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京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第23831号 / 薬科博第146号 / 新制||薬科||16(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 山下 富義, 教授 髙倉 喜信, 准教授 伊藤 美千穂 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Quaternary ammonium compounds (QACs) have long served as lead disinfectants in residential, industrial, and hospital settings. Their simple yet effective amphiphilic nature makes them an ideal class of compounds through which to explore antibacterial activity. We have developed novel multiQAC scaffolds through simple and cost-efficient syntheses, yielding hundreds of diverse compounds strategically designed to examine various aspects of antibacterial and anti-biofilm activity, as well as toxicity. Many of these bis-, tris-, and tetraQACs display antibacterial activity 10 to 100 times greater than conventional monoQACs, and are among the most potent biofilm eradicators to date. Through analyzing their activity against several strains, we have uncovered and provided further evidence for key tenets of amphiphilic QAC bioactivity: a balance of hydrophobic side chains with cationic head groups generates optimal antibacterial activity, though toxicity to eukaryotic cells needs to be mitigated. Given their ubiquitous nature and chemical robustness, the overuse of QACs has led to the development of QAC resistance genes that are spreading throughout the microbial world at an alarming rate. These resistant strains, when found in bacterial biofilms, are able to persist in the presence of lead commercial QAC disinfectants, warranting the development of next-generation biocides. Several of our scaffolds were designed with QAC resistance machinery in mind; thus, we utilized these compounds not only as antibacterial agents but also as chemical probes to better understand and characterize QAC-resistance in methicillin-resistant Staphylococcus aureus (MRSA). Our findings support previous postulations that triscationic QACs would retain potency against QAC-resistant strains. Furthermore, we have identified monocationic and aromatic moieties, as well as conformational rigidity, as being more prone to recognition by the resistance machinery. Using our chemical toolbox comprised of QACs of various charge state and scaffold, we explored both the mechanism and scope of QAC-resistance by examining their structure-resistance relationship. Our holistic findings have allowed us to better understand the dynamics of this system towards the design and development of next-generation QACs that will: (1) allow us to better probe the resistance machinery, and (2) remain efficacious against a variety of microbial pathogens. / Chemistry

Commercially important hydrogenation reactions make use of precious noble metal catalysts which are becoming increasingly scarce, and the search for capable alternative catalysts prevails. Transition metal carbides of group IV-VI metals show similar catalytic behavior to platinum and are $103/kg lower in price than the precious metal catalysts. Tungsten carbide, studied in this work, can form in different stoichiometries and phase compositions depending upon synthesis methods. Synthesis of high surface area tungsten carbide with control over its phase composition remains a challenge currently. In this work, the novel isothermal synthesis method of tungsten carbide (WC, W2C) in a CH4/H2 carburization atmosphere with synthesis temperature and presence or absence of a silica support in the catalyst precursor (WO3) as process variables was investigated. The amounts of CO and H2O formed during synthesis corresponded to the amount of oxygen in the WO3 precursor. The catalysts were further characterized by X-ray diffraction to determine phase composition and crystallite size, by scanning electron microscopy to determine morphology, and by CO chemisorption to determine metallic surface area. X-ray diffraction analysis indicated the carbide catalysts to contain W2C, WC, and metallic W phases. The use of a silica-supported precursor favored the formation of a nearly phase pure, high surface area W2C rich catalyst whereas high synthesis temperature and absence of silica precursor favored formation of a low surface area WC rich catalyst. Further, the catalysts were tested for steady state activity at a W/F (weight catalyst/toluene feed rate) of 0.20-0.30 h-1, addition of H2 to a total pressure of 21 bar absolute and 250 °C, and the effect of phase composition and surface area on the activity was studied. This work resulted in the successful synthesis of 4 tungsten carbide catalysts with varying phase compositions and surface areas and correlation of their compositions and surface areas with their corresponding toluene hydrogenation activities.

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.