Chapter 1

General introduction

1

General Introduction

Construction of heterocycles is among the most predominant synthetic activities of organic chemists, and as a major field of scientific endeavor, it continues to be a noteworthy source of variety of compounds of biological and technological interests. Getting the field more advanced by the new and efficient synthetic pathways, the activity in the area has further promised many new diverse, elegant and exciting molecules to create in a fewer number of steps.

Reasons behind accessing heterocycles are many. First is the subtle manipulation that brings them changes not only to the structure but to chemical, physical and biological properties too. Enhanced water solubility and transport of fungicide through the plant can be achieved in the fungicide by introducing more polar heterocycles. Basic nitrogen as amino substituent or part of the ring mimics desired functionality. While 1H-tetrazole is the mimic of carboxylic acid function, the furan that of masked 1,4-dicarbonyl. Chirality of heterocycles also governs outcome of asymmetric synthesis. Metal complex of chiral pyridine, imidazole and other heterocycles−based ligands are frequently used as chiral catalysts. Nature remains no longer the source of pharmaceuticals as many synthetic heterocycles act as precursors of drugs. Amoxicillin that contains β-lactum ring belongs to a family of antibiotics that have a semi-synthetic source.1Most natural scaffold-derived penicillin and cephalosporin−based drugs are key examples.2 In addition, oxycodone with both anti-cancer and anti-diabetic properties has purely synthetic source.3 Ketamine is used primarily to induceand maintain general anesthesia.4 Interaction of natural product chemistry with synthetic heterocyclic chemistry always encourages many new searches to bioactive profiles. For example, hallucinogen agent lysergic acid diethylamine (LSD) resembling natural vasoconstricting agent serotonin (5-hydroxy tryptamine) is partly synthetic. But histamine, derived in vivo from amino acid histidine, and its antagonists are now available synthetically.

In view of above, thus heterocyclic compounds have unparalleled evolution. One can tailor a heterocycle to meet particular need and application through structural modification. In addition, their ample natural occurrence, specific chemical reactivity and widespread utility have advantageously grown

Chapter 1 2

the area rapidly. About a third of recognized organic compounds are heterocycles. Today the heterocyclic synthesis, from both theoretical and practical significant points of view, has fascinated with a variety of synthetic procedures finding useful physiological and industrial applications5. Five- and six-membered heterocycles

Five- and six-membered rings containing nitrogen show a remarkable bioactivities.1,2 Five-membered pyrrolidine unit, for example, being widely used in pharmaceuticals is a structural motif of several alkaloids.3 In the last couple of decades, assemblies of many heterocycles with pyrrolidine unit appeared both naturally and synthetically as promising bioactive scaffolds. Those exploited most in the area include benzopyran, chromene and isoquinoline ring systems. Hexahydrochromeno[4,3-b]pyrrolidine is non-competitive antagonist of muscular nicotine receptor.6 Same motif exists in various natural products martinelline7 and sceletium alkaloid A.8 Naturally occurring chromene or chromane derivatives have a remarkable physiological property and some pyrrolidine−annulated benzopyrans are selective dopamine D3 receptor antagonist.9 Chromeno[4,3-b]pyrrole is effective in impulsive disorders,10parkinson’s disease,11 psychoses, memory disorders12 and anxiety.13 As a conformationally restricted nicotine or rivastigmine analogues,14 they hold great potential as acetylcholinesterase inhibitors.15 Benzopyranopyrrole and benzopyranopyridine are α-1 adrenergic antagonists useful in benign prostatic hyperplasia (BPH).16 Pyrollo-fused isoquinoline skeleton is another important unit.17 Marine alkaloids lamellarins isolated from mollusks, tunicates, and sponges18 with multidrug-resistance (MDR) phenotype have potential cytotoxic activity against cancer cell lines.19 Multiple cellular targets, such as DNA topoisomerase I,5 cancer-relevant protein kinases,20 and mitochondria21 have been also discovered. Lamellarin α 20-sulfate is a selective inhibitor of HIV integrase.22 Alkaloids such as (-)-trolline,23 one of the ingredients of Trolliuschinensis flowers and its antipode (+)-oleracein E24 from Portulaca oleracea L weed contain tricyclic hydropyrrolo[2,1-a]isoquinoline unit as a potential core structure. While former showed remarkable action against respiratory Staphylococcus aureus, Staphylcoccus pneumonia, and Klesiella pneumoniaebacteria as well as influenza viruses A and B−revealing antiviral property, later

Chapter 1 3

the DPPH-radical scavenger activity with a wide spectrum pharmaceutical properties, too. Further, (+)-crispine A, with analogous heterocyclic skeleton,has revealed cytotoxic activity against SKOV3, KB, and HeLa human cancer cell lines.25 From the above, it is therefore not surprising why a great deal of current research aims at heterocycles that incorporate a pyrrolidine unit.

1,3-DC, which involves azomethine ylide as an intermediate, is among the simple and highly efficient synthetic routes to incorporate pyrrolidine unit into heterocycles. In the area, many synthetic approaches have appeared as elegant and efficient total syntheses, which include chemo-, regio-, diastereo-, and enantioselective ones. Despite this however a modern synthetic management always sees the environmental challenges―almost running parallel to synthetic efficacy. The adaptation of green chemistry principles therefore needs to be happened in both academia and industry.1,3-DC reaction

The addition of 1,3-dipole to an alkene or alkyne (known as dipolarophile) is one of the prominent transformations in the organic synthesis.The five-membered heterocycle afforded in this way is a core unit of many complex natural products, pharmaceuticals, organocatalysts, biologically active compounds and building blocks in organic synthesis.26 This highly enantio-selective route has afforded so far large number of heterocycles. 1.1 1,3-Dipoles

There are two types of 1,3-dipoles27; one resembles allyl anion−adelocalized 4π-electron-system having one emptied and two filled orbitals, and second propargyl or allenyl anion with extra π orbital orthogonal to molecular orbital (MO). Their possible resonance structures are shown in Fig 1.1.28 A common central hetero-atom of allyl anion type dipole is generally nitrogen or oxygen, which is sp2-hybridized. Sometimes sulfur or phosphorus from higher row may replace these atoms (Fig 1.2). On the other hand, allenyl anion type dipole in which the central heteroatom is sp-hybridized nitrogen, its extra orbital doesn’t involve in the resonance (Fig 1.3).

Chapter 1 4

Fig 1.1 Types of 1,3-dipole with possible resonance structures.Oxygen in the Middle29

Carbonyl Ylide NitrosiminesCarbonyl Imines NitrosoxideCarbonyl Oxides Ozone

Nitrogen in the Middle29Azomethine Ylides AziminesAzomethine Imines Azoxy CompoundsNitrones Nitro Compounds

Sulfur in the Middle30Thiocarbonyl ylide Thiocarbonyl S-oxideThiocarbonyl S-imide Thiocarbonyl S-sulfidesphosphorous in the middle31

phosphonium ylideFig 1.2 Allyl anion type 1,3-dipoles.

Chapter 1 5

Diazonium Betaines29 Nitrillium Betaines29Diazoalkanes Nitrile OxidesAzides Nitrile IminesNitrous Oxide Nitrile Ylides

Fig 1.3 Propargyl/allenyl anion type 1,3-dipoles.1.1.1 Historical background

A transformation Buchner E. studied between diazoacetic ester and α,β-unsaturated esters, five years after its introduction by Curtius T. in 1883,32 is regarded as first 1,3-DC reaction.33 He concluded that methyl diazoacetate gave 1-pyrazoline with methylacrylate which further rearranged into 2-pyrazoline.34Nitrones, nitrile oxides and other dipoles were also discovered in the later years.35 In the first seven decades after the discovery of 1,3-DC reaction, only a few dipoles were in use. It was in 1963 that Huisgen laid out the classification of 1,3-dipoles.36 Woodward and Hoffmann also developed the theory of conservation of orbital symmetry which helped understand mechanism of concerted cycloaddition reactions.37 Houk K. N. made the prediction about the relative reactivity and regioselectivity of 1,3-DC reaction more simple, on the basis of this theory.38, 401.1.2 Dipolarophile

A component which adds to 1,3 dipole is known as dipolarophile, and alkene with a 2π-electron system is its common form say α,β-unsaturated carbonyl compounds (1), ketones (2), allylic alcohols (3), allylic halides (4), alkynes (5), vinylic ethers (6), vinylic esters (7) and imines (8) (Fig 1.4).39

Fig 1.4 Dipolarophiles.

Chapter 1 6

1.1.3 General aspects of 1,3-DC reactionsA 4π electron 1,3-dipole 9 when reacts with a 2π electron component

called dipolarophile 10 in a [π4s + π2s] fashion via concerted way, it forms five membered heterocyclic ring system 11.37

The reaction is similar to the Diels―Alder (DA) reaction, wherein a 4πelectron diene 12 reacts with a 2π electron dienophile 13 in a [π4s + π2s]-manner,yielding a six membered ring 14.

1.1.4 Effect of substituents on 1,3-dipole and dipolarophileAccording to FMO theory, dipole HOMO normally interacts with

dipolarophile LUMO in transition state. The trend, however, may reverse with the introduction of electron donating or electron withdrawing substituent on dipole and alkene components. For instance, N-methyl-C-phenylnitrone the reaction of which although controlled by dipole HOMO―dipolarophile LUMO interaction with methyl acrylate, it favors dipole LUMO―dipolarophile HOMO interaction with methyl vinyl ether, hence effecting the regio- and the diastereo-selectivity. The steric and electronic factors are additional in deciding the regioselectivity (Fig 1.5).40

Fig 1.5 Regioselectivity in 1,3-DC reactions.1.1.5 Mechanistic approaches to 1,3-DC reactions

Huisgen et al.35,36 suggested that 1,3-DC reaction proceeds through concerted mechanism41(Fig 1.6).

Chapter 1 7

Fig 1.6 Concerted vs singlet diradical mechanism of 1,3-DC reaction.According to Firestone et al., it proceeds through a singlet diradical

intermediate (Fig 1.6) that both cis- or trans-alkene results in, giving a mixture of cis- and trans- isomers via 180° rotation of C―C bond.

