1 The synthesis of aryl benzamides as potential HIV-1 non- nucleoside reverse transcriptase inhibitors (NNRTIs). LIKHOPOTSO C MOHASOA Supervised by: Prof M.L. Bode Co-supervised by: Dr. M. Zimuwandeyi A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science Submitted 03 July 2023 2 DECLARATION I Likhopotso C Mohasoa, hereby declare that this dissertation is my own work which was conducted in the School of Chemistry. It is submitted to the University of the Witwatersrand in Johannesburg for consideration of the Master of Science degree. It has never been submitted to any other university for the purpose of a degree or examination. …………………………………………….. Signature 03 July 2023 3 Abstract 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 4 second chlorine atom. Ethanethiol proved to be highly nucleophilic, leading to pyrimidine C-O bond cleavage and sulfur disubstitution, while the nitrogen nucleophiles 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. 5 DEDICATION This dissertation is dedicated to my late mother Manthomeng Mahao and my beloved family who unceasingly encouraged and supported me throughout the course of this study. 6 ACKNOWLEDGEMENTS To my father Steve Mohasoa, for not only providing me with the tools I need to succeed, but also for instilling in me the desire to do so. I have been able to get to where I am now because of your support and love. I love you daddy. My sincere gratitude goes to my supervisors, Prof. Moira L. Bode and Dr Memory Zimuwandeyi accepting me as master student, for their endless support of my MSc study and research, and for their immerse knowledge, patience, motivation, and enthusiasm. Your guidance groomed me throughout the course of this project and writing of this dissertation. I thank you for reading my dissertation and providing me with your insightful comments. I could not have imagined having wonderful supervisors for my MSc study. Thank you Dr. Kotze and Dr. Zimuwandeyi for your help in the NMR lab. Your assistance is highly appreciated. My appreciation also goes to Dr. Morifi, Thapelo, and Refilwe for the mass spec training and analyses of results. My great thanks go to Prof. Manuel for conducting crystallographic analyses for me. To Dr. Changunda who did the previous work on benzamides and pyrimidines, thank you. A big thanks to the entire organic chemistry group, you gave me a warm welcome on my first day of the MSc enrolment. I thank every one of you for the input and valuable information that I needed. Thank you Lebogang Tefu, Kabelo Zimba, Bianca Davis, Khanya Jaqeni, Nafisa, Edward, Dr. Thabo Peme, and Sandile Mkhize. I really enjoyed every single moment in the lab with your presence. Kabelo Dilebo and Aneesa Ghani, I thank you for your emotional and research support. Bongi and Tshegofatso Maboye, thank you for the funny jokes and laughter. Thank you Khonzisizwe Somnadi for sharing ideas with me. Thank you Nompumelelo Mathebula for being a good sister and a good colleague, I thank you for your willingness to help at all times. Thank you Kamegelo Butsi, the first person to help me with the NMR peak assignments. Above all, I would like to thank God, the Creator for that He never left nor forsaken me. No weapon formed against me prospered for the greater you are in me. Many thanks to my lovely sister Mamafa Mohasoa for her love, support, guidance, and motivation. To my parents’ first born Nthomeng Mohasoa, you are appreciated for your love. To mama Phikiwe Nhlapo, thanks for your love and kindness to get me a medical assistance, and for a warm welcome in your family. I appreciate and love you. Lastly, my appreciation goes to NRF and Wits University for the financial support. To Mr Kalumba Simba for the ADU opportunity to share my chemistry knowledge with the first-year engineering students. Lastly, I would like to thank Wits University for giving me the opportunity to enrol as a master student. 7 Table of Contents Abstract ................................................................................................................................................... 3 LIST OF ABBREVIATIONS AND ACRONYMS ........................................................................................... 11 CHAPTER 1: INTRODUCTION AND BACKGROUND ................................................................................ 14 1.1 HIV-AIDS .......................................................................................................................................... 14 1.1.1 Global HIV prevalence .............................................................................................................. 14 1.1.2 HIV lifecycle .............................................................................................................................. 15 1.1.3 Social and health effects of HIV/AIDS ...................................................................................... 16 1.1.4 Access, treatment, and care for people living with HIV ........................................................... 17 1.1.5 Newer tools for HIV/AIDS prevention and control .................................................................. 19 1.1.5.1 Provision of prophylactic agents ....................................................................................... 19 1.1.5.2 Implementation and monitoring of programs .................................................................. 19 1.1.6 HIV-1 reverse transcriptase inhibitors in the treatment of HIV ............................................... 21 1.1.6.1 NRTIs mechanism of action ............................................................................................... 22 1.1.6.2 NNRTIs mechanism of action ............................................................................................ 23 1.1.6.3 Drug resistance to NNRTIs ................................................................................................ 24 1.2 Synthetic targets with interesting biological activity ...................................................................... 25 1.2.1. Benzamides as structural cores .............................................................................................. 25 1.2.1.1 Biological activity of benzamides ...................................................................................... 26 1.2.1.2 Amide bond formation...................................................................................................... 29 1.2.1.5 Amide bond formation from acyl chlorides ...................................................................... 35 1.2.2 Pyrimidines as key structural components .............................................................................. 42 1.2.2.1 Biological activity of pyrimidines ...................................................................................... 43 1.2.2.2 Pyrimidine synthesis and reactions .................................................................................. 49 1.3 Research aims and objectives ......................................................................................................... 62 1.4 Aims and objectives of the project ................................................................................................. 65 1.4.1 Proposed overall synthetic approach ...................................................................................... 65 1.4.2 Proposed methodology ............................................................................................................ 65 CHAPTER 2: RESULTS AND DISCUSSION ................................................................................................ 68 2.1 Synthesis of benzamides ................................................................................................................. 68 2.1.1 Preparation of benzamides from unsubstituted benzoic acid 152 and aniline derivatives 151a-h ............................................................................................................................................... 68 2.1.2 Preparation of benzamides from methoxybenzoic acid 153 and anilines 151a-h .................. 74 2.1.2.1 Comparison between the C-8 signals of benzamide analogues derived from unsubstituted benzoic acid 152 and those derived from the methoxy-substituted benzoic acid 153 ................................................................................................................................................ 77 8 2.1.3 Preparation of benzamides from unsubstituted benzoic acid 152 and aminopyridines 151-o .......................................................................................................................................................... 77 2.1.4 Preparation of benzamides form methoxybenzoic acid 153 and aminopyridines 151i-o ....... 85 2.1.5 Preparation of benzamides 166 from 3-hydroxybenzoic acid 154 and anilines ...................... 94 2.1.5.1 The 1H NMR spectrum comparison between the appearance and disappearance of the hydroxybenzoic acid proton.......................................................................................................... 99 2.1.6 Preparation of benzamides from 3-hydroxybenzoic acid 154 and aminopyridines .............. 101 2.1.7 Synthesis of complex benzamides ......................................................................................... 103 2.1.7.1 Synthesis of 3-((2,6-dichloropyrimidin-4-yl)oxy)benzoic acid ........................................ 103 2.1.7.2 Comparison between the carbon signals of 2,4,6-trichloropyrimidine 102 and acid 155 .................................................................................................................................................... 108 2.1.6.2 Synthesis of complex benzamides from anilines 151a-h ................................................ 108 2.1.6.3 Synthesis of complex benzamides from aminopyridines 151i-o .................................... 113 2.1.6.4 Synthesis of complex benzamides from aminopyrazines 164p-q................................... 116 2.2 Nucleophilic substitution of the second chlorine atom ................................................................ 117 2.3 Third chlorine displacement by hydrolysis .................................................................................... 126 2.4 Conclusion ..................................................................................................................................... 130 Future work ......................................................................................................................................... 137 CHAPTER 3: EXPERIMENTAL PROCEDURE ........................................................................................... 143 3.1 Synthesis of benzamides from unsubstituted benzoic acid 152 and aniline derivatives 151a-h ........................................................................................................................................................ 143 3.1.1 N-(4-bromophenyl)benzamide 157a ................................................................................... 144 3.1.2 N-(3-bromophenyl)benzamide 157b ................................................................................... 145 3.1.3 N-(4-bromo-2-methylphenyl)benzamide 157c .................................................................... 145 3.1.4 N-(p-tolyl)benzamide 157d ................................................................................................. 