Synthesis and evaluation for biological activities of npyridin- 3-yl substituted [phenylsulphonamido] acetamide

Table Of Contents

Title page – – – – – – – – – – -i Approval page – – – – – – – – – -ii Dedication – – – – – – – – – – -iii Acknowledgement – – – – – – – – – -iv Abstract – – – – – – – – – – -v Table of contents – – – – – – – – – -vi

Chapter ONE

1.0. Introduction – – – – – – – – -1 1.1. Background of the study – – – – – – -1 1.1.1. Chemistry and nomenclature of sulphonamides – – – -11 1.1.2. Medicinal important or sulfonamides – – – – -12 1.2. Statement of the problem or research question – – – -16 1.3. Objective of the research – – – – – – -17 1.4. Justification of the study – – – – – – -18

Chapter TWO

Literature review 2.1. History of sulphonamide drug discovery – – – – -19 2.2. Synthesis of sulphonamides – – – – – – -21 2.2.1. Synthesis from amination of chalcones – – – – -26 2.2.2. Chloromethylsulphonylation of benzylisothiourea – – -26 2.2.3. Copper ii oxide catalytic sulphonylation method – – – -27 2.2.4. Synthesis from ionic liquid mediated approach – – – -28 2.2.5. Synthesis from heteroaryl thiols – – – – – -29 2.3. Sulphonamide as antimicrobial agents – – – – -29 vii

Chapter THREE

3.0. Experimental section – – – – – – – -31 3.1. Materials and method – – – – – – – -31 3.2 Synthesis of benzensulphonamide – – – – – -31 3.2.1. Synthesis [(phenylsulfonyl)amido]acetic acid – – – -32 3.2 .2 Synthesis of 4-methyl-2-[(phenylsulfonyl)amido]pentanoic acid -32 3.2.3 Synthesis of 2-[(phenylsulfonyl)amido]propanoic acid – – -33 3.2.4 Sythesis of 3-phenyl-2-[(phenylsulfonyl)amido]propanoic acid – -34 3.2.5 Synthesis of 4-(methylsulfanyl)-2-[(phenylsulfonyl)amido] butanoic acid – – – – – – – – -34 3.3. General method of synthesis of N-heteroaryl substituted Benzensulphonamide – – – – – – – -35 3.3.1 2-[(phenylsulfonyl)amido]-N-(pyridin-3-yl)acetamide – – -36 3.3.2. 4-methyl-2-[(phenylsulfonyl)amido]-N-(pyridin-3-yl)pentanamide. -36 3.3.3. 2-[(phenylsulfonyl)amido]-N-(pyridin-3-yl)propanamide – -37 3.3.4. 3-phenyl-2-[(phenylsulfonyl)amido]-N-(pyridin-3-yl)propanamide -38 3.3.5. 5-(methylsulfanyl)-3-[(phenylsulfonyl)amido]-N-(pyridin-3- yl)pentanamide. – – – – – – – – -39 3.4 Biological activities – – – – – – – -40 3.4.1 Antimicrobial activity – – – – – – – -40 3.5. Evaluation of antioxidant activity – – – – – -40 3.5.1. Scavenging of dpph radical – – – – – – -41 3.5.2 Reducing power – – – – – – – – -41 3.5.3. Ferrous sulphate induced lipid peroxidation scavenging – – -42 viii

