chapter-1 1. introduction - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/32217/10/10_chapter...

Heterocycles

Dept. of Chemistry, MIT, Manipal University, Manipal.

CHAPTER-1

1. INTRODUCTION

In recent years, various ailments and symptoms have been made easier to treat by the

production of drugs. New drugs are continually developed by pharmaceutical firms.

To create new products, ideas have to be created although some drugs are discovered

by accident. The idea has to be for a new drug or it has to be an idea to improve the

existing drug.

A compound can be used to treat an ailment if it has active biological activity.

Heterocyclic compounds are one of the important bioactive molecules found in

nature. These heterocyclic compounds fulfil important physiological functions.

Observations of these activities in nature led humans to the discovery many healing

materials. Among these heterocyclic compounds N-Heterocycles were found to have

good biological activity. Hence in the past few decades, the synthesis of these

heterocyclic compounds has been a subject of great interest because of their wide

applicability.

Heterocyclic compounds are organic compounds containing at least one

element other than carbon, such as sulfur, oxygen or nitrogen within a ring structure.

In addition to that, a variety of atoms such as N, O, S, Se, P, Si, B are also

incorporated in to ring structures. The name comes from the Greek word “heteros”

which means “different.” By far the most numerous and most important heterocyclic

systems are those of five and six members. Heterocyclic make up an exceedingly

important class of compounds more than half of all known organic compounds are

heterocycles. Almost all the compounds we know a drugs, vitamins, and many other

Heterocycles


natural products are heterocycles. Since in hetrocycles non-carbons usually are

considered to replace carbon atoms, they are called heteroatoms. e.g. different from

carbon and hydrogen. A ring with only heteroatoms is called heterocycles are the

counterparts of homocyclic compounds. Thus incorporation of oxygen, nitrogen,

sulfur or an atom of a related element into an organic ring structure in place of a

carbon atom gives rise to a heterocyclic compound. These structures may comprise

either simple aromatic rings or non-aromatic rings. The heterocyclic compounds

usually possess a stable ring structure which does not readily hydrolyzed or

depolymerized. Those containing one heteroatom are in general stable. Those with

two hetero atoms at more likely to occur as reactive intermediates.

Heterocyclic chemistry is one of the most interesting, applied branches of

organic chemistry and of utmost practical and theoretical importance. As a result, a

great deal of research carried out in chemistry is devoted to heterocyclic chemistry. It

is vast and expanding area of chemistry because of obvious application of compounds

derived from heterocyclic rings in pharmacy, medicine, agriculture, plastic, polymer

and other fields. Heterocyclic compounds are widely distributed in nature. By virtue

of their therapeutic properties, they could be employed in the treatment of infectious

diseases. Many heterocyclic compounds synthesized in laboratories have been

successfully used as clinical agents.

Heterocycles form by far the largest of classical organic divisions of organic

chemistry and are of immense importance biologically and industrially. The majority

of pharmaceuticals and biologically active agrochemicals are heterocyclic while

countless additives and modifiers used in industrial applications ranging from

Heterocycles


cosmetics reprography, information storage and plastics are heterocycles in nature.

One striking structural features inherent to heterocycles, which continue to be to great

advantage by the drug industry, lies in their ability to manifest substituents around a

core scaffold in defined three dimensional representations. For more than a century,

heterocyclic have constituted one of the largest areas of research in organic chemistry.

They have contributed to the development of society from a biological and industrial

point of view as well as to the understanding of life processes and to the efforts to

improve the quality of life. Among the 20 million chemical compounds identified by

the end of the second millennium, more than two-thirds at fully or partially aromatic

and approximately half are heterocycles. The presence of heterocycles in all kinds of

organic compounds of interest in electronics, biology, optics, pharmacology, material

sciences and so on is very well known.

Among heterocycles, nitrogen-containing heterocyclic compounds have

maintained the interest of researchers through decades of historical development of

organic synthesis[1].

