Synthesis and evaluation of novel functionalised indoles as anticancer agents

This thesis outlines the design and application of new routes towards a range of novel bisindolylmaleimide and indolo[2,3-a]carbazole derivatives, and evaluation of their biological effects and their chemotherapeutic potential. A key part of this work focussed on utilising a hydroxymaleimide as a replacement for the prevalent lactam/maleimide functionality and forming a series of novel derivatives through substitution on the indole nitrogens. To achieve this, a robust synthetic strategy was developed which allowed access to key maleic anhydride intermediates using Perkin-type methodology. These hydroxymaleimides were further modified via a Lossen rearrangement to furnish a series of analogues containing a 6-membered F-ring. The theme of F-ring modulation was further expanded through the utilisation of a second route involving the design and synthesis of β-keto ester intermediates, which afforded novel derivatives containing pyrazolone and isocytosine headgroups, and various N-substituents. Work on a further route involving a dione intermediate resulted in the isolation of a bisindolyl derivative with a novel imidazole F-ring. Following the synthesis of 42 novel compounds, extensive screening was undertaken using the NCI-60 cell line screen, with twelve candidates progressing to evaluation via the five dose assay. This led to the identification of several lead compounds with high cytotoxicity and excellent selectivity profiles, which included derivatives with low nanomolar GI50 values against specific cancer cell lines, and also derivatives with selective cytotoxicity. Preliminary results from a kinase screen indicated noteworthy selectivity towards GSK3α/β and PIM1 kinases, with low micromolar IC50 values being observed for these enzymes.

Design, synthesis and development of novel indolocarbazole derivatives as potential anti-cancer agents

This thesis describes work carried out on the design of new routes to a range of bisindolylmaleimide and indolo[2,3-a]carbazole analogs, and investigation of their potential as successful anti-cancer agents. Following initial investigation of classical routes to indolo[2,3-a]pyrrolo[3,4-c]carbazole aglycons, a new strategy employing base-mediated condensation of thiourea and guanidine with a bisindolyl β-ketoester intermediate afforded novel 5,6-bisindolylpyrimidin-4(3H)-ones in moderate yields. Chemical diversity within this H-bonding scaffold was then studied by substitution with a panel of biologically relevant electrophiles, and by reductive desulfurisation. Optimisation of difficult heterogeneous literature conditions for oxidative desulfurisation of thiouracils was also accomplished, enabling a mild route to a novel 5,6-bisindolyluracil pharmacophore to be developed within this work. The oxidative cyclisation of selected acyclic bisindolyl systems to form a new planar class of indolo[2,3-a]pyrimido[5,4-c]carbazoles was also investigated. Successful conditions for this transformation, as well as the limitations currently prevailing for this approach are discussed. Synthesis of 3,4-bisindolyl-5-aminopyrazole as a potential isostere of bisindolylmaleimide agents was encountered, along with a comprehensive derivatisation study, in order to probe the chemical space for potential protein backbone H-bonding interactions. Synthesis of a related 3,4-arylindolyl-5-aminopyrazole series was also undertaken, based on identification of potent kinase inhibition within a closely related heterocyclic template. Following synthesis of approximately 50 novel compounds with a diversity of H-bonding enzyme-interacting potential within these classes, biological studies confirmed that significant topo II inhibition was present for 9 lead compounds, in previously unseen pyrazolo[1,5-a]pyrimidine, indolo[2,3-c]carbazole and branched S,N-disubstituted thiouracil derivative series. NCI-60 cancer cell line growth inhibition data for 6 representative compounds also revealed interesting selectivity differences between each compound class, while a new pyrimido[5,4-c]carbazole agent strongly inhibited cancer cell division at 10 µM, with appreciable cytotoxic activity observed across several tumour types.

