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>

Investigating the Specific Interactions Between Microtubule Stabilising Agents and β-Tubulin

<p>Microtubule stabilising agents are a class of cytotoxic compounds that cause mitotic arrest through inhibition of microtubule function. They specifically target β-tubulin subunits promoting tubulin polymerisation, which eventually leads to cell death. Members of this drug class include the cancer chemotherapeutics paclitaxel and ixabepilone. However, like many cytotoxic agents, tumour cells often develop multi-drug resistance phenotypes limiting the effectiveness of such compounds. This results from the expulsion of these drugs from cells by efflux pumps, as well as mutation of their binding site. Much effort has been focused on improving the utility of this important drug class in the ongoing fight against cancer. The microtubule stabilising agents peloruside A and laulimalide originate from marine sponge species native to the South Pacific. They have similar pharmacological profiles to paclitaxel and ixabepilone, however with several unique properties. They are poor substrates for efflux pumps and target a different region on β-tubulin subunits, giving them the potential for treatment of resistant tumours. This represents a novel mechanism of action that may be exploited for drug development, and further characterisation of the binding site is warranted. The aim of this study is to investigate the contribution of two amino acids of human βItubulin to the interactions with peloruside A and laulimalide. Specifically, glu127 and lys124 have been predicted by computational modelling and analogue studies to form hydrogen bonds and other associations with the two compounds. These amino acids are located on β-tubulin subunits adjacent to the main binding pocket of peloruside A and laulimalide, and represent a potential inter-protofilament interaction that does not occur with other microtubule stabilising agents. This binding mechanism has not yet been shown by crystallography and is hence based solely on in silico work, requiring biological validation. HEK293 cells were transfected with βI-tubulin with these amino acids mutated to alanines to prevent hydrogen bond formation. Cell proliferation assays, flow cytometry, and immunoblotting were used to study the effect loss of the inter-protofilament interaction has on the bioactivity of peloruside A and laulimalide. These mutations did not significantly alter the concentration-response of cells to either drug in the cell proliferation assay. However, accumulation of cells in the G2/M phase of the cell cycle and the proportion of transfected cells showing signs of mitotic arrest significantly decreased for E127A mutant cells compared to wild type βI-tubulin transfected control cells treated with peloruside A. Furthermore, a similar reduction in cell cycle block was also seen in E127A mutant cells treated with the negative control ixabepilone, which binds to a different site on β-tubulin. No evidence seen in this study suggests that either amino acid plays a major role in peloruside A or laulimalide target binding. However, the amino acid E127 is important for inter-protofilament associations independent of drug treatment, as its mutation appeared to reduce global stability of microtubule structures. This information requires further validation, it may be useful in the design of future analogue syntheses as development of these promising drug candidates continues.</p>

Investigating the Specific Interactions Between Microtubule Stabilising Agents and β-Tubulin

<p>Microtubule stabilising agents are a class of cytotoxic compounds that cause mitotic arrest through inhibition of microtubule function. They specifically target β-tubulin subunits promoting tubulin polymerisation, which eventually leads to cell death. Members of this drug class include the cancer chemotherapeutics paclitaxel and ixabepilone. However, like many cytotoxic agents, tumour cells often develop multi-drug resistance phenotypes limiting the effectiveness of such compounds. This results from the expulsion of these drugs from cells by efflux pumps, as well as mutation of their binding site. Much effort has been focused on improving the utility of this important drug class in the ongoing fight against cancer. The microtubule stabilising agents peloruside A and laulimalide originate from marine sponge species native to the South Pacific. They have similar pharmacological profiles to paclitaxel and ixabepilone, however with several unique properties. They are poor substrates for efflux pumps and target a different region on β-tubulin subunits, giving them the potential for treatment of resistant tumours. This represents a novel mechanism of action that may be exploited for drug development, and further characterisation of the binding site is warranted. The aim of this study is to investigate the contribution of two amino acids of human βItubulin to the interactions with peloruside A and laulimalide. Specifically, glu127 and lys124 have been predicted by computational modelling and analogue studies to form hydrogen bonds and other associations with the two compounds. These amino acids are located on β-tubulin subunits adjacent to the main binding pocket of peloruside A and laulimalide, and represent a potential inter-protofilament interaction that does not occur with other microtubule stabilising agents. This binding mechanism has not yet been shown by crystallography and is hence based solely on in silico work, requiring biological validation. HEK293 cells were transfected with βI-tubulin with these amino acids mutated to alanines to prevent hydrogen bond formation. Cell proliferation assays, flow cytometry, and immunoblotting were used to study the effect loss of the inter-protofilament interaction has on the bioactivity of peloruside A and laulimalide. These mutations did not significantly alter the concentration-response of cells to either drug in the cell proliferation assay. However, accumulation of cells in the G2/M phase of the cell cycle and the proportion of transfected cells showing signs of mitotic arrest significantly decreased for E127A mutant cells compared to wild type βI-tubulin transfected control cells treated with peloruside A. Furthermore, a similar reduction in cell cycle block was also seen in E127A mutant cells treated with the negative control ixabepilone, which binds to a different site on β-tubulin. No evidence seen in this study suggests that either amino acid plays a major role in peloruside A or laulimalide target binding. However, the amino acid E127 is important for inter-protofilament associations independent of drug treatment, as its mutation appeared to reduce global stability of microtubule structures. This information requires further validation, it may be useful in the design of future analogue syntheses as development of these promising drug candidates continues.</p>