Woodward―Hoffmann37 suggested that the reaction is thermally allowed,proceeding through a concerted mechanism. The mode of interaction of all three pz orbitals of dipole is suprafacial with two pz orbitals of dipolarophile, retaining the stereochemistry (Fig 1.6). Here, dipolarophile trans-2-butene 15 furnishesexclusively trans-cycloadduct 16 with hypothetical dipole 9. Starting with the cisalkene 17 thus yields only cis-cycloadduct 18 (Fig 1.7). The reaction is therefore stereospecific, creating chiral centers.

Fig 1.7 Preservation of dipolarophile’s stereochemistry.FMO theory adequately describes the transition state of 1,3-DC reaction.

According to which, the dipole HOMO favorably interacts with the dipolarohile LUMO. The reverse is also true. On the basis of relative FMO energies, which actually take part in the reaction, Sustman described three possible interactions(Fig 1.8).42 The dominant one however is type I where the dipole HOMO

Chapter 1 8

interacts with the diloparophile LUMO, a typical for the reaction involving azomethine imines or ylides.

Energy

Fig 1.8 The classification of 1,3-DC reactions on the basis of FMOs.In type II, HOMO―LUMO interactions of both dipole―dipolarophile and

dipolarophile―dipole are important, which generally exist in the reaction of typical nitrones.

Type III involves a dominant interaction between dipolarophile HOMO and dipole LUMO. Since HOMO of nitrile oxide is low lying, its reaction is put between borderline of type II and type III, although included in type II. Another example of type III is that between nitrous oxide and ozone.

Energy

Fig 1.9 Effect of coordination of Lewis acid (LA) toward dipole and dipolarofile on their FMO energies.

Lewis acid such as metals may alter both energy and coefficient of frontier molecular orbitals of reacting partner through coordination, which depend upon electronic properties of the reagents or Lewis acid. In addition to catalyzing asymmetric 1,3-DC reactions, Lewis acid also influence regio-, diastereo-, and enantioselectivities via metal–ligand complex formation.

Chapter 1 9

1.2 Azomethine ylide and the scope of its reaction Azomethine ylide is a planar allyl anion type 1,3-dipole, containing central

nitrogen atom attached to two terminal carbons (Fig 1.10)43. With a few exceptions,44 the dipole is very reactive, unstable and short-lived. Owing to this, its stable precursor―derived―in situ generated form is often trapped in 1,3-DC reaction44 affording nitrogen containing five membered heterocycles such as pyrrolidines or pyrroles. In recent past, the strategy has become very popular and came out with a variety of methods.

Fig 1.10 The general structure of azomethine ylides.Associated with the creation of four new chiral centers, 1,3-DC reaction

involving azomethine ylide gives various stereo-isomers, leading to high levels of stereo-selectivity. Out of four possible geometries of an azomethine ylide, which determine the stereo-chemical outcome, W-(19) and U-shaped (20) ones lead to 2,5-cis-disubstituted cyclic amine product 24 via suprafacial transformation, and two S-shaped (21 and 22) 2,5-trans-disubstituted product 25 (Fig 1.11).Isomerization of ylide leads to form a mixture. In the formation of 2,5-trans-disubstituted pyrrolidines, however S-shaped geometry is general preference (21 and 22), resulted from aldehydes and unhindered secondary α-amino esters45.

Fig 1.11 Different geometries of azomethine ylide. A large number of methods have been developed for the generation of

azomethine ylide, which include the proton abstraction from imine derivatives of

Chapter 1 10

α-amino acids 26,46 thermolysis and photolysis of aziridines 28,47 condensation of aldehyde 30 with secondary amine 31,48 the decomposition of N-alkyl-N-methoxymethyl-N-(trimethylsilyl) methylamines 33,49 desilylation of various α-amino silane derivatives,50 decarboxylative condensation of amino acids,51deprotonation of iminium salts,52 and others.53 Among them, first three are most common procedures.

1.2.1 1,3-DC reactions of azomethine ylides1,3-DC reaction via in situ formed azomethine ylide is a significant

transformation in the heterocyclic preparations.54 The strategy allows the formation of two bonds and up to four stereogenic centers in a single operation (Fig 1.12).55 The reaction can be performed in two different manners;intermolecular and intramolecular. In first, azomethine ylide and dipolarophile (alkene or alkyne) are separate reacting molecules. In second approach, however both have been placed within the same molecule, which results into a considerable complexity.56

Fig 1.12 The cycloaddition reaction of the azomethine ylide.

Chapter 1 11

1.2.2 1,3-DC reactions of azomethine ylide towards diversified heterocyclesIn the literature, a variety of methodologies have been reported for the

strategy in terms of substrates, catalysts, reaction conditions, medicinal properties, industrial applications etc. Following are the relevant publications that clearly reflect the development in the area.

Purushothaman et al. reported cis-fused chromeno[3,4-b]pyrrole (37 and 39) and its analogues from substrates 36 and 38 with sarcosine, L-proline, thiazolidine-4-carboxylic acid and tetrahydroisoquinoline-3-carboxylic acid 35. Some of the candidates of the series have showed relatively good and improved antibacterial activities than standard tetracycline, along with moderate antifungal activity by a few candidates.57

Arumugam et al. coupled the intramolecular version of this strategy with Pictet―Spengler cyclization and reported chromeno[4,3-b]pyrroles 42 and indolizino[6,7-b]indoles 44. Yields were in the 50―78 % range through conventional method which under MW irradiation was improved further.Compounds revealed good antibacterial, antifungal and antioxidant activities.58

Arumugam et al. also tested unusual dipolarophiles with di- or triketones (46 or 47) and amino acids 48 in MeCN under various conditions, giving antibacterial monospiropyrrolidine/pyrrolizidine―substituted β-lactams 49 or 50.59

Chapter 1 12

Mishra et al. exploited aldehyde 51, which were derived from Morita―Baylis―Hillman adducts of acrylates, with glycine ester hydrochloride52 or methyl proline 53, affording aza-polycycles 54 or 55.60

Levesque et al. coupled the cycloaddition with Vilsmeier―Haack cyclization and prepared polycyclic alkaloid cores. Acyclic substrates 56 and 59gave bicyclic and tricyclic adducts with perfect chemoselectivity in a single operation.61

Llamas et al.62 described enantioselective Cu(MeCN)4ClO4/Taniaphos catalyzed 1,3-DC reaction with aryl vinyl sulfones 63, with nearly complete exoselectivity and enantioselectivities up to 85 % ee. The resulting 3-sulfonyl cycloadducts 65 were converted into 2,5-disubstituted pyrrolidines 66.

Chapter 1 13

Kim et al. synthesized hexahydrobenzofuro[3,2-b]pyrroles 69 from 2-vinyloxy benzaldehyde 67 and amino acid esters 68. In this work, the short tethering of dipolarophile engenders a high stereo-selectivity.63

Wang et al. employed tetrahydroisoquinoline 71 and aldehyde 70 to afford cycloadducts 72 and 73.64

Confalone et al. applied the methodology to synthesize Sceletium alkaloid A4 81.65

1.2.3 Synthesis of natural products via azomethine ylideThe total synthesis of many natural products can be achieved through 1,3-

DC reaction that involves azomethine ylide.

Chapter 1 14

Confalone and Earl66 synthesized alkaloid lycorenine 85. The intermediate azomethine ylide 83 smoothly underwent [3+2] intramolecular cycloaddition to form octahydroindole ring system 84.

Pandey et al.67 created the core structure 88 of the complex pentacyclic 5, 11-methanomorphanthridine alkaloids stereospecifically in one-pot. The success of this intramolecular [3+2] cycloaddition lies in the formal total synthesis of (±)-pancracine 89.

Banwell et al.68 obtained lamellarin K 95 via azomethine ylide derived from dihydroisoquinolinium salt. The [3+2] cycloaddition product pyrrole on de-isopropylation transformed into marine alkaloid lamellarin K 95.

Pearson and Lovering69 achieved (±)-crinine 100, in eight steps with 20% overall yield, as a single stereoisomer of the perhydroindole.

1.3 Microwave-assisted solvent-free cycloaddition reactions1.3.1 Solvent-free synthesis

Chapter 1 15

Despite the fact that solvents are associated deeply with chemical transformations, providing reactants with a favorable homogeneous medium, awareness towards the environment has imposed a tremendous pressure on developing new synthetic methodologies that control wastes.70 In this context, solvent-free protocols are often of laboratory interest, assuring of not only environmental safeguards71 but also other benefits like control over more possible products―that is unexpected part of solution phase, less handling cost―as solvent-less method involves easy work-up, possibilities of forming highly ordered structure of product― as a result of more favored host-guest interaction in solvent-free conditions and finally degree of selectivity.72

Grinding reacting partners together with each other into a fine mixture ina glass mortar is common procedure. Sophistication which allows the reaction to take place at higher temperature can also be set up and preferred. One can also monitors outcome of the reaction by TLC (thin layer chromatography).Transformations like pyrrolytic distillation of barium or calcium of carboxylic acids, few Friedel―crafts and related Fries reaction etc. undergo smoothly under solvent-free conditions. Some modified protocols like Robinson―annulation73―atandem Michael―aldol condensation, Diels―Alder reaction―a cycloaddition reaction74, and binaphthol catalyzed reaction―an important asymmetric synthesis75 also exist in this category.1.3.2 MW an alternative source of heating

Use of MW for heating purpose was realized after 1950, when Spencer P.L., during World War II76 at radar waves, noticed MW cooks the food at much quicker rate than a conventional oven. It was then led to introduction of many commercial MW ovens in use, in removing sulfur and other pollutants from coal, in rubber vulcanization, in drying product, in removing moisture and food analysis, in solvent extraction etc. In organic synthesis however it appeared in 1986, but with some limited uses.77 Today, with specifically designed multimode MW oven for large scale laboratory applications, and single mode technologywith uniform and concentrated MW power supply, the technology reflects a breakthrough in synthesis, which perhaps is a key factor in rapid expansion of the field.

