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No / While mining a data set of 554 chemicals in order to extract information on their toxicity value, we faced the problem of scaling all the data. There are numerous different approaches to this procedure, and in most cases the choice greatly influences the results. The aim of this paper is 2-fold. First, we propose a universal scaling procedure for acute toxicity in fish according to the Directive 92/32/EEC. Second, we look at how expert preprocessing of the data effects the performance of qualitative structure-activity relationship (QSAR) approach to toxicity prediction.

Mitochondrial uncouplers have the capacity of passively shuttling protons from the mitochondrial intermembrane space to the mitochondrial matrix, independent of ATP synthase. This results in the disruption of oxidative phosphorylation and increased rate of metabolism as a counter action from the mitochondria. Therefore, small molecule mitochondrial uncouplers have potential for the treatment of obesity, diabetes, non-alcoholic fatty liver disease (NAFLD), neurodegenerative disorders, amongst others. A one-pot method for the synthesis of 1H-imidazo[4,5-b]pyrazines from [1,2,5]oxadiazolo[3,4-b]pyrazines is herein disclosed. In the presence of Fe, Yb(OTf)3, and the desired electrophile partner, in situ reduction of the oxadiazole fragment followed by cyclization afforded imidazolopyrazines in moderate to good yields. The selection of different orthoesters as electrophiles also allowed functionalization on the 2-position of the imidazole ring. This new method was used to synthesize 1H-imidazo[4,5-b]pyrazines to perform structure-activity relationship studies. Thus, a library of 75 compounds was synthesized and characterized for mitochondrial uncoupling activity. The biological activity of the compounds was demonstrated in oxygen consumption rate assays affording potent mitochondrial uncouplers. The method was further applied to the synthesis of 5-alkoxy-2-(trifluoromethyl)-1H-imidazo[4,5-b]pyrazin-6-amines, with over 50 derivatives synthesized. A structure-activity relationship study was performed using a variety of substituents to fine-tune the scaffold's potency. The installation of a methoxy group at the 5-position of the scaffold resulted in the discovery of compound 4.3.20, which exhibited the best activity with an EC50 of 3.6 ± 0.4 μM in rat L6 myoblasts and a half-life of 4.4 h in mice. Compound 4.3.20 displayed potential as an anti-obesity agent in a mouse model with an effective dose of 50 mg kg-1 without changes in food intake or lean mass. Tissue distribution studies revealed predominance in the liver and both white and brown adipose tissue. In addition, 4.3.20 improved serum markers of insulin sensitivity and hyperlipidemia such as insulin, glucose, triglycerides, cholesterol, and HOMA-IR. Taken together, compound 4.3.20 and related mitochondrial uncouplers show promise for further development in the treatment of obesity and other diseases. / Doctor of Philosophy / The mitochondria, which is an organelle within our cells, is where all the nutrients ingested in the form of food are metabolized, and either used for energy or stored as fat if they are not used. The latter is the main cause of obesity, carrying with it a myriad other comorbidities, such as high blood pressure, heart disease, diabetes, certain types of cancer. Obesity has become a great concern with an incidence of 42% in the US. Mitochondrial uncouplers are molecules that target the mitochondria with a mechanism of action of converting some of the energy ingested in the form of nutrients to be lost as heat instead of being stored as fat. The potential result is a regulated form of weight-loss. Herein, we developed a method for the synthesis of a novel mitochondrial uncoupler scaffold and disclose the mitochondrial uncoupler activity of over 150 molecules. In particular, compound 4.3.20 was tested in an obesity mouse model and was shown to induce fat loss with mice fed a high fat diet. Our investigations support potential use of mitochondrial uncouplers as a mechanism for the treatment and prevention of obesity and other metabolic diseases.