145 3.1.5 N-(4-methoxyphenyl)benzamide 157e ................................................................................ 146 3.1.6 N-(2-bromo-4-methylphenyl)benzamide 157f .................................................................... 146 3.1.7 N-(2-bromo-4-methylphenyl)benzamide 157g ................................................................... 146 3.1.8 N-(4-chloro-2-methylphenyl)benzamide 157h .................................................................... 147 3.2 Synthesis of benzamides from unsubstituted benzoic acid 152 and aniline derivatives 151j-o ........................................................................................................................................................ 147 3.2.1 N-(5-bromopyridin-2-yl)benzamide 157j ............................................................................ 148 3.2.2 N-(6-methylpyridin-2-yl)benzamide 157k ........................................................................... 148 3.2.3 N-(6-bromopyridin-2-yl)benzamide 157l ............................................................................ 148 3.2.4 N-(6-chloropyridin-2-yl)benzamide 157m ........................................................................... 149 3.2.5 N-(3-bromopyridin-2-yl)benzamide 157n ........................................................................... 149 3.2.6 N-(5-chloropyridin-2-yl)benzamide 157h ............................................................................ 149 9 3.2.7 N-benzoyl-N-(5-methylpyridin-2-yl)benzamide 160i ........................................................... 150 3.2.8 N-benzoyl-N-(3-bromopyridin-2-yl)benzamide 160n .......................................................... 150 3.3 Synthesis of benzamides from 3-methoxybenzoic acid 153 and aniline derivatives 151a-h ... 150 3.3.1 N-(4-bromophenyl)-3-methoxybenzamide 158a................................................................. 151 3.3.2 N-(3-bromophenyl)-3-methoxybenzamide 158b................................................................. 151 3.3.3 N-(4-bromo-2-methylphenyl)-3-methoxybenzamide 158c ................................................. 152 3.3.4 3-methoxy-N-(p-tolyl)benzamide 158d ............................................................................... 152 3.3.5 3-methoxy-N-(4-methoxyphenyl)benzamide 158e ............................................................. 152 3.3.6 N-(4-chloro-2-methylphenyl)-3-methoxybenzamide 158h .................................................. 153 3.4 Synthesis of benzamides from 3-methoxybenzoic acid 153 and aniline derivatives 151j-o .... 153 3.4.1 3-Methoxy-N-(5-methylpyridin-2-yl)benzamide 158i .......................................................... 154 3.4.2 N-(5-bromopyridin-2-yl)-3-methoxybenzamide 158j .......................................................... 154 3.4.3 3-methoxy-N-(6-methylpyridin-2-yl)benzamide 158k ......................................................... 154 3.4.4 N-(6-bromopyridin-2-yl)-3-methoxybenzamide 158l .......................................................... 155 3.4.5 N-(6-chloropyridin-2-yl)-3-methoxybenzamide 158m ......................................................... 155 3.4.6 N-(3-bromopyridin-2-yl)-3-methoxybenzamide 158n ......................................................... 156 3.4.7 N-(5-chloropyridin-2-yl)-3-methoxybenzamide 158o .......................................................... 156 3.4.8 3-methoxy-N-(3-methoxybenzoyl)-N-(5-methylpyridin-2-yl)benzamide 165i ..................... 156 3.4.9 N-(5-bromopyridin-2-yl)-3-methoxy-N-(3-methoxybenzoyl)benzamide 165j ...................... 157 3.5 Sythesis of benzamides from 3-hydroxybenzoic acid 154 and aminopyridines ....................... 157 3.5.1 N-(5-bromopyridin-2-yl)-3-hydroxybenzamide 166j ........................................................... 158 3.5.2 N-(6-bromopyridin-2-yl)-3-hydroxybenzamide 166l ........................................................... 158 3.5.3 N-(6-chloropyridin-2-yl)-3-hydroxybenzamide 166m .......................................................... 158 3.5.4 N-(5-chloropyridin-2-yl)-3-hydroxybenzamide 166o ........................................................... 159 3.6 Preparation of methyl 3-hydroxybenzoate 141 ........................................................................ 159 3.7 Synthesis of methyl 3-((2,6-dichloropyrimidin-4-yl)oxy)benzoate 174 .................................... 159 3.8 Synthesis of 3-((2,6-dichloropyrimidin-4-yl)oxy)benzoic acid 155 ........................................... 160 3.9 Synthesis of complex benzamides from anilines 151a-h .......................................................... 160 3.9.1 N-(4-bromophenyl)-3-((2,6-dichloropyrimidin-4-yl)oxy)benzamide 168a ........................... 161 3.9.2 N-(3-bromophenyl)-3-((2,6-dichloropyrimidin-4-yl)oxy)benzamide 168b ........................... 161 3.9.3 N-(4-bromo-2-methylphenyl)-3-((2,6-dichloropyrimidin-4-yl)oxy)benzamide 168c ............ 162 3.9.4 3-((2,6-dichloropyrimidin-4-yl)oxy)-N-(p-tolyl)benzamide 168d ......................................... 162 3.9.5 3-((2,6-dichloropyrimidin-4-yl)oxy)-N-(4-methoxyphenyl)benzamide 168e ........................ 163 3.9.6 N-(4-chloro-2-methylphenyl)-3-((2,6-dichloropyrimidin-4-yl)oxy)benzamide 168h ............ 163 3.10 Synthesis of complex benzamides from aminopyridines 151j-o ............................................ 163 3.10.1 N-(5-bromopyridin-2-yl)-3-((2,6-dichloropyrimidin-4-yl)oxy)benzamide 168j ................... 164 10 3.10.2 3-((2,6-dichloropyrimidin-4-yl)oxy)-N-(6-methylpyridin-2-yl)benzamide 168k ................. 164 3.10.3 N-(6-bromopyridin-2-yl)-3-((2,6-dichloropyrimidin-4-yl)oxy)benzamide 168l ................... 165 3.10.4 N-(6-chloropyridin-2-yl)-3-((2,6-dichloropyrimidin-4-yl)oxy)benzamide 168m ................. 165 3.10.5 N-(5-chloropyridin-2-yl)-3-((2,6-dichloropyrimidin-4-yl)oxy)benzamide 168o .................. 166 3.11 Second chlorine displacement ................................................................................................ 166 3.11.1 N-(4-bromophenyl)-3-((6-chloro-2-(ethylthio)pyrimidin-4-yl)oxy)benzamide 180a .......... 166 3.11.2 3-((6-chloro-2-(ethylthio)pyrimidin-4-yl)oxy)-N-(p-tolyl)benzamide 180d ........................ 167 3.11.3 Preparation of 3-((6-chloro-2-(propylamino)pyrimidin-4-yl)oxy)-N-(p-tolyl)benzamide 184d .................................................................................................................................................... 167 3.11.4 3-((6-chloro-2-(piperidin-1-yl)pyrimidin-4-yl)oxy)-N-(p-tolyl)benzamide 185d ................. 168 3.12 Third chlorine displacement by hydrolysis ............................................................................. 168 3.12.1 3-((6-hydroxy-2-(propylamino)pyrimidin-4-yl)oxy)-N-(p-tolyl)benzamide 187d ................ 168 References .......................................................................................................................................... 170 Appendix 1: Single X-ray crystallographic data ................................................................................... 181 Appendix 2: 1H and 13C NMR data for a selected number of compounds .......................................... 266 11 LIST OF ABBREVIATIONS AND ACRONYMS HIV human immunodeficiency virus AIDS acquired immune deficiency syndrome UNAIDS Joint United Nations Programme on HIV/AIDS ART antiretroviral therapy gp41 transmembrane glycoprotein gp120 docking glycoprotein CD4 cluster of differentiation 4 RT reverse transcriptase IN integrase PR protease ds DNA double-stranded DNA WHO World Health Organization STIs sexually transmitted infections PEP post exposure prophylaxis PrEP pre-exposure prophylaxis NRTIs nucleoside reverse transcriptase inhibitors PI protease inhibitor INI integrase inhibitor NNRTIs non-nucleoside reverse transcriptase inhibitors HAART Highly active antiretroviral therapy RT reverse transcriptase NNIBP NNRTI binding pocket Å angstroms DNA deoxyribonucleic acid FDA Food and Drug Administration USA United States of America DAPYs diarylpyrimidines Wt wild-type IC50 half-maximum inhibitory concentration EC50 median effective concentration CC50 50% cytotoxicity concentration 12 AZT azidothymidine °C degree Celsius DMF N,N-dimethylformamide EDC 1-ethyl-3-(3-dimethylaminopropyl) DCC N,N-dicyclohexylcarbodiimide CDI 1,1’-carbonyldiimidazole Et3N/TEA triethylamine HOBt 1-hydroxy-7-azabenzotriazole WT wild type SI selectivity index DMAP 4-dimethylaminopyridine PCl3 phosphorous trichloride PCl5 phosphorus pentachloride POCl3 phosphorus oxychloride (COCl)2 oxalyl chloride SOCl2 thionyl chloride HCl hydrochloric acid µM micromolar nM nanomolar DABO Dihydro alkoxy benzyl oxopyrimidine SNAr nucleophilic aromatic substitution THF tetrahydrofuran NMR nuclear magnetic resonance FTIR Fourier-transform infrared spectroscopy HRMS (ES+) high resolution mass spectroscopy (electrospray ionisation) TLC thin layer chromatography m/z mass-to-charge ratio DMSO-d6 hexadeuterodimethyl sulfoxide CDCl3 deuterated chloroform EtOAc ethyl acetate NHD normal halogen dependence AlCl3 aluminium chloride 13 δ chemical shift TMS tetramethylsilane UV ultraviolet rt room temperature ppm parts per million Hz hertz NaHCO3 sodium hydrogen carbonate K2CO3 potassium carbonate Na2SO4 sodium sulphate NaOH sodium hydroxide ml millilitre(s) h hour(s) KOtBu potassium tert-butoxide DMA dimethylamine aq. aqueous 14 CHAPTER 1: INTRODUCTION AND BACKGROUND 1.1 HIV-AIDS Human immunodeficiency virus, typified by HIV-1 and HIV-2, is a member of the retroviridae family that spreads widely and causes acquired immune deficiency syndrome.1,2 While the two HIV types are similar in viral structures and mechanism of transmission, their origins, infection patterns, and ability to develop into chronic disorders and deaths differ.3–5 Compared to HIV-1, HIV-2 is less contagious and progression to AIDS is delayed, whereas infection with HIV-1 imparts high plasma viral load and deterioration of the immune system resulting in AIDS-related morbidity and mortality. Additionally, the level of viral replication is the main distinction between the two HIV strains, and host immunity is thought to have a significant role in the more effective management of HIV-2 infection.3 1.1.1 Global HIV prevalence The use of epidemiological estimates from the Joint United Nations Programme on HIV/AIDS is considered helpful for countries to report on their data and progress. As such, it is crucial for countries to provide adequate information, sufficient presumptions and interpretation that considers constraints required to generate estimates.6 The UNAIDS epidemiological estimates showed that HIV prevalence increased from 30.7 million7 to 37.7 million between 2010 and 2020 (Fig 1).7,8,9 Among the people living with HIV, 36 million were adults and 1.7 million were children below age fifteen.7 However, since the first reported case of HIV infection in the early 1980s, the HIV response has led to a tremendous success in containing the disease,6 although the responsive interventions vary between countries. One of the control strategies includes the implementation of antiretroviral therapy that is widely accessible and scaled up10 to more than 27.5 million people up to 2020.6 ART uptake has led to significant strides in the treatment of HIV infection.9 As a result, the global rates of AIDS-related fatalities declined by 47% from the peak in 2010 (Fig 1).6,7,9 