Nitrogen-containing heterocycles have been used as medicinal

compounds for many decades, and form the basis for many common drugs such as

Morphine (analgesic), Captopril (hypertension), and Vincristine, (cancer

chemotherapy). Among the drugs containing aromatic five-membered nitrogen

heterocycles are Atorvastatin (cholesterol-reducing), Celecoxib (anti-inflammatory),

Cimetidine (antiulcerative), Fluconazole (antifungal), and Losartan

(antihypertensive). Nitrogen-containing heterocycles occur in a diversity of natural

products and drugs and are of great importance in a wide variety of applications.

Aromatic nitrogen heterocycles may contain another heteroatom, such as the oxygen

in isoxazoles, oxazoles, 1,3,4-oxadiazoles, and 1,2,4-oxadiazoles.

Heterocycles


Replacement of a methylene group (-CH2-) in 1,3-cyclopentadiene by N, O or

S results in the resultant formation of pyrrole, furan and thiophene respectively. Since

nitrogen is trivalent it can be substituted only for a methane (-CH=) group in a five

membered heterocyclic ring. Five membered heterocyclic compounds with an

additional hetero atom are termed azoles. Thus azoles containing two nitrogen atoms;

one oxygen and one nitrogen atom; one sulphur and one nitrogen atom in the 1, 2-

position are designated as pyrazole, isoxazole and isothiazole respectively. When both

the heteroatoms are present in a 1, 3-relationship then they are referred to as

imidazole, oxazole and thiazole respectively. The numbering in these heterocyclic

compounds, by convention, commences from the hetero atom. But when two hetero

atoms are present in the ring then a choice is necessitated and the hetero atom which

is in the periodic table and the element of least atomic weight in that group is often

given the preference. This implies that among the three common hetero atoms, the

order of preference is oxygen, sufur and nitrogen. In other words, oxygen is assigned

position-1 in preference to others. Condensed ring systems of these heterocycles are

also known and they are named as derivatives of the parent azoles.

Many of the azoles comprise the ring system of several natural and synthetic

compounds which are important for the living systems and also as important drugs,

dyes and agriculture chemicals.

All the azoles are aromatic though resonance energies for many of them have

not been measured, and their electronic structures follow from their relationship to

pyrrole, furan and thiophene. The lone pair of electrons on the heteroatom contributes

towards the aromatic sextet. For the construction of the molecular orbital structure,

Heterocycles


each carbon atom contributes one Pz electron, the nitrogen atom gives the fourth

electron and the second heteroatom(X), i.e. nitrogen in pyrazole or oxygen in oxazole,

gives two electrons to complete the aromatic sextet. From the literature, it is obvious

that the azoles nitrogen possesses an electron pair which is situated in orthogonal

fashion to the molecular –cloud. It is this pair of electrons which permits the azoles to

behave as basic compounds and as nucleophiles. In a manner similar to other aromatic

compounds no single valence-bond structure can adequately represent these

molecules which must be considered rather as a resonance hybrid of a number of

contributing structures.

1, 2-azoles are much less reactive than 1, 3-azoles and do not usually undergo

electrophilic substitution in acidic solutions. An electrophilic reagent generally attacks

position -4 or -5 in both of these molecules[1].

In the fight against disease, some of the most significant advances are being

made by designing and testing new structures, many of which are hetero-aromatic

derivatives[2]

. Inspired by them, pharmaceutical researchers have constantly designed

and produced better pharmaceuticals for a better living.

The simple doubly unsaturated compound containing two nitrogen and three

carbon atoms in the ring, with the nitrogen atoms neighbouring, is known as pyrazole.

For a long time no pyrazole derivative had been found in nature, but in 1959 (1-

pyrazolyl) alanine was isolated from the seeds of water melons (Citurllus lanatus).

Pyrazole is a tautomeric substance; the existence of tautomerism cannot be

demonstrated in pyrazole itself, but it can be inferred by the consideration of

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pyrazolederivatives. Amongst the various heterocycles, pyrazole classes of

compounds play an important role in medicinal chemistry. Very few pyrazole

derivatives are naturally occurring may be due to the difficulty of living organisms to

construct the N-N bond. The interest in pyrazoles stemmed from their application in

drugs, dyes and as anaesthetics. Pyrazoles have also been used as antioxidants in

fuels, but their major applications have been in medicinal and agricultural fields. The

dihydropyrazoles are called pyrazolines and three of them are possible depending on

the position of the double bond. These are 1-pyrazoline, 2-pyrazoline and 1,3-

pyrazoline.

