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Bioprinting is one of the most promising technologies for future healthcare as it may benefit the repairing of wounds and injuries, disease modeling and development, transplantation of organs and reduce animal testing. This thesis aim to investigate this industry further, as there is no excessive literature on how to handle the innovation in regards to entrepreneurial and biotechnological knowledge. Hence, a research gap can be spotted and the purpose of the conducted research questions should contribute to this gap. In order to fully understand the bioprinting industry, an outline of the technology is made as part of the research. In addition to this, secondary data for patents, market valuation and annual growth rates are collected to support arguments from previous literature. Also, interviews are conducted to gather specific knowledge. As a result, bioprinting may be presented as a disruptive innovation in an uncertain market, which places certain demands on companies to act more in line with the complexity of the technology. Such companies must think more strategically and design more complex and long-term strategies. The patent data shows that there has been a decline in the technological development as patent applications have decreased significantly. Even though the technology (regarding the patents) has started to slowly decline, there is still hope for some technological improvements to come. It can be concluded that developments in bioink, scaffolds, expansion of cells and diffusion is expected, and that the use of bioprinting is increasing and will most likely continue to do so.

Today, Real World Data (RWD) is a popular topic in many studies. In particular, it is anticipated to be a significant resource for addressing issues brought on by drug development costs, lengthy development times, and safety concerns. The Swedish healthcare Quality Registries (QR) are studied to contribute to the improvement of health care with individual-based clinical data. Recorded data is used for quality improvement, guidance compliance monitoring, and research. However, the workflow for such a framework that applies RWD which is patient-related data that came from various sources to the new drug development field is currently not well-defined. Thus the main aim of this project is to establish a strategy for integrating RWD with pharmaceutical modeling. To achieve this aim, QRs were examined through an ontological approach. The data and procedures necessary for modeling the development of new drugs, as well as the correspondence of the pieces offered by QR, were studied to assess the feasibility. The modeling of new drug development was studied for three applications: Adverse Drug Event (ADE), Computer-based Simulation (CBS), and drug repurposing, and the analysis of QRs was conducted on seven diseases. After in-depth analysis, although there were differences between the registries, it showed enough feasibility in terms of how much the data provided in the studies on drug repurposing and computer-based simulation satisfied the items required for new drug development. However, in the case of rare diseases, given the lack of an automated method, the ethical ambiguity, and the speed of the process, there still seems to be potential for improvement.  Many registries have begun to support research on the development of novel medications, such as by independently recording the features of drugs. These initiatives could enable the future potential of new Real World Evidence (RWE) such as in the field of proteomics and genomics discovery. / Idag är Real World Data (RWD) ett populärt ämne i många studier. I synnerhet förväntas det vara en betydande resurs för att ta itu med problem som orsakas av kostnader för läkemedelsutveckling, långa utvecklingstider och säkerhetsproblem. Kvalitetsregistren studeras för att bidra till att förbättra hälso- och sjukvården med individbaserade kliniska data. Registrerade data används för kvalitetsförbättring, övervakning av efterlevnad av riktlinjer och forskning. Arbetsflödet för ett sådant ramverk som tillämpar RWD som är patientrelaterad data som kom från olika källor till det nya läkemedelsutvecklingsområdet är dock för närvarande inte väldefinierat. Därför är huvudsyftet med detta projekt att upprätta en strategi för att integrera RWD med läkemedelsmodellering. För att uppnå detta mål undersöktes kvalitetsregister genom ett ontologiskt tillvägagångssätt. De data och procedurer som krävs för att modellera utvecklingen av nya läkemedel, såväl som överensstämmelsen mellan de bitar som erbjuds av kvalitetsregister, studerades för att bedöma genomförbarheten. Modelleringen av utvecklingen av nya läkemedel studerades för tre tillämpningar: skadlig läkemedelseffekt, datorbaserad simulering och återanvändning av läkemedel, och analys av kvalitetsregister genomfördes på sju sjukdomar. Efter en djupgående analys, även om det fanns skillnader mellan registren, visade den tillräcklig genomförbarhet när det gäller hur mycket data som tillhandahållits i studierna om läkemedelsåteranvändning och datorbaserad simulering uppfyllde de krav som krävdes för utveckling av nya läkemedel. Men när det gäller sällsynta sjukdomar, med tanke på avsaknaden av en automatiserad metod, den etiska oklarheten och processens snabbhet, verkar det fortfarande finnas potential för förbättringar. Många register har börjat stödja forskning om utveckling av nya mediciner, till exempel genom att oberoende registrera drogens egenskaper. Dessa initiative skulle kunna möjliggöra den framtida potentialen för nya verkliga bevis, såsom inom området för proteomik och genomik.

