Human African Trypanosomiasis (HAT) is an infectious, vector-borne protozoal disease which is amongst the so-called neglected diseases. In 2000, at a summit of the United Nations, eight Millennium Development Goals (MDGs) were set, to be achieved by 2015. MDG 6 states “to combat HIV/AIDS, malaria & other diseases”. With just under 2 years to go before the end of 2015, HAT is still thriving in developing countries. The drugs currently used for the treatment of HAT are in short supply, have severe side effects and those used to treat late stages of the disease are very difficult to administer. The aforementioned challenges call for research into this neglected disease in order to develop new, safe and easy-to-use medicines. Naphthoquinones are a class of compounds shown to possess anti-parasitic activity, amongst a variety of other biological activities, and therefore this pharmacophore was selected for this study. The purpose of this study was to synthesise derivatives of 2,3-dichloro-1,4- naphthoquinone to be tested for anti-trypanosomal activity and thereafter conduct structureactivity relationship studies. A series of reactions were carried out using thiophenol, phenol and aniline nucleophiles to synthesise thioether (-S-), ether (-O-) and amino (-NH-) derivatives of 2,3-dichloro-1,4-naphthoquinone with various halogen or methyl substituents. Purification of the products was carried out by recrystallisation. Nuclear magnetic resonance (NMR), infra-red (IR) and high pressure liquid chromatography coupled to an electro-spray ionisation mass spectrometer (HPLC-ESI-MS) were the analytical methods used for structural confirmation of the products. There were eighteen 1,4-naphthoquinone derivatives that were successfully synthesised using ethanolic solutions. Unfortunately, attempts to synthesise 1,4-naphthoquinones in reactions involving 2-(trifluoro-methyl)aniline and 2-isopropyl-5-methylphenol were unsuccessful, presumably due to steric hindrance by the bulky ortho-substituents. Although the aims of the synthetic procedures were to obtain both mono- and disubstituted products by nucleophilic displacement of the chlorine atom(s) of 2,3-dichloro-1,4- naphthoquinone, only monosubstituted products were obtained from substitution with aniline and phenol nucleophiles. Thiol nucleophiles, however, selectively yielded disubstituted products only. Synthesised naphthoquinone derivatives were tested against Trypanosoma brucei and calculation of the EC₅₀ values from the obtained dose-response curves was carried out using the four parametric equation. All the 1,4-naphthoquinones showed a degree of potency, except compounds 1b, 3c and 3e, which had little or lack of potency. Structure-activity relationship studies (SARs and QSARs) were carried out to determine which structural features or functional group substituents of the naphthoquinone derivatives contribute or take away from the desired anti-trypanosomal activity. It was found that compounds with the best in vitro anti-trypanosomal potencies in the series of analogous 1,4-naphthoquinone derivatives had EC₅₀ values in the range 2.137 to 2.884 μM. The most potent compound in the series was 2-chloro-3-(4-(trifluoromethyl)phenylamino)-1,4- naphthoquinone 1e; but it was 142-fold less potent than the reference standard of melarsoprol.

Everything should be ultimately explainable in terms of the components of which it is composed. In the animal kingdom well over ninety-five percent of all recognized species are invertebrates, a distinction based on connective tissues, though most of our efforts have been directed towards vertebrates for reasons of convenience and closeness to man. Investigation of the various chemicals which make up plants and animals have been diverse, allowing us to get a general picture of what molecules to expect at each state of the evolutionary progression, but for now animal is the information complete, and for invertebrates the information is particularly sketchy. It would be convenient to have certain animals and plants completely known chemically, so that biological molecules subsequently discovered could be compared to these standards. Velella lata may be a candidate for such a standard, having a number of unique advantages. It occurs in the evolutionary chain at that point where a true multicellular animal begins, and it is the simplest animal from which genetically determined internal structural materials is conveniently isolated and purified. In plants the connective tissue is considered to be almost entirely carbohydrate polymer and in vertebrates the connective tissue is considered to be almost entirely protein (amino acid polymer), while many invertebrates have almost even mixture of protein carbohydrate in an unknown relationship. Velella presents an opportunity to examine closely the nature of the protein and its relationship to the carbohydrate. The carbohydrate of various invertebrates connective tissue matrices has been extensively studies and the structure and linkages reasonably well defined. Some questions remain regarding the length of the smallest oligosaccaride and the manner in which these are built up into higher levels of organization. Inorganic material (primarily in the form of calcium), lipid in small amounts, and moisture are also present in the invertebrate matrix. While calcium is generally considered to be in the form of the carbonate, the possibility that it is not all in this form awaits investigation, and the recent report that silicon is essential in the formation of chick connective tissues is interesting.5 Lipids are not a major component in connective tissues studied to date, and this is true of Velella also. This does not preclude, however, the possibility that lipids may play some role in the formation of membrane-like sheets which are observed in the laminations of various mollusk-arthropod matrices. Velella offers an opportunity to examine invertebrate connective tissue in an intense way to gain an understanding of the morphology and developmental dynamics which hopefully would be extensible to the vertebrate matrix.