However, the prevalence of HIV-related illnesses and deaths is consistently high in the Eastern Europe and Central Asia with noticeable increments of 43% and 32% ,respectively, from 2010 to 2020.6,7 Additionally, notwithstanding the gradual expansion of HIV testing capacity, approximately 6.1 million of HIV infected individuals were still uninformed about their status, resulting in a failure to be provided with proper care and treatment programs.7 15 Fig 1 Global HIV prevalence in 20209 1.1.2 HIV lifecycle Due to its relative importance, only the HIV-1 strain will be discussed here. HIV-1 predominantly spreads via certain body fluids and is transmitted through unprotected vaginal or anal sex, sharing contaminated needles with the infected person, and medical procedures involving piercing.11,12 Upon entry of HIV into the body, the viral envelope protein mediates insertion of the virus into a host cell membrane. This HIV-1 envelope consists of two components, transmembrane glycoprotein and the docking glycoprotein13 that make up the spikes on surface 49 of the virion.14 During the entry process, the gp120 connects to the host’s CD4 receptor, followed by interactions with one of the chemokine receptors (CCR5 or CXCR4).15 The co-receptor binding induces irreversible changes in the gp120 shape that enables the gp41 transmembrane protein to penetrate its hydrophobic fusion peptide into the host cell membrane, establishing the initial direct engagements between the virus and its target cell.16 Thereafter, the fusion of the virus with the host cell membrane occurs, and the viral core is subsequently released into the host cell cytoplasm. The HIV-1 life cycle is regarded as a concatenation of the steps detailed in Fig 2, which are controlled by three main enzymatic proteins; reverse transcriptase, integrase and protease. Since the viral genome reverse transcription and integration require both RT and IN, respectively, these enzymes are essential throughout the initial stages of the HIV viral replication cycle.17 The catalytic activities of RT enzyme that include RNA template-dependent DNA polymerization and DNA template-dependent DNA polymerization allow conversion of the single-stranded viral RNA genome into double-stranded DNA. Following the DNA-RNA hybrid strand creation, degradation of RNA strand by RNase H occurs leaving single-stranded DNA. Here, a complementary RNA strand is reverse transcribed by the viral 16 enzyme RT, generating the linear double-stranded DNA.4,13 At this stage, the IN enzyme travels through the nuclear membrane carrying the pre-integrated viral ds DNA and incorporates it into the host DNA. Using the cellular machinery of the host, the integrated viral DNA is now subjected to transcription and translation into viral polyprotein. This polyprotein is broken down into its constituent structural and functional proteins by the viral enzyme PR, resulting in viral maturation and the capacity of the mature virions to infect new host cells.4,13,17 Left untreated, HIV transitions into AIDS due to the immune system’s weakness when the infected individuals become vulnerable to destruction by the virus. Thus, these three enzymes have emerged as critical targets in the development of new drugs for HIV treatment. Fig 2 HIV life cycle14 1.1.3 Social and health effects of HIV/AIDS The time taken for HIV infection to develop into AIDS is approximately between five months to fifteen years. The first AIDS case was identified in the United States in 198118 among homosexual men, and although male-to-male sex is prevalent as a mode of HIV transmission in many parts of the developed countries, heterosexual sex is a primary element of the disease’s global transmissions responsible for 75% of infections.19 17 It has been reported that gender inequality also contributed in HIV exposure. Specifically, women are more socially vulnerable than men due to biological disparities in susceptibility, lack of sexual agency, as well as the power and privilege that men enjoy in sexual relationships.20 These effects are most recognised in sub-Saharan Africa, where women and young girls between fifteen and twenty-four years contributed 25% of infections, although this age group accounted for 10% of global infections recorded in 2020.7 Vertical HIV transmission on the other hand, can occur at any point during the pregnancy period increasing the chances of an HIV-positive mother infecting her unborn child, hampered by risk factors that include early delivery, low maternal cell-mediated immunity, prolonged labour, low CD4 counts, high viral loads, premature membrane rupture, delivery method, and untreated STDs.19,21 HIV can also be transmitted from mother to child through breast milk, although the likelihood of contracting the virus rises with lactation duration22 A number of methods have been implemented to prevent and reduce mother-to-child transmissions while breast-feeding.19 These alternative approaches involve premature discontinuation of breast-feeding and adoption of artificial feeding.23 1.1.4 Access, treatment, and care for people living with HIV There are many HIV prevention strategies in place that have not been used nearly enough with respect to the extent to which HIV infection spreads annually.27 To lower HIV transmission significantly and change the course of the epidemic, HIV typologies, transmission modes, and populations affected need to be well understood to intensify prevention activities as these factors influence how well evidence-based interventions can be combined and adapted.27 Prophylactic and antiretroviral therapies are two epidemiological strategies useful for HIV/AIDS control, with ART being appropriate to all HIV-infected individuals, whereas prophylaxis is applicable to all people at the risk of exposure to HIV infection.28 Prophylaxis shall be covered in the next section. As of 2015, the World Health Organization advises people living with HIV to begin antiretroviral therapy as soon as a positive test result is obtained for the maximum therapeutic benefits of effective viral suppression to be realized and to avoid additional transmission.29 ART development has prolonged the lifespan of HIV-infected individuals by controlling viral replication and helps immunological recovery. However, effective treatment needs ART consistency which is difficult for some people and the resulting relapses cause a burden on healthcare systems, especially in resource- constrained settings.30 Delayed treatment commencement, non-treatment adherence, and early therapy termination result in negative impact on the survival of HIV-infected people.31 ART discontinuation can either be temporary or permanent, intentional, or unplanned by health professionals or patient. Doctors might advise ART cessation either when underlying illnesses interfere 18 with oral therapy or surgery prevents oral therapy. In addition, ART discontinuation may be recommended when there is lack of antiretroviral drugs or when the medications are toxic or have severe side effects to patients.32 On the other hand, patients may decide to cease ART for reasons that include personal beliefs when a patient is virally suppressed and does not see the necessity to continue receiving treatment. Structural obstacles that include imprisonment and lack of money are additional barriers to ART discontinuation.32 Research conducted in Botswana indicated that inconsistent ART adherence accelerates the chances of seeing virus resistant to ARVs, requiring a shift to more expensive second- or third-line drug therapies.33 It was added that ART defaulters might have stopped taking their medication when financial problems made it difficult for them to pay transport to clinics where they could receive monthly treatment.33 Some participants reported that they started relying more on religion and spirituality as their coping approach.33 Coping was investigated in HIV-positive individuals in the late 1990s, and was defined as the behavioural and cognitive attempts made by an individual to change or minimize the problems brought on by a particular stressful situation.34 It was also emphasized that coping mechanisms had a significant impact on psychological effects of HIV infection. Furthermore, it was believed that HIV-infected patients who exhibit signs of psychological stress inhibit their anger by resorting to engagements in drug and alcohol use to ease their discomfort, thus, posing a significant burden to therapists.34 A decade later, another study in the US Southeast mentioned that alcohol and drug misuse is common among HIV-positive people, and is in turn linked to higher risk behaviours for secondary transmissions and lower ART adherence.35 These findings were supported by a recent study conducted in South Africa that alcohol use in people infected with HIV increases treatment inconsistency and lowers viral load suppression.36 Recently, several countries were heavily burdened by COVID-19, during which adherence to ART was disrupted in compliance to the implemented COVID-19 protocols, posing potential detrimental effects on HIV control efforts. During the COVID-19 pandemic, it was emphasized that barriers like constrained movement, prohibitive transportation costs, and stringent measures implemented to relieve the healthcare facilities and maintain social distance, and the fear of contracting and dying from COVID-19 limited access to healthcare.30,37,38 As such, many healthcare systems struggled to offer basic health services to patients due to severity of corona-virus disease.30 However, it was suggested that various medical facilities should assume leadership roles and provide care for individuals who have HIV and other immune-compromised illnesses to continue receiving HIV services.24 19 1.1.5 Newer tools for HIV/AIDS prevention and control 1.1.5.1 Provision of prophylactic agents Regular evaluation of knowledge, attitudes and behaviours that are considerate of gender roles and cultural norms is essential to help plan and administer prevention programs.39 Encouraging behaviour change, male circumcision, harm reduction initiatives for injecting substance use and HIV status awareness about testing and effectiveness of condom use are just a few of the comprehensive and successful public health programs. Many other interventions include voluntary counselling and testing, preventing vertical transmission, identifying cases of, and treating STIs and supply of post exposure prophylaxis,27 and pre-exposure prophylaxis.39 PrEP and PEP are two efficient prophylactic techniques currently accessible for use in preventing HIV infection. These methods work best when combined with non-pharmacological awareness tools like safe sex education and other synergistic and syndemic effects of the co-existing risk factors. WHO recommended PEP as a brief antiretroviral medication used to lower the risk of HIV infection after accidental exposure. It is potent if used within a 72-hour time frame, but its effectiveness depends on clinic attendance and adherence to the prescribed 28-day course of treatment. WHO advised in 2015 that regardless of gender, PrEP should be made available as a preventive measure to all those who have increased chance of HIV acquisition.40 People on PrEP are HIV negative, often young and have few comorbid conditions, if any, as opposed to those on ART. Furthermore, there are few restrictions to PrEP because it is a short-term control measure that can be stopped at any time depending on the