Dissertations / Theses on the topic 'Medicinal Chemistry'

Durante il terzo anno di dottorato mi sono occupata della sintesi finale dei glicomimetici del substrato naturale dell’enzima AGM1 GlcNAc-6P o del prodotto GlcNAc-1P. Le sintesi messe a punto sono necessarie per ottenere prodotti finali utilizzabili come strumenti molecolari chimici per studiare il ruolo dell’HBP nella regolazione della proliferazione e la sopravvivenza delle cellule tumorali. I prodotti presentati (caratterizzati mediante NMR (1H. COSY, HSQC e 13C) e Massa) sono stati preparati protetti (acetilati sugli ossidrili) per i test cellulari per favorire la loro diffusione attraverso la membrana cellulare e nella forma de-protetta per il test enzimatico, per poterne valutare la potenza inibitoria direttamente sull’enzima AGM1. La messa a punto del test enzimatico ha come obiettivo la valutazione dell’effettiva capacità inibitoria dei composti sintetizzati. Il test prevede la comparazione della quantità di UDP-GlcNAc prodotto dalla reazione accoppiata dell’enzima AGM1 e AGX1, utilizzando un estratto enzimatico cellulare, in assenza e in presenza dei potenziali inibitori. La quantificazione è stata effettuata mediante l’utilizzo della tecnica cromatografica HPLC, con un metodo ion-pair a fase inversa associato ad un rilevatore UV impostato ad una lamda di 254 nm. Inizialmente è stata preparata la retta di taratura dell’UDP-GlcNAc costruita partendo da una madre 1.5 mM e attraverso diluzioni sono state effettuate le analisi in triplicato per ogni punto della retta. Sono stati calcolati i valori del limite di rilevabilità (LOD) e di quantificazione (LOQ). Il test messo a punto prevede la quantificazione della produzione di UDP-GlcNAc a partire dai substrati GlcNAc-6P e UTP utilizzando estratti cellulari lisati. In una prima analisi si è esaminato l’estratto enzimatico per verificare la presenza di UDP-GlcNAc endogena, che non è stata riscontrata. Quindi si è verificata l’effettiva efficacia dell’estratto nel catalizzare la produzione di UDP-GlcNAc a partire dai substrati forniti e si sono messe a punto le condizioni di reazione. Infine, si è proceduto con l’analisi degli inibitori sintetizzati. Dati preliminari non hanno evidenziato una diminuzione del segnale di UDP-GlcNAc, il che farebbe escludere una buona attività inibitoria. Durante il periodo di Traineeship presso il CycloLab di Budapest ho potuto effettuare l'incapsulamento di un potenziale inibitore enzimatico in ciclodestrine funzionalizzate per il targeting di cellule tumorali. Alcuni dei composti sintetizzati possiedono caratteristiche chimico-fisiche che rendono il loro passaggio attraverso la membrana cellulare molto difficile: la presenza di gruppi solfati, solfonati e fosforamidati, che possiedono cariche negative, li rendono particolarmente polari. Lo scopo del progetto è quello di sfruttare ciclodestrine (CD) opportunamente funzionalizzate come un “cavallo di Troia” per veicolare gli inibitori enzimatici all'interno delle cellule tumorali. Al CycloLab mi sono quindi occupata della sintesi dello scaffold di interesse e dell’incapsulamento del potenziale inibitore.
The project carried out during the 3 years-Ph.D. has had the objective to identify and synthesize new glycomimetics as molecular tools to study the Hexosamine Biosynthetic Pathway (HBP), which role is to regulate the proliferation and survival of cancer cells. The project has been funded by AIRC and the principal aim was to identify the Adenocarcinoma of the Pancreatic Duct (PDAC) as the target of research. The synthesis of innovative chemical tools helps the understanding of the HBP pathway and its response in PDAC: new potential inhibitors, which are similar to the natural substrate of enzyme, can be recognized but trick the enzyme and block its activity in order to decrease the UDP-GlcNAc production and consequently modify the protein glycosylation. Due to the important role of the HBP in the cells, alteration of this pathway can bring to alteration of N- and O- glycosylation and activate the Unfolded Protein Response (UPR) during the Endoplasmic Reticulum (ER) stress. The description of the research target helps the understanding of the design of molecular tools: the focus point is the inhibition of the enzyme N-acetylglucosamine-phosphate mutase (AGM1): its inhibition could represent the way to induce apoptosis in cancer cells. Through the Molecular Design, a rational design of potential inhibitors has been done. This design is based on the similarity with the structures of the natural substrate of enzyme AGM1, with some modifications. All of the drawn structures have been used for Molecular Docking in order to get a first virtual screening on the compounds library. Starting from preliminary results of theoretical approach, the synthesis of compounds have been done following three different synthetic strategies. All the steps and reaction condition are described in details and are shown the characterization (1H, 13C NMR spectra, m/z) of all the synthetized compound. The optimization of the analytical method on High Performance Liquid Chromatography is necessary in order to achieve experimental data on the ability of the designed compounds to inhibit the target enzyme, data to be compared to those obtained through a computational theoretical approach. To this aim an HPLC method has been set-up for the quantification of UDP-GlcNAc produced using the cellular extract as enzyme source, and carrying out the reaction with the natural substrates GlcNAc-6P, UTP in the presence or not of the test molecules. Using 10 and 30 µL of extract, three compounds lead to a decrease of production of UDP-GlcNAc. The computational data ”describes” the interaction between the enzyme and the molecules. The calculation of C LogP has confirmed the most apolar character of compound 3B in the acetylated form. Some preliminary evaluation of the effect of compound 2B in a Triple Negative Breast Cancer (TNBC) cell model has been carried out. In conclusion, the study of the target of this research, the HBP pathway, and the focus on the inhibition of AGM1 are the starting point for a complete project, that includes at first the design of a library of compound based on the structural properties of the natural substrate. the “in silico” evaluation of their interaction with the target enzyme, the synthesis and the screening through an enzymatic assay.. The tuning of the strategy of synthesis is important to obtain the compound for the in vitro test. The analytical method with HPLC gives results comparable to the docking scores, and then, after a calculation of C LogP, the test on cells gives the final results of potency of compound 3B (2B the acetylated form). The last part describes the collaboration with CycloLab (Budapest): some compounds of the library possess chemical-physical characteristics that make their passage through cell membrane very harsh: they are very polar and some of them possess negative charges (sulphate, sulphonates, phosphoramidate). This preliminary work is still in progress.

In recent years, the edible biomedicinal products called nutraceuticals have been becoming more popular among the pharmaceutical industries and the consumers. In the process of developing nutraceuticals, in silico approaches play an important role in structural elucidation, receptor-ligand interactions, drug designing etc., that critically help the laboratory experiments to avoid biological and financial risk. In this thesis, three nutraceuticals possessing antimicrobial and anticancer activities have been studied. Firstly, a tertiary structure was elucidated for a coagulant protein (MO2.1) of Moringa oleifera based on homology modeling and also studied its oligomerization that is believed to interfere with its medicinal properties. Secondly, the antimicrobial efficiency of a limonoid from neem tree called ‘azadirachtin’ was studied with a bacterial (Proteus mirabilis) detoxification agent, glutathione S-transferase, to propose it as a potent drug candidate for urinary tract infections. Thirdly, sequence specific binding activity was analyzed for a plant alkaloid called ‘palmatine’ for the purpose of developing intercalators in cancer therapy. Cumulatively, we have used in silico methods to propose the structure of an antimicrobial peptide and also to understand the interactions between protein and nucleic acids with these nutraceuticals.