Design, synthesis and evaluation of novel ellipticine derivatives and analogues as anti-cancer agents

This thesis describes work carried out on the synthesis of novel 5- and 11-substituted ellipticines and derivatives of the ellipticine analogues, isoellipticine and deazaellipticine, followed by investigation of their potential as anti-cancer agents. Preparation of the key 5- and 11-substituted ellipticine targets involved the development of regiospecific, sequential alkylation reactions with alkenyllithium and Grignard reagents. Investigation of these novel reactions resulted in a new route towards 5-substituted ellipticines via Grignard reaction with vinylmagnesium bromide. These novel 5-vinylellipticine derivatives were further functionalised in an ozonolysis reaction, followed by oxidation to give a range of novel 5-substituted ellipticines. Less success was encountered in the 11-substituted ellipticine series, however preparation of these derivatives using a previously published route was accomplished, and the resulting 11-formylellipticine was further derivatised to give a panel of novel 9- and 11-substituted ellipticines, incorporating amide, carboxylate, imine and amine functionality. The successful route towards 5-substituted ellipticines was applied to the preparation of a range of novel 11-substituted isoellipticines and 6-substituted deazaellipticines, the first time substantial synthesis has been undertaken with these analogues. In addition to this, the first preparation of isoellipticinium salts is described, and a panel of novel isoellipticinium, 7 formylisoellipticinium and 7-hydroxyisoellipticinium salts were synthesised in good yields. Biological evaluation of a panel of 43 novel ellipticine, isoellipticine and deazaellipticine derivatives was accomplished with a topoisomerase II decatenation assay and submission to the NCI 60-cell line screen. Four novel isoellipticine topoisomerase II inhibitors were identified from the decatenation assay, with strong activity at 10 μM. In addition to this, NCI screening identified five highly cytotoxic ellipticine and isoellipticine compounds with remarkable selectivity profiles for different cancer types. These novel lead compounds represent new templates for further research and synthesis.

Synthesis and reactivity of α-diazosulfoxide and α-diazosulfone derivatives

The research described in this thesis involves the synthesis of α-diazo-β-oxo sulfoxides, and exploration of their reactivity. The first chapter includes an introduction to diazocarbonyl chemistry, specifically focusing on the synthesis of diazo compounds, and diazosulfoxide derivatives. The chemistry of sulfines, in particular the generation of α-oxo sulfines and their subsequent trapping as cycloadducts and dimerisation is discussed. The results of this research are discussed in the second and third chapters. The design, synthesis and reactivity of α-diazo-β-oxo sulfoxides is described in chapter 2 where diazo transfer adjacent to sulfoxides to form stable α-diazo-β-oxo sulfoxides has been achieved in cyclic systems. Decomposition of theses α-diazosulfoxides using rhodium carboxylate or carboxamide catalysts is also described. These processes proceed via a Wolff type rearrangement to form α-oxo sulfine intermediates, which were trapped as cycloadducts with dienes. In the absence of a diene trap, dimerisation of the sulfine intermediate was observed. Intramolecular C-H insertion reasctios of α-diazo-α-sulfonyl esters to form substituted sulfolane esters is described in chapter 3. The reactivity of these sulfolane esters is briefly explored. The fourth chapter contains the experimental details and the spectral and analytical data for all new compounds reported.

Synthesis and evaluation of novel azaindolocarbazole derivatives as cancer chemotherapeutics

This thesis details the design and implementation of novel chemical routes towards a series of highly propitious 7-azaindolyl derivatives of the indolocarbazole (ICZ) and bisindolylmaleimide (BIM) families, with subsequent evaluation for use as cancer chemotherapeutic agents. A robust synthetic strategy was devised to allow the introduction of a 7-azaindolyl moiety into our molecular template. This approach allowed access to a wide range of β-keto ester and β-keto nitrile intermediates. Critical analysis identified F-ring modulation as a major theme towards the advancement of ICZ and BIM derivatives in drug therapy. Thus, the employment of cyclocondensation methodology furnished a number of novel aminopyrazole, isoxazolone, pyrazolone and pyrimidinone analogues, considerably widening the scope of the prevalent maleimide functionality. Photochemical cyclisation provided for the first reported aza ICZ containing a six-membered F-ring. Another method towards achieving the aza ICZ core involved use of a Perkin-type condensation approach, with chemical elaboration of the headgroup instigated post-aromatisation. Subsequent use of a modified Lossen rearrangement allowed access to further analogues containing a six-membered F-ring. Extensive screening of the novel aza ICZ and BIM derivatives was carried out against the NCI-60 cancer cell array, with nine prospective candidates selected for continued biological evaluation. From these assays, a number of compounds were shown to inhibit cancer cell growth at concentrations of below 10 nM. Indeed, the most active aza ICZ tested is currently under assessment by the Biological Evaluation Committee of the NCI due to excellent antiproliferative activity demonstrated across the panel of cell lines, with a mean GI50 of 34 nM, a mean total growth inhibition (TGI) of 4.6 μM and a mean cytotoxicity (LC50) of 63.1 μM. Correlation to known topoisomerase I (topo I) inhibitors was revealed by COMPARE analysis, and subsequent topo I-mediated DNA cleavage assays showed inhibitory activity below 1 μM for several derivatives.

