Oxidation of acyclic alkenes and allyl and benzyl ethers with DIB/t-BuOOH/Mg(OAc)<inf>2</inf>

Oxidation of (11Z)-1′,2′-didehydrostemofoline with DIB/TBHP/Mg(OAc)2·4H2O resulted in oxidative cleavage of the C-11-C-12 double bond instead of the desired allylic oxidation of the 1-butenyl side chain. Stemofoline gave a similar result. The oxidation of more simple terminal alkenes was regioselective and gave vinyl ketones while allyl and benzyl ethers gave acrylate and benzoate esters, respectively. Allyl and benzyl ethers could be chemoselectively oxidized in the presence of a terminal alkene or benzyl group. Oxidation of an internal alkene was poorly regioselective, in contrast to the oxidation of 1-substituted cyclohexenes. © 2011 Elsevier Ltd. All rights reserved

Phytochemical, Synthetic and Biological Studies on Stemona and Stichoneuron Plants and Alkaloids: A Personal Perspective

This report is an overview of our research on phytochemical, synthetic and biological studies of the Stemona and Stichoneuron species of plants

Novel R-roscovitine NO-donor hybrid compounds as potential pro-resolution of inflammation agents

AbstractNeutrophils play a pivotal role in the pathophysiology of multiple human inflammatory diseases. Novel pharmacological strategies which drive neutrophils to undergo programmed cell death (apoptosis) have been shown to facilitate the resolution of inflammation. Both the cyclin-dependent kinase inhibitor (CDKi) R-roscovitine and nitric oxide (NO) have been shown to enhance apoptosis of neutrophils and possess pro-resolution of inflammation properties. In order to search for new multi-target pro-resolution derivatives, here we describe the design, synthesis and investigation of the biological potential of a small series of hybrid compounds obtained by conjugating R-roscovitine with two different NO-donor moieties (compounds 2, 9a, 9c). The synthesized compounds were tested as potential pro-resolution agents, with their ability to promote human neutrophil apoptosis evaluated. Both compound 9a and 9c showed an increased pro-apoptotic activity when compared with either R-roscovitine or structurally related compounds devoid of the ability to release NO (des-NO analogues). Inhibition of either NO-synthase or soluble guanylate cyclase did not affect the induction of apoptosis by the R-roscovitine derivatives, similar to that reported for other classes of NO-donors. In contrast the NO scavenger PTIO prevented the enhanced apoptosis seen with compound 9a over R-roscovitine. These data show that novel compounds such as CDKi–NO-donor hybrids may have additive pro-resolution of inflammation effects

Semisynthesis and acetylcholinesterase inhibitory activity of stemofoline alkaloids and analogues

Semisynthesis of the known Stemona alkaloids oxystemofoline (7) and methoxystemofoline (8) has been achieved starting from (11Z)-1′,2′-didehydrostemofoline (6), which confirmed their structures and absolute configurations. The synthesis of (1′R)-hydroxystemofoline (9) helped establish this compound as a natural product from Stemona aphylla. (1′S)- Hydroxystemofoline (10) and a number of related analogues were also prepared. In a TLC bioautographic assay, 9 was found to be the most active acetylcholinesterase inhibitor, but it was not as active as galanthamine

Semi-synthesis and structure-activity relationship studies of Stemona alkaloids and related analogues

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\u27,2\u27-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\u27,2\u27-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\u27R)-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\u27,2\u27-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\u27R)-hydroxystemofoline. The key aldehyde was prepared in two steps from 11(Z)-1\u27,2\u27-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\u27,2\u27- 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\u27,2\u27-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\u27,2\u27-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

Synthesis of stemofoline analogues as acetylcholinesterase inhibitors

Thirty-two new stemofoline analogues were prepared from didehydrostemofoline for studies as AChE inhibitors. C-3 Side-chain modified amino, carbamate, triazole and oxazole stemofoline derivatives were prepared. In general the amine derivatives were found to be stronger inhibitors of AChE than their alcohol analogues that we previously reported. Compounds 5 and 26, with small C-3 side-chain substituents, were two of the most active inhibitors. Preliminary molecular docking studies suggested that these compounds may inhibit AChE by binding horizontally along the passage of the active-site gorge and block access to acetylcholine. © 2012 Elsevier Ltd. All rights reserved

Anti-inflammatory, anti-bacterial and anti-acetylcholinesterase activities of two isoquinoline alkaloids-scoulerine and cheilanthifoline

Corydalis plants containing isoquinoline alkaloids are reported to possess promising pharmacological properties for the treatment of important diseases including cancer, inflammation, Alzheimer\u27s disease and microbial infections. As part of a wider program investigating Bhutanese medicinal plants, we have previously identified eight isoquinoline alkaloids from C. dubia. Out of these, we report here on two of the major alkaloids, scoulerine (1) and cheilanthifoline (2) and their inhibitory activities against acetylcholinesterase (anti-AChE), tumor necrosis factor alpha (anti TNF-α) and a bacterial strain, Helicobacter pylori. Both alkaloids showed weak anti TNF-α and antibacterial activities. However, the anti-AChE activity of scoulerine (1) was promising as it significantly inhibited AChE with a minimum inhibitory requirement (MIR) value of 0.0015 nmol, which was two-fold better than the reference drug, galanthamine (MIR value of 0.003 nmol). As there are limited anti-Alzheimer\u27s chemotherapeutics, scoulerine (1) is worthy of further exploration, including lead optimization, structure-activity-relationship studies, analog development, pharmacodynamics and in vivo animal studies

Inhibition of P-glycoprotein mediated multidrug resistance by stemofoline derivatives

Resistance to chemotherapy in cancer patients has been correlated to the overexpression of the ATP-binding cassette (ABC) drug transporters including P-glycoprotein (P-gp) that actively efflux chemotherapeutic drugs from cancer cells. We examined the mutidrug resistance reversing property of stemofoline derivatives in drug-resistance human cervical carcinoma (KB-V1) and human leukemic (K562/Adr) cell lines that overexpress P-gp. Didehydrostemofoline and eleven of its derivatives were synthesized and the cytotoxicity and their effect on doxorubicin, vinblastine and paclitaxel sensitivity in drug resistant (KB-V1 and K562/Adr) and drug sensitive (KB-3-1 and K562) cell lines by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay were determined. We found that three out of the twelve stemofoline derivatives including OH-A1, NH-B6 and NH-D6 showed commitment efficiency to increase sensitivity to doxorubicin, vinblastine and paclitaxel in KB-V1 cells and increase sensitivity to doxorubicin, and paclitaxel in K562/Adr cells whereas the effects have not been seen in their parental sensitive cancer cell lines (KB-3-1 and K562). These results indicate that stemofoline derivatives reversed P-gp-mediated multidrug resistance in vitro, and thus could be developed as effective chemosensitizers to treat multidrug-resistant cancers. The molecular mechanism of modulation of P-gp would be further determined