Purification of pharmaceuticals and nutraceutical compounds by sub- and supercritical extraction and chromatography

This thesis discusses the use of sub- and supercritical fluids as the medium in extraction and chromatography. Super- and subcritical extraction was used to separate essential oils from herbal plant Angelica archangelica. The effect of extraction parameters was studied and sensory analyses of the extracts were done by an expert panel. The results of the sensory analyses were compared to the analytically determined contents of the extracts. Sub- and supercritical fluid chromatography (SFC) was used to separate and purify high-value pharmaceuticals. Chiral SFC was used to separate the enantiomers of racemic mixtures of pharmaceutical compounds. Very low (cryogenic) temperatures were applied to substantially enhance the separation efficiency of chiral SFC. The thermodynamic aspects affecting the resolving ability of chiral stationary phases are briefly reviewed. The process production rate which is a key factor in industrial chromatography was optimized by empirical multivariate methods. General linear model was used to optimize the separation of omega-3 fatty acid ethyl esters from esterized fish oil by using reversed-phase SFC. Chiral separation of racemic mixtures of guaifenesin and ferulic acid dimer ethyl ester was optimized by using response surface method with three variables per time. It was found that by optimizing four variables (temperature, load, flowate and modifier content) the production rate of the chiral resolution of racemic guaifenesin by cryogenic SFC could be increased severalfold compared to published results of similar application. A novel pressure-compensated design of industrial high pressure chromatographic column was introduced, using the technology developed in building the deep-sea submersibles (Mir 1 and 2). A demonstration SFC plant was built and the immunosuppressant drug cyclosporine A was purified to meet the requirements of US Pharmacopoeia. A smaller semi-pilot size column with similar design was used for cryogenic chiral separation of aromatase inhibitor Finrozole for use in its development phase 2.

Synthesis, Reactivity and Biological Activity of 17β-HSD1 Inhibitors Based on a Thieno[2,3-d]pryrimidin-4(3H)-one Core

Breast cancer is the most common cancer in women in Western countries. In the early stages of development most breast cancers are hormone-dependent, and estrogens, especially estradiol, have a pivotal role in their development and progression. One approach to the treatment of hormone-dependent breast cancers is to block the formation of the active estrogens by inhibiting the action of the steroid metabolising enzymes. 17beta-Hydroxysteroid dehydrogenase type 1 (17beta-HSD1) is a key enzyme in the biosynthesis of estradiol, the most potent female sex hormone. The 17beta-HSD1 enzyme catalyses the final step and converts estrone into the biologically active estradiol. Blocking 17beta-HSD1 activity with a specific enzyme inhibitor could provide a means to reduce circulating and tumour estradiol levels and thus promote tumour regression. In recent years 17beta-HSD1 has been recognised as an important drug target. Some inhibitors of 17beta-HSD1 have been reported, however, there are no inhibitors on the market nor have clinical trials been announced. The majority of known 17beta-HSD1 inhibitors are based on steroidal structures, while relatively little has been reported on non-steroidal inhibitors. As compared with 17beta-HSD1 inhibitors based on steroidal structures, non-steroidal compounds could have advantages of synthetic accessibility, drug-likeness, selectivity and non-estrogenicity. This study describes the synthesis of large group of novel 17beta-HSD1 inhibitors based on a non-steroidal thieno[2,3-d]pyrimidin-4(3H)-one core. An efficient synthesis route was developed for the lead compound and subsequently employed in the synthesis of thieno[2,3-d]pyrimidin-4(3H)-one based molecule library. The biological activities and binding of these inhibitors to 17beta-HSD1 and, finally, the quantitative structure activity relationship (QSAR) model are also reported. In this study, several potent and selective 17beta-HSD1 inhibitors without estrogenic activity were identified. This establishment of a novel class of inhibitors is a progressive achievement in 17beta-HSD1 inhibitor development. Furthermore, the 3D-QSAR model, constructed on the basis of this study, offers a powerful tool for future 17beta-HSD1 inhibitor development. As part of the fundamental science underpinning this research, the chemical reactivity of fused (di)cycloalkeno thieno[2,3-d]pyrimidin-4(3H)-ones with electrophilic reagents, i.e. Vilsmeier reagent and dimethylformamide dimethylacetal, was investigated. These findings resulted in a revision of the reaction mechanism of Vilsmeier haloformylation and further contributed to understanding the chemical reactivity of this compound class. This study revealed that the reactivity is dependent upon a stereoelectronic effect arising from different ring conformations.

