University of Ghana http://ugspace.ug.edu.gh UNIVERSITY OF GHANA SCHOOL OF PHYSICAL AND MATHEMATICAL SCIENCES DEPARTMENT OF CHEMISTRY SYNTHESIS AND ANTIMALARIAL SCREENING OF DERIVATIVES OF BENZENESULFONAMIDES BY PLANGE FRITZ EGBERT JNR. (10310564) IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF MASTER OF PHILOSOPHY DEGREE IN CHEMISTRY JULY 2019. University of Ghana http://ugspace.ug.edu.gh DECLARATION. I, Fritz Egbert Plange Jnr. hereby declare that this research is a product of my own efforts. It contains no material previously published by another person nor material which has been accepted for the award of any degree of the University, except where due acknowledgement has been made in text. STUDENT Name: Fritz Egbert Plange Jnr. Signature: ………………………. Date: ……………………………… SUPERVISOR Name: Dr. Richard K. Amewu Signature: ………………………….. Date: ………………………………. CO-SUPERVISOR Name: Prof. Dorcas Osei-Safo Signature: ………………………….. Date: ………………………………. ii University of Ghana http://ugspace.ug.edu.gh DEDICATION. This Book is dedicated to my Father, Mr. Fritz Plange, who continues to support and encourage me and to the rest of my family. iii University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGEMENT. Thanks be to God for giving me victory through my Lord Jesus Christ. I’m immensely grateful for His wisdom which he freely gives, His mercy and grace for my life. Special Thanks goes to my parents, Mr. Fritz Plange and Mrs. Lillian Plange for their tremendous support towards my education and the rest of my family for the encouragement over the years. I am eternally grateful to my supervisor, Dr. Richard Kwamla Amewu his passion for the development of Medicinal chemistry in Ghana is evidenced through his guidance, rebuke, teaching and care throughout the duration of this project. I appreciate the Chemistry Department of the University of Ghana for providing NMR and FTIR instruments as well as Medicines for Malaria Ventures for funding the project. I also thank the H3D Drug Discovery team at UCT for the MS analysis and the Drug Discovery Unit at University of Dundee for antimalarial testing. I appreciate the lecturers and staff of Chemistry Department and the efforts of Mr. Henry Onyame- Akawffo and Justice for their assistance in this project and to the entire Level 600 class I say Thank you. iv University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENTS DECLARATION. ......................................................................................................................................... ii DEDICATION. ............................................................................................................................................ iii ACKNOWLEDGEMENT. .......................................................................................................................... iv TABLE OF CONTENTS .............................................................................................................................. v LIST OF FIGURES ................................................................................................................................... viii LIST OF TABLES. ...................................................................................................................................... ix LIST OF ABBREVIATION. ........................................................................................................................ x ABSTRACT ................................................................................................................................................ xii CHAPTER ONE. .......................................................................................................................................... 1 1.1 INTRODUCTION. ............................................................................................................................. 1 1.2 EPIDEMOLOGY. ............................................................................................................................... 1 1.3 BIOLOGY........................................................................................................................................... 3 1.4 MALARIA ERADICATION, ELIMINATION AND CONTROL. ................................................... 4 1.5 EVOLUTION OF MALARIA CONTROL. ....................................................................................... 5 1.6 VECTOR CONTROL ......................................................................................................................... 7 1.6.1 INDOOR RESIDUAL SPRAYING (IRS)................................................................................... 7 1.6.2 INSECTICIDE-TREATED NETS (ITNs). .................................................................................. 8 1.7 CHEMOPREVENTION. .................................................................................................................... 9 1.8 MALARIA VACCINE. .................................................................................................................... 10 1.9 CHEMOTHERAPY .......................................................................................................................... 11 1.9.1 ARYL AMINOALCOHOL COMPOUNDS. ............................................................................ 12 1.9.2 ANTI-FOLATE COMPOUNDS. .............................................................................................. 13 1.9.3 ARTEMISININ COMPOUNDS. ............................................................................................... 15 1.10 ANTIMALARIAL DRUG RESISTANCE. ................................................................................... 16 1.11 PROBLEM STATEMENT. ............................................................................................................ 18 1.12 JUSTIFICATION. .......................................................................................................................... 18 1.13 PROJECT AIM. .............................................................................................................................. 18 1.14 OBJECTIVES. ................................................................................................................................ 18 CHAPTER TWO. ....................................................................................................................................... 20 2.1 LITERATURE REVIEW. ................................................................................................................ 20 2.2 SULPHONAMIDES ......................................................................................................................... 21 2.3 ARYL CARBOXAMIDES. .............................................................................................................. 23 2.4 MOLECULAR HYBRIDIZATION. ................................................................................................ 25 v University of Ghana http://ugspace.ug.edu.gh CHAPTER THREE. ................................................................................................................................... 27 RESULTS AND DISCUSSION ............................................................................................................. 27 3.1 CHEMISTRY: .................................................................................................................................. 27 3.2 SPECTRAL ANALYSIS FOR FEP 005. ......................................................................................... 29 3.2.1 1H AND 13C NMR. .................................................................................................................... 29 3.2.2 DEPT 135. .................................................................................................................................. 31 3.2.3 HSQC ......................................................................................................................................... 32 3.2.4 LC-MS OF FEP005 ................................................................................................................... 33 3.2.5 FTIR OF FEP005 ....................................................................................................................... 34 3.3 SPECTRAL ANALYSIS FOR FEP 040. ......................................................................................... 35 3.3.1 H1NMR ...................................................................................................................................... 35 3.3.2 13C NMR FOR FEP040 .............................................................................................................. 36 3.4 PHYSICOCHEMICAL PROPERTIES. ........................................................................................... 37 3.5 BIOLOGICAL ACTIVITY .............................................................................................................. 38 3.6 QUANTITATIVE STRUCTURE ACTIVITY RELATIONSHIP (QSAR) ANALYSIS. ............... 39 CHAPTER FOUR ....................................................................................................................................... 42 4.1 MATERIALS .................................................................................................................................... 42 4.1.1 REAGENTS ............................................................................................................................... 42 4.1.2 CHROMATOGRAPHY ............................................................................................................ 42 4.1.3 FTIR ........................................................................................................................................... 42 4.1.4 NMR .......................................................................................................................................... 42 4.1.5 LC-MS ....................................................................................................................................... 42 4.2 Preparation of 2-chloro-5-(chlorosulfonyl) benzoic acid. ................................................................. 43 4.3 General procedure for sulfonamide formation. ................................................................................. 43 4.3.1 Preparation of 2-chloro-5-(N,N-diethylsulfamoyl)benzoic acid. ............................................... 44 4.3.2 Preparation of 2-chloro-5-(pyrrolidin-1-ylsulfonyl) benzoic acid ............................................. 44 4.3.3 Preparation of 2-chloro-5-(N,N-dimethylsulfamoyl) benzoic acid ............................................ 45 4.3.5 Preparation of 2-chloro-5-(piperidin-1-ylsulfonyl) benzoic acid. .............................................. 46 4.3.6 Preparation of 2-chloro-5-((4-phenylpiperidin-1-yl) sulfonyl) benzoic acid. ............................ 46 4.41 GENERAL PROCEDURE FOR CARBOXAMIDE FORMATION (ACID CHLORIDE METHOD). ............................................................................................................................................. 47 4.411 GENERAL PROCEDURE FOR CARBOXAMIDE FORMATION (MIXED ANHYDRIDE METHOD). ............................................................................................................................................. 47 4.4.1 Preparation of 4-chloro-N, N-dimethyl-3-(4-(pyrimidin-2-yl) piperazine-1-carbonyl) benzenesulfonamide ............................................................................................................................ 48 vi University of Ghana http://ugspace.ug.edu.gh 4.4.2 Preparation of 2-chloro-5-(N,N-dimethylsulfamoyl)-N-(pyridin-2-yl)benzamide ..................... 48 4.4.3 Preparation of 2-chloro-5-(N, N-dimethylsulfamoyl)-N-(6-methylpyridin-2-yl) benzamide .... 49 4.4.4 Preparation of 4-chloro-N,N-diethyl-3-(4-(pyrimidin-2-yl)piperazine-1- carbonyl)benzenesulfonamide ............................................................................................................ 49 4.4.5 Preparation of 2-chloro-N-(6-methylpyridin-2-yl)-5-(morpholinosulfonyl) benzamide ........... 50 4.4.6 Preparation of 2-chloro-N-(6-methylpyridin-2-yl)-5-(morpholinosulfonyl) benzamide ........... 50 4.4.7 Preparation of 2-chloro-N-(2-methylbenzo[d]thiazol-6-yl)-5-(morpholinosulfonyl) benzamide ............................................................................................................................................................ 51 4.4.8 Preparation of (2-chloro-5-(pyrrolidin-1-ylsulfonyl) phenyl) (4-(pyridin-2-yl) piperazin-1-yl) methanone ........................................................................................................................................... 51 4.4.9 Preparation of (2-chloro-5-(pyrrolidin-1-ylsulfonyl) phenyl) (4-(pyrimidin-2-yl) piperazin-1-yl) methanone. .......................................................................................................................................... 52 4.4.10 Preparation of 2-chloro-N-(2-methylbenzo[d]thiazol-6-yl)-5-(pyrrolidin-1-ylsulfonyl) benzamide ........................................................................................................................................... 53 4.4.11 Preparation of 2-chloro-N-(6-methylpyridin-2-yl)-5-(pyrrolidin-1-ylsulfonyl) benzamide .... 53 4.4.12 Preparation of 2-chloro-N-(6-methylpyridin-2-yl)-5-((4-phenylpiperidin-1-yl) sulfonyl) benzamide ........................................................................................................................................... 54 4.4.13 Preparation of 2-chloro-5-((4-phenylpiperidin-1-yl) sulfonyl)-N-(pyridin-2-yl) benzamide. . 55 4.4.14 Preparation of 2-chloro-N-(2-methylbenzo[d]thiazol-5-yl)-5-(piperidin-1-ylsulfonyl) benzamide. .......................................................................................................................................... 