Among a wide variety of aryl groups, oxadiazole is a heteroaryl group that is

often used in medicinal chemistry. Five membered ring heterocycles containing two

carbon atoms, two nitrogen atoms and one oxygen atom known as oxadiazoles are of

considerable interest in different areas of medicinal and pesticide chemistry and also

polymer and material science [3-5].

It is considered to be a bio-isoster of carboxylic functionalities and can be

used to replace an ester group to achieve compounds that are resistant to enzyme

catalysed hydrolysis [6-7].

Oxadiazoles have often been described as bio-isosteres for

amides and esters[8].

Due to increased hydrolytic and metabolic stabilities of the

oxadiazole ring, improved pharmacokinetic and in vivo performance is often

observed, which make these heterocycles an important structural motif for the

pharmaceutical industry.

Heterocycles


Thiazolidinone is considered as a biologically important active scaffold that

possesses almost all types of biological activities. Successful introduction of ralitoline

as a potent anti-convulsant, etozoline as a antihypertensive, pioglitazone as a

hypoglycemic agent and thiazolidomycin activity against Streptomyces species proved

potential of thiazolidinone moiety. This diversity in the biological response profile has

attracted the attention of many researchers to explore this skeleton to its multiple

potential against several activities. Data are presented for active compounds, some of

which have passed the preclinical testing stage. There are numerous biologically

active molecules which contain various heteroatoms such as nitrogen, sulphur and

oxygen, always drawn the attention of chemist over the years mainly because of their

biological importance. Thiazolidinones are thiazolidine derivatives and have an atom

of sulfur at position 1, an atom of nitrogen at position 3 and a carbonyl group at

position 2, 4, or 5. However, its derivatives belong to the most frequently studied

moieties and its presence in penicillin was the first recognition of its occurrence in

nature. Similarly 1,3-thiazolidin-4-ones are heterocyclic nucleus that have an atom of

sulfur and nitrogen at position 1 and 3, respectively and a carbonyl group at position 4

have been subjected to extensive study in the recent years. The 4-thiazolidinone

scaffold is very versatile and has featured in a number of clinically used drugs. They

have found uses as antitubercular, antimicrobial, anti-inflammatory and as antiviral

agents, especially as anti-HIV agents. It has been extensively reported that presence of

arylazo, sulfamoylphenylazo or phenylhydrazono moieties at different positions of the

thiazolidone ring enhanced antimicrobial activity and its antibacterial activity may be

due to its inhibitory activity of enzyme Mur B which is a precursor acting during the

biosynthesis of peptidoglycan. Numerous reports have appeared in the literature

which highlights their chemistry and pharmacological uses. In the present review,

Heterocycles


emphasis is given on diverse pharmacological properties associated with substituted

thiazolidinones and structurally related thiazolidines.

1.1 Scope and objective of current work

Infectious diseases have emerged as a serious cause of morbidity and

mortality, with 16.2 percent(equivalent to 57 million) deaths each year worldwide.

Hence, WHO has listed such diseases in 2nd

place among the lead cause of death.

Now, medicinal world has conquered many deadly infectious diseases and immensely

brought down the mortality rate to some extent. But still diseases like pneumonia,

tuberculosis (TB), typhoid, H1N1, dengue and HIV are matter of big concern at

present. Further, emerging antimicrobial resistance has created a major public health

dilemma, compounded by a dearth of new antimicrobial options. In addition, the

alarming rates of emerging and re-emerging microbial threats coupled with increasing

antimicrobial resistance, particularly in regard to multi drug resistant gram-positive

bacteria and Mycobacterium, are major concerns to the public health as well as

scientific communities worldwide.

Antimicrobial drugs have caused a dramatic change not only of the treatment

of infectious diseases but of a fate of mankind. Antimicrobial chemotherapy made

remarkable advances, resulting in the overly optimistic via that infectious diseases

would be conquered in the near future. Antimicrobial resistance is a global public

health concern that is impacted by both human and non-human antimicrobial use. The

consequences of antimicrobial resistance are particularly important when pathogens

are resistant to antimicrobials that are critically important in the treatment of human

disease. However, in reality, emerging and re-emerging infectious diseases have left

Heterocycles


us facing a counter charge from infections. Infections with drug resistant organisms

remain an important problem in clinical practice that is difficult to solve.

