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Pharmaceutical chemistry is a branch which acts as intersection of pharmacology and chemistry. Medicinal chemistry deals with designing, synthesizing and developing pharmaceutical drugs. It involves the identification, synthesis and development of new chemical entities suitable for therapeutic use. It is also includes the study of existing drugs, their biological properties, and their quantitative structure activity relationships. Pharmaceutical chemistry is focused on quality aspects of medicines and aims to assure fitness for the purpose of medicinal products.1
In the early stages of development of medicinal chemistry, scientists were primarily concerned with the isolation of medicinal agents found in plants. But recently scientists are also equally concerned with the creation of new synthetic compounds as drugs.2 The novel approach of medicinal chemistry for practice has developed from an empirical one involving organic synthesis of new compounds, based largely on modification of structures of known activity, by a more logical approach. Now computers have been pressed into the service of medicinal chemists. Computers augment the scientific process in drug discovery by assisting the chemist with collecting, storing, manipulating, analyzing and viewing the data. Further, computers provide a link to theoretical chemistry and graphic modeling, providing calculated estimates of molecular properties, models of molecules, models of biological sites and sometimes even models of drug-receptor interactions.3
CoumarinÂ (2H-chromen-2-one, 1) is a fragrantÂ chemical compoundÂ found in manyÂ plants, notably in high concentration in thetonka beanÂ (Dipteryx odorata), vanilla grass (Anthoxanthum odoratum),Â sweet woodruff (Galium odoratum),Â mulleinÂ (VerbascumÂ spp.),Â sweet grassÂ (Hierochloe odorata),Â Panicum ClandestinumÂ or 'Deers Tongue' andÂ sweet cloverÂ (FabaceaeÂ spp.). Many natural coumarins have been assigned trivial names mostly botanically derived. The main advantage of trivial names is their ease of use when contrasted with rational nomenclature. Thus coumarin itself systematically named as 2H-benzopyran-2-one. The name comes from a French word, coumarou, for theÂ tonka bean. It has a sweet odor, readily recognised as the scent of newly-mownÂ hay, and has been used in perfumes since 1882.
Coumarins are widely distributed in natural products isolated mainly from Umbelliferae, Rutaceae and Leguminosae plants. To date more than 1000 coumarin derivatives have been described. Several compounds isolated from microorganisms or animals (Novobiocin, aflotoxin) are also coumarin derivatives. Interestingly microorganisms usually produce isocoumarin derivatives.
Although coumarin itself has noÂ anticoagulantÂ properties, it is transformed into the natural anticoagulantÂ dicoumarolÂ by a number of species of fungi. This occurs as the result of the production ofÂ 4-hydroxycoumarin, then further (in the presence of naturally occurring formaldehyde) into the actual anticoagulantÂ dicoumarol, a fermentation product and mycotoxin. This substance was responsible for the bleeding disease known historically as "sweet clover disease" in cattle eating moldy sweet cloverÂ silage.4
Coumarin is used in the pharmaceutical industry as a precursor molecule in the synthesis of a number of syntheticÂ anticoagulantÂ pharmaceuticals similar to dicoumarol, the notable ones beingÂ warfarinÂ (trade named "Coumadin") and some even more potent rodenticides that work by the same anticoagulant mechanism. Coumarin has clinical medical value by itself, as anÂ edemaÂ modifier. Coumarin and other benzopyrones, such as 5,6 benzopyrone, 1,2 benzopyrone,Â diosmin, and others, are known to stimulate macrophages to degrade extracellular albumen, allowing faster resorption of edematous fluids.5 Other biological activities that may lead to other medical uses have been suggested, with varying degrees of evidence.
Coumarins form an elite class of compounds, which occupy a special role in nature. Pharmacologically, coumarins are flavonoids along with a range of other compounds. Coumarins and their derivatives have been found to exhibit a variety of biological and pharmacological activities and have raised considerable interest because of their potential beneficial effects on human health.
Natural, semisynthetic and synthetic coumarins possess a prominent place in drug research. Their utility stimulated the development of new synthetic routes for the preparation of coumarin derivatives. Some of these compounds can be utilized as useful intermediates for the synthesis of valuable heterocyclic ring systems. Among others, the 3-cinnamoyl coumarins synthesized by the reaction of 3-acetyl coumarins with aromatic aldehydes proved to be especially important.6 3-cinnamoyl coumarins have been used for synthesis of pyridines,7 isoxazolines,8 1,5-benzodiazepines,9 1,5-benzothiazepines10 and other nitrogen-containing heterocyclic compounds. Moreover, coumarins have acquired a special place in heterocyclic field because of their diversified activities such as antianxiety,9 anticonvulsant,11 anti-inflammatory,12 antioxidant,13 vasorelaxant,14 cytotoxic,15 anti-HIV,16 antitubercular17 and antimicrobial.18 As a result, coumarins and their derivatives have been the subject of extensive investigations.
