Arresting cell growth with novel functionalised indolocarbazoles

Cancer causes about 13% of all human deaths and at least one fifth of all deaths in Europe and North America. Although chemotherapy is increasingly prescribed, it is not without side effects and so new, more selective remedies for cancer sufferers must be found. Since the discovery of the anticancer properties of the indolocarbazole staurosporine, many analogues have been synthesised in order to obtain compounds that have a higher potency with respect to anticancer mechanisms. The overall objective of this project is to produce selective and highly potent novel anticancer agents through modification of the indolocarbazole structure and a focus of this work is the replacement of the lactam/maleimide heretocycle to form a series of novel indolocarbazole derivatives including the first reported synthesis of a series of novel substituted indolocarbazole uracils. Biological evaluation via the NCI 60 cell line screen has been completed for a number of these compounds with some showing significant selectivity towards individual leukaemia and melanoma cell lines.


Cancer and Chemotherapy
 Over 3.2 million people in Europe diagnosed with cancer on annual basis.  Cumulative lifetime risk of invasive cancer in Ireland is approximately 1 in 3 for men and 1 in 4 for women.  Greater need than ever to pursue targeted cancer therapies via novel drug templates.  Indolo[2,3-a]carbazole (ICZ) pharmacophore has been a major focus to medicinal chemists for over 30 years.  Staurosporine (STA) first ICZ to be isolated from a natural source; reported by Omura et al. in 1977. 1  Subsequently shown to be an extraordinarily potent inhibitor of PKC (IC 50 = 2.7 nM) and strongly cytotoxic against cancer cells. 2 1 Omura, S. et al., J. Antibiot., 1977, 30, 275 2 Tamaoki, T. et al., Biochem. Biophys. Res. Commun., 1986, 135, 397 STA in complex with CDK2  One of the largest families of proteins in humans, deregulation of protein kinases has been implicated in oncogenesis and the progression of tumours.  Oncogenic kinases continuously activate signalling pathways that regulate cell cycle progression, proliferation and cell survival.  STA found to be a nonselective inhibitor of many different kinases, such as PKA (IC 50 = 15 nM), phosphorylase kinase (IC 50 = 3 nM) and S6 kinase (IC 50 = 5 nM). 3  Crystal structures resolved for STA in complex with cyclin-dependent kinase 2 (CDK2) and PKA proved inhibition occurs in an ATP-competitive manner. 4,5  Although ATP-binding pocket is relatively conserved across pan-kinase domain, exploitation of discreet differences in active site residues and conformations can help to confer selectivity.  Rebeccamycin (REB), an ICZ with one N-glycosidic bond, was isolated in 1985 from Nocardia aerocolonigenes. 6  REB displayed considerable activity against leukemia and melanoma in mice, and inhibited the growth of A549 human lung adenocarcinoma cells, producing single strand breaks in the DNA of these cells. 7  Potent anticancer action was linked to its inhibition of topoisomerase I (topo I).
6 Clardy, J. et al., Tet. Lett., 1985, 26, 4011 7 Tomita, K. et al., J. Antibiot., 1987, 40, 668 Bisindolylmaleimides: potent ICZ precursors  Bisindolylmaleimides (BIMs) are frequently utilised as synthetic precursors to ICZs, with numerous coupling methods employed to achieve final aromatisation step.  Also found to possess uniquely potent biological activity, and a number of candidates are under consideration for the treatment of diseases such as non-small cell lung cancer, glioblastoma and diabetic peripheral retinopathy.
 A common F-ring motif in reported biologically active indolocarbazoles is the lactam/maleimide. We seek to alter the H-bonding framework to isolate new targets.
 Our work to date has focussed on utilising several novel 5and 6-membered heterocycles (X-Y-Z) to replace this ring with unique biological profiles.
 Literature suggests that there is significant scope to modify the indolocarbazole template and maintain biological activity but imbue differentiation of mode of action.  One area that has been relatively overlooked has been the F-ring and this is the focus of our current work.

M -Messenger I -Inhibitor
Kinase M M Staurosporine Modification I

Aims and objectives
• The primary aim of our program of diversity-oriented synthesis is to explore the paradigm of F-ring modulation in novel indolocarbazoles and azaindolocarbazoles. • It is envisaged that such modification can help to confer more favourable pharmacological properties and potentially increase bioavailability • Evaluation undertaken by assessment of cell growth and consequently the influence of these novel templates in the topo I-DNA complex and the exploitation of discrete differences in the kinase active site. • Initial evaluation of antiproliferative activity is followed by further investigation of discrete biological mechanism of action.  Cyclocondensation of β-keto ester to novel F-rings  Use of hydroxylamine as nucleophile yields isoxazolones which can again be functionalised further by simple alkylation.
 Guanidine is again also successful in 6-membered ring formation.

Biological evaluation of novel indolocarbazoles
Bioactivity Paradigm

NCI 60 Screen
In-house TOPO testing Kinase Screen  Biological evaluation follows a predetermined programme beginning with cellular antiproliferative activity as measured at the NCI 60 cell line screen.
 Active compounds are then profiled for Topoisomerase I and II inhibition.