Tuberculosis(TB) is a contagious disease caused by Mycobacterium tuberculosis (Mtb). According to the World Health Organization (WHO), TB resulted in the deaths of 1.8 million people from a total of 10.4 million infection cases in 2015. In order to eradicate the ongoing TB threat and combat multidrug-resistant strains (MDR-TB) new therapies are need. This dissertation covers the history of TB, Mtb cell wall structure, trehalose utilization pathways (TUP), and the design and synthesis of inhibitors related to targets in TUP; i.e. Glycosyl hydrolase GlgE, Antigen 85C (Ag85C), Pks13 and TPP2.
The second chapter describes the design and synthesis of a molecular probe to investigate the active site topology of Streptomyces coelicolor (Sco) GlgEI, a model enzyme of Mtb GlgE. Mtb GlgE is an essential enzyme which catalyzes the synthesis of cytoplasmic a-glucan by transferring maltose-1-phosphate (M1P) to a growing alpha-1,4-glucan chain. 2-Deoxy-2,2-difluoro-alpha-maltosyl fluoride (alpha-MTF) was designed as a non-hydrolysable substrate mimic of M1P to probe the active site of GlgE1 without disruption of catalytic residues. The synthesis and challenges faced during preparation of alpha-MTF are briefly described in this chapter. The key reaction in the synthesis was the use of 1-(chloromethyl)-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane ditetrafluoroborate (Selectfluor), which was used to transform peracetylated 2-fluoro-maltal into peracetylated 2,2-difluoro-a-maltosyl fluoride and peracetylate 2-deoxy-2,2-difluoro-alpha-maltosyl fluoride in a single step. The resulting compound was used by the Ronning group and co-crystallized with the Sco GlgEI-V279S. The X-ray structure of the crystals identified key interactions which may be helpful in further drug design.
The third chapter involves targeting Ag85C, in which we synthesized a library of 2-alkyl-1,2-benzisoselenazol-3(2H)-ones as potential covalent inhibitors. The synthesis used a novel thermal and photoinduced copper-promoted C-Se bond formation method and Copper and 1,10-phenantroline required for C-Se bond formation during heating (100 degree celsus) via a mechanism that likely involves atom transfer (AT); whereas, in the absence of the 1,10-phenatroline ligand, photoinduced activation, at room temperature, proceeds through a putative single electron transfer (SET) mechanism. This was the first example of a photoinduced copper-mediated C-Se cross-coupling. We synthesized a library of fifteen 2-alkyl-1,2-benzisoselenazol-3(2H)-ones which includes a biotinylated derivative with potential applications in proteomic profiling of solvent-exposed cysteines. Our collaborators evaluated the library for anti-Mtb activity by minimal inhibitory concentration (MIC) and Ag85C inhibition assay studies. All the compounds of the library were active against Mtb Ag85C and the allyl derivative (1f) possessed the most potent anti-Mtb activity with a MIC of 12.5 micro grams/mL. Compound 1f also reduced Mtb Ag85C activity to 85% of control in 40 min at 5 micro molar and showed an apparent IC50 of 8.8 micro molar.
In the fourth chapter, we synthesised 42 2-aminothiophenes (2AT) as inhibitors for Pks13. Pks13 is a validated anti-TB drug target which is essential for synthesis of mycolic acid which are found as major cell wall components trehalose monomycolate (TMM), trehalose dimycolate (TDM) and mycolyl arabinogalactan (mAG) of Mtb. The synthesis is as follows: each 2AT core was synthesized using Gewald chemistry. The library was expanded by functionalizing the 2-amino and 3-carboxylate groups on the 2AT cores. Our collaborators tested all the compounds for Mtb growth inhibition and identified the compound ethyl 6-ethyl-2-(perfluorobenzamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylate (5FT) as a lead compound. 5FT showed remarkable potency against Mtb H37RV with a MIC = 0.23 micro molar and further investigation by NIH showed an impressive potency (MIC = 0.20-0.44 micro molar) against Mtb strains resistant to isoniazid, rifampicin, and fluoroquinolones. The site of action for the 5FT compound is presumed to be Pks13 or an earlier enzyme in the mycolic acid biosynthetic pathway. This inference is based on structural similarity of the compound 5FT with known Pks13 inhibitors, which is corroborated by mycolic acid biosynthesis studies performed by Dr. Mary Jacksons lab. The Jacksons lab research shows the compound strongly inhibits the biosynthesis of all forms of mycolic acid in Mtb. In summary, these studies suggest 5FT represents a promising anti-TB lead which showed activity well below toxicity to human monocytic cells.
The fifth chapter describes the optimization studies of compound 5FT in chapter 4. In the current work, we synthesized a focused set of 14 compounds with distinct absorption, distribution, metabolism, excretion and toxicity (ADME-tox) properties. Employing a standard medicinal chemistry approach, the structure activity relationship (SAR) study on compound 5FT is divided into three parts: A) substituting the piperidine ring with its biomimetic to study and improve the potency in junction with homologation studies replacing N-ethyl group of 5FT to improve its pharmacological effect, B) replacing the ethyl ester group with an electron rich or withdrawing group to improve metabolic stability, bioavailability and longer plasma half-life (T1/2) and, C) studies on the importance and reactivity of the 2-perfluorobenzamido group. All the biological studies were done by our collaborators and the NIH (HHSN272201100009I). The synthesized compounds were subjected to Anti-TB studies against Mtb under aerobic, low oxygen and intracellular conditions as well as activity studies against drug resistant strains of Mtb. Among all the compounds, ethyl 2-(perfluorobenzamido)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxylate showed the best anti-Mtb potency of MIC = 67 nM and the compound 6-ethyl-N-methyl-2-(perfluorobenzamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide showed promising in vitro pharmacokinetic properties.
Finally, in the last chapter we describe the design and the synthesis of 7,7-difluro trehalose-7-diethyl phosphonate. This compound was designed as a non-hydrolysable substrate mimic which has two electronegative fluorine atoms on the C-7 position. The main step in the synthesis was a nucleophilic addition of (diethoxyphosphoryl)difluoromethanide on a compound with an aldehyde functional group. Upon benzyl deprotection lead to desired compound 7,7-difluro trehalose-7-diethyl phosphonate.