Sphingosine-1-phosphate (S1P) is an amino-alcohol signaling molecule produced from the intracellular phosphorylation of the lipid sphingosine. Despite possessing several identified intracellular targets, the predominant signaling functionality of S1P is derived from its activation of membrane-bound G-protein coupled receptors (GPCRs). The binding of S1P to these receptors (S1P1-5) is closely associated with immune cell development and recruitment. As such, the modulation of S1P-related pathways is of particular interest for the development of immunomodulating agents. To reach its native GPCRs, S1P must be released from the cell. This process is facilitated by the transmembrane transport protein Spinster homolog 2 (Spns2) in most vertebrates. Studies in murine species have demonstrated that the protein plays a key role in directing immune cell chemotaxis and the progression of autoimmune diseases. Consequently, Spns2 represents an attractive target for the pharmaceutical induction of immunosuppression. While several drugs that act through the modulation of S1P receptor signaling have received FDA approval for the treatment of autoimmune disorders (fingolimod, siponimod, ozanimod, and ponesimod), they typically manifest on-target cardiovascular side-effects. Therefore, the development of novel Spns2 inhibitors is a prudent alternative approach to achieve S1P-mediated lymphopenia. In this dissertation, the design, synthesis, and activities of highly potent Spns2 inhibitors are disclosed. These structures spanned several scaffolds and culminated in the discovery of a phenylurea derivative 4.11i. In vitro assessment of 4.11i demonstrated that the compound possessed an IC50 value of 92 nM, making it the most potent inhibitor of Spns2 disclosed to date. Intraperitoneal administration of 4.11i (10 mg/kg dose) into mice reduced circulating lymphocyte counts and impaired the progression of experimental autoimmune encephalomyelitis (a murine model of multiple sclerosis). Taken together, these data validated the target of 4.11i in vivo and represented the first reported instance of Spns2 inhibition as a viable multiple sclerosis treatment. Additional work is currently being undertaken to further improve in vivo activity and pharmacokinetic properties of 4.11i. / Doctor of Philosophy / White blood cells comprise a significant portion of the body's natural defense mechanisms. In healthy individuals, these white blood cells identify and destroy foreign materials and organisms. However, in patients with multiple sclerosis, immune cells can become sensitized to protein fragments lining the myelin sheath of neurons. These autoreactive immune cells recognize the body's natural neuronal proteins as antigens. Damage exerted by autoreactive cells leads to the development of neurological impairments (i.e., fatigue, muscle weakness, and slurred speech) as nerve impulses are disrupted before reaching their target. First-line treatment of multiple sclerosis often centers on the administration of immunosuppressive drugs to curtail the progression of the disease and mitigate immune cell-directed demyelination. A driving factor in white blood cell localization is the lipid sphingosine-1-phosphate (S1P). Concentrations of S1P are often not static in the body, with different tissue types and fluids possessing variable levels. Immune cells, and lymphocytes in particular, use this natural S1P gradient to dictate their movement within the body. Lymphocytes will track with the S1P gradient, going from areas of lower S1P concentration (lymph tissue) to areas of higher S1P concentration where synthetic enzyme expression is upregulated (multiple sclerosis lesions). Consequently, the development of drugs that can alter this S1P gradient represents an ideal avenue to achieve immunosuppression. One key mediator of S1P release is the transmembrane transport protein Spinster homolog 2 (Spns2). This protein directs the secretion of intracellular S1P into the extracellular space and is necessary for lymphocytes to enter circulation. However, little effort has been devoted to the development of Spns2 inhibitors. As such, the inhibition of this protein represents a novel and underexplored target for the treatment of autoimmune disorders. In this disclosure, the structures of several highly potent Spns2 inhibitors are revealed. The work around these structures led to the discovery of 4.11i. This compound proved highly potent in biological assays and animal models. Mice treated with 4.11i experienced a reduction in circulating lymphocyte counts and demonstrated less symptom manifestation in multiple sclerosis disease models.

