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My research involved in two different areas, development of novel glycosylation methodology and scope in oligosaccharide synthesis. A new scaffold for antibiotic development targeting the bacterial cell wall: Total synthesis of Albocycline and its analogs to see the mechanism of action in cell wall biosynthesis. Developed novel gem-dimethyl analogs of Fraser-Reid’s NPGs from 3,3-dimethyl 4-pentenol and 2,2-dimethyl 4-pentenol. These donors are stable toward acidic and basic conditions, which makes them step-efficient when compared to other glycosylating agents. The scope and reactivity of 3,3-dimethyl 4-pentenyl glycosides of glucose, mannose, galactose, and N-acetylglucosamine have been studied extensively for oligosaccharide synthesis. The donors are readily prepared from commercial starting materials and both glycosylation and hydrolysis yields are in the synthetically useful in oligosaccharide synthesis. NSMD methodology introduced a key step in albocycline synthesis, where (‒)-albocycline has great biological activity against “superbug” methicillin-resistant Staphylococcus aureus (MRSA). We hypothesize that albocycline inhibits the first committed step in bacterial cell wall biosynthesis. We have successfully completed two generation syntheses of albocycline. Vinylogous aldol on the left-handed fragment, aldehyde to get selectively up alcohol at the C-8 position using Davis-Ellman sulfinylimine chemistry and then oxidation with Davis oxaziridine to access requisite stereochemistry at C-4 alcohol followed by Horner-Wadsworth-Emmons to access seco-acid. Finally, a Keck macrolactonization reaction provided access to desired (‒)-Albocycline. / Chemistry

The aim of this thesis was to identify and develop anti-neuroblastoma agents via two strategies. The first involves a targeted therapy approach towards the synthesis of new drug-like PTP inhibitors (Chapter 2 and 3) and the second involved devising a new versatile synthetic route to the recently established anti-tumour natural-product lead, methyl jasmonates and its analogues (Chapter 4). From a unique proprietary screening library of 5000 drug-like compounds targeted towards PTPs, three compounds from two distinct chemical series, tetrahydroquinolines P00104 and P00341, and thiobarbituric acid P00337, were identified as PTPN22 inhibitors (IC50 = 5 μM) with moderate potency in vitro. A synthetic route to each chemical series was established and optimised and the procedure was used to synthesize a series of rationally-designed analogues for detailed structure-activity relationship (SAR) studies. The compounds were tested for PTP inhibitory activity against PTPN22 via two experimentally optimised protein assays and were tested for cytotoxicity in a number of neuroblastoma cell lines. However, none of the compounds including the resynthesized hits displayed any promising biological activity, and further investigation on these chemical series was abandoned and another strategy for developing anti-neuroblastoma agents was pursued. During the last decade, many studies have reported the cytotoxic effects of methyl jasmonate, a plant stress hormone, against various tumours both in vitro and in vivo. As the research on the anti-tumour properties of methyl jasmonate is still at early stages, and also due to the lack of a versatile synthetic procedure for the preparation of its structural derivatives, detailed SAR studies of this compound have not yet been conducted. In the course of this project, a novel versatile synthetic route to methyl jasmonate and its analogues has been developed, which allows substituents to be readily introduced at the α- and β-position of cyclopentenone. This synthetic procedure will facilitate future extensive SAR studies of methyl jasmonate in tumour cells. The cytotoxic activity of the synthesized methyl jasmonate was confirmed against a range of neuroblastoma cell lines including SK-N-SH, SHSY5Y, LAN5 and the Kelly cells, and a further study on the mechanism by which methyl jasmonate induces neuroblastoma cell death is currently underway.

La granulatimide et l'isogranulatimide, molécules naturelles isolées d'une ascidie Didemmum granulatum sont connues pour être des inhibiteurs de la Chk1, enzyme régulatrice du point de contrôle en G2 du cycle cellulaire. La première partie de ce travail est consacrée à la présentation du point de contrôle en G2 du cycle cellulaire et plus particulièrement à la checkpoint 1 kinase Chk1. Dans une seconde partie, la synthèse de nouveaux carbazoles analogues de la granulatimide et l'isogranulatimide sera décrite. Parmi ces analogues l'hétérocycle imidique sera tout d'abord remplacé par un lactame, une pyrazolinone, une pyridazine et l'hétérocycle imidazole sera quand à lui remplacé par des carbocycles à 5 ou 6 chaînons fonctionnalisés. Une troisième partie présente les résultats des tests d'inhibition de la Chk1 et les activités antiprolifératives sur trois lignées cancéreuses effectuées sur les analogues synthétisés. Tous les tests biologiques ont été réalisés au sein des laboratoires Servier.