Fig. 1: Structure of Phenprocoumon, Warfarin and Novobiocin
In addition, many coumarin derivatives have special ability to scavenge ROS and to influence processes involving free radical injury.19 The 4-hydroxycoumarin moiety, widely spread among coumarin natural products, has been the molecular template for the synthesis of a variety of analogues exhibiting important biological activity. Characteristic examples include warfarin, a synthetic compound used as a rodenticide and anticoagulant and phenprocoumon with antiviral and anti-HIV activities (Fig. 1).20 In addition, aminocoumarin analogues such as novobiocin, chlorobiocin, coumermycin and simocyclinone, which contain an amide bond at position 3 of the heterocyclic ring, are potent antibiotics.21,22
The potential of 3-acetylcoumarin derivatives as anti-inflammatory, antioxidant and antibacterial agents, prompted us to design and synthesize a series of novel coumarin analogues bearing the 3-pyrazole, 3-pyrazoline and 3-pyran functionality. Analogous pyrazole and pyrazoline based on the structure of 3-acetylcoumarin have been recently synthesized and led to compounds with combined anti-inflammatory, antioxidant and antibacterial activities.
The term pyrazole was given to this class of compounds by German ChemistÂ Ludwig KnorrÂ in 1883. Pyrazole (1H-pyrazole, 2)Â refers both to the class ofÂ simple aromatic ringÂ organic compoundsÂ of the heterocyclicÂ diazoleÂ series characterized by a 5-memberedÂ ring structureÂ composed of threeÂ carbonÂ atoms and twoÂ nitrogenÂ atoms in adjacent positions, and to the unsubstituted parent compound. Being so composed and having pharmacological effects on humans, they are classified asÂ alkaloids, although they are rare in nature.23Â In 1959, the first natural pyrazole, 1-pyrazolylalanine, was isolated from seeds ofÂ watermelons.
Pyrazole derivatives have a long history of application in agrochemicals and pharmaceutical industry as herbicides and active pharmaceuticals. The recent success of pyrazole COX-2 inhibitor has further highlighted the importance of these heterocyclic rings in medicinal chemistry. A systematic investigation of this class of heterocyclic lead revealed that pyrazole containing pharmacoactive agents play important role in medicinal chemistry. The prevalence of pyrazole cores in biologically active molecules has stimulated the need for elegant and efficient ways to make these heterocyclic lead. 23
Pyrazoles are produced synthetically through the reaction of Î±,Î²-unsaturated aldehydes/ketones with hydrazine and subsequent dehydrogenation. Fischer and Knovenagel published in the late nineteenth century, the reaction of Î±,Î²- unsaturated aldehydes/ketones with hydrazines is well method for the synthesis of pyrazole and 2-pyrazolines.24
The study of 2-pyrazoline(4,5-dihydro-1H-pyrazole, 3) derivatives has been a developing field within the realm of heterocyclic chemistry for the past several decades because of their ready accessibility through synthesis, wide range of chemical reactivity and broad spectrum of biological activity.
Chalcones and their derivatives is an attractive molecular scaffold for the search of new biologically active molecules. Studies reveal that incorporation of pyrazole moiety into various heterocyclic ring systems gives worthwhile molecules from the biological point of view.25-27 Several pharmaceutical drugs including celecoxib28 and rimonabant29 utilize the pyrazole as their core molecular entity.30,31 Many pyrazole derivatives are reported to have a broad spectrum of biological activities, such as anti-inflammatory,32 antifungal,33 herbicidal,34 antiviral,35 A3 adenosine receptor antagonists,36 etc.
Among the various derivatives of chalcones, synthesis of pyrazolines has engrossed substantial attention from organic37,38 and medicinal chemists because of their pronounced biological activities such as tranquillizing, muscle relaxant, psychoanaleptic, antidepressant, antimicrobial, etc.39-46 Keeping in view the potential biological activities of pyrazoles and pyrazolines, it was perceived that the synergistic effect of heterocyclic moieties in single nucleus might result in the formation of some worthwhile molecules from the biological point of view.
