239

Biological Activities

The work incorporated in this chapter pertains to the in vitro biological

screening tests of entire polycyclic heterocycles that are described in chapter 2, 3

and 4. The tests include the measurements of antimicrobial, antituberculosis and

antioxidant activities. The test results revealed that many compounds have

potential bioactivities, as new bioprofiels.

5.15.15.15.1 Antimicrobial Antimicrobial Antimicrobial Antimicrobial aaaactivityctivityctivityctivity

5.1.15.1.15.1.15.1.1 Introduction to the world of bacteriaIntroduction to the world of bacteriaIntroduction to the world of bacteriaIntroduction to the world of bacteria

Bacteria exist all around us and we merely look at these prokaryotic as to

be disease causing parasites. Being responsible for a large number of human

diseases, however, they help recycle certain elements such as carbon, nitrogen,

and oxygen in the atmosphere. No existence of life, if they would have failed to

decompose the waste and dead organisms. Thus, they continuously exchange

essential chemicals between the biosphere and the other environmental

segments, say atmosphere or lithosphere or hydrosphere. To decide as to

whether the bacteria are friend or enemy, it becomes harder when one has to

suppose both the positive and negative aspects in establishing their relationship

with humans.

In 1828, a German scientist C. G. Ehrenberg1 used the term “bacterium”.

Bacteria are the microscopic organisms of plant kingdom and are devoid of

chlorophyll. They are relatively simple and primitive forms of cellular organisms

known as “Prokaryotes”. Bacteriology is the 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 namely “Gram-

positive” and “Gram-negative”. Gram-positive bacteria, which retain a dark blue

color with a staining agent methyl violet, resist the discoloration by acetone and

alcohol. But, Gram-negative bacteria do not.2 Gram-positive bacteria have the cell

wall peptidoglycan layer much thicker than that of Gram-negative bacteria.

Gram-negative bacteria have an additional outer membrane. The outer

membrane is the major permeability barrier in Gram-negative bacteria. The

space between the inner and outer membranes is known as the periplasmic

Chapter 5 240

space. Gram-negative bacteria store degradative enzymes in the periplasmic

space. Gram-positive bacteria lack such periplasmic space and thus cannot store

degradative enzymes. 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.3

5.1.25.1.25.1.25.1.2 PathogensPathogensPathogensPathogens

The microorganisms, or infectious agents or more commonly germs, are

those biological agents which produce diseases in host usually known as

pathogens. There are several substrates and pathways through which pathogens

can invade a host; the principal pathways involve different episodic time frames,

but soil contamination has the longest or most persistent potential for harboring

a 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.

5.1.35.1.35.1.35.1.3 Bacterial PathogenBacterial PathogenBacterial PathogenBacterial Pathogen

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 biomolecules. Tables 5.1Tables 5.1Tables 5.1Tables 5.1 represent the list of bacteria

commonly found on the surfaces of the human body.

Chapter 5 241

Table 5.1 Table 5.1 Table 5.1 Table 5.1 Bacteria commonly found on human body surfaces.4

BacteriumBacteriumBacteriumBacterium SkinSkinSkinSkin ConConConCon----

junctivajunctivajunctivajunctiva NoseNoseNoseNose PharynxPharynxPharynxPharynx MouthMouthMouthMouth

LowerLowerLowerLower

GIGIGIGI

Ant.Ant.Ant.Ant.

UrethraUrethraUrethraUrethra VaginaVaginaVaginaVagina

Staphylococcus 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

Chapter 5 242

5.1.45.1.45.1.45.1.4 Classifications of bacteriaClassifications of bacteriaClassifications of bacteriaClassifications of bacteria

Bacterial can be classified into the following groups:

i)i)i)i) Classification based on shapeClassification based on shapeClassification based on shapeClassification based on shapes of bacterias of bacterias of bacterias of bacteria

Most of the bacteria belong to three main shapes: a rod shape bacilli, a

sphere shape cocci and spiral shaped spirilla. Some bacteria belong to different

shapes, which are more complex.

ii)ii)ii)ii) Aerobic and anaerobic bacteriaAerobic and anaerobic bacteriaAerobic and anaerobic bacteriaAerobic and anaerobic bacteria

Bacteria are also classified based on the requirement of oxygen for their

survival. Those, which need oxygen for their survival, are called aerobic bacteria

and those, which require no oxygen, are known as anaerobic bacteria. Anaerobic

bacteria cannot bear oxygen and may die off oxygenated environment. Anaerobic

bacteria are found mostly in the places like under the earth surface, deep Ocean,

and in some suitable medium.

iii)iii)iii)iii) GramGramGramGram----positive and Grampositive and Grampositive and Grampositive and Gram----negative bacterianegative bacterianegative bacterianegative bacteria

Based on gram staining method, bacteria are grouped into ‘Gram-positive’

and ‘Gram-negative’. Staining agent is used to bind it to the cell wall of the

bacteria. The following bacterial pathogens were used for antimicrobial study of

all the synthesized compounds in the present work.

GramGramGramGram----positivepositivepositivepositive

(i) Streptococcus pneumoniae5

(ii) Clostridium tetani6

(iii) Bacillus subtilis7

GramGramGramGram----negative negative negative negative

(i) Salmonella typhi8

(ii) Vibrio cholerae9

(iii) Escherichia coli10

5.1.55.1.55.1.55.1.5 Fungal PathogensFungal PathogensFungal PathogensFungal Pathogens

Fungi are plant-like organisms that lack in chlorophyll. They are one of

the five kingdoms of life. While many are beneficial and useful i.e. edible

mushrooms, others are harmful i. e. some fungi can infect plants and people.