55 4.4.15 Preparation of 2-chloro-N-(6-methylpyridin-2-yl)-5-(piperidin-1-ylsulfonyl) benzamide. ..... 56 4.4.16 Preparation of (2-chloro-5-(piperidin-1-ylsulfonyl) phenyl) (4-(trifluoromethyl) piperidin-1- yl) methanone. ..................................................................................................................................... 56 4.4.17 Preparation of (2-chloro-5-(piperidin-1-ylsulfonyl) phenyl) (4-(pyrimidin-2-yl) piperazin-1- yl) methanone ...................................................................................................................................... 57 4.5 PHYSICOCHEMICAL PROPERTIES. ........................................................................................... 57 CHAPTER FIVE ........................................................................................................................................ 58 5.1 CONCLUSIONS ............................................................................................................................... 58 5.2 RECOMMENDATION. ................................................................................................................... 58 REFERENCES. .......................................................................................................................................... 59 APPENDICIES ........................................................................................................................................... 70 APPENDIX 1: LC-MS. .......................................................................................................................... 70 APPENDIX 2: IR SPECTRA ................................................................................................................. 81 APPENDIX 3: 1H NMR SPECTRA. ...................................................................................................... 91 APPENDIX 4: 13C NMR SPECTRA. ................................................................................................... 103 vii University of Ghana http://ugspace.ug.edu.gh LIST OF FIGURES FIGURE 1.1: COUNTRIES WITH INDIGENOUS CASES AND THEIR STATUS BY 2017. ............... 2 FIGURE 1.2: LIFE CYCLE OF P. FALCIPARUM. .................................................................................. 4 FIGURE 1.3:INSECTICIDE AND ANTI-MALARIAL DRUGS USED IN THE 1930’S. ....................... 6 FIGURE 1.4: SELECTED COMPOUNDS OF THE PYRETHROID CLASS. ......................................... 8 FIGURE 1.5: PROMISING INSECTICIDES FOR THE FUTURE OF ITNS AND IRS. ......................... 9 FIGURE 1.6: ANTIMALARIAL DRUGS USED IN CHEMOPREVENTIVE THERAPIES................. 10 FIGURE 1.7: SELECTED ARYL AMINO ALCOHOL COMPOUNDS CLINICALLY USED. ........... 12 FIGURE 1.8: FOOD VACUOLE OF INTRA-ERYTHROCYTIC MALARIAL PARASITE ................ 13 FIGURE 1.9: SELECTED ANTI-FOLATE COMPOUNDS USED CLINICALLY ............................... 14 FIGURE 1.10: SIMPLIFIED SCHEME OF FOLATE METABOLISM.. ................................................ 14 FIGURE 1.11: SELECTED ARTEMISININ COMPOUNDS. ................................................................. 15 FIGURE 1.12: REACTION OF ARTEMISININ WITH HAEM. ............................................................ 16 FIGURE 1.13: GLOBAL DISTRIBUTION OF P. FALCIPARUM DRUG RESISTANCE.. ................. 17 FIGURE 2.1: STRUCTURE OF MMV019721 ........................................................................................ 20 FIGURE 2.2: CLINICALLY USED SULPHONAMIDES. ...................................................................... 21 FIGURE 2.3: ANTIMALARIAL SULFONAMIDES AND ANTI-FOLATES. ...................................... 22 FIGURE 2.4: A SELECTION OF SULPHONAMIDES UNDER DEVELOPMENT ............................. 23 FIGURE 2.5: REPRESENTATIVE ARYL CARBOXAMIDES .............................................................. 24 FIGURE 2.6: SELECTION OF CYTOTOXIC ARYL CARBOXAMIDES. ........................................... 25 FIGURE 2.7: ANTIMALARIAL BROMOBENZOTHIOPHENE CARBOXAMIDE ............................ 25 FIGURE 2.8: REPRESENTATIVE MOLECULAR HYBRIDIZED COMPOUNDS ............................. 26 FIGURE 3.1: 1H NMR FOR FEP 005 ....................................................................................................... 30 FIGURE 3.2: 13C NMR FOR FEP 005 ...................................................................................................... 31 FIGURE 3.3: DEPT 135 CHROMATOGRAM FOR FEP005 ................................................................. 32 FIGURE 3.4: HSQC CHROMATOGRAM FOR FEP005 ........................................................................ 33 FIGURE 3.5: LC-MS OF FEP005............................................................................................................. 34 FIGURE 3.6: FTIR OF FEP005. ............................................................................................................... 34 FIGURE 3.7: H1NMR FOR FEP040 ......................................................................................................... 35 FIGURE 3.8: 13C NMR FOR FEP040 ....................................................................................................... 36 FIGURE 3.9: LOG (1/C) VS LOGP AND LOG (1/C) VS PSA LINE FIT PLOTS ................................. 39 FIGURE 3.10: LOG (1/C) VS MR ............................................................................................................ 40 FIGURE 3.11: CAL. IC50 VS. IC50 ............................................................................................................ 41 viii University of Ghana http://ugspace.ug.edu.gh LIST OF TABLES. TABLE 1: IN VITRO ACTIVITY OF MMV019721 20 TABLE 2: SYNTHESISED INTERMEDIATES AND FINAL PRODUCTS WITH CORRESPONDING YIELD 28 TABLE 3: PHYSICOCHEMICAL PROPERTIES OF SYNTHESIZED BENZENE SULPHONAMIDES 37 TABLE 4: IN VITRO ANTIMALARIAL ACTIVITY OF SOME SELECTED COMPOUNDS 38 ix University of Ghana http://ugspace.ug.edu.gh LIST OF ABBREVIATION. 3D7 Plasmodium falciparum strain ACT’s Artemisinin Combination Therapies CDCl3 Deuterated Chloroform CSA Chondroitin sulfate A CSP Circumsporozoite Protein DCM Dichloromethane DDT Dichloro-diphenyl-trichloroethane DEPT Distortionless Enhancement by Polarization Transfer DHA Dihydroartemisinin DHFR Dihydrofolate reductase DHP Dihydropteroate DHPPP Dihydropteridine pyrophosphate DHPS Dihydropteroate synthase DMF Dimethyl formamide DU-145 Human prostate cancer cell line EEF Exoerythrocytic form ETA receptor Endothelin receptor type A ET3N Triethyl amine FRET Förster resonance energy transfer FTIR Fourier Transform Infrared spectroscopy GI50 Concentration for inhibiting cell growth by 50% GTP Guanosine triphosphate HT-29 Human Colorectal Adenocarcinoma Cell Line HSQC Heteronuclear single quantum coherence HT1080 Human fibrosarcoma cell line IC50 The half maximal Inhibitory Concentration IPT Intermittent preventive treatment x University of Ghana http://ugspace.ug.edu.gh IRS Indoor Residual Spraying ITN’s Insecticide-Treated Nets JNK1 c-Jun N-terminal kinase 1 LC-MS Liquid chromatography–Mass spectrometry Log P Octanol-water Partition coefficient MMP-2 Matrix metalloproteinase-2 MMV Medicine for Malaria Ventures MR Molar Refractivity NAI Naturally Acquired Immunity NF54 Transgenic Plasmodium falciparum strain 1H NMR Proton nuclear magnetic resonance 13C NMR Carbon-13 nuclear magnetic resonance PABA para-Aminobenzoic acid PfSPz Plasmodium falciparum sporozite PSA Polar Surface Area QSAR Quantitative Structure Activity Relationship RAR Retinoic acid receptor RORγt RAR-related orphan receptor gamma-t RS21/AS01 Recombinant malaria vaccine RTS, S/AS01 Recombinant malaria vaccine SP Sulfadoxine-Pyrimethamine SSV Sporozoite subunit vaccine TBV Transmission-Blocking vaccine THF Tetrahydrofuran UV Ultraviolet VAR2CSA Variant Surface antigen 2-CSA WHO World Health Organization WSV Whole Sporozoite vaccine xi University of Ghana http://ugspace.ug.edu.gh ABSTRACT Aryl carboxamides are important class of organic compounds with impressive biological activity. A small library of benzenesulphonamides were synthesized via three chemical steps; first by sulfochlorinations of chlorobenzoic acid followed by sulphonamide and amide formation to give the target compounds in high to excellent yields. Preliminary test showed the compounds possess promising in vitro antimalarial activity against the 3D7 strain of the malaria parasite. Regression analysis establish that lipophilicity has more impact on the biological activity than MR and PSA. xii University of Ghana http://ugspace.ug.edu.gh CHAPTER ONE. 1.1 INTRODUCTION. The fight against malaria has been an endless battle, stretching from vague detections as early as 2700 BCE (recorded descriptions in the canon of Medicine authored by Huang Ti, a Chinese emperor), through to the first mention, the coinage of the term, Malaria (mal aria = bad air) in Greek Literature in the Third century by Hippocrates and finally, to the 16th century, the Genesis of the era of scientific understanding of malaria (Moss, Shah, & Morrow, 2008). Through the works and discoveries of Giovanni Maria Lancisi, Charles Louis Alphonse Laveran, Patrick Manson, Ronald Ross and Giovanni Batista Grassi, the etching of a foothold in this battle had just began (Desowitz, 1991). 1.2 EPIDEMOLOGY. Malaria is a vector-borne parasitic tropical disease found in 91 countries globally (WHO, 2017b). There are more than 120 Plasmodium species infecting mammals, birds, and reptiles, however, only five including Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi infect human beings regularly (Ashley, Pyae Phyo, & Woodrow, 2018a). Plasmodium falciparum and Plasmodium vivax cause the most infections worldwide with differing geographic distributions. The contribution from other species of plasmodium parasites to the global burden is relatively low (Ashley et al., 2018a; WHO, 2016). The female Anopheles mosquito is responsible for the transmission of the malaria parasite with An. gambiae sensu stricto, An. funestus, and An. Arabiensis being the most prevalent specie in Africa (Cowman, Healer, Marapana, & Marsh, 2016). Malaria is currently autochthonous in a broad band around the equator, which includes the Americas, many parts of Asia and Africa; in Sub-Saharan Africa, 85-90 % of malaria fatalities occur (Figure 1.1) (WHO, 2017b). Plasmodium falciparum and P vivax are the predominant species worldwide with an estimated incidence of 211 million and 7·5 million cases respectively in 2017 and about 435,000 deaths in the same year (WHO, 2018). The Bulk of malaria caused by falciparum occurs in sub-Saharan Africa where approximately 190 million cases were reported. University of Ghana http://ugspace.ug.edu.gh Transmission in Africa is pretty intense in many areas, though there remain discrepancies in incidences within and between countries. Children under the age of 5 years and pregnant women are the usual casualties, making about 85-90 % of the reported cases worldwide. In 2017, Children under the age of 5 years accounted for 61% (266, 000) of all malaria deaths worldwide, bulk of this statistic was derived from endemic regions of Africa, South America and southern Asia where poverty, war and natural disasters are common. Figure 1.1: Countries with indigenous cases in 2000 and their status by 2017 (WHO, 2018). The incidence and transmission of malaria rely heavily on these climatic factors; temperature and rainfall, because vector behavior is affected by them (Dasgupta, 2018). Temperature affects the lifespan and growth of mosquitoes (Craig, Snow, & le Sueur, 2002; Mouchet & Carnevale, 1997), it is also a critical factor influencing each stage of a mosquito lifecycle (Stresman, 2010) and is a determinant in the parasite development (Russell, West, M. Anwell, & Macdonald, 1963). Stagnant water is essential for breeding by the vector and hence the effects of rainfall patterns and distribution is complicit in malaria transmission (Craig et al., 2002; Thomson, Mason, Phindela, & Connor, 2005). Though a linear relationship between transmission and climatic factors is marginally conclusive (Dasgupta, 2018), an optimum level of these climatic factors increases the behavioral activity of the vector and thus propagate the transmission of the malaria parasite (Githeko, 2009). Researchers have concluded, malaria transmission would get worse in endemic 2 University of Ghana http://ugspace.ug.edu.gh regions and a series of outbreaks may be inevitable with the advent of global warming (Reitera et al., 2004). 1.3 BIOLOGY. Plasmodium spp. are universal pathogens with a complicated life cycle oscillating between female Anopheles mosquitoes and vertebrate hosts that entails the evolution of unique zoite forms to attack different cell types at specific stages. Malaria infection begins with the bite of an inoculated female anopheles mosquito transmitting a motile infective form called the sporozoite into a vertebrate host. The infection develops through a series of stages with two major phases; one that involves the liver (exoerythrocytic phase), and one that involves red blood cells, or erythrocytes (erythrocytic phase)(Ashley et al., 2018a; Bledsoe, 2005). (A) Plasmodium sporozoite travel through the blood vessels from the dermis to the liver cells through a process known as traversal. Specialized proteins are developed for this process and it has been established by Tavares et al. that this process is important for establishing an infection by arming the sporozoite for invasion of hepatocytes, the cells in which sporozoites develop (Tavares et al., 2013). Sporozoites injected into the dermis are in ‘‘migratory mode’’ and upon interaction with hepatocytes convert to ‘‘invasive mode”. (B) Once hepatocyte infection is established, the sporozoite transforms over the subsequent 2–10 days to a liver stage or exoerythrocytic form (EEF), and development culminates in release of up to 40,000 merozoites per hepatocyte into the bloodstream by budding of parasite-filled vesicles called merosomes. After the release of the merozoites into hepatic circulation, free merozoites invade erythrocytes in a fast, dynamic, and multi-step process that is complete within 2 min. (C) After pre-erythrocyte infection, over the subsequent 48 hr., cell division (schizogony) results in 16–32 merozoites that egress when developed, which results in destruction of the erythrocyte membrane and explosive release of parasites to access new host cells for invasion and producing a rise in parasite numbers and hence human disease symptoms which commences the erythrocytic stage of the life cycle (Cowman et al., 2016). (D) During rounds of schizogony in the bloodstream, a subpopulation of intra-erythrocytic parasites switches to sexual development, producing female and male gametocytes which are 3 University of Ghana http://ugspace.ug.edu.gh subsequently taken up by the female anopheles mosquito during feeding. (E) In the gut of the mosquito, sexual reproduction takes place to produce ookinete - a fertilized, motile zygote which develops into new sporozoites that migrate to the insect's salivary glands and (F) begins the malaria infection cycle, (Figure 1.2) (Cowman et al., 2016; Vaughan, Aly, & Kappe, 2008; Walker, Nadjm, & Whitty, 2018). Figure 1.2: Life Cycle of P. Falciparum(Cowman et al., 2016). 