Caseinolytic protease (ClpP) is a cylindrical protease forming the core of protein degradation machinery in eubacteria. ClpP is tightly regulated and is non-functional without a member of the Clp-ATPases. A new class of antibiotics, termed ADEPs, bind to ClpP and allow for activation without the Clp-ATPases; leading to cell death. A more efficient synthetic route to the ADEPs utilizing solid-phase peptide synthesis was investigated. A linear peptide was synthesized, however attempts to close the depsipeptidic macrocycle via macrolactonization failed. Further attempts of assembling a branched depsipeptide for ring closure via a macrolactamization resulted in products that were not stable to cleavage conditions. A group of molecules termed Activators of Self-Compartmentalizing Proteases (ACP) were identified through a screen for activity towards ClpP. Compound ACP1 was synthesized along with twelve analogs and their activity towards ClpP evaluated. The project resulted in a compound with a higher activity than its natural product counterpart.

Alzheimer’s disease is a devastating neurodegenerative disorder and the leading cause of dementia. The disease manifests via several pathologies including neuroinflammation, oxidative stress, metal ion dyshomeostasis, and cell death. To address the multifaceted nature of this disorder, the design of several diverse compounds, targeting many pathological effects, was generated. First, a series of compounds based on curcumin and diosgenin were synthesized following the bivalent design strategy. Two compounds were discovered to have neuroprotective ability, anti-oxidative function, and anti-Aß oligomerization (AßO) properties. A second set of molecules was also designed, wherein a hybrid compound strategy was utilized. Three hybrids were to shown to protect MC65 cells from Aß-induced toxicity and to have significant anti-oxidative activity. Mechanistic studies propose that protection is through disruption of interactions between AßOs and partner proteins. Furthermore, one hybrid was also shown to be able to pass the BBB. Lastly, studies of glyburide, an anti-diabetic medication, have shown an off-target anti-inflammatory effect specific for the NLRP3 inflammasome, which has been implicated in AD development. Therefore, a series of glyburide analogs were synthesized and characterized. One analog was able to successfully inhibit the NLRP3 inflammasome and reduce IL-1ß expression without affecting blood glucose. In vivo studies demonstrated an ability to prevent or ameliorate adverse inflammation-related outcomes in murine inflammatory models. Altogether, these investigations have yielded three novel series of compounds, all capable of modifying Alzheimer’s disease pathology. These results warrant future investigations into the development, optimization, and characterization of these analogs as potential treatments for Alzheimer’s disease.

Anticoagulants are used as the first line therapy for management and prevention of thrombotic disorders. Thrombin and factor Xa have been the prime targets for regulation of the coagulation cascade. In this work, a small library of 17 benzofuran derivatives were synthesized and screened against thrombin and factor Xa. The derivatives that displayed inhibitory potential were docked on the exosite-II of factor Xa using a docking protocol that was developed in our research group. These compounds were based on the β-5 structural unit found in the oligomer -'CDSO3‘, which was prepared in our lab and was found to inhibit both thrombin and factor Xa by an exosite-II mediated allosteric disruption of the catalytic triad.The results revealed that these β-5 like derivatives are inhibitory against thrombin and factor Xa, although their potency is weak. Thrombin and factor Xa appear to recognize different structural features suggesting a significant selectivity in recognition. Furthermore, a slight preference for the benzofuran scaffold was observed with factor Xa. Probing the mechanism of inhibition using Michaelis-Menten kinetics reveal that these compounds display uncompetitive inhibition of these proteases and the mechanism of inhibition is allosteric. Docking of these compounds on factor Xa were done using GOLD (Genetic algorithm for ligand docking) and the results, explain the observed inhibition profile. The computed docked poses also give an idea of the residues on the exosite-II of factor Xa critical for inhibition. The molecules studied here are radically different in terms of structure and mechanism of inhibition from any other ligand described in literature. This represents an opportunity to discover novel molecules with a possibly different pharmacological and toxicological profile.

Sulfated low molecular weight lignins (LMWLs) have shown good activity as anticoagulants by allosterically inhibiting thrombin, as well as promising agents for treating emphysema through inhibition of elastolysis, oxidation, and inflammation. Sulfated LMWLs are chemo-enzymatically synthesized from starting monomers caffeic, ferulic, and sinapic acid into sulfated dehydropolymers known as CDS, FDS, and SDS. To further the LMWLs’ development as drugs, their structural composition and physicochemical characteristics were defined in this work. The molecular weight distribution profile of the sulfated LMWLs from size exclusion chromatography performed on a high pressure liquid chromatography system (SEC-HPLC) changed from bimodal when no surfactant is used in the mobile phase of the HPLC to unimodal when surfactant is used in the mobile phase. This indicates that some large molecular weight species, likely an aggregate of smaller molecular weight chains, are disrupted when surfactant is present. The resulting estimates of molecular weight calculated when surfactant is used in the mobile phase resulted in peak average molecular weights of 5700 Da for CDS, 7400 Da for FDS, and 4300 Da for SDS. These molecular weights are 17-45% higher and can be considered more accurate than the previously reported molecular weights (CDS: 3320 Da, FDS: 4120 Da, SDS: 3550 Da) because they were measured directly whereas previous estimates were calculated from GPC-HPLC data of the unsulfated LMWL precursors. Elemental analysis and distribution coefficient measurements were also performed on the LMWL library, revealing information about the level of sulfation and hydrophobic character of the sulfated LMWLs.

