Hydrophobic interaction chromatography (HIC) is a common purification method for biological drug substances. For continuous monitoring the quality of the purification, a column performance test is carried out to test the packing of the resin. The packing integrity is commonly tested between runs by measuring the Hight Equivalent to a Theoretical Plate (HETP) by injecting a small volume of a tracer solution (spike) that gives a change in conductivity. The column performance test provides useful information regarding the packing quality that could be used for troubleshooting before a purification is performed since a well packed gel is essential for an effective purification. During this thesis project a screening of suitable test conditions for a HETP test for a HIC column was performed. The developed HETP test were then investigated if it could be combined with the storage of the resin or if the packing is affected by the test conditions. Factors such as spike volume, flow rate and the concentration of the elute and spike solution, were considered. 0.1 M NaOH elute and 0.15 M NaCl as spike solution gave the best result based on HETP and the asymmetry of the conductivity peak. A test purification with the new HETP test conditions compared with the old method were then carried out to verify that the quality of the purification is not affected. The purification showed promising result and implementation of a more convenient storage solution and HETP test would be possible without changing the product quality.

Introduktion COVID-19 orsakas av viruset SARS-CoV-2. 3-chymotrypsin-liknande cysteinproteaset (3CLpro) är ett enzym som styr coronavirus replikation och är viktigt för viruset livscykel. Därför 3CL pro är en viktig måltavla för att hämma SARS-CoV-2. Syfte Syftet med detta projekt är att undersöka om proteashämmare kan vara en framkomlig väg för att ta fram antivirala läkemedel mot COVID-19. Metod Arbetet är en litteraturstudie och databasen PubMed har använts för att hitta vetenskapliga artiklar med hjälp av olika sökord, såsom SARS-CoV 2 and protease inhibitors, SARS-CoV-2 and 3CL-pro, MERS-CoV and 3CLpro inhibitors och SARS-CoV and 3CLpro. Totalt valdes 10 vetenskapliga artiklar ut som grund för att undersöka om några proteashämmare finns rapporterade och hur de utvärderats, dvs i prekliniska studier eller kliniska studier. Resultat  Peptidomimetiska alfa-ketoamider är effektiva bredspektrum hämmare för att hämma 3CL pro i SARS-CoV-2 och enterovirus, t.ex. alfa-ketoamiderna (13a och 13b) som även visade goda farmakokinetiska egenskaper i prekliniska studier. Även läkemedelskandidater (11a och 11b) har utvärderats i prekliniska studier och både visade hög SARS-CoV-2 3CL-pro hämmande aktivitet. Två nya studier visade att genomsekvensen för SARS-CoV-2 3CLpro är mycket lik den för SARS-CoV-1 än MERS-CoV. Herbacetin, rhoifolin pectolinarin och Biflavonoid amentoflavon 9 visade en effektiv hämning av SARS-CoV-1 3CL pro. Herbacetin, isobavachalcone, quercetin 3 ‐ β ‐ d ‐ glukosid, helikrysetin och enterovirushämmare (6b, 6c och 6d) visat sig ha hämmande aktivitet och kunde hämma den enzymatiska aktiviteten hos MERS-CoV 3CL pro. De redan framtagna HIV-proteashämmarna lopinavir och ritonavir har utvärderats i kliniska prövningar och både visade ingen skillnad mot standardbehandling samt minskade inte dödlighet. Slutsats Slutsatsen som kan dras av studien är att det finns flera proteashämmare i preklinisk fas mot SARS-COV-2 3CL-pro samt finns flera proteashämmare mot SARS-CoV-1 och MERS-CoV. Proteashämmare kan vara ett möjligt läkemedel mot COVID-19 i framtiden, men det tar lång tid för att utveckla ett nytt läkemedel.