Alzheimer’s disease (AD) drugs in therapy are limited to acetylcholine esterase inhibitors and memantine. Newly developed drugs against a single target structure have an insufficient effect on symptomatic AD patients. Results: Novel aromatically anellated pyridofuranes have been evaluated for inhibition of AD-relevant protein kinases cdk1, cdk2, gsk-3b and Fyn. Best activities have been found for naphthopyridofuranes with a hydroxyl function as part of the 5-substituent and a hydrogen or halogen substituent in the 8-position. Best results in nanomolar ranges were found for benzopyridofuranes with a 6-hydroxy and a 3-alkoxy substitution or an exclusive 6-alkoxy substituent. Conclusion: First lead compounds were identified inhibiting two to three kinases in nanomolar ranges to be qualified as an innovative approach for AD multitargeting.

Despite significant advances in cancer treatment, clinical response remains suboptimal and there is a continued requirement for improved chemotherapeutics. The attrition rate for new therapies is high, due principally to lack of in vivo efficacy and poor pharmacodynamics. Consequently better systems are required to determine in vivo preclinical efficiency and drug-target interactions. Engineering of cancer cells to express fluorescent or bioluminescent proteins, either endogenously or under the control of specific gene promoters, and their detection by noninvasive optical imaging has the potential to improve preclinical drug development. In this study, a panel of colorectal cancer cell lines were engineered to express fluorescent and luminescent proteins either constitutively or under control of gene-promoters for the DNA damage response gene p53 or the cell cycle regulator p21, both important pharmacodynamic sensors. These cell lines were characterised for their potential as in vivo models of primary and metastatic tumour therapy response, several showing significant potential. In addition to the development of these models, this study also addressed the pharmacokinetics of different luciferase substrates and identified optimal temporal and dose characteristics for each. Furthermore, a new application for bioluminescent imaging was developed and validated for use in preclinical evaluation of vascular disrupting agents, a new generation of cancer therapeutic. This study demonstrates that despite the dynamic and variable nature of fluorescent and bioluminescent imaging, reproducible results can be obtained if appropriate precautions are taken. The models developed herein will expedite cancer drug development whilst reducing and refining the use of animals in research.

In the course of this work, compounds of the natural product class of polycyclic polyprenylated acylphloroglucinols (PPAPs) were analysed. They consist of a polycyclic core structure decorated with carbonyl groups and unsaturated sidechains. The modular synthesis platform developed and optimised in the work group, facilitated the creation of a 23 compound PPAP library. This initiated the study of antibacterial properties. Further research into the derivatisation of PPAPs paved the way to a synthetic toolbox for the synthesis of new derivatives. Now a follow-up structure-activity relationship (SAR) study, aided by a novel C acylation strategy developed in the group, expanded the library with 25 new unnatural type B PPAPs. Antibiotic efficacies with minimum inhibitory concentrations (MICs) in the nanomolar range against methicillin-resistant Staphylococcus aureus (MRSA) were observed, identifying new lead compounds with significant therapeutic windows. Transitioning to cancer research, the anticancer activities of PPAPs across leukaemia, glioblastoma (GB), and prostate cancer (PCa) were investigated. A 3 phenylpropanoyl head group turned out to be crucial for proapoptotic efficacies. Optimisation efforts yielded different potent compounds for each cancer type with proapoptotic half maximal inhibitory concentration (IC50) values in the low micromolar and antiproliferative IC50 values in the nanomolar range. Flavonoid-like PPAP scaffolds were explored for enhanced biological activities through the combination of anticancer activities of PPAPs with flavonoids. A one-step synthesis method produced eleven flavonoid-like PPAPs with promising anticancer potential, particularly against GB. They exhibited strong proapoptotic effects with low toxicity on healthy cells. To expand the covalent combination of different active substances, further conjugation strategies were employed for the synthesis of PPAP-containing