Chapter FOUR

4.0 Results and discussion – – – – – – – -44 4.1 Benzene sulphonamides – – – – – – – -44 4.1.1 [(phenylsulphonyl)amido]acetic acid – – – – – -44 4.1.2. 4-methyl-2-[(phenylsulfonyl)amido]pentanoic acid – – -45 4.1.3 2-[(phenylsulfonyl)amido]propanoic acid, – – – – -46 4.1.4. 3-phenyl-2-[(phenylsulfonyl)amido]propanoic acid – – -47 4.1.5. 4-(methylsulfanyl)-2-[(phenylsulfonyl)amido]butanoic acid – -49 4.2. Synthesis of N-pyridine-3-yl substituted benzensulphonamide- -50 4.2.1. 2-[(phenylsulfonyl)amido]-N-(pyridin-3-yl)acetamide – – -51 4.2.2. 4-methyl-2-[(phenylsulfonyl)amido]-N-(pyridin-3-yl)pentanamide -51 4.2.3. 2-[(phenylsulfonyl)amido]-N-(pyridin-3-yl)propanamide – -53 4.2.4. 3-phenyl-2-[(phenylsulfonyl)amido]-N-(pyridin-3-yl)propanamide -44 4.2.5 5-(methylsulfanyl)-3-[(phenylsulfonyl)amido] -N-(pyridin-3-yl)pentanamide – – – – – – -56 4.3 Biological activities. – – – – – – – -57 4.3.1 Minimum Inhibitory Concentration(MIC) mg/ml – – – -57 4.3.2 Results of sensitivity test – – – – – – -59 4.4 Anti oxidant evaluation – – – – – – – -60 4.4.1 Invitro free radical scavenging effect of samples by dpph method -60 4.4.2. Ferrous sulphate induced lipid peroxidation – – – – -62 4.4.3 FRAP – – – – – – – – – -63

Chapter FIVE

5.1. Conclusion – – – – – – – – – -64 5.2 References – – – – – – – – – -65 ix

Thesis Abstract

In this study, a series of N-pyridin-3-yl substituted [phenylsulphonamido] acetamide has been synthesized. The reaction of phenylsulphonyl chloride with various amino acids in basic medium yielded phenylsulphonamido alkanoic acid which, on chlorination with thionyl chloride, gave acid chloride derivatives of phenylsulphonamido alkanoic acid in situ. The acid chloride derivatives on condensation with 3-aminopyridine gave corresponding acetamide in good to excellent yield. The compounds were characterized by FTIR, 1H-NMR and 13CNMR and screened for antibacterial, antifugal and antioxidant activities. The result revealed that the compounds possess antibacterial activities.One the compounds, 2- [(phenylsulfonyl)amido]propanoic acid had better antibacterial activities than ciprofloxacin the reference drug while others are less active. All the compounds has less antifungal activities than ketokonazole the reference drug. 2- [(phenylsulfonyl)amido]propanoic acid had the best antioxidant properties of all the compounds.