There are over 100,000 fungi species. Since they do not have chlorophyll, they

derive food from others. Since they don't use light to make food, fungi can live in

Chapter 5 243

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 the

skin of animals or people. This is sometimes 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 cause

watery 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 have

used following fungal pathogens for antifungal study of synthesized compounds.

(i) Candida albicans11

(ii) Aspergillus fumigatus12

5.1.6 Antimicrobial agents5.1.6 Antimicrobial agents5.1.6 Antimicrobial agents5.1.6 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

combine various antimicrobial agents for broadening the activity spectrums and

to minimize the possibility of the development of bacterial resistance. Some

Chapter 5 244

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 are

produced by living organisms which, even though in minute amounts inhibit the

growth of another organisms.

5.1.7 Antimicrobial susceptibility tests5.1.7 Antimicrobial susceptibility tests5.1.7 Antimicrobial susceptibility tests5.1.7 Antimicrobial susceptibility tests

Evaluation techniquesEvaluation techniquesEvaluation techniquesEvaluation techniques

The goal of antimicrobial susceptibility testing is to predict the in vivo

success 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

employed categories the methods. The evaluation can be done by the following

methods as given bellow.

Chapter 5 245

In present work, FDA-USA approved, WHO (world health organization)

and NCCLS13 (National Committee for Clinical Laboratory Standards)

recommended Broth Dilution methodBroth Dilution methodBroth Dilution methodBroth Dilution method, was used for antimicrobial screening tests.

Table 5.2Table 5.2Table 5.2Table 5.2 Microbial tests methodsMicrobial tests methodsMicrobial tests methodsMicrobial tests methods

DiffusionDiffusionDiffusionDiffusion DilutionDilutionDilutionDilution Diffusion & DilutionDiffusion & DilutionDiffusion & DilutionDiffusion & Dilution

Stokes method

Kirby―Bauer method

Minimum Inhibitory Concentration

E-Test method i) Broth Dilution Method

ii)Agar Dilution Method

Broth dilution methodBroth dilution methodBroth dilution methodBroth dilution method

It determines the lowest concentration of the assayed antimicrobial agent

(MIC) that under defined test conditions inhibits the visible growth of the

pathogen. This classic method gives a quantitative result in terms of the amount

of antimicrobial agents needed to inhibit the growth of specific microorganisms.

Procedure for performing the Procedure for performing the Procedure for performing the Procedure for performing the broth dilution methodbroth dilution methodbroth dilution methodbroth dilution method

a) The in vitro antimicrobial activity of the compounds under investigation

and the standard reference drugs were assessed against three Gram-

positive [MTCC (Micro Type Culture Collection), 1936 Streptococcus

pneumonia, MTCC 449 Clostridium tetani, MTCC 441 Bacillus subtilis],

three Gram-negative (MTCC 98 Salmonella typhi, MTCC 3906 Vibrio

cholerae, MTCC 443 Escherichia coli) bacteria, and two fungi (MTCC 3008

Aspergillus fumigates, MTCC 227 Candida albicans). Strains employed for

the activity were procured from MTCC, Institute of Microbial Technology,

Chandigarh, India.

b) Inoculum size for test strain was adjusted to 108 CFUmL-1 (Colony

Forming Unit per milliliter) by comparing the turbidity (turbidimetric

method).

c) Mueller―Hinton broth was used as nutrient medium to grow and dilute

the compound suspension for the test bacteria and Sabouraud-Dextrose

broth used for fungal nutrition.

Chapter 5 246

d) Ampicillin, Chloramphenicol, Ciprofloxacin, Gentamicin and Norfloxacin

were used as standard antibacterial reference drugs, whereas

Griseofulvin and Nystatin were standard antifungal drugs.

e) DMSO was used as diluents/vehicle to achieve a desired concentration of

synthesized compounds. And a standard drug to test it upon a standard

microbial strain.

f) Serial dilutions were prepared in primary and secondary screening. A

synthesized compound and a standard drug each was diluted to 2000

μgmL-1 concentration as a stock solution. In primary screening 1000, 500

and 250 μgmL-1 concentrations were taken. The active compounds found

in this primary screening were further diluted to obtain 200, 100, 62.5,

50, 25, 12.5 and 6.250 μgmL-1 concentrations for secondary screening.

g) A Control tube containing no antibiotic is immediately sub cultured

before incubation by spreading a loopful evenly over a quarter of the

plate on a medium suitable for the growth in test organism. The tubes

were incubated at 37 °C for 24 h in case of bacteria, and 48 h in case of

fungi. The highest dilution or lowest concentration showing at least 99 %

inhibition or preventing appearance of turbidity is considered as MIC

(μgmL−1). A set of tubes containing only seeded broth and the solvent

controls were maintained under identical conditions so as to make sure

that the solvent had no influence on strain growth. The results are much

affected by the size of the inoculums. The test mixture should contain 108

CFUmL-1 organisms. The protocols were summarized and compared with

standard antimicrobial and antifungal drugs in terms of MIC (μgmL-1).

5.1.85.1.85.1.85.1.8 Factors influencing antimicrobial susceptibility testingFactors influencing antimicrobial susceptibility testingFactors influencing antimicrobial susceptibility testingFactors influencing antimicrobial susceptibility testing14141414

a) Choice of media:Choice of media:Choice of media: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: Size of inoculums: Size of inoculums: Size of inoculums: Although large number of organisms does not markedly

affect many antibiotics, the ideal inoculum is one, which gives an even

Chapter 5 247

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) ppppH: H: H: H: The pHpHpHpH 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: Moisture: Moisture: 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.

e) Effects of variation in divalent cations: Effects of variation in divalent cations: Effects of variation in divalent cations: 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: Testing strains that fail to grow satisfactorily: Testing strains that fail to grow satisfactorily: 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:The conditions must be met for the antimicrobial susceptibility testing:The conditions must be met for the antimicrobial susceptibility testing: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.

