Bioactive compounds inspired by nature: synthesis, stability & delivery

Date
2020-01
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Stellenbosch : Stellenbosch University
Abstract
ENGLISH ABSTRACT: Historically, natural products (NPs) have served as a dominant contributor to the discovery of medicinal agents. Their invaluable role in modern drug discovery is emphasized by the major impact NP-inspired scaffolds and NPderivatives/mimics have had on the inception of clinically approved drugs. Evidently, Nature continues to influence human innovation in the design and development of new therapeutic agents. Since NPs have been programmed to interact with biological targets, they are thought of as evolutionary-chosen, privileged structures. In this regard, NPs serve as a structural ‘muse’ for the design of new biologically active small molecules. Moreover, the unique characteristics of NPs can be leveraged and fine-tuned to enhance potency and drug-like properties of therapeutic candidates. Owing to the tremendous impact of NPs in the field of medicinal chemistry, the inspiration gained from their scaffolds has provided a strong impetus for the development of new antiproliferative agents. The research explored in this dissertation comprised a multi-disciplinary project with the golden thread throughout the various scientific ventures being the development of bioactive anticancer agents inspired by Nature. In its entirety, the research was aimed at investigating two common challenges experienced during drug development –poor aqueous solubility and hysicochemical instability of NP/NP-inspired compounds. The two NP families whichmotivated these endeavours were the rigidins and sphaeropsidins (specifically SphA). The rigidins are marine-derived alkaloids which have been developed and optimized into a potent class of 7-deazahypoxanthines. A major breakthrough regarding the synthesis of the rigidins and their NP-inspired analogues, involved a crucial multi-component reaction (MCR) furnishing the tetrasubstituted 2-amino pyrrole precursor. The implementation of the MCR has permitted the rapid assembly of molecular diversity, serving as an attractive strategy towards the 7-deazahypoxanthines. These rigidin-inspired analogues are capable of disrupting microtubule dynamics in cancer cells and cell lines associated with MDR. However, like many other anticancer agents, the potent 7-deazahypoxanthines suffer from poor aqueous solubility. To address this impediment, two strategies were envisaged – polymeric micelle encapsulation and the introduction of solubilizing appendages. The first approach was directed towards the synthesis of a specific 7-deazahypoxanthine analogue and its covalent conjugation onto a hydrophilic polyvinylpyrrolidone (PVP) polymer via a Michael addition reaction. Although the resulting drug-polymer conjugate was successfully furnished, research efforts evolved towards the design of a more amphiphilic PVP polymer capable of loading multiple 7-deazahypoxanthine molecules. This led to the attempted synthesis of an α-substituted Nvinylpyrrolidone monomer. The next strategy aimed at enhancing the aqueous solubility of the 7-deazahypoxanthines involved incorporation of water-solubilizing groups (WSGs) within the pyrrolo[2,3-d]pyrimidine backbone. Building on a previously identified lead compound, the synthetic strategy was directed towards the addition of WSGs on the aryl rings prior to the MCR. Evaluation of the final compounds revealed that the introduction of WSGs derived from the sulfonamido acetophenone components led to a retention or improvement of nanomolar activity. Inspection of the growth inhibitory activity (GI 50 ) against HeLa cells identified a new front-runner amongst the family of rigidin-inspired analogous. This particular 7-deazahypoxanthine, 6-[4-(2-ethoxyethoxy)benzoyl]-2-(pent-4-yn-1-yl)-5-phenyl-1,7- dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one, bearing