Synthesis of Novel Pyran Fragments to Incorporate into Peloruside Analogues

<p>Cancer is currently the second largest cause of death globally, leading to a high demand for new and effective chemotherapeutics. For years, natural products have been used as a source of new bioactive compounds; of particular interest in this context, as a source of new chemotherapeutics. One chemotherapeutic candidate which has attracted significant attention in synthetic and medicinal chemistry communities, is peloruside A. Peloruside A is a bioactive secondary metabolite isolated from the New Zealand marine sponge Mycale hentscheli. Since its discovery, peloruside A has shown great promise in cancer studies both in vivo and in vitro with effects observed even at nanomolar concentrations. These chemotherapeutic effects have been shown to occur by halting cell division at the G2/M checkpoint via microtubule stabilisation. Of particular interest is that this stabilisation occurs in a manner distinct from that of the already established taxane class of microtubule stabilising drugs. This means that peloruside A is able to offer both inhibition of cell division in Taxol® resistant cells and synergistic inhibition alongside the current taxane drugs. Since peloruside A is not abundantly available from its natural source, there is a strong incentive for the development of new synthetic strategies for peloruside A production. Unfortunately attempts at aquaculture and attempts at developing an industrial scale synthesis have both proven unsuccessful thus far. In an attempt to overcome some of the difficulties with the scale up of peloruside, analogues have been developed that are intended to have similar bioactivity to peloruside A but simpler, more concise, synthetic routes. These analogues will also enable further elucidation of the binding properties of peloruside A. This project focuses on the generation of a functionalised pyran fragment, starting from a simple carbohydrate, that may be incorporated into the proposed analogues.</p>

Synthesis of Novel Pyran Fragments to Incorporate into Peloruside Analogues

<p>Cancer is currently the second largest cause of death globally, leading to a high demand for new and effective chemotherapeutics. For years, natural products have been used as a source of new bioactive compounds; of particular interest in this context, as a source of new chemotherapeutics. One chemotherapeutic candidate which has attracted significant attention in synthetic and medicinal chemistry communities, is peloruside A. Peloruside A is a bioactive secondary metabolite isolated from the New Zealand marine sponge Mycale hentscheli. Since its discovery, peloruside A has shown great promise in cancer studies both in vivo and in vitro with effects observed even at nanomolar concentrations. These chemotherapeutic effects have been shown to occur by halting cell division at the G2/M checkpoint via microtubule stabilisation. Of particular interest is that this stabilisation occurs in a manner distinct from that of the already established taxane class of microtubule stabilising drugs. This means that peloruside A is able to offer both inhibition of cell division in Taxol® resistant cells and synergistic inhibition alongside the current taxane drugs. Since peloruside A is not abundantly available from its natural source, there is a strong incentive for the development of new synthetic strategies for peloruside A production. Unfortunately attempts at aquaculture and attempts at developing an industrial scale synthesis have both proven unsuccessful thus far. In an attempt to overcome some of the difficulties with the scale up of peloruside, analogues have been developed that are intended to have similar bioactivity to peloruside A but simpler, more concise, synthetic routes. These analogues will also enable further elucidation of the binding properties of peloruside A. This project focuses on the generation of a functionalised pyran fragment, starting from a simple carbohydrate, that may be incorporated into the proposed analogues.</p>

Developing a scalable synthesis of Peloruside A

<p>Peloruside A (PelA, 1) is a marine natural product isolated from a sponge Mycale hentscheli found in Pelorus Sound, New Zealand. It is a microtubule-stabilising agent, active against various cancerous cell lines at nanomolar concentrations and offers several advantages over the current drugs on the market due to its unique mode of microtubule stabilisation, its potency and its activity in multidrug resistant cells. Since large-scale isolation of the compound from the sponge is unsustainable and an attempt to grow the sponge failed due to a sea-slug infestation, devising an efficient synthesis of peloruside A that will be able to deliver larger quantities of this compound is essential in order to conduct further studies and enable the eventual manufacture of the drug. Peloruside A is also a very interesting synthetic target as a macrolide with ten stereogenic centres, an internal pyran ring and a trisubstituted Z-double bond. Our synthetic strategy combines elements from previous total and partial syntheses with novel elements with an aim to make the synthesis more efficient. The synthesis of the side-chain fragment (C12–C20) was based on Evans' methodology1 which was also utilised to couple this fragment with the C8–C11 fragments. It was envisioned to evaluate two different end-game strategies, and to this end it was necessary to synthesise two different versions of the C8–C11 fragment. However, the synthesis of the C1–C7 fragments proved to be quite challenging and required a lot of alterations to the synthetic plan and the protecting group strategy. Various routes based on previous syntheses by Ghosh, Jacobsen and Taylor were explored.2–4 Eventually, the key intermediate was synthesised using a modified Taylor methodology. Our future work will focus on optimising and establishing fragment coupling methodologies and evaluating the two end-game approaches: macrolactonisation and a ring-closing metathesis.</p>