The greatest impact of the synthesis of heterocyclic chemistry is the

development of new pharmaceutically active and efficient compounds. Inventing and

developing a new medicine is a long, complex, costly and highly risky process that

has few peers in the commercial world. Research and development (R&D) for most of

the medicines available today has required 12-24 years for a single new medicine,

from starting a project to the launch of a drug product. In addition, many expensive,

long-term research projects completely fail to produce a marketable medicine. Each

step of a synthesis involves a chemical reaction, reagents and conditions need to be

designed to give a good yield and pure product. The discovery of new methods and

reagents grab the attention of chemists across the world. Optimization is where one or

two starting compounds are tested in the reaction under a wide variety of conditions

of temperature, solvent, reaction time etc, until the optimum conditions for product,

yield and purity are found. Then the researcher tries to extend the method to a broad

range of different starting materials to find the scope and limitations.

Heterocyclic compounds by virtue of their specific activity could be employed

in the treatment of infectious diseases. Review of literature indicated that nitrogen

containing heterocycles find a significant place in the development of

pharmacologically important molecules. The present study focussed on to explore

new molecules. Also the pharmacological activity, stability and toxicity of the

individual motifs are encouraging and well documented. The research has been

carried out by keeping in view of synthesizing new heterocyclic derivatives and the

comparative study of pharmacological activity.

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All these natural and synthetic heterocyclic compounds can and do participate

in chemical reactions in the human body. Furthermore, all biological processes are

chemical in nature. Such fundamental manifestations of life as the provision of

energy, transmission of impulses, sight, metabolism and the transfer of hereditary

information are all based on chemical reactions involving the participation of many

heterocyclic compounds, such as vitamins, enzymes, coenzymes, nucleic acids, ATP

and serotonin.

The present research work involves synthesis, characterisation and biological

studies of new nitrogen heterocycles such as pyrazoles, pyrazolines, oxadiazoles,

thiazolidinones, tetrazoles, & triazoles. Heterocyles are able to get involved in an

extraordinarily wide range of reaction types. Depending on the pH of the medium,

they may behave as acids or bases, forming anions or cations. Some interact readily

with electrophilic reagents, others with nucleophiles, yet others with both. Some are

easily oxidized, but resist reduction, while others can be readily hydrogenated but are

stable towards the action of oxidizing agents. Certain amphoteric heterocyclic systems

simultaneously demonstrate all of the above-mentioned properties. The ability of

many heterocycles to produce stable complexes with metal ions has great biochemical

significance. The presence of different heteroatoms makes tautomerism ubiquitous in

the heterocyclic series. Such versatile reactivity is linked to the electronic

distributions in heterocyclic molecules. Evidently, all the natural products and the

synthetic drugs mentioned above good examples of nature’s preference for

heterocycles whose biological activity cannot be determined by one or a combination

of two or three of the above mentioned properties.

Heterocycles


The fast growing literature on heterocycles in recent years demonstrates their

increasing significance in the pharmaceutical field. An interesting feature of many

heterocyclic compounds is that it is possible to incorporate functional groups either as

constituents or as part of the ring system itself. For example, atoms of nitrogen can be

included both as amino constituents and as part of a ring. This shows that their

structures are particularly versatile as a means of providing, or of mimicking, a

functional group.

In view of the general observation that the biological activities are invariably

associated with a large variety of nitrogen heterocyclic systems such as Pyrazole,

Oxadiazole, Thiazolidinone, Tetrazole, Triazolemoeties. A large number of their new

derivatives have been synthesized and extensively studied for antimicrobial

properties.

The study of structure-activity relationship (SAR) of the new compounds will

impart structural elements for new drug designing. Also, the results of research may

be useful in understanding the mechanism of drug action.