<p dir="ltr">Within the last decade, cancer immunotherapy, the therapeutic strategy of enhancing the body’s immune system to curb tumor growth, has reached the front lines in the war on cancer. Although common strategies such as adoptive cell transfer and immune checkpoint blockade have enjoyed success against some cancers, they regrettably lack durable efficacy across a broad patient population inflicted by heterogeneous and diverse cancer types. Moreover, application of these biological therapeutics is likewise limited due to various toxicities frequently encountered in the clinic. Taking these into account, the next generation of immunotherapies must exploit novel immunomodulatory targets and therapeutic strategies that can possess both enhanced efficacy compared to current options and more acceptable toxicity profiles in patients. Compared to biologics, small molecule inhibitors are desirable as they may circumvent concerns involving efficacy and toxicity, while allowing access to a broader arsenal of macromolecular targets. Recently, protein tyrosine phosphatase nonreceptor 22 (PTPN22), a key desensitization node in T cell signaling, has emerged as a systemic and translatable cancer immunotherapy target. Nonetheless, many of its precise functions in various immune cells is not fully resolved, thus there is a critical need for both novel chemical probes for biological interrogation and inhibitors with improved <i>in vivo </i>efficacy for further therapeutic development.</p><p dir="ltr">Built upon an overview of PTPN22’s structure, function, and value as an immunotherapy target, as well as a comprehensive assessment of reported inhibitors, this dissertation documents two separate medicinal chemistry campaigns on existing PTPN22 scaffolds. Herein, the structure activity relationships, design, and biological evaluation of a novel, superiorly selective and cell-active probe/ lead compound is disclosed. This dissertation also reports the design of a novel PTPN22 inhibitor with enhanced potency, selectivity, cellular efficacy, <i>in vivo </i>pharmacokinetics, and <i>in vivo </i>antitumor efficacy in mice. Our research efforts and the overall status and future directions of the field are also succinctly discussed.</p>

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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

Sphingosine kinase (SphK) has emerged as an attractive target for cancer therapeutics due to its role in cell proliferation. SphK phosphorylates sphingosine to form sphingosine-1-phosphate (S1P) which has been implicated as a major player in cancer growth and survival. SphK exists as two different isoforms, namely SphK1 and SphK2, which play different roles inside the cell. The dearth of isoenzyme-selective inhibitors has been a stumbling block for probing the exact roles of these two isoforms in disease progression. This report documents our efforts in developing SphK2-selective inhibitors. We provide the first demonstration of a SphK inhibitor containing a quaternary ammonium salt. We developed highly potent and moderately selective inhibitors that were cell permeable and interfered with S1P signaling inside the cell. In an effort to improve the selectivity of our inhibitors and enhance their in vivo stability, we designed and synthesized second generation inhibitors containing a heteroaromatic linker and a guanidine headgroup. These inhibitors were more potent and selective towards SphK2 and affected S1P signaling in cell cultures and various animal models. / Ph. D.