A variety of diseases including Alzheimer's disease, asthma, cancer, fibrosis, multiple sclerosis, and sickle cell disease have been associated with elevated levels of sphingosine-1-phosphate (S1P). S1P, a pleiotropic lipid mediator involved in a broad range of cellular processes, is synthesized solely by the phosphorylation of sphingosine (Sph) and is catalyzed by the two isoforms of sphingosine kinase (SphK1 and SphK2). Therefore, SphKs are a potential therapeutic target; however, the physiological role of SphK2 is still emerging. In order to determine the role of SphK2 in vivo, more potent and selective small molecule inhibitors of SphK2, as well as dual inhibitors are necessary. Herein, explorations and advancements on the second generation SphK2 selective inhibitor SLR080811 are disclosed. Investigations into the lipophilic tail region of the hSphK2 inhibitor SLR080811 are detailed. This investigation highlights the dependency of SphK2 selectivity and potency on overall compound length. More importantly, this study identified the internal aryl ring of SLR080811 as a key pharmacophore of the scaffold. To further probe the significance of the aromatic region, the phenyl ring was replaced by a 2,6-naphthyl ether skeleton. Investigations into the tail region of this scaffold are described in detail. Key discoveries from this structure-activity relationship study include SLC5111312 (hSphK2 Ki = 0.90 μM, dual hSphK inhibitor), SLC5091592 (hSphK2 Ki = 1.02 μM, > 20-fold hSphK2 selective) and SLC5121591 (hSphK2 Ki = 0.61 μM, >16-fold hSphK2 selective). Molecular modeling studies with hSphK2 indicate that the extended aromatic group is able to participate in π-π stacking interactions with Phe548. In silico docking studies indicate that a guanidine hydrogen bond to Asp211 is key for SphK2 selectivity, and incorporation of a 3'-hydroxyl group on the pyrrolidine ring increases hydrogen bonding to Asp308, thereby increasing SphK1 potency and reducing selectivity. Additionally, biological studies employing SLC5111312 have helped to further elucidate the role of SphK2, suggesting that SphK2 has a catalytic role in the regulation of blood S1P levels. The shape of the hSphK2 binding pocket was probed by introducing an indole moiety in place of the naphthyl ring and varying its substitution pattern. One key discovery from this study is SLC5101465 (hSphK2 Ki = 0.09 μM, > 111 fold SphK2 selective), which has a 1,5-indole substitution pattern with an N-nonyl "tail". Molecular docking simulations highlight the importance of rotatable bonds and a relatively linear orientation between the "head group" and "tail group" to maintain essential hydrogen bond interactions to Asp residues with the guanidine moiety while minimizing steric interactions in the middle of the binding pocket. Expanding upon the 1,5-indole scaffold of SLC5101465, a series of aryl tail derivatives are examined. This study confirms the necessity of electron withdrawing groups located at the end of the inhibitor scaffold to optimize binding in the tail region of the SphK2 binding pocket. / Ph. D.

Sphingosine 1-phosphate (S1P) is a cellular signaling molecule that has been implicated in a variety of diseases including cancer, fibrosis, Alzheimer's, and sickle cell disease. It is formed from the phosphorylation of sphingosine (Sph) by sphingosine kinase (SphK) and SphK exists as two isoforms-"SphK1 and SphK2, which differ with respect to their cellular activity and localization. As the key mediators in the synthesis of S1P, SphKs have attracted attention as viable targets for pharmaceutical inhibition. To validate their potential as therapeutic targets, we aimed to develop potent, selective, and in vivo active inhibitors of SphK. Herein, we describe the design, synthesis and biological evaluation of SphK2 inhibitors. We first describe the development of six SphK2 inhibitors that assess the utility of replacing lipophilic tail groups with heterocyclic rings. These six compounds demonstrate that the lipid binding pocket for SphK2 cannot accommodate compounds with tail groups that are conformationally restricted or positively charged. We then describe the development of aminothiazole-based analogues of an SphK1-selective inhibitor. A library of 37 aryl-substituted aminothiazole tail groups were synthesized, revealing a structure-activity relationship study that examines electronic effects on the aryl-substituted aminothiazoles and the effect of modifying the amino portion of the aminothiazole. These molecules show surprisingly good potency and selectivity for SphK2. In particular, we highlight 3.20dd (SLC4101431), a biphenyl aminothiazole that is the post potent and selective SphK2 inhibitor to date, with an SphK2 Ki of 90 nM and 100-fold selectivity for SphK2. This molecule's in vivo activity will also be discussed. Mitochondrial uncouplers are small molecules that shuttle protons from the inter membrane space to the mitochondrial matrix independent of ATP synthase, which disrupts oxidative phosphorylation and promotes increased nutrient metabolism for homeostasis to be