drug-drug conjugates (DDCs). This resulted in several PPAP–temozolomide- (TMZ) and PPAP–doxorubicin (DOX) conjugates. PPAP–TMZ showed significant anticancer potential against GB, while PPAP–DOX demonstrated substantial antileukemic activity. Challengingly, both DDCs also exhibited significant toxic effects on healthy peripheral blood mononuclear cells (PBMCs). Therefore, targeted drug delivery of PPAPs utilising PPAP hybrid drugs was investigated to enhance the selectivity of PPAP treatments. This involved the successful combination of PPAPs with linkers for antibody-drug conjugates (ADCs). To evaluate the coupling potential of the synthesised linker with monoclonal antibodies, it was reacted with cysteine. The successful reaction showed the potential of future couplings with real antibodies. For PCa specifically, prostate specific membrane antigen (PSMA)-targeting PPAPs were explored. This protein is solely expressed by PCa cells, therefore being an ideal target protein. Employing chemical structures from existing pharmaceuticals led to the design of different PSMA-targeting PPAPS. While in silico screenings indicated extraordinarily high affinity to the PSMA binding site, biological tests revealed very low anticancer activities. As the binding affinity was too strong, the PPAPs were presumably not released in the cell, which could explain the low anticancer efficacies. For this reason, cleavable linker systems were investigated as part of further research. In conclusion, this research significantly advanced the understanding of diverse PPAP derivatives, demonstrating their potential in antibacterial and anticancer applications. The outlined synthesis strategies, SAR studies, and conjugation approaches provide a foundation for future developments. Further optimisations and collaborations are needed to bring PPAPs a step closer to applications as medical treatment option.:Table of Contents Acknowledgements III Table of Contents V List of Abbreviations IX Abstract (English) XIV Abstract (German) XVII I Theoretical Part 1 Introduction 1 1.1 Medical need in modern society 1 1.2 Natural products in drug development 1 1.3 Polycyclic polyprenylated acylphloroglucinols (PPAPs) 2 1.3.1 Classifications 2 1.3.2 Biosynthesis 4 1.3.3 Biological activities 6 1.3.4 PPAP-synthesis strategies 7 1.4 Flavonoids in drug discovery 10 1.5 Hybrid drugs in cancer treatment 11 1.5.1 Antibody-drug conjugates 11 1.5.2 Drug-drug conjugates 12 1.5.3 Prostate cancer and prostate-specific membrane antigen (PSMA) 13 2 Objectives 16 2.1 Extending the PPAP library and improving biological activities 16 2.2 Examination and improvement of anticancer efficacy with unexplored PPAP scaffolds 17 2.3 Improving target selectivity for PCa using PSMA-targeting PPAPs 17 3 Expanding the PPAP library 18 3.1 Evaluation of the existing library 18 3.2 Synthesis of the PPAP22 core (PPAP26, 31) 21 3.3 Acylation of the C3 position (head group) 24 3.3.1 Established acylation method using acyl cyanides 24 3.3.2 Cyanide free acylation by PESLALZ 25 3.4 Structure-activity relationship (SAR) study of PPAP78 (50) derivatives (PPAP generation 2) 26 3.4.1 Designing the SAR study 26 3.4.2 Synthesis of PPAPs for the SAR study 28 3.4.3 Evaluation of antibacterial activity 29 3.4.4 Conclusion 32 3.5 Evaluation of anticancer activity 33 3.5.1 Synthesis of the used PPAPs 33 3.5.2 Blood cancer (leukaemia) 36 3.5.3 Brain cancer (glioblastoma) 39 3.5.4 Prostate cancer (PCa) 42 3.6 Exploring synthesis methods towards new PPAP amides 46 3.6.1 PPAP amide synthesis via isocyanates 46 3.6.2 PPAP amides from carbamates 49 3.7 Summary of the library expansion 51 4 Novel flavonoid-like PPAP scaffold 54 4.1 Exploring approaches to flavonoid-like PPAPs 55 4.1.1 Flavanones in one step from PPAP26 (31) 55 4.1.2 Conversion of flavanones to flavones 58 4.1.3 Flavone and aurone synthesis with propiolic acids 59 4.1.4 First biological activity screening 62 4.1.5 Synthesis of substituted propiolic acids 63 4.1.6 Elaborating the substitution scope with dimedone test substrates 65 4.2 Investigating flavone-like PPAPs 66 4.2.1 Synthesis of flavone-like PPAPs 66 4.2.2 Biological activity investigations 68 4.3 Exploring the synthesis of isoflavanone-like PPAPs 71 4.4 Summary of flavonoid-like PPAPs 74 5 Studies towards PPAP hybrid drugs 76 5.1 PPAP drug-drug conjugates 76 5.1.1 PPAP–TMZ conjugates 77 5.1.2 PPAP–DOX conjugates 92 5.1.3 Summary of PPAP drug-drug conjugates 96 5.2 PPAP antibody-drug conjugates 97 5.2.1 Synthesis of a linker with a Val–Cit recognition sequence 99 