InÂ chemistry,Â pyran, orÂ oxine, is a six-memberedÂ heterocyclic, non-aromatic ring, consisting of fiveÂ carbonÂ atoms and oneÂ oxygenÂ atom and containing twoÂ double bonds. TheÂ molecular formulaÂ is C5H6O. There are twoÂ isomersÂ of pyran that differ by the location of the double bonds. InÂ 2H-pyran, the saturated carbon is at position 2, whereas, inÂ 4H-pyran, the saturated carbon is at position 4.
4H-Pyran was first isolated and characterized in 1962 viaÂ pyrolysisÂ of 2-acetoxy-3,4-dihydro-2H-pyran. It was found too unstable, particularly in the presence of air. 4H-pyran easily disproportionate to the correspondingÂ dihydropyranÂ and theÂ pyryliumÂ ion, which is easily hydrolyzed in aqueous medium. Although the pyrans themselves have little significance in chemistry, many of their derivatives are important biological molecules, such as theÂ pyranoflavonoids. The term pyran is also often applied to the saturated ring analog, which is more properly referred to asÂ tetrahydropyranÂ (oxane). In this context, theÂ monosaccharidesÂ containing a six-membered ring system are known asÂ pyranoses.47
4H-Pyran is an important and common structural unit both in natural compounds and synthetic heterocyclic molecules.48,49 In recent years, polyfunctionalized 4H-pyran and its derivatives are of great synthetic interest and have been widely recognized as versatile scaffolds with diverse biological activities.50,51 These compounds showed wide pharmacological activities and some of them emerged as anti-coagulants, anti-anaphylactics, and anticancer agents.52-54 In addition, they present a potential activity on the treatment of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.55
2-Amino-4H-pyran (4) is an important scaffold of many natural products and bioactive compounds. Several methods are available for the synthesis of 4H-pyrans especially 2-amino-4H-pyrans and 2-amino-3-cyano-4H-pyrans (5) involving the bicomponent condensation of Î±-cyanocinnamonitriles with dimedone or Î²-naphthol, three-component reaction of aldehydes, malononitrile or ethyl cyanoacetate with dimedone, ethylacetoacetate, 4-hydroxycoumarin, 4-hydroxy-6-methylpyrone and active phenols. The other synthetic method for preparation of 2-amino-4H-pyran derivatives was achieved by the Michael addition-cyclization reaction of Î±, Î²-unsaturated carbonyl compound with malononitrile.56
Pyrans and fused pyrans are biologically interesting compounds with antibacterial activities,57,58 antifungal activities,59 antitumor activity60 and hypotensive effect.61 On the other hand, some pyran derivatives also have various biological properties like antiproliferation effect,62 molluscicidal activities,63 local anesthetic and antiarrhythmic activities,64 antiallergic effect65,66 and hypolipidemic activity.67
1.5 DOCKING (Molecular)
In the field ofÂ molecular modeling,Â dockingÂ is a method which predicts the preferred orientation of one molecule to a second whenÂ boundÂ to each other to form a stableÂ complex.68Â Knowledge of the preferred orientation in turn may be used to predict the strength of association orÂ binding affinity between two molecules using for exampleÂ scoring functions.
The associations between biologically relevant molecules such as proteins,Â nucleic acids,Â carbohydrates, andÂ lipidsÂ play a central role in signal transduction. Furthermore, the relative orientation of the two interacting partners may affect the type of signal produced (e.g.,Â agonism vsÂ antagonism). Therefore docking is useful for predicting both the strength and type of signal produced.
Docking is frequently used to predict the binding orientation ofÂ small moleculeÂ drugÂ candidates to their protein targets in order to in turn predict the affinity and activity of the small molecule. Hence docking plays an important role in theÂ rational design of drugs.69Â Given the biological and pharmaceuticalÂ significance of molecular docking, considerable efforts have been directed towards improving the methods used to predict docking.
1.5.1 Definition of problem
Molecular docking can be thought of as a problem ofÂ "lock-and-key", where one is interested in finding the correct relative orientation of the "key"Â which will open up theÂ "lock"Â (where on the surface of the lock is the key hole, which direction to turn the key after it is inserted, etc.). Here, the protein can be thought of as the "lock" and the ligand can be thought of as a "key". Molecular docking may be defined as an optimization problem, which would describe the "best-fit" orientation of a ligand that binds to a particular protein of interest. However, since both the ligand and the protein are flexible, aÂ "hand-in-glove"Â analogy is more appropriate thanÂ "lock-and-key".70Â During the course of the process, the ligand and the protein adjust their conformation to achieve an overall "best-fit" and this kind of conformational adjustments resulting in the overall binding is referred to asÂ "induced-fit".71
The focus of molecular docking is to computationally simulate theÂ molecular recognitionÂ process. The aim of molecular docking is to achieve an optimized conformation for both the protein and ligand and relative orientation between protein and ligand such that theÂ free energyÂ of the overall system is minimized.