Sources and concentrations of volatile organic compounds

Volatile organic compounds (VOCs) have a great influence on tropospheric chemistry; they affect ozone formation and they or their reaction products are able to take part in secondary organic aerosol formation; some of the VOCs are themselves toxic. Knowing the concentrations and sources of different reactive volatile organic compounds is essential for the development of ozone control strategies and for studies of secondary organic aerosol formation. The objective of this work was to study volatile organic compounds in urban air, develop and validate determination methods for them, characterize their concentrations and estimate the contributions of different VOC sources. Of the different compound groups detected in the urban air of Helsinki, alkanes were found to have the highest concentrations, but when the concentrations were scaled against the reactivity with hydroxyl radicals (OH), aromatic hydrocarbons and alkenes were found to have the greatest effect on local chemistry. Comparisons with rural sites showed that concentrations at Utö and Hyytiälä were generally lower than those in Helsinki, especially for the alkenes and aromatic hydrocarbons, but concentrations of halogenated hydrocarbons at Utö and carbonyls at Hyytiälä were at the same level as in Helsinki. Most halogenated hydrocarbons do not have any significant sources in Helsinki, and carbonyls are formed in the atmosphere in the reactions of other VOCs, and are therefore also produced in other than urban areas. At Hyytiälä carbonyls were found to have an important role in the local chemistry. The contribution of carbonyls as an OH sink was higher than that of the monoterpenes and aromatic hydrocarbons. Based on the emission profile and concentration measurements, the contributions of different sources were estimated at urban (Helsinki) and residential (Järvenpää) sites using a chemical mass balance (CMB) receptor model. It was shown that it is possible to apply CMB in the case of a large number of different compounds with different properties. According to the CMB analysis, the major sources for these VOCs in Helsinki were traffic and distant sources. At the residential site in Järvenpää, the contribution due to traffic was minor, while distant sources, liquid gasoline and wood combustion made higher contributions. It was also shown that wood combustion can be an important source at some locations of VOCs usually considered as traffic-related compounds (e.g., benzene).

Development of Sample Pretreatment and Liquid Chromatographic Techniques for Antioxidative Compounds

In this study, novel methodologies for the determination of antioxidative compounds in herbs and beverages were developed. Antioxidants are compounds that can reduce, delay or inhibit oxidative events. They are a part of the human defense system and are obtained through the diet. Antioxidants are naturally present in several types of foods, e.g. in fruits, beverages, vegetables and herbs. Antioxidants can also be added to foods during manufacturing to suppress lipid oxidation and formation of free radicals under conditions of cooking or storage and to reduce the concentration of free radicals in vivo after food ingestion. There is growing interest in natural antioxidants, and effective compounds have already been identified from antioxidant classes such as carotenoids, essential oils, flavonoids and phenolic acids. The wide variety of sample matrices and analytes presents quite a challenge for the development of analytical techniques. Growing demands have been placed on sample pretreatment. In this study, three novel extraction techniques, namely supercritical fluid extraction (SFE), pressurised hot water extraction (PHWE) and dynamic sonication-assisted extraction (DSAE) were studied. SFE was used for the extraction of lycopene from tomato skins and PHWE was used in the extraction of phenolic compounds from sage. DSAE was applied to the extraction of phenolic acids from Lamiaceae herbs. In the development of extraction methodologies, the main parameters of the extraction were studied and the recoveries were compared to those achieved by conventional extraction techniques. In addition, the stability of lycopene was also followed under different storage conditions. For the separation of the antioxidative compounds in the extracts, liquid chromatographic methods (LC) were utilised. Two novel LC techniques, namely ultra performance liquid chromatography (UPLC) and comprehensive two-dimensional liquid chromatography (LCxLC) were studied and compared with conventional high performance liquid chromatography (HPLC) for the separation of antioxidants in beverages and Lamiaceae herbs. In LCxLC, the selection of LC mode, column dimensions and flow rates were studied and optimised to obtain efficient separation of the target compounds. In addition, the separation powers of HPLC, UPLC, HPLCxHPLC and HPLCxUPLC were compared. To exploit the benefits of an integrated system, in which sample preparation and final separation are performed in a closed unit, dynamic sonication-assisted extraction was coupled on-line to a liquid chromatograph via a solid-phase trap. The increased sensitivity was utilised in the extraction of phenolic acids from Lamiaceae herbs. The results were compared to those of achieved by the LCxLC system.