QC 20130531

As the profession of pharmacy has transitioned from a chemistry-centered profession to a patient-centered profession, the role of medicinal chemistry in the curriculum has evolved. There is decreased emphasis on memorization of chemical structures, and priority placed on relating these structures to ADME, physical properties, and pharmacodynamics. Simultaneously, the delivery of this content has shifted from traditional lecture format to other styles. Here we discuss some new approaches to teaching medicinal chemistry.

Cell surface saccharides are involved in a variety of essential biological events. Fluorescent sensors for saccharides can be used for detection, diagnosis, analysis and monitoring of pathological processes. The boronic acid functional group is known to bind strongly and reversibly to compounds with diol groups, which are commonly found on saccharides. Sensors that have been developed for the purpose of saccharide recognition have shown great potential. However, they are very hydrophobic and this lack of essential water-solubility makes them useful in biological applications. The first section of this dissertation details the process of developing water-soluble saccharide sensors that change fluorescent properties upon binding to saccharides. The second section of the dissertation focuses on the development of DNA-minor groove binders as antiparasitical agents. Parasitical diseases comprise some of the world’s largest health problems and yet current medication and treatments for these parasitical diseases are often difficult to administer, costly to the patients, and have disruptive side effects. Worse yet, these parasites are developing drug resistance, thus creating an urgent need for new treatments. Dicationic molecules constitute a class of antimicrobial drug candidates that possess high activity against various parasites. The second section details the development of a series of di-cationic agents that were then screened in in vitro activities against parasitical species.

Le scienze omiche sono attualmente in fase di sviluppo offrendo una nuova prospettiva dell'ambiente cellulare e dell'organismo. Tra queste, la genomica e la proteomica sono tra le più sviluppate. Al contrario, la lipidomica è ancora un campo emergente. L'importanza di fornire un forte approccio metodologico abbinato ad una rigorosa interpretazione dei dati è spiegata dalla recente scoperta del ruolo chiave dei lipidi come componenti strutturali e fonte energetica ma anche in molti processi biologici (secondi messaggeri, regolatori delle interazioni intercellulari e di carica superficiale, coinvolgimento in malattie metaboliche, cancro ecc.). La spettrometria di massa, grazie ai recenti e significativi progressi, è il metodo analitico più adatto in molte delle scienze omiche nonostante la loro integrazione sia ancora agli inizi e sia necessaria un'attenta ottimizzazione dei protocolli. Tuttavia, considerata la complessità molecolare, solo una visione multi-omica può darci un quadro completo dei processi intra- ed extra-cellulari in condizioni fisiologiche e patologiche nonché in risposta ad un'esposizione ambientale o chimica per contribuire infine al campo della medicina di precisione. Il progetto di ricerca qui presentato mira quindi a fornire prospettive di proteomica e lipidomica, sia come approccio singolo che integrato, a diverse domande di ricerca mediante spettrometria di massa ad alta risoluzione. L'indagine sul proteoma cutaneo di topi glabri mi ha permesso di mostrare come il peptide endogeno β-alanil-L-istidina (carnosina) agisca in difesa dei danni UV-A. Infatti, diversi importanti sistemi proteici hanno mostrato un'alterazione del trattamento con UV-A tra cui il pathway di segnale del calcio, la funzione mitocondriale o l'espressione della sirtuina, che sono stati tutti ripristinati da un trattamento preventivo della pelle mediante un'applicazione topica di carnosina. Queste alterazioni proteomiche potrebbero derivare (almeno in parte) da ROS generati da UV-A, o/e dalla generazione di prodotti di ossidazione lipidica (HNE, acroleina) derivanti dalla perossidazione di acidi grassi polinsaturi. L'implicazione di tali agenti è enfatizzata dalla potente efficacia del trattamento nel ripristinare un normale profilo proteomico delle pelli trattate con UV-A, in accordo con la sua capacità di neutralizzare la formazione di addotti sulle proteine e la loro successiva modificazione, ripristinando così la loro funzione. Passando alla lipidomica, l'importanza biologica recentemente dimostrata dagli esteri degli acidi grassi degli idrossiacidi grassi (FAHFA) ha richiesto l'ottimizzazione di un metodo su misura per la loro identificazione e quantificazione nella matrice umana. Infatti, crescenti evidenze sui ruoli fisiologici dei FAHFA, compresi quelli antinfiammatori, antidiabetici e immunomodulatori, motivano una più ampia caratterizzazione di questi lipidi come possibili biomarcatori e bersagli terapeutici per condizioni patologiche come il diabete o l'obesità. Tuttavia, la bassa concentrazione nei tessuti umani, la grande eterogeneità della struttura e il fatto che la maggior quantità di FAHFAs nelle cellule è incorporata nei trigliceridi sfidano gli attuali metodi analitici per la loro accurata identificazione e quantificazione. La preparazione dei campioni ottenuta e l'ottimizzazione del metodo strumentale hanno tuttavia permesso con successo di isolare, rilevare e quantificare per la prima volta FAHFA endogeni sul tessuto adiposo umano indicando alterazioni significative basate sullo stato metabolico (soggetti obesi insulino-sensibili o resistenti vs soggetti magri) e porzioni di tessuto adiposo (viscerale vs sottocutaneo). Questi risultati saranno utili per comprendere meglio le potenzialità biologiche di questi lipidi bioattivi nelle patologie metaboliche. Infine, i due studi multi-omici condotti in questo percorso di dottorato hanno avuto rispettivamente lo scopo di valutare gli effetti molecolari dell'acido ialuronico a basso peso molecolare (LMW-HA) nel proteoma e lipidoma di fibroblasti dermici umani e di analizzare i cambiamenti del profilo lipidomico e proteomico indotti dal trattamento di prevenzione con γ-Orizanolo in ratti obesi. LMW-HA ha mostrato un impatto sia sul profilo proteomico che lipidico, principalmente a una concentrazione pari allo 0.50%. I risultati di proteomica non solo sono stati confermati ma anche corroborati da quelli di lipidomica e dall’integrazione dei due approcci. Infatti, la funzionalità dei mitocondri, la maturazione delle cellule e il metabolismo dei lipidi si sono stati dimostrati in linea. Per quanto riguarda le modificazioni del lipidoma, abbiamo visto un particolare aumento di ceramidi, trigliceridi ed esteri del colesterolo, tutti coinvolti nell'idratazione della pelle e nel rinnovamento dell'epidermide sostenendo il ruolo benefico dell’ acido ialuronico a basso peso molecolare come ingrediente cosmetico. Tuttavia, il corretto equilibrio tra la loro sintesi e metabolismo è fondamentale per il benessere della pelle e ulteriori studi sono necessari per l’approfondimento di queste dinamiche. La valutazione degli effetti del γ-Orizanolo (Orz), invece, ha fornito solo uno scarso numero di alterazioni significative plasmatiche sia nel proteoma che nel lipidoma probabilmente a causa della debolezza del modello animale. Sono dunque necessari ulteriori studi per confermare la rilevanza biologica di Orz mostrata finora principalmente da test biologici. Per concludere, sebbene molte domande relative a questi temi di ricerca siano ancora senza risposta e altre omiche dovrebbero essere incluse in una visione integrativa (metabolomica ad esempio), durante questo percorso multidisciplinare di dottorato ho apprezzato la complessità e il valore dell’approccio omico nell'approfondire le nostre conoscenze in diversi campi di ricerca permettendo di avvicinarsi sempre più alla medicina di precisione, obiettivo finale di tutte le scienze omiche.
The ‘omics sciences are currently in development offering a new and combined perspective of cellular and organismal environment. Among these, genomics and proteomics are among the most developed while lipidomics is still an emergent field. The importance to provide a strong methodological approach paired to a rigorous data interpretation is explained by the recent discovery of the lipids’ key role not only as structural components and energetic source but also in many biological processes (i.e., second messengers, regulators of inter-cellular interactions and of surface charge, involvement in metabolic diseases, cancer etc.). Thanks to the recent significant advances, mass spectrometry is the most suitable analytical method in many of ‘omics sciences despite their integration is still at the beginning and a careful optimization of protocols is needed. Nevertheless, considering the molecular complexity, only a multi-omics vision can give a complete picture of intra- and extra-cellular processes in physiological and pathological conditions as well as in response to an environmental or chemical exposure to finally contribute to the field of precision medicine. So, the work herein aims to provide proteomics and lipidomics perspectives, both as single and integrated approaches, to different research questions by high-resolution mass spectrometry. At first, the investigation on skin hairless mice proteome allowed me to show how an endogenous peptide, β-alanil-L-histidine (a.k.a. carnosine), is acting in defense of UV-A damages. Indeed, several major protein systems shown an alteration by UV-A treatment including calcium signaling, mitochondrial function or sirtuin expression, which were all restored by a preventive treatment of the skins by a topical application of carnosine. These proteomics alterations could result (at least in part) from ROS generated by UV-A, or/and the generation of lipid oxidation products (HNE, acrolein) resulting from the peroxidation of polyunsaturated fatty acids in the irradiated skins. The implication of such agents is emphasized by the potent efficacy of carnosine in restoring a normal proteomic profile of UV-A-treated skins, in accordance with its ability to neutralize the formation of adducts on proteins and their subsequent modification, thereby restoring their function. Moving to lipidomics, the biological importance recently demonstrated by fatty acid esters of hydroxy fatty acids (FAHFAs) asked for a tailored method optimization for their identification and quantification in human matrix. In fact, increasing evidence on the physiological roles of FAHFAs, including anti-inflammatory, anti-diabetic and immunomodulatory ones, motivates a more extensive characterization of these lipids as possible biomarkers and therapeutic targets for pathological conditions such as diabetes or obesity. Nevertheless, the low concentration in human tissues, the large structure heterogeneity and that the major amount of FAHFAs in cells is incorporated into triacylglycerols challenge current analytical methods for their accurate identification and quantification. The achieved samples’ preparation and instrumental method optimization successfully enabled to isolate, detect, and quantify endogenous FAHFAs for the first time on human adipose tissue revealing significant alterations based on metabolic status (obese insulin sensitive or resistant vs lean subjects) and adipose tissue portions (visceral vs subcutaneous). These results will be useful to better understand the biological potentiality of this bioactive lipids in metabolic pathologies. Then, the two multi-omics studies herein conducted were aimed to evaluate molecular effects of low-molecular-weight hyaluronic acid (LMW-HA) in proteome and lipidome of normal human dermal fibroblasts and to analyze lipidome and proteome profile changes induced by γ-Oryzanol (Orz) prevention treatment in obese-induced rats, respectively. LMW-HA showed an impact both on proteome and lipidome profile, mainly at 0.50 % of concentration. The proteomics results were not only confirmed but also corroborated by lipidomics and integratomics ones. Indeed, mitochondria functionality, cells maturation and lipids metabolism were concordantly demonstrated. About lipidome changes, we saw a particular increasing of ceramides, triacylglycerols and cholesterol esters involved in the skin moisturizing and epidermis renewal and so supporting the beneficial role of low-molecular weight as cosmetic ingredient. Nevertheless, the correct balance between their synthesis and degradation is essential for the skin wellness and further studies are necessary for the deepening of these dynamics. The Orz’s effects assessment, instead, provided only marginal significant alterations both in plasma’s proteome and lipidome probably due to the weakness of the animal model. Further thoughts and experiments are needed to confirm the biological relevance of Orz mainly showed in biological tests so far. To conclude, although a lot of questions related to these research topics are still unanswered and other omics should be included in an integrative vision (metabolomics for example), during this multidisciplinary PhD journey I appreciated the complexity and the value of omics sciences in deepening our knowledge about several research fields approaching more and more the precision medicine goal.