1.5.2 Docking approaches
Two approaches are particularly popular within the molecular docking community. One approach uses a matching technique that describes the protein and the ligand as complementary surfaces.72-74Â The second approach simulates the actual docking process in which the ligand-protein pairwise interaction energies are calculated.75Â Both approaches have significant advantages as well as some limitations. These are outlined below.
D:\Documents and Settings\Administrator\Desktop\Docking.gif
Fig. 2: The docking of aÂ small molecule ligandÂ (brown) to aÂ proteinÂ receptorÂ (green) to produce a complex.
Fig. 3: Small moleculeÂ docked to aÂ protein.
220.127.116.11 Shape complementarity
Geometric matching/ shape complementarity methods describe the protein and ligand as a set of features that make them dockable.76 These features may includeÂ molecular surfaceÂ /Â complementary surfaceÂ descriptors. In this case, the receptor's molecular surface is described in terms of itsÂ solvent-accessible surface areaÂ and the ligand's molecular surface is described in terms of its matching surface description. The complementarity between the two surfaces amounts to the shape matching description that may help finding the complementary pose of docking the target and the ligand molecules. Another approach is to describe the hydrophobic features of the protein using turns in the main-chain atoms. Yet another approach is to use a Fourier shape descriptor technique.77-79 Whereas the shape complementarity based approaches are typically fast and robust, they cannot usually model the movements or dynamic changes in the ligand/ protein conformations accurately, although recent developments allow these methods to investigate ligand flexibility. Shape complementarity methods can quickly scan through several thousand ligands in a matter of seconds and actually figure out whether they can bind at the protein's active site, and are usually scalable to even protein-protein interactions. They are also much more amenable to pharmacophore based approaches, since they use geometric descriptions of the ligands to find optimal binding.
The simulation of the docking process as such is a much more complicated process. In this approach, the protein and the ligand are separated by some physical distance, and the ligand finds its position into the protein's active site after a certain number of "moves" in its conformational space. The moves incorporate rigid body transformations such as translations and rotations, as well as internal changes to the ligand's structure including torsion angle rotations. Each of these moves in the conformation space of the ligand induces a total energetic cost of the system, and hence after every move the total energy of the system is calculated. The obvious advantage of the method is that it is more amenable to incorporate ligand flexibility into its modeling whereas shape complementarity techniques have to use some ingenious methods to incorporate flexibility in ligands. Another advantage is that the process is physically closer to what happens in reality, when the protein and ligand approach each other after molecular recognition. A clear disadvantage of this technique is that it takes longer time to evaluate the optimal pose of binding since they have to explore a rather large energy landscape. However grid-based techniques as well as fast optimization methods have significantly ameliorated these problems.
1.5.3 Mechanics of docking
To perform a docking screen, the first requirement is a structure of the protein of interest. Usually the structure has been determined using a biophysical technique such asÂ x-ray crystallography, or less often,Â NMR spectroscopy. This protein structure and a database of potential ligands serve as inputs to a docking program. The success of a docking program depends on two components: theÂ search algorithmÂ and theÂ scoring function.
18.104.22.168 Search algorithm
TheÂ search space [disambiguation needed]Â in theory consists of all possible orientations andÂ conformationsÂ of the protein paired with the ligand. However in practice with current computational resources, it is impossible to exhaustively explore the search space this would involve enumerating all possible distortions of each molecule (molecules are dynamic and exist in an ensemble of conformational states) and all possibleÂ rotationalÂ and translational orientations of the ligand relative to the protein at a given level ofÂ granularity. Most docking programs in use account for a flexible ligand, and several attempt to model a flexible protein receptor. Each "snapshot" of the pair is referred to as aÂ pose. A variety of conformational search strategies have been applied to the ligand and to the receptor. These include:
systematic orÂ stochasticÂ torsionalÂ searches about rotatable bonds
molecular dynamicsÂ simulations
genetic algorithmsÂ to "evolve" new low energy conformations
22.214.171.124.1 Ligand flexibility
Conformations of the ligand may be generated in the absence of the receptor and subsequently docked80Â or conformations may be generated on-the-fly in the presence of the receptor binding cavity,81Â or with full rotational flexibility of every dihedral angle using fragment based docking.82Â Force fieldÂ energy evaluation are most often used to select energetically reasonable conformations,83Â but knowledge-based methods have also been used.84
126.96.36.199.2 Receptor flexibility
Computational capacity has increased dramatically over the last decade making possible the use of more sophisticated and computationally intensive methods in computer-assisted drug design. However, dealing with receptor flexibility in docking methodologies is still a thorny issue. The main reason behind this difficulty is the large number of degrees of freedom that have to be considered in this kind of calculations. Neglecting it, however, leads to poor docking results in terms of binding pose prediction.85
Multiple static structures experimentally determined for the same protein in different conformations are often used to emulate receptor flexibility.86 AlternativelyÂ rotamer librariesÂ of amino acid side chains that surround the binding cavity may be searched to generate alternate but energetically reasonable protein conformations.87,88
188.8.131.52 Scoring function
The scoring function takes a pose as input and returns a number indicating the likelihood that the pose represents a favorable binding interaction.