Sphingosine 1-phosphate (S1P) has become a prevalent drug discovery target due to studies implicating it to several disease pathologies such as fibrosis, sickle cell disease, inflammation, diabetes, and cancer. S1P functions to induce cell proliferation and migration. S1P signaling occurs through intracellular targets or transport outside of the cell via ABC transporters, where it acts as a ligand to G-protein coupled receptors (S1P1-5). Sphingosine kinase (SphK) 1 and 2 phosphorylate sphingosine to S1P; these are the only enzymes known to mediate the phosphoryl transfer. Inhibiting either or both SphKs helps to modulate S1P, which may be useful as a therapeutic avenue for disease states where S1P signaling has gone awry. Herein, we document our efforts in profiling the structure-activity relationships (SAR) of SphK2 through an iterative process of synthesis and biological testing. First, an SAR structured around the head and linker region of our lead molecule, SLR080811, was performed. SLR080811 has a Ki of 1.3 µM and is 5-fold selective for SphK2. The modifications performed on SLR080811 yielded two promising inhibitors: SLP120701 (SphK2 selective with a Ki of 1.2 µM) and SLP7111228 (>200 fold selective for SphK1 with a Ki of 48 nM). In vitro studies in U937 cells yielded a decrease in S1P levels with the introduction of inhibitors. Mouse studies provided insight into the pharmacokinetic effect of our SphK2-selective inhibitors, revealing an increase in S1P levels in the blood. When in vivo studies were performed with the SphK1 selective inhibitor, S1P levels in blood decreased. These molecules provide the chemical biology tools to determine the effect of modulating S1P levels in vivo. We also focused our investigation on the tail region of the pharmacophore. From this study, SLM6031434 and SLM6041418 were discovered and both proved to be more potent and selective SphK2 inhibitors than SLR080811. SLM6031434 has a Ki of 370 nM and is 23-fold selective for SphK2. SLM6041418 has a Ki of 430 nM and is 24-fold selective for SphK2. Consistent with our previous observations, in vitro studies showed a decrease in S1P levels when inhibitor was introduced. Similarly, in vivo studies resulted in an increase of S1P levels in the blood. These compounds are positioned towards animal models of disease. / Master of Science

Les analogues synthétiques des nucléosides naturels constituant des acides nucléiques occupent une place importante dans le domaine du médicament comme principes actifs antiviraux ou anticancéreux. Ces nucléosides agissent comme « prodrogues » en perturbant la biosynthèse des acides nucléiques viraux ou des cellules cancéreuses après phosphorylation. Dans la recherche de nouveaux médicaments antiviraux, nous avons cherché à synthétiser de nouveaux analogues des nucléosides naturels, les 2-désoxy-adénosine et -guanosine, et de l’aciclovir et de ses dérivés (vanciclovir, ganciclovir…) qui sont très utilisés dans le traitement de l’Herpès. Des premiers travaux en série adénine et guanine, n’ont pas permis d’obtenir les dérivés cycliques recherchés dans lesquels la base et la chaîne latérale introduite en position 9 de la base sont liés par un atome d’oxygène se trouvant en position 8 pour former un nouveau cycle. Quatre analogues cycliques en série guanine ont été synthétisés dans lesquels la base et la chaîne latérale en position 8 sont liés soit par un hétéroatome (préparés par réaction de substitution nucléophile), soit par une liaison carbone-carbone (préparés par réaction radicalaire) et sont en cours d’évaluation antivirale. / The synthetic analogues of the natural 2’-deoxyribonucleosides, linked by phosphodiester groups in nucleic acids, constitute major classes of antiviral and anticancer drugs. Such nucleosides act as “prodrugs” disturbing the biosynthesis of nucleic acids after phosphorylation. Searching for new antiviral drugs, the aim of this work was the synthesis of new modified nucleosides analogues of 2’-deoxyadenosine and -guanosine also analogues of aciclovir and its derivatives (vanciclovir, ganciclovir…) widely used for Herpes treatment. In the first works in adenine and guanine series, the cyclic analogues in which the base and a side chain introduced at position 9 of the base are linked at position 8 by an oxygen atom could not be obtained. Four cyclic analogues in the guanine series were prepared in which the base and the 9-side chain are linked at position 8 are either linked by a heteroatom (synthesized by nucleophilic substitution) or by a carbon-carbon bond (synthesized by free radical reaction). The evaluation of the antiviral activity of these compounds is underway.