5.2.2 Synthesis and coupling of simplified linkers 102 5.2.3 Testing of cysteine coupling potential 106 5.2.4 Summary of PPAP antibody-drug conjugates 107 5.3 Using PSMA to increase PCa specificity of PPAPs 107 5.3.1 In silico studies for PSMA-targeting PPAPs 108 5.3.2 Synthesis of PSMA-targeting PPAPs 115 5.3.3 Biological activity of PSMA-targeting PPAPs 217 – 220 118 5.3.4 Studies towards PSMA-targeting PPAPs with cleavable hydrazide linkers 121 5.3.5 Summary of PSMA-targeting PPAPs 127 6 Summary of results 129 II Experimental Part 7 General Remarks 137 7.1 Depiction of structures 137 7.2 Substance names 137 7.3 Solvents and common chemicals 137 7.4 Analytical methods 138 7.5 Biological tests 140 7.6 Docking studies with Schrödinger© 146 8 General synthesis procedures 147 9 Syntheses expanding the PPAP library 150 9.1 Synthesis of PPAP22-core (PPAP26, 31) 150 9.2 PPAP derivatisation at C3 161 9.2.1 Standard acyl derivatisation 161 9.2.2 Standard PPAP amide synthesis 197 9.2.3 Studies towards a new PPAP amide synthesis 201 9.3 PPAP salts 205 10 Synthesis of flavonoid-like PPAPs 209 10.1 Synthesis of substituted propiolic acids 209 10.1.1 Acetylene precursors 209 10.1.2 Substituted propiolic acids 211 10.2 Synthesis of flavonoid-like dimedone test-substrates 216 10.3 Synthesis of flavonoid-like PPAPs 223 10.3.1 Aurone-like PPAPs 223 10.3.2 Flavanoid- and chalcone-like PPAPs 224 10.3.3 Flavone-like PPAPs 227 10.3.4 Dihydrocoumarin-like PPAPs 240 11 Synthesis of PPAP hybrids 242 11.1 PPAP drug-drug conjugates 242 11.1.1 PPAP-Temozolomide conjugates 242 11.1.2 PPAP-Doxorubicin conjugates 267 11.2 PPAP-antibody conjugates 276 11.2.1 Linker synthesis 276 11.2.2 Conjugate synthesis and modification 284 11.3 PSMA-targeting PPAPs 293 11.3.1 Synthesis of linkable PSMA-targeting units (PSMA-TUs) 293 11.3.2 Synthesis of a linkable PPAP amide 300 11.3.3 Linking PSMA-TUs to PPAPs 301 11.3.4 Deprotection of tBu-esters 312 III Appendix 12 X-Ray crystal structures 317 13 Exact structures of docked ligands 341 14 References 345 Curriculum vitae 355 List of publications 356 / Im Zuge dieser Arbeit wurden Verbindungen der Naturstoffklasse der polyzyklischen polyprenylierten Acylphloroglucinole (PPAPs) untersucht. PPAPs besitzen eine polyzyklische Kernstruktur, die mehrere Carbonylgruppen aufweist und mit ungesättigten Seitenketten dekoriert ist. Die in der Arbeitsgruppe entwickelte und optimierte, modulare Syntheseplattform ermöglichte die Erstellung einer ersten PPAP-Bibliothek mit 23 Verbindungen. Damit wurde die großflächige Untersuchung der antibakteriellen Aktivitäten von PPAPs eingeleitet. Weitere Erforschungen zur Derivatisierung von PPAPs ebneten den Weg zu einem synthetischen Werkzeugkasten für die Erzeugung neuer Derivate. Eine nachfolgende Struktur-Aktivitäts-Beziehungsstudie (engl. structure-activity relationship, SAR), die durch eine neuartige, in der Gruppe entwickelte C-Acylierungsstrategie ermöglicht wurde, erweiterte die Bibliothek um 25 neue nicht natürlich vorkommende Typ B PPAPs. Hierbei konnten antibiotische Wirksamkeiten mit minimalen Hemmkonzentrationen (MICs) im nanomolaren Bereich gegen Methicillin-resistenten Staphylococcus aureus (MRSA) erreicht werden und neue Leitverbindungen mit großen therapeutischen Fenstern wurden identifiziert. Die krebshemmende Wirkung von PPAPs wurde an den verschiedenen Krebsarten Leukämie, Glioblastom (GB) und Prostatakrebs (PCa) untersucht. Hierbei erwies sich eine 3 Phenylpropanoyl-Kopfgruppe als entscheidend für die proapoptotische Wirksamkeit. Strukturoptimierungen zeigten, dass für jede Krebsart jeweils unterschiedliche Verbindungen potente Wirkungen mit proapoptotischen halbmaximal inhibierenden Konzentrationen (IC50) im niedrigen mikromolaren und antiproliferativen IC50-Werten im nanomolaren Bereich aufwiesen. Flavonoid-ähnliche PPAP-Gerüste wurden im Hinblick auf verstärkte biologische Aktivitäten durch die Kombination der krebshemmenden Aktivitäten von PPAPs mit Flavonoiden untersucht. Es konnte eine einstufige Synthesemethode etabliert werden, mit welcher elf Flavonoid-ähnliche PPAPs mit vielversprechendem Krebs-bekämpfungspotenzial, insbesondere gegen GB, hergestellt wurden. Sie zeigten eine starke proapoptotische Wirkung bei geringer Toxizität gegenüber gesunden Zellen. Um die kovalente Verknüpfung verschiedener Wirkstoffe miteinander zu erweitern, wurden zusätzliche Konjugationsstrategien für die Synthese von PPAP-haltigen Wirkstoff-Wirkstoff-Konjugaten (engl.: drug-drug conjugates, DDCs) eingesetzt. Dies führte zu mehreren PPAP–Temozolomid- (TMZ) und