5.25.25.25.2 AntituberculAntituberculAntituberculAntitubercularararar propertypropertypropertyproperty

5.2.15.2.15.2.15.2.1 IntroductionIntroductionIntroductionIntroduction

Tuberculosis, MTB (Mycobacterium tuberculosis) and TB (Tubercle

bacillus) are common names, and in many cases lethal, infectious disease caused

by various strains of mycobacteria, usually MTB.15 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

Chapter 5 248

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 archaeological

evidence of human infection by M. 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.

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

positive patients are more susceptible to MTB with a 50-fold risk increase over

HIV negative patients. TB is currently to blame for 13 % of the deaths due to HIV

infection.

5.2.25.2.25.2.25.2.2 Mycobacterium tuberculosisMycobacterium tuberculosisMycobacterium tuberculosisMycobacterium 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 fungus

and 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. tuberculosis

Chapter 5 249

H37Rv 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 and coworkers.16 Other

human pathogens belonging to the Mycobacterium genus include M. avium,

which causes a TB-like disease, especially prevalent in AIDS patients, and

Mycobacterium leprae, the causative agent of leprosy.

General characteristicsGeneral characteristicsGeneral characteristicsGeneral characteristics

MTB is a fairly large non-motile rod-shaped bacterium distantly related to

the actinomycetes. Many non-pathogenic Mycobacterium 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

bacterium is a facultative intracellular parasite, usually of macrophages, and has

a slow generation time, 15―20 h, and a physiological characteristic that may

contribute 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-growing MT. It takes 4―6 weeks to get

visual colonies on either type of media. Chains of cells in smears made from in

vitro-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-positive or Gram-negative

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-positive 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

Chapter 5 250

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.

5.2.35.2.35.2.35.2.3 Antituberculosis drugsAntituberculosis drugsAntituberculosis drugsAntituberculosis drugs

Antituberculosis drugs (ATDs) are used to treat the TB and also infections

caused by nontuberculous mycobacterium (NTM). There are two types17 of

ATDs; first-line and second line drugs (a), and tuberculocidal and tuberculostatic

drugs (b).

5.2.45.2.45.2.45.2.4 Classification of ATDsClassification of ATDsClassification of ATDsClassification of ATDs

(a) First-line drugs: Isoniazid, Rifampin, Ethambutol, Pyrazinamide,

Streptomycin.

Second-line drugs: Kanamycin, Capreomycin, Cycloserine, Thiacetazone

Ethionamide, Fluoroquinolones.

(b) Tuberculocidal: Isoniazid, Rifampin, Pyrazinamide, Streptomycin,

Kanamycin, Capreomycin, Fluoroquinolones.

Tuberculostatic: Ethambutol, Thiacetazone, Cycloserine, Ethionamide.

5.2.55.2.55.2.55.2.5 AntiAntiAntiAnti----mycobacterial susceptibility Testsmycobacterial susceptibility Testsmycobacterial susceptibility Testsmycobacterial susceptibility Tests

Evolution techniquesEvolution techniquesEvolution techniquesEvolution techniques

Three 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)(1)(1)(1) The minimal inhibitory concentration (MIC)The minimal inhibitory concentration (MIC)The minimal inhibitory concentration (MIC)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

Chapter 5 251

simple and be carried out with a single drug containing slope although it is

preferable to use more than one slope.

(2) Resistance ratio(2) Resistance ratio(2) Resistance ratio(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(3) Proportion method(3) Proportion method(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.

We have used the MIC MIC MIC MIC to evaluate the anti-tuberculosis activity. It is one

of the non automated in vitro bacterial susceptibility tests. This classic method

yields a quantitative result for the amount of antimicrobial agents that is needed

to inhibit growth of specific microorganisms. It is carried out in bottle.

Determination of MIC by slope methodDetermination of MIC by slope methodDetermination of MIC by slope methodDetermination of MIC by slope method

Methods used for primary and secondary screening:

Each test compound was diluted to a 2000 μgmL-1and was used as stock solution.

Primary screen: Primary screen: Primary screen: Primary screen: In primary screening, 250 μgmL-1 concentrations of synthesized

compounds were taken. If they found active in primary screening were further

tested in a second set of dilution against all microorganisms.

Secondary screen:Secondary screen:Secondary screen:Secondary screen: The compounds that found active in primary screening were

similarly diluted to 500 μgmL-1, 250 μgmL-1, 200 μgmL-1, 125 μgmL-1, 100 μgmL-

1, 50 μgmL-1, 25 μgmL-1, 12.5 μgmL-1, 6.25 μgmL-1, 3.125 μgmL-1 and 1.5625

μgmL-1 concentrations.

A primary screening was conducted at 250 µg/ml against M. tuberculosis

H37Rv following a Lowenstein-Jensen (L-J) MIC method.18 Test compounds were

added to liquid L-J medium and then media were sterilized by inspissation

Chapter 5 252

method. A culture of M. tuberculosis H37Rv grown on L-J medium was harvested

in 0.85% saline in bijou bottles. DMSO was used as vehicle to get a desired

concentration. These tubes were then incubated at 37°C for 24 h followed by

streaking of M. tuberculosis H37Rv (5 × 104 bacilli per tube). These tubes were

then incubated at 37°C. Growth of bacilli was seen after 12, 22, and finally 28

days incubation. Tubes having the compounds were compared with control

tubes where medium alone was incubated with M. tuberculosis H37Rv. The

concentration at which complete inhibition of colonies occurred was taken as

active concentration of test compound. The standard strain M. tuberculosis

H37Rv was tested with known drugs isoniazide and rifampicin. The screening

test results are summarized as % inhibition relative to standard drugs isoniazide

and rifampicin. Compounds effecting < 90% inhibition in the primary screen

were not evaluated further. Compounds showed at least 90 % inhibition in the

primary screen was re-tested at lower concentration (MIC) in a L-J medium.