person’s preferences and risk levels of acquiring the infection, whereas antiretroviral therapy is continued throughout one’s life.41 PrEP displays 99% effectiveness when following the directed dosage, but poor compliance lowers its potency and the value to public health.42 1.1.5.2 Implementation and monitoring of programs The 2016 Political Declaration on HIV initiated a plan aimed to lower new HIV infections and AIDS- related deaths by 90% from 2010 to 2030 with at least 75% reduction by 2020. Additionally, the developments of the 90-90-90 testing and treatment targets were committed to eradicating the AIDS epidemic by 2030, which was implemented as a 90% decrease from 2010 in the number of new HIV infections and AIDS-related mortalities. This strategy states that 90% of people infected with HIV are aware of their status, 90% of HIV-positive individuals initiate ART and 90% of those on ART should be virally suppressed. The new Global AIDS Strategy and the 2021 Political declaration also incorporated extremely ambitious 95-95-95 targets.6,7 Although encouraging from a theoretical perspective, realistically these targets are quite elusive, especially in such a short period of time. The global efforts to lower new HIV infections by 75% failed.6,7 20 Since 2010, the overall number of new infections decreased by just 31% (Fig 1), far from reaching the 75% objective for the year 2020.6,7 Despite global gains being achieved by deployment of programs that use treatment as a preventative measure,43 approximately 1.5 million new infections (Fig 1) were recorded in 2020,6,7,9,10 with the majority of the infected people living in sub-Saharan Africa, contributing 67%.9 Moreover, despite the great advancements in awareness of HIV treatment and prevention to achieve epidemic control, sexual transmission, a key factor in the mode of HIV infection in sub-Saharan Africa, continues to hamper the potential of an HIV/AIDS-free generation.10 Assefa and Gilks pointed out that when the national overall number of people infected with HIV drops to the levels last observed at the beginning of the HIV pandemic, the disease can be declared over.44 Further reviewing the 90-90-90 targets, it was estimated that 84-87-90 testing and treatment targets were accomplished across the world by the end of 2020. Although promising that the third 90 target was achieved, this is only applicable to people who are informed about their HIV status and are receiving treatment. As of 2020, the overall percentage of all HIV-infected individuals with suppressed viral loads was 66% (Fig 3).6,7 Moreover, though narrow, there is still a missing gap of people infected with HIV who know their status but do not receive ART. According to literature, ART coverage was predicted to rise at a global rate of 59% in 2017 to 64.8% by 2020, also to 71.9% by 2030. Notwithstanding the significant advancements these predicted gains signify, they however missed the 81% target of the UNAIDS by the year 2020 and reflect slow progress towards 90% coverage by 2030.43 Fig 3 The 2020 global HIV testing and treatment cascade8 21 Therefore, the HIV outbreak does not seem close to its end, and it is envisioned to remain a great threat across the world. However, HIV infection may decrease if remedial action is taken, according to the International AIDS Society-Lancet Commission.44 Reaching project 2030, whilst recognizing the global developments of therapeutic programs as preventive measures, raises concerning questions about additional steps required to encourage those particularly ignorant of their HIV-positive status to initiate ART and ensure its adherence. Again, it is unclear how each country will ensure that the 3rd 90 target is maintained, given risk behaviours arising from ART defaulters. Another primary uncertainty is associated with the financial demands. Proper planning and allocation of resources will be required for PrEP and ART scale-up to achieve the project 2030 target. Chen and co-workers in their study, estimated that USD 4.7 trillion will be needed annually to fund ART supply for 90% of the global 37 million people infected with HIV.45 However, the funding for HIV/AIDS is currently stagnating because of the unwillingness to maintain funding for the disease.44 Avancena and Hutton urged the donors and recipients to alleviate the financing gaps that could jeopardize the disease progress made by contributing additional funds and increasing the impact of existing resources.46 HIV/AIDS persists in spreading across the world and there is still no viable vaccine or cure for the disease.10 Therefore, our research arises as part of the ongoing global campaign to search for novel anti-HIV therapeutic drugs that can have high anti-HIV activity, reduced toxicity, and selectivity features to help combat transmission of the concomitant pandemic. 1.1.6 HIV-1 reverse transcriptase inhibitors in the treatment of HIV The current standard regimen for HIV/AIDS treatment is a triple therapy combination, which consists of two nucleoside reverse transcriptase inhibitors plus either a protease inhibitor or an integrase inhibitor or non-nucleoside reverse transcriptase inhibitors. Highly active antiretroviral therapy is the term most frequently used to describe this combination therapy. Because monotherapies fall short in their capacity to prevent HIV-1 replication, HAART is used to block numerous viral proteins at various stages in the HIV life cycle. However, HAART does not eradicate the virus completely, but when adhered to effectively, HIV is changed from a deadly condition to a controllable chronic illness.47–49 Multiple medications can be harmful and ultimately offer health hazards due to prolonged use and drug-drug interactions. 49 HIV-1 RT is the first enzyme utilized by the virus when it enters the body of a host, and it controls the genetic replication of genetic material of HIV. RT is the most common target in the field of antiretroviral drug development. Structurally, HIV-1 RT is an asymmetric heterodimer containing two subunits (Fig 4) namely the p66 subunit which is vital for carrying out enzymatic activities and the p51 22 subunit that gives the structural and conformational support for RT. The catalytic site of HIV-1 RT is situated within the palm subdomain and is the primary target of the NRTIs whereas the hydrophobic pocket is allosterically targeted by NNRTIs.17,50 Fig 4 HIV-1 RT enzyme structure 1.1.6.1 NRTIs mechanism of action NRTIs are nucleosides that lack the 3’-hydroxyl group on the deoxyribose moiety.15 They enable proper base pairing and inclusion into DNA chains after phosphorylation, but a crucial hydroxyl group that is necessary for the insertion of the following nucleotide is missing. These competitive inhibitors act by mimicking the nucleotides that make up the viral DNA, and once incorporated into nascent viral DNA, they prevent the viral DNA chain from growing and thus, terminate the DNA chain extension.13,15,48 However, their activity depends on the ability of cellular kinases to convert them metabolically into an active triphosphate form. Such NRTIs include tenofovir 1, lamivudine 2, and emtricitabine 3 (Fig 5), which are considered as South Africa’s first-line treatment regimens.13 Fig 5 Some of the approved NRTIs: tenofovir 1, lamivudine 2, and emtricitabine 3 23 1.1.6.2 NNRTIs mechanism of action NNRTIs, which also target RT, are one of the most important components in drug combination therapies. They garnered considerable attention among the various types of anti-HIV therapeutic agents because of their low toxicity, high specificity, and high levels of antiviral activity.51 NNRTIs are non-competitive inhibitors discovered in 1990 that effectively deactivate polymerization of HIV-1 RT without the need for activation through phosphorylation. These drugs act by causing a conformational shift in the RT enzyme, through attachment to a hydrophobic region just outside the RT active site known as the NNRTI binding pocket, located 10 Å away from the DNA polymerase active site.47,51,52 Using X-ray crystal structures of NNRTIs in association with RT, studies revealed that NNRTIs retain comparable interaction modalities and binding patterns to RT, despite their structural differences. A variety of NNRTI structures could be compared to a butterfly, horseshoe, or U-like shape.48,53 They appear to be electron rich and have the potential to donate π-electrons to aromatic side chain residues near the binding pocket.48 As of September 2019, the Food and Drug Administration had approved six NNRTIs, shown in Fig 6. Nevirapine 4 was the first NNRTI drug discovered by Boehringer Ingelheim, and was approved by the FDA for use when combined with nucleoside derivatives in USA in June 199633,52,53 and in the European Union in 1998.53 This dipyridodiazepinone NNRTI15 has strong metabolic stability, excellent biological activity, and is readily able to penetrate the blood-brain barrier,53 but has unpleasant side effects and a low genetic barrier to resistance.52 Delavirdine 5 that belongs to a family of bis(heteroaryl)piperazines, was discovered by ViiV Healthcare, and was the second NNRTI to be approved by the FDA in 1997.15 Its use was however ceased owing to similar drawbacks to those of nevirapine. Efavirenz 6 is a benzoxazinone second generation NNRTI approved by the FDA in September 1998.15 It was said to drastically reduce the activity of HIV-1 in cell culture when combined with NRTIs like didanosine, zidovudine and the protease inhibitor indinavir. However, its antiviral effectiveness is often dramatically diminished after two or more mutations in HIV-1 RT. The FDA-approved NNRTIs etravirine 7 and rilpivirine 8 are diarylpyrimidines that share common structures and binding mechanisms in the NNIBP which enables both drugs to be rearranged inside RT, to compensate for the amino acid alterations caused by mutations.47,48 Doravirine 9 is the most recent NNRTI drug discovered by Merck & Co. to receive approval by the FDA in August 2018. Due to its several benefits including favourable safety profile, substantial efficacy, and low risk of developing resistance, it is currently administered to treatment naïve patients.15 24 Fig 6 FDA approved NNRTIs: Nevirapine 4, Delavirdine 5, Efavirenz 6, etravirine 7, rilpivirine 8, and doravirine 9 1.1.6.3 Drug resistance to NNRTIs During drug therapy, resistance to NNRTIs quickly develops because of the low fidelity of HIV-1 RT and high level of HIV-1 replication47 that results in mutations that change the amino acids surrounding the NNIBP. This reduces the potency of NNRTIs, leading to treatment failures.48 NNRTIs 4, 5, and 6 can successfully halt the reproduction of HIV-1 wild-type virus but are less active against several frequently seen mutant viruses which include Y181C, L100I, Y188C, and K103N.54 Among the reported HIV-1 mutations, K103N is the most prevalent NNRTI resistant mutation. The K103N is a non-polymorphic mutation present on the loop that links β5 and β6 in the p66 subunit of RT situated near the binding pocket, and is positioned farther away from the RT active site.55 The X- ray crystal structure of a K103N mutant comprising Tyr181 bound to efavirenz in exchangeable positions is represented in Fig 7.13 The K103N mutation blocks the entrance of efavirenz to the NNIBP causing the unliganded conformation of NNIBP to remain unchanged. The presence of the K103N mutation also lowers the activity of these first generation NNRTIs, resulting in the IC50 values ranging from 20 to 55 times higher.56 Thus, evaluation of challenges related to the evolving drug resistance is imperative for examining different scaffolds as potential pathways towards the development of alternative NNRTI therapeutic agents. 