Degree Name

Doctor of Philosophy


The Stemona species are monocotyledonous plants that belong to the Stemonaceae family. These plants are widely spread throughout South East Asia and northern Australia. The crude extracts from the roots of Stemona sp. have been used in China and South East Asia for agricultural and medicinal purposes. Stemona alkaloids have been reported for their insecticidal activities which may be associated with their ability to inhibit the enzyme acetylcholinesterase (AChE). Inhibitors of this enzyme are currently used to treat patients with Alzheimer’s disease and therefore the discovery of new AChE inhibitors is of medical importance. During the course of this study stemofoline was reported to increase sensitivity of anticancer drugs in treatment of multidrug resistance (MDR) cervical cancer cells.

The aims of the project were to use the known stemofoline-type alkaloid, 11(Z)-1',2'-didehydrostemofoline, as a template to prepare rare Stemona alkloids and their analogues for testing as AChE inhibitors and structure-activity relationship (SAR) studies and to test their abilities to increase the sensitivity of anticancer drugs to MDR cancer cells.

11(Z)-1',2'-Didehydrostemofoline was isolated in grams quantities from the root extracts of unknown Stemona sp. which had been collected in Amphur Mae Moh, Lampang, Thailand.

From this starting material, four Stemona alkaloids, included stemoburkilline, oxystemofoline, methoxystemofoline and (1'R)-hydroxystemofoline, were prepared along with many analogues that incorporated hydroxyl and amino groups at the C-3 side chain position on the stemofoline back bone. The semi-synthesis of stemoburkilline was reported in Chapter 3. Using 11(Z)-1',2'-didehydrostemofoline as the starting material, stemoburkilline was prepared in four steps, these included the hydrogenation of the side chain alkene to produce stemofoline. Hydrogenation of the C-11-C-12 alkene moiety of stemofoline followed by a base catalysed ringopening reaction of 11,12-dihydrostemofoline in the presence of TMSCl to give the TMS protected version of stemoburkilline. Mild TMS deprotection then gave stemoburkilline. The ring-opening process was proposed to occur through an E1cB mechanism. The NMR spectroscopic data of the synthesised stemoburkilline, which indicated the formation of the Z-isomer, was identical to those of the natural product. This study led to the revision of the stemoburkilline structure from an E-isomer to a Z-isomer.

Chapter 4 of this thesis reports the preparation of the key aldehyde intermediate which was used to prepare several stemofoline derivatives that contained a hydroxyl group in the C-3 butyl side chain. These included the natural products, oxystemofoline, methoxystemofoline and (1'R)-hydroxystemofoline. The key aldehyde was prepared in two steps from 11(Z)-1',2'-didehydrostemofoline via an asymmetric dihydroxylation reaction of the C-3 1-butenyl side chain and then oxidative cleavage of the subsequence diol. A modified Julia olefination reaction on the aldehyde was employed as a key step in the preparation of oxystemofoline and methoxystemofoline, using sulfone reagents which had 4-hydroxybutyl and 4- methoxybutyl side chains, respectively. The synthesis of oxystemofoline and methoxystemofoline allowed reassignment of the 13C NMR signals for C-6 and C-1′ from those reported for the natural products. Allylation of the aldehyde under indium-mediated conditions or using chiral allylborane reagents provided (1′R) and (1′S)-homoallylic alcohol products that lead to (1′R)-hydroxystemofoline and its (S)- epimer, respectively. Surprisingly, the synthesised (1′R)-hydroxystemofoline proved to be identical to a natural product that was later isolated from the root extracts of Stemona aphylla. The Wittig reaction of the aldehyde with (triphenylphosphoranylidene)acetaldehyde provided a mixture of three aldehyde products formed from consecutive Wittig reactions. These aldehyde products were later reduced to three different alcohols, included the enol, the dienol and the trienol. The A, B, C ring core structure of stemofoline was also provided in two steps via an Upjohn dihydroxylation reaction of stemofoline followed by an oxidative cleavage of the corresponding C-11, C-12 diol. The oxidation reaction of 11(Z)-1',2'- didehydrostemofoline using Na2WO4·2H2O as a catalyst provided its N-oxide product in a shorter reaction time than the formerly reported method.

