Chapter 2 Ascididemin
The pyridoacridones are a naturally occurring class of alkaloids typically isolated from marine sources. The class is characterised by a tricyclic core (Figure 2.1) typically fused to one or more additional ring systems.
This class of alkaloids are known for their cytotoxicity with many showing strong cytotoxic activity in vitro. This activity is generally thought to be a consequence of their potential as DNA intercalators, suggested by their planar core structure. Due to this cytotoxic nature, extensive studies on the antitumor activities of pyridoacridones have been reported.48-50 In addition, various other biological activities have been found for members of this class of alkaloid including antibacterial, antifungal and antiviral properties.49
Amphimedine (2.1), the first reported pyridoacridone,51 was isolated from an Amphimedon sp. sea sponge. It has been reported to have cytotoxicity against p388 murine leukemia cell lines at 0.3-0.4 mg mL-1.52 This level of cytotoxicity, however, has been reported not to be useful for the development of anti-cancer agents.53
Sampangine (2.2), isolated from the plant Cananga odorata,54 is one of the simplest pyridoacridones of this class, and is reported to posess potent anti-microbial activity against Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus and Mycobacterium intracellulare.55 The marine alkaloid meridine (2.3) has been shown to have considerable antifungal activity.56
Kuanoniamine A (2.4), isolated from a marine tunicate,57 is an example of a sulfur containing pyridoacridone alkaloid which exhibits potent cytotoxicity with an IC50 of 0.03 mM against the human colon tumour cell line HCT-ll6.58 The structurally related marine alkaloid ascididemin (2.5) also exhibits numerous biological activities and will be discussed in greater detail in the following sections
Ascididemin: Discovery and biological activity
In 1987 Schmitz et al. reported the structure of 2-bromoleptoclinidinone 2.6 from an ascidian tentatively identified as Leptoclinides species.59 The alkaloid was shown to have mild cytotoxic properties with an ED50 towards the PS lymphocytic leukemia cell line of 0.4 mg mL-1. The fused pentacyclic structure was considered a very rare motif for marine organisms with only two other examples reported at that time.
In 1988 the marine alkaloid ascididemin 2.5 was isolated by Kobayashi et al.,32 from the Okinawan tunicate Didemnum sp., and found to be cytotoxic with an IC50 of 0.39 mg mL-1 against L1210 murine leukemia cells in vitro. The reported structure prompted a structural revision of 2-bromoleptoclinidinone to 2-bromoascididemin 2.7.60 This structure had originally been discounted due to the apparent lack of Fe (II) chelation expected with nitrogen atoms at the 7- and 8- positions. 1,10-Phenanthroline changes colour to deep red upon the addition of Fe(II) whereas 2-bromoascididemin did not give a visible colour change and thus was believed to be non-chelating towards Fe(II).60 The lack of a visible colour change is presumably due to the extended conjugation of the system which would lead to a non-visible (UV) change in absorbance on chelation.
A third member of the ascididemin series, 11-hydroxyascididemin 2.8, isolated from Leptoclinides sp.61,62 was reported in 1990. This molecule was also found to exhibit cytotoxicity towards cultures of murine leukemia cells (P388) with an IC50 of 0.3-0.4 mg mL-1, later seperately reported as 2.3 mM.63 In 1994 a meridine-like alkaloid, cystodamine, was reported but the structure was subsequently revised to 11-hydroxyascididemin.64,65
Ascididemin 2.5 exhibits a range of different biological activities, including the ability to inhibit the DNA processing enzyme topoisomerase II at a concentration of 30 mM.62 In 1995 Copp et al. reported additional cytotoxicity data and antimicrobial activities for ascididemin as part of a preliminary structure-activity study.66 Cytotoxicity towards mouse leukemia (P388), human colon (HCT-116) and human breast (MCF7) tumour cell lines was reported with an IC50 of 0.4, 0.3 and 0.3 mM, respectively. Selective cytotoxicity towards the DNA double strand break repair deficient Chinese Hamster Ovary (CHO) cell line xrs-6 versus the repair competent wild type CHO cell line BR1 was reported.66 Toxicity towards non-malignant African Green Monkey kidney cell-line BSC-1, bacteria Bacillus subtilis (Gram-positive) and Eschericha coli (Gram-negative) and fungi Candida albicans and Cladisporium resinae has also been reported.66 In 1995 Duncan et al. reported ascididemin as an example of a hit, yielded by a newly developed high-throughput antimycobacterial screen, with an IC90 of 0.21 mg mL-1 and a MIC of 0.25 mg mL-1.67 Further to this, additional biological data has been progressively reported: a lack of antiviral potential,58,63 antimicrobial activity,63,68 antiparasitic activity,49,69 selective topoisomerase II inhibitory activity,70 anti-tumour activity,32,48,58,68,71-75 telomerase inhibitory76 and antitubercular activity.33,77 By way of comparison, 2-bromoascididemin 2.7 has been shown to have antitumour activity with a similar profile to ascididemin,48 and antiparasitic activity,69 but no data is available for other biological activities. Similarly, 11-hydroxyascididemin showed antitumour activity but with a significantly reduced (~10 fold) potency compared to the other two natural products.48 In contrast to ascididemin, 11-hydroxyascididemin exhibits moderate activity against both DNA (Herpes simplex (ATCC VR 733)) and RNA (Polio virus 1 (Pfizer vaccine strain) viruses but no protective activity against HIV.63