The thesis comprises five chapters. Chapter one, the introduction, starts with a brief discussion of the more famous Pd catalysed reactions and their relevance to Pd as a catalyst in allene chemistry. The main part of the introduction reviews the recent work in Pd catalysed allene chemistry (formation of C-C, C-O and C-N bonds) and its importance in both synthetic and natural product syntheses. The second chapter “results and discussion” discusses the author own work including the selection of a broad series of novel substrates chosen to enable a wide range of multicomponent cascades to be designed. These cascades enable the combination of 3, 4, 5, 7 and 9 substrates in a regio and stereoselective manner delivering novel products that enabled exploration of “biochemical space”. In all cases 1-4 Z-double bonds are created stereoselectively. These strategies are applied to the novel synthesis of potentially bioactive heterocycles including those derived from reactions of the rigid adamantyl tecton involving formation of eight new bonds. The third chapter “results and discussion” summarises preliminary work on 1,3-dipolar cycloaddition generating pyrimidinylpyrrolidine. The fourth chapter contains the experimental details of all new compounds.

It is well known boronic acids have its unique chemistry and related applications in organic synthesis. The boronic acid functionally group also plays very important roles in medicinal chemistry and chemical biology. For example, boronic acids have been developed as potential therapeutic agents, chemical biology tools. All these applications are directly related to the unique electronic and chemical properties of the boronic acid group. Herein, several application of boronic acids have been studied: 1) several groups of compounds were found as bacterial quorum sensing inhibitors; 2) a boronate compound was developed as a probe for detecting reactive oxygen species (ROS); and 3) boronic acid-modified aptamers can be used for glycoprotein recognition.