Sphingosine kinase 1 (SphK1) is the key enzyme catalyzing the formation of sphingosine-1-phosphate (S1P), which is an important signaling molecule that regulates multiple biological process including inflammatory responses. Elevated SphK1 activity as well as upregulated S1P levels is linked to various diseases such as cancer, fibrosis and sickle cell disease. Therefore, there is a growing interest in studying SphK1 as a potential target for these diseases. Through high-throughput screening, various SphK1 inhibitors have been discovered, among which PF-543 is the most potent and selective inhibitor reported to date (Ki=3.6 nM, >100 fold selectivity for SphK1). Previous research indicated that SphK1 inhibitor PF-543 is effective in reducing S1P levels and slowing down the development of sickle cell disease in vivo. However, the lack of in vivo stability of PF-543 still makes it necessary to develop inhibitors with an improved pharmacokinetic profile. In this study, PF-543 was employed as the lead compound, and the influence of different tails groups and head groups on binding affinity and in vivo stability were investigated. In brief, (R)-prolinol-based derivatives with various tail groups including alkyl, alkoxy and biphenyl groups were synthesized. Their inhibition potency was tested in a broken-cell assay, and hit compounds were further evaluated in a yeast cell assay to determine EC50 values. The U937 cell line and mice model were utilized for hit compounds to quantify S1P reduction in vitro and in vivo. Our preliminary results indicated compound 2.14d was the best hit discovered, with 88% SphK1 inhibition at 1 μM. In addition, compound 2.14d with a Ki of 0.68 μM and an EC50 of 0.15 μM, reduced the S1P of U937 cells by 90% at 1 μM. Its analog with a shorter tail group, 2.14a, reduced plasma S1P levels by 20% in mice (10 mg/kg, 3 h). Further modification of the head group of 2.14d produced compound 3.14c bearing a secondary benzylamine head group, with an EC50 value of 0.39 μM and less in vivo activity (14% plasma S1P reduction at 10 mg/kg, 6 h). / Doctor of Philosophy / Sphingosine-1-phosphate (S1P) is a molecule related to various diseases, such as cancers and inflammatory diseases. Elevated levels of S1P promote the development of these diseases, thus making it necessary to reduce the production of S1P in patients. Since S1P is generated in human body by an enzyme called sphingosine kinase (SphK), inhibiting the activity of SphK can be beneficial for reducing S1P levels. Developing inhibitors for SphK is also a promising strategy for curing such diseases. A very potent inhibitor has been reported, but it could be metabolized quickly into other inactive metabolites in human, which renders it ineffective. To develop better drug candidates, a series of compounds with similar structure has been synthesized and tested for their potency and metabolic stability. Based on analysis of the relationship between the compound structures and activities, several compounds with less potency and different metabolic stability has been prepared and their efficacy in reducing S1P levels has been tested.

A sol-gel process has been utilized to develop novel hybrid materials incorporating organic and inorganic species. The objectives of this project were to study the feasibility of this new bridging route and, if successful, study the structure-property relationships of these new materials. In this thesis, tetraorthosilicate was used to combine with one of three types of oligomers, which included silanol terminated polydimethylsiloxane (PDMS), triethoxysilane endcapped polytetramethyleneoxide (PTMO), and PTMO with multiple triethoxysilane functional groups. The general reaction scheme was to first generate silanols from both components through the hydrolysis reaction, and then form the network structure by a co-condensation reaction with the silanol groups. The preparation of these hybrid materials was successful. Most of these hybrid materials were obtained without significant cracking problems with an initial TEOS loading up to 80 wt%, and the final products were always transparent. For the PDMS containing systems, the tensile strength was always lower than 8 MPa. and the elongation at break was in the range of 5-25%. Dynamic mechanical results showed a bimodal tan<i>δ</i> behavior, which was attributed to two different physico-chemical environments of the oligomers: one was PDMS rich phase and one represented more dispersed PDMS oligomers. The dispersion of the oligomers increased with acid and silicate content of the system, and this postulation of better PDMS dispersion was strongly supported by the SAXS results which showed a systematic decrease in the mean square electron density fluctuation. The mechanical properties of PTMO containing materials were considerably enhanced compared to the PDMS hybrid systems. Depending on the composition and oligomeric molecular weight, the tensile strength could reach 33 MPa. Also, the range of the elongation at break increased up to ca. 100%. The tan<i>δ</i> spectra showed a single, broad maximum at temperatures much higher than the T_g of pure PTMO oligomers, which indicated the absence of a pure oligomer phase. A broad maximum in the SAXS profile was observed in most cases, implying the existence of a correlation distance in these PTMO containing materials. To rationalize all the experimental observations, a schematic model was suggested which contains highly condensed TEOS clusters and mixed regions of partially condensed TEOS and PTMO. This model was further supported by swelling data and by agreement between the SAXS correlation length and the estimated PTMO end-to-end distance. The systems prepared with PTMO possessing multiple triethoxysilane groups showed the most promising results in terms of mechanical properties. The tensile strength ranged from ca. 30 to 55 MPa., and the ambient modulus was nearly 10⁸ Pa. Also, a yield point was observed in some cases and was postulated to be an indication of partial continuity of the silicate phase. / Ph. D.

