No description available.

This thesis describes two main programmes: the synthesis of a new class of homochiral amines and the synthesis of ketone analogues of 3a-esterified tropane alkaloids. In chapter one, a scaled-up synthesis of (5)-a-(diphenylmethyl)pyrrolidine 1 is described. The key hydrogenation step of the oxazolidinone intermediate 2 was extended to the synthesis of the other chiral amines 70, 73, 76, 79 and 82. Hydrogenation of the oxazolidinones proceeded in good yields (71 - 87 %) and was not susceptible to racemisation. Thus, a convenient route from amino acid ester hydrochlorides (S)-valine 65, (S)-phenylalanine 66, (S)-alanine 67, (S)-isoleucine 68 and (S)-leucine 69 allowed a range of novel chiral amines to be prepared. In chapters two and three, a new route to ketone analogues of tropane esters is described. In chapter two, results of an attempt to prepare ketone 110 are outlined. Ketone 110 is an analogue of the tropane alkaloid littorine 101, where the bridging ester O atom is replaced by a CH2 group. The first approach to ketone 110 involved the Wittig reaction of acetylmethylenephosphorane 118 and the Homer-Wadsworth- Emmons reaction of diethylbenzoylmethanephosphonate 122 with tropinone 116. Tropinone 116 was found to be particularly unreactive in both cases. The second approach to ketone 110 involved the coupling reactions of both N-troc-3a- tosyloxymethyltropane 170 and N-troc-3 a-iodomethyltropane 185 with 2-phenylacetyl- 1,3-dithiane 147 and 1,3-ditihiane 142. These were also unreactive and as a result the synthesis of ketone 110 remains unresolved. In chapter three, the synthesis of other ketone analogues of naturally occurring 3 a- esterified tropane alkaloids is described. A six-step route to the ketones was devised and in this route the Grignard reactions of tropan-3 -ylacetaldehyde 227 emerged as the key to the success of the strategy. Three ketone analogues 218, 219 and 220 of tropate esters were successfully prepared. Ketone 220, the analogue of apoatropine 201, was found to be a muscarinic acetylcholine receptor antagonist (EC(_50) 1.9x10(^-7) M) in guinea-pigileum, showing a 500-fold less activity than atropine 202. However the activity is still within the clinical range.

No description available.

<p> Pramanicin (1) is a natural product that was first isolated as an anti-fungal agent: it possesses bioactivity against both the acapsular and capsular forms of Cryptococcus neoformans. 1 was found to act on smooth muscle in an endotheliumdependent manner. Further, studies of Ca2+ release from the endoplasmic reticulum of endothelial cells were consistent with an influence on the endothelial nitric oxide synthase (eNOS) pathway. Recently, we showed that 1 activates caspases 3 and 9. In order to determine which functional groups in 1 are required for this activity, several analogues were prepared (2~7). These included 2 and 3 prepared through fermentation and modification of 1, as well as 4-7 which were prepared by chemical synthesis. A test of bioactivity for 1-7 indicates that all compounds are active. The small differences in activity are discussed. </p> / Thesis / Master of Science (MSc)

No description available.

No description available.

<p>Histone deacetylases (HDACs) have been shown to play key roles in tumorigenesis, and</p><p>have been validated as effective enzyme target for cancer treatment. Largazole, a marine natural</p><p>product isolated from the cyanobacterium Symploca, is an extremely potent HDAC inhibitor that</p><p>has been shown to possess high differential cytotoxicity towards cancer cells along with excellent</p><p>HDAC class-selectivity. However, improvements can be made in the isoform-selectivity and</p><p>pharmacokinetic properties of largazole.</p><p>In attempts to make these improvements and furnish a more efficient biochemical probe</p><p>as well as a potential therapeutic, several largazole analogues have been designed, synthesized,</p><p>and tested for their biological activity. Three different types of analogues were prepared. First,</p><p>different chemical functionalities were introduced at the C2 position to probe the class Iselectivity profile of largazole. Additionally, docking studies led to the design of a potential</p><p>HDAC8-selective analogue. Secondly, the thiol moiety in largazole was replaced with a wide</p><p>variety of othe zinc-binding group in order to probe the effect of Zn2+ affinity on HDAC</p><p>inhibition. Lastly, three disulfide analogues of largazole were prepared in order to utilize a</p><p>different prodrug strategy to modulate the pharmacokinetic properties of largazole.</p><p>Through these analogues it was shown that C2 position can be modified significantly</p><p>without a major loss in activity while also eliciting minimal changes in isoform-selectivity. While</p><p>the Zn2+-binding group plays a major role in HDAC inhibition, it was also shown that the thiol</p><p>can be replaced by other functionalities while still retaining inhibitory activity. Lastly, the use of</p><p>a disulfide prodrug strategy was shown to affect pharmacokinetic properties resulting in varying</p><p>functional responses in vitro and in vivo.</p><p>v</p><p>Largazole is already an impressive HDAC inhibitor that shows incredible promise.</p><p>However, in order to further develop this natural product into an anti-cancer therapeutic as well as</p><p>a chemical probe, improvements in the areas of pharmacokinetics as well as isoform-selectivity</p><p>are required. Through these studies we plan on building upon existing structure–activity</p><p>relationships to further our understanding of largazole’s mechanism of inhibition so that we may</p><p>improve these properties and ultimately develop largazole into an efficient HDAC inhibitor that</p><p>may be used as an anti-cancer therapeutic as well as a chemical probe for the studying of</p><p>biochemical systems.</p> / Dissertation