The aim of this project was to investigate a potential connection between a molecule's capacity to penetrate the blood-brain barrier and its electrostatic potential on the surface of the molecule. A molecule's capacity to penetrate the blood-brain barrier is quantified by their log BB value. This was done by creating a set of 58 molecules, divided into a test set and a calibration set, in Gaussian 16. The molecules were then optimized and their quantum chemical properties were calculated by using HS-95. By using stepwise regression in both Matlab and Excel these different parameters were then used to search for a correlation between the parameters and experimental log BB values. This resulted in an equation with 10 variables with an adjusted R2 value of 0,6456. When tested against the test set the mean fault was 0,1443. Outliers were then identified and removed and stepwise regression was executed once more. This resulted in an equation with 10 variables with an adjusted R2 0,8749. When this was tested against the test set the mean fault was 0,0798. The results showed that the variables that were important were the electrostatic potential, surface area and volume.

This thesis investigates the anti-leukemic activity of terpenoids isolated from medicinal mushrooms and marine sponges, as well as their possible targets and mechanisms of action. In the first section, we focused on studying the triterpenoidal components of three triterpenoid-enriched medicinal mushrooms Antrodia cinnamomea, Ganoderma lucidum, and Poria cocos, which have been used in folk medicine for centuries and also developed into several contemporary marketed products. We isolated the major and characteristic triterpenoids from these mushrooms, together with six new lanostanoids (II-1–II-6). The anti-leukemic activity of the isolates was evaluated in vitro using MTT proliferative assay and seven of them exhibited potential anti-leukemic effect. The active lead compounds were further subjected to computational analyses utilizing the ChemGPS-NP tool. We established a database for the anti-leukemic relevant chemical space of triterpenoids isolated from these three medicinal mushrooms, which could be used as a reference database for further research on anti-leukemic triterpenoids. Our results indicated that the anti-leukemic effect of the active lead compounds was mediated not only through topoisomerases inhibition but also through inhibiting DNA polymerases. The second and third sections focused on isolation of anti-leukemic sesterterpenoids from sponges. The investigation of Carteriospongia sp. led to the isolation of two new scalarane-type sesterterpenoids (III-1 and III-2) and one known tetraprenyltoluquinol-related metabolite (III-3). All isolates exhibit an apoptotic mechanism of action against Molt 4 cells, found to be mediated through the disruption of the mitochondrial membrane potential (MMP) and inhibition of topoisomerase IIα expression. Detailed investigation of the apoptotic mechanism of action using molecular docking analysis revealed that compound III-1 might target Hsp90 protein. The apoptotic-inducing effect of III-3 was supported by in vivo experiment by suppressing the volume of xenograft tumor growth (47.58%) compared with the control. In the final section of this thesis we studied manoalide and its derivatives, sesterterpenoids isolated from the sponge Luffariella sp.. Manoalide has been studied as a potential anti-inflammatory agent for the last thirty years with more than 200 publications and 40 patents. However, the configurations at positions 24 and 25 were never revealed. In the current study, ten manoalide-type sesterterpenoids (IV-1–IV-10) were isolated from Luffariella sp. and their stereoisomers at positions 24 and 25 were identified and separated for the first time. The configuration at positions 24 and 25 showed to have a significant effect on the anti-leukemic activity of manoalide derivatives, with the 24R,25S-isomer exhibiting the most potent anti-leukemic activity. The apoptotic mechanism of action of compound IV-7 against Molt 4 cells was investigated, and the compound was found to trigger MMP disruption and intracellular reactive oxygen species (ROS) generation. Compound IV-7 also inhibited activity against both human topoisomerases, I and II. The in vivo experiment further supported the anti-leukemic effect of IV-7 with a 66.11% tumor volume suppression compared to the control.