1.4 MALARIA ERADICATION, ELIMINATION AND CONTROL. The vision of a malaria-free world has been of great urgency to the global community. The definition and guidelines of malaria elimination and eradication have developed gradually over the years while the control of it has somewhat remained unchanged (Li et al., 2019). To attain this vision of a malaria free world, these strategies must be intricately woven, not prematurely prioritize one over the other but a smooth transition between strategies without an enfeebling effect on its 4 University of Ghana http://ugspace.ug.edu.gh progress. Malaria control is the strategy that takes precedence and is described as the curtailment of the diseases to ubiquity where it is no longer considered a global health threat, implying the perpetual nature of the control programs (Li et al., 2019). Malaria elimination as defined by W.H.O is the interference of local transmission (reduction to zero incidence of indigenous cases) of a specified malaria parasite species in a defined geographic area; continued measures are required to avoid re-establishment of transmission (World Health Organization & Global Malaria Programme, 2017). Malaria eradication is defined as the permanent lowering to zero of the global incidence of malaria infection caused by all species of human malaria parasites, it can be regarded as a series of elimination programs. Once a complete wipeout of malaria incidences has been achieved, intervention measures become redundant (World Health Organization & Global Malaria Programme, 2017). 1.5 EVOLUTION OF MALARIA CONTROL. Since Ross’s and Grassi’s findings in 1902 that established mosquitoes as the vector responsible for the transmission of malaria, a conundrum emerged on how best to control it. Two schools of thought emerged with those who believed that malaria transmission was as a result of poverty and underdevelopment and opted for improvements in economic and social conditions as an effective way to control malaria on one side whilst those who believed in the steady scientific advances and that more focus should be given to public health interventions such as chemotherapy and advanced vector control methods are on the other side (Packard & Gadehla, 1997). In 1955, the Great Malaria Eradication Campaign, launched by W. H. O was designed purposely to permanently wipe out the incidence of infection and transmission by all malaria parasite species (Hamoudi & Sachs, 1999). This campaign was encouraged by the efficacy, technical and economic feasibility of Dichloro-diphenyl-trichloroethane (DDT) as an insecticide and other effective antimalarial drugs such as Chloroquine, pamaquine and mepacrine all discovered at the latter end of the 1930s (Figure 1.3) (Packard, 1998). 5 University of Ghana http://ugspace.ug.edu.gh Figure 1.3:Insecticide and anti-malarial drugs used in the 1930’s. The program was reviewed and described as a major failure, but not without some notable successes in eliminating malaria from most of Europe, North America, the Caribbean and parts of Asia and South-Central America. However, in Africa, where malaria control was of greatest importance, virtually nothing was attempted there (Moss et al., 2008). The overemphasis of insecticide-based approach on malaria eradication, may have been the most successful anti-malaria strategy of all time. Yet it contributed to a legacy of insecticide-resistant vectors and a poorly developed system for fast tracking elimination and eradication efforts as was the case in some parts of Asia. (Packard, 1998) (Moss et al., 2008). The future of malaria its control, elimination and eradication would no longer take a single- approached method after that debacle during the mid-20th century (Moss et al., 2008). Successful control will require an in-depth understanding of all parameters, epidemiological and ecological interactions between host, parasites and vector. After the 1969 World Health Assembly (WHA), a global resolution was reached, hinting at the enforcement of measures of infinite duration aimed at axing the incidence of malaria to a point where it would no longer be considered as a major global health threat (Li et al., 2019; Moss et al., 2008). In 2015 a Global Technical Strategy for Malaria developed by WHO and partners was adopted by the World Health Assembly with the purpose of advancing endeavors with a complete wipe out as the goal for perpetuity, but with a pragmatic view of the strenuous difficulties we must overcome and the threats to current gains, especially the development of artemisinin and insecticide resistance. In the entirety of malaria’s history, promising advances have been made in the key areas of Vector control, Chemoprevention, and Chemotherapy (Cowman et al., 2016; WHO, 2015). 6 University of Ghana http://ugspace.ug.edu.gh 1.6 VECTOR CONTROL The 1980’s and 90’s saw a horrifying and devastating rise in incidences and transmission of malaria in Africa, endemic parts of Asia, South and Central America, after the formal declaration which ended the global eradication program (Moss et al., 2008). The drastic increase in investments towards the malaria elimination campaign in the year 2000, saw the introduction of advanced vector control strategies and artemisinin based therapies that concomitantly halved the burden of malaria globally particularly in Sub Saharan Africa (Cowman et al., 2016; Nkumama, O’Meara, & Osier, 2017). The two core, broadly applicable measures for malaria vector control are the insecticide-treated nets (ITNs) and indoor residual spraying (IRS)(Raghavendra, Barik, Reddy, Sharma, & Dash, 2011). 1.6.1 INDOOR RESIDUAL SPRAYING (IRS). The patronage of DDT was discouraged over the years due to environmental and health concerns coupled with increased vector resistance (Moss et al., 2008). This led to the discovery of Pyrethroids, a class of compounds potent against insects and other invertebrates and lauded for its low toxicity (to mammals exclusively) which was first isolated from an East African Chrysanthemum flower (Soderlund et al., 2002). They have been the compounds of choice for ITN’s and IRS up on till 2017 (Pichichero, 2018), however, several mechanisms of resistance to these insecticides have been identified and are predominant among malaria vectors (Alout, Labbé, Chandre, & Cohuet, 2017; Butler, 2013). 7 University of Ghana http://ugspace.ug.edu.gh Figure 1.4: Selected compounds of the pyrethroid class. Although the effect of those compounds on the vectors is diminishing, it isn’t completely redundant (Churcher, Lissenden, Griffin, Worrall, & Ranson, 2016); In the WHO African Region, the percentage of the population at risk protected by IRS declined from 10.1% (80 million) in 2010 to a low point of 5.4% (51 million) in 2016, before rising to 6.6% (64 million) in 2017 (WHO, 2018).This advocates that pyrethroid-based vector control interventions remain effective against malaria transmission regardless of the insecticide resistance (Alout et al., 2017). 1.6.2 INSECTICIDE-TREATED NETS (ITNs). In sub-Saharan Africa, where the onslaught of malaria was at its highest, preventive and curative interventions halved the prevalence of Plasmodium falciparum, concomitantly averting more than half a billion clinical cases (Raghavendra et al., 2011). The introduction of ITNs was undoubtedly the reason for this progress, they still are, and are extensively dispensed in indigenous areas. The 2017 World Malaria Report indicated that advancement in lowering the burden of malaria has stalled and, in some countries, malaria cases and deaths are increasing (WHO, 2018). In response, there is an ongoing research on potent non-pyrethroid compounds that could eventually replace them. For instance, Chlorfenapyr, a non-neurotoxic insecticide with a different mode of action to the pyrethroid compounds was found to be 2-5 times more toxic to pyrethroid resistant vectors (N’Guessan et al., 2007). Also WHO are reviewing the efficacy of dual treated insecticide nets; that is combining the pyrethroid compound with an insect growth regulator (pyriproxyfen) or a 8 University of Ghana http://ugspace.ug.edu.gh synergist (piperonyl butoxide) which has shown promising results in their early trials (Ashley, Pyae Phyo, & Woodrow, 2018b; Pichichero, 2018). Figure 1.5: Promising insecticides for the future of ITNs and IRS. 1.7 CHEMOPREVENTION. As the saying goes, “Prevention is better than cure”, drugs and treatment (which includes vaccines) are employed in combating malaria infections. This indeed is necessary for the population highly susceptible to malaria and they include pregnant women, children under the age of 5 and travelers in high endemic areas (Desai et al., 2018; Walker et al., 2018). The WHO recommends the intermittent preventive treatment (IPT) to protect pregnant women and children in areas of moderate and high malaria transmission. The antimalarial drug chosen for this is sulfadoxine-pyrimethamine (SP)(White, 1991; WHO, 2016, 2017b). Additionally, seasonal malaria chemoprevention programs are organized, targeting children between 3-59 months old however, the impact of these measures are threatened by its frequency (not enough) and the increasing resistance to SP drug. The use of alternative antimalarial drugs are being explored, one such is dihydroartemisinin–piperaquine currently under evaluation (Ashley et al., 2018a; WHO, 2017b). 9 University of Ghana http://ugspace.ug.edu.gh Figure 1.6: Antimalarial Drugs used in Chemopreventive therapies. 1.8 MALARIA VACCINE. Another effective tool for prevention is vaccine development. However, due to the complexity of the malaria parasite as compared to bacteria and viruses, it is more difficult to develop a vaccine. That is why for over 50 years since malaria vaccine development began with seminal studies in mice using irradiated sporozoites, there hasn’t been any licensed product (Draper et al., 2018). The silver lining though, is during that time, steady progress has been made in the understanding of cellular and molecular mechanisms in the life cycle of the parasite, and that effort has sought of paid off with a number of development pursuing for licensure (Draper et al., 2018; Hoffman, Vekemans, Richie, & Duffy, 2015). The complexity of the malaria parasite has made the development of a vaccine rather evasive. Hence in their development, emphasis must be given to each stage of infection and life-cycle to accommodate an effective target (Hoffman & Miller, 1996). These targets include the production of antigen specific protective antibodies of which some are focused on the sporozoite and/or liver stages of the life cycle (pre-erythrocytic stages), some on the asexual erythrocytic stages (Blood stages), and some on the sexual erythrocytic and mosquito stages (Alonso et al., 2011). The pre-erythrocytic stage has seen two classes of vaccine developed. The sporozoite subunit vaccine (SSV) which is fabricated as a virus-like particle and the whole sporozoite vaccine (WSV), as the name suggests, uses a whole live-attenuated sporozoite that ceases to develop at various stages prior to the symptomatic blood stage. These vaccines directs immune responses against the major circumsporozoite protein (CSP), first malaria protein to be cloned, which covers the surface of the infecting sporozoite (Draper et al., 2018). So a couple of vaccines in each class have been developed at varying stages. For SSVs, RTS, S/AS01 vaccine has reached it pilot implementation stage and the vaccine RS21/AS01 in phase I and II of clinical trials (Rts, 2015). The WSV which includes PfSPZ Vaccine among others has reached its 10 University of Ghana http://ugspace.ug.edu.gh second stage of clinical trials (Seder et al., 2013). Another target of interest for vaccine development is at the blood stage. There is a keen interest at this stage because, there is evidence of naturally acquired immunity (NAI) to malaria among adult patients in high transmission areas due to repeated exposure to blood-stage parasite(Draper et al., 2018). Chemically attenuated whole-parasite blood-stage vaccine (Raja, Stanisic, & Good, 2017) and subunits against pregnancy-associated malaria using VAR2CSA (Pehrson, Salanti, Theander, & Nielsen, 2017) were recently developed and they were programmed to function by replicating aspects of NAI with results still pending. The sexual erythrocytic and mosquito stages has seen the development of Transmission-Blocking Vaccines (TBV). TBV is unique among malaria vaccine strategies, because they do not directly prevent infection or clinical symptoms within the host, but rather affects the parasite’s life cycle in the mosquito vector, purposefully preventing sporozoite development and onward transmission (Draper et al., 2018). However, previously developed vaccines had major issues with reactogenicity (Wu et al., 2008). Intense research is being done on TBV’s and so far has yielded some promising results with the discovery of good antigens (Kapulu et al., 2015). 