Electronic Theses and Dissertations (Masters)

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Design and synthesis of triazine derivatives as non-nucleoside reverse transcriptase inhibitors
(University of the Witwatersrand, Johannesburg, 2024) Munetsi, Wendy; Bode, Moira; Ngwira, Kennedy
This research work was carried out to investigate the properties of different groups that can be used to modify the triazine core with the aim of designing a new library of possible HIV non- nucleoside reverse transcriptase inhibitors (NNRTIs). Triazine derivatives have been used extensively in the synthesis of numerous classes of drugs due to their significant biological activity. In this project, the specific focus was to synthesize 1,3,5-triazine derivatives by successive nucleophilic substitution reactions of the Cl atoms from cyanuric chloride. In the first step of the substitution reactions, 2,4,6-trichloro-1,3,5-triazine was reacted with various anilines, phenols and thiophenols which acted as nucleophiles to displace one of the Cl atoms upon reaction completion. The yields varied from 28% -90% with the best yields being observed when the anilines were used as a nucleophile and most of the substituents in this first step were anilines. The substituents used at each step of the substitution were vital in terms of determining the order of the reaction to enable a successful reaction. The introduction of different linkers to the triazine core such as -NH, -S, -O yielded compounds with different properties expected to provide significant interactions in the NNRTI binding pocket. We expected better binding properties from the -NH bearing compounds due to hydrogen bond formations with amino acid residues inside the allosteric binding pocket of the HIV-1 RT. The success of the second step of the substitution reactions was identified to be dependent on the substituent attached to the triazine ring from the first step. Some reactions were not successful when a stronger nucleophile was used in the first step and a weaker nucleophile was being used as the incoming nucleophile substituting the second Cl atom. Therefore, these reactions were repeated and the order of the reaction rearranged. Temperatures were increased and reaction times were increased at this stage as the reactivity of the triazine ring was reduced and therefore higher kinetic energy was required for successful reactions. In general, the synthesized triazine derivatives bearing two aromatic substituents exhibited the most significant presence of tautomers. The final stage in the synthesis of the trisubstituted triazine derivatives was relatively complex and required much higher temperatures and longer reaction times. The reactions were also performed at smaller scales and difficulties with the purification processes also contributed to the loss of product thereby resulting in lower yields, with one of the compounds giving a yield of 11%. The results obtained from the anti-HIV assay studies from the selected compounds tested, showed that antiviral activity was observed in triazine derivatives with electron withdrawing groups attached to the aromatic substituent as well as -NH and -O linkers at the right and left wing of the triazine core, respectively.
Development of a Commercial Manufacturing Process of 9-[(R)-2- (phosphonomethoxy)propyl] adenine (PMPA): A Key Intermediate for the Production of Tenofovir-based HIV Medicines
(University of the Witwatersrand, Johannesburg, 2023) Mbutho, Banele; Gohain, Mukut; De Koning, Charles
South Africa runs the largest antiretroviral (ARV) program in the world and yet 99% of the active pharmaceutical ingredients (APIs) used to make ARVs are imported from China. Dependence on imported APIs has major cost implications and influences the medication’s security of supply. This project was concerned with making it possible to produce the APIs tenofovir, a precursor for tenofovir disoproxil fumarate and tenofovir alafenamide locally and at a lower cost. A new synthetic route recently introduced by Medicines 4 All (M4ALL) was studied and used in this dissertation. The four-step process that produces an adenine derivative was optimized and scaled into a commercial industrial process producing tenofovir intermediates in repeatable yield and purity. This route was determined to be the most cost-effective since it utilized low cost and commercially available diaminomaleonitrile and triethyl orthoformate as starting materials—contrary to the synthetic routes currently used by the 17 largest tenofovir manufacturers. Key process improvements included a decrease in the number of solvents used and the minimization of by-product formation. Results showed that high yields of tenofovir intermediates were successfully synthesized using this new route. As such, the chemical company we conducted this research in, Chemical Process Technology Pharma will be able to employ this synthetic methodology to affordably produce the APIs used in the manufacturing of ARVs locally improving access to affordable medication.
Metal Pincers as Antiviral Agents Targeting SARS-CoV-2 Spike Protein
(University of the Witwatersrand, Johannesburg, 2023-08) Bracken, Matthew Lee; Munro, Orde Q.