<p>Despite major advances in the understanding of the biology of chronic lymphocytic leukaemia (CLL), progress in improving its treatment has been limited and it still remains an incurable disorder. In the present research, we have performed <i>in vitro</i> drug sensitivity testing of primary CLL cells for preclinical evaluation of cytotoxic drugs, using the fluorometric microculture cytotoxicity assay (FMCA).</p><p>The tumour type-specific activities of 14 standard drugs, evaluated <i>in vitro</i> on tumour cells from patients with CLL and acute leukaemias, were in good agreement with their known clinical activities. A correlation between drug treatment and development of cellular drug resistance was demonstrated in CLL, but not in the acute leukaemias. Moreover, the nucleoside analogues fludarabine, cladribine, cytarabine and gemcitabine, as well as the anthracycline idarubicin, were highly active in CLL cells.</p><p>A new cytotoxic drug candidate, CHS 828, was evaluated in primary cell cultures from a broad spectrum of tumours. CHS 828 was highly active against haematological malignancies <i>in vitro</i>, especially CLL, but also against some solid tumours. The drug appeared to be non cross-resistant with standard drugs.</p><p>In addition, the relationship between drug sensitivity <i>in vitro</i> and a recently described prognostic factor in CLL, the mutational status of the immunoglobulin variable heavy chain (IgV_H) gene, was evaluated. Interestingly, cells with unmutated IgV_H genes were more chemosensitive than the mutated cells. </p><p>In summary, our results indicate that <i>in vitro</i> studies on tumour cellsfrom leukaemia patients may yield considerable information regarding the activity, mechanisms of action and cross-resistance of cytotoxic drugs, as well as concerning the relationship between drug sensitivity and prognostic factors, which can be useful in the preclinical evaluation of new cytotoxic drugs. Furthermore, the results suggest that the pyrimidine analogues cytarabine and gemcitabine, as well as the anthracycline idarubicin, may have a role in the treatment of CLL. The new cyanoguanidine CHS 828 is highly active in CLL cells and appears to be non cross-resistant with standard drugs. The poorer prognosis in patients with CLL cells with unmutated IgV_H genes can not be explained by increased chemoresistance.</p>

Despite major advances in the understanding of the biology of chronic lymphocytic leukaemia (CLL), progress in improving its treatment has been limited and it still remains an incurable disorder. In the present research, we have performed in vitro drug sensitivity testing of primary CLL cells for preclinical evaluation of cytotoxic drugs, using the fluorometric microculture cytotoxicity assay (FMCA). The tumour type-specific activities of 14 standard drugs, evaluated in vitro on tumour cells from patients with CLL and acute leukaemias, were in good agreement with their known clinical activities. A correlation between drug treatment and development of cellular drug resistance was demonstrated in CLL, but not in the acute leukaemias. Moreover, the nucleoside analogues fludarabine, cladribine, cytarabine and gemcitabine, as well as the anthracycline idarubicin, were highly active in CLL cells. A new cytotoxic drug candidate, CHS 828, was evaluated in primary cell cultures from a broad spectrum of tumours. CHS 828 was highly active against haematological malignancies in vitro, especially CLL, but also against some solid tumours. The drug appeared to be non cross-resistant with standard drugs. In addition, the relationship between drug sensitivity in vitro and a recently described prognostic factor in CLL, the mutational status of the immunoglobulin variable heavy chain (IgVH) gene, was evaluated. Interestingly, cells with unmutated IgVH genes were more chemosensitive than the mutated cells. In summary, our results indicate that in vitro studies on tumour cellsfrom leukaemia patients may yield considerable information regarding the activity, mechanisms of action and cross-resistance of cytotoxic drugs, as well as concerning the relationship between drug sensitivity and prognostic factors, which can be useful in the preclinical evaluation of new cytotoxic drugs. Furthermore, the results suggest that the pyrimidine analogues cytarabine and gemcitabine, as well as the anthracycline idarubicin, may have a role in the treatment of CLL. The new cyanoguanidine CHS 828 is highly active in CLL cells and appears to be non cross-resistant with standard drugs. The poorer prognosis in patients with CLL cells with unmutated IgVH genes can not be explained by increased chemoresistance.