1.9 CHEMOTHERAPY The design of antimalarial drugs is purposed to cure malaria. These drugs are used to treat malaria in people suspected to have the disease or diagnosed with it, prevent infection in people travelling to malaria-autochthonous regions who have no immunity, and routine intermittent treatment of certain malaria prone groups in autochthonous regions (Ashley et al., 2018a). Since the Era of scientific understanding of malaria began, effective solutions to treatment of malaria was sought after till the discovery of cinchona bark in the 16th century in the forest of South America (Moss et al., 2008). 200 years later, the active ingredients of cinchona bark, quinine (Figure 1.6), was isolated (Harrison, 2015). This revolutionized the search for an effective chemotherapeutic drugs for malaria. Antimalarial medications used in the treatment of malaria rests on the nature and seriousness of the disease (Lindner, Miller, & Kappe, 2012). Monotherapeutic treatment of malaria has long since faded out due to multiple drug resistance specie of malaria parasite subsequently leading to 11 University of Ghana http://ugspace.ug.edu.gh treatment failure in patients. To curtail this problem with some added benefits, the concept of combination therapy has been employed and is the current practice in the treatment of malaria today (Walker et al., 2018)(Plewes, Leopold, Kingston, & Dondorp, 2019). Majority of the antimalarial drugs are effective against the asexual erythrocytic stages of the parasite, therefore are called blood schizonticidal drugs. Tissue schizonticidal drugs target the hypnozoites (dormant stage of the parasite) in the liver whereas gametocytocidal drugs are more effective in at clearing sexual erythrocytic forms of the parasite in the bloodstream and thus severs transmission of the malaria parasite to the mosquito. Sporontocides prevent or inhibit evolution of malarial oocysts and sporozoites in infected mosquito (Kumar, Bhardwaj, Prasad, & Singh, 2018). Presently, available antimalarial drugs can be segregated into “three” broad categories with respect to their chemical structure and mechanism of action. These include Aryl aminoalcohols, Anti- folates and the Artemisinins. 1.9.1 ARYL AMINOALCOHOL COMPOUNDS. Aryl aminoalcohol compounds: quinine, quinidine, halofantrine, lumefantrine, chloroquine, amodiaquine, mefloquine, cycloquine etc. (Figure 1.6). Figure 1.7: Selected Aryl Amino Alcohol compounds clinically used. This class of compounds are examples of quick acting schizonticidal drugs. Within the food vacuole of the blood stage parasite, hemoglobin is degraded by the parasite to provide a myriad of amino acids needed for it growth and development (Kumar et al., 2018). However, formation of a large quantity of Heme as a byproduct is dangerous to the parasite. This damages the membrane due it’s per oxidative properties. The parasite converts the heme to hematin and finally to hemozoin 12 University of Ghana http://ugspace.ug.edu.gh as a detoxification route. Aryl aminoalcohol compounds act at the hematin stage by inhibiting the hemozoin biocrystallization, thus facilitating an aggregation of cytotoxic heme in the food vacuoles of the parasites thereby poisoning it (Alam et al., 2009; Foley & Tilley, 1998; Kumar et al., 2018). Figure 1.8: Food vacuole of intra-erythrocytic malarial parasite as drug target (Golan, Tashjian, & Armstrong, 2011). 1.9.2 ANTI-FOLATE COMPOUNDS. Antifolate compounds are another category which broadly classifies the mechanism of action of the compounds at its active site. Examples include proguanil, pyrimethamine, trimethoprim, dapsone etc. (Figure 1.8) shows the structures of some clinically used antifolates. 13 University of Ghana http://ugspace.ug.edu.gh Figure 1.9: Selected Anti-folate compounds used clinically The cytoplasm of the parasite cell harbors hundreds of enzymes essential for it survival. Among them, seven of these enzymes facilitates folate metabolism which is critical for the parasites growth (Kumar et al., 2018). Only two enzymes of the seven are known targets, they are dihydropteroate synthase (DHPS), and dihydrofolate reductase (DHFR). DHPS enzyme is responsible for de novo biosynthesis of folates from guanosine triphosphate (GTP), p-aminobenzoic acid (pABA) and glutamate. Sulfa-based anti-folate compounds like dapsone and sulfadoxine are schizonticidal by inhibiting the enzyme DHPS responsible for the synthesis of dihydrofolate in malaria parasites (Hyde, 2005). DHFR is a ubiquitous enzyme that participates in the recycling of folates by reducing dihydrofolate to tetrahydrofolate which eventually facilitates a biosynthetic reaction that leads to the synthesis of DNA. Anti-folate compounds pyrimethamine and proguanil inhibits DHFR enzyme, disrupting the biosynthetic reaction which leads to an arrest in DNA synthesis and subsequent parasite death (Foley & Tilley, 1998; Hyde, 2005; Ouellette, 2001). Figure 1.10: Simplified scheme of folate metabolism. The malaria parasite synthesizes folates de novo, but cannot utilize preformed folates. The two primary targets of antimalarial drugs which target folate metabolism are denoted with the boxed arrows (WISER, 2005). 14 University of Ghana http://ugspace.ug.edu.gh 1.9.3 ARTEMISININ COMPOUNDS. Artemisinin compounds as shown in Figure 1.10 (artemisinin, dihydroartemisinin, artesunate, artemether, arteether etc.). They are derivatives of the compound isolated from the plant Artemisia annua. Artemisinin and its derivatives are widely used throughout the world and are the mainstay for treating malaria (Alam et al., 2009; Kumar et al., 2018). However, due to increase in resistance and risk of resistance of newer antimalarial drugs, these compounds are usually combined to increase its effectiveness and reduce the risk of resistance to individual compounds (Cowman et al., 2016; Plewes et al., 2019). Figure 1.11: Selected Artemisinin Compounds. The exact mechanism of action of artemisinin compounds isn’t entirely straightforward as compared to other anti-malarial drugs (Krieger et al., 2018). They do not directly attack malarial parasites or cells but rather, upon activation of the endoperoxide ring, reacts with haem of the red blood cells, which results in the generation of free radicals that destroys vulnerable proteins, resulting in the death of the parasite (Figure 1.11) (Robert, Dechy-Cabaret, Cazelles, & Meunier, 2002; Tilley, Straimer, Gnädig, Ralph, & Fidock, 2016; J. Wang et al., 2019). Additionally, artemisinin compounds binds to a large number of targets. This promiscuous nature aids it ability to kill the parasite at all stages of its life cycle (Aweeka & German, 2008; J. Wang et al., 2015). Hence, it is effectively used in combination with other antimalarials as a schizonticidal (blood and tissue), gametocytocidal and sporontocidal drug with varying efficacy depending on the derivative (Kumar et al., 2018; WHO, 2017a). 15 University of Ghana http://ugspace.ug.edu.gh Figure 1.12: Reaction of Artemisinin with Haem (Robert et al., 2002). 1.10 ANTIMALARIAL DRUG RESISTANCE. Infectious microbial disease remains a pressing problem worldwide, because microbes have resisted prophylaxis or therapy longer than any other form of life (Wanger et al., 2017). In recent decades, drug resistance has been the focal hurdle among many hurdles in the battle against malaria with the first recorded instance in 1957, where standard treatment of malaria with chloroquine in some patients in the regions of Southeast Asia and Latin America had failed (Wellems & Plowe, 2001). By the 1980’s this resistance had spread to Africa. Problems of multidrug-resistant malaria specie have reached an alarming level in many countries around the world. Currently, three out of five malaria species known to affect humans are registered to be drug resistant ( P. falciparum, P. vivax and P. Malariae) (Bloland, 2001). WHO defined ‘drug resistance’ as the ability of a parasite to thrive or prosper regardless of the method of drug intake and absorption of a drug given in amounts equal to or higher than what is normally recommended but within the tolerance of the subject. The form of the drug active against the parasite must be able to gain entry to the parasite or the infected erythrocyte for the period of the time essential for its normal action (WHO, 2010). The development of resistance can be observed as happening in two phases. The first phase is as a result of an initial genetic event that sparks the birth of a resistant mutant (de novo mutation) that 16 University of Ghana http://ugspace.ug.edu.gh hands the parasite a great chance to survive the drug. In the second phase, the resistant parasites are chosen for survival and starts to multiply, ultimately resulting in a parasite population that is immune to treatment (J Le Bras, Basco, & de Pecoulas, 1996; Jacques Le Bras & Durand, 2003; WHO, 2010). A study by Rathod, McErlean & Lee suggested that P. falciparum in South-East Asia has an inherent propensity to develop drug resistance through genetic mutation (Rathod, McErlean, & Lee, 2002). Drug counterfeiting and non-adherence to treatment regimen have played a key role in the development of resistance specie (Ashley et al., 2018a; Bloland, 2001; Tettey, 1966). Chloroquine and Quinidine which seem to be partially ineffective in treatment of P. falciparum malaria in Sub-Saharan and East and West Africa originated from South Asia, specifically from the Thai-Cambodia border. There is evidence of decreasing sensitivity of P. falciparum to artemisinin derivatives in the Cambodia region where patients had significantly longer clearance times after monotherapy, and this has developed further into decreasing clinical efficacy and potential partner drug resistance (Ashley et al., 2014; Dondorp et al., 2009; WHO, 2017a). This has become a potential threat to the continued efficacy of ACTs. Figure 1.13: Global distribution of P. falciparum drug resistance. Countries shown by level of antimalarial resistance of local P. falciparum. Countries approaching malaria elimination also shown. (Plewes et al., 2019). 17 University of Ghana http://ugspace.ug.edu.gh 1.11 PROBLEM STATEMENT. The consistent efforts for the radical cure of malaria has been hampered by the evolution of drug resistance, the most difficult hurdle for antimalarial therapy. Concerted efforts within the scientific community towards obtaining the upper hand on the emanation and distribution of resistance to existing drugs is yet to be achieved. More recently, attention has been focused on exploiting the various metabolic and biochemical pathways (genomics, bioinformatics and structural biology) of the parasite in anticipation of identifying and exploiting novel drug targets with novel mechanism of action. 1.12 JUSTIFICATION. The discovery of drug and insecticide resistance since 1930’s has kept researchers and medical practitioners on their toes. A single approach method to curb an outbreak has proven rather redundant and a multi-faceted approach is on the brink of retrogressing. The rate at which drug resistance is increasing, in about a decade if there hasn’t been any new potent antimalarial drug, there would be outbreaks of catastrophic proportions. Hence, the need to develop new antimalarials with new and effective mechanism of action. 1.13 PROJECT AIM. To synthesize a library of benzene sulfonamides and evaluate them for antimalarial activity. 1.14 OBJECTIVES. 1. Synthesize a library of benzene sulfonamides. 2. Characterize the compounds using NMR, FTIR, LC-MS, Melting point etc. 3. Evaluate them for their antimalarial activity. 4. Perform a comprehensive QSAR to develop an optimized lead. 5. Evaluate any Structure Activity Relationship or Structure Property Relationship. 18 University of Ghana http://ugspace.ug.edu.gh 19 University of Ghana http://ugspace.ug.edu.gh CHAPTER TWO. 2.1 LITERATURE REVIEW. The Medicine for Malaria Ventures (MMV) recently published the Pathogen box, containing approximately 400 diverse drug-like molecules, representing families of structures identified in phenotypic screens of pharmaceutical and academic libraries against the plasmodium falciparum malaria parasite as well as other neglected diseases. It was established to accelerate drug development in addressing diseases of poverty by way of discovering new leads for innovative drugs against neglected tropical diseases (Duffy et al., 2017; Van Voorhis et al., 2016). One of the compounds in the pathogen box MMV019721, a benzothiozoyl benzamide, showed activity in vitro towards two sensitive strains 3D7 and NF54 of the malaria parasite and was found not to be toxic to living cells of the host. Figure 2.1: Structure of MMV019721 Table 1: In vitro activity of MMV019721 IC50 (nM) Erythrocyte Gametocytes Sporozoites Cytotoxicity 3D7 NF54 P. berghei HepG2 cell line 552.2 310.7 42.73 4035 In the figure above, the key pharmacophores identified are the sulphonamide and the benzamide. Hence we explore their benefits. 20 University of Ghana http://ugspace.ug.edu.gh 2.2 SULPHONAMIDES The sulphonamides are a group of synthetic antimicrobial agents that are structural analogues of para-aminobenzoic acid (PABA) and thus act as competitive antagonists in microbial cells (Dorn & Volcheck, 2018). The sulfonamide functional group has an impressive pharmaceutical pedigree due to it being the most effective anti-folate compound. It is used across a spectrum of therapeutic areas such as antibacterials (Aminov, 2017; Wanger et al., 2017), protease inhibitors (Jain, Saravanan, & Singh, 2013; Supuran, Casini, & Scozzafava, 2003), diuretics (Carta, Scozzafava, & Supuran, 2012), hypoglycaemic (Supuran et al., 2003) among others. Figure 2.2 Show the structures of some commercially produced sulfonamides used in treatment of various ailments sulfamethoxazole (1), Furosemide (2) and Darunavir (3). Figure 2. 