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 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.

Anti-AIDS agents 81. Design, synthesis, and structure-activity relationship study of betulinic acid and moronic acid derivatives as potent HIV maturation inhibitors.

In our continuing study of triterpene derivatives as potent anti-HIV agents, different C-3 conformationally restricted betulinic acid (BA, 1) derivatives were designed and synthesized in order to explore the conformational space of the C-3 pharmacophore. 3-O-Monomethylsuccinyl-betulinic acid (MSB) analogues were also designed to better understand the contribution of the C-3' dimethyl group of bevirimat (2), the first-in-class HIV maturation inhibitor, which is currently in phase IIb clinical trials. In addition, another triterpene skeleton, moronic acid (MA, 3), was also employed to study the influence of the backbone and the C-3 modification toward the anti-HIV activity of this compound class. This study enabled us to better understand the structure-activity relationships (SAR) of triterpene-derived anti-HIV agents and led to the design and synthesis of compound 12 (EC(50): 0.0006 microM), which displayed slightly better activity than 2 as a HIV-1 maturation inhibitor.

Changing epidemiology of serious bacterial infections in febrile infants without localizing signs.

OBJECTIVE: Historically, management of infants with fever without localizing signs (FWLS) has generated much controversy, with attempts to risk stratify based on several criteria. Advances in medical practice may have altered the epidemiology of serious bacterial infections (SBIs) in this population. We conducted this study to test the hypothesis that the rate of SBIs in this patient population has changed over time. PATIENTS AND METHODS: We performed a retrospective review of all infants meeting FWLS criteria at our institution from 1997-2006. We examined all clinical and outcome data and performed statistical analysis of SBI rates and ampicillin resistance rates. RESULTS: 668 infants met criteria for FWLS. The overall rate of SBIs was 10.8%, with a significant increase from 2002-2006 (52/361, 14.4%) compared to 1997-2001 (20/307, 6.5%) (p = 0.001). This increase was driven by an increase in E. coli urinary tract infections (UTI), particularly in older infants (31-90 days). CONCLUSIONS: We observed a significant increase in E. coli UTI among FWLS infants with high rates of ampicillin resistance. The reasons are likely to be multifactorial, but the results themselves emphasize the need to examine urine in all febrile infants <90 days and consider local resistance patterns when choosing empiric antibiotics.

The inoculum effect and band-pass bacterial response to periodic antibiotic treatment.

The inoculum effect (IE) refers to the decreasing efficacy of an antibiotic with increasing bacterial density. It represents a unique strategy of antibiotic tolerance and it can complicate design of effective antibiotic treatment of bacterial infections. To gain insight into this phenomenon, we have analyzed responses of a lab strain of Escherichia coli to antibiotics that target the ribosome. We show that the IE can be explained by bistable inhibition of bacterial growth. A critical requirement for this bistability is sufficiently fast degradation of ribosomes, which can result from antibiotic-induced heat-shock response. Furthermore, antibiotics that elicit the IE can lead to 'band-pass' response of bacterial growth to periodic antibiotic treatment: the treatment efficacy drastically diminishes at intermediate frequencies of treatment. Our proposed mechanism for the IE may be generally applicable to other bacterial species treated with antibiotics targeting the ribosomes.

Bacteriophages as anti-infective agents: recent developments and regulatory challenges.

The biennial meeting on 'Exploiting Bacteriophages for Bioscience, Biotechnology and Medicine', held in London, UK, on 20 January 2012, and chaired by George Salmond (University of Cambridge, UK) hosted over 50 participants representing 13 countries. The highly multidisciplinary meeting covered a diverse range of topics, reflecting the current expansion of interest in this field, including the use of bacteriophages as the source of biochemical reagents for molecular biology, bacteriophages for the treatment of human and animal diseases, bacteriophage-based diagnostics and therapeutic delivery technologies and necessity for, and regulatory challenges associated with, robust clinical trials of phage-based therapeutics. This report focuses on a number of presentations from the meeting relating to cutting-edge research on bacteriophages as anti-infective agents.

IQ-motif peptides as novel anti-microbial agents

The IQ-motif is an amphipathic, often positively charged, a-helical, calmodulin binding sequence found in a number of eukaryote signalling, transport and cytoskeletal proteins. They share common biophysical characteristics with established, cationic a-helical antimicrobial peptides, such as the human cathelicidin LL-37. Therefore, we tested eight peptides encoding the sequences of IQ-motifs derived from the human cytoskeletal scaffolding proteins IQGAP2 and IQGAP3. Some of these peptides were able to inhibit the growth of Escherichia coli and Staphylococcus aureus with minimal inhibitory concentrations (MIC) comparable to LL-37. In addition some IQ-motifs had activity against the fungus Candida albicans. This antimicrobial activity is combined with low haemolytic activity (comparable to, or lower than, that of LL-37). Those IQ-motifs with anti-microbial activity tended to be able to bind to lipopolysaccharide. Some of these were also able to permeabilise the cell membranes of both Gram positive and Gram negative bacteria. These results demonstrate that IQ-motifs are viable lead sequences for the identification and optimisation of novel anti-microbial peptides. Thus, further investigation of the anti-microbial properties of this diverse group of sequences is merited.

NO-loaded Zn2+-exchanged zeolite materials: A potential bifunctional anti-bacterial strategy

Nitric oxide (NO) is important for the regulation of a number of diverse biological processes, including vascular tone, neurotransmission, inflammatory cell responsiveness, defence against invading pathogens and wound healing. Transition metal exchanged zeolites are nanoporous materials with high-capacity storage properties for gases such as NO. The NO stores are liberated upon contact with aqueous environments, thereby making them ideal candidates for use in biological and clinical settings. Here, we demonstrate the NO release capacity and powerful bactericidal properties of a novel NO-storing Zn2+-exchanged zeolite material at a 50 wt.% composition in a polytetrafluoroethylene polymer. Further to our published data showing the anti-thrombotic effects of a similar NO-loaded zeolite, this study demonstrates the antibacterial properties of NO-releasing zeolites against clinically relevant strains of bacteria, namely Gram-negative Pseudomonas aeruginosa and Gram-positive methicillin-sensitive and methicillin-resistant Staphylococcus aureus and Clostridium difficile. Thus our study highlights the potential of NO-loaded zeolites as biocompatible medical device coatings with anti-infective properties. (C) 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.