This thesis describes work done to design and synthesize protease inhibitors, with the intention of developing therapeutic agents for Alzheimer’s disease (AD) and the chronic liver condition caused by infection of the hepatitis C virus (HCV). AD is the most common form of dementia, and HCV infection is the primary reason for liver transplantation in industrialized countries. Today, these two illnesses affect 24 and 170 million people, respectively. It has been shown that the human aspartic protease BACE-1 plays an important role in the development of AD, and thus inhibition of BACE-1 may offer a way to improve the quality of life of individuals afflicted with the disease. Furthermore, it is known that the serine protease NS3 is a vital component in the replication of HCV. Several novel potent BACE-1 inhibitors encompassing different transition state mimics were prepared. First, a hydroxyethylene moiety encompassing a secondary hydroxyl group was evaluated as a transition state analogue, producing inhibitors in the low nanomolar range. Various tertiary hydroxyl isosteres were also investigated as the central core, with the aim of shielding the pivotal hydroxyl group. These transition state isosteres consisted of tertiary hydroxyl analogues of previously used secondary hydroxyl containing norstatine, statine, and hydroxyethylamine isosteres. Several tertiary hydroxyl-containing inhibitors were found to be active in the low micromolar range. In addition, two inhibitors were co-crystallized with the BACE-1 enzyme to provide X-ray crystal structures, which furnished valuable binding information for further design of improved BACE-1 inhibitors. The goal in the HCV NS3 protease inhibitor project was to design, synthesize and evaluate a novel hydroxycyclopentene bioisostere to the previously used acyl-hydroxyproline moiety. The investigation revealed that it was possible to synthesize inhibitors containing this new bioisostere that were potent in the low nanomolar range. Further optimization by rigidification of the most active inhibitor resulted in equipotent macrocyclic compounds.

Palladium-catalysed carbonylative multicomponent reactions have proven useful for the synthesis of structurally diverse compounds. Carbon monoxide serves as an atom-efficient, one-carbon building block, which allows for further structural elaboration of the carbonyl compound. By varying the components of the carbonylative multicomponent reaction, considerable product diversity can readily be attained. However, due to the reluctance to use toxic CO gas, considerable efforts have been directed at exploring non-gaseous approaches. The work described in this thesis has mainly focused on the development of palladium(0)-catalysed, carbonylative multicomponent synthetic methodology, using the non-gaseous CO source molybdenum hexacarbonyl, in the synthesis of heterocycles and other biologically relevant functional groups. The first part of this work describes the development of a non-gaseous carbonylative Sonogashira cross-coupling of bifunctional ortho-iodoanilines and terminal alkynes. Where 4-quinolones were synthesised via a carbonylation/cyclisation sequence. Using a similar synthetic strategy, three different N-cyanobenzamide intermediates were prepared by palladium-catalysed carbonylative couplings of various aryl halides and bromides and cyanamide. The formed intermediates provided a basis for further chemical transformations. First, ortho-iodoanilines were carbonylatively coupled with cyanamide and subsequently cyclised to yield heterocyclic 2-aminoquinazolinones. Next, building on those findings, the same synthetic strategy was applied to ortho-halophenols to provide a highly convenient domino carbonylation/cyclisation method for the preparation of benzoxazinones. The developed method was used to evaluate the efficiency of various non-gaseous CO sources. Third, the palladium-catalysed carbonylative synthesis of N-cyanobenzamides, was used to produce biologically relevant N-acylguanidines with considerable product diversity. Finally, one of the developed carbonylative methodologies was used in the preparation of potential NDH-2 inhibitors based on a quinolinyl pyrimidine scaffold. The prepared compounds were biologically evaluated in terms of inhibition of oxidoreductase NDH-2 and antibacterial activity on Gram-negative bacteria, S. aureus and Mtb. The biological evaluation revealed that some of the quinolinyl pyrimidines exerted inhibitory activity on the NDH-2 enzyme and possessed antibacterial properties. The work described in this thesis has been devoted to the development of non-gaseous one-pot, multicomponent carbonylation/cyclisation and carbonylation/amination reactions. The described methods offer highly attractive synthetic strategies that can be of great value to synthetic and medicinal chemists.

Introduction Oligonucleotides are short nucleic acid chains, usually 19-27mer long. They bind to their corresponding chain, making a specific inhibition possible. In pharmaceuticals, this can be used to inhibit the expression of a gene or protein of interest. Oligonucleotides are usually analyzed based on separation using both hydrophobic and ion-exchange properties. In this project, the possibility to use a mixed-mode column to separate these oligonucleotides and their impurities were explored. Method Liquid chromatography is used as the separation method and the method of detection is both mass spectrometry and UV. Three different columns are evaluated; C18, DNAPac RP, and mixed-mode RP/WAX. Results and discussion Different compositions of mobile phases and gradients are evaluated based on a literature study. Triethylamine, triethylammonium acetate, ammonium formate, hexafluoroisopropanol is used along with both methanol and acetonitrile. Phosphate buffer is evaluated on LC-UV. The results from the C18 column displays a good separation of the oligonucleotides, whilst the DNAPac RP is not as sufficient using the same mobile phases. The mixed-mode column provides good separation and selectivity using phosphate buffer and UV detection. Conclusion Mixed-mode column has the potential to be used for separation of oligonucleotides and one future focus would be to make the mobile phase compatible with mass spectrometry. Phosphate buffer and UV detection seems to be the go-to mobile phase using mixed-mode column even though MS is a more powerful tool for the characterization and identification of oligonucleotides. This provides a hint about the challenge in making the mobile phase MS compatible.