2: Clinically used Sulphonamides. Compound 1 is a commercially produced antibiotic employed in the treatment of bacterial infections such as urinary tract infections, bronchitis, and prostatitis and is effective against both gram negative and positive bacteria (Masters, O’Bryan, Zurlo, Miller, & Joshi, 2003). Compound 2 on the other hand is a sulfonamide loop diuretic used medically to treat fluid build-up due to heart failure, liver scarring, or kidney disease (Wargo & Banta, 2009). Whereas compound 3, is a protease inhibitor used to treat HIV infection (Adkins & Faulds, 1998). Sulfonamides are also known to possess some anti-malarial activity. In (Figure 2.4), Sulfadoxine (4), act on the schizont stages of the erythrocytic cycle as a specific inhibitor of the enzyme dihydropteroate synthetase in the tetrahydrofolate synthesis pathway (P. Wang, Wang, Sims, & Hyde, 2007). Monotherapy isn’t employed with sulfonamides in treating malaria because they are not potent enough on their own but co-administration or combining them with antifolate compounds like pyrimethamine or 21 University of Ghana http://ugspace.ug.edu.gh trimethoprim most commonly as fixed-doses, produce synergistic effects sufficient to cure sensitive strains of malaria (Kumar et al., 2018; WHO, 2010). Figure 2.3: Antimalarial Sulfonamides and Anti-folates they are usually combined with. M. Zareef et al. reported on the antimalarial activity of derivatives of compound 5 which were found to be a potent inhibitor of the degradation of hemoglobin from Plasmodium berghei (Zareef et al., 2007). The relevance of this functional group as an important pharmacophore is amplified by its target specificity being folic acid and its derivatives. These targets are important for normal cell growth and function in most microbes by acting as cofactors in several biosynthetic reactions involved in amino acids synthesis. The absence of this pathway in humans makes it an important microbial target (Hansen & Inselman, 2014; ROLLO, 1955). Generally, sulfonamides act as inhibitors to cell growth and function but they are also usually biphasic; that is at concentrations lower than what is required for inhibition in the target, stimulation of the host’s tissues may occur, which usually produces a synergistic effect between the sulfonamide and a specific antibody within the host (Carta et al., 2012; Klotz, 1944; ROLLO, 1955). According to an extensive review by Richard J. Henry, the activity of sulfonamide action is directly related to its concentration, temperature at which it is administered, and is inversely related to inoculum size and is greatly influenced by the structure of the sulfonamide and the pH (ROLLO, 1955). These parameters affect the mechanism of action of the sulfonamide. Supporting this claim, Patel et al. discovered in some eukaryotic cells (E. coli, Yeast, P. falciparum) that sulfa drugs compete with p-aminobenzoate (pABA) as well as condense with the substrate required for the precursor of the folate synthesis, dihydropteridine pyrophosphate (DHPPP), to form (Sulfa-DHP) an adduct of the sulfa drug and the substrate required for folate synthesis, dihydropteroate (DHP). This adduct, particularly in P. falciparum was found to have a superior in vitro antimalarial activity compared to that exhibited by the parent drug. 22 University of Ghana http://ugspace.ug.edu.gh In addition, the activity of the sulfa adduct was not alleviated by physiological levels of folate, hinting at a novel mechanism of action (Patel, Mberu, Nzila, & Macreadie, 2004). Figure 2.4 represent examples of sulfonamides that possess very interesting therapeutic applications. Compound 6 is reported to significantly reduced the number of lung metastasis, and also effectively suppresses the micro metastatic disease in a rat mammary carcinoma model (Lagente, Le Quement, & Boichot, 2009). Rossello and co-workers also reported a novel compound, 7, which has selectivity towards MMP- 2. This compound showed significant anti-invasive properties in fibro sarcoma HT1080 cells (Rossello et al., 2004) while 8 was reported to kill tumor cells in vitro by activating JNK1 pathways (Reddy et al., 2004), hence displaying anti-tumor and anti- cancer properties of sulphonamides. Figure 2.4: A Selection of Sulphonamides under development 2.3 ARYL CARBOXAMIDES. Aryl carboxamide derivative are important class of organic compounds that consist of an amide functionality directly bonded to an aromatic ring. Examples are benzamides, thiophene carboxamides, pyridine carboxamides etc. Aryl carboxamide derivative possess different kinds of pharmacological activities such as antimicrobial (He, Alian, Stroud, & Ortiz De Montellano, 2006; Sharshira & Mahrous Hamada, 2011), analgesic (Ukrainets et al., 2018), anti-inflammatory (Caliendo et al., 2001) anticancer (Reddy et al., 2005), cardiovascular (Dableh, Yashpal, Rochford, & Henry, 2005) as well as inhibition of various receptors and other biological activities. Due to 23 University of Ghana http://ugspace.ug.edu.gh these biological significances, scientists have developed a wide range of interesting aryl carboxamide derivatives and explored their therapeutic usefulness. Figure 2.5 represent examples of clinically used carboxamides. Compound 9 is a common analgesic and anti-inflammatory drug used for the relief of fever, headaches, and other minor aches and pains (Darias et al., 1992). Y. Wang et al., discovered a series of N-(4-aryl-5-aryloxy-thiazol- 2-yl)-amides as potent RORγt (RAR-related orphan receptor-gamma-t) inverse agonists. RORγt is responsible for controlling the differentiation of Th17 cells where overexpression can lead to human autoimmune inflammatory diseases, including multiple sclerosis. Compounds 10 and 11 showed excellent RORγt activity with 10 exhibiting a pIC50 of 8.0 and 7.7 in FRET assay and dual FRET assay, respectively (Huang et al., 2018). Figure 2.5: Representative aryl carboxamides Figure 2.5 A novel series of 6-N-arylcarboxamidopyrazolo [4.3-d] pyrimidin-7-one derivatives, were synthesized and evaluated for their in vitro anticancer activities against HT-29 colon and DU-145 prostate cancer cell lines by V. N. Devegowda et al. (Figure 2.6) The in vitro anticancer activity tests indicated that compound 12 was the most cytotoxic agent against both colon and prostate cancer cell lines with GI50 values of 0.44 µM and 1.07 µM respectively. While 13 was highly selective towards colon (HT-29) cancer cell line, the other compounds such as 14 and 15 exhibited significant anticancer activity against HT-29 (Devegowda et al., 2010). 24 University of Ghana http://ugspace.ug.edu.gh Figure 2.6: Selection of cytotoxic aryl carboxamides. The antimalarial potential of the Aryl carboxamide derivatives have also been reported. Banerjee et al. reported the antimalarial activity for thiophene carboxamides. In their study, bromo- benzothiophene 16, possess potent inhibitory activity for the blood schizont stage of the mouse model of the plasmodium parasite in vitro as well as in vivo. (Figure 2.7) The compounds were found to specifically impair the development of metabolically active trophozoite stage of intraerythrocytic cycle and the intravenous administration of 16 enhances the longevity of P. berghei infected mice by 2 weeks compared to disease control animals (Banerjee et al., 2011). Figure 2.7: Antimalarial bromobenzothiophene carboxamide 2.4 MOLECULAR HYBRIDIZATION. Molecular Hybridization is defined as a strategy of rational design of new ligands or prototypes based on the recognition of pharmacophoric sub-unities in the molecular structure of two or more known bioactive derivatives which, through the adequate fusion of these sub-unities, lead to the 25 University of Ghana http://ugspace.ug.edu.gh design of new hybrid architectures that maintain pre-selected characteristics of the original templates (Claudio Viegas-Junior, Eliezer J. Barreiro, & Carlos Alberto Manssour Fraga, 2007). The issue of drug resistance is as a result of, among other factors, the consistent use of the drug to treat diseases (Cheesman, Ilanko, Blonk, & Cock, 2017). For instance, Sulfonamides have been used both as a monotherapy drug and combinatory drug in the treatment of multiple diseases and health conditions for approximately 86 years (Cheesman et al., 2017). Several biological activities studied by different researchers in organic scaffolds are based on the aryl carboxamides. The effects of substitutions on carboxamides are either by aliphatic, aromatic or heteroaromatic systems that have led to various types of biological activities (Asif, 2016). 17, Sulpiride, is an atypical antipsychotic medication used purposely to treat psychosis connected with schizophrenia and major depressive disorder, and sometimes used in low dosage to treat anxiety and mild depression. (Figure 2.8) Figure 2.8: Representative molecular hybridized compounds Andrew A. Mortlock and co synthesized a series of novel sulfonamides which have shown high affinity and selectivity for the endothelin ETA receptor. 18, a potent ETA-selective endothelin antagonist which showed a pIC50 value of 9.3 and a high in vivo oral potency, when dosed at 2.5 mgkg-1 in conscious rat, with a duration of action in excess of 4 hours (Mortlock et al., 1997). 26 University of Ghana http://ugspace.ug.edu.gh CHAPTER THREE. RESULTS AND DISCUSSION 3.1 CHEMISTRY: The synthesis of the titled compounds was achieved in three chemical steps in moderate to high yields. First, 4-chlorobenzoic acid was refluxed in chlorosulfonic acid at 140 oC for three hours according to the method developed by Gerald F. Holland to furnish Sulfonyl chloride intermediate. This was followed by the formation of the key sulphonamide intermediates by treating the sulfonyl chloride with a number of amines. Finally, the titled compounds were made by amide bond formation via acyl chloride or the mixed anhydride intermediates (Scheme 1) (Holland, 1983). Scheme 1: Synthesis of the sulphonamide intermediate compounds and Synthetic route to the titled compounds via the mixed anhydride and acid chloride routes. a) DCM, Et3N, ClCOC2H5, 0 0C, 90 min. d) DCM, oxalyl chloride, DMF, r.t, 1 h. b) and c) NHR1R2 r.t, 1 h. 27 University of Ghana http://ugspace.ug.edu.gh Table 2: Synthesised intermediates and final products with corresponding yields. R1R2 4 5 1 2 3 6 R3R4 80% 78% 70% 84% 82% 75% 7 10 18 FEP003 FEP018 FEP040 30% 8% 35% 15 22 FEP012 FEP037 27% 21% 11 14 FEP020 FEP008 15% 15% 8 12 16 19 23 FEP005 FEP021 FEP011 FEP041 FEP038 35% 6% 25% 30% 30% 9 13 17 20 FEP006 FEP019 FEP022 FEP056 23% 16% 29% 30% 21 FEP044 29% All the synthesized compounds were characterized by 2D and 3D NMR, FTIR, LC-MS (refer to Appendices 3, 2 and 1 respectively). The NMR was used to ascertain the purity and to confirm that the titled compounds were duly synthesized. The analysis was done with Bruker 500 MHz spectrophotometer. 28 University of Ghana http://ugspace.ug.edu.gh 3.2 SPECTRAL ANALYSIS FOR FEP 005. 3.2.1 1H AND 13C NMR. The 1HNMR for compound FEP005 (Figure 3.1) gave a singlet at chemical shift of 2.51 ppm corresponding to the methyl signal (H1). A doublet of doublets at 3.07 ppm and a multiplet at 3.79-3.73 ppm correspond to the morpholinyl protons (H9) and (H10) respectively. The aromatic and pyridinyl protons occurred between 8.51-6.91 ppm. The broad, almost invisible singlet at the chemical shift of 8.88 corresponds to the H5 peak while doublets at 8.20 ppm, 8.09 ppm and 7.80 ppm correspond to H6, H8 and H7 respectively. The triplet peak at 7.74 ppm and doublets at 7.67 ppm and 7.02 ppm correspond to H3, H4 and H2 respectively. 29 University of Ghana http://ugspace.ug.edu.gh Aromatic Protons H10 H9 H1 Expanded Aromatic Protons H8 H4 H7 H2 H3 H6 H5 Figure 3.1: 1H NMR for FEP 005 The 13C NMR (Figure 3.2) on the other hand gave 14 distinct peaks suggesting there are 14 equivalent carbons. The signal at the chemical shift of 23.7 ppm (C1) corresponds to the methyl carbon while those at 46.2 ppm (C14) and 65.7 ppm (C15) are those of the morpholinyl carbons. The peak at 163 (C7) correspond to the carbonyl carbon while the rest at 157.2 (C6), 150.1 (C2), 138.9 (C4), 136.3 (C9, C12), 135.0 (C8), 131.4 (C10), 130.5 (C11), 128.9 (C13), 120.4 (C3), 111.1 (C5) correspond to the pyridyl and aromatic carbons. 30 University of Ghana http://ugspace.ug.edu.gh C15 C14 C11 C1 C4 C10 C3 C5 C12 C9/4 C6 C2 C8 C7 Figure 3.2: 13C NMR FOR FEP 005 3.2.2 DEPT 135. In the DEPT 135 NMR analysis (Figure 3.3), the methyl and the methine carbons are positive whilst the methylene carbons show negative signals. The quartenary carbons do not show at all in the chromatogram. The DEPT 135 made it possible to group the carbons into methyl, methylene, methane and quaternary carbons. The results for FEP005 suggest we have one methyl (CH3) carbon, two methylene (CH2) carbon emanating from the morpholinyl group and six methine (CH) carbons. 31 University of Ghana http://ugspace.ug.edu.gh Figure 3.3: DEPT 135 Chromatogram for FEP005 3.2.3 HSQC This is a 2-Dimensional (2D) experiment which maps the chemical shift of the proton with the chemical shift of the carbons directly attached to it. The proton spectrum is on one axis while the carbon is on the other. The least intense cross peaks give the shift of the corresponding proton and the carbon as well as the one-bond coupling between methane and a quaternary carbon. Cross peaks in the aromatic region of the HSQC spectrum for FEP005 confirms the 6 aromatic protons and their corresponding carbons. The two most intense cross peaks in the spectrum corresponds to 8 protons on the 4 carbons in the morpholino substituent. The next intense cross peak is that of the 3 protons on the methyl carbon attached to the aromatic ring (Figure 3.4). 