Most scoring functions are physics-basedÂ molecular mechanicsÂ force fieldsÂ that estimate the energy of the pose; a low (negative) energy indicates a stable system and thus a likely binding interaction. An alternative approach is to derive a statistical potential for interactions from a large database of protein-ligand complexes, such as theÂ Protein Data Bank, and evaluate the fit of the pose according to this inferred potential.
There are a large number of structures fromÂ X-ray crystallographyÂ for complexes between proteins and high affinity ligands, but comparatively fewer for low affinity ligands as the later complexes tend to be less stable and therefore more difficult to crystallize. Scoring functions trained with this data can dock high affinity ligands correctly, but they will also give plausible docked conformations for ligands that do not bind. This gives a large number ofÂ false positiveÂ hits, i.e., ligands predicted to bind to the protein that actually doesn't when placed together in a test tube.
One way to reduce the number of false positives is to recalculate the energy of the top scoring poses using (potentially) more accurate but computationally more intensive techniques such asÂ Generalized BornÂ orÂ Poisson-BoltzmannÂ methods.
A binding interaction between aÂ small moleculeÂ ligand and anÂ enzymeÂ protein may result in activation orÂ inhibitionÂ of the enzyme. If the protein is a receptor, ligand binding may result inÂ agonismÂ orÂ antagonism. Docking is most commonly used in the field ofÂ drug designÂ most drugs are smallÂ organicÂ molecules, and docking may be applied to:
Hit identification- docking combined with aÂ scoring functionÂ can be used to quickly screen large databases of potential drugsÂ in silicoÂ to identify molecules that are likely to bind to protein target of interest.
Lead optimization- docking can be used to predict in where and in which relative orientation a ligand binds to a protein (also referred to as the binding mode or pose). This information may in turn be used to design more potent and selective analogs.
BioremediationÂ - Protein ligand docking can also be used to predict pollutants that can be degraded by enzymes.89
Inflammation is a complex stereotypical reaction of the body expressing the response to damage of its cells and vascularized tissues and it has two main components - cellular and humoral. The cellular component involves the movement of leukocytes from blood vessels into the inflamed tissue. Various types of leukocytes are involved in the initiation and maintenance of inflammation. The discovery of the detailed processes of inflammation has revealed a close relationship between inflammation and the immune response. Once leukocytes have arrived at a site of infection or inflammation, they release mediators which control the later accumulation and activation of other cells. There are four major plasma enzyme systems which have an important role in haemostasis and control of inflammation. These are the complement system, the clotting system, the fibrinolytic (plasmin) system and the kinin system. Inflammatory mediators are soluble, diffusible molecules that act locally at the site of tissue damage and infection, and at more distant sites. The major constituents of cell membranes are phospholipids. Cellular phospholipases, especially phospholipase A2 and C, are activated during inflammation and degrade phospholipids to arachidonic acid. Virtually all cellular arachidonic acid is esterified in membrane phospholipids where its presence is tightly regulated through multiple interconnected pathways. Free arachidonic acid is a transient, critical substrate for the bio-synthesis of eicosanoid second messengers. Receptor-stimulated release, metabolism, and re-uptake of free arachidonate are all important aspects of cell signalling and inflammation. Acute inflammation is mediated by granulocytes or polymorphonuclear leucocytes, while chronic inflammation is mediated by mononuclear cells such as monocytes and macrophages which can be further stimulated to maintain inflammation through the action of an adaptive cascade involving lymphocytes (T cells and B cells) and antibodies.90,91 Two types of free radicals are produced by neutrophils, macrophages, endothelial and other cells. The first type is represented by reactive oxygen intermediates which are formed in neutrophils by the activity of NADPH oxidase, the enzyme of the respiratory burst. The second type includes reactive nitrogen intermediates. The first member of them, nitric oxide, is produced by nitric oxide synthase.