25 Fig 7 Efavirenz interacting with Tyr 181 in the wild type The DAPY NNRTI drugs etravirine 7 and rilpivirine 8 were developed using structure-based multidisciplinary strategies to prevent the activity of HIV-1 bearing similar NNRTI-resistant mutations. They have a higher genetic barrier to resistance than the former NNRTI drugs 4, 5, and 6.55 They retain their efficacy against the wild type virus and additionally, unlike the first and second generation NNRTIs 4, 5, and 6, the DAPYs are not sensitive to the K103N mutation, and substantially preserve almost all of their effectiveness.13 The intrinsic torsional freedom of the DAPYs enables them to shift into various conformational modes through orientation (wiggling) and reposition (jiggling) to fit in the NNIBP alterations induced by different RT resistance mutations.57,58 Again, compared to nevirapine that is structurally rigid, the inhibitors’ relative flexibility may allow the DAPYs to easily adapt to a drug pocket that has undergone mutation.57 Despite that, Changunda59 suggested that caution should be exercised while exploring the idea of more conformational flexibility to preserve the best possible balance between the benefits that could be obtained from either the improved torsional mobility or less constrained rotation. He added that forsaking limited rotation at the expense of increased torsional flexibility might offer excessive amount of entropic energy to the ligand. Consequently, this could restrict the ligand from locking itself inside the enzyme’s binding site, and in turn negate the purpose of boosting the ligand binding affinity.59 1.2 Synthetic targets with interesting biological activity 1.2.1. Benzamides as structural cores Continuous efforts made in the development of new NNRTIs in our laboratory led to the identification of benzamide compounds possessing anti-HIV activity.59 Benzamide compounds are classified as carbonic acid amide derivatives of benzoic acids.60 An amide (CONR) commonly referred to as 26 carboxamide or organic amide, is a chemical molecule having a generic formula RC(C=O)NR’R”, where R, R’, and R” represent the organic groups or hydrogen atoms. Carboxamides in their classical configuration, have a central carbon skeleton attached simultaneously to the nitrogen and oxygen atoms through single and double bonds respectively.61,62 Based on the number of substituents bonded to the nitrogen atom, amides are categorized into primary, secondary, and tertiary subclasses. The primary amide is obtained when the hydroxyl group (OH) of a carboxylic acid is displaced by the NH2 amino group. When other groups substitute one or both hydrogen atoms in the primary amides, the resulting molecule is referred to as a secondary or tertiary carboxamide.60 1.2.1.1 Biological activity of benzamides Benzamide-based derivatives have been identified as a valuable class of compounds that inhibit histone deacetylase both in in vivo and in vitro anticancer studies.63 Two examples of the benzamide- containing drugs that have undergone clinical trials include entinostat 9 and mocetinostat 10 (Fig 8). Entinostat 9 is an effective class I selective histone deacetylase (HDAC) inhibitor that can be used alone or in conjunction with other medications due to its high level of tolerability. The combination of entinostat and 13-cis-retinoic acid was examined in a phase I trial to ascertain their tolerability, safety, pharmacodynamics/pharmacokinetic characteristics in advanced solid tumours. Despite their lack of activity, the combination therapy was well tolerated, and patients with kidney, pancreatic, and prostate cancer experienced prolonged stable illness. In breast cancer patients, the effects of entinostat alone or in combination with the aromatase inhibitor exemestane were assessed in a placebo-controlled and randomized phase II research. The results highlighted the safety, and clinical efficacy of this combination therapy in ER+ advanced breast cancer patients. Mocetinostat 10 is an HDAC inhibitor that targets both Class I and IV histone deacetylases with high specificity. The safety and antileukaemic efficacy of mocetinostat were both demonstrated in a phase I trial in individuals with myelodysplastic syndrome or leukaemia. An effective treatment with a controllable side effect profile was shown in a phase II trial in individuals infected with chronic lymphocytic leukaemia.63 Fig 8 Structures of entinostat 9 and mocetinostat 10 27 Other significant biological characteristics of benzamides are shown by N-(4-chlorophenyl)-4- methoxy-3-(methylamino)benzamide, IMB-0523 11 (Fig 9), which is a drug candidate for inhibiting Hepatitis B virus (HBV), with a promising selectivity index and anti-HBV profile.64 Fig 9 Chemical structure of IMB-0523 11 Sorafenib 12 (Fig 10) is also a benzamide-containing drug that has pharmacophoric profiles such as H- bond donor/acceptor, terminal lipophilic moiety, central aromatic linker, and heteroatomic ring,65 which received approval for use in Europe as a treatment for hepatocellular carcinoma.66 Fig 10 Pharmacophoric features of sorafenib 12 and related analogues 13 and 14 Eissa and co-workers assessed benzoxazole benzamide compounds related to sorafenib as potential anti-proliferative inhibitors of VEGFR-2 (vascular endothelial growth factor receptor-2) and apoptotic inducers.66 Compounds 13 and 14 (Fig 10), were deemed most effective and displayed pharmacophoric features that are indistinguishable from those of 12, with superior docking scores 28 relative to that of 12. One feature of interest is that of the amide group, which is crucial as a hydrogen- bond donor/acceptor present in both 12 and compounds 13 and 14. These compounds exhibited different activities against the VEGFR enzyme, with 13 (IC50= 0.268 µM) being more active than 14 (IC50= 0.361 µM) and 12 (IC50= 0.352 µM).66 Al-Masoudi and colleagues examined a range of thiazol-2-yliedene-benzamide derivatives for anti- HIV-1 and anti-HIV-2 activities based on a Quantitative Structure-Activity Relationship (QSAR).67 In reference to their results, twenty of their compounds were deduced to have adopted a butterfly-like conformation, which is significant for RT inhibitory efficacy against HIV-1. Among the twenty compounds, analogues 15 and 16 (Fig 11) were hypothesized to act as potential NNRTIs because of their extraordinary antiviral effectiveness.67 Fig 11 Structures of compounds 15 and 16 Scala and co-workers also evaluated the anti-HIV potency of compounds 17 and 18 in vitro in a classic MTT-MT4 test.68 Compound 18 with a benzamide moiety at the N1 position of the pyrazole ring was found to be effective at blocking HIV proliferation with low cytotoxicity and a considerable selectivity index (EC50= 2.77 µM, CC50= 116.7 µM, and SI= 68). Even though compound 17 also consists of a benzamide tail, it did not appear to exhibit the antiviral activity in comparison to scaffold 18, thus, highlighting the crucial role displayed by the position of the benzamide motif present in 18 (Fig 12). Impressively, it exhibited the RT inhibitor properties, dramatically lowering RT DNA synthesis, albeit to a lesser extent than shown by AZT.68 Fig 12 Structures of compounds 17 and 18 29 1.2.1.2 Amide bond formation One of the most significant organic processes involves the formation of amide bonds since these bonds frequently occur in both natural and industrial products69 such as plastics, fabrics, insecticides, and fertilizers.70 The amide functionality is also present in various synthetic structures and serves as the protein’s structural backbone. Polymers containing the amide bond are fundamental for use in a vast number of applications from materials that include Kevlar and nylon to more sophisticated uses for biological applications like drug administration.71 Given that the amide bond is present in 25% of all medications now available on the market,71,72 amide synthesis is equally important in small scale and large-scale processes,73 with amidation reactions the most widely employed techniques in medicinal chemistry.72 1.2.1.2.1 Induced thermal and microwave-assisted amidation reactions The amidation reaction is characterized by a condensation process between carboxylic acids 19 and amines 20 (Scheme 1). Usually, the amidation reactions are nonspontaneous74 and require conditions like high temperatures above 100 °C to overcome the formation of unreactive carboxylate ammonium salt 21 (Scheme 1). The benefits of thermal conditions include the ease of use and great atom economy.71 However, substrates sensitive to high reaction temperatures like amino acids should not be exposed to such harsh conditions.75 In addition, the reactants must exhibit particular features that include excellent thermal stability, low volatility, and melting points under 200 °C.74 Furthermore, the preparative utility of thermal amidation can be restricted by the amide’s dehydration at increased reaction temperatures to produce a corresponding nitrile or tar.71 Scheme 1 Thermal amidation v/s coupling reagent amidation75 Mitchell and Reid were the first authors to publish primary amide synthesis from ammonia and aliphatic carboxylic acids under thermal conditions at approximately 190 °C in 1931.76 Although good yields were obtained, the products were often contaminated with either nitrile or ammonium salt or both, and in some instances, autoclaving was necessary to dehydrate the ammonium salts.76 A 30 commercial production of dimethylacetamide was carried out following the similar method, with the product being isolated as an azeotrope after the reaction of dimethylamine and acetic acid.77 It appeared that with the optimization of reaction conditions to pressures above 6200 kPa and high temperatures between 250 and 325 ˚C, the formation of azeotrope could be lowered by reacting excess amounts of dimethylamine with acetic acid.77 Using 4 Å molecular sieves, a variety of amides synthesized without using catalysts or coupling reagents at 140 ˚C were reported by Cossy and Pale-Grosdemange in 1989.78 At temperatures ranging from 120 °C to 160 ˚C, amides were formed by reacting neat mixtures of carboxylic acid and amines under 3 Å molecular sieves. Compared to benzylamine which gave 75% yield, the reaction between benzoic acid and N-benzylamine only provided trace amounts of the product at 160 ̊ C after 24 hours.79 Thus, direct amide bond formation from uncatalyzed amines and carboxylic acids at temperatures below those previously reported was explored.80 Conducting the reaction in a sealed vessel in the absence of molecular sieves promoted a favourable equilibrium without the efforts to eliminate water, and the use of non-polar solvents like toluene facilitated the formation of amides at 110 °C for 22 h. Although the conversion rates were unsatisfactory in most cases, at least N-benzylpropionamide 22 was isolated in 79% yield under similar conditions (Scheme 2).80 Scheme 2 Reagents and conditions: (a) Toluene (2.0M), 110 °C, 22 h In 2008, Herrero and colleagues reported the effects of nonthermal microwave and conventional thermal conditions on the direct amidation reaction of methacrylic acid 23 with (R)-1- phenylethylamine 24 in the absence of activating or coupling reagents (Scheme 