1.2Antimicrobials and their importance

An antimicrobial is a substance that kills or inhibits the growth of

microorganisms such as bacteria, fungi or protozoans. This capability makes them

unique for the control of deadly infectious diseases caused by a large variety of

pathogenic microorganisms.

Today, more than 15 different classes of antimicrobials are known. They differ

in chemical structure and mechanism of action. Specific antimicrobials are necessary

for the treatment of specific pathogens.

Heterocycles


Since their discovery, antimicrobial agents have substantially reduced the

threat posed by infectious diseases. The use of these ‘’wonder drugs’’ combined with

improvements in sanitation, housing, nutrition and the advent of widespread

immunization programmes has led to a dramatic drop in deaths from diseases that

were previously widespread, untreatable and frequently fatal. Also, these drugs have

contributed to the major gains in life expectancy by helping to control many serious

infectious diseases.

Antimicrobials can be divided into two classifications based upon their effects

on target cells. Substances that actually kill microorganisms are termed ‘bactericidal’.

Compounds that only inhibit the growth of microorganisms are termed

‘bacteriostatic’. The decision to use a bactericidal or bacteriostatic drug to treat

infection depends entirely upon the type of infection. Some examples of bactericidal

and bacteriostatic drugs are streptomycin, Aminoglycosides, Penicilln, Sulfonamides,

Tetracycline etc.

Also, based on their range of activity, antimicrobial drugs can be classified as

(i) narrow spectrum drugs, which are only active against a relatively small number of

gram-positive organisms. (ii) moderate spectrum drugs, which are effective against

gram positive and the most systemic, enteric and urinary tract gram-negative

pathogens (iii) narrow and moderate spectrum drugs like beta-lactam antibiotics (iv)

broad spectrum drugs which are active almost all microorganism i.e., antibiotic is

ampicillin.

http://en.wikipedia.org/wiki/Ampicillin

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1.3 Antimicrobial agents and their mechanism of action

In medication of microbial infections different types of antimicrobial dressings

(AMDs) are used. Before the discovery of Penicillin, in the early 1940’s, no true cure

for gonorrhea, strep throat, or pneumonia existed. Patients with infected wounds often

had to have a wounded limb removed, or face death from infections. Now, most of

these infections can be cured easily with a short course of many exiting active

antimicrobials.

The old antimicrobial technology was based either on poisons or heavy metals,

which may not have killed the microbe completely, allowing the microbe survive,

change, and become resistant to the poisons and/or heavy metals. However, it has

been observed that with the development of new antimicrobials, microorganisms have

adapted and become resistant to previous antimicrobial agents. A schematic

representation of the history of antimicrobials has been captured in the following

figure.

Fig- Discovery of antimicrobials in the past years

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The antimicrobial agents function by attacking various cellular targets which include

ceil wall, plasma membrane, nucleic acids and proteins synthesis of the microbe. The

precise mechanisms of action of antimicrobial drugs are still not clear, but the

following possible views were proposed for their mode of action.

1.3.1 Inhibition of cell wall synthesis: Certain antimicrobials work by inhibiting the

cell wall synthesis. Therefore, they have little effect on host cells, which do not

contain peptidoglycan. Penicillin, Bacitracin, Cephalosporine and Vancomycin act in

this way.

1.3.2 Inhibition of protein synthesis: Several antimicrobial agents like

Chloramphenicol, Erythromycin, Streptomycin, Tetracyclines etc. act by inhibiting

protein synthesis. As ribosomes of prokaryotic cells are slightly different from those

of eukaryotes, they can be used as a target.

1.3.3 Injury to the plasma membrane: This is a mode of action for certain

antibacterials and antifungals. Antifungals are able to work mostly against fungus cell

membranes because they contain ergosteol instead of cholesterol. However, these

antimicrobials are potentially quite toxic to the host. The examples include

Polymixins (antibacterial), and Amphotericin B, Miconazole, and Ketoconazole

(antifungal).

1.3.4 Inhibition of nucleic acid synthesis: These drugs interfere with DNA

replication and transcription, but their selective toxicity varies. Rifampin and certain

quinolone derivatives are the examples under this mode of action.

1.3.5 Inhibition of the synthesis of essential metabolites: Generally sulfas and

Trimethoprim functions by this way. They interfere with the pathway on which

bacteria synthesize folic acid. Since humans produce folic acid by a different

pathway, these drugs have less effect on humancells.