PPAP–Doxorubicin (DOX) Konjugaten. PPAP–TMZ zeigte eine signifikante krebshemmende Wirksamkeit hinsichtlich GB, während PPAP–DOX eine beträchtliche antileukämische Aktivität zeigte. Problematisch ist, dass beide DDCs auch erhebliche toxische Wirkungen auf gesunde periphere mononukleare Blutzellen (PBMCs) zeigten. Aus diesem Grund wurden Untersuchungen zur Verbesserung der Selektivität von PPAP-Behandlungen unter Zuhilfenahme von Wirkstoffhybriden für den gezielten Wirkstofftransport (engl.: targeted drug delivery) durchgeführt. Dazu wurden PPAPs mit Linkern für Antikörper-Wirkstoff-Konjugate (engl.: antibody-drug conjugate, ADCs) verknüpft. Um das Kopplungspotenzial des hergestellten Linkers mit Antikörpern zu testen, wurde dieser mit Cystein umgesetzt. Die erfolgreiche Reaktion zeigte das Potenzial von zukünftigen Kopplungen mit echten Antikörpern. Speziell für PCa wurden gegen das prostataspezifische Membranantigen (PSMA) gerichtete PPAPs erforscht. Dieses Protein wird ausschließlich von PCa-Zellen exprimiert und stellt daher eine ideale Zielstruktur dar. Die Verwendung chemischer Strukturen aus bereits eingesetzten Arzneimitteln führte zur Entwicklung verschiedener PSMA-gerichteter PPAPs. Während in-silico-Screenings auf eine außergewöhnlich hohe Affinität zur PSMA-Bindungsstelle hinwiesen, ergaben biologische Tests nur eine sehr geringe krebshemmende Wirkung. Aufgrund der zu starken Bindungsaffinität wurden die PPAPs in der Zelle vermutlich nicht freigesetzt, was die niedrige krebshemmende Wirkung erklären könnte. Deshalb wurden im Rahmen weiterer Forschung spaltbare Linkersysteme untersucht. Insgesamt hat diese Forschungsarbeit das Verständnis für verschiedene PPAP-Derivate erheblich verbessert und ihr Potenzial für antibakterielle und krebsbekämpfende Anwendungen aufgezeigt. Die skizzierten Synthesestrategien, SAR-Studien und Konjugationsansätze bilden eine Grundlage für zukünftige Weiterentwicklungen dieses Forschungsprojektes. Weitere Optimierungen und Kooperationen sind notwendig, um PPAPs einen Schritt näher an die Anwendung als medizinische Behandlungsoption zu bringen.:Table of Contents Acknowledgements III Table of Contents V List of Abbreviations IX Abstract (English) XIV Abstract (German) XVII I Theoretical Part 1 Introduction 1 1.1 Medical need in modern society 1 1.2 Natural products in drug development 1 1.3 Polycyclic polyprenylated acylphloroglucinols (PPAPs) 2 1.3.1 Classifications 2 1.3.2 Biosynthesis 4 1.3.3 Biological activities 6 1.3.4 PPAP-synthesis strategies 7 1.4 Flavonoids in drug discovery 10 1.5 Hybrid drugs in cancer treatment 11 1.5.1 Antibody-drug conjugates 11 1.5.2 Drug-drug conjugates 12 1.5.3 Prostate cancer and prostate-specific membrane antigen (PSMA) 13 2 Objectives 16 2.1 Extending the PPAP library and improving biological activities 16 2.2 Examination and improvement of anticancer efficacy with unexplored PPAP scaffolds 17 2.3 Improving target selectivity for PCa using PSMA-targeting PPAPs 17 3 Expanding the PPAP library 18 3.1 Evaluation of the existing library 18 3.2 Synthesis of the PPAP22 core (PPAP26, 31) 21 3.3 Acylation of the C3 position (head group) 24 3.3.1 Established acylation method using acyl cyanides 24 3.3.2 Cyanide free acylation by PESLALZ 25 3.4 Structure-activity relationship (SAR) study of PPAP78 (50) derivatives (PPAP generation 2) 26 3.4.1 Designing the SAR study 26 3.4.2 Synthesis of PPAPs for the SAR study 28 3.4.3 Evaluation of antibacterial activity 29 3.4.4 Conclusion 32 3.5 Evaluation of anticancer activity 33 3.5.1 Synthesis of the used PPAPs 33 3.5.2 Blood cancer (leukaemia) 36 3.5.3 Brain cancer (glioblastoma) 39 3.5.4 Prostate cancer (PCa) 42 3.6 Exploring synthesis methods towards new PPAP amides 46 3.6.1 PPAP amide synthesis via isocyanates 46 3.6.2 PPAP amides from carbamates 49 3.7 Summary of the library expansion 51 4 Novel flavonoid-like PPAP scaffold 54 4.1 Exploring approaches to flavonoid-like PPAPs 55 4.1.1 Flavanones in one step from PPAP26 (31) 55 4.1.2 Conversion of flavanones to flavones 58 4.1.3 Flavone and aurone synthesis with propiolic acids 59 4.1.4 First biological activity screening 62 4.1.5 Synthesis of substituted propiolic acids 63 4.1.6 Elaborating the substitution scope with dimedone test substrates 65 4.2 Investigating flavone-like PPAPs 66 4.2.1 Synthesis of flavone-like PPAPs 66 4.2.2 Biological activity investigations 68 4.3 Exploring the synthesis of isoflavanone-like PPAPs 71 4.4 Summary of flavonoid-like PPAPs 74 5 Studies towards PPAP hybrid drugs 76 5.1 PPAP drug-drug conjugates 