maintained. Consequently, small molecule mitochondrial uncouplers have been pursued as probes for mitochondrial function and as potential therapeutics for the treatment of obesity and type 2 diabetes. Herein, we describe the design, synthesis, and biological evaluation of small molecule mitochondrial uncouplers. We report a library of 52 compounds that have good mitochondrial uncoupling activity over a wide therapeutic range, including 5.16t (SHC4111522) and 5.17i (SHC4091665), which have EC50 values of 0.63 uM and 1.53 uM, respectively, and achieve at least 2-fold increase in oxygen consumption rates relative to basal levels. With these molecules, we demonstrate that pKa and cLogP significantly contribute to uncoupling activity and must be accounted for when developing new generation small molecule mitochondrial uncouplers. / Ph. D. / Sphingosine kinase 1 and 2 (SphK1 and SphK2) are enzymes that facilitate the production of the biomolecule sphingosine 1-phosphate (S1P), which plays an essential role in cell growth and survival. However, overproduction of S1P has been linked to a number of diseases including cancer, Alzheimer’s, and sickle cell disease. Therefore, because S1P is involved in these diseases, the amount of available S1P must be controlled. This work describes the design, development, and biological study of over 40 compounds that could be used as potential inhibitors of SphK2 to help control S1P levels and, therefore, hopefully alleviate the effects of disease. In particular, this work describes molecules that probe the SphK2 binding pocket and demonstrates that the molecules cannot be rigid or positively charged when binding to the hydrophobic portion of the SphK2 binding pocket. Additionally, this work describes the most potent and selective reported SphK2 inhibitor to date, 3.20dd (SLC4101431). Mitochondrial uncouplers are compounds that target our body's mitochondria and aim to make ATP production challenging, causing the mitochondria to burn extra energy in the form of glucose and fatty acids to allow normal levels of ATP to be produced. By making the mitochondria burn extra energy, mitochondrial uncouplers have the potential to be treatments for diseases such as obesity and diabetes. This works describes the design, development, and biological study of over 50 mitochondrial uncouplers that are capable of increasing mitochondrial activity over a wide concentration range, including 5.16t (SHC4111522) and 5.17i (SHC4091665), which are very potent and effective uncouplers.

Malaria is a severe and deadly mosquito-borne disease. Although treatable, the continuous emergence of multi-drug resistant parasite strains urgently calls for the development of novel antimalarial agents. P. falciparum parasites synthesize essential isoprenoid precursors, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), via a non-mevalonate pathway: the methylerythritol phosphate (MEP) pathway. This pathway is not utilized by humans. Thus, compounds that target the MEP pathway and disrupt isoprenoid biosynthesis in P. falciparum hold promise as potent and safe new antimalarial agents, that engage new targets. Previously, we and others identified MMV008138 from the Malaria Box as a MEP pathway targeting compound. Later work revealed that it targets the IspD enzyme within the MEP pathway. Work in the Carlier group has established preliminary structure-activity relationship (SAR) of MMV008138: 1) (1R,3S)-configuration is required; 2) 2', 4'-disubstitution of the D-ring with small, electronegative substituents; 3) functional importance of carboxylate acid at C3. In this work, I aim to gain further insight into the C3 SAR and A-ring SAR of lead compound MMV008138. Synthesized acid bioisosteres and A-ring analogs of MMV008138 were evaluated in their ability to inhibit P. falciparum parasite growth. We showed that the C3 substituent of MMV008138 has a very tight SAR, and likely interacts with a very constricted pocket within the PfIspD enzyme. A-ring modifications are limited to certain positions of MMV001838 and need to be sterically small. However, we have yet to identify a modification that significantly improves drug lead potency. Future work will continue towards understanding the A-ring SAR of MMV008138, as well as D-ring SAR and C1-SAR. Efforts will also be directed towards finding analogs with improved potency, transport and metabolic stability. / MS / Malaria is a severe and deadly mosquito-borne disease, caused by malaria parasites. Although treatable, the continuous emergence of drug resistance urgently calls for the development of novel antimalarial agents. Research in the Carlier group is aimed at finding drug molecules that can selectively target and kill the malarial parasite, and at the same time be safe to humans. The Carlier group has identified MMV008138 from the Malaria Box as a promising drug lead. In this work, I aim to understand the how the structure of MMV008138 play a role in its ability to kill malaria parasites. These results will help identify modification strategies that may significantly improve drug lead potency.