Chapter 1 16

Located between IR and radio waves in the EMR spectrum, wavelengthsof MW fall inthe1mm―1m or 30 MHz―3GHzrangewithin which molecules can rotate only. Because of its right penetration depth to interact with sample, and generation by available power sources, 2450 MHz is among the various available frequencies preferred for laboratory scale samples. Out of its electric and magnetic fields, only the electric field transfers energy into heat. Magnetic field interactions normally have no effect on chemical synthesis. MWs move at the speed of light (300,000 km/sec). The energy in MW photons (0.037 kcal/ mol) is very low relative to the typical energy required to cleave molecular bonds (80―120 kcal/mol); thus, MWs doesn’t affect the molecular structure. In the excitation of molecules, the effect of MW absorption is purely kinetic.

In traditional way of synthesis, the heat first passes through the vesseland then to solvent. Due to thermal conductivity, which may vary with the materials, it makes the energy transfer way slow and inefficient. Further, the conductive heating also hinders the chemist’s control over the reaction. In contrast, MWs couple directly with the molecules in the reaction mixture, leading to a rapid rise in temperature. Thus, no thermal conductivity of the vessel materials affects the heating process. On the contrary, it allows an instantsuperheating of anything that will react to dipole rotation or ionic conduction; the two fundamental mechanisms to transfer energy to the substance being heated. MWs heating also offers facile reaction control and can be described as ‘instant on-instant off’. Dipole rotation is an interaction in which polar molecules try to align themselves with the rapidly changing electric field of the MWs. The second way to transfer energy is ionic conduction, which results if there are free ions or ionic species present in the substance being heated.

In a typical reaction coordinate, the process begins with reactants with a certain energy level. To complete the transformation, reactants must collide in the correct geometrical orientation to become activated to a higher-level transition state. The difference between these levels is the activation energy required to reach this higher state. With enough energy absorbed, the reactants quickly react and return to a lower energy state―the products of the reaction. MW irradiation doesn’t affect the activation energy instead provides the

Chapter 1 17

momentum to overcome this barrier and complete the reaction more quickly than conventional heating. 1.3.3 MW-assisted, solvent-free organic synthesis

Majority of publications that highlight accelerations for a wide range of organic reactions describe the significant role of solvent-free conditions and MWs irradiations, leading to large reductions in times and enhancements in the transformation.78 MWs organic syntheses are categorized mainly into three ways;79 in water (a), in organic solvents (b) and in solvent-free environment (c). The solvent-free procedure can be used in following three different ways.80

i) As liquid-liquid or liquid-solid systems provided at least one of the reactants is liquid polar molecule. Due to the direct absorption of radiation by reagents, effect of MW is remarkable.81

ii) When reagents are supported on solid supports in dry media, e.g. alumina, silicas or clays.82

iii) Phase transfer catalysts (PTC) mediated systems wherein a liquid reagent acts both as a reactant and an organic phase.83

Coupling of MW activation thus offers many advantages in the heterocyclic synthesis which includes shorter reaction times and high conversions to products. With80 potentialities of working with open vessels and enhancing the possibility of up-scaling the reactions on preparative scale, this eco-friendly approach can effectively be applied to the rapid generation of assembly of many hetero atom organic compounds. Combinatorial heterocyclic synthesis for example has allowed many pharmaceuticals to create efficiently in a short reaction time. Number of publications in this area is increasing rapidly with approximately more than 2000 since the pioneering work of Gedye et al.started in 1986.84 It is noted that the country which the technique seems to be developed in is India. From the literature survey below, it clearly shows there are many reports on MW-assisted reactions.85

Arrieta et al.86 reported bipyrazole 104 via in situ generated azomethine imine intermediate 102 from pyrazolyl hydrazones 101 and alkyne derivatives103 as dipolarophile.

Chapter 1 18

Pospisil et al. developed intramolecular 1,3-DC reaction of aldehydes 105with amines 106, giving cyclic products 108, 109 and 110 in a short time with higher yields, and stated that % yields were influenced by steric factor R2.87

Dinica et al.88 described solvent-free-KF/alumina supported reaction of 4,4’-bipyridinium ylides 112, in situ generated from 4,4’-bipyridinium diquaternary salts 111, with activated alkynes 113 in MW irradiations. It yielded7,7’-bis-indolizines 114 in the 81–93% range.

Katritzky and Singh89 showed that the 1,3-DC reaction of organic azides 115 to acetylenic amides 116 afforded corresponding N-substituted C-carbamoyl-1,2,3-triazoles 117 in excellent yields.

Chapter 1 19

Bortolini et al.90 reported environmentally friendly procedure for 4’-aza-2’,3’-dideoxy nucleosides 120 from vinyl nucleo-bases 118 and nitrones 119. The vinyl nucleo-bases have been first time used.

Fraga-Dubreuil et al. developed91 imidate, from ethyl α-cyano-α-amino acetate 121, as potential azomethine ylide, which undergoes a very fast, highly regioselective cycloaddition with a wide range of dipolarophiles particularly aldehydes 122 and Schiff’s base 125. This environmentally-friendly methodology also afforded new oxazoles 123―124 and diazoles 126―127 with good yields (81―98 %).

Taherpour and Kheradmand92 used trimethylsilyl azide 129 (Me3Si-N3) with alkylpropiolates 128 and DMAD (dimethyl acetylenedicarboxylate) to construct alkyl 1,2,3-triazole-4-carboxylate derivatives 130. This one-pot 1,3-DC method afforded products in higher yields.

Ramesh et al.93 developed Baylis―Hillman adduct 131 as typical dipolarophile from ninhydrin which gave interesting spiropyrrolidine133/pyrrolizidine 134 heterocycles with sarcosine 80/proline 77 and various activated ketones 132.

Chapter 1 20

Arrieta et al.94 demonstrated very fast 1,3-DC of imines 136, derived from α-aminoesters, with β-nitrostyrenes 135. Three isomeric pyrrolidines 137,yielded in the 81―87 % range, were further aromatized and give the mixture of pyrrole and 4-nitropyrrole 138.

O2NR2

R1 N CO2MeNH

R1

R2

CO2Me

O2N

NH

R1 CO2Me

R2XMWsolvent-free

MWaromatization

135

136 137 138

Oritani et al. 95 described the highly stereoselective intramolecular cycloadditions of unsaturated N-substituted azomethine ylides. The mixture of aldehyde 139 and N-methyl- or N-benzylglycine ethyl esters 140 impregnated on silica gel, and then exposed to MW without solvent. Corresponding tricyclic products 142 were obtained in 79 and 81% yields, respectively.

NO

O

CHO

NO

O

NH

NO

N

O

H

H

H

CO2EtR

CO2EtR

RNHCH2CO2Et

MW / 15 min

142139

140

141 79-81 %R Me, Bn

Jayashankaran et al. synthesized dispiropyrrolo 146/pyrrolizidino 145ring systems. Azomethine ylides formed via decarboxylative step afforded typical products with dipolarophile 9-arylidine-fluorene 144 in shorter reaction time, in higher yields.96

Chapter 1 21

Guezguez et al. reported CuI and DIPEA catalysts in the 1,3-DC reaction, that dramatically enhanced reaction between azido-2’ deoxyribose 147 and alkynes 148, which are absorbed on silica, affording corresponding 1,2,3-triazolyl-nucleosides 149α or 149β, within few minutes.

Abdel-Aziz et al.97 described regioselective version of 1,3-DC reaction viain situ generation of nitrilimines, derived from substrate 150 and 5-arylidene-2-arylimino-4-thiazolidinones 151 or 2-(4-arylidene)thiazolo[3,2-a]benzimidazol -3(2H)-ones 153. It yielded corresponding 1,3,4-triaryl-5-N-arylpyrazole-carboxamides 152 and pyrazolylbenzimidazoles 154 in higher yields.

Bashiardes et al.98 synthesized pyrrolidines 159 and pyrroles 160 from activated and nonactivated, alkenes 156 and alkynes 157. When compared with heating in both presence and absence of solvent, the reaction in MW-irradiation improved not only in terms of the yields but reaction times, too.

Chapter 1 22

1.4 Biological screening tests1.4.1 Antimicrobial study1.4.1.1 Bacteria

Existing all around us are bacteria that we can merely look upon, belongs to prokaryotic group as disease causing parasites. Although responsible for a large number of human diseases, they help recycle certain elements like carbon, nitrogen, and oxygen in the atmosphere. There is no existence of life, if they fail to decompose the waste and dead organisms. So, they continuously exchange the essential elements of biosphere with other environmental segments like atmosphere, lithosphere and hydrosphere. Therefore it becomes harder to decide as to whether the bacteria are friend or enemy as they suppose both the positive and negative aspects to establish a relationship with humans.

