The conformation of paclitaxel in the bound state on the protein has been proposed to be the T-taxol conformation, and paclitaxel analogs constrained to the T-taxol conformation proved to be significantly more active than paclitaxel in both cytotoxicity and tubulin polymerization assays, thus validating the T-taxol conformation as the tubulin-binding conformation. In this work, eight compounds containing an aza-tricyclic moiety as a mimic of the baccatin core of paclitaxel have been designed and synthesized as water-soluble simplified paclitaxel analogs, among which 3.50-3.52 and 3.55 were conformationally constrained analogs designed to bind to the paclitaxel binding site of tubulin, based on their similarity to the T-taxol conformation. The open-chain analogs 3.41-3.43 and 3.57 and the bridged analogs 3.50-3.52 and 3.55 were evaluated for their antiproliferative activities against the A2780 cell lines. Analogs 3.50-3.52 and 3.55 which were designed to adopt the T-taxol conformation showed similar antiproliferative activities compared to their open-chain counterparts. They were all much less active than paclitaxel. In the second project, a series of paclitaxel analogs with various thio-containing linkers at C-2′ and C-7 positions were designed and synthesized in our lab. These analogs were attached to the surfaces of gold nanoparticles by CytImmune Sciences for the development of mutifunctional tumor-targeting agents. The native analogs and the gold bound analogs were evaluated for their antiproliferative activities against the A2780 cell line. All the compounds tested showed comparable or better activities than paclitaxel. Stability studies were performed for selected analogs in hydrolysis buffer, which showed that the analogs released paclitaxel in buffer over time. In the third project, the synthesis of a conformationally constrained paclitaxel analog which was designed to mimic the REDOR-taxol conformation was attempted. Two synthetic routes were tried and significant progress was made toward the synthesis of the conformationally constrained analog. However, both of the current synthetic routes failed to produce the key intermediate that would serve as the precursor for a ring-closing metathesis reaction to furnish the macrocyclic ring. / Ph. D.

<p>Rational conversion of biologically active peptides to nonpeptide compounds with retained activity is an appealing approach in drug development. One important objective of the work presented in this thesis was to use computational modeling to aid in such a conversion of the peptide angiotensin II (Ang II, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe). An equally important objective was to gain an understanding of the requirements for ligand binding to the Ang II receptors, with a focus on interactions with the AT₂ receptor.</p><p>The bioactive conformation of a peptide can provide important guidance in peptidomimetic design. By designing and introducing well-defined secondary structure mimetics into Ang II the bioactive conformation can be addressed. In this work, both γ- and β-turn mimetic scaffolds have been designed and characterized for incorporation into Ang II. Using conformational analysis and the pharmacophore recognition method DISCO, a model was derived of the binding mode of the pseudopeptide Ang II analogues. This model indicated that the positioning of the Arg side chain was important for AT₂ receptor binding, which was also supported when the structure–activity relationship of Ang II was investigated by performing a glycine scan.</p><p>To further examine ligand binding, a 3D model of the AT₂ receptor was constructed employing homology modeling. Using this receptor model in a docking study of the ligands, binding modes were identified that were in agreement with data from point-mutation studies of the AT₂ receptor.</p><p>By investigating truncated Ang II analogues, small pseudopeptides were developed that were structurally similar to nonpeptide AT₂ receptor ligands. For further guidance in ligand design of nonpeptide compounds, three-dimensional quantitative structure–activity relationship models for AT₁ and AT₂ receptor affinity as well as selectivity were derived. </p>

Rational conversion of biologically active peptides to nonpeptide compounds with retained activity is an appealing approach in drug development. One important objective of the work presented in this thesis was to use computational modeling to aid in such a conversion of the peptide angiotensin II (Ang II, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe). An equally important objective was to gain an understanding of the requirements for ligand binding to the Ang II receptors, with a focus on interactions with the AT2 receptor. The bioactive conformation of a peptide can provide important guidance in peptidomimetic design. By designing and introducing well-defined secondary structure mimetics into Ang II the bioactive conformation can be addressed. In this work, both γ- and β-turn mimetic scaffolds have been designed and characterized for incorporation into Ang II. Using conformational analysis and the pharmacophore recognition method DISCO, a model was derived of the binding mode of the pseudopeptide Ang II analogues. This model indicated that the positioning of the Arg side chain was important for AT2 receptor binding, which was also supported when the structure–activity relationship of Ang II was investigated by performing a glycine scan. To further examine ligand binding, a 3D model of the AT2 receptor was constructed employing homology modeling. Using this receptor model in a docking study of the ligands, binding modes were identified that were in agreement with data from point-mutation studies of the AT2 receptor. By investigating truncated Ang II analogues, small pseudopeptides were developed that were structurally similar to nonpeptide AT2 receptor ligands. For further guidance in ligand design of nonpeptide compounds, three-dimensional quantitative structure–activity relationship models for AT1 and AT2 receptor affinity as well as selectivity were derived.