Several studies have explored the mechanism of action of ascididemin. In addition to cytotoxicity and topoisomerase II inhibition, ascididemin was shown to have the ability to induce reductive DNA cleavage.78 Dirsch et al. demonstrated ascididemin causes mitrochondrial dysfunction leading to apoptosis.79 DNA microarray data has shown that ascididemin has antimycobacterial activity that is partially dependant on iron scavenging but also has additional activity not present in other iron scavenging molecules.77 More recently reports of ascididemin as a chemical defence for the ascidian Cystodytes sp. have been published.80-82 López-Legentil et al. have shown that ascididemin deters predation of the ascidian. The addition of sulfuric acid eliminated the deterrent activity, which was believed to be due to protonation of the alkaloid
Syntheses of pyridoacridone ascididemin
In 1989 Bracher reported the first synthesis of ascididemin.83 His method proceeded by the oxidative amination of quinoline-5,8-quinone with o-aminoacetophenone in the presence of cerium trichloride and air (Scheme 2.1). The diaryl amine adduct was cyclised with concentrated sulphuric acid in glacial acetic acid to give rings tetracyle 2.9. Ring E was furnished via enamine formation with dimethylformamide diethyl acetal followed by reflux with ammonium chloride in acetic acid to give ascididemin (2.5).
A second route was reported in 1990.84,85 This route (Scheme 2.2) starts with rings A, B and E from either of two different starting materials. Introduction of ring D was achieved by a novel aza Wadsworth-Emmons reaction [Scheme 2.2, step (i)] however, due to low yields an alternative route was favoured. This second approach starts with epoxide ring opening followed by oxidation of the resultant alcohol (Scheme 2.2, steps (ii) and (iii)). Finally photocyclisation was used to close ring C and give ascididemin.
In 1993, a biomimetic synthesis of ascididemin was reported (Scheme 2.3).86 As with Bracher’s methodology, this method used oxidative amination of quinoline-5,8-quinone in the presence of cerium trichloride and air, but this time using a more functionalised amine. The resultant adduct could be converted to ascididemin in two steps; an annulation using base or acid to form two rings, followed by oxidation with air.
In 2000 a significantly different synthesis of ascididemin was reported.71 This method started with the introduction of a triflate group followed by palladium cross-coupling with a protected acetylene group. In turn the acetylene group was deprotected and annulated to form ring E. Subsequent demethylation and bromination produced a regiospecific dienophile which finally underwent hetero Diels-Alder cycloaddition to give ascididemin 2.5 (Scheme 2.4).
Copp et al. reported an alternative annulation of tetracycle 2.9, generated via Bracher’s methodology, utilising paraformaldehyde (Scheme 2.5).58 This method provides for the facile introduction of substituents at the six position via the use of alternative aldehydes.
Each of the described synthetic approaches to ascididemin offers the ability to introduce different R groups at different positions of the molecule. For analogues varying at the 6-position, the last approach (Scheme 2.5) offers the greatest flexibility and simplicity
Previous SAR studies of ascididemin
Several studies on the structure-activity of ascididemin have been reported. These studies have primarily focused on the antitumour activity of this class of compounds, with some studies investigating antimicrobial, and more recently, antimycobacterial activity.
In 1995 Copp et al. reported numerous assay results for ascididemin, 8-deaza-ascididemin 2.11 and their synthetic precursors 2.9 and 2.10.
The synthetic precursors had little of the biological activity exhibited by ascididemin which indicated the importance of the E ring to the observed biological activity. 8-Deaza-ascididemin (2.11) exhibited reduced cytotoxicity compared to ascididemin with a four fold decrease in activity towards the mouse leukemia tumour cell line (P388) and a 1000+ fold drop in activity against human colon (HCT116) and human breast (MCF7) tumour cell lines. This result indicated the presence of multiple mechanisms of action in mammalian cell lines. The antimicrobial activity was found to be modulated by the presence or absence of N-8 with some antimicrobial activities requiring the presence of the nitrogen and others requiring the absence.66
These results were expanded upon with a report of a more expansive library of analogues.68 This library covered synthetic precursors, simplified structures (one or more of the rings removed) and six-substituted ascididemin analogues. The biology reported covered antiviral, antimicrobial and antitumour activity. Analogues substituted at the six position possessed reduced cytotoxicity and antimicrobial activity
Chapter One Introduction
1.1 Third world disease
Chapter Two Ascididemin
2.2 Results and Discussion
2.4 Future studies
Chapter Three 2-Pyranones
3.2 Results and discussion
3.4 Future Work
Chapter Four Experimental
4.1 General laboratory procedures
4.2 Experimental details for Chapter 2
4.3 Experimental details for Chapter 3
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