5.35.35.35.3 Antioxidant Antioxidant Antioxidant Antioxidant activityactivityactivityactivity

5.3.15.3.15.3.15.3.1 IntroductionIntroductionIntroductionIntroduction

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

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’’.19 The term antioxidant originally was used to refer

specifically to a chemical that prevents the oxygen consumption. In the late 19th

and 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

Chapter 5 253

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.

5.3.25.3.25.3.25.3.2 Free radicals (Free radicals (Free radicals (Free radicals (FRsFRsFRsFRs))))20

They are atomic or molecular species with unpaired electrons on an

otherwise 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 of

bacteria 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

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.

5.3.35.3.35.3.35.3.3 Reactive oxygen species Reactive oxygen species Reactive oxygen species Reactive oxygen species (ROS)(ROS)(ROS)(ROS)21

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

Chapter 5 254

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.

5.3.45.3.45.3.45.3.4 Clasification and general characteristic of antioxidantsClasification and general characteristic of antioxidantsClasification and general characteristic of antioxidantsClasification and general characteristic of antioxidants

Antioxidant can be clasified as

(a) Enzymatic and Non(a) Enzymatic and Non(a) Enzymatic and Non(a) Enzymatic and Non----Enzymatic antioEnzymatic antioEnzymatic antioEnzymatic antioxidantxidantxidantxidant

(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(b) Sources of the antioxidants(b) Sources of the antioxidants(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)

Characteristics of antioxidantsCharacteristics of antioxidantsCharacteristics of antioxidantsCharacteristics of antioxidants

The 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

Chapter 5 255

due to its stability. The antioxidant free radicals can also react with lipid free

radicals to form stable complex compounds.

Mechanism of antioxidantsMechanism of antioxidantsMechanism of antioxidantsMechanism of antioxidants

Mechanism 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).

Factors affecting the efficiency of antioxidantFactors affecting the efficiency of antioxidantFactors affecting the efficiency of antioxidantFactors 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.

Fig 5.1Fig 5.1Fig 5.1Fig 5.1 Reaction of antioxidants with radicals.

Ideal antioxidantsIdeal antioxidantsIdeal antioxidantsIdeal 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

� Economical

� Not absorbable by the body

5.3.55.3.55.3.55.3.5 Methods of antioxidant assayMethods of antioxidant assayMethods of antioxidant assayMethods of antioxidant assay

Various methods have been reported for measuring total antioxidants

power such as the total peroxy radical trapping parameter assay,22 the ferric

reducing antioxidant power (FRAP) method,23 the phycoerythrin fluorescence-

Chapter 5 256

based assay,24 the enhanced chemiluminescence assay,25 oxygen radical

absorbance capacity (ORAC) assay,26 trolox-equivalent antioxidant capacity

(TEAC) assay,27 1,1-diphenynl-2-picryl-hydrazyl (DPPH) antioxidant assay28 and

ABTS+ radical scavenging activity.29

5.3.65.3.65.3.65.3.6 Ferric reducing antioxidant power (FRAP)Ferric reducing antioxidant power (FRAP)Ferric reducing antioxidant power (FRAP)Ferric reducing antioxidant power (FRAP)23 assayassayassayassay

In present work, we have employed a FRAP assay, which measure the

capacity of test compound to reduce ferric tripyridyl-s-triazine (Fe(III)-TPTZ)

complex to the ferrous tripyridyltriazine (Fe(II)-TPTZ) at low pH. (Fe(II)-TPTZ)

has an intensive blue colour and can be monitored at 593 nm.

General Procedure for General Procedure for General Procedure for General Procedure for in vitroin vitroin vitroin vitro antioxidant activity by FRAP assayantioxidant activity by FRAP assayantioxidant activity by FRAP assayantioxidant activity by FRAP assay

Reagents:Reagents:Reagents:Reagents:

1) Acetate buffer, pH 3.6, 300 mmol/L; (3.1g sodium acetate trihydrate and 16

mL glacial acetic acid per liter of buffer solution).

2) 10 mmol/L, 2,4,6-tripyridyl-s-triazine (TPTZ) (MW 312.34) solution in 40

mmol/L HCl.

3) 20 mmol/L, FeCl3∙6H2O (MW 270.30) in distilled water (DW).

FRAP working solution: FRAP working solution: FRAP working solution: FRAP working solution: It can be prepared by mixing of 25 mL acetate buffer (1),

2.5 mL TPTZ solution and 2.5 mL FeCl3∙6H2O solution. The working solution

must always be a freshly prepared.

Sample solutionSample solutionSample solutionSample solution: 0.005 g dissolved in 25 mL DMF.

Standard solution: Standard solution: Standard solution: Standard solution: 1mmol of ascorbic acid (MW 176.13 g/mol) in 100 mL DW.

Procedure:Procedure:Procedure:Procedure: 400 µL sample or 200 µL standard was mixed with 3.0 mL FRAP

working solution and incubated the resulted mixture at 37˚C for 10 min. Its

absorbance was measured at 593 nm using respective blank solutions. The

antioxidant activity is expressed as ascorbic acid equivalent (mM/100 g of dried

compound).