The purpose of this work was to prove the concept that complexes of bioavailable metal ions may be designed to target specific solvent-exposed amino acid residues on therapeutic protein targets. The complexes synthesized and studied were novel Zn(II) and Cu(II) NNN amide pincers. The chelates were designed by in silico methods to target solvent-exposed tyrosine residues on the receptor binding domain of SARS-CoV-2. These tyrosine residues are crucial for binding host cell receptors and by targeting these groups, the metal pincers may potentially act as antiviral fusion inhibitors for the treatment of COVID-19. Biophysical studies were carried out to determine the binding affinity between the chelate and phenolic residues. These studies identified the most likely binding site for the metal complex on the SARS-CoV-2 spike protein epitope. The novel chelates were crystalized and found to adopt hexameric metallocycle architecture.
The Design and Synthesis of Anti-Tubercular Lariatin a Peptidomimetics
(University of the Witwatersrand, Johannesburg, 2023-07) Nyembe, Priscilla Lebohang; Makatini, Maya Mellisa
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is one of the major causes of death and morbidity worldwide. Approximately 10 million people worldwide are infected with Mtb annually, with an estimated 1.5 million deaths. However, potent anti-TB drugs with a new mechanism of action have not been developed in the last thirty years, and only 5 anti-TB drugs are still clinically used. Currently, available drugs and vaccines have failed to control its spread. Furthermore, the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mtb is a significant public health concern because most of the anti-TB drugs that have been in use for over 40 years are no longer effective for the treatment of these infections. Thus, there is an increased demand for novel anti-tubercular drugs with a different mode of action directed at new Mtb targets. Lariatin A, an anti-mycobacterial peptide, has received interest in the synthesis field due to its distinctive threaded structure which consist of a linear and cyclic portions and its unique bactericidal mechanism toward Mtb. This research focuses on designing and synthesizing derivatives of Lariatin A and investigating their binding properties to the mycobacterium caseinolytic protease (ClpP), a protein essential for the growth of Mtb. A simpler synthetic route for derivatizing Lariatin A peptides was achieved by incorporating two cysteine amino acid residues onto the sequence for cyclization of the peptide via the formation of a disulfide bond instead of a lactam bond. To further simplify the synthetic procedure, derivatives with shorter sequences as well as peptide-peptoids hybrids were also designed. Eight mimetics of Lariatin A were synthesized [Pep_PNL1 (1), Pep_PNL2 (2), Pep_HA (3), Pep_TA (4), Pep_TAA (5), Pep_HAP (6), Pep_PTA (7), Pep_PHA (8)]. The proposed derivatives were synthesized using the solid phase peptide synthesis technique and a sub-monomeric approach was followed to synthesize the peptide-peptoid hybrids. Purification of the peptides was achieved by utilizing semi-preparative High-Pressure Liquid Chromatography and they were characterized by Liquid Chromatography-Mass Spectrometry. The peptides were obtained in low to moderate yields, and the linear tail portion derivative (4) showed 70% ClpP inhibition, while the linear tail derivative coupled to the adamantane moiety (5) showed a 49% inhibition factor. NMR (nuclear magnetic resonance spectroscopy) and CD (circular dichroism) were utilized to determine the secondary structural features. The CD experiments indicated that peptide 1 adopts stable conformations while its separate tail (4) and cyclic (3) regions loss conformity. Pep_PTA (7) displayed the characteristics of both peptide and peptoid as seen from its formation of beta sheets. NMR and CD experiments confirmed that 4 exist in a helical conformation. Hence helical Lariatin A derivatives targeting the Mycobacterium tuberculosis caseinolytic protease can be synthesized using the solid phase peptide synthesis strategy.
The synthesis of aryl benzamides as potential HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs)
(University of the Witwatersrand, Johannesburg, 2023-07) Mohasoa, Likhopotso Cecilia; Zimuwandeyi, Memory; Bode, Moira L.