Developing a scalable synthesis of Peloruside A

<p>Peloruside A (PelA, 1) is a marine natural product isolated from a sponge Mycale hentscheli found in Pelorus Sound, New Zealand. It is a microtubule-stabilising agent, active against various cancerous cell lines at nanomolar concentrations and offers several advantages over the current drugs on the market due to its unique mode of microtubule stabilisation, its potency and its activity in multidrug resistant cells. Since large-scale isolation of the compound from the sponge is unsustainable and an attempt to grow the sponge failed due to a sea-slug infestation, devising an efficient synthesis of peloruside A that will be able to deliver larger quantities of this compound is essential in order to conduct further studies and enable the eventual manufacture of the drug. Peloruside A is also a very interesting synthetic target as a macrolide with ten stereogenic centres, an internal pyran ring and a trisubstituted Z-double bond. Our synthetic strategy combines elements from previous total and partial syntheses with novel elements with an aim to make the synthesis more efficient. The synthesis of the side-chain fragment (C12–C20) was based on Evans' methodology1 which was also utilised to couple this fragment with the C8–C11 fragments. It was envisioned to evaluate two different end-game strategies, and to this end it was necessary to synthesise two different versions of the C8–C11 fragment. However, the synthesis of the C1–C7 fragments proved to be quite challenging and required a lot of alterations to the synthetic plan and the protecting group strategy. Various routes based on previous syntheses by Ghosh, Jacobsen and Taylor were explored.2–4 Eventually, the key intermediate was synthesised using a modified Taylor methodology. Our future work will focus on optimising and establishing fragment coupling methodologies and evaluating the two end-game approaches: macrolactonisation and a ring-closing metathesis.</p>

Design and Synthesis of a Highly Simplified Pateamine Analogue

<p>Pateamine (1) is a natural product from the marine sponge Mycale hentscheli that exhibits potent anticancer properties, and has potential as an antiviral agent, and in preventing the muscle wasting disorder cachexia. This biological activity of pateamine is due to its ability to inhibit the eukaryotic initiation factor eIF4A, which leads to the formation of stress granules, the inhibition of protein synthesis, and ultimately cell death. Unfortunately, pateamine is obtained in very small amounts from Mycale hentscheli; thus, it is necessary to synthesise pateamine and novel structural analogues in the laboratory. Previously a separate binding and scaffolding domain of pateamine was proposed, which led to the synthesis of a simplified des-methyl des- amino analogue that reduced the number of synthetic steps compared to pateamine while retaining its biological activity. This was followed by the synthesis of a simplified triazole- containing analogue 9 6 ; unfortunately, this exhibited substantially reduced bioactivity compared to pateamine, and it is therefore necessary to determine if the reduction in bioactivity was due to the replacement of the thiazole ring with a triazole ring, or due to the removal of key methyl groups of pateamine. Thus, the thiazole-containing analogue of 96 is deemed to be an important synthetic target. In this Master’s project a highly simplified side chain-free analogue 130 was synthesised, which laid the groundwork for future synthesis of a thiazole-containing analogue of 96. The synthesis of 130 was achieved through a convergent synthesis with one commercially available and two prepared fragments. Particular attention was paid to the development of an efficient thiazole formation methodology, as well as optimising fragment synthesis and coupling reactions. Determination of the binding of analogue 130 with eIF4A using a competitive bioactivity assay in the presence of pateamine was then undertaken, which showed that either 130 does not bind to eIF4A or that it binds non-covalently and is then displaced by pateamine.</p>