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Fig-Schematic representation of mechanism of action on bacterial cell

During the past 80 years, antimicrobial drugs have been critical in the fight

against infectious diseases caused by bacteria and other microbes. However, in the

past decade these ease-causing microbes have become resistant to the antimicrobial

drug therapy causing severe public health problem. Wound infections, gonorrhea,

tuberculosis, pneumonia, septicemia and childhood ear infections are just a few of the

diseases that have become hard to with antimicrobials. One part of the problem is

those bacteria and other microbes that infections are remarkably resilient and have

developed several ways to resist antibiotics and other antimicrobial drugs. Currently,

resistance to first-line antimicrobial agents is further aggravated. Infections caused by

these resistant microbes fail to respond to treatment resulting in prolonged illness and

greater risk of death. Nowadays, the alarming rates of emerging and re-emerging

microbial threats coupled with increasing antituberculosis, antibacterial and antifungal

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resistance; particularly in regard to multi drug-resistant microbes are major concerns

to the public health as well as scientific communities worldwide.

The new and more expensive drugs have developed; their cost is beyond the

common’s reach. As a consequence, these trends have emphasized the pressing need

for new, more effective, cheaper and safe antimicrobial agents. This has instigated the

scientific community to carry out extensive research activities on design and

development of new antimicrobials.

1.3.6 Antimicrobial screening

In general, antimicrobial activity of any substance can be investigated by

various methods (WHO/CDS/CSR/RMD, 2003). Both in vivo and in vitro methods

are used for screening of compounds for antimicrobial activity. Amongst them, in

vitro are extensively used for the preliminary evaluation of antifungal, antibacterial

and antituberlosis activities. Further, in vivo studies are employed on animal models

of the condition necessary to elucidate the mechanisms of antimicrobial action and to

drugs that can control infection caused by pathogenic microbes. Furthermore, the

(IC50) studies of these molecules are performed on a mammalian Vero cell line in

pass them into phase trials.

During the last decades, several experimental procedures were developed for

Antimicrobial Susceptibility Testing (AST) by CLSI (Clinical and Laboratory

Standards institute). That created standards to perform ASTs. These methods are

extensively being used in the molecular potency against microbes.

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Generally, in vitro antimicrobial susceptibility testing methods are divided

mainly types, viz. (i) Diffusion, (ii) Dilution and (iii) Diffusion and Dilution methods.

The important antimicrobial testing methods have been discussed in the following

1.3.7 Diffusion methods

Diffusion method involves two important techniques, viz. Stokes method and

Kirby method. These methods are typically used for antimicrobial susceptibility

testing, which are being well recommended by the National committee for clinical

standards (NCCLS).

1.3.8 Stokes method

In this method a known quantity of bacteria is grown on agar plates in the

presence of relevant standard antibiotics. If the bacteria are susceptible to a particular

antimicrobial, an area of clearing surrounds the wafer where bacteria are not of

growing (called a zone of inhibition). Also, the rates of antimicrobial diffusion are

determined and these values are used to estimate the bacteria’s sensitivity to that

particular antimicrobial agent. In general, larger zones correlate with smaller

concentration of test compounds for a specific microorganism. This information can

be used to choose antimicrobials to combat a particular infection.

1.3.9 Kirby method

Kirby-Bauer test[9],

known as the disk-diffusion method, is the most widely

used antibacterial susceptibility test in determining the precise antibiotics used to treat

the exact infection.

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This method relies on the inhibition of bacterial growth measured under

standard conditions. This test, a culture medium, specifically the Mueller-Hinton agar,

is uniformly and asceptically inoculated with the test organism and then filter paper

discs, which impregnated with a specific concentration of a particular antimicrobial, is

placed on the medium. The organism will grow on the agar plate while the

antimicrobial ‘works’ to inhibit the growth. If the organism is susceptible to a specific

antimicrobial drug, there will be no microorganism around the disc containing the

antibiotic. Thus, a ‘zone of inhibition’ can be served and measured to determine the

susceptibility to an antimicrobial for that particular drug.