In the work presented this thesis, two different conditions that are classified as protein misfolding diseases: Alzheimer's disease and lysozyme amyloidosis and proteins that could have a beneficial effect in these diseases, have been studied using Drosophila melanogaster, commonly known as the fruit fly. The fruit fly has been used for over 100 years to study and better understand fundamental biological processes. Although the fruit fly, unlike humans, is an invertebrate, many of its central biological mechanisms are very similar to ours. The first transgenic flies were designed in the early 1980s, and since then, the fruit fly has been one of the most widely used model organisms in studies on the effects of over-expressed human proteins in a biological system; one can regard the fly as a living, biological test tube. For  most proteins, it is necessary that they fold into a three-dimensional structure to function properly. But sometimes the folding goes wrong; this may be due to mutations that make the protein unstable and subject to misfolding. A misfolded protein molecule can then aggregate with other misfolded proteins. In Alzheimer's disease, which is the most common form of dementia, protein aggregates are present in the brains of patients. These aggregates are composed of the amyloid-β (Aβ) peptide, a small peptide of around 42 amino acids which is cleaved from the larger, membrane-bound, protein AβPP by two different enzymes, BACE1 and γ-secretase. In the first part of this thesis, two different fly models for Alzheimer’s disease were used: the Aβ fly model, which directly expresses the Aβ peptide, and the AβPP-BACE1 fly model, in which all the components necessary to produce the Aβ peptide in the fly are expressed in the fly central nervous system (CNS). The two different fly models were compared and the results show that a significantly smaller amount of the Aβ peptide is needed to achieve the same, or an even greater, toxic effect in the AβPP-BACE1 model compared to the Aβ model. In the second part of the thesis, these two fly models for Alzheimer’s disease were again used, but now to investigate whether lysozyme, a protein involved in our innate immune system, can counteract the toxic effect of Aβ generated in the fly models. And indeed, lysozyme is able to save the flies from Aβ-induced toxicity. Aβ and lysozyme were found to interact with each other in vivo. The second misfolding disease studied in this thesis is lysozyme amyloidosis. It is a rare, dominantly inherited amyloid disease in which mutant variants of lysozyme give rise to aggregates, weighing up to several kilograms, that accumulate around the kidneys and liver, eventually leading to organ failure. In the third part of this thesis, a fly model for lysozyme amyloidosis was used to study the effect of co-expressing the serum amyloid P component (SAP), a protein that is part of all protein aggregates found within this disease class. SAP is able to rescue the toxicity induced by expressing the mutant variant of lysozyme, F57I, in the fly's CNS. To further investigate how SAP was able to do this, double-expressing lysozyme flies, which exhibit stronger disease phenotypes than those of the single-expressing lysozyme flies previously studied, were used in the fourth part of this thesis. SAP was observed to reduce F57I toxicity and promote F57I to form aggregates with more distinct amyloid characteristics. In conclusion, the work included in this thesis demonstrates that: i) Aβ generated from AβPP processing in the fly CNS results in higher proteotoxicity compared with direct expression of Aβ from the transgene, ii) lysozyme can prevent Aβ proteotoxicity in Drosophila and could thus be a potential therapeutic molecule to treat Alzheimer’s disease and iii) in a Drosophila model of lysozyme amyloidosis, SAP can prevent toxicity from the disease-associated lysozyme variant F57I and promote formation of aggregated lysozyme morphotypes with amyloid properties; this is important to take into account when a reduced level of SAP is considered as a treatment strategy for lysozyme amyloidosis.