Calculation:Calculation:Calculation:Calculation:

FRAP value can be calculated by following equation:30

Chapter 5 257

5555.4.4.4.4 AntiproliferativeAntiproliferativeAntiproliferativeAntiproliferative activityactivityactivityactivity

5.4.15.4.15.4.15.4.1 Proliferation and cancerProliferation and cancerProliferation and cancerProliferation and cancer::::31313131

Cellular life involves two main processes DNA synthesis accompanied

with mitosis (1) and cell differentiation (2) that produce new and specialized

cells, respectively. Normally, these processes undergo smoothly to occur, and

operate all cell cycle stages effectively with controlled mechanism, catalyzed by

chemical signals such as cellular growth factors and growth inhibitors. In a

damaged organ, due to decrease in inhibitor production, the cell proliferation

rate though continues to increase but until the replacement of lost cells only. In

cancer cells, however, above regulated processes are aberrant and cell may over

produce growth factors, under express growth inhibitors, or over growth factor

receptors that leads to a loss of normal growth control, which ultimately results

in increased cell proliferation. It is believed that proto-oncogenes, which control

normal proliferation and differentiation, are transformed into oncogenes that in

turn alter the cellular control mechanism and so it stimulates processes that

support cellular proliferation. Epidermal growth factor receptor (EGFR) is often

over-expressed in squamous carcinomas (breast, lung or bladder).

There are four important phages of the cell cycle known as G1, S, G2 and

M. In the first phase called G1, newly created cell is born. The tissue type

however decides how long it will remain in this stage. In normal proliferation

cell, it will quickly move into second stage S or synthesis. Here, nuclear DNA is

replicated into two copies. The next, third phase called G2 in which preparations

are made for final cycle M or mitosis phase. In cell cycle, the control point G1/S is

of major importance in understanding cancer and cancer treatment, relative to

other control point G2/M. Cell in G1 stage undergoes possible three processes,

i.e. it may enter the third phase, or enter into fifth stage Go (termed as quiescent,

or undergo terminally differentiation and die). In normal cell proliferation, cells

may undergo all these transformations but, there is no net change in number of

cells. However, in tumor, fraction of cells proliferating increases at expense of

quiescent or terminally differentiation such that there is a net increase in

number of cells.

Cells can be born into a proliferative state or nonproliferative state. In

tumors, new cells that are produced in hypoxic regions, i.e. areas typically nearer

Chapter 5 258

the centre of tumor and poorly perfuse, may be born into non-proliferative state.

These cells are not sensitive to drugs. Thus, when tumor is exposed to

chemotherapeutic agents, the outer most cells that are mainly in proliferative

state are destroyed. Those cells which were in non-proliferative state may then

be recruited into a proliferative state. Thus, several round of chemotherapeutic

agent are required in order to eradicate the tumor.

The water soluble protein called growth factors can be secreted by cells

capable of binding to the membrane of other cells at glycoprotein receptor. This

binding will initiate a series of several biochemical reactions, leading ultimately

in gene expression of growth factors. There are three main groups of growth

factors, which include endocrine growth factors, paracrine growth factors and

autocrine growth factors. First represents hormones such as thyroxine or

insulin, produced at one site but distributed throughout the body. Second have

multiple locations of production but act on nearby cell due to short plasma-half

life. Growth factors of third type are produced by cell and simulate the same cell.

Major role of this growth factor is to control the number of cells either in

proliferative or nonproliferative state.

The term neoplasm is defined as a new and diseased form of tissue

growth. Benign neoplasms are not cancerous but malignant neoplasms are,

called malignant tumor, a group of diseases involving abnormal cell growth with

the potential to invade or spread to other parts of the body. Benign tumors do

not spread to other parts of the body.32 Possible signs and symptoms include: a

new lump, abnormal bleeding, a prolonged cough, unexplained weight loss, and a

change in bowel movements, etc. While these symptoms may indicate cancer,

they may also occur due to other issues. 33 There are over 100 different known

cancers that affect humans.32

Common factors responsible for inviting cancer are tobacco use, obesity, a

poor diet, lack of physical activity, and addiction to alcohol34 Others include

certain infections, exposure to ionizing radiation, and environmental

pollutants.35 In the developing world nearly 20% of cancers are due to infections

such as hepatitis B, hepatitis C, and human papillomavirus. These factors act, at

least partly, by changing the genes of a cell. Typically many such genetic changes

are required before cancer develops.36 Approximately 5–10% of cancers are due

Chapter 5 259

to genetic defects inherited from a person's parents.37 Cancer can be detected by

certain signs and symptoms or screening tests. It is then typically investigated

further by medical imaging and confirmed by biopsy.38

In 2012 about 14.1 million new cases of cancer occurred globally. It

caused about 8.2 million deaths or 14.6% of all human deaths.39 The most

common types of cancer in males are lung cancer, prostate cancer, colorectal

cancer, and stomach cancer, and in females, breast cancer, colorectal cancer, lung

cancer, and cervical cancer. Skin account for at least 40% of cases.40 In

children, acute lymphoblastic leukaemia and brain tumors are most common.41

The risk of cancer increases significantly with age and many cancers occur more

commonly in developed countries.