1.3.10 Dilution Methods

Dilution technique mainly includes minimum inhibition concentration (MIC)

method, be further classified as broth dilution and agar dilution methods.

1.3.11 Minimum Inhibitory Concentration (MIC) method

MIC method[10]

is generally used to determine the minimal concentration of

antimicrobial to inhibit or kill the microorganisms completely. This can be achieved

by dilution of antimicrobial solution in either agar or broth media. The dilutions are

normally expressed in log2 serial dilutions (i.e. two fold). In this method, a pure

culture of a single microorganism is grown in appropriate broth. The culture is

standardized using standard microbiological techniques (nearly I million cells per

milliliter). The compound under screening is diluted a number of times, 1:1, using a

sterile diluent. After dilution, a volume of the standardized inoculum equal to the

volume of the diluted compound is added to each vessel bringing the microbial

concentration to approximately 500,000 cells per milli liter. The inoculated, serially

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diluted antimicrobial agent is incubated. After incubation, dilution vessels are

observed for microbial growth, the results of which are usually by turbidity or colour

change. The last tube in the dilution series that does not demonstrate growth

corresponds to the minimum inhibitory concentration (MIC) of the microbial agent.

1.3.12 Broth dilution method

The Broth dilution method is a simple technique for testing a small number of

isolates even single isolate. It involves serial dilution of the antimicrobial agent in a

liquid medium, which is then inoculated with a standardized number of organisms and

incubated in a prescribed time. The lowest concentration of antibiotic preventing

appearance of turbidity is considered to be the minimal inhibitory concentration. It

has the added advantage the same tubes can be taken for Minimum Bactericidal

Concentrations (MBC) tests also.

1.3.13 Agar dilution method

In this method, the compounds under screening are diluted on log2 dilution

intervals here each petri dish contains 50 per cent of the concentration of the given

compound in the previous dilution. The diluted solution is incorporated into the agar

medium and mixed by rotation and poured into petri dish. A control plate without any

antimicrobial agent into the medium is also used along with each compound tested, to

check for E-test and control strains. Readings are recorded after the petri dishes have

been incubated. The main advantage of the method is that it is possible to test several

organisms in the same plate.

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1.3.14 Dilution and Diffusion method

Dilution and diffusion method is a convenient method to screen the

antimicrobial susceptibility of any substance. It is also known as Epsilometer test (E

test). This exponential gradient’ testing methodology is generally used for the

quantitative antimicrobial screening wherein both dilution of antimicrobial and

diffusion of antimicrobial the medium involve. In this method, a thin inert carrier strip

containing a predefined antimicrobial gradient is used. It is then applied onto an

inoculated agar plate. Then, there is an immediate release of the drug. On incubation

for 24 hours, a symmetrical inhibition ellipse is produced. The intersection of the

inhibitory zone edge and the calibrated strip indicates the MIC value over a wide

concentration range (>10 dilutions) with inherent precision and accuracy. Epsilometer

test is simple, easy to perform and is a reliable method for MIC. Also, it has been

shown to be a good alternative to the agar and broth dilution tests, particularly for the

strains such as Haemophilus influenza. However its cost and limited availability is a

concern.

The latest ‘genotypic’ technique for detection of antimicrobial resistance

genes has been promoted as a way to increase the speed and accuracy of susceptibility

testing. Numerous DNA based assays are being developed to detect bacterial

antibiotic resistance at level. These methods, when used in conjunction with

phenotypic analysis, offer the promise of increased sensitivity, specificity, and speed

in the detection of specific known resistance genes and can be used in tandem with

traditional laboratory AST methods.

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Although a variety of methods exist, the goal of in vitro antimicrobial

susceptibility testing is to provide a reliable predictor of how an organism is likely to

respond to microbial therapy in the infected host. This type of information aids the

clinician in selecting the appropriate antimicrobial agent, aids in developing

antimicrobial use policy, and provides data for epidemiological surveillance. Such

epidemiological surveillance data is a base to choose the appropriate empirical

treatment (first-line therapy) and to detect the emergence and/or the dissemination of

resistant bacterial strains or resistance determinants in different bacterial species. The

selection of a particular AST method is based many factors such as validation data,

practicality, flexibility, automation, cost, reproducibility, accuracy, and individual

preference.

