Synthesis and Evaluation of the Diaminoquinazoline Series as Anti-Tubercular Agents
Abstract
The 2,4-diaminoquinazoline class of compounds has previously been identified as an effective inhibitor of Mycobacterium tuberculosis growth. We conducted an extensive evaluation of the series for its potential as a lead candidate for tuberculosis drug discovery. Three segments of the representative molecule N-(4-fluorobenzyl)-2-(piperidin-1-yl) quinazolin-4-amine were examined systematically to explore structure–activity relationships influencing potency. We determined that the benzylic amine at the 4-position, the piperidine at 2-position and the N-1 (but not N-3) are key activity determinants. The 3-deaza analog retained similar activity to the parent molecule. Biological activity was not dependent on iron or carbon source availability. We demonstrated through pharmacokinetic studies in rats that good in vivo compound exposure is achievable. A representative compound demonstrated bactericidal activity against both replicating and non-replicating M. tuberculosis. We isolated and sequenced M. tuberculosis mutants resistant to this compound and observed mutations in Rv3161c, a gene predicted to encode a dioxygenase, suggesting that the compound may act as a pro-drug.
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Introduction
Tuberculosis (TB), caused by Mycobacterium tuberculosis, is an infectious disease for which there is still a great need for discovery and development of novel drugs to improve therapy.1 In 2010 alone, the World Health Organization reported 8.8 million new cases and 1.4 million deaths from the disease.2 In addition, billions of people harbor latent infections with no clinical symptoms, but with the potential to advance to active form. Current TB treatment requires a combination of four drugs, isoniazid (INH), rifampicin (RIF), pyrazinamide (PZA), ethambutol (ETH) for 2 months followed by an additional 4 months of INH and RIF. These drugs have been in use for many decades, contributing to a rise in the emergence of multidrug resistant (MDR) and extensively drug-resistant (XDR) strains of M. tuberculosis. New drugs are needed urgently to shorten the duration of therapy and to treat drug-resistant strains.
Diaminoquinazolines (DAQ) have been reported with activity against a diverse range of biological diseases including lupus, rheumatoid arthritis, malaria and hypertension.3
Conclusion
Based on previous reports of sub-micromolar MIC activity of the DAQ series in TB assays, the main goal of our work was to deter- mine whether the series is a viable starting point for drug lead development. Surprisingly, we found the activity of the series in liquid medium was limited and anti-tubercular activity was weaker than previously reported. We hypothesized that this dis- parity was a result of variation in assay conditions, the most noticeable difference being a change in carbon source. However, we saw no improvement in anti-tubercular activity of these compounds when tested using palmitate as a carbon source. In previous work, cell viability was determined by metabolic activity (using Alamar blue), whereas we measured bacterial growth (by OD and fluorescence). This may be indicative of mode of action, since compounds might be able to reduce metabolic activity more efficiently than prevent growth.
However, good activity was seen on solid medium for these compounds where the MICs were lower than in liquid medium (even using the same carbon source, glucose). This was surprising, since in our experience MICs on solid medium are normally higher by several-fold. This may reflect a true difference in bacterial physiology or metabolism between the two states. Culture on solid medium may reflect better the physiological state of bacteria dur- ing infection, particularly in granulomas or in biofilms.
We conducted a systematic exploration of a diaminoquinazoline compound series for inhibitory activity against M. tuberculosis. SAR efforts around N-3 replacements led to a fused ring in the form of an imidazoquinoline (103) with an improvement in biological activity and an opportunity for SAR development on an alternate scaffold. PK evaluation indicated there is no obvious barrier to property optimization. Our data demonstrated that the DAQ series had an encouraging microbiological profile with bactericidal activity against replicating and non-replicating M. tuberculosis, suggesting that the target of the series is important for bacterial viability. Future work to identify the target could allow screening for alter- native scaffolds with improved properties and biological activity. These properties may have implications in the application of these compounds as anti-TB agents. In addition, the DAQ series could be useful tools for probing cell death mechanisms and for target identification studies.