Thesis Overview

INTRODUCTION 1.1 Background of the study The growing incidence of microbial resistance to currently used antibiotics represents a serious medical problem. Therefore, there is an urgent need to develop new classes of therapeutic agents to treat microbial infectious. Such new therapeutic agent has to exhibit a wide spectrum of biological activities. Sulphonamides, an important class of pharmaceutical compounds, exhibit a wide spectrum of biological activities1. The basic sulphonamide group [SO2, NHR] occurs in various biologically active compounds including antimicrobial drugs, antithyroid agents, antitumor, antibiotics and inhibitors of carbonic anhydrase2 . Sulphonamides are widely used to treat microbial infections by inhibiting the growth of Gram negative and Gram positive bacteria, some protozoa and fungi3 . Clinically, sulphonamides are used to treat several urinary tract infections and gastrointestinal infections4. Sulphonamides5 that are aromatic or heteroaromatic are responsible for the inhibition of the growth of tumor cells. They act as antitumor agents by inhibiting carbonic anhydrase activity. They are structurally similar to paminobenzoic acid (PABA) which is a cofactor that is needed by the bacteria for the synthesis of folic acid. Sulphonamide antibiotics inhibit the conversion of x PABA into folic acid and thus ultimately inhibit the synthesis of DNA. They are also used in veterinary medicine to treat infections in livestock6. In primary care medicine, sulphonamides are widely used in various conditions including gastrointestinal7 and urinary tract infections8. Sulphonamide is the organic framework of main focus in this research and it belongs to the family of suphurcontaining compounds9, which are earlier referred to as sulpha drugs . Some of these sulpha drugs that have performed “healing magic” in the world of chemotherapy include; sulphanilamide(1), sulphadiazine(2), sulphacetamide(3), sulphamonomethoxine(4), sulphasalazine(5), sulphadoxine(6), among others. Sulphonamides have long been the subject of pharmaceutical interest as a result of their potent biological activities10 H2N S O O NH2 H2N S O O N+ N N 1 2 H2N S O O NH O CH3 3 xi S O O NH N N O CH3 H2N 4 S O O NH N N N N O HO OH O CH3 5 S O O NH H2N N N O CH3 O CH3 6 Furthermore, the sulphonamide group has been proven to have remarkable utility in medicinal chemistry and features in a number of clinically relevant small molecules11. For instance, some currently approved drugs with sulphonamide structural skeletons include the antihypertensive agent bosentan(7)12, the antiviral HIV protease inhibitor amprenavir(8)13,the phosphodiesterase-5 inhibitor sildenafil14, antidiabetic drug glibenclamide(9)15, antidiabetic nonantibiotic glimepiride and the diuretic drug torasemide16 xii S O O NH H C CH3 3 H3C N N O N N O OH O CH3 7 S O O N H2N CH3 H3C NH OH O O O O 8 O NH S O O NH O NH OMe Cl 9 In addition, suphonamides are also highly relevant both in the animal world and the plant life cycle. For example, the breakdown of cyclic guanosine monophosphate is retarded by sildenafil, a substituted guanine analog, which keeps cut flowers fresh for another week and also strengthens plant stems to stand straight even in the midst of storm and wind17. A preserving effect on fruits and vegetable was also found, making sildenafil , a promising agent18. Today, it is xiii marketed under the trade name of Viagra19, which is a potent drug used in erectile dysfunction in man20. It has been reported that more than 150 million men worldwide have this dysfunction21. In another discovery, sulphonamide has been documented as highly efficient candidate with high inhibitory activity22 Similarly, some sulphonamides have been established as potent drugs in the treatment of insomnia and other sleep challenges in man by antagonizing orexin neural activity. Activation of orexin neurons contribute to the promotion and maintenance of wakefulness23. Conversely, relative inactivity of orexin neurons allows the onset of sleep24. Consequently, blocking orexin signaling with receptor antagonists may provide a new mechanism for decreasing wakefulness. Thus , a novel therapeutic opportunity for the treatment of insomnia was reported using a dual receptor antagonists almorexant25. Also, a low molecular weight aryl containing N-glycine-suphonamide (2-[(4-tert-butyl-benzenesulphonyl)-p-tolyamino]- N,N-dimethyl-acetamide(10), was discovered as a potent and selective antagonist with very poor oral bioavailability (F=1%) in wister rats.26 S O O N N N CH3 O CH3 MeO Cl N CH3 H3C MeO 10 xiv The mode of action of sulphonamide is by mimicking p-aminobenzoic acid (PABA), which is needed in bacteria as a substrate of the enzyme dihydropteroate synthetase for the