Synthesis and Evaluation of Imidazopyridinones, Pyrrolopyrimidines and Tetrahydrocarbazoles as Antibacterial Agents
Abstract
Mankind is in a continual arms race against bacteria, as we develop new antibiotics whilst the bacteria develop resistance against them. The need to produce novel antibiotics is now dire, as most new commercial antibiotics have the same modes of action as previous generations. Since bacteria rapidly become resistant against these, it is important we develop novel antibiotic compounds with novel modes of actions. The goal of this thesis has been the development of antibiotic compounds. To achieve this, we have synthesized three different compound classes targeting two bacterial proteins; thymidylate monophosphate kinase (TMPK), a key enzyme in DNA synthesis, and the β sliding clamp, a protein in the replisome which increases the rate of DNA replication.
Inspired by an imidazopyridinone with high activity towards P. aeroginosa TMPK, we developed a new synthetic pathway towards a methoxylated analog in 17% yield. The trityl protected key imidazopyridinone was prepared in four steps. The most challenging reaction was the regioselective installation of a methyl group at position 3 on 2,3-diamino-5-bromo-pyridine. This was solved by formation of the corresponding benzyl carbamate at -98 °C, followed by reduction. In the following carbocyclization, the use of carbonyl diimidazole gave the highest yield of the methods evaluated, and reduces the hazards as compared to using triphosgene. The end-game involves Suzuki cross-coupling, amide formation and deprotection. Unfortunately, the amide coupling step was inefficient without protection of the N3 position. However, this was solved by a direct amidation from a methyl ester by the use of bis(trimethylaluminum)-1,4-diazabicyclo[2.2.2]-octane as promotor. The imidazopyridinone produced was a potent inhibitor of E. coli TMPK. However, due to the lack of cell activity, and our inability to explain the results by molecular docking, this project was discontinued.
Whilst testing the minimum inhibitory concentration (MIC) of a library of heterocycles, we came across a 4-amino-6-aryl-pyrrolo[2,3-d ]pyrimidine with bactericidal properties against S. aureus. Based on this compound, we synthesized a library of pyrrolo- and thienopyrimidines, which we used to explore the structure-activity relationship against S. aureus. This revealed that a large substituent in the para position of the 4-amino benzyl group and a para-phenol in the C6 were important for activity. The most active compound also had 8-fold lower MIC when combined with the antimicrobial peptide betatide. Metabolomics and signalomics revealed that the compound kills bacteria by nitrosative stress or inhibition of the tricarboxylic acid cycle. The synergistic effect with betatide was caused by excessive stress.
Spurred on by a failure of in silico docking and high-throughput screening, we synthesized a library of tetrahydrocarbazoles for use in NMR screening against the β sliding clamp. The first step of the synthesis, a Fischer indolization, proved challenging due to the formation of regioisomers. Initially racemization occurred during a later step, but careful tuning of reaction conditions lowered racemization significantly. Several compounds produced were active against the protein in an STD NMR-based assay. The intended use of these compounds is as spy-molecules for screening compounds against the β sliding clamp by 19F NMR. The initial results are promising; however, the robustness and reproducibility of the method(s) must be further evaluated.