Dihydro-alkoxybenzyloxopyrimidines are heteroaryl-containing compounds that have previously been shown to exhibit excellent activity against HIV-1 reverse transcriptase (RT) enzyme. In our own laboratory, 2-chloro-N-(6-(piperidin-1-yl)pyridin-2-yl)benzamide was identified as a compound with activity against wild-type HIV-1. Using these two structural types as a guide, as part of our ongoing studies to search for anti-HIV therapeutic agents that target the RT enzyme, a library of arylbenzamide compounds bearing a pyrimidine ring as a central core was synthesized. These compounds contained an oxygen linker to allow flexible rotation of the molecule in the RT active site, with the aim of achieving activity against wild-type and mutant HIV-1. As a starting point, in order to first identify a suitable synthetic method and then apply it for our target novel compounds, four different carboxylic acids and two classes of amines were tested. Amidation reactions were carried out on unsubstituted benzoic acid, 3-methoxybenzoic acid, 3-hydroxybenzoic acid, and 3-((2,6-dichloropyrimidin-4-yl)oxy)benzoic acid. In this last case, the 3-hydroxybenzoic acid moiety had already been linked to the pyrimidinyl core in order to test which order of reaction worked best: linking followed by amidation, or the reverse. Reaction of these benzoic acid derivatives with anilines and aminopyridines gave the resulting benzamides in 22-99% yields after optimization. When triethylamine was used as a base in amidation reactions involving 2-amino-3-bromopyridine, 2-amino 5-bromopyridine and 2-amino-5-methylpyridine, diacylation was favoured, while when pyridine was used, monoacylation predominated. The reactions to link benzoic acid derivatives to the pyrimidinyl core were carried out by displacement of chlorine on 2,4,5-trichloropyrimidine. The displacement of the first chloride was tested using three types of nucleophiles. The first nucleophile was methyl 3-hydroxybenzoate, effectively a protected benzoic acid, which afforded methyl 3-((2,6-dichloropyrimidin-4-yl)oxy)benzoate in 81% yield. Problems with subsequent hydrolysis of the ester made this route impractical. The second nucleophile was 3-hydroxybenzoic acid which provided 3-((2,6-dichloropyrimidin-4-yl)oxy)benzoic acid in 81% yield. The third nucleophile was N-(5-bromopyridin-2-yl)-3-hydroxybenzamide, where amidation had already been performed, which transformed into the desired compound N-(5-bromopyridin-2-yl)-3- ((2,6-dichloropyrimidin-4-yl)oxy)benzamide in 28%. The low yield obtained from reaction of the amidated nucleophile identified the most promising route to be linking of 3-hydroxybenzoic acid to 2,4,5-trichloropyrimidine first, followed by amidation. After the successful displacement of the first chlorine atom, two of the resulting analogues 3-((2,6-dichloropyrimidin-4-yl)oxy)-N-(p-tolyl)benzamide and N-(4-bromophenyl)-3-((2,6-dichloropyrimidin 4-yl)oxy)benzamide were functionalized with sulfur and nitrogen nucleophiles by displacement of a second chlorine atom. Ethanethiol proved to be highly nucleophilic, leading to pyrimidine C-O bond cleavage and sulfur disubstitution, while the nitrogen ucleophiles propylamine and piperidine afforded their corresponding derivatives in good yields without breaking the carbon-oxygen bond. The newly coupled propyl compound was further derivatized by means of hydrolysis with sodium hydroxide to yield the desired novel 3-((6-hydroxy-2-(propylamino)pyrimidin-4-yl)oxy)-N-(p tolyl)benzamide or 3-((6-oxo-2-(propylamino)-1,6-dihydropyrimidin-4-yl)oxy)-N-(p-tolyl)benzamide compound.
The Synthesis of Pyrido-fused 8-Methoxy Carbazoles by Using a Light-Assisted, Base Mediated Cyclization Reaction
(University of the Witwatersrand, Johannesburg, 2023) Magagula, Bongi Florence; Ntsimango, Songeziwe; De Koning, Charles B.