In 1828, a German scientist C. G. Ehrenberg99 used the term “bacterium”. Bacteria are microscopic organisms in plant kingdom, devoid of chlorophyll, relatively simple and primitive forms of cellular organisms known as “Prokaryotes”. Bacteriology is science that deals with the study of bacteria. Christian Gram, a Danish physician, in 1884, discovered a stain named as Gram stain, which can divide all bacteria into two classes; “Gram +ve” and “Gram –ve”. A dark blue color the Gram +ve bacteria retained with staining agent methyl violet couldn’t be removed by acetone and alcohol washes. But, the Gram –ve bacteria couldn’t resist such discoloration.100 Gram +ve bacteria have cell wall of peptidoglycan layer much thicker than that of Gram –ve bacteria. Gram –ve bacteria have an additional outer membrane. The outer membrane is major permeability barrier in Gram –ve bacteria. The space between the inner and outer membranes is known as the periplasmic space. Gram –ve bacteria store degradative enzymes in the periplasmic space. Gram +ve bacteria lack such periplasmic space. In both the cases, digestive enzymes perform extra-cellular digestion. Digestion is needed since large molecules do not readily pass across the outer membrane (if present) or cell membrane.1011.4.1.2 Pathogens

The microorganisms, or infectious agents or more commonly germs, are the biological agents that produce diseases in the host, usually known as pathogens. There are several substrates and pathways through which pathogens

Chapter 1 23

can invade a host; the principal pathways include different episodic time frames, but soil contamination has longest, most persistent potential to harbor pathogen. Pathogens have certain characteristics that they need and use to cause a disease. These so-called virulence factors have specific functions in the successive steps that result in an infection. An infection can be seen as a miniature battle between pathogen and host, the first trying to remain present and to feed and multiply, while the host is trying to prevent this. The resulting infection is a process which have three possible outcomes; the host wins and the pathogens are removed possibly with the help of medication so that the host can recover; the pathogens win the ultimate battle and kill host; or an equilibrium is reached in which a host and pathogens live involuntarily together and damage is minimized.1.4.1.3 Bacterial PathogenTable 1.1 Bacteria commonly found on human body surfaces.102

Bacterium Skin Con-junctiva Nose Pharynx Mouth LowerGI Ant.Urethra VaginaStaphylococcus epidermidis (1) ++ + ++ ++ ++ + ++ ++Staphylococcus aureus* (2) + +/- + + + ++ +/- +Streptococcus mitis + ++ +/- + +Streptococcus salivarius ++ ++Streptococcus mutans* (3) + ++Enterococcus faecalis* (4) +/- + ++ + +Streptococcus pneumoniae* (5) +/- +/- + + +/-Streptococcus pyogenes* (6) +/- +/- + + +/- +/-Neisseria sp. (7) + + ++ + + +Neisseria meningitidis* (8) + ++ + +Enterobacteriaceae*(Escherichia coli) (9) +/- +/- +/- + ++ + +Proteus sp. +/- + + + + + +Pseudomonas aeruginosa* (10) +/- +/- + +/-Haemophilus influenzae* (11) +/- + + +Bacteroides sp.* ++ + +/-Bifidobacterium bifidum (12) ++Lactobacillus sp. (13) + ++ ++ ++Clostridium sp.* (14) +/- ++Clostridium tetani (15) +/-Corynebacteria (16) ++ + ++ + + + + +Mycobacteria + +/- +/- + +Actinomycetes + +Spirochetes + ++ ++Mycoplasmas + + + +/- +++ = nearly %, + = common (about 25 %), +/- = rare (less than 5 %), * = potential pathogen

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Bacteria that cause disease are called pathogenic bacteria. Bacteria can cause diseases in humans, in animals and also in plants. Some bacteria can only make a particular host ill; others cause trouble in a number of hosts depending on the host specificity of the bacteria. The diseases caused by bacteria are almost as diverse as the bugs themselves and include infectious diseases such as pneumonia, food borne illnesses, tetanus, typhoid fever, diphtheria, syphilis and leprosy and even certain forms of cancer. Bacterial cells grow and divide, replicating repeatedly to form a large numbers present during an infection or on the surfaces of the body. To grow and divide, organisms must synthesize or take up many types of bio-molecules. Tables 1.1 represent the list of bacteria commonly found on the surfaces of the human body.1.4.1.4 Classifications of bacteriai) Classification based on shapes of bacteria

There are three main shapes of bacteria: a rod shape bacilli, a sphere shape cocci and spiral shaped spirilla. Others are more complex.ii) Aerobic and anaerobic bacteria

Bacteria that use oxygen for their survival are aerobic bacteria and those, which don’t are anaerobic ones. Later one cannot bear oxygen and may die off oxygenated environment, and found mostly in the places under the earth surface, the deep ocean or some suitable mediums.iii) Gram +ve and Gram –ve bacteria

Based on gram staining method, bacteria are grouped into ‘Gram +ve’ and ‘Gram –ve’. Staining agent is used to bind it to the cell wall of the bacteria. The following pathogens were used for antimicrobial study in the present work. Gram +ve

(i)Streptococcus pneumonia,103 (ii) Clostridium tetani,104(iii) Bacillus subtilis105

Gram –ve(i) Salmonella typhi,106 (ii) Vibrio cholerae,107(iii) Escherichia coli108

1.4.1.5 Fungal PathogensFungi are plant-like organisms that lack in chlorophyll, existing in over

100,000 different species as one of the five kingdoms of life. While many are

Chapter 1 25

beneficial and useful i.e. edible mushrooms, others are harmful i. e. some fungi can infect plants and people. Lacking in chlorophyll, they derive food from others. They don't use light to prepare food and so can live in damp and dark places. They are also known as saprophytic organisms, as they grow on dead organic matter. Most commonly, fungi grow as pathogen on skin of animals or people. Sometimes they are called ringworm symptom. Fungus cause irritation to nose and causes allergies. Over 37 million people have allergies including those mainly caused by fungus.

Buildings and other constructive houses can also get sick from some fungi known as Penicillium and Stachybotrys. They float in the air and can causewatery eyes and breathing problems.

Fungi also cause a number of plant and animal diseases; in humans, ringworm, athlete's foot, and several more serious diseases. Since, fungi are similar to animals more chemically and genetically than other organisms, it makes fungal diseases very difficult to treat. Plant diseases caused by fungi include rusts, smuts, and leaf, root, and stem rots, and may cause severe damage to crops. Most antibiotics that function on bacterial pathogens cannot be used to treat fungal infections due to the fact that fungi and their hosts both have eukaryotic cells. The typical fungal spore size is 1―40 μm in length.We haveused following fungal pathogens for antifungal study of synthesized compounds.

(i) Candida albicans109, (ii) Aspergillus fumigatus1101.4.1.6 Antimicrobial agents

The modern antimicrobial chemotherapy era began with Fleming's discovery of powerful bactericidal substance penicillin in 1929, and Domagk's discovery of broad spectrum antimicrobial synthetic sulfonamides in 1935. For his work on first synthetic antibacterial agent “prontosil”, this German bacteriologist and pathologist Gerhard Domagk received the Nobel Prize in 1939. Antimicrobial agents may either be bactericidal, killing the target bacterium or fungus or bacteriostatic, inhibiting their growth. Though bactericidal agents are more effective, bacteriostatic agents are extremely beneficial since they permit the normal defense of the host to destroy microorganisms. Antimicrobial agents may be classified according to the type of organisms they kill i.e. antibacterial, antiviral, antifungal, antiprotozoal and anthelmintic. It could be also useful to

Chapter 1 26

combine various antimicrobial agents for broadening the activity spectrums and to minimize the possibility of the development of bacterial resistance. Some antibiotic combinations are more effective than single agent. This is termed as synergism. Combination therapy has proved latest therapy more effective. Some bacteriostatic agents on a novel combination give bactericidal activity. Independently, both the drugs sulphamethoxazole and trimethoprime are bacteriostatic but their combination is widely used as a bactericidal combination. Two such bactericidal drugs used in combination therapy include refampin plus dapsoneb in leprosy, and refampin and isoniazide in Tuberculosis. World Health Organization has also approved such combinations.

Most microbiologists explain that the antimicrobial agents are used in the treatment of infectious disease; antibiotics, coming from natural source are produced by certain groups of microorganisms. A hybrid substance is a semi synthetic antibiotic, wherein a molecular version produced by the microbe is subsequently modified by the chemist to achieve desired properties. Furthermore, some antimicrobial compounds, originally discovered as metabolic products of microorganisms, can be synthesized entirely by chemical means. In the medical and pharmaceutical worlds, all these antimicrobial agents used in the treatment of disease are referred to as antibiotics―chemicals that areproduced by living organisms which, even though in minute amounts inhibit the growth of another organism. 1.4.1.7 Antimicrobial susceptibility testsEvaluation techniques

The goal of antimicrobial susceptibility testing is to predict the in vivosuccess or failure of antibiotic therapy. Tests are performed in vitro to measure the growth response of an isolated organism towards a particular drug. The tests are performed under standardized conditions so that the results are reproducible. The raw data are either expressed in terms of a microorganism zone size or minimum inhibitory concentration (MIC). Antimicrobial susceptibility testing methods are divided based on the principle applied in each system. All techniques involve either diffusion of antimicrobial agent in agar or dilution of antibiotic in agar or broth. The types of automated techniques

Chapter 1 27

employed categories the methods. The evaluation can be done by the following methods as given bellow. Table 1.2 Microbial tests methodsDiffusion Dilution Diffusion & DilutionStokes method Minimum Inhibitory Concentration E-Test methodKirby―Bauermethod i) Broth Dilution Methodii)Agar Dilution Method

In the present work, we have used, FDA-USA approved, WHO (world health organization) and NCCLS111 recommended Broth Dilution method, for antimicrobial screening tests. Broth dilution method

It determines the lowest concentration of an assayed antimicrobial agent, in terms of MIC that under defined test conditions inhibits the visible growth of the pathogens. Results are quantitative in terms of the amount of antimicrobial agents to inhibit the growth of specific microorganisms. 1.4.1.8 Factors influencing antimicrobial susceptibility testing112

a) Choice of media: Consistent and reproducible results were obtained in media prepared especially for sensitivity testing. Satisfactory media provide essentially clear, distinct zones of inhibition say 20 mm or greater in diameter. Unsatisfactory media produce no zone of inhibition, growth within the zone, or a zone of less than 20 mm.

b) Size of inoculums: Although large number of organisms does not markedly affect many antibiotics, the ideal inoculum is one, which gives an even dense growth without being confluent. Overnight broth cultures of organisms and suitable suspensions from solid media were diluted appropriately to give optimum inoculum for sensitivity testing.

c) pH: The pH of the medium was kept in between 7.2 and 7.4 at rt after gelling. If the pH is too low, certain drugs may appear to lose potency (e.g., aminoglycosides, quinolones and macrolides). Sometimes other agents may appear to have excessive activity (e.g. tetracyclines). Higher pH produces opposite effects.

d) Moisture: The surface should be moist, but no moisture droplets should be apparent on the surface of the medium or on the petri dish covers when the plates are inoculated.

