Modification of protein inhibitor antibiotics to improve efficacy/safety

In antibiotics research, a key goal is to understand the role played by each structural component in determining the biological activity, efficacy, and safety. This exploration not only aids in the quest for novel and more effective therapeutic agents but also serves as a crucial step in interpreting the mechanism of antibiotic action. This involves understanding how the antibiotic molecule interacts with specific enzymes or substances within microbial cells, thereby inhibiting various metabolic reactions. Through comprehensive chemical investigation, particularly through stereospecific synthesis, to identify which functional groups of the antibiotic are responsible for its activity and gain insights into the nature of its interaction with specific components of microbial cells.

This research has focused on the antibiotic chloramphenicol, a seldom-prescribed drug due to its toxicity, particularly towards human mitochondria. The primary objectives of this study encompass the strategic design and synthesis of novel chloramphenicol derivatives aimed at enhancing its antibacterial efficacy while concurrently mitigating its potential toxicity. Modifications included the incorporation of nitrogen into the aromatic ring as well as the replacement of the nitro group with bioisosteric functional groups such as an imidazole and difluoro substitution.

In the first part of this project, a library of structurally designed analogues of the broad-spectrum antibiotic chloramphenicol were generated and simulated by molecular docking (AutoDock vina) in the crystal structure of the E. coli ribosome bound to chloramphenicol. The work aims to identify the best positions of chloramphenicol orientation with the 3D chemical models (ligands) generated using Chimera software with the template crystal structure of E. Coli ribosome protein bound to the chloramphenicol binding sites (PDB file: 3OFC, superseded by 4V7T). Evaluation of the best orientation, affinity, and interaction of the ligand – protein complex which can lead to optimization and finding the potential drugs against the protein target in less time and an affordable way. The final target analogues were further investigated in SwissADME that predicts properties of potential drug candidates.

The second part of this project was the attempted chemical synthesis of the novel analogues of chloramphenicol from the first part of the project. The targeted molecules are the pyridine analogue of chloramphenicol (aza-chloramphenicol), and the bioisosteric replacement of the nitro group in chloramphenicol by the imidazole analogue, and the difluoro substituted.

The syntheses were based on short routes via stable intermediate. Sonogashira, Hiyama, Minisci, Grignard, and Stille coupling reactions were carried out. Various chemical mechanisms and column chromatography techniques were done to reach to the desired products. The intermediates were fully characterised using NMR, IR, and mass spectrometry. The microbiological evaluation was assessed with the disk diffusion assay and MIC determination assay. Two strains of Gram-positive and two strains of Gram-negative bacteria were tested against the synthesised intermediates. The diffusion assay showed that all 18 compounds had reduce growth against Gram-positive and p. aeruginosa at 100 µg/ml. The intermediates showed MIC values of 12.5 µg/ml for Gram-negative bacteria and for the Gram-positive ranged from (1.56 µg/ml – 12.5 µg/ml). Neutral red assay was used for assessing cell viability and cytotoxicity of the synthesised intermediates on HUVEC cells. The results showed that the phthalimide derivative 94 observed no cytotoxic effects on the HUVEC cells at 10 µg/ml with cell viability at 94.42%. Other intermediates showed that cell viability decreased as the concentration increased suggesting that it might exhibit some cytotoxic effects. The structure activity relationship (SAR) showed that the intermediates with nitropyridine rings, dilfuorobenzene, phthalimide group, methoxy, and imidazole exhibited varied efficacy against different bacteria strains, with some showing moderate to significant antibacterial activity.