O câncer é a segunda maior causa de mortes no mundo, sendo os tumores de mama os mais prevalentes e letais entre as mulheres. Apesar de vasta quimioterapia disponível, os tratamentos apresentam problemas como alta toxicidade e resistência. Dentre os tumores de mama, o subtipo triplo negativo (TNBC) apresenta o pior prognóstico e a maior limitação de tratamentos. O presente trabalho de doutorado visa o desenvolvimento de novos candidatos para tratamento de tumores de mama triplo-negativos. Fármacos que têm como alvo a proteína tubulina estão entre as terapias anticâncer mais bem-sucedidas e representam a primeira linha de tratamento para tumores do tipo TNBC. Neste contexto, foram desenvolvidas acridinonas que inibem a polimerização da tubulina e são capazes de impedir os principais mecanismos de resistência aos fármacos desta classe. O composto líder não interage com a bomba de efluxo glicoproteína-P, além de ser igualmente potente contra a linhagem celular superexpressando a tubulina βIII, uma isoforma clinicamente relevante. O mecanismo de ação revelou a interação desses compostos com o sítio da colchicina da proteína alvo. Os compostos apresentaram valores de IC50 entre 10 e 12.000 nM contra a linhagem tumoral TNBC MDA-MB-231. Em contrapartida, não foi observada citotoxicidade na linhagem normal de fibroblastos humanos (HFF1). Ensaios de imunofluorescência reforçaram a ação seletiva dos compostos, mostrando que os mesmos perturbaram a rede de microtúbulos nas células MDA-MB-231, mas não nas células HFF1. As substâncias também inibiram a migração celular e a angiogênese in vitro. Os enantiômeros do composto líder foram separados, levando a identificação de um eutômero 10 vezes mais potente contra as células tumorais e 2 vezes mais potente contra a tubulina quando comparado com a mistura racêmica. Um candidato a fármaco, eficaz e seguro, deve apresentar um balanço favorável entre a sua potência e seus parâmetros farmacocinéticos. Dessa forma, o metabolismo e a farmacocinética das acridinonas bioativas foram investigados. Em geral, os compostos foram metabolicamente estáveis, mas requerem otimização da solubilidade e permeabilidade para o desenvolvimento de fármacos administrados por via oral. A avaliação do composto líder revelou propriedades promissoras que justificam a sua consideração em modelos pré-clínicos de prova de conceito. O objetivo é a geração de candidatos a novos fármacos moduladores da tubulina com ação anticâncer. / Cancer is the second most common cause of death globally, being the breast tumors the leading cause of death in women. The number of anticancer medicines grows yearly but still exhibits problems such as high toxicity and resistance. The breast cancer has a number of subtypes and the one presenting the poorest prognosis and biggest therapeutic limitation is called triple negative breast cancer (TNBC). This PhD work aims to develop new small molecules as candidates to the treatment of the triple negative breast cancer. Drugs that target the protein tubulin are among the most successful anticancer therapies and represent the first line treatment to TNBC tumors. In this context, we developed a series of acridinones as tubulin inhibitors that can circumvent common resistance mechanisms to tubulin modulators. The lead compound did not interact with the P-glycoprotein and presented same effectiveness against cell lines overexpressing a clinically relevant tubulin isotype (βIII). In this work, we determined the mechanism of action of these compounds, that bind to the colchicine site in the tubulin. The compounds presented IC50 cytotoxicity values between 10 – 12000 nM against the TNBC cell line MDA-MB-231, with no cytotoxicity against a normal fibroblast cell line (HFF1). Immunofluorescence studies reinforced the compounds selectivity showing they disrupted the microtubules network on MDA-MB-231 cells but not on the HFF1 ones. The substances also inhibited cell migration and angiogenesis in vitro. The enantiomers of the lead compound were purified, and we identified an eutomer 10-fold more potent against the tumor cells and 2-fold more potent against the tubulin when compared to the racemic mixture. The metabolism and pharmacokinetics of the compounds were also studied aiming the development of agents with a favourable balance between potency and pharmacokinetic parameters, essential feature of an effective and safe drug. In general, the compounds were metabolically stable but need an improvement in the solubility and permeability properties to be developed as oral drugs. In summary, the lead compound presents promising properties to be assessed in preclinical proof of concept studies for further development as next-generation antitubulin drugs.