Chapter 1 28

e) Effects of variation in divalent cations: Variations in divalent cations may affect results. Excessive cation will reduce zone sizes, whereas low cation may result in unacceptably large zones of inhibition.

f) Testing strains that fail to grow satisfactorily: Only aerobic or facultative bacteria that grow well on unsupplemented media should be tested. Certain fastidious bacteria do not grow sufficiently on unsupplemented media. These organisms require supplements or different media to grow and they should be tested on the media.

The conditions must be met for the antimicrobial susceptibility testing:a) Intimate contact between the test organisms and substance.b) Required conditions for the growth.c) Same conditions throughout the study.d) Aseptic/sterile environment.

1.4.2 Antituberculosis study1.4.2.1 Introduction

Tuberculosis, MTB (Mycobacterium tuberculosis) and TB (Tuberclebacillus) are common names, and in many cases lethal, infectious disease caused by various strains of mycobacteria, usually MTB.113 Mycobacteria are Gram-resistant (waxy cell walls), non-motile, pleomorphic rods, related to the actinomyces. Most mycobacteria are found in habitats such as water or soil. However, few are intracellular pathogens of animals and humans. MTB, along with M. bovis, M. africanum, and M. microti all cause the disease known as tuberculosis, and are members of the tuberculosis species complex. Each member of the TB complex is pathogenic, but M. tuberculosis is pathogenic for humans while M. bovis is usually pathogenic for animals. M. bovis causes TB in the animal kingdom long before invading humans. However, after the domestication of cattle during 8000―4000 BC, there was archaeologicalevidence of human infection byM. bovis probably through milk consumption. MTB probably a human-specialized form of M. bovis developed among milk-drinking. The Indo-European speeded the disease during their migration to the Western Europe, and the Eurasia. By 1000 BC, MTB and pulmonary TB had started spreading throughout the world.

Chapter 1 29

TB is a chronic infectious disease caused by MTB, which is responsible for deaths of about 1 billion people during last two centuries. It usually attacks the lungs but can also affect the central nervous system, the lymphatic system, the circulatory system, the genitourinary system, the gastrointestinal system, bones, joints and even the skin. It spreads through the air when people who have an active MTB infection cough, sneeze, or otherwise transmit their saliva through the air. According to the WHO, approximately 1.86 billion people, that is, 32 % of the world population is infected with MTB. WHO estimates about 8 million new active cases of TB per year and nearly 2 million deaths each year, that is, 5000 people every day. In India alone, one person dies of TB every minute. HIV +ve patients are more susceptible to MTB with a 50-fold risk increase over HIV –ve patients. TB is currently to blame for 13 % of the deaths due to HIV infection.1.4.2.2 Mycobacterium tuberculosis

MTB is pathogenic bacteria species in the genus Mycobacterium and the causative agent of most cases of TB the Latin prefix "myco—" means both fungusand wax; its use here relates to the "waxy" compounds in the cell wall.

MTB, then known as the “Tubercle bacillus”, was first described on 24 March 1882 by Robert Koch, who subsequently received the Nobel Prize in physiology or medicine for this discovery in 1905; the bacterium is also known as "Koch's bacillus". The MTB genome was sequenced in 1998. M. tuberculosisH37Rv was first isolated in 1905, remained pathogenic and is the most widely used strain in tuberculosis research. The complete genome sequence and annotation of this strain was published in 1998 by Cole et al.114 Other human pathogens belonging to the Mycobacterium genus include M. avium, whichcauses a TB-like disease, especially prevalent in AIDS patients, andMycobacterium leprae, the causative agent of leprosy.General characteristics

MTB is a fairly large non-motile rod-shaped bacterium distantly related to the actinomycetes. Many non-pathogenic Mycobacteriums are components of the normal flora of humans, found most often in dry and oily locales. The rods are 2―4 micrometers in length and 0.2―0.5 micrometers in width. MTB is an obligate aerobe. For this reason, in the classic case of tuberculosis, MTB complexes are always found in the well-aerated upper lobes of the lungs. The

Chapter 1 30

bacterium is a facultative intracellular parasite, usually of macrophages, and has a slow generation time, 15―20 h, and a physiological characteristic that maycontribute to its virulence.

Two media that are used to grow MTB; an agar based Middlebrook's medium and egg based Lowenstein-Jensen (L-J) medium. MTB colonies are small and buff colored when grown on either medium. Both types of media contain inhibitors to keep contaminants from out-growingMT.Ittakes4―6weekstogetvisual colonies on either type of media. Chains of cells in smears made from invitro-grown colonies often form distinctive serpentine cords. This observation was first made by Robert Koch who associated cord factor with virulent strains of the bacterium. MTB is not classified as either Gram +ve or Gram –ve because it does not have the chemical characteristics of either, although the bacteria do contain peptidoglycan (murein) in their cell wall. If a Gram stain is performed on MTB, it stains very weakly Gram +ve or not at all (cells referred to as "ghosts").

Mycobacterium species, along with members of a related genus Nocardia, are classified as acid-fast bacteria due to their impermeability by certain dyes and stains. Despite this, once stained, acid-fast bacteria will retain dyes when heated and treated with acidified organic compounds. One acid-fast staining method for MTB is the Ziehl-Neelsen stain. When this method is used, the MTB smear is fixed, stained with carbol-fuchsin (a pink dye), and decolorized with acid-alcohol. The smear is counterstained with methylene-blue or certain other dyes. Acid-fast bacilli appear pink in a contrasting background.1.4.2.3 Antituberculosis drugs

Antituberculosis drugs (ATDs) are used to treat the TB and also infections caused by nontuberculous mycobacterium. There are two types115 of ATDs; first-line and second-line drugs (a), and tuberculocidal and tuberculostatic drugs (b).1.4.2.4 Classification of ATDs(a) First-line drugs: Isoniazid, Rifampin, Ethambutol, Pyrazinamide,Streptomycin.Second-line drugs: Kanamycin, Capreomycin, Cycloserine, ThiacetazoneEthionamide, Fluoroquinolones.(b) Tuberculocidal: Isoniazid, Rifampin, Pyrazinamide, Streptomycin, Kanamycin, Capreomycin, Fluoroquinolones.Tuberculostatic: Ethambutol, Thiacetazone, Cycloserine, Ethionamide.

Chapter 1 31

1.4.2.5 Anti-mycobacterial susceptibility testsEvolution techniquesThree well-known measures of sensitivity test:

1) Minimal inhibitor concentration (MIC)2) Resistance ratio (RR)3) Proportion method

These tests are set up on solid media. (1) The minimal inhibitory concentration (MIC)

MIC is defined as the minimal concentration of the drug required to inhibit the growth of the organisms, where growth is defined as 20 colonies or more. This definition of growth is chosen so that only a small proportion (e.g. 1%) of wild strains would be classified as resistant by its use. This method is simple and be carried out with a single drug containing slope although it is preferable to use more than one slope.(2) Resistance ratio

It is the resistance as a ratio of the MIC of a test strain to that of control strain. This procedure calls for a rigid standardization since the inherent technical errors usually make it less efficient than the MIC method in distinguishing sensitive and resistant strains. A further disadvantage of the use of RR is that there may be more variation in sensitivity of the control strain than in wild strain resulting in increase in the error. However, the RRs’ are more than one slope.(3) Proportion method

This method has a high degree of precision. The inoculum suspension is standardized by weight of the bacilli and serial ten-fold dilution of the suspension are made for seeding onto drug free and drug containing slopes.1.4.3 Antioxidant activity1.4.3.1 Introduction

An antioxidant is an agent capable of slowing or preventing the oxidation of other molecules. Oxidation reactions can produce free radicals, which start chain reactions that damage cells. Antioxidants terminate these chain reactions by removing free radical intermediates, and inhibit other oxidation reactions by being oxidized themselves. As a result, antioxidants are often reducing agents

Chapter 1 32

such as thiols, ascorbic acid or polyphenols. In addition, natural antioxidants in medicine have many industrial uses, such as preservatives in food and cosmetics and preventing the degradation of rubber and gasoline.

An antioxidant is ‘‘any substance that, when present at low concentrations compared to those oxidisable substrate, significantly delays or prevents oxidation of that substrate’’.116 The term antioxidant originally was used to refer specifically to a chemical that prevents the oxygen consumption. In the late 19thand early 20th century, an extensive study was devoted to uses of these agents in important industrial processes such as the prevention of metal corrosion, the vulcanization of rubber, and the polymerization of fuels in the fouling of internal combustion engines. Early research on antioxidants in biology focused on preventing the oxidation of unsaturated fats, which cause rancidity. Antioxidant activity could be measured simply by placing the fat in a closed container with oxygen and measuring the rate of oxygen consumption. However, it was the identification test of vitamins A, C, and E as antioxidants that revolutionized the field and led to the realization of the importance of antioxidants in the biochemistry of living organisms. The possible mechanism of their action was first explored when it was recognized that a substance with anti-oxidative activity is likely to be one that is itself readily oxidized. Research into how vitamin-E prevents the process of lipid peroxidation led to the identification of antioxidants as reducing agents that prevent oxidative reactions, often by scavenging reactive oxygen species before they can damage cells.1.4.3.2 Free radicals (FRs)117

They are atomic or molecular species with unpaired electrons on anotherwise open shell configuration. These unpaired electrons are usually very unstable and highly reactive, so radicals are likely to take part in the chemical reactions. Free radicals play an important role in a number of biological processes, some of which are necessary for life, such as the intracellular killing ofbacteria by neutrophil granulocytes. FRs have also been implicated in certain cell signalling processes. The two most important oxygen-centered free radicals are superoxide and hydroxyl radical. They are derived from molecular oxygen under reducing conditions. However, because of their reactivity, these same free radicals can participate in unwanted side reactions resulting in cell damage and

Chapter 1 33

under certain conditions that can be highly toxic to the cells. Well known examples of antioxidants include superoxide (O2), hydroxyl radical (OH), peroxyl (ROO), alkoxyl (RO), hydroperoxyl (HO2) etc.1.4.3.3 Reactive oxygen species (ROS)118

