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Marine natural products isolation, screening and analogue synthesis

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posted on 2024-11-11, 21:59 authored by Ana Zivanovic
Most drugs used today demonstrate their bioactivity by acting as receptor antagonists or as enzyme inhibitors. Due to the significant role of enzymes in the activity of a number of disease processes, and their structural conformation that is easily accessible to inhibition by small drug like molecules, they are widely investigated as drug targets. The first step in the preparation of an enzyme inhibitor is defining the target enzyme whose function is going to be interrupted or blocked in a particular disease or infection. c-AMP Dependent protein kinase A (PKA) is an important enzyme involved in the regulation of a large number of physiological processes. As such, the inhibition of PKA has become an attractive drug target in a number of areas, in particular in immune function and for memory disorders. Interestingly, despite PKA being one of the most well characterised of all protein kinases, there are as yet no PKA inhibitors reported from marine sources. The natural environment has provided a wealth of chemically diverse, bioactive compounds that are responsible for over half of the medications currently available for a multitude of diseases. Herein, ten algal species from South East Australia assigned as Prionitis linearis, Corallina vancouveriensis, Codium fragile, Codium dimorphum, Colpomenia sinuosa, Padina sp., Phyllospora comosa, Sargassum vestitum, Sargassum sp. and Ulva lactuca were analysed. Extracts of different polarity were generated from each sample and tested for their PKA inhibitory activity, haemolytic activity and brine shrimp cytotoxicity giving novel results on bioactivity from marine algae. There are no literature data so far on the kinase inhibitory activities of extracts isolated from these algal samples and for most of the samples haemolytic data are also reported for the first time. Furthermore, the chemical (fatty acid and sterol) composition of the ten algal extracts were also analysed and compared. The bioactivity profiles of the alga samples were strongly species dependent, with no apparent correlation between PKA inhibition, haemolysis and/or brine shrimp lethality. In general, the non-polar fractions isolated from all alga samples were found to give potent biological results and displayed more activity, in particular greater PKA inhibitory activity, compared with the polar fractions. The biological activity is reflected in the different algal divisions, with the order of PKA inhibitory activity identified as follows: brown seaweeds > red seaweeds > green seaweeds. The main constituents of the non-polar fractions were identified as fatty acids (FAs) and sterols in all algal samples. The FA composition was dominated by saturated FAs, hexadecanoic acid and unsaturated 9,12-octadecadienoic acid were the most predominant acids, while cholesterol was found to be the major sterol of all ten algal species investigated. The deep sea is one of the most biodiverse habitats on the planet and deep-sea sponges are the largest source of new deep-water natural products reported and are found to contain a rich diversity of new and unusual sterols. In this study four deep-sea sponges from North Western Australia assigned to the genera Haliclona, Sarcotragus, Ircinia and Geodia were investigated for their biological activities in a variety of assays (for PKA inhibition, haemolytic activity and brine shrimp toxicity assay). The chemical composition of the extracts of different polarity obtained from all three samples were also determined and compared. Overall the sponge Haliclona sp. displayed greater haemolytic activity than PKA inhibitory activity, along with low levels of brine shrimp lethality with the bioactivity mainly found in the non-polar fractions. The Ircinia/Sarcotragus sponge species exhibited higher PKA inhibitory activity than the activity found in the other two assays, with the two non-polar fractions displaying activity greater than 80%. The trend of the bioactive non-polar fractions continued for the Geodia sponge species where the non-polar extracts showed both potent PKA inhibitory activity (97%) and brine shrimp lethality (88%), with modest haemolytic activity observed for several fractions. Most of the identified compounds in those fractions were fatty acids and sterols and contributed to a partial fatty acid and sterol profile of this deep-sea demosponge. A total of 29 fatty acids and 23 sterols were identified, with the highest proportions found in Ircinia/Sarcotragus sp. > Haliclona sp. > Geodia sp. There was no apparent correlation amongst the sterol composition of the three deep-sea sponges. The sterol profile varied across the sponge species either in type of sterols or in relative percentages. This work, has however uncovered the rich potential of deep-sea sponges as sources of bioactive metabolites. In addition, this study has laid the foundations for the future investigation and assessment of kinase inhibitory potential of metabolites from both shallow and deep water invertebrates. Another aspect of drug discovery, in addition to isolating natural products, is modification of the isolated metabolites from nature and their structural modification towards improved bioactivity. Development of natural products includes modification of functional groups, their removal, introduction of novel groups and stereocenters to the molecule, or occasionally radical changes of the basic scaffold. Although the structural diversity in nature is extraordinary, synthetic chemistry methods can provide novel structure types which cannot be obtained from natural biosynthetic methods. Isatin derivatives were previously investigated in our research group for cytotoxicity against a range of human cell lines and found to display potent activity, causing decreased cell proliferation in all cell lines studied. Previous literature research of isatin-indole compounds has also demonstrated that these scaffolds are also important for kinase inhibitory activity, as evidenced by the large number of indole based kinase inhibitors approved for use and currently in clinical trials. Hence, it was of interest to further develop our isatin-based structures and test them for potential protein kinase A inhibitory activity. The aim was to determine the structural requirements and the position of the different groups on the isatin molecule necessary for PKA inhibitory activity. Herein, the synthesis of 41 novel imino isatin derivatives and 14 alkenyl isatin analogues and evaluation of their cytotoxicity, PKA and PKB inhibitory activity is presented, followed by a detailed analysis of their structure-activity relationships. Of a total of 77 isatin derivatives prepared four isatin derivatives displayed potent PKA inhibitory activity (65-86%) and six displayed potent PKB inhibitory activity (57-84%). Compounds 178, 208, 209 and 246 displayed the highest PKA inhibitory activities and their IC50 values were determined as 43.5, 51.8, 38.5, 52.2 μM respectively (at 100 μg/mL). Four isatin derivatives (175, 176, 177 and 208) were active in both PKA and PKB assay. Three isatins (163, 187, 206) displayed selective activity over PKB than PKA, while two compounds (178, 209) were more selective towards PKA. In addition the newly developed isatin derivatives also exhibited potent cytotoxicity against human leukemic monocyte lymphoma cell lines U937 in the micromolar range. This work suggests that isatin derivatives possess a real potential to be further developed into PKA and PKB inhibitors in the future.

History

Year

2012

Thesis type

  • Doctoral thesis

Faculty/School

School of Chemistry

Language

English

Disclaimer

Unless otherwise indicated, the views expressed in this thesis are those of the author and do not necessarily represent the views of the University of Wollongong.

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