to give better retention and separation than use of conventional reversed-phase columns. To improve the selectivity and detectability further, selected ion monitoring (SIM) and selected reaction monitoring (SRM) acquisition modes were used for quantification of the investigated compounds. In summary, the aim of this work was to contribute to the knowledge of the recognition mechanisms of molecularly imprinted polymers in aqueous matrices, which is important for extending the use of MISPE for several types of bioanalytical applications.

The first part of this thesis addresses the design and synthesis of amine building blocks accomplished by applying two different synthetic procedures, both of which were developed using solid-phase chemistry. Chapter 1 presents the first of these methods, entailing a practical solid-phase parallel synthesis route to N-monoalkylated aminopiperidines and aminopyrrolidines achieved by selective reductive alkylation of primary and/or secondary amines. Solid-phase NMR spectroscopy was used to monitor the reactions for which a new pulse sequence was developed. The second method, reported in Chapter 2, involves a novel approach to the synthesis of secondary amines starting from reactive alkyl halides and azides. The convenient solid-phase protocol that was devised made use of the Staudinger reaction in order to accomplish highly efficient alkylations of N-alkyl phosphimines or N-aryl phosphimines with reactive alkyl halides. The second part of the thesis describes the design and synthesis of three classes of protease inhibitors targeting the cysteine proteases cathepsins S and K, and the serine protease hepatitis C virus (HCV) NS3 protease. Chapter 4 covers the design, solid-phase synthesis, and structure-activity relationships of 4-amidofurane-3-one P1-containing inhibitors of cathepsin S and the effects of P3 sulfonamide groups on the potency and selectivity towards related cathepsin proteases. This work resulted in the discovery of highly potent and selective inhibitors of cathepsin S. Two parallel solid-phase approaches to the synthesis of a series of aminoethylamide inhibitors of cathepsin K are presented in Chapter 5. Finally, Chapter 6 reports peptide-based HCV NS3 protease inhibitors containing a non-electrophilic allylic alcohol moiety as P1 group and also outlines efforts to incorporate this new template into low-molecular-weight drug-like molecules.

The asymmetric hydrogenation of olefins is a facile and popular method of reaching chiral products. Whereas ruthenium- and rhodium-catalyzed asymmetric hydrogenations have a long history, the use of iridium in this area is new but fast-growing. Since the first chiral N,P-ligated iridium catalyst was created in the late 1990s, the growing pool of N,P ligands has filled up rapidly, but most have been tested with a limited range of standard olefins. To extract the full potential of these complexes, new methods using substrates having many possible applications must be developed. This thesis focuses on the iridium-catalyzed asymmetric hydrogenation of three different new substrate classes to yield very high conversions and enantiomeric excesses (ee's). As the use of fluorine has recently become common in many different fields of chemistry, the asymmetric reduction of fluoroolefins to reach chiral products having fluorine at the stereogenic centers is highly interesting. We studied this reaction and eventually obtained very high ee values and lower degree of defluorination (Paper I and Paper II). The hydrogenations of trifluoromethylated olefins to reach products useful in applications reaching from pharmaceuticals to additives in liquid crystal displays (LCDs) were also challenging, but fruitful (Paper III). As asymmetric hydrogenation usually demands differences in the substituents of the double bond, the highly selective reduction of 1,1-diaryl olefins having similar aryls give a new perspective on the broad scope of substrates that N,P-ligated iridium complexes can reduce selectively (paper IV).

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