Nitrogen-containing compounds such as indoles and carbazoles are significant classes of the N-heterocycles that show great promise as anti-cancer compounds. Indoles such as 2,3-diarylindole, 3-pyranyl indole and carbazoles such as 9-methoxyellipticine are compounds which possess anticancer or antitumor properties. Due to the favourable biological activities of N-heterocyclic compounds, medicinal and synthetic chemists have developed numerous methodologies for their synthesis. In this research project, the broad aim was to synthesize pyrido-fused carbazoles from 5-methoxyindole using methodologies that have been previously used in our laboratories and by other chemists while changing the position of the nitrogen atom on the pyrido-fused carbazoles. The first step in the synthesis of these carbazoles was the treatment of 5-methoxyindole with di-tert-butyl dicarbonate in the presence of 4-dimethylaminopyridine (DMAP) which gave the desired protected indole, tert-butyl 5-methoxy-1H-indole-1-carboxylate in excellent yields (90-99%). Exposure of the tert-butyl 5-methoxy-1H-indole-1-carboxylate to lithium 2,2,6,6-tetramethypiperidide followed by quenching with triisopropyl borate and hydrochloric acid gave (1-(tert-butoxycarbonyl)-5-ethoxy-1H-indol-2-yl)boronic acid. Using this and various halogen substituted pyridines, for example 3-bromo-4-methylpyridine in the Suzuki-Miyaura coupling reaction gave tert-butyl 5-ethoxy-2-(4methylpyridin-3-yl)-1H-indole-1-carboxylate (83% yield). This was further reacted with paraformaldehyde and iron (III) chloride or phosphorus oxychloride and DMF. After the removal of tert-butoxycarbonyl protecting group utilizing various methods this produced 5-methoxy-2-(4-methylpyridin-3-yl)-1H-indole-3-carbaldehyde (48% yield). 5-Methoxy-2-(4-methylpyridin-3-yl)-1H-indole-3-carbaldehyde possesses all the carbons of the final compounds and is suitably functionalized to partake in the key photo-induced and ase-mediated cyclization reaction. Previous studies pointed to the necessity of an alkyl protecting group on the indole-N atom. As a result, the indole nitrogen atom was then protected again with a methyl or a benzyl group; where the N-benzyl could be removed at a later stage. For example, reaction of 5-methoxy-2-(4-methylpyridin-3-yl)-1H-indole-3-carbaldehyde dissolved in THF, potassium hexamethyldisilazide and benzyl bromide furnished 1-benzyl-5-methoxy-2-(4-methylpyridin-3-yl)-1H-indole-3-carbaldehyde (83% yield). The key step in this synthesis was the light-assisted, base-mediated cyclization reaction which has been reported by de Koning and co-workers, where a solution of 1-benzyl-5-methoxy-2-(4-methylpyridin-3-yl)-1H-indole-3-carbaldehyde dissolved in dry DMF and potassium tert-butoxide was heated and irradiated with medium mercury lamp yielding the desired pyrido fused carbazole, 11-benzyl-8-methoxy-11H-pyrido[3,4-a]carbazole in a good yield of 70%. Following the outlined synthetic procedure depicted above, we were able to synthesize 5 analogues of 11-benzyl-8-methoxy-1H-pyrido[3,4-a]carbazole.
Use of transaminases for the biosynthesis of enantiopure building blocks of two essential medicines: Ethambutol and Dolutegravir
(University of the Witwatersrand, Johannesburg, 2023) Maboya, Josephine; Pienaar, Daniel
(S)-2-Amino butan-1-ol and (R)-3–amino butan-1-ol play an important role as intermediates in the synthesis of the anti-tuberculosis drug ethambutol and HIV integrase inhibitor drug dolutegravir respectively. The current industrial preparation of these enantioenriched amino alcohols is quite a challenging process; it typically involves the use of harsh chemicals, results in low yields, and generates hazardous waste materials. Consequently, these methods tend to be expensive, and it has been demonstrated that the cost of these intermediates has a significant impact on the overall costs of the synthesis of the entire drug. Therefore, it is not surprising that the convenient, cost–effective, and environmentally benign production of these optically pure amino alcohols is still the subject of ongoing investigations. The chemo-enzymatic approach holds great potential to replace the conventional routes for the synthesis of enantiopure amines. Transaminase enzymes (ATAs), in particular, have gained much attention over time due to their remarkable capability to transform inexpensive ketone starting materials into valuable enantiopure amino alcohols. Through the utilization of the isopropyl amine donor system, pro-chiral ketone starting materials were effectively transformed into the desired (S)-isopropyl 2-aminobutanoate and (R)-isopropyl 3-aminobutanoate using transaminase biocatalysis. These reactions proceeded well under milder conditions such as ambient temperature and pressure conditions, and impressively under an aqueous environment. Three (S)-enantiomer selective “hit “enzymes were discovered (ATA-189, ATA-194, and ATA-254) for the biotransformation of alpha-keto ester substrate into an enantio-enriched amino ester product, with enantiomeric excess ranging between 95-99% and the yield was 15-73% depending on the enzyme and reaction conditions. However, when it came to dolutegravir intermediate, a different scenario unfolded. In this case, the majority of the ATA enzymes in our enzyme library fortuitously exhibited selectivity for the (R)-enantiomer. In particular, four highly enantioselective enzymes (ATA-254, ATA-261, ATA-262, and ATA-234) were discovered, demonstrating % e.e ranging from 93% to 99.99%, with corresponding yields from 38% to 45%. The successful biotransformation of an inexpensive pro-chiral starting material into highly valuable enantioenriched amino ester intermediates represents a significant achievement. Coupled with an effective reduction method to convert these intermediates into the corresponding amino alcohols, this biotransformation process holds immense potential for enabling the sustainable and cost- effective production of both of the valuable ethambutol and dolutegravir amine intermediates

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