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

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