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Permanent link (DOI): https://doi.org/10.7939/R3DZ03G17

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Design, Synthesis and Antimycobacterial Evaluation of a Novel Class of Chemotherapeutic Agents Open Access

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Other title
Subject/Keyword
Chemotherapeutic Agents, Mycobacterium tuberculosis
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Garg, Saurabh
Supervisor and department
Kumar, Rakesh (Laboratory Medicine and Pathology)
Rennie, Robert (Laboratory Medicine and Pathology)
Examining committee member and department
Kaur, Kamaljit (School of Pharmacy)
Kunimoto, Dennis (Medicine and Dentistry)
EL-Kadi, Ayman (Pharmacy and Pharmaceutical Science)
Department
Medical Sciences-Laboratory Medicine and Pathology
Specialization

Date accepted
2017-05-18T14:27:35Z
Graduation date
2017-11:Fall 2017
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
Mycobacterium tuberculosis (Mtb), one of the deadliest known human pathogens, infects ~ 2 billion people (33% of the world's population) and remains the leading infectious cause of death. According to the World Health Organization's (WHO) report, 225 million new infections will occur and ~79 million people worldwide will die of tuberculosis (TB) between the years 1998-2030. Worldwide, TB accounts for 9.3 million new cases and 2 million deaths every year. HIV infection has contributed to a significant increase in the incidence of TB globally. Further, emergence of multi-drug resistant (MDR), extensively-drug resistant (XDR) and totally-drug resistant (TDR) strains of mycobacteria has made this disease almost incurable. Therefore, there is an urgent need to discover novel antimycobacterial agents and effective therapeutic regimens. The genome of Mtb encodes several enzymes involved in nucleic acid synthesis, and pyrimidine and purine biosynthesis, which differ significantly in selectivity towards their substrates and/or potential inhibitors from those in mammals. Therefore, modified nucleosides could act as selective inhibitors of mycobacterial RNA and DNA synthesis and replication. In this study, I have designed and synthesized nineteen novel deoxy, dideoxy, ribo and arabino pyrimidine nucleoside analogs containing 5-ethynyl, 5-(2-propynyloxy) and 5-hydroxymethyl uracil bases. These compounds were evaluated for their activity against several mycobacterial species (Mtb, M. bovis and M. avium). Combination therapy has been the most successful approach in the treatment of mycobacterial infections, and therefore selected compounds were investigated in combination with isoniazid and rifampicin in 2 and/or 3 drug combinations. Inhibition of intracellular mycobacterial growth within macrophages and in vivo activity of the novel agents in an infection model of Mtb was also carried out. Although, several compounds exhibited modest inhibitory activity at higher concentrations against Mtb and M. bovis they displayed unexpected synergistic interactions at lower concentrations with isoniazid and rifampicin. Interestingly, the active analogs were also found to inhibit intracellular mycobacterial replication in a human monocytic cell line infected with H37Ra. From this work, 5-hydroxymethyl-3-N-(2-propynyl)-3’-azido-2’,3’-dideoxyuridine and 5-hydroxymethyl-3-N-(2-propynyl)-2’,3’-dideoxyuridine emerged as the most potent compounds. Oral treatment of mice infected with Mtb (H37Ra) with these compounds demonstrated promising antimycobacterial effect. I have also designed and investigated novel drug conjugates by integrating a pyrimidine nucleoside effective against mycobacteria with an existing TB drug working by a different mechanism as a new class antimycobacterial agent. To test this unique approach, 5’-mono-pyrazinoate and 3’, 5’-di-pyrazinoates of 5-fluoro-2’-deoxyuridine were synthesized. Both mono- and di-pyrazinoated conjugates were evaluated for their antimycobacterial activity alone and in combination with isoniazid and rifampicin in in vitro and in vivo experiments. In a mouse model of Mtb infection, a di-pyrazinoated nucleoside compound significantly reduced mycobacterial loads upon oral administration alone and when combined with a low dose of isoniazid or rifampicin, when compared to parent drugs alone, and their individual 2- or 3-drug combinations with isoniazid and rifampicin. The mono-pyrazinoated compound was less effective than di-pyrazinoated compound in mice infected with Mtb. In these studies, I also investigated a new strategy for TB and/or TB-HIV co-infection by designing and synthesizing a novel co-drug incorporating an anti-HIV nucleoside analog 3’-azidothymidine (AZT) with an anti-TB drug p-aminosalicylic acid (PAS). This novel synthesized 5’-para-aminosalicylate-AZT co-drug was evaluated in vitro and in vivo for its antimycobacterial effects alone and in combinations with existing drugs, isoniazid and rifampicin. Strikingly, the results demonstrated that oral treatment with the designed co-drug provided significant inhibition of Mtb in the mouse model in lungs, liver and spleen at 1/20th of the dose of parent drug p-aminosalicylic acid. Intriguingly, the co-drug when co-administered with isoniazid or rifampicin, also furnish enhanced effects over isoniazid or rifampicin alone or their combinations as expected. In vitro cytotoxicity of the identified compounds, conjugates and co-drug was not observed up to the highest concentration tested. In the in vivo studies, none of the mice became sick, lost weight or died in all of the treatment groups with the new class of compounds. From this project, new classes of effective antimycobacterial agents have emerged that have potential to be developed as a new generation of therapeutic regimens for the treatment of TB and TB-HIV co-infection. The importance of my initial finding is that they open new avenues of research to augment current therapy, shorten the duration of treatment, and avoid drug-resistance problems.
Language
English
DOI
doi:10.7939/R3DZ03G17
Rights
This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for the purpose of private, scholarly or scientific research. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
Citation for previous publication
Garg S et al. 2016. Bioorg Med Chem. 24:5521-5533

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