MenD, a thiamin diphosphate (ThDP)-dependent enzyme, catalyzes the reaction from isochorismate (ISC) to 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC), and thus is also called SEPHCHC synthase. This conversion is the first committed step in the classical menaquinone (Vitamin K2) biosynthetic pathway, requiring 2-ketoglutarate (2-KG), ThDP and Mg²⁺. Since the biosynthesis of menaquinone is essential in some bacterial pathogens, for example <i>Mycobacterium tuberculosis</i>, MenD or the menaquinone pathway could be a target for drug development.<p> The method for the kinetic assay of the MenD-catalyzed reaction was evaluated by comparing UV spectrophotomeric measurements and HPLC analysis. It was validated that the steady-state kinetics of the MenD-catalyzed reaction can be determined by monitoring UV absorbance of ISC at 278 nm and 300 nm.<p> Phosphonate analogues of 2-KG were synthesized and assayed as inhibitors of the MenD reaction. It was found that the phosphonate analogues of 2-KG are competitive inhibitors with varied affinity for MenD. Of the inhibitors, monomethyl succinyl phosphonate (MMSP) was the most effective, with a <i>K</i>_i of 700 nM. However, the potent MenD inhibitors show no effectiveness against mycobacterial growth.<p> An analogue of isochorismate, trans-(±)-5-carboxymethoxy-6-hydroxy-1,3-cyclohexadiene-1-carboxylate ((±)-CHCD), was synthesized. The (+)-CHCD was found to be an alternative substrate for the MenD-catalyzed reaction. When CHCD was utilized in the MenD reaction, 5-carboxymethoxy-2-(3-carboxy-propionyl)-6-hydroxy-cyclohex-2-enecarboxylate (CCHC) was isolated and characterized, which was believed to be the product of spontaneous isomerization of the SEPHCHC-like analogue. The kinetic study of MenD reaction using (±)-CHCD, in association with the kinetics pattern probed by MMSP, demonstrated for the first time that the MenD-catalyzed reaction has a Ping Pong bi bi kinetic mechanism.<p> The analysis of sequence and structure of MenD from E. coli allowed the investigation of the active site residues and their catalytic functions by mutation of the individual residues. S32A, S32D, R33K, R33Q, E55D, R107K, Q118E, K292Q, R293K, S391A, R395A, R395K, R413K and I418L were prepared and assayed kinetically with respect to 2-KG, ISC, (±)-CHCD, ThDP and Mg²⁺. The values of <i>K</i>_m^a and <i>k</i>_cat^a/<i>K</i>_m^a for the mutants, in comparison with that of wild type MenD, provide valuable insight into the catalytic mechanism of MenD.

MenD, a thiamin diphosphate (ThDP)-dependent enzyme, catalyzes the reaction from isochorismate (ISC) to 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC), and thus is also called SEPHCHC synthase. This conversion is the first committed step in the classical menaquinone (Vitamin K2) biosynthetic pathway, requiring 2-ketoglutarate (2-KG), ThDP and Mg²⁺. Since the biosynthesis of menaquinone is essential in some bacterial pathogens, for example <i>Mycobacterium tuberculosis</i>, MenD or the menaquinone pathway could be a target for drug development.<p> The method for the kinetic assay of the MenD-catalyzed reaction was evaluated by comparing UV spectrophotomeric measurements and HPLC analysis. It was validated that the steady-state kinetics of the MenD-catalyzed reaction can be determined by monitoring UV absorbance of ISC at 278 nm and 300 nm.<p> Phosphonate analogues of 2-KG were synthesized and assayed as inhibitors of the MenD reaction. It was found that the phosphonate analogues of 2-KG are competitive inhibitors with varied affinity for MenD. Of the inhibitors, monomethyl succinyl phosphonate (MMSP) was the most effective, with a <i>K</i>_i of 700 nM. However, the potent MenD inhibitors show no effectiveness against mycobacterial growth.<p> An analogue of isochorismate, trans-(±)-5-carboxymethoxy-6-hydroxy-1,3-cyclohexadiene-1-carboxylate ((±)-CHCD), was synthesized. The (+)-CHCD was found to be an alternative substrate for the MenD-catalyzed reaction. When CHCD was utilized in the MenD reaction, 5-carboxymethoxy-2-(3-carboxy-propionyl)-6-hydroxy-cyclohex-2-enecarboxylate (CCHC) was isolated and characterized, which was believed to be the product of spontaneous isomerization of the SEPHCHC-like analogue. The kinetic study of MenD reaction using (±)-CHCD, in association with the kinetics pattern probed by MMSP, demonstrated for the first time that the MenD-catalyzed reaction has a Ping Pong bi bi kinetic mechanism.<p> The analysis of sequence and structure of MenD from E. coli allowed the investigation of the active site residues and their catalytic functions by mutation of the individual residues. S32A, S32D, R33K, R33Q, E55D, R107K, Q118E, K292Q, R293K, S391A, R395A, R395K, R413K and I418L were prepared and assayed kinetically with respect to 2-KG, ISC, (±)-CHCD, ThDP and Mg²⁺. The values of <i>K</i>_m^a and <i>k</i>_cat^a/<i>K</i>_m^a for the mutants, in comparison with that of wild type MenD, provide valuable insight into the catalytic mechanism of MenD.

No description available.