3).81 In a single mode microwave reactor, placing equivalent amounts of the starting materials at 180 °C monitored by a calibrated IR sensor, and applying simultaneous cooling afforded methacrylamide 25 in 90% yield after 15 minutes. The calibrated technique used estimated the real internal temperature to be around ca. 200 °C. Carrying out a similar experiment at the same reaction temperature and time by applying conventional heating afforded only 12% of 25, while increasing the reaction time two-fold proved ineffective in improving the yields. It was further demonstrated that at the same temperature range, different product distributions were isolated when comparing the results from the microwave reactions and conventional heating.81 31 Scheme 3 Amidation reaction between 23 and 24 by thermal and microwave energy Khadse and co-workers outlined the ring opening of dimethylaminobenzylidene oxazolone 26 (Scheme 4), based on thermal effects, by employing conventional heating and induced microwave- assisted irradiation for the synthesis of benzamides.82 It was reported that the conventional method was time consuming and resulted in decomposition of materials in the reaction vessel. Additionally, getting the desired product was difficult even when different solvents were used. Furthermore, a variety of by-products were obtained when performing similar reactions using polar solvents like dimethylsulfoxide or dimethylformamide, resulting in failure to separate the target compounds from the resultant mixtures. In contrast, when repeating similar reactions using microwave conditions, the reaction time was significantly decreased without decomposition and co-products, while minimal work-up was necessary for the product purity. Although direct interaction of the microwave energy with the reaction mixture accelerates the reaction, this method, however, suffers if the energy penetration depth is poor, thus, impeding the reaction scale-up.82 R= X= F, Cl, Me, OMe Scheme 4 Reagents and conditions: (a) p-Dimethylaminobenzaldehyde, (CH3CO)2O, CH3COONa, heat; (b) R-NH2, acetonitrile, triethylamine, MW, 420-560 Watt, 8-20 min, 65% 32 1.2.1.2.2 Amide synthesis using peptide coupling reagents Direct amide synthesis can easily be performed from various commercially available reagents under mild reaction conditions.74 The OH¯ group is a strong base and a poor leaving group, so the OH¯ from carboxylic acid needs to be converted into a reactive intermediate to create a better leaving group.75 To circumvent the high energy barrier for making amides, these activating agents are commonly used to promote efficient amide bond formation, though the selection of an effective coupling reagent is imperative. As demonstrated by library-based synthesis in medicinal chemistry, a wide variety of substrates with different reactivities such as bulky substrates, secondary amines and anilines are often employed to produce amides. This broad range of reactivity would need to be well tolerated by the coupling reagents.83 Forty-five percent of amidation reactions are reportedly conducted by employing carbodiimides that include 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide 27, N,N- dicyclohexylcarbodiimide 28, and uronium salts like (2-(1H-benzotriazol-1-yl)-1,1,3,3- tetramethyluronium hexaflurophosphate 29, and 1-[Bis(dimethylamino)methylene]-1H-1,2,3- triazolo[4,5-b]-pyridinium 3-oxide hexafluorophosphate 30, while 4% are performed by using 1,1’- carbonyldiimidazole 31 and n-propylphosphonic acid anhydride 32 (Fig 13).73 Fig 13 Structures of carbodiimides and uronium salts DCC 28 was first documented in 1955 by Sheehan and Hess for coupling reactions of carboxylic acids and amines.84a Scheme 5 illustrates the coupling mechanism for which the two reagents combine. In the first step, carboxylic acid interacts with 28 to produce an active O-acylisourea intermediate 33 which in turn reacts readily with nucleophiles such as amines. Following the formation of this intermediate, a variety of products are produced; 33 (i) the amide formation through the amine coupling resulting in the establishment of dicyclohexylurea 34. This by-product is a precipitate that can be eliminated by filtration. (ii) a by-product referred to as N-acylurea 35.83 Ideally, the temperature should be low to prevent its production.84a (iii) the carboxylic acid anhydride production, which upon interaction with the amine (often requires 2 equivalents of acid) provides the amide.83 The reaction is usually carried out at equal stoichiometric amounts of all the necessary reagents which would need to be dissolved in various aprotic solvents that include but are not limited to DMF and chloroform. For the formation of the desired amide product, the mixture is then subjected to ice-cold conditions and left overnight to reach room temperature. Where relevant, to lower the broad racemization that is observed with DCC alone in peptide and protein synthesis, N-hydroxysuccinimide (HOSu) is normally introduced to mediate the DCC coupling process84a due to its high nucleophilicity towards the carbonyl carbon of the carboxylic acid.84c The resulting HOSu ester rapidly reacts with the amino group of the protein84b preserving the chirality of the peptide activated compounds susceptible to racemization.84c Scheme 5 Mechanism of amide synthesis using DCC 83,85 For the synthesis of amide prodrugs 36, Makhija et al86 examined the reactivity of various nonsteroidal anti-inflammatory drugs and sulfonamides exemplified by ibuprofen 37 and suphamethoxazole 38, respectively, employing DCC 28 in the absence of additives (Scheme 6). To reduce the levels of N- acylurea 35, the reaction of carboxylic acid 37 and 28 was allowed to proceed for 2 hours under the pre-cold conditions and left stirring overnight at ambient temperature. Once the reaction was 34 complete, elimination of dicyclohexylurea by filtration was applicable to afford derivative 36 in 76% yield.86 Scheme 6 Reaction conditions: (a) DCC 28, DCM, 0 °C, 2h, rt, overnight, 76% Since the unveiling of DCC 28 as a coupling agent in amidation reactions, several other carbodiimides such as CDI 31 have also been studied. For instance, Lee and Kim presented the efficient coupling of ammonium acetate (NH4OAc) and carboxylic acid in the presence of 31 in ionic liquid medium for the preparation of primary amides 39 (Scheme 7).87 It was reported that the amide bond was successfully formed by one-pot sequential addition of the various carboxylic acids (Represented by 40) and 31 in 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM]BF4. Subsequent treatment with NH4OAc and triethylamine (Et3N) at 80 °C yielded the corresponding amide 39 in 92%. Scheme 7 Reaction conditions: (a) 31, [BMIM]BF4, 80 °C, 2h; (b) NH4OAc, Et3N, 80 °C, 3 h, 92% Another popular peptide coupling reagent EDC 27, also attracted a great deal of attention due to the ease with which its side product, dimethylaminopropyl-3-ethylurea is separable from the product. This urea is highly soluble and can effortlessly be removed during aqueous extraction.88 In 1993, Desai and Stramiello performed amidation reactions using a polymer-bound EDC, where single coupled products with yields ranging from 77-97% were isolated. Chloroform was selected as a solvent of choice since performing the reactions in the presence of either tetrahydrofuran or ether led to insufficient swelling of reagents, resulting in slow reaction.89 In 1995, Nakajima and Ikanda examined the function of 27 in an aqueous system to study the impact of dissociation and pH of the amino and carboxyl groups in amidation reactions.90 In this study, hydrogels (Gel-A and Gel-B) containing carboxyl groups were used to simply separate the products from the reaction batch. In comparison to Gel-A, the amide formation was substantially elevated in Gel-B due to different positions where the carboxyl groups were situated along the polymer chains.90 35 Recently, Ghosh and Shahabi demonstrated that EDC 27 can effectively promote the coupling of Boc- protected acid 41 and electron deficient amine 42 in the presence of both the HOBt and DMAP coupling reagents.91 It was shown that 0.1 equivalents of DMAP and HOBt were insufficient to promote the reaction since only 38% yield of 43 could be obtained. Instead, excess molar quantities of DMAP (1 equiv) were required to enhance the reaction yield to 72%. The general reaction mechanism to which both additives mediate the coupling is illustrated in Scheme 8. The reactive HOBt ester 44 is produced during the interaction of acid 41 and 27 together with HOBt, followed by the acyl transfer formation of the highly reactive acyl iminium ion 45 driven by DMAP. The reaction between 44 and less reactive amines like aniline was anticipated to proceed slowly. In the last step, the reaction between 42 and intermediate 45 ought to proceed more rapidly, allowing the generation of DMAP and the formation of product 43. Scheme 8 Mechanism of amide formation using EDC/HOBt coupling Despite the significance of the peptide coupling reagents to improve the yields and allow mild reaction conditions, excess stoichiometric quantities of these substances are needed. Consequently, each product molecule generates at least one comparable amount of waste, which is rendered strenuous and is time-consuming to eliminate from the reaction mixture, thus, raising transformation costs.70,71,73,74,75,92 1.2.1.5 Amide bond formation from acyl chlorides The preparation of therapeutic agents by three pharmaceutical companies was examined in 2006, and it was discovered that out of 128 inspected compounds 65% contained a minimum of one amide unit. 36 It was reported that 36% of these compounds were synthesized through the application of coupling reagents, whilst 44% were made by employing acyl chloride precursors.71 Acyl chlorides also known as acid chlorides, are one of the most common and straightforward intermediates convenient for the production of amide bonds which do not involve peptides, and are usually used when there is no risk for racemization, hydrolysis, or cleavage of the protecting groups. Acid chlorides are normally prepared using a vast array of chlorinating reagents such as phosphorous trichloride, phosphorus pentachloride, phosphorus oxychloride, oxalyl chloride, thionyl chloride,88,93 or cyanuric chloride75 (Fig 14). The phosphorus, carbon, or sulphur atoms of these reagents activate the carbonyl carbon from the carboxylic acid93 by providing the chloride which substitutes the hydroxyl group. Fig 14 Structures of chlorinating reagents (COCl)2 is often used for the production of acyl chlorides in process chemistry. Its application is advantageous due to its low boiling point (61 °C) that makes it easier to remove from the reaction mixture by distillation.94 However, owing to the stoichiometric liberation of three gases, carbon monoxide, carbon dioxide, and hydrochloric acid, the use of oxalyl chloride is somewhat toxic and potentially harmful. The phosphorus-containing reagents are often used in circumstances where the acid is incompatible with the thionyl chloride. 