In Chapter 5 of this thesis, the reductive amination reactions of the aldehyde were reported to prepare 17 secondary amine derivatives in yields ranging from 25- 93%. Some secondary amines were used in further methylation and carbamylation reactions to prepare tertiary amine and carbamate derivatives, respectively. A guanidine derivative was also prepared as its HCl salt.

In Chapter 6 of this thesis, an examination of CuI-catalysed click reactions was studied around a C-3 ethyne stemofoline analogue. The alkyne was prepared in a one step reaction from the key aldehyde and the Bestmann-Ohira reagent. Four triazoles were prepared using two different methods, a classical method using azides and a microwave-assisted method in which the azides were generated in situ. While two isoxazoles were prepared using chlorooximes as precursors to the corresponding nitrile oxides. The Sonogashira coupling of the alkyne gave the phenylalkyne product and the alkyne dimer which was more efficiently and directly prepared under Eglington coupling conditions. A hydrogenation reaction of the phenylalkyne product provided a compound with a flexible C-3 2-phenylethyl side chain.

In Chapter 7 the AChE inhibitory activities of the synthesised stemofoline alkaloids and analogues are reported using a TLC bioautographic method which measured the activity as a minimum inhibitory requirement (MIR) in ng or nmol against electric eel AChE (eeAChE). Galanthamine was used as a positive control having a MIR of 1 ng (0.003 nmol). The four Stemona alkaloids synthesised from this study, stemoburkilline, oxystemofoline, methoxystemofoline and (1′R)- hydroxystemofoline, showed MIRs of 50, 50, 50 and 5 ng, respectively. (1′R)- Hydroxystemofoline also showed the highest activity among the alcohol derivatives. Compared to 11(Z)-1',2'-didehydrostemofoline (MIR = 5 ng), its N-oxide had similar activity (MIR = 5 ng) while that of stemofoline, which lacked the side chain alkene functionality, showed less activity with a MIR of 10 ng. Lacking the lactone ring of the stemofoline, the A, B, C ring core structure derivative was 10 times less active than stemofoline (MIR = 100 ng). The cyclopentyl amino carbamate, the dimethylamine and the alkyne derivatives had the highest activities in the group with MIR values of 1 ng. In general it was found that most of the amine derivatives were more active than the alcohol derivatives. While the click products showed moderate activities in the range of 50-100 ng except for the benzyl triazole derivative which had a higher activity with a MIR value of 5 ng. Compared to the alkyne (MIR = 1 ng), the phenylalkyne and the alkyne dimer were less active (MIRs = 50 and 100 ng, respectively). However, the compound with a flexible 2-phenylethyl side chain was 10 times more active than the phenylalkyne with a MIR of 5 ng. Some compounds were tested for their IC50 values against eeAChE and human AChE (hAChE) using a colorimetric assay (known as Ellman’s method). 11(Z)-1',2'-Didehydrostemofoline and an isopropylamine analogue showed good activities against eeAChE (IC50 values = 19.2 and 12.9µM, respectively) and hAChE (IC50 values = 25.0 and 19.9µM, respectively) but were not as potent as galanthamine (IC50 values = 0.9 and 0.6µM for eeAChE and hAChE, respectively). While other Stemona derivatives showed lower activities against eeAChE and hAChE with IC50 values in the range of 52.5- 302.3 µM and 28.7-52.4 µM, respectively. The MDR-reversing properties of some Stemona compounds were performed using the colorimetric MTT assay. Among the tested compounds, stemofoline showed the highest modulating effect on resistant KB-V1 cells by decreasing the IC50 of paclitaxel from 10.06±1.56 µM to 1.4±0.45 µM and that of vinblastine from 0.61±0.05 µM to 0.09±0.01 µM. Stemofoline had the highest modulating effect on the resistant KB-V1 cells.

In Chapter 8 of this thesis, SAR studies are described using pharmacophore generation and molecular docking. The best seven different pharmacophore models were generated in order to search for the binding mode of the Stemona compounds. Unfortunately, based on the results from pharmacophore mapping alone we could not confirm the exact binding site. Thus, protein-ligand docking was performed using three different AChEs. The results from molecular docking suggested that the Stemona compounds were more likely to fit vertically in the active-site gorge of AChEs and bind between the active site and the PAS of AChEs. These computational studies showed that Stemona compounds may inhibit AChEs by allosterically binding at the PAS and blocking acetylcholine from reaching the active site.