76 5.1.1 PPAP–TMZ conjugates 77 5.1.2 PPAP–DOX conjugates 92 5.1.3 Summary of PPAP drug-drug conjugates 96 5.2 PPAP antibody-drug conjugates 97 5.2.1 Synthesis of a linker with a Val–Cit recognition sequence 99 5.2.2 Synthesis and coupling of simplified linkers 102 5.2.3 Testing of cysteine coupling potential 106 5.2.4 Summary of PPAP antibody-drug conjugates 107 5.3 Using PSMA to increase PCa specificity of PPAPs 107 5.3.1 In silico studies for PSMA-targeting PPAPs 108 5.3.2 Synthesis of PSMA-targeting PPAPs 115 5.3.3 Biological activity of PSMA-targeting PPAPs 217 – 220 118 5.3.4 Studies towards PSMA-targeting PPAPs with cleavable hydrazide linkers 121 5.3.5 Summary of PSMA-targeting PPAPs 127 6 Summary of results 129 II Experimental Part 7 General Remarks 137 7.1 Depiction of structures 137 7.2 Substance names 137 7.3 Solvents and common chemicals 137 7.4 Analytical methods 138 7.5 Biological tests 140 7.6 Docking studies with Schrödinger© 146 8 General synthesis procedures 147 9 Syntheses expanding the PPAP library 150 9.1 Synthesis of PPAP22-core (PPAP26, 31) 150 9.2 PPAP derivatisation at C3 161 9.2.1 Standard acyl derivatisation 161 9.2.2 Standard PPAP amide synthesis 197 9.2.3 Studies towards a new PPAP amide synthesis 201 9.3 PPAP salts 205 10 Synthesis of flavonoid-like PPAPs 209 10.1 Synthesis of substituted propiolic acids 209 10.1.1 Acetylene precursors 209 10.1.2 Substituted propiolic acids 211 10.2 Synthesis of flavonoid-like dimedone test-substrates 216 10.3 Synthesis of flavonoid-like PPAPs 223 10.3.1 Aurone-like PPAPs 223 10.3.2 Flavanoid- and chalcone-like PPAPs 224 10.3.3 Flavone-like PPAPs 227 10.3.4 Dihydrocoumarin-like PPAPs 240 11 Synthesis of PPAP hybrids 242 11.1 PPAP drug-drug conjugates 242 11.1.1 PPAP-Temozolomide conjugates 242 11.1.2 PPAP-Doxorubicin conjugates 267 11.2 PPAP-antibody conjugates 276 11.2.1 Linker synthesis 276 11.2.2 Conjugate synthesis and modification 284 11.3 PSMA-targeting PPAPs 293 11.3.1 Synthesis of linkable PSMA-targeting units (PSMA-TUs) 293 11.3.2 Synthesis of a linkable PPAP amide 300 11.3.3 Linking PSMA-TUs to PPAPs 301 11.3.4 Deprotection of tBu-esters 312 III Appendix 12 X-Ray crystal structures 317 13 Exact structures of docked ligands 341 14 References 345 Curriculum vitae 355 List of publications 356

新藥研發為一高風險、高報酬的產業，股票或債券投資人通常不願承擔那麼高的風險。本研究利用類似Mortgage Backed Securities的概念，發行RBO（Research Backed Obligation），募集50億美金的Megafunds，投資在200個藥物上。RBO包含了Senior bond、Junior bond，以及Equity三個tranche。Senior與Junior的利率分別為5%與8%，在4年與6年後到期，Equity不發利息或股利，而是於投資期間結束後，領回基金總額扣除Bond後的價值。 本研究使用2003-2011年共5820個新藥臨床實驗結果，依照藥物型式、對應疾病分為59類，將各種藥物於各階段臨床實驗的成功機率帶入模型。每種藥物模擬5000次。最後得到投資各種藥物時，Senior bond與Junior bond的違約機率與違約時的期望損失，以及Equity報酬率的期望值與標準差。 分析模擬的結果，可得到除了其中三種藥物之外，Senior與Junior bond的違約機率皆分別在5 bp與2.5 %以下。至於Equity的部分，除了其中四種藥物的報酬率較低外，多數藥物年化報酬率的期望值在8-16%間、標準差在14-15.5%間。各藥物的風險並不會差太多，然而各種藥物報酬率的期望值彼此間有所差距。故以RBO投資新藥產業時，仍需注意投資的藥物種類。 / Biomedical innovation has become riskier and more difficult to finance with traditional sources such as private and public equity. Here we propose a financial structure called RBO (Research Backed Obligations) which is similar to Mortgage Backed Securities. In RBO structure, a $5 billion ‘Megafunds’ is financed by issuing Senior bonds, Junior bonds, and Equity. Senior bond and Junior bond tranches yield 5% and 8% annually, due within 4 and 6 years, respectively. Equity tranche does not pay any interest and obtain the residual asset after all debt obligations have been satisfied. ‘Megafunds’ will be invested on the 200 biomedical programs at various development stage to reduce the portfolio’s risk. We use the historical clinical trail data of 5820 new drug programs from 2003 to 2011. These drugs are classified into 59 groups by molecular type and disease area. Success rates of each development stage are imported into our simulation model, 5000 simulations for each drug group. The simulation result included the default rate of the Senior bonds and Junior bonds, loss of the bonds when the bonds default, and the expected value and standard deviation of the Equity return. We show that except for 3 drug groups, the default rate of Senior bond and Junior bond are less than 5 bp and 2.5% respectively for all the drugs. The expected return of Equity are between 8-16% of almost all the drug, although 4 drug groups show poorer performance. The standard deviations are between 14-15.5% for all drug groups. Consequently, almost all the drug groups have similar risks, but the expected return of the Equity tranche of these drug groups are quite different.