synthesis of tetrahydrofolic acid, a basic growth factor essential for the metabolic process of bacteria27. Sulphonamide are preferred due to the ease of administration28, wide spectrum of antimicrobial activity29, non-interference with host defence mechanism and relative freedom from problems of superinfection30- 31 Sulphonamides have been classified using various parameters: For instance, by using the antibiotic properties, it can be classified into antibiotic (antimicrobial) sulphonamides and non antibiotic (non antimicrobial) sulphonamides32. Although, almost all therapeutically useful sulphonamides are aromatic linked as in Ar- SO2NH2 or Ar-SO2NHR33, yet, there are important distinctions between sulphonylarylamines (antimicrobial/suphonamides), nonarylamine (non antimicrobial) sulphonamides, and sulphones, with regard to allergy and other drug reactions34. Sulphone , unlike sulphonamide, has its sulphonyl unit incorporated between two carbon systems which may be part of either aliphatic or aromatic moieties 35. Most reactions to sulphonylarylamines probably results from multifactorial immunologic and toxic metabolic mechanisms, whereas less is known about the precise mechanism of reaction of other sulphur-containing drugs. Some xv sulphonamides such as the anticonvulsante, sultiames are devoid of antibacterial activity. The sulphonylurea and thiazide diuretics are newer drug groups used on the antibacterial sulphonamides36. One of the sulphonamides of paramount importance is benzenesulphonamide derivatives. Benzenesulphonamide moiety is an integral part of many drugs and drug-like scaffolds37. Many derivatives of benzenesulphonamide have been explored as important starting materials and reactive intermediates in various organic synthesis. For example, 2- hydroxyalkylbenzene sulphonamides have been reported as the important starting materials 38 for the structure activity relationship (SAR) study during the search for cycloxygenase-2 (COX-2) inhibitors as analgesic39 and anti-inflammatory agents40. The chemistry of sulphonamides has recently shown them to be highly efficient synthons in the preparation of various valuable biologically active compounds41. In exploiting the chemistry of sulphonamides as ligands, various researchers have attempted and embarked upon designing and synthesizing various novel metal based sulphonamides42. The chiral ligands of sulphonamides designed as first , second and third generation ligands for catalyzing stereoselective synthesis of highly relevant asymmetric compounds43 has been years odyssey. In a similar manner, a new series of sulphonamides derived Schiff bases has been synthesized by a condensation reaction of various sulphonamides with aromatic aldehydes44. xvi The sulphonamides obtained were further investigated for their metal complexing potential in terms of chelation and biological properties45 . On the issue of side effect, it is important to make a distinction between sulpha drugs and other sulphur containing drugs and additives, such as sulphates and sulphites, which are chemically unrelated to the sulphonamide group, and do not cause the same hypersensitivity reaction seen in the sulphonamides. Although there are reported cases of allergy to sulphonamide drug but the term sulpha allergy is imprecise and misleading and therefore should be discouraged46. Sulpha allergies are commonly reported as side effect of sulphonamide drugs, hence medications containing sulphonamides are prescribed carefully. In fact, issues in understanding the clinical evidence and drug dynamics as they relate to individuals and populations have been explored47. Statistics have shown that approximately 3% of the general populations show sulpha drug allergies, when treated with sulphonamides and other similar antibiotics48. The potential utilization of sulpha drug compounds as corrosion inhibitors has also been established49. The inhibitory effect of four sulpha drug compounds namely, sulphamethazine , sulphaguanidine , sulphamethoxazole and sulphadiazine , on mild steel corrosion in 1.0 M HCl solution was evaluated using galvanostatic polarization weight loss technique50. All the sulpha drug compounds examined were reported to reduce the corrosion of mild steel indicating their high potency as xvii anti-corrosion agents. Among the compounds studied, sulphadiazine was shown to have the best inhibition efficiency51. Some quantum mechanical studies have successfully linked the inhibition efficiency with molecular properties for different kinds of organic compounds52. Sulphonmide derivatives of azo dyes achieve improved light stability, water solubility and fixation to fibre. The sulphonamide dyes, especially secondary sulphonamide dyes, exhibited superior dye exhaustion and