Measurements of Volatile Organic Compounds - from Biogenic Emissions to Concentrations in Ambient Air

Volatile organic compounds (VOCs) affect atmospheric chemistry and thereafter also participate in the climate change in many ways. The long-lived greenhouse gases and tropospheric ozone are the most important radiative forcing components warming the climate, while aerosols are the most important cooling component. VOCs can have warming effects on the climate: they participate in tropospheric ozone formation and compete for oxidants with the greenhouse gases thus, for example, lengthening the atmospheric lifetime of methane. Some VOCs, on the other hand, cool the atmosphere by taking part in the formation of aerosol particles. Some VOCs, in addition, have direct health effects, such as carcinogenic benzene. VOCs are emitted into the atmosphere in various processes. Primary emissions of VOC include biogenic emissions from vegetation, biomass burning and human activities. VOCs are also produced in secondary emissions from the reactions of other organic compounds. Globally, forests are the largest source of VOC entering the atmosphere. This thesis focuses on the measurement results of emissions and concentrations of VOCs in one of the largest vegetation zones in the world, the boreal zone. An automated sampling system was designed and built for continuous VOC concentration and emission measurements with a proton transfer reaction - mass spectrometer (PTR-MS). The system measured one hour at a time in three-hourly cycles: 1) ambient volume mixing-ratios of VOCs in the Scots-pine-dominated boreal forest, 2) VOC fluxes above the canopy, and 3) VOC emissions from Scots pine shoots. In addition to the online PTR-MS measurements, we determined the composition and seasonality of the VOC emissions from a Siberian larch with adsorbent samples and GC-MS analysis. The VOC emissions from Siberian larch were reported for the fist time in the literature. The VOC emissions were 90% monoterpenes (mainly sabinene) and the rest sesquiterpenes (mainly a-farnesene). The normalized monoterpene emission potentials were highest in late summer, rising again in late autumn. The normalized sesquiterpene emission potentials were also highest in late summer, but decreased towards the autumn. The emissions of mono- and sesquiterpenes from the deciduous Siberian larch, as well as the emissions of monoterpenes measured from the evergreen Scots pine, were well described by the temperature-dependent algorithm. In the Scots-pine-dominated forest, canopy-scale emissions of monoterpenes and oxygenated VOCs (OVOCs) were of the same magnitude. Methanol and acetone were the most abundant OVOCs emitted from the forest and also in the ambient air. Annually, methanol and mixing ratios were of the order of 1 ppbv. The monoterpene and sum of isoprene 2-methyl-3-buten-2-ol (MBO) volume mixing-ratios were an order of magnitude lower. The majority of the monoterpene and methanol emissions from the Scots-pinedominated forest were explained by emissions from Scots pine shoots. The VOCs were divided into three classes based on the dynamics of the summer-time concentrations: 1) reactive compounds with local biological, anthropogenic or chemical sources (methanol, acetone, butanol and hexanal), 2) compounds whose emissions are only temperaturedependent (monoterpenes), 3) long-lived compounds (benzene, acetaldehyde). Biogenic VOC (methanol, acetone, isoprene MBO and monoterpene) volume mixing-ratios had clear diurnal patterns during summer. The ambient mixing ratios of other VOCs did not show this behaviour. During winter we did not observe systematical diurnal cycles for any of the VOCs. Different sources, removal processes and turbulent mixing explained the dynamics of the measured mixing-ratios qualitatively. However, quantitative understanding will require longterm emission measurements of the OVOCs and the use of comprehensive chemistry models. Keywords: Hydrocarbons, VOC, fluxes, volume mixing-ratio, boreal forest

2,1,3-Bentsoksadiatsolirakenteiset molekyylit antibakteerisina ja antiparasiittisina yhdisteinä