32 University of Ghana http://ugspace.ug.edu.gh Figure 3.4: HSQC Chromatogram for FEP005 3.2.4 LC-MS OF FEP005 The LC-MS of compound FEP005 measured in the positive mode using electrospray ionization (Figure 3.5). While the LC show that the compounds was pure, the mass obtained was at m/z of 396 which represents the molecular ion [M + H] + from the compound of molar mass C17H18ClN3O4S =395.86g/mol. DAD1 A, Sig=280,4 Ref=550,10 (D:\DATA\2018-05-13\2018-05-11 2018-05-13 17-12-50\1AA-0101.D) mAU 2500 2000 1500 1000 500 0 0.5 1 1.5 2 2.5 3 3.5 4 min MSD1 TIC, MS File (D:\DATA\2018-05-13\2018-05-11 2018-05-13 17-12-50\1AA-0101.D) MM-APCI, Pos, Scan, Frag: 200, "sc 8000000 6000000 33 4000000 2000000 0 0.5 1 1.5 2 2.5 3 3.5 4 min 2.418 2.658 University of Ghana http://ugspace.ug.edu.gh *MSD1 SPC, time=2.620:2.717 of D:\DATA\2018-05-13\2018-05-11 2018-05-13 17-12-50\1AA-0101.D MM-APCI, Pos, Scan, Frag 100 Max: 564821 + [M+H] = 396.0 80 60 40 20 0 300 325 350 375 400 425 450 475 500 m/z Figure 3.5: LC-MS of FEP005 3.2.5 FTIR OF FEP005 FEP005 showed characteristic absorption frequencies (Figure 3.6) at 3322.5cm-1 characteristic of NH stretch. Also, bands appearing at 2855.4, 1698.8, 1453.8, 1152.33 and 1112.50 are characteristic of C-H, C=O, C=C, S=O and CN stretch vibrations respectively. Other bands at 1301.15, 1073.3 and 720.95 are characteristic of C-H bending and C-O, C-Cl bond vibrations. Similar chromatograms were obtained for the rest of the synthesized compound. C-Cl C-H O-H -1 -1 -1 720cm 2855cm 3322cm C-N -1 1164cm C=O -1 S=O 1698cm C=C -1 AR 1152cm -1 1453.8cm Figure 3.6: FTIR OF FEP005. 34 396.0 399.1 398.0 University of Ghana http://ugspace.ug.edu.gh 3.3 SPECTRAL ANALYSIS FOR FEP 040. 3.3.1 H1NMR The H1NMR for compound FEP040 gave diagnostic peaks at the aromatic and sp3 regions of the spectrum (Figure 3.7). A singlet at chemical shift 2.84 ppm corresponds to the methyl group (HJ) attached to the benzothiazole ring. A triplet at 3.03 ppm corresponding to the protons (HI) attached to Nitrogen in the piperidine ring. A multiplet occurring at 1.65 ppm and 1.45 ppm corresponds to the protons (HK) and (HL) in the piperidine ring respectively. A singlet in the aromatic region at 8.21 ppm signaling the amide proton (HB). The aromatic protons with differing multiplicities (HA to HG) occurring between 8.53 ppm to 7.41 ppm. Figure 3.7: H1NMR for FEP040 35 University of Ghana http://ugspace.ug.edu.gh 3.3.2 13C NMR FOR FEP040 The 13C spectrum of FEP040 confirmed 18 distinct Carbons (Figure 3.8). The signal at 167.20ppm identifies the carbonyl carbon at C1. The chemical shift from (163.18 to 112.96) ppm corresponds to the 13 distinct aromatic carbons C2 to C14. The methyl group C18, attached to the benzothiazole is represented by the chemical shift at 20.13ppm while the peaks at 46.96ppm, 25.11ppm and 23.36ppm signifies the 5 carbons C15, C16 and C17 on the piperidine ring respectively. C15 C16 EXPANDED AROMATIC CARBONS C18 C17 C9 C12 C13 C14 C11 C1 C5/6 C10 C2 C3 C7 C4 C8 Figure 3.8: 13 C NMR for FEP040 36 University of Ghana http://ugspace.ug.edu.gh 3.4 PHYSICOCHEMICAL PROPERTIES. Studies on three physicochemical proprieties of the series of benzene sulphonamides analogues were obtained both by experimental analysis and by calculation through high-end software. QSAR proprieties such as octanol-water partition coefficient (log P), molar refractivity (MR) and Polar Surface Area (PSA) were investigated. The Molar Refractivity of the compound is a measure of the total polarizability of a mole of a compound. It is a steric factor that relates the dispersion forces of the compound to its density and molar mass (Ghose, Viswanadhan, & Wendoloski, 1999). The MR of the analogues ranges from 66.07 to 115.98. Lipophilicity is a key property that has a major effect on solubility, absorption, distribution, metabolism, and excretion properties as well as pharmacological activity. High lipophilicity may cause the compound to have low solubility, metabolic instability and poor absorption (Waring, 2010). According to Lipinski’s rule, for a compound to be drug like, the Log P value should be less than 5. The Log P for these synthesized compounds ranges from 1.2 to 4.5 hence they aren’t too lipophilic and the probability of giving bioavailability issues would be low. The Polar Surface Area is defined as the amount of molecular surface arising from polar atoms (nitrogen and oxygen atoms) together with their attached hydrogen atoms. PSA is commonly used in drug discovery for the maximization of a drug's potential to permeate cells. Molecules with a polar surface area of greater than 140 angstroms squared tend to be poor at permeating cell membranes (Veber et al., 2002). From the table, the PSA of the synthesized compounds have the ability to permeate cell membranes as they do not exceed the drug-able limit. Table 3: Physicochemical properties of synthesized benzene sulphonamide derivatives. No. COMPOUND LogP MR PSA No. COMPOUND LogP MR PSA 7 FEP 003 2.43 112.93 125.22 16 FEP 011 2.07 91.1 87.75 8 FEP 005 1.76 100.17 96.78 17 FEP 022 2.46 106.39 82.2 9 FEP 006 1.2 113.92 104.32 18 FEP 040 3.58 115.98 114.11 10 FEP 018 3.21 111.4 115.98 19 FEP 041 3.08 87.75 103.24 37 University of Ghana http://ugspace.ug.edu.gh 11 FEP 020 2.51 112.39 82.19 20 FEP 056 2.27 95.09 116.99 12 FEP 021 2.53 96.64 87.75 21 FEP 044 3.19 66.07 101.38 13 FEP 019 1.86 113.93 95.08 22 FEP 037 4.37 87.75 123.29 14 FEP 008 2.1 114.34 95.68 23 FEP 038 4.5 87.75 127.88 15 FEP 012 2.06 86.5 87.75 MMV019721 3.44 113.36 115.99 3.5 BIOLOGICAL ACTIVITY The synthesized compounds were first screened in vitro for their antimalarial activity and the results obtained were examined to establish a trend (Table 3). Based on that, optimized leads can be synthesized in order to identify a potential drug candidate. Preliminary results obtained for nine of the compounds showed promising activity in the micro molar range against the 3D7 strain of the malaria parasite. One of the compounds synthesized FEP040, showed a superior activity of 286.1 nM compared to the control compound MMV019721 with IC50 value of 552 nM. Table 4: In vitro antimalarial activity of some selected compounds COMPOUNDS STRUCTURE IC50(µM) COMPOUNDS STRUCTURE IC50(µM) FEP011 8.69 FEP008 25.11 MMV019721 0.552 FEP018 3.086 FEP003 1.23 FEP019 25.11 38 University of Ghana http://ugspace.ug.edu.gh FEP005 25.11 FEP020 25.11 FEP021 7.79 FEP040 0.27 3.6 QUANTITATIVE STRUCTURE ACTIVITY RELATIONSHIP (QSAR) ANALYSIS. Biological activity is often marked as a physiological response in the host or the parasite that arises from the interaction with a foreign entity. A response can be generated as a result of a characteristic of that foreign entity. Therefore, in order to establish a correlation between activity and that characteristic, in this case, the physicochemical properties, a liner regression analysis was done between the activity and each calculated physicochemical property and their line fit graphs plotted. y = 0.0389x - 4.6868 y = 0.9574x - 3.1742 LogP Line Fit Plot PSA Line Fit PlotR² = 0.6287 1 R² = 0.749 1 R= 0.793 R= 0.865 0.5 0.5 0 0 0 1 2 3 4 0 50 100 150 -0.5 -0.5 -1 -1 -1.5 -1.5 -2 -2 PSALog(1/ LogP Log(1/C) Linear (Log(1/C))C) Figure 3.9: Log (1/C) vs LogP and Log (1/C) vs PSA line fit plots From the line fit plots, lipophilicity (Log P) (Figure 3.9) had the best correlation of 0.87 as compared to PSA (0.79) (Figure 3.9) and MR (0.28) (Figure 3.10). 39 Log(1/C) Log(1/C) University of Ghana http://ugspace.ug.edu.gh y = 0.0242x - 3.3408 MR Line Fit Plot 1 R² = 0.0803 R= 0.283 0.5 0 0 50 100 150 200 -0.5 -1 -1.5 -2 MR Log(1/C) Linear (Log(1/C)) Figure 3.10: Log (1/C) vs MR The activity obtained for the lead and nine of the compounds and their respective physicochemical properties showed from the line fit plots that lipophilicity of the compounds had a resounding impact on the biological activity. Moreover, compounds with the benzothiozoyl benzamide moiety as seen in table 4 showed the best activity, hinting at the importance of this scaffold for bioactivity. Hence, the observed optimum Log P should be around 4.4. Additionally, to further grasp the full extent on how each physicochemical property affects the biological activity, a multiple regression analysis was done and a model was developed. Therefore, the activity can be expressed as: Log (1/C) = 0.712LogP + 0.026PSA - 0.021MR - 2.955 n=10; r= 0.95; r2= 0.91; Adj. r2= 0.87; S= 0.27 The equation above reveals the polarizing extent to which lipophilicity has an effect on activity relative to the polar surface area and molar refractivity. To determine the accuracy of this model, the activity of the lead and nine compounds were calculated using the model. Then a line fit graph of the calculated activity was plotted against the experimental activity of compounds. 40 Log(1/C) University of Ghana http://ugspace.ug.edu.gh Table 4: Calculated IC50 and Experimental IC50 COMPOUND Cal.IC50 IC50 IC50 Line Fit Plot (µM) (µM) y = 0.9572x + 0.3491 R² = 0.8836 FEP 003 2.03 1.23 R= 0.9440 FEP 005 18.28 25.11 35 FEP 008 22.07 25.11 30 FEP 011 12.24 8.69 25 FEP 018 0.92 3.09 20 FEP 019 33.25 25.11 15 FEP 020 23.08 25.11 10 5 FEP 021 7.51 7.79 0 FEP 040 0.50 0.27 0 10 20 30 40 IC50 MMV019721 0.69 0.55 PIC50 Linear (PIC50) Figure 3.11: Cal. IC50 vs. IC50 Analysis of Table 4 reveals the closeness of the calculated value and the experimental value. The line fit plot in Figure 3.11 confirms this with a near perfect correlation of 0.94. 41 Cal.IC50 University of Ghana http://ugspace.ug.edu.gh CHAPTER FOUR 4.1 MATERIALS 4.1.1 REAGENTS With the exception of those stated, all reagents were obtained from commercial suppliers. Pure and sealed DCM, Et3N and THF were used throughout the experiments. 4.1.2 CHROMATOGRAPHY Thin layer chromatography was performed on pre-coated silica gel aluminium sheets. UV light (254 nm) was used for all visualizations and flash column chromatography was performed using silica gel 60Å particle size 35-70 micron, Davisil® Chromatography grade. 4.1.3 FTIR FTIR was performed using FTIR spectrophotometer spectrum. Solid samples were applied neat onto sodium chloride discs. 4.1.4 NMR 1H and 13C NMR spectra were recorded using a Bruker 500 MHz NMR spectrophotometer situated at the Department of Chemistry. Spectra were referenced to the residual solvent peak and chemical shifts are expressed in ppm. The NMR experiments were performed at 25 oC. The following annotations are used to describe multiplicity; s for singlet, bs for broad-singlet, d for doublet, t for triplet, q for quartet, m for multiplet and coupling constants (J) are expressed in Hertz (Hz). 4.1.5 LC-MS Mass spectra were recorded using the Liquid Chromatography Mass Spectrometers Agilent HPLC system. The ionization technique used was the Electron Spray (ESI) with Agilent Jet Stream Electrospray (AJS-E) machine. The ionization was done in positive mode and the mobile phase of the column was 10 nM NH4OAc in 90 % CH3OH in H2O. The stationary phase was Kinetex Cores C18, 2.6 µM, 3 x 50nm, 100Å maintained at 40 oC. 42 University of Ghana http://ugspace.ug.edu.gh 4.2 Preparation of 2-chloro-5-(chlorosulfonyl) benzoic acid. To chlorosulfonic acid (10 mL, as solvent) cooled in an ice-bath was slowly added 2-chlorobenzoic acid (5.0 g, 31.9 mmol) and the resulting mixture was heated to 140 oC for 3 h. The mixture was then cooled to room temperature and added dropwise to stirred ice-water (150 mL). The resulting precipitate was filtered beige solid. Yield obtained was 90%. V max (neat) cm 1: 3428.17, 3085.24, 1684.24, 1025.58, 543.10. 1H NMR (500 MHz, CDCl3) δH 9.1 (bs, 1H, OH), 8.70 (dd, J = 9.8, 2.4 Hz, 1H), 8.16 (dd, J = 8.6, 2.4 Hz, 1H), 7.81 (d, J = 8.6 Hz, 1H). 13C NMR (126 MHz, Chloroform-d) δ 168.0, 142.8, 142.5 133.3, 131.1, 129.9. Melting point. (61-62) oC. 4.3 General procedure for sulfonamide formation. To a solution of 2-chloro-5-chlorosulfonyl benzoic acid in THF (20 mL) was added an amine (1.5 equiv) and stirred for 24 h at room temperature. After stirring, the reaction was quenched with 20 mL 1M HCl. The aqueous mixture was extracted with either chloroform or ethyl acetate (20 mL x 3), the organic phase washed with brine and dried over anhydrous magnesium sulphate. The solvent was evaporated to give the crude product as white solid. 43 University of Ghana http://ugspace.ug.edu.gh 4.3.1 Preparation of 2-chloro-5-(N,N-diethylsulfamoyl)benzoic acid. This compound was prepared according to the general procedure for sulfonamide formation in 50 % yield as a mild yellowish amorphous solid. V max (neat) cm1: 2976.63, 1682.85, 1333.380, 1164.49, 941.71, 703.75. 1H NMR (500 MHz, CDCl3) δH 8.44 (s, 1H), 8.27 (d, J = 2.3 Hz, 1H) 7.80 (dd, J = 8.4, 2.3 Hz, 1H), 7.62 (dd, J = 8.3, 2.3 Hz, 1H), 3.30 (q, J = 6.8 Hz, 4H), 1.18 (t, J = 6.8 Hz, 6H). 13C NMR (126 MHz, CDCl3) δC 168.5, 139.4, 138.5, 132.4, 131.4, 130.7, 129.5, 42.1, 14.3. Melting pt. (145-147) oC 4.3.2 Preparation of 2-chloro-5-(pyrrolidin-1-ylsulfonyl) benzoic acid This compound was prepared according to the general procedure for sulfonamide formation in 70 % yield as a yellowish solid. V max (neat) cm1: 2970.26, 1679.63, 1344.11, 1167.52, 1011.81, 595.77. 1H NMR (500 MHz, CDCl3) δH 8.35 (s, 1H), 7.84 (d, J = 8.4 Hz, 1H), 7.59 (d, J = 8.4 Hz, 1H), 3.21 (t, J = 5.9 Hz, 4H), 1.75 (t, J = 5.9 Hz, 4H). 13C NMR (126 MHz, CDCl3) δC 167.8, 139.0, 136.3, 132.3, 131.6, 131.0, 48.2, 25.2. Melting pt. (205-207) oC 44 University of Ghana http://ugspace.ug.edu.gh 4.3.3 Preparation of 2-chloro-5-(N,N-dimethylsulfamoyl) benzoic acid This compound was prepared according to the general procedure for sulfonamide formation in 80 % yield as a pink-white solid. 1H NMR (500 MHz, Chloroform-d) δ 8.41 (d, J = 2.2 Hz, 1H), 8.07 (s, 1H), 7.89 (dd, J = 8.5, 2.2 Hz, 1H), 7.71 (d, J = 8.4 Hz, 1H), 2.79 (s, 6H). 13C NMR (126 MHz, CDCl3) δC 168.0, 139.5, 135.1, 132.4, 132.0, 131.4 129.5, 37.8. Melting pt. (175-176) oC 4.3.4 Preparation of 2-chloro-5-(morpholinosulfonyl) benzoic acid. This compound was prepared according to the general procedure for sulfonamide formation in (85) % yield as a whitish solid. 