This thesis investigates new methods for the environmentally sustainable synthesis of heterocyclic scaffolds for application in medicinal chemistry. Chapter I introduces general principles of sustainability in synthetic organic chemistry. This includes the characterization and application of sustainable solvents and the use of biomass feedstocks in synthesis. Chapter II explores the synthesis of substituted isoindolinones via a ruthenium-catalyzed alkyne cyclotrimerization. The introduction details the synthesis and medicinal application of isoindolinones and describes previous research involving alkyne cyclotrimerizations. Following this, the development of a regioselective alkyne cyclotrimerization reaction in a sustainable solvent is reported. The optimized alkyne cyclotrimerization conditions are then used to synthesize a selection of isoindolinone products. Chapter III describes the application of a kinetically-controlled furan-Diels–Alder reaction to the synthesis of heterocyclic scaffolds, including the endo-cantharimide. The study and application of furan-Diels–Alder reactions are introduced. Following this, the Diels–Alder reaction of a 3-alkoxyfuran under sustainable reaction conditions is explored experimentally and applied to the diastereoselective synthesis of endo-cantharimides. The potential application of endo-cantharimides in medicinal chemistry is discussed with the aid of biological testing and the Diels–Alder reactions of 3-alkoxyfurans is probed with the aid of computational calculations. Chapter IV concerns the cyclization of reducing sugars to prepare chiral tetrahydrofurans. The role of tetrahydrofurans in medicinal chemistry, the synthesis of tetrahydrofurans from sugar derivatives and the application of hydrazones in synthetic chemistry are introduced. Following this the development of a hydrazone-mediated cyclization of L-arabinose under sustainable reaction conditions is reported. The optimized conditions are applied to prepare tetrahydrofurans from other sugars. The manipulation of the tetrahydrofuran products is also explored. Chapter V draws some general conclusions from the thesis and describes potential future directions for the research. Chapter VI contains the details of experimental procedures and compound characterization for the results discussed in Chapters II–IV.

In this thesis the successful synthesis of a novel tricyclic scaffold containing a ring fused triazole and piperidine will be discussed and furthermore its utility as a new potential privileged structure will be evaluated. As starting material for the synthesis of the considered scaffolds the commercially available ethyl 4-oxopiperidine-1-carboxylate 108 was used. Piperidone 108 was converted into scaffolds 96, 97 and 98 alternating the ring size (n=1, 2, 3) of the fused bicyclic ring. This tricyclic triazole scaffold was incorporated into known biologically active molecules (Sitaglitpin, a DPP-4 inhibitor; Maraviroc, a CCR-5 receptor antagonist and GDC-0941, a pi3K inhibitor) to test its potential to serve as a new possible Privileged Scaffold. Through scaffold hopping, analogues with excellent biological activity against the chosen biological targets were achieved. Additionally, the stereoselective synthesis of one of the four possible isomers of the tricyclic triazole 178 was obtained in 4 steps to give the synthetically access towards the synthesis of all feasible enantiomeric and diastereomeric analogues.

Nous avons synthétisé des composés d'étain avec des acides undécylénique, ricinoléique et caprylique. Ils ont une activité importante contre certaines souches de microrganismes, puisque ils agissent pour certains d’entre eux à des concentrations nanomolaires. Staphylococcus aureus semble être 4000 fois plus sensible à leur toxicité que les cellules de mammifères. Nous avons obtenu des composés du ruthénium qui présentent cytotoxicité contre des cellules cancéreuses suivant un mécanisme d'action différent de ceux observés pour le Cisplatine ou d'autres composés de Ru, grâce à leur grande stabilité dans les réactions de substitution. Enfin, pendant la vectorisation des composés dérivés du Ru avec une Affitine nous avons pu acquérir des connaissances importantes sur un éventuel mécanisme d'action de ce type de molécules dont le potentiel redox très abaissé par rapport aux composés correspondants pourrait être responsable de la polymérisation de protéines cibles par transfert d’électron
Related to antimicrobial research, we synthesized tin compounds derived from undecylenic, ricinoleic and caprylic acids and we found that they show very high activity against some strains of bacteria and yeast, even in nM range, being up to four thousand times more potent against Staphylococcus aureus than against mammalian cells. For ruthenium compounds, in turn, we have confirmed that the mode of action of some compounds that were synthesized recently is undoubtedly different from Cisplatin or other ruthenium compounds, because of their high stability toward substitution reactions. Finally, during the vectorization of compounds derived from Ru with Affitin we have gained important knowledge about a possible mechanism of action of this type of molecule: it could indeed be possible that these compounds which have a very reduced redox potential compared to corresponding compounds can cause polymerization of proteins by electron transfer

This study involves using methyllithium to synthesize Fischer carbene complexes as precursors for metal templated α,β-unsaturated complexes with potential as acceptors in the Baylis Hillman reaction as well as in Dötz benzannulation. Fischer carbene complexes contain low oxidation state metal centers, are electrophilic in nature and are stabilized by π-donating substituents such as alkoxy and amino groups. The increased electron withdrawing nature of the metal carbonyl moiety was expected to improve the rates of reaction compared to organic carbonyls. Four Fischer carbenes were synthesized via nucleophilic addition of MeLi to chromium and tungsten hexacarbonyl at low temperatures followed by alkylation using either a Meerwein salt (Me₃OBF₄) to give the desired Fischer metal methyl methoxy carbenes or Et₄NBr/alkylhalide to make the corresponding ethoxy and allyloxy carbenes. Characterization was by means of ¹³C NMR, ¹H NMR, and IR. In silico studies were carried out looking at the effect of substituents on the carbene bond. Synthesis of α,β-unsaturated complexes was effected via the aldol condensation route and found to be unfavorable using enolizable aldehydes, although the use of two aryl aldehydes resulted in successful preparation of two α,β-unsaturated complexes. Difficulty in the purification of these complexes hindered their full characterization. Computational studies looked at the effect of substituents on the system as well as variation of the metal from Cr to Mo and W. Synthesis of Baylis Hillman adducts using α,β-unsaturated complexes as acceptors was unsuccessful due to the ease of product oxidization. One potential product was obtained in its crude form although purification was not possible due to oxidation. Computational studies suggested that the oxygen on the ligand negatively impacts the stability of these Fischer carbene derived Baylis Hillman adducts promoting intramolecular oxidation of the metal. The α,β-unsaturated complexes and Baylis Hillman adducts were considered to be candidates to undergo Dötz benzannulation methodology. The use of the α,β-unsaturated complexes in this reaction was generally unsuccessful, both in the microwave and in conventional reflux conditions. Computational studies of these compounds were carried out to facilitate understanding of their stability and configuration.