Investigation on the synthesis of the C-13 side chain of taxol was carried out in order to prepare modified taxol derivatives by coupling of the side chain acid chloride to a suitably protected baccatin III. The side chain was prepared by the Darzens condensation. Acylation of baccatin III was performed with simple acylating agents and extensive studies of the ¹H NMR and ¹³C NMR spectra of various acylbaccatins III were carried out aided by homonuclear and heteronuclear COSY experiments. This work led to the unambiguous assignment of the ¹H NMR and ¹³C NMR spectra of these compounds. Coupling of more bulky side chains to 7-(2,2,2-trichloroethyloxycarbonyl) baccatin III was difficult and yields were poor. Conventional methods, using triethylamine or pyridine with 4-dimethylaminopyridine in the coupling reaction of 3- phenylpropanoyl chloride and 7-(2,2,2-trichloroethyloxycarbonyl) baccatin III led to the desired coupled product in low yield together with two coupled compounds possessing more than one phenylpropanoyl group on the C-13 side chain. When the coupling reaction was performed in the presence of silver cyanide in refluxing toluene, only 13-(3-phenylpropanoyl) baccatin III was obtained. However, these two methods were not successful in the coupling reaction of 2-acetyl-3- phenyllactyl chlorlde with 7-(2,2,2-trichloroethyloxycarbonyl) baccatin III. Preliminary studies on the cleavage of the N-acyl group at the C-3' position of taxol and cephalomannine were performed. Taxol reacted with zinc bromide in chloroform-methanol solution to produce 10- deacetyl-7-epitaxol and 10-deacetyltaxol. No cleavage of the N-acyl group was detected in this case and in other reactions in which taxol was treated with various selective reagents. Other attempts involved the conversion of cephalomannine to its ozonolysis products with a pyruvyl group at the 3’-NH group. A method of cleavage of the N-pyruvyl group has not yet been found, however. / Ph. D. / incomplete_metadata

A flavoenzima diidroorotato desidrogenase (DHODH) catalisa a quarta reação da via de novo de biossíntese de pirimidinas, se destacando como alvo molecular chave para parasitos causadores de Doenças Negligenciadas (DNs). Tendo em vista a demanda por novas alternativas terapêuticas para estas doenças, o objetivo deste trabalho foi a triagem in vitro de inibidores da enzima DHODH de Leishmania major (LmDHODH) em Asteraceae. Esta triagem foi acompanhada por abordagem metabolômica em UHPLC-ESI-HRFTMS para os extratos vegetais e estudos de QSAR (Quantitative Structure-Activity Relationships) para as substâncias isoladas. As etapas experimentais realizadas e os resultados obtidos foram: 1) Ensaios enzimáticos: os valores de IC50 foram determinados para 59 extratos (?IC50: 148,0 ?g.mL-1 a 9,4 mg.mL-1) e 57 substâncias isoladas (?IC50: 27,0 ?M a 2,6 mM). As substâncias mais ativas frente à LmDHODH apresentaram seletividade, ao exercer inibição irrelevante sobre DHODH humana. Adicionalmente, estudos de termoestabilidade confirmaram que as lactonas sesquiterpênicas (STLs) são, de fato, capazes de se ligar a esta enzima; 2) Estudos metabolômicos: as impressões digitais metabólicas por LC-MS foram obtidas com sucesso para os 59 extratos e o processamento dos dados forneceu 3.694 substâncias. A desreplicação por meio de uma biblioteca de padrões identificou com segurança 49 metabólitos secundários. Por meio da correlação in silico com os dados de inibição enzimática, foram determinados com êxito os principais biomarcadores dos extratos e obteve-se um modelo de regressão confiável para predição do potencial de inibição de novos extratos provindos de espécies ainda não testadas frente à LmDHODH, classificando-os como ativos ou inativos com base exclusivamente na sua impressão digital por UHPLC-ESI-HRFTMS; 3) Estudos de QSAR com 21 STLs: o modelo de QSAR baseado em descritores moleculares apresentou robustez e confiabilidade (R2 / Q2 / P2 > 0,6 e RMSE < 0,3), revelando que uma maior inibição desta enzima requer a distribuição balanceada das regiões hidrofóbicas