Resistance to microtubule-stabilising agents following point mutation of human βI-tubulin

<p>Marine environments represent a rich source of bioactive secondary metabolites that may be harnessed for use in a therapeutic context. Two novel compounds, peloruside A and laulimalide, isolated from the marine sponges Mycale hentsheli and Cacospongia mycofijiensis, respectively, both demonstrate useful pharmacological properties in mammalian cells. These compounds share major similarities with microtubule-stabilising agents. Like other agents in this class, peloruside A and laulimalide bind to the β-tubulin subunit of microtubules, the primary cytoskeletal element of eukaryotic cells. These compounds enhance polymerisation dynamics between ternary microtubule structures and severely hinder necessary cytoskeletal rearrangements within the cell. Over the course of a patient’s treatment, cancerous cells may develop multi-drug resistance phenotypes. P-glycoprotein drug efflux pumps play a major role in the development of therapy resistance in many cancers, as the current generation microtubule-stabilising agents are easily removed from diseased cells by upregulated efflux mechanisms. Unlike agents already in clinical application, both peloruside A and laulimalide are poor substrates for removal by these mechanisms, making them and their synthetic derivatives interesting as potential treatments for drug-resistant tumours. Peloruside A and laulimalide exhibit potent nanomolar anti-mitotic activities in vitro and arrest cell cycle progression in G₂/M phase, leading to cell death – a characteristic mode of action among microtubule-stabilising agents. Unlike all known agents in this class, peloruside A and laulimalide share a secondary, unique binding region in β-tubulin. In the past decade our understanding of this region has developed, revealing a second, unique mechanism for stabilisation of microtubules. Using mammalian cells to model physiological tubulin, the present study investigates the predicted role of aspartic acid 297 of human βI-tubulin in the binding association of both peloruside A and laulimalide. This particular amino acid is predicted to hydrogen bond with both compounds, contributing to their activity as stabilisers. It was revealed that the introduction of a point mutation in D297 resulted in a small but highly consistent resistance phenotype to both compounds, but not to microtubule-stabilising agents that bind to the traditional, taxoid site on β-tubulin. It was concluded that aspartic acid 297 is likely to be one of the amino acids directly involved in the binding association of peloruside A and laulimalide to β-tubulin, contributing partial compound stabilisation. The rational synthesis of future analogues may benefit from these findings in the design of molecules with enhanced interactions at this particular amino acid residue.</p>

Resistance to microtubule-stabilising agents following point mutation of human βI-tubulin

<p>Marine environments represent a rich source of bioactive secondary metabolites that may be harnessed for use in a therapeutic context. Two novel compounds, peloruside A and laulimalide, isolated from the marine sponges Mycale hentsheli and Cacospongia mycofijiensis, respectively, both demonstrate useful pharmacological properties in mammalian cells. These compounds share major similarities with microtubule-stabilising agents. Like other agents in this class, peloruside A and laulimalide bind to the β-tubulin subunit of microtubules, the primary cytoskeletal element of eukaryotic cells. These compounds enhance polymerisation dynamics between ternary microtubule structures and severely hinder necessary cytoskeletal rearrangements within the cell. Over the course of a patient’s treatment, cancerous cells may develop multi-drug resistance phenotypes. P-glycoprotein drug efflux pumps play a major role in the development of therapy resistance in many cancers, as the current generation microtubule-stabilising agents are easily removed from diseased cells by upregulated efflux mechanisms. Unlike agents already in clinical application, both peloruside A and laulimalide are poor substrates for removal by these mechanisms, making them and their synthetic derivatives interesting as potential treatments for drug-resistant tumours. Peloruside A and laulimalide exhibit potent nanomolar anti-mitotic activities in vitro and arrest cell cycle progression in G₂/M phase, leading to cell death – a characteristic mode of action among microtubule-stabilising agents. Unlike all known agents in this class, peloruside A and laulimalide share a secondary, unique binding region in β-tubulin. In the past decade our understanding of this region has developed, revealing a second, unique mechanism for stabilisation of microtubules. Using mammalian cells to model physiological tubulin, the present study investigates the predicted role of aspartic acid 297 of human βI-tubulin in the binding association of both peloruside A and laulimalide. This particular amino acid is predicted to hydrogen bond with both compounds, contributing to their activity as stabilisers. It was revealed that the introduction of a point mutation in D297 resulted in a small but highly consistent resistance phenotype to both compounds, but not to microtubule-stabilising agents that bind to the traditional, taxoid site on β-tubulin. It was concluded that aspartic acid 297 is likely to be one of the amino acids directly involved in the binding association of peloruside A and laulimalide to β-tubulin, contributing partial compound stabilisation. The rational synthesis of future analogues may benefit from these findings in the design of molecules with enhanced interactions at this particular amino acid residue.</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>