1H NMR (500 MHz, Chloroform-d) δ 8.36 (d, J = 2.3 Hz, 1H), 8.18 (s, 1H), 7.84 (dd, J = 8.4, 2.3 Hz, 1H), 7.70 (d, J = 8.4 Hz, 1H), 3.79 (m, 4H), 3.07 (m, 4H). 13C NMR (126 MHz, Chloroform-d) δ 167.2, 139.8, 134.4, 132.5, 131.8, 131.4, 130.1, 66.0, 45.9. Melting point. (95-96) oC 45 University of Ghana http://ugspace.ug.edu.gh 4.3.5 Preparation of 2-chloro-5-(piperidin-1-ylsulfonyl) benzoic acid. This compound was prepared according to the general procedure for sulfonamide formation but with a change of solvent being benzene, used as the solvent in this synthesis. (75-80) % yield was obtained as a creamy or yellowish solid. V max (neat) cm1: 2927.44, 1681.65, 1337.89, 1168.93, 749.58, 587.29. 1H NMR (500 MHz, Chloroform-d) δ 8.33 (d, J = 2.3 Hz, 1H), 7.83 (dd, J = 8.4, 2.3 Hz, 1H), 7.65 (d, J = 8.4 Hz, 1H), 3.03 (t, J = 5.5 Hz, 4H), 1.66 (p, J = 5.7 Hz, 4H), 1.46 (tq, J = 8.9, 5.6, 4.5 Hz, 2H).13C NMR (126 MHz, Chloroform-d) δ 167.1, 139.0, 135.7, 132.2, 131.6, 131.1, 130.0, 46.9, 25.1, 23.4. Melting pt. (203-205) oC. 4.3.6 Preparation of 2-chloro-5-((4-phenylpiperidin-1-yl) sulfonyl) benzoic acid. This compound was prepared according to the general procedure for sulfonamide formation in (75) % yield as a white flakey solid. V max (neat) cm1: 2923.70, 1694.25, 1343.73, 1164.92, 929.34, 571.07. 1H NMR (500 MHz, Chloroform-d) δ 8.64 (d, J = 2.4 Hz, 1H), 8.10 (dd, J = 8.6, 2.3 Hz, 1H), 7.88 (d, J = 8.6 Hz, 1H), 7.76 (m, 2H), 7.68 (dd, J = 8.3, 1.9 Hz, 2H), 7.30 (m, 1H), 4.06 – 3.85 (m, 4H), 2.52 – 2.37 (m, 1H), 1.97 – 1.79 (m, 4H). 13C NMR (126 MHz, Chloroform- 46 University of Ghana http://ugspace.ug.edu.gh d) δ 167.8, 144.6, 139.4, 135.5, 133.1, 132.4, 131.9, 131.4, 129.6, 128.7, 126.7, 46.9, 41.7, 32.5. Melting pt. (166-170) oC. 4.41 GENERAL PROCEDURE FOR CARBOXAMIDE FORMATION (ACID CHLORIDE METHOD). To a suspension of the sulfonamide intermediates (1 equiv, 0.20g) in dry DCM (20 mL), was added oxalyl chloride (2equiv.) at room temperature followed by 2 drops of DMF and stirred for 1 hour. The solvent and excess oxalyl chloride were removed by evaporation to afford acid chloride. Acid chloride of sulfonamide intermediates is re-dissolved in DCM (20 mL) and then added to (2 equiv) of amine and (1.2 equiv) Et3N and stirred for 1 h at room temperature. After stirring, the reaction was quenched with 20 mL 1M HCl. The aqueous mixture was extracted with either chloroform or ethyl acetate (20 mL x 3) and the organic phase washed with brine and dried over anhydrous magnesium sulphate. After concentration, the crude was purified through silica gel column. 4.411 GENERAL PROCEDURE FOR CARBOXAMIDE FORMATION (MIXED ANHYDRIDE METHOD). To a solution of sulfonamide intermediates (1 equiv., 0.20g) in dry DCM (20mL) was added (1.5 equiv.) Et3N and (0.9 equiv.) ethyl chloroformate. The reaction was stirred for 60 min at 0 oC. (1.5 equiv.) Amine was added and stirred. After 30 min, the reaction was warmed to room temperature and stirred for a further 90 min. The reaction mixture was concentrated and purified through silica gel column to afford the target molecule. 47 University of Ghana http://ugspace.ug.edu.gh 4.4.1 Preparation of 4-chloro-N, N-dimethyl-3-(4-(pyrimidin-2-yl) piperazine-1-carbonyl) benzenesulfonamide This compound was synthesized according to the general procedure for carboxamide formation (mixed anhydride method) in 29 % yield as a white flakey solid. V max (neat) cm 1: 2921.06, 1642.62, 1588.62, 1435.75, 1340.16, 1164.00, 714.93. 1H NMR (500 MHz, CDCl3) δH 8.34 (d, J = 4.3 Hz, 2H), 7.79 (d, J = 10.2 Hz, 2H), 7.58 (d, J = 8.2 Hz, 1H), 6.57 (t, J = 4.3 Hz, 1H), 4.06 – 3.91 (m, 3H), 3.85 (d, J = 4.9 Hz, 3H), 3.40 – 3.19 (m, 6H), 1.17 (t, J = 7.0 Hz, 6H). 13C NMR (126 MHz, CDCl3) δC 165.6, 161.4, 157.8, 140.0, 136.6, 134.4, 130.6, 128.5, 126.4, 110.8, 43.9, 43.4, 42.0, 41.6, 14.1. Melting pt. (120-123) oC. 4.4.2 Preparation of 2-chloro-5-(N,N-dimethylsulfamoyl)-N-(pyridin-2-yl)benzamide This compound was synthesized according to the general procedure for carboxamide formation (acid chloride method) in 27 % yield as a pale white amorphous solid. V max (neat) cm 1: 3299.99, 3073.41, 1695.91, 1523.65, 1434.90, 1310.53, 1151.61, 781.84. 1H NMR (500 MHz, CDCl3) δH 9.13 (s, 1H), 8.36 (d, J = 8.3 Hz, 1H), 8.16 (d, J = 4.7 Hz, 1H), 8.12 (s, 1H), 7.83 (d, J = 9.2 Hz, 1H), 7.80 (d, J = 7.9 Hz, 1H), 7.65 (d, J = 8.4 Hz, 1H), 7.15 – 7.08 (m, 1H), 2.77 (s, 6H). 13C NMR (126 MHz, CDCl3) δC 163.4, 150.9, 147.8, 138.8, 136.1, 135.7, 135.6, 131.4, 130.5, 128.9, 120.6, 114.4, 37.8. MS (ES) [M + H] + = 340.0 m/z 100 % C14H14ClN3O3S =339.8. Melting pt. (278- 280) oC. 48 University of Ghana http://ugspace.ug.edu.gh 4.4.3 Preparation of 2-chloro-5-(N, N-dimethylsulfamoyl)-N-(6-methylpyridin-2-yl) benzamide This compound was synthesized according to the general procedure for carboxamide formation (acid chloride method) in 25 % yield as a pale white amorphous solid. V max (neat) cm 1: 3338.85, 3081.91, 1699.31, 1575.89, 1451.01, 1333.66, 1149.98, 703.70. 1H NMR (500 MHz, Chloroform- d) δ 8.59 (s, 1H), 8.15 (d, J = 8.2 Hz, 1H), 8.09 (d, J = 2.2 Hz, 1H), 7.82 (dd, J = 8.4, 2.2 Hz, 1H), 7.70 (d, J = 7.8 Hz, 1H), 7.65 (d, J = 8.4 Hz, 1H), 6.99 (d, J = 7.5 Hz, 1H), 2.77 (s, 6H), 2.46 (s, 3H). 13C NMR (126 MHz, Chloroform-d) δ 163.2, 157.3, 150.0, 138.9, 136.2, 135.7, 135.5, 131.3, 130.4, 128.8, 120.2, 111.2, 37.9, 24.0. MS (ES) [M + H]+ = 354.0 m/z 100 % C15H16ClN3O3S =353.82. Melting pt. (260-261) oC. 4.4.4 Preparation of 4-chloro-N,N-diethyl-3-(4-(pyrimidin-2-yl)piperazine-1- carbonyl)benzenesulfonamide This compound was synthesized according to the general procedure for carboxamide formation (acid chloride method) in 15 % yield as a yellowish-white amorphous solid. V (neat) cm1max : 3063.60, 1639.11, 1597.29, 1435.13, 1326.07, 1151.87, 695.96. 1H NMR (500 MHz, CDCl3) δH 8.34 (d, J = 4.4 Hz, 2H), 7.80 (s, 1H), 7.78 (s, 1H), 7.58 (d, J = 8.2 Hz, 1H), 6.57 (t, J = 4.2 Hz, 1H), 4.11 – 3.91 (m, 3H), 3.88-3.80 (m, 3H), 3.45 – 3.18 (m, 6H), 1.17 (t, J = 7.0 Hz, 6H). 13C NMR (126 MHz, CDCl3) δC 165.8, 161.0, 158.1, 139.5, 136.2, 134.7, 130.3, 128.5, 126.4, 110.8, 49 University of Ghana http://ugspace.ug.edu.gh 46.5, 43.9, 43.4, 42.3, 41.5, 14.2. MS (ES) [M + H]+ = 438.1 m/z 100 % C19H24ClN5O3S =437.94. Melting pt. (145-148) oC 4.4.5 Preparation of 2-chloro-N-(6-methylpyridin-2-yl)-5-(morpholinosulfonyl) benzamide This compound was synthesized according to the general procedure for carboxamide formation (acid chloride method) in 23 % yield as a brownish amorphous solid. V max (neat) cm 1: 2857.36, 1641.07, 1435.83, 1352.47, 1172.96, 695.96. 1H NMR (500 MHz, CDCl3) δH 8.35 (d, J = 4.7 Hz, 2H), 7.76 (d, J = 2.1 Hz, 1H), 7.73 (dd, J = 5.8, 2.0 Hz, 1H), 7.64 (d, J = 8.3 Hz, 1H), 6.58 (t, J = 4.7 Hz, 1H), 4.08–3.91 (m, 3H), 3.91–3.79 (m, 3H), 3.76 (t, J = 4.7 Hz, 4H), 3.38 – 3.25 (m, 2H), 3.12–2.99 (m, 4H). 13C NMR (126 MHz, CDCl3) δC 165.2, 161.5, 157.9, 137.1, 135.7, 135.0, 130.87, 129.4, 127.3, 110.6, 66.0, 46.7, 45.9, 43.9, 43.4, 41.8. MS (ES) [M + H] + = 451.1 m/z 100 % C19H22ClN5O4S =451.1. Melting pt. (128-130) oC. 4.4.6 Preparation of 2-chloro-N-(6-methylpyridin-2-yl)-5-(morpholinosulfonyl) benzamide This compound was synthesized according to the general procedure for carboxamide formation (acid chloride method) in 35 % yield as a pale white amorphous solid. V (neat) cm1max : 3322.45, 2855.35, 1698.79, 1577.79, 1453.82, 1152.33, 720.34. 1H NMR (500 MHz, CDCl3) δH 8.51 (s, 1H), 8.06 (d, J = 8.1 Hz, 1H), 7.99 (d, J = 2.1 Hz, 1H), 7.71 (dd, J = 8.4, 2.1 Hz, 1H), 7.65 – 7.60 50 University of Ghana http://ugspace.ug.edu.gh (m, 1H), 7.58 (d, J = 8.4Hz, 1H), 6.91 (d, J = 7.5 Hz, 1H), 3.74 – 3.62 (m, 4H), 3.02 – 2.92 (m, 4H), 2.37 (s, 3H). 13C NMR (126 MHz, CDCl3) δC 163.0, 157.2, 150.1, 138.9, 136.3, 135.0, 131.4, 130.5, 128.9, 120.4, 111.1, 65.7, 46.2, 23.7. MS (ES) [M + H]+ = 396 m/z 100 % C17H18ClN3O4S =395.860. Melting pt. (158-159) oC 4.4.7 Preparation of 2-chloro-N-(2-methylbenzo[d]thiazol-6-yl)-5-(morpholinosulfonyl) benzamide This compound was synthesized according to the general procedure for carboxamide formation (acid chloride method) in 30 % as a white solid. V max (neat) cm 1: 3232.87, 3063.67, 1683.07, 1325.72, 1168.87, 799.95. 1H NMR (500 MHz, CDCl3) δH 8.55 (s, 1H), 8.18 (s, 1H), 8.11 (s, 1H), 7.93 (d, J = 8.5 Hz, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.67 (d, J = 8.3 Hz, 1H), 7.42 (d, J = 8.6 Hz, 1H), 3.77 (s, 4H), 3.06 (s, 4H), 2.86 (s, 3H). 13C NMR (126 MHz, CDCl3) δC 167.3, 162.9, 150.7, 136.8, 136.26, 136.0, 135.0, 134.0, 131.5, 130.5, 129.2, 122.5, 118.9, 113.0, 65.9, 46.2, 20.1. MS (ES) [M + H] + = 452.0 m/z 100 % C18H18ClN3O4S = 451.95. Melting pt. (254) oC. 4.4.8 Preparation of (2-chloro-5-(pyrrolidin-1-ylsulfonyl) phenyl) (4-(pyridin-2-yl) piperazin-1-yl) methanone. 51 University of Ghana http://ugspace.ug.edu.gh This compound was synthesized according to the general procedure for carboxamide formation (mixed anhydride method) in 15% yield as a white foam. V max (neat) cm 1: 3069.65, 1633.53, 1587.30, 1434.97, 1239.05, 1159.61, 619.83. 1H NMR (500 MHz, CDCl3) δH 8.22 (dd, J = 4.8, 1.2 Hz, 1H), 7.83 (dd, J = 8.4, 2.1 Hz, 1H), 7.80 (d, J = 2.1 Hz, 1H), 7.61 (d, J = 8.4 Hz, 1H), 7.57 – 7.51 (m, 1H), 6.73 – 6.66 (m, 2H), 4.08 (m, 1H), 4.08 – 3.99 (m, 1H), 3.93 – 3.85 (m, 1H), 3.77 – 3.63 (m, 2H), 3.59 (t, J = 5.2 Hz, 2H), 3.37 (qd, J = 13.1, 8.0 Hz, 2H), 3.27 (dd, J = 11.2, 6.0 Hz, 4H), 1.89 – 1.78 (m, 4H). 13C NMR (126 MHz, CDCl3) δC 165.5, 159.1, 148.0, 137.9, 136.9, 136.6, 135.3, 130.6, 129.0, 126.9, 114.2, 107.4, 48.0, 46.5, 45.5, 45.2, 41.6, 25.3. MS (ES) [M + H] + = 435.1 m/z 100 % C20H23ClN4O3S = 434.94. Melting pt. (176-177) oC. 4.4.9 Preparation of (2-chloro-5-(pyrrolidin-1-ylsulfonyl) phenyl) (4-(pyrimidin-2-yl) piperazin-1-yl) methanone. This compound was synthesized according to the general procedure for carboxamide formation (mixed anhydride method) in 16 % yield as a white foam. V max (neat) cm 1: 2985.19, 1702.66, 1552.46, 1434.34, 1235.10, 1123.63, 765.69. 1H NMR (500 MHz, CDCl3) δH 8.33 (t, J = 4.4 Hz, 2H), 7.81 (dd, J = 8.4, 2.1 Hz, 1H), 7.79 (d, J = 2.0 Hz, 1H), 7.60 (d, J = 8.3 Hz, 1H), 6.54 (dt, J = 17.6, 4.7 Hz, 1H), 4.07 – 3.90 (m, 2H), 3.89 – 3.78 (m, 6H), 3.61 – 3.52 (m, 4H), 3.36 – 3.20 (m, 4H). 13C NMR (126 MHz, CDCl3) δC 165.6, 161.0, 157.6, 155.1, 136.8, 130.5, 129.01, 126.8, 110.6, 110.1, 61.5, 48.5, 46.4, 43.6, 41.8, 25.3. MS (ES) [M + H]+ = 436.1 m/z 100 % C19H22ClN5O3S = 435.93. Melting pt. (69-70) oC. 52 University of Ghana http://ugspace.ug.edu.gh 4.4.10 Preparation of 2-chloro-N-(2-methylbenzo[d]thiazol-6-yl)-5-(pyrrolidin-1-ylsulfonyl) benzamide This compound was synthesized according to the general procedure for carboxamide formation (mixed anhydride method) in 8% yield as a white foam. V max (neat) cm 1: 3287.62, 2964.30, 1661.13, 1517.10, 1339.55, 1155.11, 816.08. 1H NMR (500 MHz, CDCl3) δH 8.55 (s, 1H), 8.13 (d, J = 36.6 Hz, 2H), 7.90 (d, J = 34.1 Hz, 2H), 7.66 (s, 1H), 7.43 (s, 1H), 3.30 (s, 4H), 2.86 (s, 3H), 1.84 (s, 4H). 13C NMR (126 MHz, CDCl3) δC 167.2, 163.2, 150.9, 136.7, 136.2, 135.5, 134.0, 131.2, 130.1, 128.8, 122.5, 118.83, 112.9, 48.0, 25.2, 20.1. MS (ES) [M + H]+ = 436.1 m/z 100 % C H ClN O S = 435.95. Melting pt. (244) o19 20 3 3 2 C. 4.4.11 Preparation of 2-chloro-N-(6-methylpyridin-2-yl)-5-(pyrrolidin-1-ylsulfonyl) benzamide This compound was synthesized according to the general procedure for carboxamide formation (mixed anhydride method) in 6 % yield as a white foam. V max (neat) cm 1: 3298.64, 1700.53, 1451.11, 1150.15, 793.69, 581.07. 1H NMR (500 MHz, CDCl3) δH 8.66 (s, 1H), 8.16 (d, J = 8.2 Hz, 1H), 8.13 (d, J = 2.0 Hz, 1H), 7.87 (dd, J = 8.4, 2.1 Hz, 1H), 7.70 (t, J = 7.9 Hz, 1H), 7.63 (d, J = 8.4 Hz, 1H), 6.99 (d, J = 7.5 Hz, 1H), 3.28 (t, J = 6.7 Hz, 4H), 2.48 (s, 3H), 1.88 – 1.75 (m, 4H). 13C NMR (126 MHz, CDCl3) δC 163.2, 150.0, 139.3, 136.9, 135.9, 135.7, 131.3, 130.2, 128.4, 53 University of Ghana http://ugspace.ug.edu.gh 120.3, 111.2, 65.8, 48.2, 25.2, 15.3. MS (ES) [M + H]+ = 380.1 m/z 100 % C17H18ClN3O3S =379.86. Melting pt. (200) oC. 4.4.12 Preparation of 2-chloro-N-(6-methylpyridin-2-yl)-5-((4-phenylpiperidin-1-yl) sulfonyl) benzamide This compound was synthesized according to the general procedure for carboxamide formation (mixed anhydride method) in 30 % yield as a white amorphous solid. . V max (neat) cm 1: 3301.76, 2923.08, 1693.37, 1452.56, 1155.91, 700.82, 607.93. 1H NMR (500 MHz, Chloroform-d) δH 8.73 (s, 1H), 8.20 (d, J = 8.3 Hz, 1H), 8.10 (d, J = 2.2 Hz, 1H), 7.83 (dd, J = 8.4, 2.2 Hz, 1H), 7.73 (t, J = 8.0 Hz, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.30 (t, J = 7.5 Hz, 2H), 7.27 – 7.19 (m, 2H), 7.19 – 7.13 (m, 2H), 7.02 (d, J = 7.5 Hz, 1H), 3.96 (d, J = 11.6 Hz, 1H), 2.51 (s, 3H), 2.45 (dd, J = 12.3, 9.4 Hz, 3H), 2.17 (s, 1H), 1.93 (d, J = 13.5 Hz, 2H), 1.88 (dd, J = 12.0, 4.0 Hz, 1H), 1.86 – 1.80 (m, 1H), 1.28 (s, 1H), 1.34 – 1.23 (m, 2H), 0.91 – 0.81 (m, 2H). 13C NMR (126 MHz, Chloroform-d) δC 157.32, 149.93, 144.60, 138.90, 136.16, 135.89, 135.76, 131.33, 130.45, 128.83, 128.66, 126.71, 126.69, 120.25, 111.16, 46.90, 41.72, 32.51, 24.01. Melting pt. (219-220) oC 54 University of Ghana http://ugspace.ug.edu.gh 4.4.13 Preparation of 2-chloro-5-((4-phenylpiperidin-1-yl) sulfonyl)-N-(pyridin-2-yl) benzamide. This compound was synthesized according to the general procedure for carboxamide formation (mixed anhydride method) in 21 % yield as a white amorphous solid. . V max (neat) cm 1: 3305.56, 2933.88, 1699.42, 1434.53, 1147.84, 937.69, 696.93. 1H NMR (500 MHz, Chloroform-d) δH 9.05 (s, 1H), 8.37 (d, J = 8.4 Hz, 1H), 8.24 (d, J = 4.9 Hz, 2H), 8.12 (s, 1H), 7.82 (m, 1H), 7.74 (d, J = 8.7 Hz, 1H), 7.67 (m, 1H), 7.30 (t, J = 7.3 Hz, 2H), 7.22 (t, J = 7.3 Hz, 2H), 7.14 (dd, J = 17.8, 6.8 Hz, 1H), 3.96 (t, J = 11.8 Hz, 4H), 3.14 (q, J = 8.8, 6.2 Hz, 1H), 1.95 – 1.88 (m, 4H). 