在各大藥廠明星藥品專利到期、新藥開發數量銳減的當下，全球首例植物新藥MediGene Veregen™ 的核准上市為製藥產業帶來新的希望，雖然製藥價值鏈與商品化模式已為人所熟知，但針對植物新藥特殊性所架構之商品化模式還是一個全新的議題，為了架構一個適合台灣中小型藥廠的植物新藥開發模式，本研究嘗試以技術層面為根基，從法規面、產品面與產業面深入探討MediGene Veregen™ 關鍵的成功因素，從中獲取值得台灣藥廠參考的經驗，同時考量台灣植物新藥開發的大環境限制因素，包括法規與健保，給予台灣藥廠一些植物新藥商品化策略建議。 本研究之架構以實務觀點出發，首先整理參考文獻以探討植物新藥包含的範圍與藥品開發流程，幫助藥廠了解植物新藥商品化需要具備的條件與資訊；接著針對台灣與美國在植物新藥方面審查上市之法規、流程與審核成果進行研究，結果顯示目前台灣有兩種植物新藥審查系統「中藥新藥」與「植物抽取新藥」，對廠商而言並不如美國單一系統來得便利；再者藉由探討植物新藥的價值鏈結構、法規結構、產品結構與產業結構，試圖架構植物新藥商品化模式；接下來以兩家新藥開發廠商為例進行實際個案研究，一家為成功推出植物新藥商品的德國藥廠MediGene AG，一家為台灣藥廠中天生技/合一生技，主要藉由分析MediGene Veregen™ 商品化過程的關鍵成功因素，比較中天生技/合一生技WH-1商品化模式的異同，探討是否有足以借鏡與改進之處。最後，歸納整理上述的研究做出結論，並且對於台灣藥廠提出策略建議，希望能對於台灣新藥開發公司有實質上的幫助。 / While the star drug patents of each big pharmaceutical company are expired one after another and the quantity of their newly developed drugs sharply declines in these years, MediGene Veregen™, the first botanical new drug in the world, enters the market and therefore brings a new hope for the pharmaceutical industry. Although the value chain and the commercialization model of pharmaceutical industry have been known and researched a lot, the specific construction of commercialization model for botanical drugs is still a brand new subject. In older to construct the model that is suitable for Taiwanese middle and small pharmaceutical companies for the process from development to commercialization of botanical new drugs, this research, based on the technical analysis, attempts to discuss the key success factors of MediGene Veregen™ through analyzing the aspects of the laws, regulations, industry and product itself. With the case study about the environmental limited factors of new drug development in Taiwan, including the laws and regulations as well as the health insurance, this research tries to offer Taiwan pharmaceutical companies some strategic suggestions for the development and commercialization of botanical new drugs. The structure of this research is based on the practical viewpoints. First, we reorganize the reference in order to define the scope of botanical new drugs and the processes of drug development. It can help pharmaceutical companies understand the conditions and information needed for botanical new drug commercialization. Then, our studies focus on the laws and regulations in Taiwan and the United States, as well as the application processes and approvals for botanical new drugs. The results show that there are two evaluation systems in Taiwan for botanical new drug applications. For those pharmaceutical companies, the dual system is not as convenient as the sole evaluation system in the States. Furthermore; based on the discussion on the structure of the value chain, laws, regulations, product and industry, this paper makes an attempt to construct a better commercialization model of botanical new drugs. Next, two pharmaceutical companies are chosen as case study in this paper. One is a German pharmaceutical company, MediGene AG, which launched the first botanical new drug. The other is a Taiwan pharmaceutical company, MicroBio Co., Ltd/Oneness Bio-Tech Co., Ltd. By analyzing the key success factors in the commercialization process of MediGene Veregen™ and comparing its commercialization model with MicroBio Co., Ltd/Oneness Bio-Tech Co. WH-1, we try to get any valuable idea and insight. Finally, our conclusion and strategic suggestions may have solid help for Taiwan botanical new drug pharmaceutical companies.