através da superfície molecular, maior largura das moléculas e menor hidrofobicidade. O modelo 3D baseado em descritores farmacofóricos também foi útil (R2: 0,79; Q2: 0,55) e confirmou a importância da orientação adequada dos ligantes, propriedades superficiais e formato das moléculas, refletindo propriedades de um possível sítio de ligação para as STLs na enzima. Portanto, alguns dos produtos naturais testados nesta triagem in vitro são, de fato, capazes de inibir seletivamente a enzima LmDHODH, tratando-se de uma descoberta relevante, visto que uma infinidade de metabólitos secundários leishmanicidas já foram descritos, porém, para a maioria deles, o mecanismo de ação segue desconhecido. Os resultados evidenciaram (1) as espécies de Asteraceae como importante fonte na busca por novos inibidores desta enzima e (2) as substâncias mais ativas como ponto de partida para novas estruturas guias (lead compounds) visando novos fármacos antiparasitários para o tratamento de DNs, especialmente a leishmaniose. / The flavoenzyme dihydroorotate dehydrogenase (DHODH) catalyzes the fourth reaction of the de novo pyrimidine biosynthetic pathway, standing out as a key molecular target for trypanosomatid parasites causing Neglected Diseases (NDs). In view of the global demand for new therapies for such diseases, this study aimed for the in vitro screening of inhibitors of Leishmania major DHODH (LmDHODH) in Asteraceae, accompanied by metabolomic approach with UHPLC-ESI-HRFTMS for plant extracts and QSAR studies (Quantitative Structure-Activity Relationships) for isolated compounds. The experimental steps performed and the results obtained were: 1) Enzymatic assays: the IC50 values were determined for 59 plant extracts (?IC50: 148.0 ?g.mL-1 a 9.4 mg.mL-1) and 57 natural compounds (?IC50: 27.0 ?M a 2.6 mM). The most active compounds showed selectivity against LmDHODH by exercising irrelevant inhibition of human DHODH. In addition, thermostability studies have confirmed that sesquiterpene lactones (STLs) are indeed capable of binding to this enzyme; 2) Metabolomic studies: the metabolic fingerprints by LC-MS were successfully obtained for the 59 extracts and 3,694 peaks/compounds were provided after data processing. The dereplication using a library of natural compounds has safely identified 49 secondary metabolites. By means of in silico correlation with the enzymatic inhibition data, the main biomarkers of the extracts were successfully determined and a reliable regression model was obtained to predict the inhibition potential of new extracts from species not yet tested against LmDHODH, classifying it as active or inactive based solely on their fingerprint by UHPLC-ESI-HRFTMS; 3) QSAR studies with 21 STLs: a reliable QSAR model based on molecular descriptors was obtained (R2 / Q2 / P2 > 0.6 and RMSE < 0.3), which indicated that stronger inhibition requires a balanced distribution of the hydrophobic regions across the molecular surface, as well as higher width and lower hydrophobicity of the molecules. A pharmacophore-based 3D-QSAR approach also afforded a useful model (R2: 0.79; Q2: 0.55), which confirmed the importance of proper orientation of the ligands, molecular surface features and shape for stronger inhibition, reflecting properties of a putative common binding site. Thus, some of the natural products tested in this in vitro screening are actually capable of selectively inhibiting LmDHODH. This constitutes a relevant finding, since an infinity of leishmanicidal compounds have been described, however, for most of them, the mechanism of action remains unknown. The results highlighted (1) Asteraceae species as important sources of new LmDHODH inhibitors and (2) the most active metabolites as promising starting points for new led compounds aiming new antiparasitc drugs for treatment of NDs caused by trypanosomatids, especially leishmaniasis.

Xu, Xiao-ping. / "March 1999." / Thesis (Ph.D.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (p. 152-166). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.