Aikaisemman tutkimuksen perusteella tiedettiin tiettyjen 2,1,3-bentsoksadiatsolirakenteisten molekyylien olevan aktiivisia Chlamydia pneumoniae –bakteeria vastaan. Tutkimusta lähdettiin jatkamaan ja 2,1,3-bentsoksadiatsolimolekyylien rakenne-aktiivisuusuhteista haluttiin saada lisätietoa. Tarkoituksena oli kehittää 2,1,3-bentsoksadiatsolimolekyyleille ja sen avulla muodostaa molekyylikirjasto. Syntetisoidut molekyylit haluttiin testata sekä Chlamydia pneumoniae -bakteeria että Leishmania donovani –parasiittia vastaan. Chlamydia pneumoniae –bakteeri aiheuttaa akuutteja ylä- ja alahengitystieinfektiota, kuten keuhkoputkentulehdusta. Akuutissa tulehduksessa oireet vaihtelevat huomattavasti. Chlamydia pneumoniae –bakteerilla on myös taipumus aiheuttaa kroonisia tulehduksia. Nämä ovat useissa tutkimuksissa yhdistetty kansantaloudellisesti merkittäviin sairauksiin, kuten ateroskleroosiin ja astmaan. Leishmanioosi on toiseksi yleisin loissairaus ihmisellä malarian jälkeen. Leishmania donovani –parasiitti voi aiheuttaa tappavaa viskeraalista leishmanioosia. Vuodessa leishmanioosiin kuolee yli 50 000 ihmistä. Viime vuosina leishmanioosin lääkehoidossa on esiintynyt monenlaisia ongelmia. Osat lääkkeistä ovat menettäneet tehonsa ja osalla esiintyy vakavia haittavaikutuksia. 2,1,3-Bentsoksadiatsolirakenteisille yhdisteille saatiin kehitettyä toimiva synteesireitti. Lähtöaineena käytettiin 4-amino-2-nitrobentsoehappoa, josta saatiin hapettavalla renkaansulkeutumisreaktiolla 2,1,3-bentsoksadiatsoli-5-karboksyylihappoa. Karboksyylihaposta syntetisoitiin amidi-välituotteen kautta 2,1,3-bentsoksadiatsoli-5-karbonitriiliä. Hydroksyyliamiini hydrokloridin avulla 2,1,3-bentsoksadiatsoli-5-karbonitriilistä muodostettiin vastaavaa karboksimidamidia, joka oli synteesireitin yhteinen välituote kaikille molekyyleille. Viimeisessä vaiheessa N´-hydroksidi-2,1,3-bentsoksadiatsoli-5-karboksimidamidin annettiin reagoida joko fenyyli-isosyanaatin tai fenyyli-isotiosyanaatin kanssa, jolloin saatiin lopputuotetta. Synteesireitin kehittäminen osoittautui haastavaksi ja loppujen lopuksi saatiin ainoastaan kolme lopputuotetta syntetisoitua. Yksi lopputuotteista testattiin C. pneumoniae –bakteeria vastaan Åbo akademissa Turussa. Testattavaa yhdiste ei sisältänyt 2,1,3-bentsoksadiatsoliarengasta ja bioaktiivisuuskokeen tulos oli odotusten mukainen. Yhdiste ei ollut aktiivinen C. pneumoniae –bakteeria vastaan alhaisilla konsentraatioilla ja tuloksesta voitiin todeta 2,1,3-bentsoksadiatsolirengaan olevan tärkeä aktiivisuuden kannalta. Kaksi lopputuotetta saatiin testaukseen Leishamania donovani –parasiittia vastaan Israeliin. Ainoastaan toinen molekyyleistä sisälsi 2,1,3-bentsoksadiatsolirakenteen. Bioaktiivisuuskokeiden tulokset olivat erittäin rohkaisevia. Yhdisteet olivat aktiivisia parasiittia vastaan jo alhaisilla konsentraatioilla. Kuitenkin 2,1,3-bentsoksadiatsolirakenteinen molekyyli oli aktiivisempi, joten tämäkin aktiivisuuskokeen perusteella huomattiin rengasrakenteen olevan tärkeä aktiivisuuden kannalta.