Industrial chemicals in European Union produced or imported in volumes above 1 tonne annually, necessitate a registration within REACH. A common problem, concerning these chemicals, is deficient information and lack of data for assessing the hazards posed to human health and the environment. Animal studies for the type of toxicological information needed are both expensive and time consuming, and to that an ethical aspect is added. Alternative methods to animal testing are thereby requested. REACH have called for an increased use of in silico tools for non-testing data as structure-activity relationships (SARs), quantitative structure-activity relationships (QSARs), and read-across. The main objective of the studies underlying this thesis is related to explore and refine the use of in silico tools in a risk assessment context of industrial chemicals. In particular, try to relate properties of the molecular structure to the toxic effect of the chemical substance, by using principles and methods of computational chemistry. The initial study was a survey of all industrial chemicals; the Industrial chemical map was created. A part of this map was identified including chemicals of potential concern. Secondly, the environmental pollutants, polychlorinated biphenyls (PCBs) were examined and in particular the non-dioxin-like PCBs (NDL-PCBs). A set of 20 NDL-PCBs was selected to represent the 178 PCB congeners with three to seven chlorine substituents. The selection procedure was a combined process including statistical molecular design for a representative selection and expert judgements to be able to include congeners of specific interest. The 20 selected congeners were tested in vitro in as much as 17 different assays. The data from the screening process was turned into interpretable toxicity profiles with multivariate methods, used for investigation of potential classes of NDL-PCBs. It was shown that NDL-PCBs cannot be treated as one group of substances with similar mechanisms of action. Two groups of congeners were identified. A group including in general lower chlorinated congeners with a higher degree of ortho substitution showed a higher potency in more assays (including all neurotoxic assays). A second group included abundant congeners with a similar toxic profile that might contribute to a common toxic burden. To investigate the structure-activity pattern of PCBs effect on DAT in rat striatal synaptosomes, ten additional congeners were selected and tested in vitro. NDL-PCBs were shown to be potent inhibitors of DAT binding. The congeners with highest DAT inhibiting potency were tetra- and penta-chlorinated with 2-3 chlorine atoms in ortho-position. The model was not able to distinguish the congeners with activities in the lower μM range, which could be explained by a relatively unspecific response for the lower ortho chlorinated PCBs. / Den europeiska kemikalielagstiftningen REACH har fastställt att kemikalier som produceras eller importeras i en mängd över 1 ton per år, måste registreras och riskbedömmas. En uppskattad siffra är att detta gäller för 30 000 kemikalier. Problemet är dock att data och information ofta är otillräcklig för en riskbedömning. Till stor del har djurförsök använts för effektdata, men djurförsök är både kostsamt och tidskrävande, dessutom kommer den etiska aspekten in. REACH har därför efterfrågat en undersökning av möjligheten att använda in silico verktyg för att bidra med efterfrågad data och information. In silico har en ungefärlig betydelse av i datorn, och innebär beräkningsmodeller och metoder som används för att få information om kemikaliers egenskaper och toxicitet. Avhandlingens syfte är att utforska möjligheten och förfina användningen av in silico verktyg för att skapa information för riskbedömning av industrikemikalier. Avhandlingen beskriver kvantitativa modeller framtagna med kemometriska metoder för att prediktera, dvs förutsäga specifika kemikaliers toxiska effekt. I den första studien (I) undersöktes 56 072 organiska industrikemikalier. Med multivariata metoder skapades en karta över industrikemikalierna som beskrev dess kemiska och fysikaliska egenskaper. Kartan användes för jämförelser med kända och potentiella miljöfarliga kemikalier. De mest kända miljöföroreningarna visade sig ha liknande principal egenskaper och grupperade i kartan. Genom att specialstudera den delen av kartan skulle man kunna identifiera fler potentiellt farliga kemiska substanser. I studie två till fyra (II-IV) specialstuderades miljögiftet PCB. Tjugo PCBs valdes ut så att de strukturellt och fysiokemiskt representerade de 178 PCB kongenerna med tre till sju klorsubstituenter. Den toxikologiska effekten hos dessa 20 PCBs undersöktes i 17 olika in vitro assays. De toxikologiska profilerna för de 20 testade kongenerna fastställdes, dvs vilka som har liknande skadliga effekter och vilka som skiljer sig åt. De toxicologiska profilerna användes för klassificering av PCBs. Kvantitativa modeller utvecklades för prediktioner, dvs att förutbestämma effekter hos ännu icke testade PCBs, och för att få ytterligare kunskap om strukturella egenskaper som ger icke önskvärda effekter i människa och natur. Information som kan användas vid en framtida riskbedömning av icke-dioxinlika PCBs. Den sista studien (IV) är en struktur-aktivitets studie som undersöker de icke-dioxinlika PCBernas hämmande effekt av signalsubstansen dopamin i hjärnan.