Objective: To integrate process-oriented guided-inquiry learning (POGIL) team-based activities into a 1-semester medicinal chemistry course for doctor of pharmacy (PharmD) students and determine the outcomes.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2019
Cataloged from PDF version of thesis.
Includes bibliographical references.
Modern molecular biology, biochemical, and chemical techniques have made it possible to identify individual natural products that possess pharmacological activity from medicinal plants. While approximately 50% of all new FDA-approved small molecule therapeutics in the past 40 years were natural products or natural product analogs, challenges including limited natural resources and the difficulty of solving the total synthesis or semi-synthesis of natural products has limited our ability to harness the full diversity of chemical structures provided by nature to treat human diseases. One solution to these challenges is the elucidation of plant specialized metabolite biosynthetic pathways. Identifying and characterizing the enzymes involved in specialized metabolite biosynthesis will provide insight into the evolution of enzymes performing interesting chemistries and provide new tools for the enzymatic production of therapeutically promising natural products. The goal of this dissertation is to explore the aspects of both medicinal chemistry and the elucidation of biosynthetic pathways that can contribute to the development of novel therapeutics. First, we analyzed the structure-activity relationship of analogs of the the flavonoid icariin and identified analogs with improved potency in inhibiting human phosphodiesterase-5. We subsequently identified and characterized a novel flavonoid prenyltransferase and O-methyltransferase from the medicinal herb Epimedium sagittatum that is known to produce many bioactive prenylated and methylated flavonoids.
by Yasmin-Pei Chau.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

In this thesis I described the theory and application of several computational methods in solving medicinal chemistry and biophysical tasks. I pointed out to the valuable information which could be achieved by means of computer simulations and to the possibility to predict the outcome of traditional experiments. Nowadays, computer represents an invaluable tool for chemists. In particular, the main topics of my research consisted in the development of an automated docking protocol for the voltage-gated hERG potassium channel blockers, and the investigation of the catalytic mechanism of the human peptidyl-prolyl cis-trans isomerase Pin1.

In this thesis I described the theory and application of several computational methods in solving medicinal chemistry and biophysical tasks. I pointed out to the valuable information which could be achieved by means of computer simulations and to the possibility to predict the outcome of traditional experiments. Nowadays, computer represents an invaluable tool for chemists. In particular, the main topics of my research consisted in the development of an automated docking protocol for the voltage-gated hERG potassium channel blockers, and the investigation of the catalytic mechanism of the human peptidyl-prolyl cis-trans isomerase Pin1.

The active antimalarial principles of three traditional medicinal plants, Passerina obtusifolia (Thymelaeaceae), Tetradenia riparia (Labiatea) and Xerophyta retinervis (V elloziaceae) were investigated by employing bioassay guided fractionation. Two novel compounds and five known constituents were isolated from the active fractions of these three plants. The types of compounds isolated included: three triterpenoids (20(29)-Lupene-3α,28- diol (30), 20(29)-Lupene-3α, 16β,28-triol (32) and 3β-Hydroxy-20(29)-Lupen-28-oic acid (42)); two diterpenoids (8-Abietene-7 β,13 β -diol (45) and cariocal (51)); one flavonoid ( 5-Hydroxy:.4' ,6, 7-trimethoxyflavone ( 44)) and one flavonolignan ( 11-0- acetyl hydnocarpin (62)). In addition, one analogue of 7α-hydroxyroyleanone (41) (which was previously isolated from T riparia and was found to be the active antimalarial principle of the plant) was prepared.

Cyclic dipeptides are widely used as models for larger peptides because of their simplicity and limited conformational freedom. Some cyclic dipeptides have been shown to produce antiviral, antibiotic and anti-tumour activity (Milne et al., 1998). In this study the cyclic dipeptides, cyclo(Ser-Ser) and cyclo(Ser-Tyr), were synthesised from their corresponding linear precursors using a modified phenolinduced cyclisation procedure. The phenol-induced cyclisation procedure resulted in good yields and purity of the cyclic dipeptides. Quantitative analysis and evaluation of the physicochemical properties of the cyclic dipeptides was achieved by using high-performance liquid chromatography, scanning electron microscopy, thermal analysis and X-ray powder diffraction. The structures of the synthesised cyclic dipeptides were elucidated using infrared spectroscopy, mass spectrometry, nuclear magnetic resonance spectroscopy and molecular modelling. The study aimed to determine the biological activity of cyclo(Ser-Ser) and cyclo(Ser-Tyr) with respect to their anticancer, antimicrobial, haematological and cardiac effects. Anticancer studies revealed that cyclo(Ser-Ser) and cyclo(Ser- Tyr) inhibited the growth of HeLa (cervical cancer), HT-29 (colon cancer) and MCF (breast cancer) cancer cell lines. Both cyclic dipeptides also inhibited the growth of certain selected Gram-positive, Gram-negative and fungal microorganisms in the antimicrobial study. Although the inhibition of growth in the anticancer and antimicrobial studies was statistically significant, the clinical relevance is questionable, since the inhibition produced by both cyclic dipeptides was very limited compared to other pre-existing anticancer and antimicrobial agents. Cyclo(Ser-Tyr) exhibited significant activity in the haematological studies, where it increased the rate of calcium induced-coagulation, and decreased the rate of streptokinase-induced fibrinolysis. Both cyclic dipeptides, however, failed to produce any significant effects on thrombin-substrate binding and ADPinduced platelet aggregation. Cardiac studies revealed that cyclo(Ser-Ser) and especially cyclo(Ser-Tyr) reduced the heart rate, coronary flow rate and ventricular pressure of isolated rat hearts.

Computational methods have become an integral part of drug development and can help bring new and better drugs to the market faster. The process of predicting the biological activity of large compound collections is known as virtual screening, and has been instrumental in the development of several drugs today in the market. Computational methods can also be used to elucidate the energies associated with chemical reactivity and predict how to improve a synthetic protocol. These two applications of computational medicinal chemistry is the focus of this thesis. In the first part of this work, quantum mechanics has been used to probe the energy surface of palladium(II)-catalyzed decarboxylative reactions in order to gain a better understating of these systems (paper I-III). These studies have mapped the reaction pathways and been able to make accurate predictions that were verified experimentally. The other focus of this work has been to develop virtual screening methodology. Our first study in the area (paper IV) investigated if the results from several virtual screening methods could be combined using data fusion techniques in order to get a more consistent result and better performance. The study showed that the results obtained from data fusion were more consistent than the results from any single method. The data fusion methods also for several target had a better performance than any of the included single methods. Next, we developed a dataset suitable for evaluating the performance of virtual screening methods when applied to large compound collection as a replacement or complement for high throughput screening (paper V). This is the first benchmark dataset of its kind. Finally, a method for using computationally derived reaction coordinates as basis for virtual screening was developed. The aim was to find inhibitors that resemble key steps in the mechanism (paper VI). This initial proof of concept study managed to locate several known and one previously not reported reaction mimetics against insulin regulated amino peptidase.

Room temperature iodocyclisation of homoallylamines stereoselectively delivers functionalised 2- (iodomethyl)azetidine derivatives in high yield. Increasing reaction temperature from 20 °C to 50 °C switches the reaction outcome to realise the stereoselective formation of functionalised 3-iodopyrrolidine derivatives. It was shown that these pyrrolidines are formed via thermal isomerisation of the aforementioned azetidines. Primary and secondary amines could be reacted with iodomethyl azetidine derivatives to deliver stable methylamino azetidine derivatives. With subtle changes to the reaction sequences homoallyl amines could be stereoselectively converted to either cis- or trans- substituted 3-amino pyrrolidine derivatives at will. The stereochemical divergent synthesis of cis and trans substituted pyrrolidines supports an ion part, aziridinium, isomerisation pathway for azetidine to pyrrolidine isomerisation. Six azetidine derivative were probed in a zebrafish embryo developmental assay for capacity to illicit morphological changes. The range of effects across the probed molecules demonstrates the suitability of this assay for screening azetidine derivatives. One of the probed molecules, rac-(((cis)-1-benzyl-4-phenylazetidin-2-yl)methyl)piperidine, exhibited particularly promising effects in the developmental assay.