ROS includes oxygen ions, free radicals and peroxides both inorganic and organic. They are generally very small molecules and are highly reactive due to the presence of unpaired valence shell electrons. ROSs forms as a natural by-product of the normal metabolism of oxygen and have important roles in cell signalling. However, during times of environmental stress ROS levels can increase dramatically, which can result in significant damage to cell structures.This cumulates into a situation known as oxidative stress. Cells are normally able to defend themselves against ROS damage through the use of enzymes such as superoxide dismutase and catalases. Small molecule antioxidants such as ascorbic acid (vitamin C), uric acid, and glutathione also play important roles as cellular antioxidants. Similarly, polyphenol antioxidants assist in preventing ROS damage by scavenging free radicals. Examples of ROS includes hydrogen peroxide, H2O2 (Fenton´s reaction); hypochlorous acid, HClO; ozone, O3; singlet oxygen, 1O2. 1.4.3.4 Clasification and general characteristic of antioxidantsAntioxidant can be clasified as (a) Enzymatic and Non-Enzymatic antioxidant(i) Non-Enzymatic antioxidant (ii) Enzymatic Antioxidant Alpha tocopherol (vitamin E) Superoxide dismutase (SOD) Beta Carotene Glutathione peroxidase enzyme Ascorbic acid (vitamin C) The catalase enzyme

Other antioxidants Alpha tocopherol (vitamin E)(b) Sources of the antioxidants

(i) Natural antioxidants (ii) Synthetic antioxidants Tocopherols Butylated Hydroxy Anisole(BHT) Nordihydroguaiaretic acid Butylated Hydroxy Toluene Sesamol Tertiary Butyl Hydroquinone Gossypol Propyl Gallate (PG)

Chapter 1 34

Characteristics of antioxidantsThe major antioxidants currently in use in foods are monohydroxy or

polyhydroxy phenols with various ring substitutions. They have low activation energy to donate hydrogen. The resulting antioxidant free radical does not initiate another free radical due to the stabilization of delocalization of radical electron. The resulting antioxidant free radical is not subject to rapid oxidation due to its stability. The antioxidant free radicals can also react with lipid free radicals to form stable complex compounds.Mechanism of antioxidantsMechanism of antioxidant involves two steps. Hydrogen donation to free radicals by antioxidants. Formation of a complex between the lipid radical and the antioxidant

radical (free radical acceptor).R

RO

ROO

+ AH

+ AH

+ AH

RH

ROH

ROOH

R

RO

ROO

RA

ROA

ROOA

+ A

+ A

+ A

Antioxidant + O2 Oxidized antioxidant

+ A

+ A

+ A

Fig 1.13 Reaction of antioxidants with radicals.Factors affecting the efficiency of antioxidant Activation energy of antioxidants to donate hydrogen should be low Oxidation potential should be high Reduction potential should be low Stability to pH and processing.

Ideal antioxidants No harmful physiological effects Not contribute an objectionable flavor, odor, or color to the fat Effective in low concentration Fat-soluble Carry-through effect No destruction during processing Readily-available

Chapter 1 35

Economical Not absorbable by the body

1.4.3.5 Methods of antioxidant assayVarious methods have been reported for measuring total antioxidants

power such as the total peroxy radical trapping parameter assay,119 the ferric reducing antioxidant power (FRAP) method,120 the phycoerythrin fluorescence-based assay,121 the enhanced chemiluminescence assay,122 oxygen radical absorbance capacity (ORAC) assay,123 trolox-equivalent antioxidant capacity (TEAC) assay,124 1,1-diphenynl-2-picryl-hydrazyl (DPPH) antioxidant assay125and ABTS+ radical scavenging activity.1261.5 Present work

Looking to the biological and technical significances of pyrrolidine-annulated heterocyclic systems, it was therefore planned to synthesize these interesting scaffolds in Part-I.

Part-I of the thesis is composed of two Chapters. Chapter-1 (present one) introduces to the chemistry of azomethine ylide, MW-assisted, solvent-free synthesis and biological screening tests. Chapter-2 deals with the synthesis and biological evaluation of pyrrolidine-incorporated heterocycles (Section-I) and their amino analogues frameworks (Section-II).

Rest of the work of the thesis has been described in Part-II, which demonstrates domino strategies for constructing pyran-annulated heterocycles.

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References 1. Michael, E.; Pichichero, M. D. Pediatrics 2005, 115, 1048. 2. Demain, A. L.; Sanchez, S. Microbial drug discovery: 80 Years of progress. J.Antibiot. 2009, 62, 5.3. Riley, J; Eisenberg, E; Müller-Schwefe, G; Drewes, AM; Arendt-Nielsen, L. Curr.Med. Res. Opin. 2008, 24, 175.4. Peck, T. E.; Hill, S. A. Williams M. Pharmacology for anaesthesia and intensive care(3rd edition). Cambridge: Cambridge university press. p. 111. 2008.5. Simon, C.; Peyronel, J. F.; Rodriguez, J. Org. Lett. 2001, 3, 2145. 6. Rossini, M.; Budriesi, R.; Bixel, M. G.; Bolognesi, M. L.; Chiarini, A.; Hucho, F.; Krogsgaard-Larsen, P.; Mellor, A.; Minarini, I. R.; Tumiatti, V.; Usherwood, P. N. R.; Melchiorre, C. J. Med. Chem. 1999, 42, 5212.7. (a) Witherup, K. M.; Ransom, R. W.; Graham, A. C.; Bernard, A. M.; Salvatore, M. J.; Lumma, W. C.; Anderson, P. S.; Pitzenberger, S. M.; Varga, S. L. J. Am. Chem. Soc.1995, 117, 6682; (b) Mahmud, H.; Lovely, C. J.; Rasika Dias, H. V. Tetrahedron2001, 57 , 4095.8. (a) Gong, Y.-D.; Najdi, S.; Olmstead, M. M.; Kurth, M. J. J. Org. Chem. 1998, 63, 3081; (b) Bashiardes, G.; Safir, I.; Mohamed, A. S.; Barbot, F.; Laduranty, J. Org. Lett. 2003, 5, 4915.9. (a) Dubuffet, T.; Newman-Tancerdi, A.; Cussac, D.; Audinot, V.; Loutz, A.; Millan, M. J.; Lavielle, G. Bioorg. Med. Chem. Lett. 1999, 9, 2059; (b) Dubuffet, T.; Muller, O.; Simonef, S. S.; Descomhes, J. -J.; Laubie, M.; Verheuren, T. J.; Lavielle, G. Bioorg. Med.Chem. Lett. 1996, 6, 349.10. Caine, B. Science 1993, 260, 1814.11. Carlson, J. Neurol. Transm. 1993, 94, 11.12. Sokoloff, P.; Giros, B.; Martres, M. P.; Bouthenet, M. L.; Schwartz, J. C. Nature 1990, 347, 147.13. Wilner, P. Clin. Neuropharm. 1985, 18, 549.14. (a)Yang, X. B.; Luo, S. J.; Fang, F.; Liu, P.; Lu, Y.; He, M. Y.; Zhai, H. B. Tetrahedron2006, 62, 2240; (b) Bolognesi, M. L.; Bartolini, M.; Cavalli, A.; Andrisano, V.; Rosini, M.; Minarini, A.; Melchiorre, C. J. Med. Chem. 2004, 47, 5945.15. Bolognesi, M. L.; Andrisano, V.; Bartolini, M.; Minarini, A.; Rosini, M.; Tumiatti, V.; Melchiorre, C. J. Med. Chem. 2001, 44, 105.16. (a) Pars, H. G.; Granchelli, F. E.; Razdan, R. K.; Keller, J. K.; Teiger, D. G.; Rosenberg,F. J.; Harris, L. S. J. Med. Chem. 1976, 19, 445; (b) Meyer, M. D,; Altenbach, R. J.; Basha, F. Z.; Carroll, W. A.; Drizin, I.; Kerwin, J. F.;Wendt, M. D. 1999, US 5891882.17. (a) Battersby, A. R.; Binks, R.; Huxtable, R. Tetrahedron Lett. 1968, 58, 6111; (b) Lundstrom, J. In The Alkaloids; A. Brossi, Ed.; Academic: New York, NY, 1983, Vol. 21, p 312; (c) Bentley, K. W. Nat. Prod. Rep. 2003, 20, 342. 18. (a) Andersen, R. J.; Faulkner, D. J.; Cun-heng, H.; Van Duyne, G. D.; Clardy, J. J. Am.Chem. Soc. 1985, 107, 5492; (b) Urban, S.; Butler, M. S.; Capon, R. J. Aust. J. Chem.1994, 47, 1919; (c) Urban, S.; Capon, R. J. Aust. J. Chem. 1996, 49, 711; (d) Ham, J.; Kang, H. Bull. Korean Chem. Soc. 2002, 23, 163; (e) Krishnaiah, P.; Reddy, V. L. N.; Venkataramana, G.; Ravinder, K.; Srinivasulu, M.; Raju, T. V.; Ravikumar, K.; Chandrasekar, D.; Ramakrishna, S.; Venkateswarlu, Y. J. Nat. Prod. 2004, 67, 1168.