En Si Wang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 126-140 (2nd gp.)). / Acknowledgements --- p.i / Contents --- p.ii / Abstract --- p.iv / List of Acronyms and Abbreviations --- p.vi / introduction --- p.1 / Chapter I. --- "Platelet Activating Factor (PAF)´ؤPast, Present, and Future" --- p.1 / Chapter I-1. --- What is PAF? --- p.1 / Chapter I-2. --- Biochemistry of PAF --- p.2 / Chapter I-2-1. --- Metabolic Cycle of PAF --- p.3 / Chapter I-2-1-A. --- Biosynthesis of PAF --- p.4 / Chapter I-2-1 -B. --- Inactivation of PAF --- p.6 / Chapter I-2-2. --- Role of Endogenous PAF in Cell --- p.7 / Chapter I-3. --- Chemistry of PAF --- p.8 / Chapter I-4. --- Pathobiology of PAF --- p.9 / Chapter II. --- PAF Receptor --- p.10 / Chapter II-1. --- Presence and Characteristics of PAF Receptor --- p.10 / Chapter II-l-l. --- Solubilization of PAF Receptor --- p.10 / Chapter II-1-2. --- G-Protein Involvement --- p.11 / Chapter II-1-3. --- Species Differences --- p.11 / Chapter II-1-4. --- Multiple Conformational States of PAF Receptor --- p.12 / Chapter II-1-5. --- PAF Receptor Heterogeneity --- p.12 / Chapter II-2. --- Putative Conformation of PAF Membrane Binding Sites --- p.13 / Chapter II-3. --- Recent Progress in PAF Receptor Research --- p.15 / Chapter III. --- PAF Receptor Antagonist --- p.18 / Chapter III-1. --- Classification of PAF Antagonists --- p.18 / Chapter III-2. --- Inhibition Types of PAF Receptor Antagonists --- p.19 / Chapter III-2-1. --- Nonspecific Inhibition of the Effects of PAF --- p.21 / Chapter III-2-2. --- Specific Inhibition of PAF --- p.22 / Chapter III-3. --- Recent Progress in PAF Receptor Antagonist Research --- p.22 / Chapter IV. --- Pharmacology and Syntheses of Spiro-Ether Structural Units --- p.26 / Chapter IV-1. --- Natural Products Containing Spiro-Ether and Related Structural Units --- p.30 / Chapter IV-1-1. --- Labdane Diterpenoids Containing Spiro-Ether Structural Units --- p.30 / Chapter IV-1-2. --- Leucodrin and Related Derivatives --- p.32 / Chapter IV-2. --- Synthetic Methods of Spiro-Ethers and Related Derivatives --- p.34 / Chapter V. --- Aim of the Present Work --- p.45 / RESULTS AND DISCUSSION --- p.47 / Chapter I. --- Isolation and Structure Elucidation of Prehispanolone (1) and Preleoheterin (3) --- p.47 / Chapter I-1. --- Material and Isolation --- p.47 / Chapter I-2. --- Structure Elucidation of Prehispanolone (1) and Preleoheterin (3) --- p.47 / Chapter II. --- Synthesis of Model Compounds --- p.53 / Chapter II-l. --- "Synthesis of 2-Methyl-1,7-dioxaspiro[4.4]nonane (137)" --- p.53 / Chapter II-2. --- "Synthesis of 2,2-Dimethyl-l,7-dioxaspiro[4.4]nonane (139)" --- p.68 / Chapter II-3. --- "Synthesis of 2,2-Diphenyl-1,7-dioxaspiro[4.4]nonane (141) and 2,2-Diphenyl-l,7-dioxaspiro[4.4]non-8-ene (142)" --- p.72 / Chapter III. --- "Partial Synthesis of 13R, 14,15-Dihydroprehispanolone (5)，13S,14,15-Di- hydroprehispanolone (135) and prehispanolone (1)" --- p.76 / CONCLUSION --- p.89 / EXPERIMENTAL SECTION --- p.91 / REFERENCES --- p.126 / APPENDIX --- p.141

Hung Sau Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 131-137). / Table of Contents --- p.1 / Acknowledgements --- p.V / Abbreviations --- p.VI / Aim of investigation --- p.IX / Abstract --- p.XI / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Cancer Chemotherapy --- p.2 / Chapter 1.2 --- Plants as sources of useful drugs --- p.4 / Chapter 1.3 --- Potent antitumor compounds found in Goniothalamus giganteus --- p.7 / Chapter 1.4 --- Brief introduction of GONIOTRIOL --- p.8 / Chapter 1.5 --- The study on the antitumor activities of the antitumor compounds --- p.9 / Chapter 1.6 --- Biochemistry study of the anticancer agents --- p.10 / Chapter Chapter 2 --- Materials and Methods --- p.18 / Chapter 2.1 --- Materials --- p.19 / Chapter 2.1.1 --- Animals --- p.19 / Chapter 2.1.2 --- "Buffers, Culture Media and Chemicals" --- p.19 / Chapter 2.1.3 --- Cell lines --- p.20 / Chapter 2.1.4 --- Dye solutions --- p.21 / Chapter 2.1.5 --- Reagents and buffers for Agarose gel --- p.21 / Chapter 2.1.6 --- Synthetic goniotriol and its derivatives --- p.21 / Chapter 2.2 --- Methods --- p.23 / Chapter 2.2.1 --- Radioactive Precursor Incorporation Assays --- p.23 / Chapter 2.2.2 --- MTT assay --- p.24 / Chapter 2.2.3 --- Neutral Red assay --- p.24 / Chapter 2.2.4 --- Isolation and preparation of cells --- p.25 / Chapter 2.2.5 --- Assay for the solvent effect --- p.25 / Chapter 2.2.6 --- Assay for the in vitro antitumor activity THC88 on different cell lines --- p.27 / Chapter 2.2.7 --- Assay of the effect of THC86 on solid sarcoma Scl80 in vivo --- p.28 / Chapter 2.2.8 --- Assay of the effect of THC86 on peritoneal Scl80 in vivo --- p.28 / Chapter 2.2.9 --- Assay of the effect of THC89 on peritoneal EAT in vivo --- p.28 / Chapter 2.2.10 --- Assay of synthetic compound (THC89 and THC87) on the mitogenic activity of spleen lymphocytes --- p.29 / Chapter 2.2.11 --- Assay of synthetic compound (THC87) on the proliferation of murine bone marrow cells from compound- treated mice --- p.30 / Chapter 2.2.12 --- "Assay of synthetic compounds (Ml, P51 and P1) on nonmalignant cell-line" --- p.31 / Chapter 2.2.13 --- Assay of antitumor activity of synthetic compound (THC86)on PU5-1.8 --- p.31 / Chapter 2.2.14 --- Assay of the cytocidal effect of THC86 --- p.32 / Chapter 2.2.15 --- "Assay on the effect of THC86 on the synthesis of DNA, RNA and protein" --- p.32 / Chapter 2.2.16 --- Direct DNA cleavage by THC86 --- p.33 / Chapter 2.2.17 --- DNA fragmentation assay / Chapter 2.2.18 --- Assay of the effect of the synthetic compound (THC86) on different growth fraction of the cells / Chapter 2.2.19 --- Mitosis Study / Chapter 2.2.20 --- Assay for the stability of the synthetic compounds / Chapter Chapter 3 --- Structure / activity relationship of the synthetic compounds --- p.36 / Chapter 3.1 --- Results --- p.37 / Chapter 3.1.1 --- In vitro antitumor activity of the synthetic compounds --- p.37 / Chapter 3.2 --- Discussion --- p.45 / Chapter Chapter 4 --- Antitumor activities of the synthetic compounds --- p.63 / Chapter 4.1 --- Results --- p.64 / Chapter 4.1.1 --- Solvent effect in the screening process --- p.64 / Chapter 4.1.2 --- The effect of the synthetic compound (THC88) on different cell lines --- p.69 / Chapter 4.1.3 --- In vivo anti-tumor activities of the synthetic compounds --- p.71 / Chapter 4.1.3a --- Effect of THC86 on solid sarcoma Sc180 in vivo --- p.71 / Chapter 4.1.3b --- Effect of THC86 on peritoneal Scl80 in vivo --- p.71 / Chapter 4.1.3c --- Effect of THC89 on peritoneal EAT in vivo --- p.72 / Chapter 4.1.4 --- Cytotoxic effect of the tested compounds on normal cells --- p.77 / Chapter 4.1.4a --- Cytotoxic effect of THC89 on normal splenocytes in vitro --- p.77 / Chapter 4.1.4b --- Effect of THC87 on the proliferation of splenocytes --- p.77 / Chapter 4.1.4c --- Effect of THC87 on the proliferation of murine bone marrow cells --- p.78 / Chapter 4.1.4d --- Cytotoxic effect on non-malignant cell-line BALB/c 3T3/A31 --- p.78 / Chapter 4.2 --- Discussion --- p.85 / Chapter Chapter 5 --- The study on the antiproliferative mechanisms of the synthetic compounds --- p.88 / Chapter 5.1 --- Results --- p.89 / Chapter 5.1.1 --- "Effect of the synthetic compounds on Cell Growth, DNA, RNA and Protein" --- p.89 / Chapter 5.1.1a --- Effect of THC86 on PU5-1.8 (macrophage-like tumor) --- p.89 / Chapter 5.1.1b --- Cytocidal effect of THC86 on EAT --- p.89 / Chapter 5.1.1c --- "Effect of the synthetic compounds on synthesis of DNA, RNA and protein" --- p.90 / Chapter 5.1.2 --- Study of the synthetic compounds on the interactions of DNA --- p.101 / Chapter 5.1.2a --- DNA cleavage assay --- p.101 / Chapter 5.1.2b --- DNA fragmentation assay --- p.101 / Chapter 5.1.3 --- Effect of the synthetic compounds on different growth fraction of the cells --- p.104 / Chapter 5.1.4 --- Mitosis study of the synthetic compounds --- p.106 / Chapter 5.1.5 --- Investigation of the stability of the synthetic compounds in culture medium --- p.112 / Chapter 5.2 --- Discussion --- p.117 / Chapter Chapter 6 --- General Discussion --- p.122 / References --- p.131