HIV-1 is undoubtedly one of the viruses that have characterized the last half century in human history. This virus, found to be the AIDS etiological cause, is still one of the biggest challenges for drug therapy, since there are still neither a vaccine nor a definitive cure. Therefore, despite more than 25 marketed drugs administered in several combinations in HAART (Highly Active Anti-Retroviral Therapy), there is still the need of molecules active through new mechanisms, in order to overcome the many resistances developed by the virus to counter the pharmacological treatment. The main purpose of this thesis is to identify new active molecules able to inhibit viral replication with innovative mechanisms. Also, considering that the AIDS patients easily develop secondary infections, another part of the work is focused on the study of antifungal agents previously developed in our laboratories. The first chapter contains the research carried out directly on the HIV-1 enzyme Integrase. This one results to be particularly interesting because against it, at the moment, only three marketed drugs with the same mechanism of action are available. Moreover, it has not a homologue protein in human cells, eliminating the problem of poor selectivity. Starting with the study of Integrase, after computational analysis and biological tests, we discovered a very interesting dual-inhibitor with unique activity profile and active in vitro with no toxic effects at the maximum tested concentration. The second chapter is focused on the study of molecules with antifungal activity able to block candida infections, which easily occur in immunocompromised patients. This study was carried out on macrocyclic compounds developed in our laboratory, revealing a very unusual mechanism of entrance into fungal cells. Specific biological experiments were performed to investigate in depth the behaviour of these molecules, leading to a potential new treatment to overcome the problem of antifungal resistance. The third and last chapter is focused on the development of a new part of code for the program KNIME, a data-pipelining software used in CADDD (Computer-Aided Drug Design and Development). Leaning on LigandScout, new code (nodes) was programmed, and the existing one was implemented. In particular, we focused on two topics: chirality and fingerprints. Two nodes were programmed to address chirality issues (Standard Property and Chirality Enumerator node) and four based on fingerprints (Fingerprint Calculator node, Fingerprint Similarity node, Fingerprint Clustering node and the Diversity Picker node).

Tuberculosis (TB) is a life-threatening infectious disease caused by Mycobacterium tuberculosis (Mtb). Globally, TB is a major public health burden with an estimated 10.4 million new cases and 1.8 million deaths reported in 2015. Although TB is curable, the treatment options currently available are beset by numerous shortcomings such as lengthy and complex treatment regimens, drug-drug interactions, drug toxicities, as well as emergence of widespread multi-drug resistance. Therefore, there is an urgent and compelling need to develop new, more effective, safer drugs with novel mechanisms of action, and which are capable of shortening treatment duration. This study focused on hit-to-lead optimization of two new classes of compounds with potential anti-TB properties: 2-aminoquinazolinones (AQZs) and benzoxazole-based oximes (BZOs). A hit compound for each of these classes with low micromolar antimycobacterial activity had previously been identified through phenotypic whole-cell in vitro screening.

Tuberculosis (TB) and malaria continue to be major public health concerns, globally claiming 2-3 million deaths every year. A number of efficacious drugs are available for the treatment of TB and malaria, which, through various combination therapies, are fully effective in treating these diseases. However, the wide spread resistance in M. tuberculosis (Mtb) and P. falciparum, the causative agents of TB and malaria, respectively, has made the existing therapies less effective. Thus novel agents able to circumvent drug resistance and other challenges associated with existing TB and malaria treatments are urgently needed. The development of a new drug is a lengthy and costly process; therefore, approaches that can save both time and money need to be emphasised. Drug repositioning is one such approach that has been applied in this project. Drug repositioning basically involves a situation where a drug active in one disease is derivatised or used as a template for the synthesis of derivatives active in another disease. This approach has the potential to significantly shorten the drug discovery process. This study focused on the repositioning of two drugs, the antibacterial agent fusidic acid and the antipsychotic agent metergoline, in TB and/or malaria via medicinal chemistry approaches. New semisynthetic derivatives of fusidic acid and metergoline were synthesized and evaluated for antimycobacterial activity against the H37Rv strain of Mtb and antiplasmodial activity against the NF54 strain of P. falciparum.

This thesis describes phytochemical and biological/pharmacological studies on six medicinal plants. Plant materials were extracted by ethanol, tested for their biological activity and then subjected to "bioassay-guided fractionation" to yield active and inactive compounds. The compounds were identified by standard physico-chemical techniques including UV, IR, NMR and ElMS. The crude ethanol extract of the leaves of Premna shcimperi was found to be antibacterial against gram positive bacteria (Staphylococcus aureus and Bacillus subtilis). An antibacterial bioassay-guided fractionation of the extract resuited in the isolation of the active principle, (5R,8R,9S,10R)-12-oxo-ent-3,13(16)- clerodien-15-oic acid (SHM-l). This novel compound was bactericidal against S. aureus and B. subtilis at concentrations of 25 and 50 µg ml⁻¹. Studies on the structural-activity relationship of SHM-l showed that the α,β unsaturated moiety played a major role in its antibacterial activity. Investigation of the inactive fractions of P. schimperi extract afforded three flavonoid aglycones: luteolin, quercetin and kaempferidej three flavonoid glycosides: luteolin-4" -,β-D-glucoside, quercetin-3-,β-D-galactoside and quercetin-α-L-arabinopyranoside and five cinnamate and benzoate derivatives. The ethanol extract of the leaves of Premna oligotricha was also found to be antibacterial against a range of gram-positive bacteria. A bioassay-directed isolation afforded three novel active principles, two diterpenes: 16-hydroxy-clerod- 3,13(14}-diene-15,16-olide (SHM-3) and ent-12-oxolabda-8,13(16}-dien-15-oic acid (SHM-S) and a sesquiterpene, 7-α, hydroxy-2-oxo-6,11-cyclofarnes-3(15)-ene (SHM-19). While the activity of SHM-5 and SHM-19 was marginal that of SHM-3 was good, almost comparable with streptomycin. The antimicrobially inactive fractions of P. oligotricha extract yielded a novel diterpene, ent-8,β12α-epidioxy- 12,β-hydroxlabda-9(11), 13-dien-15-oic acid γ lactone and two novel flavonoids: 3,5,5'-trihydroxy-6,7 ,3' ,4'-tetramethoxyflavone and 3,5,7 ,5'-tetrahydroxy-6,3',4'- trimethoxyflavone. Investigation of the leaves of Premna recinosa afforded six flavonoids: quercetin, luteolin, pachypodol, chrysosplenol-D, naringenin and eriodictyol and three lignans: {+)-8-hydroxy-pinoresinol, {+)-lariciresinol and {-)-seco-isolariciresinol. Portulaca oleracea and Pentas schimperiana are used in the folk medicine as local anaesthetics. Studies on the isolated nerve and/or muscle preparations showed that extracts do block nerve conduction in these excitable tissues. A subsequent systematic bioassay-guided fractionation of crude extracts showed that excess potassioum ions in the extracts were responsible for the in vitro pharmacological activity of P. oleracea and P. schimperiana. Extracts from the leaves of Leonotis ocymifolia var raineriana showed no antibacterial activity. Three novel diterpenes: ent- {13S)-9,13α-epoxylabda- 6{ 19),β,16{15)-diol dilactone, (13R)-6,β-acetoxy-9, 13α-epoxylabda-19(20),8,16{ 15)- diol dilactone and 20,8-acetoxy-9α,13e-dihydroxy-15(16)-epoxy-Iabd-14-en-6,19,(β- O-lactone together with a known compound: {13S)-20,β-acetoxy-9,13α-epoxylabda- 6(19),β,16{15)-diol dilactone were isolated. Investigation of the root afforded two known compounds 3-methoxy-4-hydroxy-{trans)-cinnamaldehyde and 3-methoxy- 4-hydroxy-bezaldehyde.