13C NMR (126 MHz, Chloroform-d) δC 147.9, 144.6, 138.8, 136, 131.4, 130.5, 130, 128.7, 126.7, 126.7, 120.7, 114.6, 46.9, 41.7, 32.5. Melting pt. (177-180) oC 4.4.14 Preparation of 2-chloro-N-(2-methylbenzo[d]thiazol-5-yl)-5-(piperidin-1-ylsulfonyl) benzamide. This compound was synthesized according to the general procedure for carboxamide formation (mixed anhydride method) in 35 % yield as a white amorphous solid. V max (neat) cm 1: 2921, 1642, 1588, 1453.82, 1164, 1152.33, 714. 1H NMR (500 MHz, Chloroform-d) δH 8.53 (d, J = 2.1 Hz, 1H), 8.21 (s, 1H), 8.05 (d, J = 2.2 Hz, 1H), 7.91 (d, J = 8.7 Hz, 1H), 7.75 (dd, J = 8.4, 2.3 Hz, 1H), 55 University of Ghana http://ugspace.ug.edu.gh 7.61 (d, J = 8.4 Hz, 1H), 7.41 (dd, J = 8.7, 2.2 Hz, 1H), 3.03 (t, J = 5.5 Hz, 4H), 2.84 (s, 3H), 1.65 (t, J = 5.7 Hz, 4H), 1.49 – 1.41 (m, 2H). 13C NMR (126 MHz, Chloroform-d) δC 167.20, 163.18, 150.72, 136.72, 136.20, 136.05, 135.61, 134.15, 131.23, 130.27, 128.78, 122.54, 118.93, 112.96, 46.96, 25.11, 23.36, 20.13. Melting pt. (202) oC. 4.4.15 Preparation of 2-chloro-N-(6-methylpyridin-2-yl)-5-(piperidin-1-ylsulfonyl) benzamide. This compound was synthesized according to the general procedure for carboxamide formation (mixed anhydride method) in 30 % yield as a white amorphous solid. . V (neat) cm1max : 3317.84, 2926.69, 1698.68, 1455.87, 1152.49, 722.74, 580.43. 1H NMR (500 MHz, Chloroform-d) δH 8.56 (s, 1H), 8.13 (d, J = 8.3 Hz, 1H), 8.04 (d, J = 2.3 Hz, 1H), 7.78 (dd, J = 8.4, 2.2 Hz, 1H), 7.67 (t, J = 7.9 Hz, 1H), 7.62 (d, J = 8.4 Hz, 1H), 6.97 (d, J = 7.5 Hz, 1H), 3.02 (t, J = 5.5 Hz, 4H), 2.45 (s, 3H), 1.68 – 1.62 (m, 4H), 1.49 – 1.40 (m, 2H). 13C NMR (126 MHz, Chloroform-d) δC 163.2, 157.3, 150, 138.9, 136.1, 135.6, 131.2, 130.4, 128.7, 120.2 111.2, 47.0, 25.1, 24.0, 23.4. Melting pt. (206-208) oC 4.4.16 Preparation of (2-chloro-5-(piperidin-1-ylsulfonyl) phenyl) (4-(trifluoromethyl) piperidin-1-yl) methanone. 56 University of Ghana http://ugspace.ug.edu.gh This compound was synthesized according to the general procedure for carboxamide formation (mixed anhydride method) in 29 % yield as a creamy white powder. V max (neat) cm 1: 2925.85, 1680.95, 1337.60, 1168.64, 1045.36, 749.61, 567.21. 1H NMR (500 MHz, Chloroform-d) δ 8.41 (t, J = 1.7 Hz, 1H), 7.90 (dt, J = 8.4, 1.8 Hz, 1H), 7.65 (dd, J = 8.4, 1.3 Hz, 1H), 3.27 (tt, J = 4.2, 2.1 Hz, 4H), 2.98 (m, 1H) 2.18 (t, J = 1.3 Hz, 4H), 1.82 (tt, J = 4.1, 2.1 Hz, 4H), 1.32 – 1.24 (m, 6H). 13C NMR (126 MHz, Chloroform-d) δC 167.29, 139.02, 135.66, 135.1, 132.18, 131.61, 131.14, 129.97, 46.94, 29.68, 25.12, 23.41, 21.20. Melting pt. (215-217) oC 4.4.17 Preparation of (2-chloro-5-(piperidin-1-ylsulfonyl) phenyl) (4-(pyrimidin-2-yl) piperazin-1-yl) methanone This compound was synthesized according to the general procedure for carboxamide formation (acid chloride method) in 30% yield as a yellow oil. . V max (neat) cm 1: 2942.92, 1641.22, 1583.63, 1340, 1159.48, 795.49, 606.43. 1H NMR (500 MHz, Chloroform-d) δH 8.35 (dd, J = 4.8, 3.1 Hz, 2H), 7.79 – 7.70 (m, 2H), 7.62 (dd, J = 8.4, 3.2 Hz, 1H), 7.29 (s, 0H), 6.58 (td, J = 4.8, 3.1 Hz, 1H), 4.08 – 3.91 (m, 2H), 3.87 (s, 1H), 3.88 – 3.80 (m, 2H), 3.39 – 3.24 (m, 2H), 3.10 – 2.97 (m, 5H), 1.72 – 1.60 (m, 10H), 1.47 (ddt, J = 8.1, 4.9, 2.7 Hz, 2H). 13C NMR (126 MHz, Chloroform- d) δC 165.46, 161.45, 157.84, 136.67, 136.15, 135.14, 130.55, 129.22, 127.01, 110.75, 46.92, 46.68, 43.90, 43.41, 41.80, 25.12, 23.34. Melting pt: (170-171) oC 4.5 PHYSICOCHEMICAL PROPERTIES. Lipophilicity (Log P), Polar Surface Area (PSA) and Molar Refractivity (MR) were calculated using CHEMAXON. 57 University of Ghana http://ugspace.ug.edu.gh CHAPTER FIVE CONCLUSIONS AND RECOMMENDATIONS 5.1 CONCLUSIONS A series of benzene sulfonamides have been successfully synthesized in three chemical steps, first by refluxing 2-chlorobenzoic acid with chlorosulfonic acid to form sulfonyl chloride followed by amine coupling to form the sulfonamide intermediates. The sulfonamides then condensed with various amines to form the titled benzamides. Characterization of pure compounds after column chromatography were done by spectroscopic methods such as IR, NMR and LC-MS. Some Physicochemical properties have also been calculated by CHEMAXON software. Calculated LogP of the synthesized compounds range from 1.2-4.5 suggesting the compounds were not too lipophilic. Calculated PSA in the range of 68.31-125.22 were obtained suggesting the compound will permeate membranes well. A few of the compounds had shown good antimalarial activity toward 3D7 strain of the malaria parasite in micro molar range with Compound FEP040 returning an activity of 0.268 nM superior to the lead compound MMV019721 (0.552 nM). Regression analysis shows that lipophilicity has the most significant effect on the biological activity. 5.2 RECOMMENDATION. • Due to the wide medicinal benefits of both pharmacophoric moieties, biological screening should not be limited to only antimalarial activities. • Additional analogues should be synthesized to ensure a comprehensive Structure-Activity Relationship (SAR) study. • Analogues should be tested against other strains of the malaria parasite. • Further screening of the compounds should be undertaken to establish the molecular target of this class of compounds. 58 University of Ghana http://ugspace.ug.edu.gh REFERENCES. Adkins, J. C., & Faulds, D. (1998). Amprenavir. Drugs, 55(6), 837–842; discussion 843-4. 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Journal of Enzyme Inhibition and Medicinal Chemistry, 22(3), 301–308. https://doi.org/10.1080/14756360601114569 69 University of Ghana http://ugspace.ug.edu.gh APPENDICIES APPENDIX 1: LC-MS. FEP 003 DAD1 A, Sig=280,4 Ref=550,10 (D:\DATA\2018-05-23\2018-05-21 2018-05-23 19-33-22\1FA-0301.D) mAU 2500 2000 1500 1000 500 0 0.5 1 1.5 2 2.5 3 3.5 4 min MSD1 TIC, MS File (D:\DATA\2018-05-23\2018-05-21 2018-05-23 19-33-22\1FA-0301.D) MM-APCI, Pos, Scan, Frag: 200, "sc 5000000 7 00 e+ 4000000 52 5 2.7: ea A r 3000000 2000000 1000000 0 0.5 1 1.5 2 2.5 3 3.5 4 min *MSD1 SPC, time=2.611:2.743 of D:\DATA\2018-05-23\2018-05-21 2018-05-23 19-33-22\1FA-0301.D MM-APCI, Pos, Scan, Frag 100 Max: 269956 80 60 40 20 0 420 430 440 450 460 470 480 m/z 70 452.0 453.0 454.0 455.0 2.641 University of Ghana http://ugspace.ug.edu.gh FEP 005 DAD1 A, Sig=280,4 Ref=550,10 (D:\DATA\2018-05-13\2018-05-11 2018-05-13 17-12-50\1AA-0101.D) mAU 2500 2000 1500 1000 500 0 0.5 1 1.5 2 2.5 3 3.5 4 min MSD1 TIC, MS File (D:\DATA\2018-05-13\2018-05-11 2018-05-13 17-12-50\1AA-0101.D) MM-APCI, Pos, Scan, Frag: 200, "sc 8000000 6000000 4000000 2000000 0 0.5 1 1.5 2 2.5 3 3.5 4 min *MSD1 SPC, time=2.620:2.717 of D:\DATA\2018-05-13\2018-05-11 2018-05-13 17-12-50\1AA-0101.D MM-APCI, Pos, Scan, Frag 100 Max: 564821 80 60 40 20 0 300 325 350 375 400 425 450 475 500 m/z 71 398.0 396.0 399.1 2.418 2.658 University of Ghana http://ugspace.ug.edu.gh FEP006 DAD1 A, Sig=280,4 Ref=550,10 (D:\DATA\2018-05-13\2018-05-11 2018-05-13 17-12-50\1AB-0201.D) mAU 2500 2000 1500 1000 500 0 0.5 1 1.5 2 2.5 3 3.5 4 min MSD1 TIC, MS File (D:\DATA\2018-05-13\2018-05-11 2018-05-13 17-12-50\1AB-0201.D) MM-APCI, Pos, Scan, Frag: 200, "sc 7000000 6000000 5000000 4000000 3000000 2000000 1000000 0 0.5 1 1.5 2 2.5 3 3.5 4 min *MSD1 SPC, time=2.611:2.892 of D:\DATA\2018-05-13\2018-05-11 2018-05-13 17-12-50\1AB-0201.D MM-APCI, Pos, Scan, Frag 100 Max: 278036 80 60 40 20 0 360 380 400 420 440 460 480 500 m/z 72 2.419 2.657 453.1 452.1 455.1 454.1 3.097 3.169 University of Ghana http://ugspace.ug.edu.gh FEP 008 DAD1 A, Sig=280,4 Ref=550,10 (D:\DATA\2018-05-13\2018-05-11 2018-05-13 17-12-50\1AC-0301.D) mAU 2500 2000 1500 1000 500 0 0.5 1 1.5 2 2.5 3 3.5 4 min MSD1 TIC, MS File (D:\DATA\2018-05-13\2018-05-11 2018-05-13 17-12-50\1AC-0301.D) MM-APCI, Pos, Scan, Frag: 200, "sc 7000000 6000000 008 e+ 5000000 3 .09 16 a: 1 4000000 A re 3000000 2000000 1000000 0 0.5 1 1.5 2 2.5 3 3.5 4 min *MSD1 SPC, time=2.691:3.094 of D:\DATA\2018-05-13\2018-05-11 2018-05-13 17-12-50\1AC-0301.D MM-APCI, Pos, Scan, Frag 100 Max: 414263 80 60 40 20 0 360 380 400 420 440 460 480 500 m/z 73 439.1 438.1 441.1 440.1 2.790 University of Ghana http://ugspace.ug.edu.gh FEP 011 DAD1 A, Sig=280,4 Ref=550,10 (D:\DATA\2018-05-13\2018-05-11 2018-05-13 17-12-50\1AD-0401.D) mAU 2500 2000 1500 1000 500 0 0.5 1 1.5 2 2.5 3 3.5 4 min MSD1 TIC, MS File (D:\DATA\2018-05-13\2018-05-11 2018-05-13 17-12-50\1AD-0401.D) MM-APCI, Pos, Scan, Frag: 200, "sc 8000000 7000000 6000000 5000000 4000000 3000000 2000000 1000000 0 0.5 1 1.5 2 2.5 3 3.5 4 min *MSD1 SPC, time=2.620:2.901 of D:\DATA\2018-05-13\2018-05-11 2018-05-13 17-12-50\1AD-0401.D MM-APCI, Pos, Scan, Frag 100 Max: 352890 80 60 40 20 0 250 275 300 325 350 375 400 425 m/z 74 2.362 354.0 357.0 356.0 2.657 3.275 University of Ghana http://ugspace.ug.edu.gh FEP 012 DAD1 A, Sig=280,4 Ref=550,10 (D:\DATA\2018-05-13\2018-05-11 2018-05-13 17-12-50\1AE-0501.D) mAU 2500 2000 1500 1000 500 0 0.5 1 1.5 2 2.5 3 3.5 4 min MSD1 TIC, MS File (D:\DATA\2018-05-13\2018-05-11 2018-05-13 17-12-50\1AE-0501.D) MM-APCI, Pos, Scan, Frag: 200, "sc 8000000 7000000 6000000 5000000 4000000 3000000 2000000 1000000 0 0.5 1 1.5 2 2.5 3 3.5 4 min *MSD1 SPC, time=2.524:2.620 of D:\DATA\2018-05-13\2018-05-11 2018-05-13 17-12-50\1AE-0501.D MM-APCI, Pos, Scan, Frag 100 Max: 366954 80 60 40 20 0 280 300 320 340 360 380 400 m/z 75 0.083 304.1 322.0 340.0 343.0 342.0 2.565 2.689 University of Ghana http://ugspace.ug.edu.gh FEP 018 DAD1 A, Sig=280,4 Ref=550,10 (D:\DATA\2018-05-13\2018-05-11 2018-05-13 17-12-50\1AF-0601.D) mAU 2500 2000 1500 1000 500 0 0.5 1 1.5 2 2.5 3 3.5 4 min MSD1 TIC, MS File (D:\DATA\2018-05-13\2018-05-11 2018-05-13 17-12-50\1AF-0601.D) MM-APCI, Pos, Scan, Frag: 200, "sc 5000000 4000000 3000000 2000000 1000000 0 0.5 1 1.5 2 2.5 3 3.5 4 min *MSD1 SPC, time=2.726:2.805 of D:\DATA\2018-05-13\2018-05-11 2018-05-13 17-12-50\1AF-0601.D MM-APCI, Pos, Scan, Frag 100 Max: 261062 80 60 40 20 0 420 430 440 450 460 m/z 76 436.1 437.0 438.0 439.0 2.765 University of Ghana http://ugspace.ug.edu.gh FEP 019 DAD1 A, Sig=280,4 Ref=550,10 (D:\DATA\2018-05-13\2018-05-11 2018-05-13 17-12-50\1AG-0701.D) mAU 1400 1200 1000 800 600 400 200 0 0.5 1 1.5 2 2.5 3 3.5 4 min MSD1 TIC, MS File (D:\DATA\2018-05-13\2018-05-11 2018-05-13 17-12-50\1AG-0701.D) MM-APCI, Pos, Scan, Frag: 200, "sc 7000000 6000000 5000000 4000000 3000000 2000000 1000000 0 0.5 1 1.5 2 2.5 3 3.5 4 min *MSD1 SPC, time=2.647:2.761 of D:\DATA\2018-05-13\2018-05-11 2018-05-13 17-12-50\1AG-0701.D MM-APCI, Pos, Scan, Frag 100 Max: 316646 80 60 40 20 0 380 400 420 440 460 480m/z 77 436.1 438.1 2.588 439.1 2.678 University of Ghana http://ugspace.ug.edu.gh FEP 020 DAD1 A, Sig=280,4 Ref=550,10 (D:\DATA\2018-05-23\2018-05-21 2018-05-23 19-33-22\1FB-0401.D) mAU 2000 1500 1000 500 0 0.5 1 1.5 2 2.5 3 3.5 4 min MSD1 TIC, MS File (D:\DATA\2018-05-23\2018-05-21 2018-05-23 19-33-22\1FB-0401.D) MM-APCI, Pos, Scan, Frag: 200, "sc 6000000 7 5000000 00e+ 84 2.3 6 : 4000000 rea A 3000000 060 e+ 14 1 97 2000000 a: 1. A re 1000000 0 0.5 1 1.5 2 2.5 3 3.5 4 min *MSD1 SPC, time=2.576:2.761 of D:\DATA\2018-05-23\2018-05-21 2018-05-23 19-33-22\1FB-0401.D MM-APCI, Pos, Scan, Frag 100 Max: 234123 80 60 40 20 0 410 420 430 440 450 460 470 m/z 78 435.1 436.1 437.1 438.1 2.534 2.614 University of Ghana http://ugspace.ug.edu.gh FEP 021 DAD1 A, Sig=280,4 Ref=550,10 (D:\DATA\2018-05-13\2018-05-11 2018-05-13 17-12-50\1AH-0801.D) mAU 2500 2000 1500 1000 500 0 0.5 1 1.5 2 2.5 3 3.5 4 min MSD1 TIC, MS File (D:\DATA\2018-05-13\2018-05-11 2018-05-13 17-12-50\1AH-0801.D) MM-APCI, Pos, Scan, Frag: 200, "sc 8000000 7000000 6000000 5000000 4000000 3000000 2000000 1000000 0 0.5 1 1.5 2 2.5 3 3.5 4 min *MSD1 SPC, time=2.682:2.760 of D:\DATA\2018-05-13\2018-05-11 2018-05-13 17-12-50\1AH-0801.D MM-APCI, Pos, Scan, Frag 100 Max: 403026 80 60 40 20 0 340 360 380 400 420 m/z 79 380.1 383.0 382.1 2.708 3.167 University of Ghana http://ugspace.ug.edu.gh FEP 022 DAD1 A, Sig=280,4 Ref=550,10 (D:\DATA\2018-05-23\2018-05-21 2018-05-23 19-33-22\1FC-0501.D) mAU 2500 2000 1500 1000 500 0 0.5 1 1.5 2 2.5 3 3.5 4 min MSD1 TIC, MS File (D:\DATA\2018-05-23\2018-05-21 2018-05-23 19-33-22\1FC-0501.D) MM-APCI, Pos, Scan, Frag: 200, "sc 7000000 6000000 5000000 4000000 3000000 2000000 1000000 0 0.5 1 1.5 2 2.5 3 3.5 4 min *MSD1 SPC, time=2.506:2.997 of D:\DATA\2018-05-23\2018-05-21 2018-05-23 19-33-22\1FC-0501.D MM-APCI, Pos, Scan, Frag 100 Max: 214409 80 60 40 20 0 360 380 400 420 440 460 m/z 80 410.1 413.1 412.1 2.581 3.024 3.117 University of Ghana http://ugspace.ug.edu.gh APPENDIX 2: IR SPECTRA 81 University of Ghana http://ugspace.ug.edu.gh 82 University of Ghana http://ugspace.ug.edu.gh 83 University of Ghana http://ugspace.ug.edu.gh 84 University of Ghana http://ugspace.ug.edu.gh 85 University of Ghana http://ugspace.ug.edu.gh 86 University of Ghana http://ugspace.ug.edu.gh 87 University of Ghana http://ugspace.ug.edu.gh 88 University of Ghana http://ugspace.ug.edu.gh 89 University of Ghana http://ugspace.ug.edu.gh 90 University of Ghana http://ugspace.ug.edu.gh APPENDIX 3: 1H NMR SPECTRA. 91 University of Ghana http://ugspace.ug.edu.gh 92 University of Ghana http://ugspace.ug.edu.gh 93 University of Ghana http://ugspace.ug.edu.gh 94 University of Ghana http://ugspace.ug.edu.gh 95 University of Ghana http://ugspace.ug.edu.gh 96 University of Ghana http://ugspace.ug.edu.gh 97 University of Ghana http://ugspace.ug.edu.gh 98 University of Ghana http://ugspace.ug.edu.gh 99 University of Ghana http://ugspace.ug.edu.gh 100 University of Ghana http://ugspace.ug.edu.gh 101 University of Ghana http://ugspace.ug.edu.gh 102 University of Ghana http://ugspace.ug.edu.gh APPENDIX 4: 13C NMR SPECTRA. 103 University of Ghana http://ugspace.ug.edu.gh 104 University of Ghana http://ugspace.ug.edu.gh 105 University of Ghana http://ugspace.ug.edu.gh 106 University of Ghana http://ugspace.ug.edu.gh 107 University of Ghana http://ugspace.ug.edu.gh 108 University of Ghana http://ugspace.ug.edu.gh 109 University of Ghana http://ugspace.ug.edu.gh 110 University of Ghana http://ugspace.ug.edu.gh 111 University of Ghana http://ugspace.ug.edu.gh 112 University of Ghana http://ugspace.ug.edu.gh 113 University of Ghana http://ugspace.ug.edu.gh 114