Un composé de type N-aryle pyridinium (PF) a montré un réel potentiel en tant qu’inducteur chimique de la maladie de Parkinson (MP). Son administration in vitro et in vivo a permis d’induire l’apparition des marqueurs cellulaires caractéristiques de la MP mais suivant une cinétique d’action beaucoup plus lente que les inducteurs parkinsoniens actuellement disponibles. Ces travaux préliminaires ont suggéré l’existence d’un lien entre le motif N-aryle pyridinium et la capacité pour la molécule qui le porte à induire chimiquement la MP. Ces résultats ont dès lors laissé envisager la préparation de composés structurellement inspirés, capables de mimer plus fidèlement les mécanismes biochimiques impliqués lors de l’apparition de la MP. Partant du même composé de départ – un dérivé de l’aniline – deux voies de synthèse ont été suivies et ont permis d’aboutir à des N-aryle pyridiniums diversement fonctionnalisés. La première voie d’accès, exploitant la réaction de Zincke, a permis d’aboutir à des composés ne portant pas de groupement hydroxyle sur le noyau pyridinium. La deuxième voie d’accès, comprenant une étape-clé d’activation anodique et inspirée de la préparation du PF, a permis d’aboutir à des N-aryle pyridiniums 3-hydroxylés. Pour ces deux voies de synthèse, la nature du composé de départ a contraint à la modification des protocoles opératoires décrits dans la littérature afin de pouvoir accéder aux molécules ciblées. L’évaluation toxicologique de ces N-aryle pyridiniums a donné lieu à une étude de relation structure/activité. Les résultats de cette étude ont permis d’identifier les éléments structuraux permettant à la fois l’inhibition du complexe I de la chaine respiratoire mitochondriale et l’induction d’une neurotoxicité in vitro. Il a également été possible d’identifier, pour la première fois, un paramètre permettant la modulation de la pénétration cellulaire dopaminergique et par suite, de l’activité de ces inducteurs chimiques de la MP / A N-aryl pyridinium derivative (PF) has shown promising results as a chemical inducer of Parkinson's disease (PD). Its in vitro and in vivo administration has induced the appearance of characteristical cellular markers of PD but following a much slower kinetics than currently available PD inducers. These preliminary studies have suggested the existence of a link between the N-aryl pyridinium moiety and the ability of the molecule bearing it to chemically induce PD. These results have therefore led to the preparation of structurally related compounds, able to mimic more closely the biochemical mechanisms involved in the onset of PD.Using the same starting material - an aniline derivative - two synthetic pathways have been followed and have led to diversely functionalized N-aryl pyridiniums. The first pathway, based on the Zincke reaction, afforded compounds without hydroxyl group on the pyridinium ring. The second pathway, including a key step of anodic activation and inspired the preparation of PF, led to 3-hydroxy-N-aryl pyridiniums. For both synthetic routes, the nature of the starting material has implied the modification of previously protocols described to gain access to the targeted molecules. The toxicological evaluation of these N-aryl pyridiniums gave rise to a structure / activity relationship study. The results of this study have led to the identification of structural elements allowing both the inhibition of complex I of the mitochondrial respiratory chain and the induction of in vitro neurotoxicity. It was also possible to identify for the first time, a parameter for the modulation of dopaminergic cell penetration and hence the activity of these chemical inducers of PD

L’objectif de cette thèse, réalisée en collaboration avec la société Mérial, concerne la santé animale et s’oriente vers la synthèse de molécules actives à visée préventive ou thérapeutique. Deux sujets distincts sont abordés. Dans un premier temps, le but recherché est la synthèse d’une série de molécules présentant une activité anti-coccidienne pour le traitement préventif des poulets. Quarante-trois composés originaux, issus d’une synthèse courte à deux ou trois étapes, ont été préparés par hémi-synthèse de la monensine. Quinze molécules ont été testées in vitro et trois d’entre elles montrent une activité importante vis-à-vis du parasite Eimeria tenella. Dans un second temps, nous nous sommes intéressés à la synthèse de nouveaux répulsifs pour un large panel d’insectes. Après avoir fait une étude