colour fastness to washing, sublimation and rubbing on fine denier PP fabrics53. They have been used as protecting groups of hydroxyl or amino functionalities for easy removal under mild conditions54. Even though many synthetic methods have been reported for the preparation of sulphonamides55, the sulphonylation of ammonia56 or primary and secondary amines with sulphonyl chlorides in the presence of a base is still the method of choice because of high efficiency and simplicity of the reaction . Nevertheless, various acceptable techniques involve the need to reduce the amount of toxic waste and by-products arising from chemical processes required thereby increasing emphasis on the use of less toxic and environmentally compatible materials in the design of new synthetic methods57. One of the most promising approaches is using water as the reaction media58 while others include microwave irradiation technique, heterogeneous catalytic approach, solvent free media usage, nontoxic solid support resin59 etc. Sulphonamides an ionizable, polar antimicrobial xviii compounds, may reach the environment in substantial amount by the spreading of manure60 or other means. In a similar manner, amide formation is a fundamental reaction of great interest in organic chemistry61. The development of efficient methods for the synthesis of amides remains a great challenge because of their importance in chemistry and biology, with a wide range of industrial and pharmaceutical applications and as valuable intermediates in organic synthesis62. Hence, it is convincing to design the synthetic route in such a way to have a second amide functionality, being incorporated within the frame work of the synthesized sulphonamides, this will help in the comparative study of the antimicrobial activity of the ordinary sulphonamide with amide bearing sulphonamide derivatives. Despite the various hazardous effects posed by deadly microbes63, it is highly surprising to know that no new antimicrobial drug has been discovered in the last few years. Therefore, there is a continuous need for the design and synthetic formulation of new class of antimicrobial drugs in order to control rapid spread of harmful microbes. There has been a frequent failure in treatment of infections using antibiotics. Biofilm effect is the mechanism responsible for the frequent failure of antibiotic treatment to cure infections of medical devices and other prosthetic materials64. In xix the biofilm stage, a phenotypic change occur in which the bacteria require generally much higher concentrations of antibiotics to inhibit their growth. In fact, it has been recently discovered that the comparative study of the minimum inhibitory concentration (MIC) and minimum biofilm eliminating concentration (MBEC) is a potential factor determining the changes in the pattern of antibiotic sensitivity of gram negative bacilli from the planktonic to the biofilm stage of growth65. In addition, the rapid emergence of drug resistance has become the most urgent concern because it renders current treatments ineffective and therefore compels the scientific community to continue efforts in the design of inhibitory agents that can efficiently combat drug resistance 1.1.1 Chemical classification and Nomenclature of sulphonamides Sulphonamides are derivatives of sulfuric acids. Sulphonamides are chemically quite stable, these are weak acids compared to carboxylic acid amides. The sulphonamide functional group is – (SO2-NH2, a sulfonyl group connected to an amine group). The general formula is RSO2NH2. where R is some organic group such as alkyl, benzyl etc.. Any sulfonamide can be considered as derived from a sulfonic acid by replacing a hydroxyl group with an amine group. In medicals, the term xx “sulfonamide” is sometimes used as a synonym for sulfa drug, a derivative or sulfamilamide. 1.1.2 Medicinal properties of sulfonamide The sulphonamide scaffold is well known for the design of many sulphonamide compounds with diverse medicinal properties. The presence of benzene ring allows sulfonamides to partition through bacterial cell walls. Once inside of the bacteria cells, sulfonamides must be ionizable and contain both a positive and negative charge on opposite sides of its structure, which further resembles the structure of PABA and result in binding of sulfonamide. Antibacterial activity Kumar and coworkers66 have reported the synthesis of some Schiff bases or sulfonamides by condensing 4–aminobenzene sulphonamide with different aromatic aldehydes in the presence or glacial acitic acid and ethanol at 50-600C. The antimicrobial activity of these compounds was evaluated by agar diffusion xxi method. Several Schiff bases derived from sulphonamide have shown good antibacterial activity. Anti-inflammatory