Development of drugs and natural products

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Chapter 3: Pyridoacridines

Introduction: Pyridoacridines

Pyridoacridines are a class of coloured marine natural products that contain an 11H-pyrido[4,3,2-mn]acridine core (3.1). Calliactine (3.2) and amphimedine (3.3) were the first compounds isolated in this class (in 1940 and 1983, respectively) and there are now well over 100 examples reported.68 Pyridoacridines are generally planar in structure and have different side chains and various rings fused to the core structure.69
Many of the reported pyridoacridine alkaloids exhibit cytotoxicity. Inhibition of cell growth has been linked to the ability of the planar alkaloid structures to intercalate into DNA to effect anti-cancer activity.70 The biological activities of pyridoacridines are wide-spread with reports of compounds exhibiting anti-bacterial, anti-fungal, anti-viral, anti-parasitic and insecticidal activities.70
The amphimedine-related compounds are paticularly interesting given that a number of examples related to 3.3 have recently been isolated as well as interesting trends in bioactivities reported (vide infra). Previous access to the amphimedine scaffold by way of total syntheses are generally long and difficult, requiring harsh reaction conditions and aim to synthesise one paticular compound at a time (vide infra). Paticularly appealing is the biomimetic synthesis of the amphimedine alkaloids, as such a method can prove to be a quick and effective way to prepare a library of analogues as well as having the potential for the synthesis of undiscovered natural products. The biosynthetic origins of this subclass of alkaloids has been speculated upon (vide infra), but the field remains unexplored.
As mentioned above, the pyridoacridines represent a large family of compounds where subclasses can be found in relation to various sidechains and fused rings present in the structure. Natural products isolated in 2011-2014 are discussed as well as compounds that are directly related to the amphimedine scaffold. Previous biomimetic synthetic efforts to the pyridoacridine scaffold are highlighted and previous syntheses of relevant pyridoacridines are also discussed.

Recently isolated pyridoacridines

In the years 2011-2014 alone, eight new pyridoacridines were discovered and their biological activities reported. In 2014, Quinn’s group reported the isolation of ancorine A (3.4) and cnemidine A (3.5) from an Australian sponge Ancorina geodides and a tunicate Cnemidocarpa stolonifera, respectively.68 Both compounds were tested against a human prostate cancer line (PC3) and a neonatal foreskin fibroblasts noncancer cell line (NFF). It was observed that 3.4 was weakly cytotoxic (IC50 against PC3, 17 µM) whereas 3.5 was found to be potent with an IC50 of 1.1 µM aginst PC3 cell line. Cnemidine A also exhibited 68% inhibition of NFF cells (at a concentration of 10 µM).68
A study on the bioactive extract of the ascidian Cystodytes violatinctus, collected from the Solomon Islands, led to the isolation of four new natural products, dehydrokuanoniamine D (3.6), shermilamine F (3.7) and arnoamines C (3.8) and D (3.9).71 The arnoamine compounds were the first examples of a pyridoacridine that contained a modified acrylamide sidechain as a substituent on the pyrrole ring. All three compounds were tested against the HCT116, SW480 and A375 cell lines wherein arnoamine D appeared the most active (IC50 of 4.3–8.5 µM) and dehydrokuanoniamine F was selectively active against the SW480 cell line (IC50 3.3 µM).71
Extraction of the purple morph of the ascidian Cystodytes dellechiajei collected in Catalonia yielded two pyridoacridines, 13-didemethylaminocycloshermilamine D (3.10) and demethyldeoxyamphimedine (3.11).72 These compounds were isolated alongside shermilamine, kuanoniamine and styelsamines C (3.12) and D (3.13). The two compounds 3.10 and 3.11 were tested against marine and terrestrial bacterial strains (Listonella anguillarum and Microccocus luteus, respectively) and both compounds exhibited micromolar activity against both strains of bacteria (compounds were less active against the marine strain).72 It was proposed that the biosythetic origin of 3.11 is styelsamine D, whereby Bry et al. reported that stirring of the organic extract of the C. dellechiajei purple morph with formaldehyde (10%) resulted in the increase of the concentration of 3.11 in parallel to the decrease of concentration of styelsamine D (3.13).72

Pyridoacridines and the 8H-pyrido[2,3,4-mn]acridone core.

