Development of new drug leads for tuberculosis

Abstract

Tuberculosis (TB), caused by infection with the bacterium Mycobacterium tuberculosis, has re-emerged as a global health risk with a significant proportion of new TB cases classified as multi-drug resistant (MDR) or extensively drug resistant (XDR). As such, there is a desperate need for the development of TB therapies which operate via novel modes of action. This thesis outlines the development of new drug leads for tuberculosis employing a target-based approach and by synthesis and derivatization of natural product scaffolds. Specifically, the first and second section of the thesis describes the target-based approach towards the development of inhibitors against enzymes in the shikimate and peptidoglycan biosynthetic pathways such as type II dehydroquinase (type II DHQase) and N-acetylglucosamine-1-phosphate (GlmU) uridyltransferase. These enzymes have been demonstrated to be essential for the survival and virulence of M. tuberculosis in vitro. The third section of the thesis describes the development of synthetic derivatives of sansanmycins, uridylpeptide antibiotics isolated from Streptomyces spp. SS. Inhibition of M. tuberculosis type II DHQase is the subject of chapter 3 of this thesis. Initial synthetic efforts were directed at the preparation of inhibitors possessing an anhydroquinate core which served as a mimic for the enol intermediate of the type II DHQase catalyzed reaction. A library of inhibitors with triazole and alkyne linkers designed to bridge the anhydroquinate core with various aryl and heteroaryl groups were synthesized from a common ene-yne intermediate employing Cu(I) azide-alkyne cycloaddition and palladium catalyzed Sonogashira cross-coupling. The majority of compounds exhibited potent inhibition of M. tuberculosis type II DHQase (Ki = 0.039-3.2 M). Despite the potent inhibition against M. tuberculosis type II DHQase, anhydroquinate-based compounds exhibited poor inhibition of M. tuberculosis (H37Rv) in vitro. The lack of antitubercular activity was mainly attributed to the hydrophilicity of the anhydroquinate core which makes it difficult for these compounds to traverse the waxy, hydrophobic M. tuberculosis cell wall. This problem was addressed by substituting this core with functionally simpler, more hydrophobic fragments. A fragment elaboration approach was subsequently employed to devise novel type II DHQase inhibitors. This fragment elaboration study led to the identification and development of catechol and acetonide-based compounds which exhibited low micromolar inhibition against M. tuberculosis type II DHQase (Ki = 5-86 μM) and markedly improved antitubercular activity relative to anhydroquinate-based inhibitors (MIC50 = 10-850 μM against the avirulent H37Ra strain of M. tuberculosis). Chapter 5 of this thesis outlines the successful development of a continuous enzyme kinetic assay for M. tuberculosis GlmU uridyltransferase. It also features the design of inhibitors based on the substrates and putative transition-state of the GlmU uridyltranferase reaction. Three classes of substrate-based inhibitors based on aminothiazole, sulfonamide and chromone scaffolds were synthesized along with transition-state mimics. Unfortunately, these compounds only demonstrated weak to moderate inhibition of M. tuberculosis GlmU uridyltransferase. This chapter also outlines the successful synthesis of the first inhibitors of M. tuberculosis GlmU uridylransferase based on an aminoquinazoline scaffold which exhibited micromolar inhibitory activity. The structure-activity relationships (SARs) elucidated from this series of compounds will aid the design of more potent compounds in the future. Chapter 6 of this thesis focuses on the development of a low-epimerization fragment condensation strategy to assemble analogues of sansanmycins with high optical purity. A total of nine sansanmycin analogues were synthesized and evaluated for in vitro growth inhibition of M. tuberculosis H37Rv. Most compounds exhibited excellent antitubercular activity with MIC50 values in the high nanomolar to low micromolar range (0.3-2.2 μM). Preliminary data indicated that these compounds demonstrated intracellular killing of M. tuberculosis in infected human THP-1 macrophages. The SARs elucidated for the first-generation sansanmycin analogues now form the foundation for the design and synthesis of more potent second-generation compounds. The activity of these compounds in macrophage killing assays holds promise for the in vivo evaluation of these analogues.