Iminosugars are a class of small molecules defined by substitution of a sugar’s ring oxygen with nitrogen. Various chemical modifications of these basic structures (e.g. alkyl chain addition off of the ring nitrogen) have been developed during the last several decades. These molecules have been considered as therapeutics for a number of pathologies including viral infection, congenital disorders of glycosylation (of both glycoproteins and glycolipids), and diabetes. This thesis focuses on the application of a small subset of iminosugars, known as deoxynojirimycin derivatives, as therapeutics against dengue virus induced pathology. Dengue virus infection predominates in tropical climates, but autochthonous infection has recently emerged in areas of both southern Europe and the southern United States. With 390 million people infected annually, dengue is the most prevalent arthropod-borne viral infection worldwide, and the possibility of severe pathology including haemorrhage, shock, and/or death, necessitates development of effective antiviral therapies. Although the molecular mechanisms responsible for progression to severe dengue disease are not completely understood, there is considerable evidence for the role of both the innate and the adaptive immune responses in development of life-threatening complications. Excessive activation of the innate immune response, a phenomenon known as cytokine storm, has been hypothesised to explain development of symptoms related to vascular permeability, whereas the adaptive immune response has been implicated in severe disease through two hypotheses – the antibody dependent enhancement and original antigenic sin hypotheses. The evidence regarding each of these potential mechanisms of severe pathology is discussed throughout this thesis principally with respect to how iminosugar treatment could alter any detrimental effects of the immune response to dengue virus infection. The principal aim of this thesis is to consider the potential of deoxynojirimycin iminosugars as antiviral therapeutics in dengue infection with a focus on how these molecules exert their antiviral effects in primary human cells. I first consider the contributions of glycoprotein inhibition and glycolipid inhibition on production of infectious dengue virus. These experiments suggest that inhibition of glycoprotein folding is responsible for inhibition of infectious dengue virus production. I next consider the impact of treatment of a promising clinical candidate iminosugar, N9-methoxynonyl-deoxynojirimycin (MON-DNJ), on the primary human macrophage transcriptome. In uninfected macrophages as well as macrophages infected with dengue virus or treated with lipopolysaccharide to model bacterial sepsis, iminosugar treatment results in activation of the unfolded protein response and inhibition of several elements of the inflammatory response including signalling by the cytokines IFN-γ and TNF-α, and the inflammatory cascade mediated by NF-κB. Activation of the unfolded protein response as a result of treatment with MON-DNJ can be confirmed by analysis of phosphorylated (activated) NFE2L2, a transcription factor that functions principally to control oxidative stress in response to ER stress signals. Modulation of the inflammatory response of macrophages to dengue infection and bacterial sepsis is confirmed by analysis of secreted cytokines. As predicted by my transcriptomic experiments, levels of TNF-α and IFN-γ produced in response to dengue or lipopolysaccharide are reduced by treatment with MON-DNJ. Finally, I attempted to extend these observations to an animal model of dengue infection with a particular focus on TNF receptor and ligand superfamily members. Unfortunately, heterogeneity of cells types from tissue samples as well as limitations of the animal model complicate interpretation of these findings. Nevertheless, this thesis demonstrates that MON-DNJ is an effective dengue antiviral therapeutic and that this therapeutic activity may be related to both reduction of infectious virus as a consequence of inhibition of glycoprotein processing and as a result of changes to the host’s response to the pathogen. These results have been used in part to justify recently initiated clinical trials of MON-DNJ as a dengue antiviral therapy.

Pharmaceutical drug development is a costly and time consuming process. Reportedly, it takes about 10-15 years and ~900 million dollars of investment to launch a new drug in the world market. Any measure that increases the power and also decreases uncertainty about that power also increases drug net present value. For some time now, it has been argued that judicious utilization of available data might lead to more efficient use of resources during drug development. Conventionally, assessment of effectiveness has been based on comparing change from baseline at some pre-specified time for the control and test treatment (SPA). The last observation carry forward (LOCF) is a widely used technique if the data are missing due to any reason. Although, LOCF is known to introduce bias, the direction and magnitude is debatable.The primary aim of the proposed simulation experiments was to assess the properties of the random effects model (REM) and mixed model repeated measures (MMRM) methods that utilize all the data collected during pivotal trials. A total of 43 scenarios based on disease progression, magnitude of drug effect, between and within subject variability and patient drop-outs were analyzed. Three analysis methods, viz. SPA, REM and MMRM, were investigated. For the SPA method, the missing data were imputed with four different methods, such as LOCF, mean imputation, population and individual regression. The false-positive, false-negative inferences and bias in estimating the effect size for each method was assessed.The most important finding of this report is that the REM and MMRM methods are efficient alternatives to the SPA methods with ~50% savings on sample size. These methods are based on sound scientific principles and provide stronger evidence against the null hypothesis. The choice of the REM versus MMRM method is dependent on the purpose of the analysis and data gathered from the experimental design. The results support the use of likelihood-based MMRM methods for regulatory decision making. The REM methods are useful in understanding the time course of the disease and drug effect, making predictions based on the data and gaining insights into time to steady state effect for rational decision making. The SPA methods are less powerful across all the scenarios. The SPA-LOCF yielded anticonservative results in some cases with type-1 error rate exceeding 15% if data were missing due to toxicity. On the other hand, the drug effect was consistently underestimated (~40%), if data were missing due to lack of effectiveness. The results demonstrate that the SPA-LOCF methods make it practically impossible to establish effectiveness in these areas with a reasonable sample size.