5.4.25.4.25.4.25.4.2 Methods of antioxidant assayMethods of antioxidant assayMethods of antioxidant assayMethods of antioxidant assay

Various methods have been available for measuring in vitro

antiproliferative activity such as MTT assay 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyl-2H-tetrazolium bromide,42 MTS assay MTS-based cell titer 96

nonradioactivity cell proliferation assay (3-(4,5-dimethylthiazol-2-yl)-5-(3-

carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium),43 statistical

analysis, Cell culture, ATP and DNA Assays,44 and sulforhodamine B (SRB)

assay.45

5.4.5.4.5.4.5.4.3333 SulforhodamineSulforhodamineSulforhodamineSulforhodamine B (SRB) assayB (SRB) assayB (SRB) assayB (SRB) assay::::

Antiproliferative assays for antitumor activity were performed in 96-well

plates using the National Cancer Institute (NCI) protocol.46 As a model to study

the anticancer activity of the synthetic compounds, we used six human solid

tumor cell lines A549 (non-small cell lung), HBL-100 (breast), HeLa (cervix),

SW1573 (non-small cell lung), T-47D (breast), and WiDr (colon). The in vitro

antiproliferative activity was evaluated after 48 h of drug exposure using the

sulforhodamine B (SRB) assay.45 According to NCI protocol; only compounds

soluble in DMSO at 40 mM were tested. The results expressed as GI50 (50%

growth inhibition) are given in Table 5.Table 5.Table 5.Table 5.10101010....

Chapter 5 260

5.5.5.5.5555 Biological sBiological sBiological sBiological screening tests resultscreening tests resultscreening tests resultscreening tests results

5.5.5.5.5555.1.1.1.1 AnAnAnAntimicrobial tests results:timicrobial tests results:timicrobial tests results:timicrobial tests results:

All newly synthesized compounds were tested for their in Vitro

antimicrobial, antituberculosis and antioxidant activities, and the results were

compared with that of standard drugs as listed below.

Table 5.3 Antimicrobial tests Table 5.3 Antimicrobial tests Table 5.3 Antimicrobial tests Table 5.3 Antimicrobial tests results of reference standard drugs (MIC, results of reference standard drugs (MIC, results of reference standard drugs (MIC, results of reference standard drugs (MIC, μμμμgmLgmLgmLgmL----1).1).1).1).

DrugsDrugsDrugsDrugs

GramGramGramGram----positive bacteriapositive bacteriapositive bacteriapositive bacteria GramGramGramGram----negative negative negative negative

bacteriabacteriabacteriabacteria FungiFungiFungiFungi

B.sB.sB.sB.s....

MTCCMTCCMTCCMTCC

441441441441

C.t.C.t.C.t.C.t.

MTCCMTCCMTCCMTCC

449449449449

S.pS.pS.pS.p. . . .

MTCCMTCCMTCCMTCC

1936193619361936

E.c.E.c.E.c.E.c.

MTCC MTCC MTCC MTCC

443443443443

S.t.S.t.S.t.S.t.

MTCCMTCCMTCCMTCC

98989898

V.c.V.c.V.c.V.c.

MTCCMTCCMTCCMTCC

3906390639063906

A.f.A.f.A.f.A.f.

MTCC MTCC MTCC MTCC

3008300830083008

C.aC.aC.aC.a

MTCC MTCC MTCC MTCC

227227227227

Gentamicin 1 5 0.5 0.05 5 5 − −

Ampicillin 250 250 100 100 100 100 − −

Chloramphenicol 50 50 50 50 50 50 − −

Ciprofloxacin 50 100 50 25 25 25 − −

Norfloxacin 100 50 10 10 10 10 − −

Nystatin − − − − − − 100 100

Griseofulvin − − − − − − 100 500

B.s.: Bacillus subtilis, C.t.: Clostridium tetani, S.p.: Streptococcus pneumoniae,

E.c.: Escherichia coli, S.t.: Salmonella typhi, V.c.: Vibrio cholerae,

A.f.: Aspergillus fumigatus, C.a.: Candida albicans

MTCC: Microbial Type Culture Collection

MIC: Minimum Inhibition Concentration

“−” represent drugs not tested.

Table 5.Table 5.Table 5.Table 5.4 % 4 % 4 % 4 % GGGGrowth inhibition rowth inhibition rowth inhibition rowth inhibition values values values values of standard antitubercular reference drugsof standard antitubercular reference drugsof standard antitubercular reference drugsof standard antitubercular reference drugs....

DrugsDrugsDrugsDrugs % Inhibition% Inhibition% Inhibition% Inhibition MIC (MIC (MIC (MIC (µµµµg/mL)g/mL)g/mL)g/mL)

Isoniazide 99 0.2

Rifampicin 98 40

� Drugs used for Drugs used for Drugs used for Drugs used for antioxidantantioxidantantioxidantantioxidant studiesstudiesstudiesstudies

Chapter 5 261

Ascorbic acid was used as a standard to determine total antioxidant

power of the compounds. Some 200 µL, 1 mmol solution of ascorbic acid was

tested. Optical density of solution was 1.457 at 593 nm, which was used to

calculate the FRAP value of other compounds.

5.5.5.5.5555.1.1.1.1.1.1.1.1 Biological screening test results of Biological screening test results of Biological screening test results of Biological screening test results of tetrahydrotetrahydrotetrahydrotetrahydro----/dihydrochromen/dihydrochromen/dihydrochromen/dihydrochromenoooo----

[4',3':4,5]pyrano[2,3[4',3':4,5]pyrano[2,3[4',3':4,5]pyrano[2,3[4',3':4,5]pyrano[2,3----cccc]pyrazoles]pyrazoles]pyrazoles]pyrazoles ((((Chapter 2Chapter 2Chapter 2Chapter 2, Section I, Section I, Section I, Section I).).).).