93 Although the phosphorus chlorinating reagents are relatively affordable and easily accessible, they are less frequently utilized on large-scale for the synthesis of the acid chlorides.94 The most frequently reported methods involve the small-scale syntheses. For example, Boyer and co-workers reported the formation of picryl chloride using POCl3 on a milligram scale.95 Zeng and colleagues synthesized itaconyl chloride from itaconic acid and PCl5 on a milliliter scale.96 Various benzoyl chlorides were also produced on a millimole scale by Xiao and Han employing PCl3.97 Cyanuric chloride is readily accessible on the market, non-expensive,98 and can be used on large-scale processes. Its advantages include functional group tolerance, and reduced side-product generation. Additionally, the insoluble cyanuric acid by-product can be easily eliminated by filtration.99 Senier heated cyanuric chloride together with sodium salts of the carboxylic acid at 100 °C for eight hours to prepare benzoyl and acetyl chlorides in 1886.100 Venkataraman and Wagle performed similar reactions at room temperature for three hours. The acid chloride rapidly formed upon treating cyanuric chloride with two equivalents of the acid and triethylamine, through the sigma complex 46 (Scheme 9), 37 occurring from a nucleophilic attack by the oxygen atom of the acid on cyanuric chloride. The resulting insoluble precipitate 47 described as a corresponding monoanilino or dianilino derivative, is termed chlorodihydroxy-s-triazine or dichlorohydroxytriazine.100 Scheme 9 Acyl chloride formation from cyanuric chloride In 2002, Luo and colleagues prepared amino resin-bound dichlorotriazine (DCT) from a reaction of cyanuric chloride with the Wang alcohol resin as a chlorinating agent for the synthesis of acyl chlorides (Scheme 10).101 In 2008, Betti and co-workers demonstrated that cyanuric chloride can be used in ionic liquids for the Beckmann rearrangement of oximes to prepare amides.102 Recently, Yadav and Awashthi prepared a triazine-linked covalent organic polymer from cyanuric chloride and p- aminophenol to effectively promote the coupling of carboxylic acids with amines.103 Scheme 10 Acid chloride synthesis through DCT Thionyl chloride is the most popular reagent employed for the activation of carboxylic acids because it is volatile, inexpensive, and among the most cost-effective methods for making acyl chlorides.93 The conversion of carboxylic acids into acyl chlorides through SOCl2 can be done neat or using a solvent.88 The most frequently reported solvents employed when carrying out reactions following the thionyl chloride route include n-heptane, acetonitrile, dichloromethane, toluene, and tetrahydrofuran.94 Additionally, the application of thermal energy is required, and in some instances, pyridine can be added to speed up the process.88 The thionyl chloride approach liberates sulfur dioxide and 38 hydrochloric acid gases as the only by-products present in the reaction vessel. These gases are considered non-carcinogenic although SOCl2 and the generated HCl can practically pose significant challenges due to high corrosive nature of the acid. Owing to their low molecular masses can effortlessly be eliminated through distillation.71 A schematic representation of amide bond formation is detailed below in Scheme 11, where the carboxylic acid attacks thionyl chloride 47 to yield unstable and highly electrophilic intermediate 48. Subsequently, protonation of 48 by the generated HCl gives rise to carbonium ion 49 which is attacked by a weakly nucleophilic chloride ion. The resulting intermediate 50 is a tetrahedral structure that can disintegrate into the acid chloride 51, sulfur dioxide, and HCl. Scheme 11 Mechanism of the acyl chloride formation104 Upon the formation of the acid chloride 51, subsequent in situ aminolysis can be carried out while using an organic base to prevent the deprotonation of an amine into its unreactive HCl salt and absorb the resultant HCl. Inert anhydrous solvents are typically used for the couplings with non-nucleophilic tertiary amines such as diisopropylmethylamine (Hunig’ base), triethylamine, N-methylmorpholine,88 or pyridine 52. In rare instances, pyridine can be employed as a solvent to enhance solubility and accelerate the reaction. Since 52 reacts more effectively with 51 than amine 20, it functions as an acyl transfer catalyst in the amination process via nucleophilic acyl substitution reaction resulting in the formation of acyl-pyridinium salt adduct 53 (Scheme 12).93 This behavior is attributable to the robust Lewis basic nature and comparative soft character of the nitrogen atom on 52. Relative to the acyl chloride, the resulting ionic complex 53 is highly electrophilic and susceptible to nucleophilic attack by an amine, exacerbated by the nitrogen’s positive charge.93 Although acid chlorides are water sensitive, they can react with amines under aqueous conditions in the presence of sodium hydrogen carbonate, 39 potassium carbonate, potassium phosphate, or sodium hydroxide.94 This transformation is commonly known as the Schotten-Baumann reaction.70,71 Scheme 12 Mechanism amide bond formation by acyl-transfer with pyridine Kwolek and colleagues outlined the synthesis of poly(1.4-benzamide) 54 starting from the reaction of p-aminobenzoic acid with two equivalents of thionyl chloride under reflux for 3 h (Scheme 13). 105 The resulting acyl chloride 55 was obtained in 95% yield and used for further derivatization to afford polymer 54. Scheme 13 Synthesis of 54 via thionyl chloride route Acrylol chloride was prepared by reacting acrylic acid with thionyl chloride, and reacted without prior isolation with stoichiometric quantities of anilines using N,N-dimethylacetamide (DMAC) as a solvent in the presence of aqueous NaOH to give corresponding acrylamides in 88-98%.106 It was observed that the use of DMAC offered benefits over DMF in terms of stability and conversion rates. However, because DMAC appeared to react with thionyl chloride, cautious addition was necessary to circumvent the reactivity problem. Similar reactions were carried out by heating acyl chlorides with stoichiometric amounts of benzylamine without the presence of a base resulting in 80-90% yields of benzylamides exemplified by 56 (Scheme 14).106 Scheme 14 Reagents and conditions: (a) SOCl2, DMAC, -5 °C; (b) benzylamine, 0-60 °C, 80-90% 40 The synthesis of benzamides 57 as possible antiprofiliferative agents was reported by Asaki and colleagues.107 In this experiment, various carboxylic acids were first esterified by ethanol, α- brominated, and coupled with N-bromosuccinimide. The resultant esters were hydrolyzed, and subsequently chlorinated with thionyl chloride under reflux. Successive amination of the corresponding acyl chlorides resulted in the formation of the desired benzamides 57 (Scheme 15).107 R= F, Cl, Br, I, CF3 Scheme 15 Reagents and conditions: (a) EtOH, H2SO4, reflux; (b) NBS (N-bromosuccinamide), CCl4, cat. (PhCO)2O2, reflux; (c) K2CO3, 1-methylpiperazine, THF, rt; (d) 1 N NaOH, reflux, then aq. HCl; (e) SOCl2, reflux; (f) 6-methyl-N1-(4-(pyridin-3-yl)pyrimidin-2-yl)benzene-1,3-diamine, 52, rt, The antibacterial N-(3-hydroxy-2pyridinyl)benzamides 58 with the electron withdrawing R-groups, were synthesized by Mobinikhaledi and co-workers through the activation of carboxylic acids 59 with thionyl chloride (Scheme 16).108 A drop-wise addition of the resultant acid chlorides 60 to an ice-cold solution of 2-amino-3-pyridinol, sodium carbonate, diethyl ether, and water afforded benzamide derivatives 58 in 40-65% yields. R= 4-nitro, 3,5-dinitro, 4-chloro-3-nitro 41 Scheme 16 Reagents and conditions: SOCl2, benzene, reflux, 1-2 h; (b) 2-amino-3-pyridinol, K2CO3, diethyl ether, H2O, rt, overnight, 40-65% Davi and colleagues reported the preparation of mono-amide ligands through the conversion of picolinic acid and pyridine-2,6-dicarboxlic acid into their corresponding acid chlorides using thionyl chloride.109 During the picolinic acid activation, a pair of products 61 (31-54% yield) was isolated after the in situ reaction of picolinoyl chlorides with the corresponding anilines. Each pair contains a minor product 62 (10-13% yield) substituted with a chlorine atom at the para-position to the ring nitrogen (Scheme 17).109 It was assumed that the ring was chlorinated when the pyridine ring was activated during a nucleophilic attack by the chloride anion. This was suggested to have occurred either at the time of the coupling process or during the acid chloride formation. R= Me, Et, Ph Scheme 17 Reagents and conditions: (a) SOCl2, reflux, 16 h; (b) N-methylaniline or N-ethylaniline or N- diphenylaniline, DCM, rt, 16 h, 10-54% Jagtap and colleagues reported a one-pot synthesis of benzamides by combining benzoic acid 63 with different amines induced by thionyl chloride under catalyst, solvent, and ligand free conditions at room temperature.110 The reaction proceeded for 2-4 hours, and the products were isolated in 80- 94% yields. The reaction shown below (Scheme 18) proceeds in a similar manner as previously described. 42 Scheme 18 Reported formation of the amide bond118 Leggio and co-workers also outlined a one-pot synthesis of amides from various benzoic acids and amines employing thionyl chloride.111 Initially, the reaction between benzoic acid and diethylamine was put to test in the presence of triethylamine as a base, and thionyl chloride as a promoter (Scheme 19). A complete reaction conversion was realized within 5 minutes, the corresponding N,N- diethylbenzamide 54 was isolated in 86% yield. A second experiment was carried out to examine how the reaction would proceed without the use of triethylamine. The reaction was conducted by combining equimolar amounts of all the reagents involved in the reaction such as thionyl chloride, benzoic acid, and diethylamine in dichloromethane at room temperature. The reaction did not go to completion after 20 minutes in the absence of a base resulting in the recovery of benzoic acid 40 in 65 % and isolation of 54 in 31% yield. The order in which the reagents were added also was shown to have a significant impact on the reaction’s outcome. A series of analogues were therefore synthesized using triethylamine, which occurred according to the reaction scheme illustrated below (Scheme 19).111 Scheme 19 One-pot amide bond formation 1.2.2 Pyrimidines as key structural components The pyrimidine nucleus is an azaheterocyclic structure112 related to the benzene ring, which is characterized by the replacement of two ring carbons in the first and third positions of its six- membered ring with electronegative heteroatoms nitrogen atoms. This results in an increased electron density on the nitrogen atom, accompanied by greater electron deficiency on the remaining carbon atoms. Due to this lack of electron density, the carbon atoms are susceptible to nucleophilic 43 attack, followed by nucleophilic displacement of a suitable leaving group, particularly at the α- and ƴ- positions to the ring nitrogens.113 Pyrimidine-containing heterocycles are of tremendous interest because they present a vital class of both natural and synthetic compounds.114 These aza-substituted arenes are also useful as electron donating ligands in coordination chemistry that serve as suitable substitutes for pyridines.112 1.2.2.1 Biological activity of pyrimidines In a wide array of natural products and biologically active scaffolds, heteroatoms and heterocyclic compounds are typically found as familiar cores. According to statistics, 85% of all bioactive substances contain heterocycles or are heterocyclic compounds themselves, with nitrogen-based heterocycles constituting the most common backbone in these complex structures.115 The pyrimidine moiety can be found in a broad range of commercially marketed medications with adenine 55 and thiamine 56 amongst the