a glycol ether solubilizing appendage, exhibited an exceptional GI 50 of 15 nM against HeLa cancer cell lines. Although the aqueous solubility of the most potent 7-deazahypoxanthines were assessed based on their hydrophobicity (log D), it was suspected that the poor solubility was rather attributed to the solid-state characteristics arising from the network of strong intermolecular forces. Building on this work, a collaborative project on the prodrug behaviour of N9-substituted 7-deazahypoxanthines was undertaken. Stability studies conducted in cell medium via LC-MS analysis revealed the conversion of the N9-substituted 7-deazahypoxanthines into their unsubstituted potent variants. Thereby, substitution at the N9 position of the 7-deazahypoxanthine presented an additional potential tactic for aqueous solubility enhancement. The other NP which motivated this project is the fungal metabolite SphA. This tetracyclic diterpenoid demonstrates potent cytotoxicity towards apoptotic- and multidrug-resistant cancer cell lines. SphA’s unique mechanism of action has been ascribed to the disruption of ion homeostasis in cancer cells through the impairment of regulatory cell volume increase (RVI). Unfortunately, the potential success of SphA as an anticancer agent is hindered due to its poor physicochemical stability in cell medium. As such, the research of this section was directed towards two objectives – analysis of the degradation behaviour of SphA and the impetus towards improved stability. Initially, SphA was incubated in cell medium with sample analysis taken at varying time intervals using LC-MS. Owing to the complexity of the LC-MS data, a complementary simplified buffer solution was implemented. Extensive LCMS analysis revealed possible SphA degradation metabolites based on their molecular ions, with several corresponding to SphA degradation product-amino acid adducts. Most noteworthy was the presence of a major SphA degradation metabolite (SphA-Met), which increased in concentration over extended incubation periods. Subsequently, scale-up degradation experiments and use of preparative HPLC resulted in the isolation of SphA-Met. Finally, successful structural elucidation of this major degradation metabolite was achieved via extensive 2D NMR spectroscopy and innovative NMR spectroscopy protocols. Bioactivity evaluation of SphA-Met revealed a loss in activity compared to the parent SphA NP, thereby further motivating the necessity to address its undesired degradation behaviour. With the hope of enhancing antiproliferative activity, as well as stability, a semi-synthetic SphA derivative was furnished using olefin cross-metathesis. Although significant improvement in potency was established by our collaborators, stability issues of the semi-synthetic derivative in cell medium persisted. Thus, a final research thrust was directed towards the synthesis of an amphiphilic block copolymer capable of physical encapsulation and protection of the SphA derivative. Pleasingly, the desired PVP-b-PDLLA copolymer was successfully generated and polymeric micelle formation was confirmed. Ultimately, this dissertation encompassed several scientific disciplines focussed on the advancement of rigidin- and SphA-inspired research. In addition to contributions made to the small-molecule synthetic arena, integrated knowledge from separation science, structural elucidation and polymer chemistry was applied towards the different research ventures. Considering the findings, the development of these NP-inspired compounds as efficacious anticancer agents holds much promise.