Table 5.5Table 5.5Table 5.5Table 5.5 displays in vitro antimicrobial screening test results, in MIC, of

all chromeno[4’,3’:4,5]pyrano[2,3-c]pyrazole derivatives BLCBLCBLCBLC1111‒25, ‒25, ‒25, ‒25, described

previously in Section I Section I Section I Section I of Chapter 2,Chapter 2,Chapter 2,Chapter 2, along with their antioxidant activity in FRAP

(ferric reducing antioxidant power). For claiming the biological potential of the

compound, moderate, good or excellent, MIC values of the compound were

directly compared at least with that of any of the standard reference drugs used

in the study, against respective bacterial species, refereeing Table 5.3Table 5.3Table 5.3Table 5.3 on page

260. It shows that compounds are rather anti-bacterial, and not anti-fungal, in

nature, as those having at least Ampicillin–like potency are relatively larger in

number, against each bacterial type. For example, all compounds except BLCBLCBLCBLC13131313

showed good resistivity against two Gram +ve Bacillus subtilis and Clostridium

tetani bacteria. In addition, about 30% of them have good activity against Gram

+ve Streptococcus pneumoniae and two Gram –ve Escherichia coli and

Salmonella typhi bacteria, with similar potency. The compound BLCBLCBLCBLC13131313 is although

very poor against Gram +ve Bacillus subtilis bacteria, it has noticeable potency

against Clostridium tetani bacteria, even better than that of standard Ampicillin,

reaching to that of Chloramphenicol and Norfloxacin. Same is true for BLCBLCBLCBLC3333––––5555 and

BLCBLCBLCBLC20,20,20,20, as they have relatively a good resistivity against Clostridium tetani

bacteria, resembling standard drug Ciprofloxacin in the activity. BLCBLCBLCBLC15151515 on the

other hand resembles standard drug Chloramphenicol in activity, but against

Gram –ve Escherichia coli bacteria. Other compounds with potency higher than

Chapter 5 262

Ampicillin include Bacillus subtilis bacteria resistive BLCBLCBLCBLC1 1 1 1 and Streptococcus

pneumoniae bacteria resistive BLCBLCBLCBLC21212121 & BLCBLCBLCBLC25252525, with MIC close to that of two

standard drugs; Chloramphenicol and Ciprofloxacin. Compounds with the

Norfloxacin–equivalent potency were also found in the screening tests which

include BLCBLCBLCBLC2222, BLC, BLC, BLC, BLC19, 19, 19, 19, BLCBLCBLCBLC21, 21, 21, 21, BLCBLCBLCBLC23232323, , , , against Gram +ve Bacillus subtilis bacteria.

Compounds with potency more than Ampicillin but less than that of other

standard drugs were also found. For example, compounds BLCBLCBLCBLC4, 4, 4, 4, BLCBLCBLCBLC6,7, 6,7, 6,7, 6,7, BLCBLCBLCBLC16, 16, 16, 16,

BLCBLCBLCBLC20,2220,2220,2220,22 are active against Bacillus subtilis bacteria, and BLCBLCBLCBLC2,8, 2,8, 2,8, 2,8, BLCBLCBLCBLC12, 12, 12, 12, BLCBLCBLCBLC14,15, 14,15, 14,15, 14,15,

BLCBLCBLCBLC17, 17, 17, 17, BLCBLCBLCBLC21,25 21,25 21,25 21,25 against Clostridium tetani bacteria.

Although poor resistivity has been registered by majority of compounds

against fungi, BLCBLCBLCBLC1,6, 1,6, 1,6, 1,6, BLCBLCBLCBLC8,11,8,11,8,11,8,11, BLCBLCBLCBLC16,19, 16,19, 16,19, 16,19, BLCBLCBLCBLC23,2423,2423,2423,24 are not disappointing in this regard,

as they have at least standard drug Griseofulvin―equivalent activity against

Candida albicans fungi. The inhibition of M. tuberculosis H37Rv bacteria by all

compounds was also determined. Compounds BLCBLCBLCBLC9, 9, 9, 9, BLCBLCBLCBLC13131313 and BLCBLCBLCBLC19191919 showed

good antitubercular activity, with more than 90% growth inhibition.

Finally, compounds were tested for their ferric reducing antioxidant

power (FRAP). Among the compounds tested, BLCBLCBLCBLC14,1514,1514,1514,15 were distinguished with

higher FRAP values (>300 mM/100gm). FRAP values of other compounds

nevertheless don’t go below 200 mM/100gm, indicating that they also possess

good antioxidant power.

Examining all compounds structurally, it can be related that

polyheterocycles derived from 1-Ph/1-(4-MePh)-3-pyrazol-5-ones are relatively

good in antibacterial activity, except BLCBLCBLCBLC15151515. Compound BLCBLCBLCBLC15151515 was obtained using

N-(3-Cl-Ph)-3-methyl-pyrazol-5-one, but surprisingly it showed excellent results

against Escherichia coli bacteria.

5.5.5.5.5555....1.2 1.2 1.2 1.2 Biological screening test results ofBiological screening test results ofBiological screening test results ofBiological screening test results of aminoaminoaminoaminochromenchromenchromenchromen----annulated pyranoannulated pyranoannulated pyranoannulated pyrano––––

pyrazoles BLCpyrazoles BLCpyrazoles BLCpyrazoles BLC26―41 26―41 26―41 26―41 ((((ChaChaChaChapter 2, Section IIpter 2, Section IIpter 2, Section IIpter 2, Section II).).).).

