Noruega

3.3.1 Marrubiin from White Horehound (Marrubium vulgare) 3.3.1.1 Background

M. vulgare (L.) is known to yield the compound marrubiin (78), which is known as a component of traditional medicine and shows analgesic properties. Studies into the structure of this compound were undertaken as early as the 1900’s,219,220 _{but the}

structure was not completely determined until 1953.221 _{An X-ray study in 1983 provided}

conclusive evidence of the structure including stereochemistry of the product, which aligned with previous determination through synthetic studies.222 _{Since then it has been}

extensively studied and shown that this compound and simple derivatives thereof have a number of different biological activities.223-225 _{Numerous publications into the synthesis}

of this compound exist, including a total synthesis paper published in 2016.226 _This

compound is not thought to be the true natural product, but an artefact of isolation. Premarrubiin (78b) is the true natural product which readily converts to marrubiin (78) on exposure to water and heat.227

Figure 3.37– Marrubiin (78) and premarrubiin (78b)

3.3.1.2 Extraction and Isolation

The extraction of this plant material with this PHWE method, and subsequent heptane extraction gave an extract enriched in the compound marrubiin (78). A fraction from the

O OH O H O H 78 78b

chromatographic separation was taken which contained a high proportion of the compound, but was still not pure. However, trituration with hexane resulted in the isolation of marrubiin (78) as an amorphous solid. The spectral data obtained was consistent with that reported in the literature.226

Figure 3.38 – 1_{H NMR spectrum of the crude extract from M. vulgare (below) and the}

purified compound (above).

In spite of the low isolated yield (0.06 % w/w, much lower than a reported yield of 0.4 % w/w)228_{, analysis of the} 1_{H NMR spectrum of the crude extract (862 mg from 15 g of M.}

vulgare, 5.75 % w/w) indicated a much higher content of 78, which appears to decompose. This is demonstrated by significant reduction in the intensity of the furan resonances in the 1_{H NMR spectrum at 6.27, 7.24 and 7.36 ppm (Figure 3.39). This}

reflects the importance of a method such as this novel PHWE method which is rapid, and has been shown to require less chromatography compared with other extraction methods for isolation of the compounds within the extract.

O OH O H O H 78 (after trituration)

Figure 3.39 – 1_{H NMR spectrum flash chromatographic fractions containing marrubiin,}

showing significant decomposition had occurred during purification.

In spite of these low yields, this material is of interest for synthetic applications within the research group, targeting natural products and analogues based on this scaffold. Commercial sourcing of the compound is not feasible, as with many of the examples detailed in this thesis.* _{Investigations are continuing into the improved isolation of this}

compound.

3.3.2 Castanospermine from Moreton Bay Chestnuts (Castanospermum australe) 3.3.2.1 Background

The toxic seeds of C. australe (A.Cunn & C.Fraser ex Hook.),also known as Moreton Bay Chestnuts, are known to contain the neurotoxic alkaloid castanospermine (79), which was first identified in 1981.229 _{It has been extensively studied since then and shown}

diverse biological activity, including anti-HIV activity, anti-diabetic activity, and inhibition of various enzymes.230-236

Figure 3.40– Castanospermine, isolated from C. australe

N HO HO OH H OH 79

The initial report details a procedure where 3 kg of the seeds are extracted with litres of 75% EtOH:H2O, and separated by means of multiple ion exchange resin columns, and

multiple crystallisation steps. The final yield of crystalline compound was 1.69 g, for an overall yield of 0.056% w/w. The X-ray analysis of the compound only gave relative stereochemistry, and the absolute stereochemistry was determined upon total synthesis of the compound from glucose, reported in 1984.237

Subsequently, the extraction and isolation of this compound was patented, with an improvement on the reported yield of 1.2 % w/w, representing the isolation of 3.5 g of crystals from 100 g of the fresh seeds. 238 _{A summary of the procedure from the patent is}

as follows:

“(a) extracting the ground seeds with a mixture of water and 2-propanol;

(b) washing the extract with petroleum ether;

(c) chromatographing the solution on an acid resin with elution with ammonium hydroxide;

(d) rechromatographing the eluate on a basic resin with elution with water; and

(e) concentration of the aqueous eluate followed by trituration with acetone to give castanospermine.”

Therefore, it was investigated if PHWE could be applied to this important compound.

3.3.2.2 Extraction and Isolation

For extraction of castanospermine (79) by espresso machine PHWE, the fresh seeds were grated before extraction of the wet material. It was observed that the seeds were unable to be extracted if allowed to dry out as they went very hard and could not be ground up. Amberlyst® 15 acidic ion exchange resin was added to bind to the castanospermine, and the supernatant was then filtered away from the resin. The resin was then eluted with a methanol:ammonia solution, to provide an extract enriched in castanospermine. Purification by automated gradient flash chromatography yielded the compound as a white amorphous solid, in a yield of 0.56 % w/w (565 mg). This is 10x higher than the

the patent, though in much less time (which may be due to variation of concentration of the compound within the seeds).229,238 _{This is another valuable natural product, and}

therefore this represents another example of viable extraction of compounds that are expensive to source commercially. *

Figure 3.41–1_{H NMR spectrum of castanospermine in D} 2O.

3.3.3 Summary

The extraction of marrubiin (78) and castanospermine (79) demonstrate the application of the method for rapid extraction of known valuable natural products which may be used as starting materials for synthesis. The extraction of marrubiin from M. vulgare

showed that in spite of the sensitive nature of the target compound, the PHWE extract contained a high proportion of this valuable natural product. Purification of this compound proved problematic, with significant decomposition observed, highlighting the viability of the extraction method for extraction of sensitive compounds.

The extraction of castanospermine from C. australe provided quantities of this material sufficient for small-scale synthetic studies. However, this method may not be as efficient as other methods for extracting this compound, particularly on a larger scale. This further N HO HO OH H OH 79

demonstrated that traditional natural product extraction techniques are still important for various plants, and that the PHWE method is a complementary extraction technique that like all extraction methods has its limitations.