The main theme of this thesis is that there is a common structural basis for drugs acting on the central nervous system (CNS), and that this concept may be used to design new CNS-active drugs which have greater specificity and hence less side-effects. To develop these ideas, the biological basis of how drugs modify CMS neurotransmission is described, and illustrated using dopaminergic pathways. An account is then given of the use of physicochemical concepts in contemporary drug design. The complete conformational analysis of several antipsychotic drugs is used to illustrate some of these techniques in the development of a model for antipsychotic drug action. After reviewing current structure-activity studies in several classes of CNS drugs (antipsychotics, anti-depressants, stimulants, hal1ucinogens, anticonvulsants and analgesics), a hypothesis for a common structural basis of CNS drug action is proposed- This is based on a topographical comparison of the X-ray structures of eight representative CNS-active drugs, and consists of three parts: 1.there is a common structural basis for the activity of many different CNS-active drug classes; 2. an aromatic ring and a nitrogen atom are the primary binding groups whose topographical arrangement is fundamental to the activity of these drug classes; 3. the nature and placement of secondary binding determines different classes of CNS drug activity. A four-Point model for this common structural basis is then defined using 14- CNS-active drug structures that include the original eight used in proposing the hypothesis. The coordinates of this model are: R1 (0. 3.5, 0), R2 (0, -3.5, O), N (4.8. -0.3, 1.4), and R3 (6.3, 1.3, 0), where R1 and R2 represent the point locations of a hydrophobic interaction of the common aromatic ring with a receptor, and R3 locates the receptor point for a hydrogen bond involving the common nitrogen, N. Extended structures were used to define the receptor points R1, R2 and R3, and the complete conformational space of each of the 14 molecules was considered. It is then shoun that the model may be used to predict whether a given structure is likely to show CNS activity: a search over 1,000 entries in the current Merck Index shows a high probability (82%) of CNS activity in compounds fitting the structural model. Analysis of CNS neurotransmitters and neuropeptides shows that these fit the common model well. Based on the available evidence supporting chemical evolution, protein evolution, and the evolution of neurotransmitter functions, it is surmised that the aromatic ring/nitrogen atom pharmacophore proposed in the common model supports the idea of the evolution of CNS receptors and their neurotransmitters, possibly from an aromatic amine or acety1cho1ine acting as a primaeval communicating molecule. The third point in the hypothesis trilogy is then addressed. The extensive conformation-activity analyses that have resulted in well-defined models for five separate CNS drug classes are used to map out the locations of secondary binding groups relative to the common model for anti-psychotics, antidepressants, analgesics, anticholinergics, and anticonvulsants. With this information, and knowledge derived from receptor-binding data, it is postulated that drugs having specified activity could be designed. In order to generate novel structures having a high probability of CNS-activity, a process of drug design is described in which known CNS structures are superimposed topographically using the common model as a template. Atoms regarded as superfluous may be selectively deleted and the required secondary binding groups added in predicted locations to give novel structures. It is concluded that this process provides the basis for the rational design of new lead compounds which could further be optimized for potent and specific CNS activity.

Oxide materials exhibit a wide variety of structures and properties. In particular, transition metal oxides tend to be highly stable while exhibiting a wide range of properties that can be used for numerous applications. This work focuses on investigating how the structures��� of 4d and 5d transition metal oxides influences their properties. Specifically oxides of Ru, Rh and Ir were investigated. A complete solid solution was found between isostructural Pb���Mn���O������ and Pb���Rh���O������. Pb���Rh���O������ shows a Verwey-type transition at 185 K. This transition remains with a 3 % substitution of Mn for Rh but disappears with a 4 % substitution of Mn for Rh. The structure was found to expand in the direction perpendicular to the layers of the structure, which is the c-axis, despite a contracting unit cell. Bi for Pb substitution in Pb���Mn���O������ was found to be limited as compared to in Pb���Rh���O������. Alkali metal substitution on the A-site of the orthorhombic perovskite SrRuO��� showed only low substitution levels were possible. Nonetheless, the substituted phases showed decreased ferromagnetic Curie temperatures, increased electrical resisitivity and relatively unchanged Seebeck coefficients. Thermoelectric studies of Sr[subscript 2-x]La[subscript x]CoRuO��� perovskite phases showed Sr���.���La���.���CoRuO��� with the best thermoelectric performance. This system showed possible correlations between cation ordering on the B-site and the charge carrier transport. A similar thermoelectric study of (RhV)[subscript 1+x]Ti[subscript 1-2x]O��� phases crystallizing in a disordered trirutile structure was done. Electron carriers were found to be dominant and dependent on Ti content. The electron carriers appear to become diminished at higher temperatures. Sr���IrO��� crystallizes in a K���NiF���-type structure. Effects of Ti, Fe and Co substitution for Ir were investigated. A complete Sr���Ir[subscript 1-x]Ti[subscript x]O��� solid solution was synthesized and characterized while limited solubility was found for Fe and Co substitutions. All substitutions showed a decrease in the c-cell parameter coupled with a decrease in octahedral tilting. All substitutions also showed a decrease in magnetic susceptibility and an increase in the paramagnetic effective moment was observed for Co and Fe doped samples. An incomplete solid solution was formed for Sr���Ti[subscript 1-x]Rh[subscript x]O��� phases; however effects of increased octahedral tilting with higher Rh content were observed. / Graduation date: 2013