The enzymatic oxidation of arachidonic acid has been shown to yield potent pathological agents by two major pathways. Those of the prostaglandin (PG) pathway, particularly PGE2, have been implicated as inflammatory mediators for many years. The discovery and biological activities of thromboxane A2 and prostacyclin as well as a destructive oxygen-centered radical as additional products of this bio-synthetic pathway now require these to be considered as potential inflammatory mediators. Like PGE2, their biosynthesis is prevented by nonsteroidal anti-inflammatory agents (NSAIDs). More recently, the alternative metabolic route, the lipoxygenase pathway, has been shown to yield a new class of arachidonic acid oxygenation products, the leukotrienes, which also appear to be important inflammatory mediators. Unlike the prostaglandins, some of which play important roles as biological regulators, the actions of the lipoxygenase products appear to be exclusively of a pathological nature.92,93
Cyclooxygenase (COX), the rate limiting enzyme of the prostanoid biosynthetic pathway catalyzes the conversion of arachidonic acid to important inflammatory mediators such as prostaglandins (PGs), prostacyclins and thromboxanes.94 The existence of enzyme cyclooxygenase in its two distinct isoforms and thus non-selective action of classical non-steroidal anti-inflammatory drugs (NSAIDs) results in certain mechanism based side effects including dyspepsia, gastrointestinal ulcerations, bleeding and nephrotoxicity.95 Both the isoforms differ in their regulation and expression. The constitutive COX-1 is responsible for the biosynthesis of PGs, which involves the cytoprotection of gastrointestinal tract and platelet aggregation.96 COX-2 is induced by pro-inflammatory molecules such as interleukin-1 (IL-1), tumor necrosis factor-a (TNF-a), lipopolysaccharide (LPS), carrageenan, etc. that leads to inflammation.97 COX-2 levels are undetectable in most tissues under normal physiological conditions, but are significantly elevated in acute and chronic inflammations. Inhibition of both isoforms by classical NSAIDs with preferential binding affinity for enzyme COX-1 causes serious side effects. The association of COX-2 with induced inflammation has led to the hypothesis that selective inhibition of COX-2 over COX-1 might provide good anti-inflammatory agents. Therefore, development of novel compounds having anti-inflammatory activity with an improved safety profile is still a necessity. In addition, inflammation is known not only as a symptom of great deal of common diseases but also as an early phase of some life-threatening diseases such as cancer, heart vascular diseases and Alzheimer's dementia. Thus the discovery of novel anti-inflammatory agents has been attracting a lot of interests.
1.6.1 Non-steroidal Anti-inflammatory drugs (NSAIDs)
Nonsteroidal anti-inflammatory drugs (NSAIDs) have been recognized as important class of therapeutic agents for the alleviation of pain and inflammation associated with a number of pathological conditions. However, long term use of NSAIDs has been associated with several side effects such as gastrointestinal mucosal damage, bleeding, intolerance and renal toxicity.98-101 Consequently, extensive research has been directed towards improving their pharmacological profile that led to the discovery of multiple isoforms of cyclooxygenase (COX) that are differently regulated.102,103 The discovery of the inducible isoform of cyclooxygenase enzyme (COX-2) spurred the search for anti-inflammatory agents devoid of the undesirable effects associated with classical NSAIDs. Recently, a novel class of selective COX-2 inhibitors has been discovered.
Among this class, celecoxib (6) was shown to be a potent and gastrointestinal (GI) safe anti-inflammatory agent. It is considered a typical model of pyrazole containing, diaryl-heterocyclic template that is known to selectively inhibit COX-2.104 Furthermore, several other compounds containing pyrazole functionality were also reported to exhibit anti-inflammatory activity.
1.7 Antioxidant activity
The knowledge of free radicals and reactive oxygen species is producing a revolution in the field of medicine that promises a new age of health and disease management. The formation and activity of a number of compounds, known as reactive oxygen species, which have a tendency to donate oxygen to other substances are producing various potential harmful effects. Evidences show that free radical damage contributes to the etiology of many chronic health problems such as cardiovascular and inflammatory disease, cataract and cancer. Antioxidants can prevent free radical induced tissue damage by preventing the formation of radicals, scavenging them, or by promoting their decomposition.