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19. Quesada, A. R.; Grávalos, M. D. G.; Puentes, J. L. F. Br. J. Cancer 1996, 74, 677.20. Baunbaek, D.; Trinkler, N.; Ferandin, Y.; Lozach, O.; Ploypradith, P.; Rucirawat, S.; Ishibashi, F.; Iwao, M.; Meijer, L. Mar. Drugs 2008, 6, 514 .21. (a) Kluza, J.; Gallego, M. -A.; Loyens, A.; Beauvillain, J. -C.; Sousa - Faro, J. -M. F.; Cuevas, C.; Marchetti, P.; Bailly, C. Cancer Res. 2006, 66, 3177; (b) Ballot, C.; Kluza, J.; Martoriati, A.; Nyman, U.; Formstecher, P.; Joseph, B.; Bailly, C.; Marchetti, P. Mol.Cancer Ther. 2009, 8, 3307; (c) Ballot, C.; Kluza, J.; Lancel, S.; Martoriati, A.; Hassoun, S. M.; Mortier, L.; Vienne, J. -C.; Briand, G.; Formstecher, P.; Bailly, C.; Nevière, R.; Marchetti, P. Apoptosis 2010, 15, 769.22. Kamiyama, H.; Kubo, Y.; Sato, H.; Yamamoto, N.; Fukuda, T.; Ishibashi, F.; Iwao, M.Bioorg. Med. Chem. 2011, 19, 7541.23. Wang, R. F.; Yang, X. W.; Ma, C. M.; Cai, S. Q.; Nong, J. J.; Shoyama, Y. Heterocycles2004, 63, 1443. 24. Xiang, L.; Xing, D.; Wang, W.; Wang, R.; Ding, Y.; Du, L. Phytochemistry 2005, 66, 2595.25. Zhang, Q.; Tu, G.; Zhao, Y.; Cheng, T. Tetrahedron 2002, 58, 6795. 26. (a) Cheng, Y.; Huang, Z. -T.; Wang, M.-X. Curr. Org. Chem. 2004, 8, 325; (b) Notz, W.; Tanaka, F.; Barbas III, C. F. Acc. Chem. Res. 2004, 37, 580; (c) Felpin, F.-X.; Lebreton, J. Eur. J. Org. Chem. 2003, 3693; (d) Pearson, W. H.; Stoy, P. Synlett 2003, 903. (e) Pearson, W. H. Pure Appl. Chem. 2002, 74, 1339; (f) Pharmaceuticals, Vol. 1–4 (Ed.: McGuire, J. L.), Wiley-VCH, Weinheim, 2000.27. (a) Smith, M. B.; March, J. March's Advanced Organic Chemistry; 5 ed.; John Wiley & Sons, Inc.: New York, 2001; (b) Stanley, L. M.; Sibi, M. P. Chem. Rev. 2008, 108,2887.28. (a) Cox, A. P.; Thomas, L. F.; Sheridan, J. Nature 1958, 181, 1000; (b) Hiberty, P. C.; Leforestier, C. J. Am. Chem. Soc. 1978, 100, 2012.29. Gothelf, K. V.; Jørgensen, K. A. Chem. Rev. 1998, 98, 863.30. Padwa, A.; Pearson, W. H. Synthetic applications of 1,3-dipolar cycloaddition chemistry toward heterocycles and natural products. John Wiley & Sons, Inc. 2002.31. Takeda, T. Modern Carbonyl Olefination.Wiley-VCH Verlag GmbH & Co. KGaA,Weinheim, 2004.32. Curtius, T. Ber. Dtsch. Chem. Ges. 1883, 16, 2230.33. Buchner, E. Ber. Dtsch. Chem. Ges. 1888, 21, 2637.34. Buchner, E.; Fritsch, M.; Papendieck, A.; Witter, H. Liebigs Ann. Chem. 1893, 273, 214.35. Huisgen, R. In 1,3-Dipolar Cycloaddition Chemistry; Padwa, A., Ed.; Wiley: New York, 1984; Vol. 1, p 1.36. Huisgen, R. Angew. Chem. 1963, 75, 604.37. (a) Woodward, R. B.; Hoffmann, R. The Conservation of Orbital Symmetry; Verlag Chemie: Weinheim, 1970; (b) Woodward, R. B.; Hoffmann, R. J. Am. Chem. Soc.1965, 87, 395.38. (a) Houk, K. N.; Sims, J.; Duke, R. E.; Strozier, R. W., Jr.; George, J. K. J. Am. Chem.Soc. 1973, 95, 7287; (b) Houk, K. N.; Sims, J.; Watts, C. R.; Luskus, L. J. J. Am. Chem.Soc. 1973, 95, 7301.

Chapter 1 38

39. (a) Atkinson, R. S. Stereoselective Synthesis, Wiley and Sons: England, 1995, 173, 231; (b) Padwa, A.; Weingarten, M. D. Chem. Rev. 1996, 96, 223; (c) Muthusamy, S. Tetrahedron 2002, 58, 9477.40. Houk, K. N.; Yamaguchi, K. In 1,3-Dipolar Cycloaddition Chemistry; Padwa, A., Ed.; John Wiley & Sons, Inc: New York, 1984, Vol. 2, pp. 407. 41. Houk, K. N.; Gonzales, J.; Li, Y. Acc. Chem. Res 1995, 28, 81.42. (a) Sustmann, R. Tetrahedron Lett. 1971, 12, 2717; (b) Sustmann, R. Pure Appl.Chem. 1974, 40, 569.43. Husinec, S.; Savic, V. Tetrahedron: Asymmetry 2005, 16, 2047.44. (a) Grigg, R.; Malone, J. F.; Mongkolaussavaratana, T.; Thianpatanagul, S.Tetrahedron 1989, 45, 3849; (b) Fleury, J. P.; Schoeni, J. P.; Clerin, D.; Fritz, H. Helv.Chim. Acta 1975, 58, 2018; (c) Seidl, H.; Huisgen, R.; Knorr, R. Chem. Ber. 1969, 102, 904.45. Coldham, I.; Hufton, R. Chem. Rev. 2005, 105, 2765.46. Tsuge, O.; Kanemasa, S.; Yoshioka, M. J. Org. Chem. 1988, 53, 1384.47. (a) Huisgen, R.; Cheer, W.; Huber, H. J. Am. Chem. Soc. 1967, 89, 1753; (b) DeShong, P.; Kell, D. A. Tetrahedron Lett. 1986, 27, 3979.48. Panja, S. K.; Karmakar, P.; Chakraborty, J.; Ghosh, T.; Bandyopadhyay, C. Tetrahedron Lett. 2008, 49, 4397.49. Harwood, L. M.; Vickers, R. J. In Synthetic Applications of 1,3-Dipolar CycloadditionChemistry Toward Heterocycles and Natural Products; Padwa, A.; Pearson, W. H., Eds.; John Wiley & Sons, Inc., New York, 2002, vol 59, 170.50. Carruthers, W.; Coldhanm, I. Modern Methods of Organic Synthesis, Cambridge Universty Press, 2004, 228.51. Döndaş, A. H.; Fishwick, C. W. G.; Grigg, R.; Kilner, C. Tetrahedron 2004, 60, 3473.52. Kraus, G. A.; Nagy, J. O. Tetrahedron Lett. 1983, 24, 3427.53. Pearson, W. H.; Stoy, P.; Mi, Y. J. Org. Chem. 2004, 69, 1919.54. (a) Lown, J. W. In 1,3-Dipolar Cycloaddition Chemistry; Padwa, A., Ed.; Wiley: New York, 1984; Vol. 1, p 653; (b) Vedejs, E. In Advances in Cycloaddition; Curran, D. P., Ed.; JAI Press: Greenwich, CN, 1988; Vol. 1, p 33; (c) Grigg, R. Chem. Soc. Rev. 1987, 16, 89; (d) Kanemasa, S.; Tsuge, O. In Advances in Cycloaddition; Curran, D. P., Ed.; JAI Press: Greenwich, CN, 1993; Vol. 3, p 99; (f) Najera, C.; Sansano, J. M. Curr. Org.Chem. 2003, 7, 1105.55. Grigg, R.; Malone, J. F.; Mongkolaussavaratana, T.; Thianpatanagul, S. Tetrahedron1989, 45, 3849.56. (a) Wade, P. A. In Comprehensive Organic Synthesis; Trost, B. M., Fleming, I., Eds.; Pergamon: Oxford, U.K., 1991; Vol. 4, p 1111; (b) Namboothiri, I. N. N.; Hassner, A. Top. Curr. Chem. 2001, 216, 1.57. Purushothaman, S.; Prasanna, R.; Niranjana, P.; Raghunathan, R.; Nagaraj, S.; Rengasamy, R. Bioorg. Med. Chem. Lett. 2010, 20, 7288.58. Arumugam, N.; Raghunathan, R.; Almansour, A. I.; Karama, U. Bioorg. Med. Chem.Lett. 2012, 22, 1375. 59. Arumugam, N.; Periyasami, G.; Raghunathan, R.; Kamalraj, S.; Muthumary, J. Eur. J.Med. Chem. 2011, 46, 600.60. Mishra, A.; Rastogi, N.; Batra, S. Tetrahedron 2012, 68, 2146.