AFRIKAANSE OPSOMMING: Histories het natuurlike produkte (NPte) 'n dominante bydrae gelewer tot die ontdekking van medisinale middels. Hul onskatbare rol in die uitvinding van moderne medisyne word beklemtoon deur die groot impak wat NPgeïnspireerde strukture en NP-afgeleides/nabootsers al gehad het op die ontstaan van klinies goedgekeurde middels. Duidelik beïnvloed die natuur die mens se innovasie in die ontwerp en ontwikkeling van nuwe terapeutiese middels. Aangesien NPte geprogrammeer is om met biologiese teikens in wisselwerking te wees, word dit beskou as evolusiegekose, bevoorregte strukture. In hierdie verband, dien NPte as 'n strukturele ‘muse’ vir die ontwerp van nuwe biologies aktiewe klein molekules. Die unieke eienskappe van NPte kan ook aangewend word en fyn ingestel word om die sterkte en geneesmiddelagtige eienskappe van terapeutiese kandidate te verbeter. As gevolg van die geweldige impak van NPte op die gebied van medisinale chemie, het die inspirasie vanaf hul strukture die ontwikkeling van nuwe antiproliferatiewe middels sterk aangemoedig. Die navorsing wat in hierdie proefskrif ondersoek is, het uit 'n multidissiplinêre projek bestaan. Die verwantskap tussen die verskillende wetenskaplike ondernemings was die ontwikkeling van bioaktiewe teenkankermiddels wat geïnspireer is deur die Natuur. Die navorsing was in die geheel daarop gemik om twee algemene uitdagings wat tydens medisyne-ontwikkeling ervaar word, te ondersoek – swak oplosbaarheid in water en fisiochemiese onstabiliteit van NP/NP-geïnspireerde verbindings. Die twee NP-families wat hierdie pogings gemotiveer het, was die rigidiene en spheropsidiene (spesifiek SphA). Die rigidiene is van marien afgeleide alkaloïede wat ontwikkel en geoptimaliseer is tot 'n kragtige klas van 7- deazahipoxantiene. 'n Groot deurbraak ten opsigte van die sintese van die rigidiene en hul NP-geïnspireerde analoë, het 'n belangrike multikomponentreaksie (MKR) behels wat die tetrasubstitueerde 2-amieno-pirroolvoorloper voorsien het. Die implementering van die MKR het die vinnige samestelling van molekulêre diversiteit moontlik gemaak, en dien dus as 'n aantreklike strategie vir die skepping van 7-deazahipoxantiene. Hierdie rigidiengeïnspireerde analoë kan die mikrotubule-dinamika in kankerselle en sellyne wat met multi-middelweerstandigheid geassosieer word, ontwrig. Net soos baie ander teenkankermiddels, ly die kragtige 7-deazahipoxantiene aan 'n swak oplosbaarheid in water. Om hierdie kwessie aan te spreek, is twee strategieë voorgestel – polimeer-miselle inkapseling en die byvoeging van oplosbare groepe. Die eerste benadering was gerig op die sintese van 'n spesifieke 7-deazahipoxantien-analoog en die kovalente binding daarvan op 'n hidrofiliese polivinielpirolidoon (PVP) polimeer via 'n Michael-addisiereaksie. Alhoewel die resulterende geneesmiddel-polimeer-konjugaat suksesvol voorsien was, het navorsingspogings ontstaan om 'n meer amfifiliese PVP-polimeer te ontwikkel wat in staat is om verskeie 7-deazahipoxantien molekules te verbind. Dit het dus gelei tot die sintese pogings van 'n α-gesubstitueerde N-vinielpirolidoon-monomeer. Die volgende strategie wat daarop gemik was om die wateroplosbaarheid van die 7-deazahipoxantiene te bevorder, het die insluiting van wateroplosbare groepe (WOGe) in die pirrool[2,3-d]pirimidien raamwerk behels. As gevolg van 'n voorheen geïdentifiseerde voorloper-verbinding, was die sintetiese strategie gerig op die toevoeging van WOGe op die arielringe voor die MKR. Evaluering van die finale verbindings het aangedui dat die byvoeging van WOGe, afgelei van die sulfonamiedo-asetofenoonkomponente, gelei het tot die behoud of verbetering van die nanomolêre aktiwiteit. Inspeksie van die groei-beperkende aktiwiteit (GI 50 ) teen HeLa-selle het gelei tot die identifisering van 'n nuwe voorloper onder die familie van rigidien-geïnspireerde analoë. Hierdie spesifieke 7-deazahipoxanthien, 6-[4-(2-etoksie-etoksie)bensoiel]-2-(pent-4-in-1-iel)-5-feniel-1,7-dihidro-4H-pirrool[2,3-d]pirimidien-4-one, met 'n glykoleteroplosbare groep, het 'n uitsonderlike GI 50 van 15 nM teen HeLa-kanker-sellyne getoon. Alhoewel die water oplosbaarheid van die kragtigste 7-deazahipoxantiene op grond van hul hidrofobisiteit (log D) beoordeel was, word dit vermoed dat die swak oplosbaarheid eerder toegeskryf moet word aan die vaste toestand eienskappe a.g.v. die netwerk van sterk intermolekulêre kragte. Verdere verrigtinge op hiedie navorsingsgebied behels 'n samewerkingsprojek oor die voor-medisyne-gedrag van N9-gesubstitueerde 7-deazahipoxantiene. Stabiliteitstudies wat in selmedium via vloeistofchromatologie-massaspektrometrie VC-MS analise uitgevoer was, het die omskakeling van die N9-gesubstitueerde 7-deazahipoxantiene tot hul ongesubstitueerde sterk aktiewe variante aan die lig gebring. Sodoende het substitusie op die N9-posisie van die 7-deazahipoxantiene 'n addisionele potensiële taktiek vir die verbetering van hul oplosbaarheid in water gelewer. Die ander NP wat hierdie projek gemotiveer het, is die swammetaboliet SphA. Hierdie tetrasikliese diterpenoïed toon 'n kragtige sitotoksisiteit teenoor apoptotiese en multi-middelweerstandige kankerselle. SphA se unieke werkingsmeganisme word toegeskryf aan die ontwrigting van ioon-homeostase in kankerselle deur die verswakking van die toename in regulerende selvolume (TRV). Ongelukkig word die potensiële sukses van SphA as 'n teenkankermiddel verhinder as gevolg van die swak fisiochemiese stabiliteit in die selmedium. Dus, was die navorsing van hierdie afdeling op twee doelstellings gerig: ontleding van die afbrekingsgedrag van SphA en die aanmoediging tot verbetering van stabiliteit. Gedurende aanvanklike studies is SphA in selmedium geïnkubeer met monsteranalise by verskillende tydsintervalle met behulp van VC-MS. Vanweë die kompleksiteit van die VC-MS data, is 'n komplimentêre vereenvoudigde bufferoplossing geïmplementeer. Uitgebreide VC-MS ontledings het moontlike SphA-afbrekingsmetaboliete op grond van hul molekulêre ione aan die lig gebring, met verskeie ooreenstemmings met die SphA-afbraakprodukaminosuuraddukte. Die opvallendste waarneming was die teenwoordigheid van 'n hoof SphA-afbreekmetaboliet (SphA-Met), wat gedurende langer inkubasietydperke in konsentrasie toegeneem het. Daarna het opskaling van die afbrekings-eksperimente en gebruik van voorbereidende hoë-druk vloeistofchromatologie (HDVC) gelei tot die isolasie van SphA-Met. Uiteindelik is die suksesvolle strukturele verklaring van hierdie hoof afbreekmetaboliet (SphAMet) deur middel van uitgebreide 2D-KMR spektroskopie en innoverende KMR spektroskopie-protokolle verkry. Bio-aktiwiteitsevaluering van SphA-Met het 'n verlies aan aktiwiteite in vergelyking met die oorspronklike SphA NP getoon. Die noodsaaklikheid om die ongewenste afbreekgedrag van SphA aan te spreek word hierdeur verder gemotiveer. Met die hoop om antiproliferatiewe aktiwiteit te verbeter, sowel as stabiliteit, is 'n semi-sintetiese SphA-afgeleide met behulp van olefien-kruismetatese voorsien. Alhoewel 'n beduidende verbetering in sterkte deur ons medewerkers vasgestel was, het stabiliteitsprobleme van die semi- sintetiese afgeleide in selmedium voortgeduur. 'n Finale navorsingspoging was dus gerig op die sintese van 'n amfifiele blok-copolymeer, wat in staat is om fisiese inkapseling en beskerming van die SphA-afgeleide, te maak. Aangenaam was die gewenste PVP-b-PDLLAkopolimeer suksesvol gegenereer en polimeer-misellevorming was bevestig. Oor die algeheel, het hierdie proefskrif verskeie wetenskaplike dissiplines wat gerig was op die bevordering van rigidien- en SphA-geïnspireerde navorsing, omvat. Benewens die bydraes wat tot die sintetiese arena van klein molekules gelewer het, is geïntegreerde kennis van skeidingswetenskap, strukturele verklaring en polimeerchemie op die verskillende navorsingsondernemings toegepas. As u die bevindings in ag neem, hou die ontwikkeling van hierdie NP-geïnspireerde verbindings as effektiewe teenkankermiddels, baie belofte in.
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Thesis (PhD)--Stellenbosch University, 2020.
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