Activity Benzene sulfonamide derivatives carrying a pyrozole moity were prepared by Abddaal et al67 which where structurally related COX-2 inhibitor celecoxib (celebrex®) and were found to be potent anti-inflammatory agents with little or no tendency to evoke gastric ullercities. In these derivatives, N-Ethyl -2- (3-merthyl – 5- oxo-4, 5- dihydro-pyazile -1- carboxyl)-benzensulfonamide (11) was found to be more active than the celebrex. S O O NHC2H5 O N N CH3 O 11 Growth Hormone scretagogue Receptor High specific activity sulfur -35- labeled sulphonamide radioligand has been developed by Dean and co-workers68 for the identification of a GH secretagogue receptor [355] – MK – 0677 was found to posses the necessary concentration of high selectivity, affinity and specific activity required for utilization as a radioligand in the study of this nearly discovered receptor. xxii Cancer-Associated carbone anhytrases (CAs) Neogluco conjugate a new class of sulfonamide link was designed by Moore and co-workers69 to selectively target and inhibit the extracellular domains of the cancer-relevant CA isozymes. The carbohydrate fragment in these compounds is linked to the classical aromatic sulfonamide CA pharmacore to target inhibition of cancer associated CAs. The CA inhibitors design were very good CA IX inhibitors and potent CA XII inhibitors. Antimicrobial activity Iqbal and coworkers70 synthesized benzene sulphonamides bearing 2,5 disubstituted 1,3, 4- oxadiazole moiety and tested these compounds for antimicrobial and antifugal activity. Compound (12) exhibited significant antibacterial and significant antifugal activities due to the presence of a chloro group on position 4 of the phenyl substitutent, the free SH group at position 5 of the oxadiazole ring, and the free – NH2 group of the sulphonamido moiety. Cl S O H2N O N N O SH 12 xxiii B – Lactamase inhibitors Eidam71 synthesized new sulfonamide boronic acids, derived from the conversion of the canonical R1 caboxamide. This has substantial inhibition activity against B- Lactamases, they also rescued antibiotic resistance when used in combination with third generation antibiotics in bacterial cell cultures. This superficially modest substitution changes the geometry of the inhibitors enough to scramble the SAR observed in the analogue carboxamides. R S O O NH B HO OH 13 Protein kinase inhibitors The isoquinoline sulphonamide (14) derivatives synthesized by Hidaka et al72 had ability to inhibit protein kinases, some of the derivatives (15-20) exhibited selective inhibition towards certain protein kinase. The inhibtors where freely reversible and of the noncompetitive type with respect to the phosphate acceptor. The isoquinoline sulphonamides which are structurally unrelated to ATP, compete with ATP for free emzyme but do not interact with same enzyme form as does the phosphate acceptor. xxiv N S O O R 14 R= N H3C H3C CH3 NH N CH3 CH3 H3C NH H3C NH H3C H2N HN CH3 NH N H3C CH3 15 16 17 19 20 1.2 STATEMENT OF THE PROBLEM Sulphonamides are a class of broad antibacterial compounds that have a wide range of application. The group belongs to distinctive class of compounds that constitute at least five different classes of pharmacologically active agents73. The basic sulfonamide group, SO2NH-, occurs in various biologically active compounds, including antimicrobial drugs, antithyroid agents, antitumor, antibiotics and inhibitors of carbonic anhydrase74. xxv There is a vast increase in resistance of infectious disease to antibiotics, which negates treatment by almost all the known classes of antimicrobial compounds. Under these circumstances, the development of novel classes of antimicrobial compound becomes imperative75. Literature survey reveales that minor modifications in the structure of sulphonamide can lead to quantitative as well as qualitative changes in the biological activity.76 Inspite of the wide biological application of benzene sulphonamide derivatives, nothing is known of the N-heteroaryl substituted derivatives. The biological activities of these class of compound are yet unexploited. 1.3 OBJECTIVE OF THE RESEARCH The objectives of these research are as follows; Ø To synthesize benzene sulphonamide from benzene sulphonyl chloride and amino acids. Ø To synthesize the N-pyridine-3-yl derivatives of the Ø benzenesulphonamides. Ø To characterize the synthesizes compounds using FTIR,1HNMR and 13CNMR. Ø To screen the new compounds for antibacterial, antifungal and antioxidant activities. 1.4 Justification Of The Study xxvi The wide application of sulphonamides and the need to synthesize new functionalized derivatives with probable improved biological activities justifies this research.

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