Chapter 1 39

61. Levesque, F.; Belanger, G. Org. Lett. 2008, 10, 4939.62. Llamas, T.; Arraya´s, R. G.; Carretero, J. C. Org. Lett. 2006, 8, 1795.63. Kim, I; Na, H. –K.; Kim, K. R.; Kim, S. G.; Lee, G. H. Synlett. 2008, 13, 2069.64. Wang, B.; Mertes, M. P.; Mertes, K. B.; Takusagawa, F. Tetrahedron Lett. 1990, 31, 5543.65. Confalone, P. N.; Huie, E. M. J. Am. Chem. Soc. 1984, 106, 7175.66. Confalone, P. N.; Earl, R. A. Tetrahedron Lett. 1986, 27, 2695.67. Ganesh Pandey, Prabal Banerjee, Ravindra Kumar, and Vedavati G. Puranik Org.Lett. 2005, 7, 3713.68. Banwell, M.; Flynn, B.; Hockless, D. Chem. Commun. 1997, 2259.69. Pearson, W. H.; Lovering, F. E. J. Org. Chem. 1998, 63, 3607.70. (a) István, T.; Anastas, P. T. Chem. Rev. 2007, 107, 2167; (b) Adams, D. J.; Dyson, P. J.; Tavener, S. J. Chemistry in Alternative Reaction Media, John Wiley & Sons, Chichester, UK, 2004; (c) Candeias, N. R.; Branco, L. C.; Gois, P. M. P.; Afonso, C. A. M.; Trindade, A. F. Chem. Rev. 2009, 109, 2703. 71. (a) Varma, R. S. Green Chem. 1999, 1, 43; (b) Lidstr¨om, P.; Tierney, J.; Wathey, B.; Westman, J. Tetrahedron 2001, 57, 9225.72. Bala, B. D.; Rajesh, S. M.; Perumal, S. Green Chem. 2012, 14, 2484. 73. Rajagopal, D.; Narayanan, R.; Swaminathan, S. Proc. Indian Acad. Sci. 2001, 113, 197.74. Huertas, D.; Florscher, M.; Dragojlovic, V. Green Chem. 2009, 11, 91.75. Tanaka, K.; Toda, F. Chem. Rev. 2000, 100, 1025. 76. (a) Neas, E. D.; Collins, M. J. Introduction to MW Sample Preparation Theory andPractice, Kingston, H. M.; Jassie, L. B. Eds., American Chemical Society 1988, ch. 2, 7; (b) Mingos, D. M. P.; Baghurst, D. R. MW Enhanced Chemistry Fundamentals,Sample Preparation, and Applications, Kingston, H. M.; Haswell, S. J.; Eds., American Chemical Society, 1997, ch. 1, 3.77. Giguere, R. J.; Bray, T. L.; Duncan, S. M.; Majetich, G. Tetrahedron Lett. , 1986, 27, 4945.78. Bougrin, K.; Loupy, A.; Soufiaoui, M.J. Photochem. Photobiol. C Photochem. Rev.2005, 6, 139.79. Ahluwalia, V. K.; Kidwai, M. New trends in green chemistry, 2004, 61.80. (a) Loupy, A.; Petit, A.; Hamelin, J.; Texier-Boullet, F.; Jacquault, P.; Math´e, D. Synthesis 1998, 1213; (b) Toda, F. Synlett. 1993, 303; (c) Toda, F. Acc. Chem. Res.1995, 28, 480; (d) Loupy, A. in: Knochel, P. (Ed.), Modern Solvents in Organic Synthesis, vol. 206, Springer-Verlag, Berlin, Heidelberg, 1999, pp. 154–207.81. (a) Tanaka, K. Solvent-free Organic Synthesis, Wiley-VCH, Weinheim, Germany, 2003; (b) Vidal, T.; Petit, A.; Loupy, A.; Gedye, R. N. Tetrahedron 2000, 56, 5473; (c) Perreux, L.; Loupy, A. (Eds.), Microwaves in Organic Synthesis, Wiley-VCH, Weinheim, Germany, 2002 (Chapter 3). 82. (a) Smith, K. Solid Supports and Catalysts in Organic Synthesis, Ellis Horwood, PTR Prentice Hall, Chichester, 1992; (b) Loupy, A.; Bram, G.; Sansoulet, J. New J. Chem.1992, 16, 233; (c) Clarck, J. H. Chem. Rev. 1980, 80, 1059.83. (a) Loupy, A.; Luche, J. L. Sonochemical and MW activation in phase transfer catalysis, in: Sasson, Y; Neumann, R. (Eds.), Handbook of Phase Transfer Catalysis,

Chapter 1 40

Blackie, 1996, p. 369; (b) Deshayes, S.; Liagre, M.; Loupy, A.; Luche, J. L.; Petit, A. Tetrahedron 1999, 55, 10851; (c) Bram, G.; Loupy, A. Sansoulet, J. Isr. J. Chem.1985, 26, 291.84. Gedye, R. N.; Smith, F.; Westaway, K.; Ali, H.; Baldisera, L.; Laberge, L.; Roussell, J. Tetrahedron Lett. 1986, 27, 279.85. (a) Devi, I. ; Bhuyan. P. J. Tetrahedron Lett. 2004, 45, 8625; (b) Ferro, S.; Rao, A.; Zappala, M.; Chimirri, A.; Barreca, M. L.; Witvrouw, M.; Debyser, Z.; Monforte, P.Heterocycles 2004, 63, 2727; (c) Adib, M.; Jahromi, A. H.; Tavoosi, N.; Mahdavia, M.;Bijanzadehc, H. R. Tetrahedron Lett. 2006, 47, 2965; (d) El Ashry, E. H.; Awada, L. F.; Ibrahim, E. I.; Bdeewya, O. K. Arkivoc 2006, 2, 178.86. Arrieta, A.; Carrillo, J. R.; Cossio, F. P.; Diaz-Ortiz, A.; G´omez- Escalonilla, A.; De la Hoz, A.; Langa, F.; Moreno, A. Tetrahedron 1998, 54, 13167.87. (a) Pospisil, J.; Potacek, M. Eur. J. Org. Chem. 2004, 710; (b) Pospisil, J.; Potacek, M. Tetrahedron 2007, 63, 337.88. Dinica, R. M.; Druta, I. I.; Pettinari, C. Synlett 2000, 1013.89. Katritzky, A. R.; Singh, S. K. J. Org. Chem. 2002, 67, 9077.90. Bortolini, O.; D’Agostino, M.; Nino, A. D.; Maiuolo, L.; Nardi, M.; Sindona, G. Tetrahedron 2008, 64, 8078.91. Fraga-Dubreuil, J.; Cherouvrier, J. R.; Bazureau, J. P. Green Chemistry, 2000, 2, 226.92. Taherpour, A. A.; Kheradmand, K. J. Heterocyclic Chem. 2009, 46, 131.93. Ramesh, E.; Kathiresan, M.; Raghunathan. R. Tetrahedron Lett. 2007, 48, 1835.94. Arrieta, A.; Otaegui, D.; Zubia, A.; Cossıo, F. P.; Dıaz-Ortiz, A.; Hoz, A. D. L.; Herrero, M. A.; Prieto, P.; Foces-Foces, C.; Pizarro, J. L.; Arriortua, M. I. J. Org. Chem. 2007, 72, 4313.95. Cheng, Q.; Zhang, W.; Tagami, Y.; Oritani, T. J. Chem. Soc. Perkin Trans. 2001, 1, 452.96. Jayashankaran, J.; Durga, R.; Manian, R. S.; Raghunathan, R. Tetrahedron Lett.2004, 45, 7303.97. Abdel-Aziz, H. A.; El-Zahabi, H. S. A.; Dawood, K. M. Eur. J. Med. Chem. 2010, 45, 2427.98. Bashiardes, G.; Safir, I.; Mohamed, A. S.; Barbot, F.; Laduranty, J. Org. Lett. 2003, 5, 4915.99. Ehrenberg's Symbolae physioe. Animalia evertebrata. Decas prima. Berlin, 1828. 100. Gram, H. C. Fortschr. Med. 1884, 2, 185.101. Murray, P. R.; Pfaller, M. A.; Rosenthal, K. S. Medical Microbiology. 5th Ed.; 2009.102. URL: http://www.textbookofbacteriology.net/normalflora.html (27/07/2013).103. URL: http://en.wikipedia.org/wiki/Streptococcus_pneumoniae(27/07/2013).http://www.textbookofbacteriology.net/S.pneumoniae.html (27/07/2013).104. URL: http://en.wikipedia.org/wiki/Clostridium_tetani (27/07/2013).http://www.textbookofbacteriology.net/clostridia.html (27/07/2013).105. URL: http://en.wikipedia.org/wiki/Bacillus_b vsubtilis (27/07/2013).http://www.textbookofbacteriology.net/Bacillus.html (27/07/2013).106. URL: http://en.wikipedia.org/wiki/Salmonella (27/07/2013).

Chapter 1 41

http://www.sanger.ac.uk/Projects/S_typhi (27/07/2013).http://www.textbookofbacteriology.net/salmonella.html (27/07/2013).107. URL: http://en.wikipedia.org/wiki/Vibrio_cholerae (27/07/2013).http://www.textbookofbacteriology.net/cholera.html (27/07/2013).108. URL: http://en.wikipedia.org/wiki/Escherichia_coli (27/07/2013).http://emedicine.medscape.com/article/217485-overview(27/07/2013).109. URL: http://en.wikipedia.org/wiki/Candida_albicans (27/07/2013).110. URL: http://en.wikipedia.org/wiki/Aspergillus_fumigatus (27/07/2013). http://www.aspergillus.org.uk (27/07/2013).111. National Committee for Clinical Laboratory Standards (NCCLS), 940, West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA. Performance Standardsfor Antimicrobial Susceptibility Testing; Twelfth Informational Supplement.NCCLS document (2002) M100-S12 (M7) [ISBN 1-56238-454-6].112. Cruickshank, R. Medical Microbiology; 12th Ed.; Chaurchi-Livingstone, Deinburgh, London, 1975; Vol. II.113. (a) Kumar, V; Abbas, A. K.; Fausto, N.; Mitchell, R. N. Robbins Basic Pathology; 8th Ed.; Saunders Elsevier, 2007; p 516–522; (b) Ryan, K. J.; Ray, C. G. Sherris MedicalMicrobiology; 4th Ed.; McGraw Hill, 2004.114. Cole, S. T.; Brosch, R.; Parkhill, J. Nature 1998, 393, 537.115. (a) Seth, S. D.; Seth, V. Text Book of Pharmacology; 3rd Ed.; Elsevier, 2009; p X.74; (b) Janin, Y. L.; Bioorg. Med. Chem. 2007, 15, 2479.116. Halliwell, B.; Gutteridge, J. M. C. Free Radicals Biol. Med. 1995, 18, 125.117. Halliwell, B.; Gutteridge, J. M. C. Methods in Enzymology 1990, 186, 1.118. Halliwell, B. Nutr. Rev. 1994, 53, 253.119. Wayner, D. D. M.; Burton, G. W.; Ingold, K. U. FEBC Lett. 1985, 187, 33.120. Benzie, I. F. F.; Strain, J. J. Anal. Biochem. 1996, 239, 70.121. Glazer, A. N. Methods Enzymol. 1990, 186, 161.122. Lewis, S. E.; Boyle, P. M. Fertil. Steril. 1995, 64, 868.123. Cao, G.; Prior, R. L. Clin. Chem. 1998, 44, 1309.124. Miller, N. J.; Davies, M. J. Milner, A. Clin. Sci. 1993, 84, 407.125. Mensor, L.L.; Fabio, S. M.; Alexandre, S. R. Phytother. Res. 2001, 15, 127.126. Re, R.; Pellegrini, N.; Pannala, A. Free Radical Biol. Med. 1999, 26, 1231.