The majority of pyridoacridines are derivatives of the 8H-pyrido[2,3,4-mn]acridone core (3.14) with amphimedine (3.3) being the first example that contained this subunit.69 The alkaloid was isolated from a Pacific sponge Amphimedon sp. collected at Guam Island.73 Since its isolation, new compounds related to amphimedine have been isolated, namely the aforementioned demethyldeoxyamphimedine (3.11), neoamphimedine (3.15) and deoxyamphimedine (3.16).
The first of the ‘new’ amphimedines to be discovered was neoamphimedine. It was isolated in 1999 from a Xestospongia sp. collected from the Philippines and it was also found in a Xestospongia cf. carbonaria collected from Micronesia.74 Amphimedine (3.3) was also isolated alongside 3.15 and they both underwent quantitative DNA cleavage tests, where it was found that 3.15 stimulates topoisomerase II (TopoII) dependant cleavage of DNA.74 Neoamphimedine was observed to induce TopoII to cause catenation of DNA, suggestive of antineoplastic potential of the compound. Recent studies on the biological activities of neoamphimedine reported it to inhibit Metnase-enhanced cell growth (IC50 of 0.5 µM), where Metnase is known to enhance TopoII activiy and increase resistance to TopoII poisons.75 Li et al. also found that 3.15 displayed potent cytotoxicity (nM IC50 values) in human cancer cells lines (breast, colorectal, lung and leukemia) with significant potency against colorectal cancer (IC50 = 6 nM). From the reported results, the mechanism of cytotoxicity of 3.15 was characterised as G2-M cell cycle arrest and apoptosis.76
A study on another Xestospongia sp. sponge collected from Baler, Philippines, allowed for the isolation of deoxyamphimedine (3.16).77 This compound was reported to exhibit cytotoxic activity against human colon tumor cells (HCT-116, IC50 = 335 nM), Chinese hamster ovary cells, AA8 (wild type, IC50 = 25 µM) and EM9 cells (sensitive to single strand DNA breaking, IC50 = 6 µM).77 The alkaloid also causes damage to DNA in vitro independently of topoisomerase enzymes through the generation of ROS.78
A bioassay directed isolation study on Xestospongia cf. carbonaria collected in Palau identified three new natural products, the hydroxylated analogues of deoxyamphimedine 3.17 and 3.18 as well as debromopetrosamine (3.19).79
Alongside the new compounds, deoxyamphimedine, amphimedine and neoamphimedine were also isolated. A zebrafish phenotypic assay was used to assess bioactivity, where the zebrafish were treated with the compounds listed above. All compounds except for amphimedine induced death of the treated zebrafish.79 Amphimedine (3.3) exhibited a phenotype at 30 µM, and the variety of the phenotypic responses induced by 3.3 suggested that interference was occuring at a fundamental process but no specific target could be pinpointed.79
Another bioassay-directed isolation study on Indo-Pacific collections of Xestospongia cf. carbonaria and cf. exigua led to the isolation of three neoamphimedine-type compounds, 5-methoxy-neoamphimedine (3.20), neoamphimedine Y (3.21) and Z (3.22).80 A completely new acridine was also isolated and was named alpkinidine (3.23) for its similarity to the natural product plakinidine family of compounds (eg. plakinidine A (3.24)). Compounds 3.20 and 3.23 were found to be selective for solid tumors in a disk diffusion soft agar colony assay with both compounds being selective against murine cell lines versus human cell lines.80
Styelsamine D (3.13), which is speculated to be the biosynthetic precursor to deoxyamphimedine and demethyldeoxyamphimedine, was originally isolated with three other compounds, styelsamines A-C from the Indonesian ascidian Eusynsteyla latericius.81 Styelsamines A-D (3.25, 3.26, 3.12, 3.13) exhibited mild cytotoxicity toward the human colon tumor cell line (HCT-116) with IC50 values of 33, 89, 2.6 and 1.6 µM repectively. It was observed that styelsamine A (3.25) would readily transform to the oxidised 3.27 which is analogous to the cystodytin family of compounds.81

General Introduction
1.1. Development of drugs and natural products
1.2. Marine natural products in the drug pipeline
1.3. Cancer
1.4. Biomimetic synthesis of natural products
1.5. Aims of the projects
Chapter 2: Thiaplidiaquinone A and B 
2.1. Introduction: Thiaplidiaquinone A and B
2.2. Natural products containing the 1,1-dioxo-1,4-thiazine structural feature
2.3. Synthetic strategy: Thiaplidiaquinone A and B
2.4. Synthesis of 2-geranyl-1,4-benzoquinone
2.5. Dimerisation and cyclisation of 2-geranyl-1,4-benzoquinone
2.6. Synthesis of thiaplidiaquinone A/B and relevant regioisomers
2.7. UV-Vis analysis of thiaplidiaquinones A and B and their regioisomers
2.8. Biological evaluation of thiaplidiaquinone A/B, analogues and precursors
2.9. Summary and future works
Chapter 3: Pyridoacridines 
3.1. Introduction: Pyridoacridines
3.2. Proposed biomimetic approach to various pyridoacridines
3.3. Previous synthetic strategies to the amphimedines
3.4. Synthesis of styelsamine B and styelsamine D
3.5. Alternative synthetic route to styelsamine D
3.6. Synthesis of demethyldeoxyamphimedine and deoxyamphimedine
3.7. Selective synthesis of deoxyamphimedine
3.8. Oxidation of deoxyamphimedine for the synthesis of amphimedines
3.9. Alternative synthetic route to neoamphimedine
3.10. Synthesis of analogues to the amphimedine natural products
3.11. Synthetic efforts towards the arnoamine scaffold
3.12. Synthetic efforts towards the alpkinidine scaffold
3.13. NCI evaluations of synthesised amphimedines
3.14. Summary of pyridoacridine studies
3.15. Future work
Chapter 4: Experimental Procedures 
4.1. General methods and instrumentation
4.2. Chapter two experimental procedures
4.3. Chapter three experimental procedures
Inspirations from Nature: Biomimetic Synthesis of Bioactive Marine Natural Products

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