In our present study, serial dilutions method has been followed for the

investigation of antimicrobial properties of newly synthesized compounds, since this

method is popular in laboratory due to low cost, reproducibility in results, convenient

to perform and accuracy. The detailed experimental procedures along with screening

results have been discussed in this chapter. Substitution of active functional groups

have been showed enhanced antibacterial activity. Remaining compounds have

showed moderate antimicrobial activity.

1.4 The main objectives of the present research work are as follows:

Synthesis of new 1,5-disubstituted pyrazolines, 1,5-disubstituted pyrazole-4-

carbaldehyde, 1,2,4-triazole derivatives (Schiff and Mannich bases), 1,5-

disubstituted 1,3,4-oxadiazoles, Tetrazoles, and 2,3-disubstituted thiazolidin-

4-ones.

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Characterization of new compounds by IR, 1HNMR, Mass spectral studies and

also by elemental analysis.

Evaluation of pharmacological screening such as antimicrobial activity using

different bacterial and fungal strains.

1.5 References

1. Xian Zhang, Hongliang Wang, YuhuanLi Ruiyuan Cao, Novel Substituted

Heteroaromatic Piperazine and Piperidine Derivatives as Inhibitors of Human

Enterovirus 71 and Coxsackievirus A16; Molecules, 2013; 18: 5059-5071.

2. Balwinder Singh, Vishal Sharma, Gagandeep Singh, Synthesis and In Vitro

Cytotoxic Activity of Chromenopyridones, Int J of Med Chem, 2013, Article

ID 984329, 7-14.

3. Toyoharu Kobayashi, Taku Sakaguchi, and Shigeo Katsumura, One-Pot

Asymmetric 6π-Azaelectrocyclization as a New Strategy for Alkaloid

Synthesis, An International Journal for Reviews and Communications in Het

Chem(material science);2013; 87(4): 729-761.

4. Lekh Nath Gautam, Qiaoyi Wang, Novruz G. Akhmedov, Jeffrey L,

Stereoselectivesynthesis of N-heterocycles through amine addition to

nitroalkenes, Org. Biomol. Chem;2013;11:1917-1920.

5. Yonghong Song, Zhaozhong J. Jia, Robert M. Scarborough, track new patents

and technologies;2013; 514341 (USPTO) Class 514.

6. StephanReck, Christian Näther, and Willy Friedrichsen, Synthesis and

Reactions of a Novel Furo[3,4-d]thiazole, An International Journal for

Reviews and Communications in Het Chem;1998; 48(5):853-860

http://www.hindawi.com/40436797/



http://www.freshpatents.com/Yonghong-Song-FosterCity-invdirs.php

http://www.freshpatents.com/Zhaozhong-J-Jia-SanMateo-invdirj.php

http://www.freshpatents.com/Robert-M-Scarborough-HalfMoonBay-invdirs.php

http://www.freshpatents.com/Drug-bio-affecting-and-body-treating-compositions-dtnewntc514.php

Heterocycles


7. Jordi Garcia and Jaume Vilarrasa, Fluoroazoles. An MNDO SCF MO Study,

An International Journal for Reviews and Communications in Het Chem,

1988; 27(8):1803-1807.

8. Ahmed El-Ziaty, Abdelaal Abdalh, Ashraf Hamed, SayedShiba, Abdelhafed

Abdullha Eur J of Chem,(2012);3 (1): 65-70

9. Adnan A. Bekhita, Ola A. El-Sayed, Elsayed Aboulmagd, JiYoung Park,

Tetrazolo[1,5-a]quinoline as a potential promising new scaffold for the

synthesis of novel anti-inflammatory and antibacterial agents, Eur J of Med

Chem,2004;39: 249–255.

10. Umarani Natrajan et al reported A facile design and efficient synthesis of

schiff's bases of tetrazolo [1,5-a] quinoxalines as potential anti-inflammatory

and anti-microbial agents, Der Pharma Chemica, 2010; 2(1):159-167.

chapter-1 1. introduction - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/32217/10/10_chapter...

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