In the recent years, antioxidant research has expanded dramatically due to its potential benefit in disease prevention and health promotion. Many research models have been established for the studies of mechanisms of action of antioxidants as well as identification of new antioxidants especially from natural sources. Most of the natural antioxidants come from fruits, vegetables, spices, grains and herbs which contain a wide variety of antioxidant compounds that may help to protect cellular damages from oxidative stress and also lower the risk of chronic diseases.105
The antioxidant molecules may directly react with the reactive radicals and destroy them, while they may become new free radicals which are less active, longer-lived and less dangerous than those radicals they have neutralized. Small molecules, such as vitamin C, vitamin E, uric acid and glutathione play important roles as cellular antioxidants. Many synthetic antioxidants have been widely used in the food industry to retard lipid oxidation. However, lots of them are not preferred for pharmacological use due to toxicological concerns. Thus, more and more interest has focused on identifying plant products and their synthetic analogs to use as dietary antioxidant supplements.
Antioxidants are the compounds that prevent oxidative damage induced by free radicals and Reactive Oxygen Species (ROS). The association of antioxidants with inflammation stems from the recognition that free radicals are produced during the inflammatory process by macrophages. It has been reported that ROS are involved in the cyclooxygenase- and lipoxygenase-mediated conversion of arachidonic acid into proinflammatory intermediates. On this basis, several natural and synthetic antioxidants have been tested and shown to possess anti-inflammatory properties.106,107
1.8 Bacterial infection
Pathogenic (disease-causing) microorganisms have repeatedly altered the course of human history. From the earliest examples of art, literature and scientific writing, the devastating consequences for the populations gripped by diseases of different kinds and severity have been documented in great detail. For example, the influenza pandemic between 1918 and 1920 resulted in an estimated 70 million deaths worldwide. Even today, the overall burden of infectious disease remains high. In 2011, infectious diseases accounted for an estimated 26% of deaths worldwide.
One of the more significant achievements of the 20th century has been the discovery and commercial development of the numerous therapeutic agents that now provide reliably effective treatments for many infectious diseases that have previously caused extensive mortality, morbidity and fear. Medicinal chemist has played an important role in discovering and synthesizing antibacterials. The remarkable success in this endevor is that this century may be regarded as the "Antimicrobial Era", although concerns about the increasing prevalence of antibiotic resistant pathogens have suggested that we may already enter a "Post Antimicrobial Era".
Modern medicine is dependent on chemotherapeutic agents, chemical agents that are used to treat disease. Chemotherapeutic agents destroy pathogenic microorganisms or inhibit their growth at concentrations low enough to avoid undesirable damage to the host. Most of these agents are antibiotics, microbial products or their derivatives that can kill susceptible microorganisms or inhibit their growth. Drugs such as the sulfonamides are sometimes called antibiotics although they are synthetic chemotherapeutic agents, not microbially synthesized.
Antibiotics are chemical substances excreted by some microorganism which inhibit the growth and development of other microbes. Some of these drugs that were obtained naturally were put to chemical modifications in attempts to enhance beneficial effects while minimizing the toxic effects. The resultant modified product is termed as semi synthetic antibiotics. Most antibiotic currently used are semi synthetic. The chemist has synthesized many drugs that have got the antibacterial property and less toxicity. These drugs are called synthetic antibiotic drugs. Naturally occurring antibiotic, their semisynthetic derivatives and synthetic antibiotics have got the same target. i.e. antimicrobial action. Hence all these drugs were put together to be called antimicrobial agents.
In spite of available potent antimicrobials for management of infections, development of bacterial resistance to existing drugs is a major concern in antibacterial therapy and necessitates continuing research for new classes of antibacterial. One of the main driving forces for development and spread of resistance is high antibiotic consumption, reflected in resistance rates directly correlating with the prescription of antimicrobial drugs.
Of particular concern are severe infections caused by multi drug-resistant Gram-positive pathogens, which result in high mortality rates especially in the hospital settings. The individual organisms responsible include Staphylococcus aureus and penicillin-resistant Streptococcus pneumoniae. There are several antibiotics effective against these organisms but these organisms easily acquire resistance against them. Resistant development is inherent in the mode of action of all antibiotics and hence posses challenges in the development of new antimicrobial agents to overcome it. Some bacterial strains have been found to be resistant to all currently available antibacterial agents.108
The antibiotics prescribed are also having the different adverse effects, such as; sulphonamides, fluroquinolones and Î²-lactum antibiotics are allergic to some individuals, cephalosporin and polyenes antibiotics share common nephrotoxicity, aminoglycoside antibiotics are ototoxicity, antitubercular drugs are progressive liver damage and antiviral drugs largely in response to human immunodeficiency viruses (HIV) infection are hematologic toxicity.109 Therefore it is imperative to design and develop new antimicrobial agents with novel chemical structures possibly having modes of action rather than analogues of the existing ones.
