Discoveries from the alpine daisy Celmisia viscosa: unique diterpenes and flavones, and variation within the species

Abstract

This thesis focuses on the natural product chemistry of the native Alpine daisy, Celmisia viscosa. Previous work on C. viscosa has shown the presence of the diterpenes epimanool and the rare halimadienol, also present were these compounds with a new glycoside attached. There were indications of these and related compounds in other New Zealand Celmisia species. This study aimed to investigate variation among Celmisia and to isolate and characterise the major novel compounds associated with variation. Using C. viscosa, a repeatable method of CDCl3 extraction and NMR and GC analyses from small quantities of leaf material has been established. This allowed for the investigation of chemical variation using portions of stored botanical voucher specimens, extracts of which were subjected to Principal Component Analysis (PCA). Variation among eight species of Celmisia was investigated, with results indicating that C. viscosa stands out, having generally the highest secondary metabolite concentrations and intraspecific variation. Investigation of variation within a C. viscosa plant sourced from End Peak, Harris Mountain found the leaves to contain the highest concentration of diterpenes. Further investigation, of C. viscosa leaves from three plants from the same region indicated only variation in diterpene concentration, not diterpene type. C. viscosa leaf material was sourced from herbaria, giving access to nearly the full geographic range of C. viscosa from different regions around South Island, New Zealand. Results indicated the presence of two distinct chemotypes. Isolation of the major compounds from each chemotype gave epimanool and the reported epimanool glycoside, 2’,6’-dideoxy-hexopyran-3’-ulose isolated from CHCl3 leaf extracts from the Kakanui Ranges. Halimadienol plus two novel derivatives, halimadienol 4’-acetyl-2’,6’-dideoxy-hexopyran-3’-ulose and halimadienol-4’-hydroxy-2’,6’-dideoxy-hexopyran-3’-ulose were isolated from CHCl3 leaf extracts from the Rock and Pillar Ranges. Halimadienol has been also reported from Jungermannia infusca and Plagiochila barteri, but it was not fully characterised. The data herein presents the full characterisation of halimadienol with X-ray crystallography revealing the absolute stereochemistry. NMR Spectroscopic data indicated halimadienol to be conformationally mobile, but subjecting the compound to 13C NMR at various temperatures failed to give access to the slow exchange conformation. However, computational modelling revealed two significant conformers, the major of which matched that of the crystal structure. 2D NMR and spectroscopic data for the halimadienol 4’-acetyl-2’,6’-dideoxy-hexopyran-3’-ulose, previously isolated, but not published, is presented herein. Isolation of halimadienol-4’-hydroxy-2’,6’-dideoxy-hexopyran-3’-ulose gave the opportunity for the absolute stereochemistry of the glycoside moiety to be deduced using Mosher ester analysis. Halimadienol-4’-hydroxy-2’,6’-dideoxy-hexopyran-3’-ulose was acetylated to further confirm the stereochemistry. Halimadienol and epimanool have quite different retention indices and times with halimadienol eluting earlier from GC-FID-MS column. This behaviour suggested that the halimadienol could be thermally degrading upon injection onto the GC column. A study of the behaviour of these compounds upon injection onto a column at various temperatures failed to verify this assertion, so halimadienol was chemically dehydrated, with the products being reinjected onto GC, thereby refuting the hypothesis. Three novel acylated trimethoxyflavones: 8-(3”-methylbutanoyl), 5-hydroxy-5,7,4’-trimethoxyflavone, 8-(2”-methylbutanoyl), 5-hydroxy-5,7,4’-trimethoxyflavone and 8-(2”-methylpropanoyl), 5-hydroxy-6,7,4’-trimethoxyflavone were discovered in a bulk CHCl3 extract of leaves from the Rock and Pillar Ranges. Characterisation of these compounds was achieved by 2D NMR, comparison to model compounds and X-ray crystallography. Subsequent structural elucidations of a mixture of four novel acylated dimethoxyflavones: 8-(2”-methybutanoyl), 5,4’-dihydroxy-6, 7-dimethoxyflavone; 8-(3”-methybutanoyl), 5,4’-dihydroxy-6,7-dimethoxyflavone; 8-(2”-methybutanoyl), 5,7-dihydroxy-6,4’-dimethoxyflavone; 8-(3”-methybutanoyl), 5,7-dihydroxy-6,4’-dimethoxyflavone and a mixture of two novel acylated monomethoxyflavones: 8-(2”-methylbutanoyl),5,7,4’-trihydroxy-6-methoxyflavone and 8-(3”-methylbutanoyl),5,7,4’-trihydroxy-6-methoxyflavone were made using the trimethoxyflavones as model compounds. X-ray crystallography of 8-(2”-methylbutanoyl),5,7,4’-trihydroxy-6-methoxyflavone confirmed the proposed structures. An ethanol extract of C. viscosa leaf material from Mount Harris gave the two major dimethoxyflavones; 8-(2”-methybutanoyl), 5,4’-dihydroxy-6,7, dimethoxyflavone; 8-(3”-methybutanoyl), 5,4’-dihydroxy-6,7, dimethoxyflavone in a 2:1 mixture allowing for partial 2D NMR assignment of both compounds. Analysis of the seven other species of Celmisia indicated novel flavone profiles and analysis of the full range of C. viscosa leaf extracts by HPLC indicated the presence of flavone chemotypes. While this work has increased the chemical knowledge of C. viscosa a range of further work remains for both the species and the genus. Further analysis of the chemical variation of viscid species of Celmisia regionally may uncover further species exhibiting chemotypic variance, while bulk extraction may lead to the discovery of other novel diterpenes. More within site leaf extract analysis may uncover differing chemotypes within plants in proximity. If more bulk C. viscosa from other regions is sourced, extraction and isolation may provide evidence of more novel diterpene derivatives. Ecological studies with halimadienol and epimanool may provide insight around the function of these compounds. Assessment of the contents of trichomes, found on the flowers of C. viscosa, would confirm whether these structures contain flavones or diterpenes.