Synthesis and evaluation of novel quinolines and quinazolinediones as potential anti-cancer agents

This thesis outlines the design and effectuation of novel chemical routes towards a nascent class of functionalised quinoline-5,8-diones and the expansion of a series of contemporary quinazolinediones towards an innovative family of pyridinoquinazolinetetrone derivatives. This fragment based approach is envisaged to lead to advancements in the three scaffolds, expanding the SAR pool of both quinolines and quinazolinediones with subsequent evaluation of chemotherapeutic potential as well as furnishing a new class of tricycle for biological investigation. Development of novel quinoline-5,8-diones is provided for by expanding on existing methodology. Using a variety of nucleophiles on a critical intermediate, a broad range of novel compounds was afforded allowing chemotherapeutic potential to be assessed, while also serving as intermediates for accomplishing novel pyridinoquinazolinetetrone congeners. In order to incorporate functionality into our quinazolinedione template, an efficient synthetic strategy was constructed which provided a robust route to effectuate a highly derivatised pyrimidinedione ring. As derivatisation of this template is unreported our chief priority was to synthesise a range of diverse quinazolinediones. The application of annulation methodology using functionalised precursors provided a library of N-3 derivatised quinazolinedione analogues. These, along with their N-1 functionalised derivatives provide a wide scope from which to construct a series of pyridinoquinazolinetetrone derivatives while also serving as a unique class of molecules whose biological potential is uncharted. Although the actualisation of the pyridinoquinazolinetetrone was ultimately unsuccessful, our work has led to the development of novel quinoline-5,8-diones which were found to possess excellent anti-cancer activity when assessed by the NCI screen. Of the quinazolinediones synthesised eight compounds were accepted for screening by the NCI. Results from the single-dose tests however indicated that these compounds possessed little cytotoxic activity at 10 μM. The development of this novel template in conjunction with the highly active quinolinediones serves as an excellent rostrum for future synthetic endeavours.

The application of aryne chemistry and use of α-diazo carbonyl derivatives in indazole synthesis with biological evaluation

This thesis outlines the synthetic chemistry involved in the preparation of a range of novel indazole compounds and details the subsequent investigation into their potential as biologically active agents. The synthetic route utilised in this research to form the indazole structure was the [3+2] dipolar cycloaddition of diazo carbonyl compounds with reactive aryne intermediates generated in situ. The preparation of further novel indazole derivatives containing different functional groups and substituents was performed by synthesising alternative 1,3- dipole and dipolarophile analogues and provided additionally diverse compounds. Further derivatisation of the indazole product was made possible by deacylation and alkylation methods. Transformation reactions were performed on alkenecontaining ester side chains to provide novel epoxide, aldehyde and tertiary amine derivatives. The first chapter is a review of the literature beginning with a short overview on the structure, reactivity and common synthetic routes to diazo carbonyl derivatives. More attention is given to the use of diazo compounds as 1,3-dipoles in cycloaddition reactions or where the diazo group is incorporated into the final product. A review of the interesting background, structure and reactivity of aryne intermediates is also presented. In addition, some common syntheses of indazole compounds are presented as well as a brief discussion on the importance of indazole compounds as therapeutic agents. The second chapter discusses the synthetic routes employed towards the synthesis of the range of indazoles. Initially, the syntheses of the diazo carbonyl and aryne precursors are described. Next, the synthetic methods to prepare the indazole compounds are provided followed by discussion on derivatisation of the indazole compounds including N-deacylation, N-benzylation and ester side-chain transformation of some alkene-containing indazoles. A series of novel indazole derivatives were submitted for anti-cancer screening at the U.S National Cancer Institute (NCI). A number of these derivatives were identified as hit compounds, with excellent growth inhibition. The results obtained from biological evaluation from the NCI are provided with further results pending from the Community for Open Antimicrobial Drug Discovery. The third chapter details the full experimental procedures, including spectroscopic and analytical data for all the compounds prepared during this research.