Bastadins and Related Compounds from the Marine Sponges Ianthella basta and Callyspongia sp:

Structure Elucidation and Biological Activities

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Sponges are soft bodied and sessile animals, which lack spine and shell (Armstrong & Quigley 1999). For their defence against fouling organisms, predators, and neighbours competing for space, sponges rely on bioactive natural products instead (Proksch et al. 2002). In this project the marine sponge Ianthella basta Oken, 1815, belonging to the order Verongida Bergquist, 1978, was investigated. Species of the order Verongida are biochemically characterised by the production of brominated tyrosine derivatives. (Bergquist & Cook 2002) Several bastadins and a brominated phenyl acetic acid were
isolated. One other sponge was investigated too, Callyspongia “blue”, which yielded a brominated diphenyl ether (compound 5). Bastadins are characteristic secondary metabolites of the sponge I. basta. They are tyrosine-derived, brominated, oxime-bearing macro bis-diaryl ether tetrapeptides, which can be either cyclic or linear. 36 bastadin and hemibastadin derivatives are
known; additional to these one new congener named bastadin-24 was isolated during this work. The bastadins do not only inhibit barnacle larval settlement, thrombocyte aggregation, display cytotxicity, inhibit bacterial growth as well as protein kinases, as described in this thesis, but do also show for example antagonistic effects on the ryanodin receptor and calcium channels in the endoplasmatic reticulum (Mack et al. 1994, Chen et al. 1999)
In this project ten bastadins (bastadin-2, 3, -4, -6, -7, -9, -10, -11, -16, -24), one
hemibastadin (hemibastadin-1), one phenyl acetic acid and one brominated diphenyl ether were isolated and elucidated. In addition to these three hemibastadin-1 congeners, were synthesized (L-tyrosine-tyramide, norbromohemibastadin-1 and 6,6’ dibromohemibastadin-1) for SAR studies.
All compounds (isolated and synthesized) were tested in following assays: i) barnacle antifouling assay, ii) human thrombocyte aggregation, iii) cytotoxicity, iv) antibiotic activity, and v) inhibition of different protein kinases.
Biofouling causes significant economical and environmental losses worldwide through reducing boat speeds and increasing fuel consumption (Wahl 1989). Until today organotin, copper oxide, and herbicide coatings have been commercially used for preventing on-growth. These coatings were, however recently banned IMO with a complete prohibition by 2008 (IMO 2001). Barnacles are severe macro-fouling organisms. Attempts are now being made to understand the physiology of barnacle larvae, their settlement behaviour, and their ability to metamorphose (Fusetani 2004, Sjögren et al. 2004a, Dahlström et al. 2005).
This is important not only for understanding ecological processes,
but also for finding environmentally sound non-toxic antifouling agents.
As sponges are filter feeders, over-growth by fouling organisms would be devastating for their survival as inhaling pores would likely be clogged. A reliable chemical defence is therefore decisive for these organisms. And they therefore have become prime candidates in the search for antifouling entities.
The bastadins isolated here were screened in the barnacle assay together with the synthesized hemibastadin analogues as well as other previously isolated brominated compounds of sponge origin. All bastadins, hemibastadin-1, norbromohemibastadin-1, 2,2’-dibromo-hemibastandin-1, and psammaplin A, turned out to be active against barnacle cyprid settlement. Bastadin-16, psammaplin A, hemibastadin-1, and norbromohemibastadin-1 also showed to be toxic against the larvae. The conclusion was drawn that an oxime group is decisive for the antifouling activity in these compounds. In contrast to the lipophilic psammaplin A, the hydrophilic sulphated psammaplin A derivative showed no antifouling activity. It was therefore hypothesized that the compound needed to cross membranes and that the target for psammaplin A lied intracellularly. The brominated diphenyl ether (also called compound 5) was active without any toxicity in the barnacle antifouling assay. This compound was taken into screening studies together with other diphenyl ethers against bacterial growth, diatom activity, and mollusc settlement. Compound 5 turned out to be the most active substance in all of the three assays. The other active brominated diphenyl ethers did all carry four bromines, just like compound 5. The non-brominated diphenyl ether did not display any activity at all.
The knowledge that oxime bearing compounds possess thrombocyte anti-aggreagtion properties took the bastadins to be tested against human thrombocyte aggreagation with various results. Bastadin-9 and -16 showed the most pronounced activites followed by norbromohemibastadin-1 as well as bastadin-6 and -7. Neither of these inhibited the thromboxane formation to an effeicient extent. L-tyrosine-tyramide did not show any activity and the activity of the other hemibastadin-1 analogues decreased with increasing amounts of bromines. Bastadin-16 and norbromohemibastadin-1 were taken further to studies of the mechanism of action and tested against thrombocyte aggregation stimulated with collagen, ADP, collagen+SC 560 (COX-1 antagonist), and the thromboxane receptor (TP) agonist U 46619. The results pointed in the direction of a TP antagonistic effect. Bastadin-16 and norbromohemibastadin-1 as well
as the inactive bastadins 3 and 11 were tested in a TP displacement assay. Interestingly all the tested compounds turned out to be active against TP. Further tests remain to be done to evaluate why some bastadin-3 and -11 do not inhibit the aggregation but indeed TP. Binding to the plasma proteins may be a reason for this observation. All compounds were tested for their cytotoxicity against the mouse lymphoma, L5178Y. Active bastadins were bastadin-2, -4, -6, -11, and -16. Compound 5 was the most active substance and interestingly the hemibastadin-1 analogue L-tyrosinetyramide, was the only synthezised derivative displaying activity. This compound was not active in any other assays. It is difficult to draw any SAR conclusions to the cytotoxicity data, but obviously the bromination does not play a major role. The compounds tested for their protein kinase inhibition were bastadin-3, -6, -7, -11, -16, and -24 as well as the full set of synthesized hemibastadin-1 analogues. Bastadin-3 exhibited the strongest inhibition against most protein kinases followed by bastadin-24. The least active bastadins turned out to be bastadin-6 and -16, which only inhibited
a few protein kinases. Bastadin-7 showed a broader inhibitory effect than these, but not as strong as bastadin-3 and bastadin-24. None of the successfully synthesized hemibastadin-1 analogues were active. A linear structure seems to advocate a protein kinase inhibition. None of the compounds isolated or synthesized in this project showed any activity against S.epidermidis biofilm formation. However, activity against several bacterial stains including S. epidermidis, S. aureus, and E. faecium was shown by bastadin-3
and -4 as well as hemibastadin-1 and compound 5. Activity against L. major and T. brucei was observed for compound 5. Bastadin-4 was also active against T. brucei. As stated in the beginning of this thesis, sponge-derived metabolites possess a great vary of biological activities. This is probably because of their defensive function in their original environment. This defence mechanism can also be seen by the antifouling activity displayed by the bastadins. The novel bastadin activities found here may contribute to the understanding of the mode of action of these entities as well as increase the comprehension of the biological and pharmacological systems as such.