La détermination de la conformation d’une petite molécule liée à sa cible biologique nous permet de concevoir des drogues de propriété biologique améliorée. Cette détermination peut être difficile dû aux limitations techniques, comme indiqué par le débat sur la conformation de microtubule-lié d’une drogue anticancéreuse – paclitaxel. Les études utilisant la cristallographie des rayons X et la RMN du liquide ne peut pas fournir les informations détaillées sur la conformation espérée. La RMN du solide est un choix raisonnable en mesurant précisément des distances interatomiques de la molécule, et le marquage sélectif au tritium permet de mesurer une distance longue jusqu’à 14,4 Å avec une précision haute grâce au rapport gyromagnetique élevé de ce noyau. Aucune modification structurale n’a été rendue par le marquage au tritium. Ainsi notre sujet ayant pour l’objectif de déterminer la conformation bioactive du paclitaxel comporte la synthèse des 6 isotopomères de paclitaxel ditritiés sur les sites particuliers, suivie par la préparation des complexes de microtubule-paclitaxel marqué. L’analyse de RMN du tritium à l’état solide fournira les distances clés pour la détermination. 2 isotopomères ont été synthétisés par tritier le paclitaxel dibromé et coupler la baccatine tritiée et la chaîne latérale tritiée, respectivement. La stratégie synthétique conçue permet de réaliser la synthèse avec un rendement généralement satisfaisant et une bonne stéréosélectivité. Différentes méthodes de tritiation ont été testées, dont un enrichissement isotopique supérieur à 92% a été obtenu. La synthèse des autres isotopomères ainsi que des complexes de microtubule-paclitaxel est en cours de réaliser dans notre laboratoire. / The determination of the conformation of small molecule bound to its biological target would facilitate people to design improved drugs. This determination can be difficult due to technical limitations, as exemplified by the long standing debate on the microtubule-binding conformation of a natural anticancer drug – paclitaxel. Previous studies using X-ray crystallography and solution-state NMR failed to furnish direct information on the expected conformation. Solid-state NMR may help in this task by providing precise interatomic distances, and the selective labeling on different sites with tritium atoms enables accurate measurement of long-range distances (up to 14.4 Å) owing to the high gyromagnetic ratio of this nucleus, without any structural modification of the molecule. So our project aiming at illustrating the bioactive conformation of paclitaxel consists the syntheses of 6 different paclitaxel isotopomers bearing a pair of tritiums at specified positions, flowing by the preparations of corresponding microtubule-labeled paclitaxel complexes. The solid-state tritium NMR analyses of these complexes would provide key distances for determining the expected conformation. Up to now, 2 paclitaxel isotopomers have been prepared from labelling the dibrominated paclitaxel precursor and from coupling the tritiated taxane rings and the tritiated side chains, respectively. The synthetic strategy allowed us to realize the syntheses in generally high yield and good stereoselectivity. Different tritiation methods have been used, from which an isotopic enrichment of higher than 92% was obtained. The syntheses of other 4 isotopomers, together with the microtubule complexes are currently underway in our lab.

<p>This study addresses the issue of how to convert peptides into drug-like non- peptides with retained biological activities at peptide receptors. Angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, Ang II) was used as a model peptide. </p><p>Knowledge of the bioactive conformations of endogenous peptides is invaluable for the conversion of peptides into less peptidic analogues. Effectively constrained cyclic analogues, with retained pharmacological activities, may provide valuable information about the bioactive conformations of the peptide in question. </p><p>This thesis describes the development of synthesis for a number of protected, aldehyde-functionalized building blocks for standard solid phase peptide synthesis, and their use for the preparation of cyclic peptide analogues. The effect of variations in the side-chain lengths of the building blocks, on the outcome of the cyclizations was studied. Incorporation of a building block derived from L-aspartic acid afforded bicyclization towards the C-terininal end of the peptide, while for the corresponding L-glutamic acid derived building block, N-terminal directed bicyclization was achieved. A building block derived from L-2-aminoadipic acid was exploited for monocyclization furnishing <i>cis-</i> and <i>trans-</i> vinyl sulfide bridged peptide analogues. </p><p>The described cyclization methods have been applied to the synthesis of a number of conformationally constrained Ang II analogues, for which the pharmacological properties have been evaluated. Two of the Ang II analogues synthesized displayed high affinities and full agonist activities at the AT₁ angiotensin receptor, and have proven to be useful tools in the search for the bioactive conformation of Ang II.</p>

This study addresses the issue of how to convert peptides into drug-like non- peptides with retained biological activities at peptide receptors. Angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, Ang II) was used as a model peptide. Knowledge of the bioactive conformations of endogenous peptides is invaluable for the conversion of peptides into less peptidic analogues. Effectively constrained cyclic analogues, with retained pharmacological activities, may provide valuable information about the bioactive conformations of the peptide in question. This thesis describes the development of synthesis for a number of protected, aldehyde-functionalized building blocks for standard solid phase peptide synthesis, and their use for the preparation of cyclic peptide analogues. The effect of variations in the side-chain lengths of the building blocks, on the outcome of the cyclizations was studied. Incorporation of a building block derived from L-aspartic acid afforded bicyclization towards the C-terininal end of the peptide, while for the corresponding L-glutamic acid derived building block, N-terminal directed bicyclization was achieved. A building block derived from L-2-aminoadipic acid was exploited for monocyclization furnishing cis- and trans- vinyl sulfide bridged peptide analogues. The described cyclization methods have been applied to the synthesis of a number of conformationally constrained Ang II analogues, for which the pharmacological properties have been evaluated. Two of the Ang II analogues synthesized displayed high affinities and full agonist activities at the AT1 angiotensin receptor, and have proven to be useful tools in the search for the bioactive conformation of Ang II.