Protein Kinase C and Anaplastic Lymphoma Kinase Targeted Compounds

The protein kinases (PKs) belong to the largest single family of enzymes, phosphotransferases, which catalyze the phosphorylation of other enzymes and proteins and function primarily in signal transduction. Consequently, PKs regulate cell mechanisms such as growth, differentiation, and proliferation. Dysfunction of these cellular mechanisms may lead to cancer, a major predicament in health care. Even though there is a range of clinically available cancer-fighting drugs, increasing number of cancer cases and setbacks such as drug resistance, constantly keep cancer research active. At the commencement of this study an isophthalic acid derivative had been suggested to bind to the regulatory domain of protein kinase C (PKC). In order to investigate the biological effects and structure-activity relationships (SARs) of this new chemical entity, a library of compounds was synthesized. The best compounds induced apoptosis in human leukemia HL-60 cells and were not cytotoxic in Swiss 3T3 fibroblasts. In addition, the best apoptosis inducers were neither cytotoxic nor mutagenic. Furthermore, results from binding affinity assays of PKC isoforms revealed the pharmacophores of these isophthalic acid derivatives. The best inhibition constants of the tested compounds were measured to 210 nM for PKCα and to 530 nM for PKCδ. Among natural compounds targeting the regulatory domain of PKC, the target of bistramide A has been a matter of debate. It was initially found to activate PKCδ; however, actin was recently reported as the main target. In order to clarify and to further study the biological effects of bistramide A, the total syntheses of the natural compound and two isomers were performed. Biological assays of the compounds revealed accumulation of 4n polyploid cells as the primary mode of action and the compounds showed similar overall antiproliferative activities. However, each compound showed a distinct distribution of antimitotic effect presumably via actin binding, proapoptotic effect presumably via PKCδ, and pro-differentiation effect as evidenced by CD11b expression. Furthermore, it was shown that the antimitotic and proapoptotic effects of bistramide A were not secondary effects of actin binding but independent effects. The third aim in this study was to synthesize a library of a new class of urea-based type II inhibitors targeted at the kinase domain of anaplastic lymphoma kinase (ALK). The best compounds in this library showed IC50 values as low as 390 nM for ALK while the initial low cellular activities were successfully increased even by more than 70 times for NPM-ALK- positive BaF3 cells. More importantly, selective antiproliferative activity on ALK-positive cell lines was achieved; while the best compound affected the BaF3 and SU-DHL-1 cells with IC50 values of 0.5 and 0.8 μM, respectively, they were less toxic to the NPM-ALK-negative human leukemic cells U937 (IC50 = 3.2 μM) and BaF3 parental cells (IC50 = 5.4 μM). Furthermore, SAR studies of the synthesized compounds revealed functional groups and positions of the scaffold, which enhanced the enzymatic and cellular activities.

Non-enzymatic Degradation of (1→3)(1→4)-β-D-glucan in Aqueous Processing of Oats

Cereal water-soluble β-glucan [(1→3)(1→4)-β-D-glucan] has well-evidenced health benefits and it contributes to the texture properties of foods. These functions are characteristically dependent on the excellent viscosity forming ability of this cell wall polysaccharide. The viscosity is affected by the molar mass, solubility and conformation of β-glucan molecule, which are further known to be altered during food processing. This study focused on demonstrating the degradation of β-glucan in water solutions following the addition of ascorbic acid, during heat treatments or high pressure homogenisation. Furthermore, the motivation of this study was in the non-enzymatic degradation mechanisms, particularly in oxidative cleavage via hydroxyl radicals. The addition of ascorbic acid at food-related concentrations (2-50 mM), autoclaving (120°C) treatments, and high pressure homogenisation (300-1000 bar) considerably cleaved the β-glucan chains, determined as a steep decrease in the viscosity of β-glucan solutions and decrease in the molar mass of β-glucan. The cleavage was more intense in a solution of native β-glucan with co-extracted compounds than in a solution of highly purified β-glucan. Despite the clear and immediate process-related degradation, β-glucan was less sensitive to these treatments compared to other water-soluble polysaccharides previously reported in the literature. In particular, the highly purified β-glucan was relatively resistant to the autoclaving treatments without the addition of ferrous ions. The formation of highly oxidative free radicals was detected at the elevated temperatures, and the formation was considerably accelerated by added ferrous ions. Also ascorbic acid pronounced the formation of these oxidative radicals, and oxygen was simultaneously consumed by ascorbic acid addition and by heating the β-glucan solutions. These results demonstrated the occurrence of oxidative reactions, most likely the metal catalysed Fenton-like reactions, in the β-glucan solutions during these processes. Furthermore, oxidized functional groups (carbonyls) were formed along the β-glucan chain by the treatments, including high pressure homogenisation, evidencing the oxidation of β-glucan by these treatments. The degradative forces acting on the particles in the high pressure homogenisation are generally considered to be the mechanical shear, but as shown here, carbohydrates are also easily degraded during the process, and oxidation may have a role in the modification of polysaccharides by this technique. In the present study, oat β-glucan was demonstrated to be susceptible to degradation during aqueous processing by non-enzymatic degradation mechanisms. Oxidation was for the first time shown to be a highly relevant degradation mechanism of β-glucan in food processing.