Design and Synthesis of a Highly Simplified Pateamine Analogue

<p>Pateamine (1) is a natural product from the marine sponge Mycale hentscheli that exhibits potent anticancer properties, and has potential as an antiviral agent, and in preventing the muscle wasting disorder cachexia. This biological activity of pateamine is due to its ability to inhibit the eukaryotic initiation factor eIF4A, which leads to the formation of stress granules, the inhibition of protein synthesis, and ultimately cell death. Unfortunately, pateamine is obtained in very small amounts from Mycale hentscheli; thus, it is necessary to synthesise pateamine and novel structural analogues in the laboratory. Previously a separate binding and scaffolding domain of pateamine was proposed, which led to the synthesis of a simplified des-methyl des- amino analogue that reduced the number of synthetic steps compared to pateamine while retaining its biological activity. This was followed by the synthesis of a simplified triazole- containing analogue 9 6 ; unfortunately, this exhibited substantially reduced bioactivity compared to pateamine, and it is therefore necessary to determine if the reduction in bioactivity was due to the replacement of the thiazole ring with a triazole ring, or due to the removal of key methyl groups of pateamine. Thus, the thiazole-containing analogue of 96 is deemed to be an important synthetic target. In this Master’s project a highly simplified side chain-free analogue 130 was synthesised, which laid the groundwork for future synthesis of a thiazole-containing analogue of 96. The synthesis of 130 was achieved through a convergent synthesis with one commercially available and two prepared fragments. Particular attention was paid to the development of an efficient thiazole formation methodology, as well as optimising fragment synthesis and coupling reactions. Determination of the binding of analogue 130 with eIF4A using a competitive bioactivity assay in the presence of pateamine was then undertaken, which showed that either 130 does not bind to eIF4A or that it binds non-covalently and is then displaced by pateamine.</p>

Towards the Synthesis of Hybrid Peloruside A and Laulimalide Analogues

<p>(+)-Peloruside A is a novel cytotoxic marine natural product isolated from the New Zealand sponge Mycale hentscheli(42). Peloruside A is a potential anticancer agent that has a similar mode of action to that of the successful drug paclitaxel. Biological analysis indicated that (+)-peloruside A promotes tubulin hyperassembly and cellular microtubule stabilisation which lead to mitosis blockage in the G2/M phase of the cell cycle and consequent cell apoptosis(43),(47). (-)-Laulimalide is also a cytotoxic natural product with microtubule stabilising bioactivity, and is a potential anticancer agent(47). Biological analysis showed that (+)-peloruside A and (-)-laulimalide are competitive, suggesting that they bind to the same active site(47). (+)-Peloruside A and (-)-laulimalide also display synergy with taxoids(47). Due to the structural complexity of peloruside A, our research has focused on developing an analogue 151 for ease of synthesis. Thus, the simplified C5-C9 dihydropyran moiety of (-)-laulimalide, with fewer stereocentres than that of (+)-peloruside A, has been incorporated into analogue 151 whilst retaining the 16- membered ring backbone of (+)-peloruside A. The proposed synthesis of 151 involves a Yamaguchi macrolactonization, a 1,5-anti-aldol coupling, and a ring closing metathesis as key reactions. This thesis reports on the synthesis of key fragments of analogue 151 and the crucial 1,5-anti-aldol coupling reaction for the assembly of the carbon backbone.</p>

Towards the Synthesis of Hybrid Peloruside A and Laulimalide Analogues

<p>(+)-Peloruside A is a novel cytotoxic marine natural product isolated from the New Zealand sponge Mycale hentscheli(42). Peloruside A is a potential anticancer agent that has a similar mode of action to that of the successful drug paclitaxel. Biological analysis indicated that (+)-peloruside A promotes tubulin hyperassembly and cellular microtubule stabilisation which lead to mitosis blockage in the G2/M phase of the cell cycle and consequent cell apoptosis(43),(47). (-)-Laulimalide is also a cytotoxic natural product with microtubule stabilising bioactivity, and is a potential anticancer agent(47). Biological analysis showed that (+)-peloruside A and (-)-laulimalide are competitive, suggesting that they bind to the same active site(47). (+)-Peloruside A and (-)-laulimalide also display synergy with taxoids(47). Due to the structural complexity of peloruside A, our research has focused on developing an analogue 151 for ease of synthesis. Thus, the simplified C5-C9 dihydropyran moiety of (-)-laulimalide, with fewer stereocentres than that of (+)-peloruside A, has been incorporated into analogue 151 whilst retaining the 16- membered ring backbone of (+)-peloruside A. The proposed synthesis of 151 involves a Yamaguchi macrolactonization, a 1,5-anti-aldol coupling, and a ring closing metathesis as key reactions. This thesis reports on the synthesis of key fragments of analogue 151 and the crucial 1,5-anti-aldol coupling reaction for the assembly of the carbon backbone.</p>