The isomers of cyclo(Trp-Pro) (cyclo(L-Trp-L-Pro), cyclo(L-Trp-D-Pro), cyclo(D-Trp-LPro) and cyclo(D-Trp-D-Pro)) have been successfully synthesized and screened for biological activity. High percentage yields were obtained by using the three phase synthesis system, which involves the synthesis of the intermediate protected linear dipeptides, followed by the removal of the protecting Boc groups. This step is followed by cyclization and crystallization of the isomers. The diketopiperazines rings of cyclo(L-Trp-L-Pro) and cyclo(D-Trp-D-Pro) contain cisamide bonds, while cyclo(L-Trp-D-Pro) and cyclo(D-Trp-L-Pro) contain trans-amide bonds. These bonds govern the conformation of the diketopiperazines ring. The isomers have shown different degrees of biological activity, possibly as a result of the orientation of the side chain of tryptophan and this difference in conformation, leading to varying interactions between isomer and a range of receptors. Under experimental conditions, 10-3 M cyclo(L-Trp-D-Pro) and cyclo(D-Trp-L-Pro) showed effective anticancer activity against the cervical cancer cell line, HeLa, resulting in a <50% reduction in cell viability. Cytotoxicity screening with cyclo(D-Trp-L-Pro) indicated that it was hepatocyte-specific in its toxicity, whilst the other isomers were cytotoxic against the other cell types tested. At 1mg/ml, cyclo(L-Trp-L-Pro) proved to be an effective antimicrobial agent against Gram positive bacteria, while cyclo(L-Trp-DPro) effectively inhibited the growth of the Gram negative bacteria, Esherichia coli. Cyclo(D-Trp-L-Pro) proved to be effective against Streptococcus, while cyclo(D-Trp-DPro) effectively reduced viability of the yeast, Candida albicans. Cyclo(D-Trp-L-Pro) was the only isomer to show Ca2+-channel antagonism, whilst the other isomers resulted in opening of the Ca2+-channel. No effects were observed on K+-channel activity for all the isomers tested. The isomers also proved to be valuable antiarrhythmic agents by effectively reducing the time spent in ventricular tachycardia and arrhythmia, as well as decreasing the time for the heart rate to return to a normal sinus rhythm. Furthermore, cyclo(L-Trp-D-Pro) showed positive chronotropic activity, while cyclo(D-Trp-L-Pro) ii showed negative chronotropic activity. In addition, cyclo(L-Trp-D-Pro) and cyclo(D-Trp- L-Pro) also increased the coronary flow rate. 0.125 –1 mM Cyclo(L-Trp-D-Pro) decreased aggregation in washed platelets induced by thrombin. All isomers increased adhesion to an artificial surface when the platelets were stimulated by ADP, yet caused reduced adhesion when the platelets were stimulated by thrombin. These results prove the potential of these compounds as novel agents in a range of biological fields, indicating that a combination of L- and D- amino acids may prove more effective than an agent consisting solely of L-amino acids.

This thesis examines ways in which the chemical reactivity of benzimidazole-, pyrrole- or pyrrolizinone-based heterocycles can be modulated. The latent reactivity of these systems has been used to develop enzyme inhibitors, antitumour compounds and a novel amino acid derivatisation technique. Chapter One introduces the concept of modulated chemical reactivity, with illustrative examples from biochemistry, synthesis and medicinal chemistry. The application of latent reactivity towards the inhibition of serine proteases, a class of proteolytic enzymes, is then reviewed. Two inhibitor classes are discussed, the first of which require an inorganic cofactor, e.g. a metal ion, for enhanced activity. Secondly, mechanism-based inhibitors, which rely on activation by the protease to covalently derivatise the enzyme, are discussed. The application of latent reactivity to effect DNA alkylation, and hence inhibit tumour cell growth, is also reviewed. This section places particular emphasis on alkylators that arc attached to an intercalator to enhance their affinity for DNA. Chapter Two describes the inhibition of -chymotrypsin, a representative serine protease, by compounds that were proposed to exhibit enhanced activity upon the coordination of Zn2+. The attempted synthesis of the putative inhibitors 2.1a-b was hindered by the instability of the diamine precursors 2.2a-b. The model compound 2.1c was prepared, however this compound was prone to deuteration and oxidation at its methylene bridge. Benzimidazoles 2.14a-c were subsequently prepared, in addition to the dihydrochloride salt of 2.14a. These compounds were moderate to weak inhibitors of a-chymotrypsin, and no Zn2+-mediated enhancement of inhibition was observed. Benzimidazole 2.14b was shown to coordinate Zn(OAc)2 by 1H NMR spectroscopy, which illustrated that the lack of Zn2+-enhanced inhibition of a-chymotrypsin by compounds of type 2.14 did not result from an inability to complex metal ions. Chapter Three describes the inhibition of a-chymotrypsin by a new series of C2-acyl-C5- (hydroxyalkyl)pyrroles. These compounds are postulated to be mechanism-based inhibitors. The pyrrole-based peptidomimetics 3.13a and 3.14a were prepared and assayed against a-chymotrypsin. Minimal difference in activity was observed between these compounds, despite the bulky hydrophobic moiety at C5 of 3.14a, which was proposed to bind more tightly to the S1 subsite than the 5-(hydroxymethyl) group of 3.13a. Mass spectrometry of 3.13a incubated with a-chymotrypsin suggested that inhibition was noncovalent in nature. Modifications at the C2 position of 3.13a led to the preparation of 3.13b, 3.24 and 3.25, of which the latter was the optimal inhibitor, and an IC50 value of 108 M was determined for this compound. The attempted solid phase synthcsis of 3.13a was ineffectual due to the instability of the 5-(hydroxymethyl)pyrrole moiety to TFA. Solid phase synthesis of a 5-formylpyrrole precursor to 3.13a gave the desired product 3.36 as an inseparable mixture with pyrrole acid 3.16b. Solution phase synthesis of the 5- (hydroxymethyl)pyrrole 3.35, which possessed a carboxylic acid moiety, afforded an unstable compound. Chapter Four details a novel derivatisation method for amino acids, whereby the amine is first "capped" by pyrrole acid 3.16b. Subsequent reaction with hydrocinnamoyl chloride releases the latent spectrophotometric properties of the pyrrole-amino acid adduct. A previous study afforded pyrrolizinones 4.11a-c, and this study expanded the range of pyrrolizinones available for analysis to include 4.11d-i and 4.17a-b. Initial UV-vis spectroscopic analysis revealed that methanol degraded pyrrolizinone 4.11e. UV-vis spectroscopy of 4.11a-i/4.17a-b in acetonitrile revealed that the variable R1 and R2 groups did not affect the Amax and 8 values in a systematic manner. Analysis of the LRMS fragmentation patterns for 4.11a i/4.17a-b, and their pyrrole precursors 3.18a-i/3.29a-b, revealed that a production was generated whose mass was dependent on the R1 group. The abundance of the diagnostic product ion was dependent on the nature of R1 and R2. Fragmentation was increased by the technique of electron impact, compared to electro spray ionisation. Chapter Five describes the application of 5-(hydroxymethyl)pyrroles towards the development of DNA alkylators with latent reactivity. Attachment of an intercalator to the pyrrole moiety via an alkyl linker was proposed to increase cytotoxicity. Model studies afforded 5.21 (naphthyl-based) and 5.31 (quinolyl-based), however the synthetic methodology was not amenable to compounds that incorporated anthryl or acridine moieties, i.e. "true intercalators". Compounds 5.21 and 5.31 displayed moderate activity against the P388 cancer cell line. The 5-(hydroxymethyl)pyrrole substructure analogue (5.41) elicited no activity in the P388 assay. Compound 5.31 did not react with aniline, which suggested that compounds of this type would not effect DNA alkylation in vivo. A DNA binding assay revealed that 5.31 had a low affinity for the genetic material, which rationalised its moderate P388 activity. Compounds 5.21 and 5.31 displayed minimal antiviral and antimicrobial activity.