bibliographique approfondie, nos recherches se sont concentrées sur la réactivité de la coumarine. Environ soixante-dix molécules ont été synthétisées par modifications fonctionnelles des 4-, 6- et 7- hydroxycoumarines et de la coumarine. La grande majorité a été testée en présence de drosophiles. Six molécules présentant une activité répulsive équivalente à celle du DEET, produit de référence, ont été identifiées / This thesis was completed in collaboration with the Animal Health Company Merial. The objective of the thesis is the synthesis of preventive and therapeutic bioactive molecules. Two separate subjects were treated. First, the aim was the synthesis of a series of molecules having an anticoccidial activity for the preventive treatment of chickens. Forty-three original compounds, issued from a short synthesis with two or three steps, were prepared from monensin by hemi-synthesis. Among them, fifteen were tested in vitro and three of them showed a significant activity against Eimeria tenella. Thereafter, we have worked on the synthesis of new repellents for a wide range of insects. After a comprehensive bibliographic study, our research was focused on the coumarin reactivity. More than seventy molecules were synthesized by structural modifications of 4-, 6- and 7-hydroxycoumarins and of coumarin. Almost all the compounds were tested in the presence of drosophila. Six molecules showing a repellent activity equivalent to DEET, used as a reference product, were identified

This thesis develops an integrated methodology based on the desirability index and QSAR models to virtually optimise molecules. Statistical and algorithmic tools are proposed to search in huge collections of compounds obtained by combinatorial chemistry the most promising ones. First, once the drugability properties of interest have been precisely defined, QSAR models are developed to mimic the relationship between those optimised properties and chemical descriptors of molecules. The literature on QSAR models is reviewed and the statistical tools to validate the models, analyse their fit and their predictive power are detailed. Even if a QSAR model has been validated and sounds highly predictive, we emphasise the importance of measuring extrapolation by the definition of its applicability domain and quantifying the prediction error for a given molecule. Indeed, QSAR models are often massively applied to predict drugability properties for libraries of new compounds without taking care of the reliability of each individual prediction. Then, a desirability index measures the compromise between the multiple estimated drugability properties and allows to rank the molecules in the combinatorial library in preference order. The propagation of the models prediction error on the desirability index is quantified by a confidence interval that can be constructed under general conditions for linear regression, PLS regression or regression tree models. This fulfills an important lack of the desirability index literature that considers it as exact. Finally, a new efficient algorithm (WEALD) is proposed to virtually screen the combinatorial library and retain the molecule with the highest desirability indexes. For each explored molecule, it is checked if it belongs to the applicability domain of each QSAR models. In addition, the uncertainty of the desirability index of each explored molecule is taken into account by gathering molecules that can not be distinguished from the optimal one due to the propagation of QSAR models prediction error. Those molecules do not have a significantly smaller desirability than the optimal molecule found by WEALD. This constitutes another important improvement in the use of desirability index as a tool to compare solutions in a multicriteria optimisation problem. This integrated methodology has been developed in the context of lead optimisation and is illustrated on a real combinatorial library provided by Eli Lilly and Company. This is the main application of the thesis. Nevertheless, as the results on desirability index uncertainty are applicable under general conditions, they can be applied to any multicriteria optimisation problem, like it often occurs in industry.