It has been reported that scavenging of ROS might be beneficial in reducing the development of inflammation. Multi-drug treatment of inflammatory conditions associated with bacterial infections poses a unique problem especially for patients with impaired liver or kidney functions. Hence, mono therapy with a drug having anti-inflammatory, antioxidant and antibacterial activities is highly desirable, both from the pharmaco-economic and patient compliance points of view, which has been the goal of our ongoing research program.
In drug-designing programmes, an essential component of the search for new leads is the synthesis of molecules, which is novel yet resembles known biologically active molecules by virtue of the presence of critical structural features. Certain small heterocyclic molecules act as highly functionalized scaffolds and are known pharmacophore of a number of biologically active and medicinally useful molecules
Widespread interest in the chemistry of coumarins in a large number of natural and synthetic products has attracted the interest due to their biological activities and their potential applications as pharmacological agents. Several coumarin ring systems bearing various substituents at the C-3 position are widely distributed in nature. Furthermore, most of compounds prepared from 3-acetyl coumarin have anti-microbial, anti-inflammatory and antioxidant activities. The heterocyclic systems encompassing pyrazoles, pyrazoline and 4H-pyran are explored to the maximum extent owing to their wide spectrum of pharmacological activities. Special attention is warranted towards the synthetic design and development of pyrazoles, pyrazoline and 4H-pyran because of their high demand in academic and pharmaceutical sectors in the synthesis of biologically active heterocycles. The combination of the distinct pharmacophores of two different biologically active compounds in the same structure has been previously reported in literature and is highly likely to lead to hybrid compounds with significant activity.
Keeping in view of this, we hereby report the synthesis of novel, coumarin containing pyrazole, pyrazoline and 4H-pyran nucleus. These compounds were evaluated for their anti-inflammatory, antioxidant and antibacterial activities.
1.9 Rationale for synthesis
Rationale for synthesizing substituted pyrazole (7), pyrazoline (8) and pyran (9) derivatives from 3-acetylcoumarin as anti-inflammatory, antioxidant and antibacterial agents.
The title compounds were synthesized as anti-inflammatory, antioxidant and antibacterial agents because number of compounds with similar structures was reported to possess anti-inflammatory, antioxidant and antibacterial activities.
3-[3-(substituted phenyl)-1-isonicotinoyl-1H-pyrazol-5-yl]-2H-chromen-2-one (10) have been synthesized from 3-acetylcoumarin and shown encouraging in-vitro antibacterial activity against Gram-positive and Gram-negative bacteria.110
5-(substituted)aryl-3-(3-coumarinyl)-1-phenyl-2-pyrazolines (11) have synthesized from 3-aryl-1-(3-coumarinyl)propan-1-ones and exhibited significant anti-inflammatory activity. 111
2-amino-5-oxo-4-phenyl-5,10b-dihydropyrano[3,4-c]-chromene-1-carbonitrile (12) reported as antibacterial agent. Compound 12 is 2-amino-4H-pyran derivatives.112
In present study substituted coumarinyl-pyrazole (7), coumarinyl-pyrazoline (8) and coumarinyl-pyran (9) were synthesized. The structures (7, 8, 9) incorporates many structural features, which associated with anti-inflammatory, antioxidant and antibacterial activities. As physiochemical properties influence the biological activity; to a great extent; various substituted analogs or derivatives were synthesized.
These compounds were tested for acute toxicity and acute anti-inflammatory activity. Further the compounds which showed promising acute anti-inflammatory activity were also tested for chronic anti-inflammatory and ulcerogenic activities. It has been reported that scavenging of ROS might be beneficial in reducing the development of inflammation. In order to determine the extent of scavenging effect, all compounds were tested for antioxidant activity using 1,1-diphenyl-2-picryl hydrazyl (DPPH) free radical and Hydrogen peroxide-scavenging activity.
Further these compounds were tested for antibacterial activity against gram positive and gram negative bacteria by zone of inhibition using cup-plate-agar-diffusion method and minimum inhibition concentration was determine by conventional agar-dilution method.