Neuroinflammatory based-diseases are a very challenging area for medicinal chemists. Several efforts have been made during the years; however, an effective treatment for these diseases, such as Alzheimer’s disease (AD) and multiple sclerosis (MS), does not exist yet. Neuroinflammatory -based diseases are multifactorial in nature with still unclear pathogenic mechanisms and scarce information on how neuroinflammation is interconnected with other concomitant events, such as neurodegeneration. Polypharmacology is one of the milestones for the development of therapies able to combat multifactorial diseases. Particularly, the development of multitarget compounds through different strategies (linking, fusing, merging) has permitted to expand the potential arsenal to treat multifactorial diseases. Based on these considerations, this thesis was focused on the development of multitarget molecules for combating neuroinflammatory diseases through different and innovative polyphamacological approaches in four projects. Projects 1 & 2 focused on the development of fatty acids (FAs)/drug conjugates for the treatment of MS and AD, respectively. Particularly, we applied a conjugation strategy among omega-3 FAs and valproic acid (project 1) or P2Y6-agonists (project 2), in order to obtain innovative multitarget molecules with potentially increased property in terms of efficacy and pharmacokinetics, and less cytotoxicity. Projects 3 & 4 focused on the design and synthesis of multitarget molecules derived from food byproduct (cashew nut-shell liquid) for the treatment of AD. Particularly, we developed different series of molecules as potentially globally accessible drugs, by applying a hybridization strategy. Notably, we developed a small library of dual sustainable HDAC/ferroptosis inhibitors (project 3), and a library of cholinergic inhibitors with potential anti-inflammatory profile (project 4) as multitarget hybrids for the treatment of AD.

In living systems, proteins usually team up into “molecular machinery” implementing several protein-to-protein physical contacts – or protein-protein interactions (PPIs) – to exert biological effects at both cellular and systems levels. Deregulations of protein-protein contacts have been associated with a huge number of diseases in a wide range of medical areas, such as oncology, cancer immunotherapy, infectious diseases, neurological disorders, heart failure, inflammation and oxidative stress. PPIs are very complex and usually characterised by specific shape, size and complementarity. The protein interfaces are generally large, broad and shallow, and frequently protein-protein contacts are established between non-continuous epitopes, that conversely are dislocated across the protein interfaces. For this reason, in the past two decades, PPIs were thought to be “undruggable” targets by the scientific research community with scarce or no chance of success. However, in recent years the Medicinal Chemistry frontiers have been changing and PPIs have gained popularity amongst the research groups due to their key roles in such a huge number of diseases. Until recently, PPIs were determined by experimental evidence through techniques specifically developed to target a small group of interactions. However, these methods present several limitations in terms of high costs and labour- and time-wasting. Nowadays, a large number of computational methods have been successfully applied to evaluate, validate, and deeply analyse the experimentally determined protein interactomes. In this context, a high number of computational tools and techniques have been developed, such as methods designed to construct interaction databases, quantum mechanics and molecular mechanics (QM/MM) to study the electronic properties, simulate chemical reactions, and calculate spectra, and all-atom molecular dynamics simulations to simulate temporal and spatial scales of inter- and intramolecular interactions. These techniques have allowed to explore PPI networks and predict the related functional features. In this PhD work, an extensive use of computational techniques has been reported as valuable tool to explore protein-protein interfaces, identify their hot spot residues, select small molecules and design peptides with the aim of inhibiting six different studied PPIs. Indeed, in this thesis, a success story of in silico approaches to PPI study has been described, where MD simulations, docking and pharmacophore screenings led to the identification of a set of PPI modulators. Among these, two molecules, RIM430 and RIM442, registered good inhibitory activity with IC50 values even within the nanomolar range against the interaction between MUC1 and CIN85 proteins in cancer disease. Furthermore, computational alanine scanning, all-atom molecular dynamics simulations, docking and pharmacophore screening were exploited to (1) rationally predict three potential interaction models of NLRP3PYD-ASCPYD complex involved in inflammatory and autoimmune diseases; (2) identify a potentially druggable region on the surface of SARS-CoV-2 Spike protein interface and select putative inhibitors of the interaction between Spike protein and the host ACE2 receptor against COVID-19 (CoronaVIrus Disease 2019); (3) investigate intramolecular modifications as a consequence of a point mutation on C3b protein (R102G) associated with the age-related macular degeneration (AMD) disease; (4) design non-standard peptides to inhibit transcriptional events associated with HOX-PBX complex involved in cancer diseases; and (5) to optimise a patented peptide sequence by designing helix-shaped peptides embedded with the hydrogen bond surrogate approach to tackle cocaine abuse relapses associated with Ras-RasGRF1 interaction. Although all the herein exploited techniques are based on predictive calculations and need experimental evidence to confirm the findings, the results and molecular insights retrieved and collected show the potential of the computer-aided drug design applied to the Medicinal Chemistry, guaranteeing labour- and time-saving to the research groups. On the other hand, computing ability, improved algorithms and fast-growing data sets are rapidly fostering advances in multiscale molecular modelling, providing a powerful emerging paradigm for drug discovery. It means that more and more research efforts will be done to invest in novel and more precise computational techniques and fine-tune the currently employed methodologies.

Thesis (Ph.D.)--Indiana University, Dept. of Chemistry, 2009.
Title from PDF t.p. (viewed on Jul 22, 2010). Source: Dissertation Abstracts International, Volume: 70-12, Section: B, page: 7549. Adviser: Mu-Hyun Baik.