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The preparation of intermediates for the production of etoposide by the use of bio-transformations with plant cell cultures and plant cell culture extracts

University of British Columbia

This thesis investigates the use of plant cell cultures in combination with synthetic chemistry to provide a new and inexpensive route to etoposide, an anti-cancer drug (1). Two facets of plant cell culture technology were investigated The first of these involved the use of cell cultures as a reaction medium in which synthetic precursors were added to the cultures and the bio-transformation products analyzed. The second facet investigated was the use of cell cultures of Podophyllum peltatwn to directly produce podophyllotoxin (3) and 4'-demethylpodophyllotoxin (5) in the culture medium. These compounds could then be utilized as intermediates in the production of etoposide (1). The synthetic studies involved the use of the readily available aromatic aldehydes, for example 3, 4-dihydroxybenzaldehyde (89), as starting materials. In an efficient route employing a tandem conjugate addition of 86 and 88 to the butenolide 87, the precursor 85 was prepared. By a similar route an efficient synthesis of the precursors 63 and 64 were performed. Biotransformation of the precursors 62, 63 and 64 with the whole cell culture of Catharanthus roseus resulted in the production of 71 as the only identified product. Compounds 62, 63 and 64 were treated with horseradish peroxidase (HRP) to compare plant cell enzymatic processes, with a commercially available peroxidase enzyme. The compound 63 was found to undergo bio-transformation to give the cyclized product 69 in 15-19% yield. Bio-transformation of the precursor 63 using a CFE (cell free extract) prepared from the cell culture of Catharanthus roseus.also produced the compound 69. Optimization of the conditions for this cyclization afforded 69 in 70% yield. The bio-transformation of (62) with the CFE prepared from the cell culture of Catharanthus roseus afforded 79. The precursor 85 was treated with the CFE prepared from the cell culture of Catharanthus roseus and found to cyclize to yield the product 115. The same cyclization was achieved by treatment of the precursor 85 with the CFE prepared from the cell culture of Podophyllum peltatum. The precursor 116 was bio-transformed using both Podophyllum peltatum whole cells and the CFE of Catharanthus roseus to produce the compound 118. The compounds 69, 115 and 118 all have the necessary fused ring structure present in the podophyllotoxins, however they do not have the correct stereochemistry at position 1. Further work is in progress in our laboratory to address this problem. The second part of this thesis is concerned with the hydroxylation of the butanolide 69 and deoxypodophyllotoxin (6) to produce an intermediate suitable for conversion into etoposide (1). Reactions were carried out with the CFE prepared from the cell culture of Catharanthus roseus using various co-factors. Procedures were employed to stabilize and characterize the enzyme preparations. Hydroxylation experiments were carried out with the cell culture of Tripterygium wilfordii using the precursors 6 and 69. Hydroxylation, using the CFE prepared from the cell cultures of Catharanthus roseus, of the precursors 133 and 134 obtained fron 6, afforded the hydroxylated product 139. The final part of the thesis involves the isolation of the lignans podophyllotoxin (3), deoxypodophyllotoxin (6), podophyllotoxone (60) and 4'-demethylpodophyllotoxin (5), from the cell suspension culture of Podophyllum peltatum. This is the first time that these lignans have been isolated from the cell cultures of Podophyllum peltatum.

Text

2011-01-28

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http://hdl.handle.net/2429/30929

Chemistry

University of British Columbia

Graduate