Chemoenzymatic Synthesis of Chiral Organosulfur Compounds

This thesis is primarily concerned with the enzyme- catalysed synthesis of sulfoxides using reductase and dioxygenase enzymes. Chapter 1 provides an introduction to the topic of redox chemistry with particular emphasis on the application of reductase and dioxygenase enzymes in organosulfur chemistry. Earlier literature methods for the production of enantiopure sulfoxides are reviewed. A brief discussion of the methods used for the determination of enantiomeric excess and absolute configuration is provided. Chapter 2 contains results obtained using a range of whole-cell bacteria each using a dimethyl sulfoxide reductase enzyme. The synthesis of a series of racemic sulfoxides and the development of appropriate CSPHPLC analytical methods is discussed. Kinetic resolutions of a series of sulfoxides have been achieved. Chapter 3 contains a presentation of results using dioxygenase enzymes as biocatalysts for the asymmetric sulfoxidation of dialkyl sulfoxides including thioacetal sulfoxides. A new range of monosulfoxides, cis-dihydrodiols and cis- dihydrodiol sulfoxides have been isolated in enantiopure form. Chapter 4 is focussed on the application of chiral sulfoxides in synthesis. A new chemoenzymatic route to diol sulfoxide enantiomers and the derived enantiopure phenols and catechols is discussed. The application of chemically synthesised sulfoxide enantiomers in the production of hydroxy sulfoxides is reported. Chapter 5 provides a full experimental section where the synthesis of sulfides and racemic sulfoxides is included. The methods used in the isolation and characterisation of bioproducts from the biotransformation are discussed and full experimental details given.

Putative neuroprotective compounds in in vitro models of dopaminergic neurodegeneration

Parkinson´s disease (PD) is a debilitating age-related neurological disorder that affects various motor skills and can lead to a loss of cognitive functions. The motor symptoms are the result of the progressive degeneration of dopaminergic neurons within the substantia nigra. The factors that influence the pathogenesis and the progression of the neurodegeneration remain mostly unclear. This study investigated the role of various programmed cell death (PCD) pathways, oxidative stress, and glial cells both in dopaminergic neurodegeneration and in the protective action of various drugs. To this end, we exposed dopaminergic neuroblastoma cells (SH-SY5Y cells) to 6-OHDA, which produces oxidative stress and activates various PCD modalities that result in neuronal degeneration. Additionally, to explore the role of glia, we prepared rat midbrain primary mixed-cell cultures containing both neurons and glial cell types such as microglia and astroglia and then exposed the cultures to either MPP plus or lipopolysaccharide. Our results revealed that 6-OHDA activated several PCD pathways including apoptosis, autophagic stress, lysosomal membrane permeabilization, and perhaps paraptosis in SH-SY5Y cells. Furthermore, we found that minocycline protected SH-SY5Y cells from 6-OHDA by inhibiting both apoptotic and non-apoptotic PCD modalities. We also observed an inconsistent neuroprotective effect of various dietary anti-oxidant compounds against 6-OHDA toxicity in vitro in SH-SY5Y cells. Specifically, quercetin and curcumin exerted neuroprotection only within a narrow concentration range and a limited time frame, whereas resveratrol and epigallocatechin 3-gallate provided no protection whatsoever. Lastly, we found that molecules such as amantadine may delay or even halt the neurodegeneration in primary cell cultures by inhibiting the release of neurotoxic factors from overactivated microglia and by enhancing the pro-survival actions of astroglia. Together these data suggest that the strategy of dampening oxidative species with anti-oxidants is less effective than preventing the production of toxic factors such as oxidative and pro-inflammatory molecules by pathologically activated microglia. This would subsequently prevent the activation of various PCD modalities that cause neuronal degeneration.