Mechanistic Investigation of 1,5-Anti Stereoinduction in Aldol Reactions

<p>Peloruside A (+)-1 is a novel secondary metabolite isolated from a New Zealand marine sponge (Mycale hentscheli) by Northcote and West of Victoria University. Because it has a polyketide backbone, aldol reactions have been widely employed for its total synthesis. Aldol reactions displaying 1,5-anti stereoinduction mediated by the C₁₅ stereocenter (according to peloruside A numbering) have proven useful for the synthesis of the C₁₁–C₁₂ bond of peloruside A and analogues. This project is the continuation of Stocker's and Turner's studies on the excellent stereoinduction of 2 in boron-mediated aldol reactions. The relative stereochemistry of the corresponding aldol product is consistence with the expectations of Kishi's C database for a 1,5-anti product. Furthermore, the diphenylsilyl acetal tethered eight-membered ring of 2 has proven to be essential for its stereoinduction, while the homoallylic oxygen does not appear to play a significant role. Although 1,5-anti aldol reactions have been used frequently in the syntheses of polyketidederived natural products, the underlying mechanism for the 1,5-anti-stereoinduction remains inconclusive. Three models have been proposed, including Hoberg's π-stacking model, Goodman's hydrogen-bonding model, and a modification of Abiko's diborylated model. The underlying mechanism for the stereoinduction of 2 was investigated using variable temperature NMR, 1D NOESY and 1D ROESY experiments. It was found that Hoberg's and Abiko's models are not able to explain the stereoinduction of 2 and that Goodman's model used for explaining the transition states of the aldol reaction of β-trimethylsilyloxy methyl ketones is also not suitable. A modification of Goodman's model has been proposed to explain the excellent 1,5-anti stereoinduction of 2. While attempts to couple 2 and 3 to a variety of bulky aldehydes bearing groups with different steric and electronic factors in boron-mediated aldol reactions were unsuccessful, the reaction of 3 with 4-bromobenzaldehyde using TiCl₄ and DIPEA afforded an excellent yield (>99%) of the aldol product. This revealed the six-membered ring in the TS of the boron-mediated aldol reaction is too compact for 2 and 3. However, it was found that 2 is incompatible with TiCl₄. Key questions regarding the 1,5-anti-stereoinduction of 2 have been answered and a modified procedure for the NMR investigation of an aldol reaction is described in this thesis.</p>

Mechanistic Investigation of 1,5-Anti Stereoinduction in Aldol Reactions

<p>Peloruside A (+)-1 is a novel secondary metabolite isolated from a New Zealand marine sponge (Mycale hentscheli) by Northcote and West of Victoria University. Because it has a polyketide backbone, aldol reactions have been widely employed for its total synthesis. Aldol reactions displaying 1,5-anti stereoinduction mediated by the C₁₅ stereocenter (according to peloruside A numbering) have proven useful for the synthesis of the C₁₁–C₁₂ bond of peloruside A and analogues. This project is the continuation of Stocker's and Turner's studies on the excellent stereoinduction of 2 in boron-mediated aldol reactions. The relative stereochemistry of the corresponding aldol product is consistence with the expectations of Kishi's C database for a 1,5-anti product. Furthermore, the diphenylsilyl acetal tethered eight-membered ring of 2 has proven to be essential for its stereoinduction, while the homoallylic oxygen does not appear to play a significant role. Although 1,5-anti aldol reactions have been used frequently in the syntheses of polyketidederived natural products, the underlying mechanism for the 1,5-anti-stereoinduction remains inconclusive. Three models have been proposed, including Hoberg's π-stacking model, Goodman's hydrogen-bonding model, and a modification of Abiko's diborylated model. The underlying mechanism for the stereoinduction of 2 was investigated using variable temperature NMR, 1D NOESY and 1D ROESY experiments. It was found that Hoberg's and Abiko's models are not able to explain the stereoinduction of 2 and that Goodman's model used for explaining the transition states of the aldol reaction of β-trimethylsilyloxy methyl ketones is also not suitable. A modification of Goodman's model has been proposed to explain the excellent 1,5-anti stereoinduction of 2. While attempts to couple 2 and 3 to a variety of bulky aldehydes bearing groups with different steric and electronic factors in boron-mediated aldol reactions were unsuccessful, the reaction of 3 with 4-bromobenzaldehyde using TiCl₄ and DIPEA afforded an excellent yield (>99%) of the aldol product. This revealed the six-membered ring in the TS of the boron-mediated aldol reaction is too compact for 2 and 3. However, it was found that 2 is incompatible with TiCl₄. Key questions regarding the 1,5-anti-stereoinduction of 2 have been answered and a modified procedure for the NMR investigation of an aldol reaction is described in this thesis.</p>