most popular examples of biological or pharmaceutical molecules (Fig 15).112 Thymine 57, uracil 58, and cytosine 59 are other relevant pyrimidine-based compounds which are vital as components of DNA and RNA nucleic acids.114 Fig 15 Structures of pyrimidine-containing substances The pyrimidine skeleton has additional significant chemical and pharmacological activities as a compound of veterinary and agrochemical products.116 It was discovered to possess antihistaminic, antioxidant, anticonvulsant, antidiabetic, antipyretic, antihypertensive, analgesic, anti-inflammatory, antileishmanial, antifungal, herbicidal and antibacterial properties.114,117 A number of pyrimidine derivatives some of which are shown in Fig 16, are said to exhibit anticancer (Compounds 60-63) and antiviral (Compounds 64-67) characteristics.116 For this reason, pyrimidine synthesis is a crucial area of attention to synthetic chemists and novel approaches are reported.112 44 Fig 16 Anti-cancer and anti-HIV drugs containing the pyrimidine pharmacophore Adding to the antiviral activity of the pyrimidine derivatives, dihydrothiopyrano[4,3-d]pyrimidine 68118 and dihydrofuro[3,4-d]pyrimidine compounds 69 and 70 have been evaluated as potential NNRTIs with promising antiviral properties.119 Kang and colleagues designed a library of thiophene[3,2- d]pyrimidine compounds as possible HIV-1 NNRTI drugs (Fig 17).120 All the compounds were proven to show moderate or superior activity against the wild-type virus in MT-4 cells. The sulphonamide derivatives 71 (Fig 17), were particularly found to be active against the entire viral panel, excluding REFS056. Both compounds demonstrated excellent antiviral effectiveness against the K103N mutant at EC50 values of 0.032 and 0.070 µM respectively, and potent against the E138K with EC50 values up to 0.035 and 0.045 µM respectively. Moreover, they functioned as conventional NNRTIs and displayed substantial affinity for the wild-type virus at IC50 values of 1.041 and 1.138 µM respectively. Wang and co-workers assessed a range of hydrazone-substituted thiophene[3,2-d] compounds and tested them for their anti-HIV-1 potential .121 The results indicated that compound 72 (Fig 17), exhibited the best anti-HIV potency (EC50= 21.2 nM), with more than 10-fold increased effectiveness over nevirapine (EC50= 281 nM). Furthermore, compared to nevirapine, etravirine, and azidothymidine, 72 displayed significantly less cytotoxicity (CC50= 183 M) with a high selectivity index (8632). 45 R= 4-NHCOCH2, 4-CN Fig 17 Reported pyrimidine derivatives potent against HIV Dihydro alkoxy benzyl oxopyrimidine 73 (Fig 18) compounds were identified as an intriguing class of NNRTI agents introduced over the past three decades.48,51,58 DABOs belong to a family of heterocyclic compounds that share the pyrimidine backbone as a prevalent structural feature with the HEPTs 74 and DAPYs (represented by 7, Fig 18), that allows them to directly interact with the NNIBP.58 The 4- pyrimidinone-based DABOs are structurally related to HEPTs when the N-1 substituent on the HEPTs is shifted to the second carbon atom of the pyrimidine ring, resulting in considerable anti-HIV potency.48 46 Fig 18 Structural similarities of DAPY 7, DABO 73 and HEPT 7448 Following the initial publication of the DABO compounds in 1992, several structural modifications have been made throughout the years leading to the discovery of numerous DABO analogues which include N-DABOs, S-DABOs, and other similar derivatives to improve their antiviral efficacy.51 2-Alkylthio-6- benzylpyrimidin-4(3H)-ones (S-DABOs) have since then sparked the extensive curiosity of researchers, resulting in the revelation of various novel analogues with outstanding antiviral activities as NNRTIs.51 Modifications on S-DABO compounds 75 (Fig 19), led to the synthesis of various analogues based on SAR for the purpose of enhancing their antiviral activity.122 It was revealed that the alkylthio side chains of S-DABOs are essential structural features necessary for anti-HIV-1 efficacy of these compounds, whereas eliminating the C-2 linker resulted in reduced antiviral activity. Displacing a benzyl moiety at the C-6 position of the pyrimidine ring with the phenyl or n-propyl (derivatives 76 and 77 respectively), resulted in substantial loss of the anti-HIV-1 activity. While the introduction of the bulkier groups like 1-naphthylmethyl and 2-naphthylmethyl motifs afforded compounds (78 and 79 respectively) with reduced activity concentrations, reasonable anti-HIV-1 potencies were observed in systematic modifications with the phenylethyl, phenoxymethyl, and (phenylthio)methyl (80) substituents at the 6-position. It was further emphasized that the antiviral activity was lost when exchanging the substituents at the C5 and C6 positions as represented by scaffolds 75 and 81.122 47 R= sec-butyl, cyclopentyl, cyclohexyl; R1= H, Me; X= CH2, O, S Fig 19 S-DABO lead compounds Vig and colleagues reported structural alterations by introducing alkyl groups on the 6-benzyl ring and C-5 position of the thymine ring, while keeping the C-2 linker fixed.123 In cell-free inhibition, compounds with bulkier substituents displayed superior IC50 values with better composite binding pocket filling. A good agreement was demonstrated by the improvement in the inhibitory efficacy that occurred as isopropyl, ethyl, and methyl groups were introduced to the thymine ring at the C-5 position (Table 1). Compound 82 bearing the methyl motifs at the 6-benzyl ring improved hydrophobic interaction with the NNIBP and was marginally more active against recombinant HIV RT than its related analogues 93-85 containing hydrogen atoms at the same position. Despite the lower IC50 concentrations in recombinant RT in cell free tests, compound 82 lacked the capacity to reduce the replication of HIV in cells infected with HTLVIIIB, presumably due to their distinct intracellular metabolism and cellular absorption.123 48 Table 1: The results of S-DABO effects on p24 synthesis in peripheral blood mononuclear cells infected with HIV, purified recombinant HIV RT enzymatic potency, and peripheral blood mononuclear cells feasibility.123 Compound Number R1 R2 IC50 [rRT] (µM) 82 83 84 85 i-Pr i-Pr Me Et Me H H H 4.8 6.1 18.8 9.7 Manetti and co-workers explored the role played by the expansion of the alkylarylthio linker on anti- HIV efficacy, given that this type of substitution at the second position of the S-DABO compounds is rarely studied.124 Therefore, while maintaining 2,6-dichlorobenzyl at the 6-position and p- methoxyphenyl attached to the S-alkyl n-linker, the antiviral activity of compounds 86 varied depending on the length of the extension (n= 1, n= 2, and n= 3 respectively) of the C-2 alkyl chain. The scaffold linked by an ethyl core was the most effective S-DABO derivative with respect to its shortened n= 1 and lengthened n= 3 analogues and showed superior anti-HIV activity in cell assays infected with pluriresistant (IRLL98) and WT viruses. However, a shortened linker gave a compound with the improved activity against the WT-RT, with notable efficacy against IRLL98. The reduced activity of the expanded derivatives against the WT virus was attributed to diminished hydrophobic contact of the benzyl motif at C-2, including the solvent and the methyl group adverse interactions. Molecular modelling calculations demonstrated that the Leu234, Leu100, Trp229, Phe227, and Tyr188 aromatic side chains that construct the hydrophobic region, allow the 6-benzyl ring of 86 (n= 1) to fit properly within the location (Fig 20). Conversely, expansion of the benzyl group, caused the 4-methoxy moiety to exceed the Pro225 and Pro236 apertures, leading to undesirable interactions with the solvent, thus, resulting in restricted hydrophobic engagements between the phenyl ring and Val106.124 49 Fig 20 Structure for S-DABO derivative 106 and surrounding reported mutations 1.2.2.2 Pyrimidine synthesis and reactions 1.2.2.2.1 Pyrimidine core formation Gabriel and Colman have been widely acknowledged by various researchers for their breakthrough in isolating the parent compound 87 in 1899112,125,126 through decarboxylation of pyrimidine-4-carboxylic acid 88 (Fig 21).112 Fig 21 Formation of 87 via decarboxylation of 88 Oostveen et al127 published a paper on the synthesis of unsubstituted pyrimidine 87 by dissolving 1- methylpyridinium methyl sulfate in liquid ammonia under extremely cold conditions (-33 °C) for one hour. After the second process of ring opening and ring closure, 6-amino-1-methyl-1,6- dihydropyrimidine 88 transformed into 2-methylamino-1,2-dihydropyrimidine 89, which then aromatized to afford pyrimidine 87 in 55-60% yield (Scheme 20). Scheme 20 Formation of unsubstituted pyrimidine 50 1.2.2.2.2 Synthesis of simple pyrimidine derivatives A quest for a quick transformation to produce more substituted pyrimidines remains fundamental among organic chemists since these templates can be employed to synthesise important intermediates or final compounds. Brugnatelli128 prepared the first pyrimidine derivative, alloxan 90 in 1818 through the oxidative degradation of uric acid by nitric acid (Scheme 21).128 Scheme 21 Brugnatelli synthesis of alloxan 90 The Frankland and Kolbe synthesis is also a popular method known112,129 for the preparation of a pyrimidine derivative, cyanalkine 91 (Scheme 22) is obtained via treatment of propionitrile with potassium metal under thermal conditions.129 Scheme 22 Frankland and Kolbe synthesis of cyanalkine 91 The Pinner reaction evolved as the preferred method for making pyrimidine derivatives.130 This method entails the condensation reaction of amidine 92 and 1,3-dicarbonyl 93 (Scheme 23), which enables the synthesis of several substituted pyrimidines 94. However, this strategy is frequently impeded by harsh reaction conditions and occasionally results in low yields.112 Scheme 23 Pinner synthesis Consequently, a variety of techniques have also been researched to broaden the application and increase the chemical effectiveness of the pyrimidine syntheses. Several of the widely used methods rely on the nitrogen-containing fragments starting from either urea, guanidine, or thiourea. Acetyl 51 acetone is a great illustration of this approach since it reacts well with the nitrogen-based fragments for the synthesis of dimethyl-substituted pyrimidines (Scheme 24).131 Scheme 24 Production of dimethylpyrimidines Nitriles are considered as valuable nitrogen units of the nitrogen-containing fragments to create various pyrimidine derivatives. Cyanamide 95 (Scheme 25) is specifically a helpful nitrile source that allows the formation of the pyrimidines.129 Scheme 25 Synthesis of pyrimidine derivatives from nitriles The synthesis of several pyrimidine compounds, 6-methylthiouracils was published by Tominaga and colleagues using N-bis(methylthio)methylenecyanamide 96 as a nitrogen source. Subsequent reaction of 96 with various methylene derivatives employing potassium carbonate or potassium hydroxide at room temperature provided pyrimidine derivatives 97 in 15 to 80% yields (Scheme 26).132 52 X= CN, COOMe, COMe, C6H5 Scheme 26 Synthesis of pyrimidines from N-bis(methylthio)methylenecyanamide 96 Within biological synthesis, the pyrimidine ring is naturally formed by the reactions of aspartate, bicarbonate, and glutamine. Four enzymatic processes transform these initial ingredients into orotate 97 (Scheme 27), which is a ribonucleotide biosynthesis precursor.129,133 Glutamine and bicarbonate