Chapter 5 263

Reduction of nitro products BLCBLCBLCBLC1111––––16161616 into corresponding amino analogs has

been described in SectionSectionSectionSection----IIIIIIII of CCCChapter 2hapter 2hapter 2hapter 2. All these amino products, thus

obtained, were screened for their antibacterial and antifungal activities, in

addition to anti-oxidant activity. The results are shown in TableTableTableTable 5.65.65.65.6. The

reduction showed improvement in activity of many compounds. For example,

compound BLCBLCBLCBLC36 36 36 36 with amino group displayed better resistivity against both

Gram –ve Escherichia coli and Vibrio cholera bacteria than did their

corresponding NO2–derivative BLCBLCBLCBLC11111111, against same bacteria. Other examples

include BLCBLCBLCBLC29, 30 29, 30 29, 30 29, 30 (against Escherichia coli and Salmonella typhi bacteria), BLCBLCBLCBLC35,3835,3835,3835,38

(against Escherichia coli and Salmonella typhi bacteria), BLCBLCBLCBLC39393939 (against Bacillus

subtilis, Streptococcus pneumoniae and Escherichia coli bacteria), BLCBLCBLCBLC40 40 40 40 (against

Bacillus subtilis and Clostridium tetani bacteria), which showed better activity in

comparison with their corresponding nitro derivatives. Compounds with activity

improved against a single bacterial type include BLCBLCBLCBLC41414141 from BLCBLCBLCBLC16161616 (against

Bacillus subtilis bacteria), BLCBLCBLCBLC33333333 from BLCBLCBLCBLC8 8 8 8 (against Clostridium tetani bacteria),

BLCBLCBLCBLC28282828 from BLCBLCBLCBLC3333(against Streptococcus pneumoniae bacteria), BLBLBLBLCCCC29,3029,3029,3029,30 from

BLCBLCBLCBLC4,54,54,54,5 (against Escherichia coli bacteria) and BLCBLCBLCBLC31313131 from BLCBLCBLCBLC6666 (against Vibrio

cholerae bacteria). In case of nitro derivative BLCBLCBLCBLC13131313, however, the reduction

didn’t show any changes in activity against Clostridium tetani bacteria. The

similar trend in MIC values can be seen in case of antifungal activity on reduction

of some nitro derivatives. For examples, BLCBLCBLCBLC31,3231,3231,3231,32, BLC, BLC, BLC, BLC34343434 and BLCBLCBLCBLC37373737––––39393939 with lower

MICs (in comparison with corresponding nitro derivatives BLCBLCBLCBLC6,76,76,76,7, BLC, BLC, BLC, BLC9,9,9,9, BLCBLCBLCBLC12121212––––14141414)

emerged as promising antifungal agents, against Candida albicans fungi.

Chapter 5 264

25

25

25

25

BL

CB

LC

2

50

25

02

00

20

06

2.5

62

.5

20

02

00

BL

CB

LC

–

H

H

P

h

25

02

50

2

00

20

0

62

.56

2.5

2

50

2

50

2

00

20

0

25

0

>5

00

6

9

25

7.3

24

24

24

24

2B

LB

LCC

20

02

00

12

51

25

BL

BL

CC

–

NO

H

4

-Me

Ph

2

50

2

50

2

50

2

00

20

0

12

51

25

2

50

>

50

0

50

0

54

2

66

.5

23

23

23

23

2B

LC

BL

C1

25

12

51

00

10

01

00

10

02

00

20

0B

LC

BL

C

–

NO

H

3

-ClP

h

12

51

25

2

50

2

50

1

00

10

0

10

01

00

2

00

20

0

50

0

50

0

87

2

14

.8

22

22

22

22

2B

LC

BL

C2

00

20

0B

LC

BL

C

–

NO

H

P

h

20

02

00

2

50

2

50

2

50

5

00

2

50

>

50

0

>5

00

5

6

23

0.4

21

21

21

21

2B

LC

BL

C1

00

10

02

00

20

06

2.5

62

.5

20

02

00

20

02

00

BL

CB

LC

–

H

N

O

4-M

eP

h

10

01

00

2

00

20

0

62

.56

2.5

2

50

2

50

2

00

20

0

20

02

00

>

50

0

40

2

63

.5

20

20

20

20

2B

LC

BL

C2

00

20

01

25

12

51

00

10

02

00

20

0B

LC

BL

C

–

H

NO

3

-ClP

h

20

02

00

1

25

12

5

25

0

10

01

00

2

00

20

0

25

0

25

0

>5

00

1

2

25

3.0

19

19

19

19

2B

LC

BL

C1

00

10

01

00

10

02

00

20

09

39

3

BL

CB

LC

–

H

N

O

Ph

1

00

10

0

10

01

00

5

00

5

00

5

00

2

00

20

0

>5

00

5

00

9

39

3

21

7.8

18

18

18

18

BL

CB

LC

20

02

00

12

51

25

20

02

00

BL

CB

LC

M

e

H

H

Ph

2

50

2

50

2

00

20

0

12

51

25

2

00

20

0

25

0

>5

00

>

50

0

56

2

20

.4

17

17

17

17

BL

CB

LC

20

02

00

10

01

00

20

02

00

BL

CB

LC

H

H

H

P

h

25

0

20

02

00

1

00

10

0

25

0

50

0

20

02

00

2

50

>

50

0

38

2

50

.4

16

16

16

16

22

BL

CB

LC

20

02

00

12

51

25

20

02

00

BL

CB

LC

M

e

NO

H

2

,5-C

lP

h

20

02

00

2

50

1

25

12

5

20

02

00

2

50

5

00

5

00

5

00

3

9

21

9.3

15

15

15

15

2B

LC

BL

C2

00

20

06

2.5

62

.5

10

01

00

12

51

25

33

9.5

33

9.5

B

LC

BL

C

Me

N

O

H

3-C

lPh

2

50

2

00

20

0

25

0

62

.56

2.5

1

00

10

0

12

51

25

5

00

>

50

0

57

3

39

.53

39

.5

14

14

>5

00

1

41

42

BL

CB

LC

20

02

00

31

0.4

31

0.4

B

LC

BL

C

Me

N

O

H

4-M

eP

h

25

0

20

02

00

5

00

5

00

5

00

2

50

5

00

2

3

31

0.4

31

0.4

13

13

13

13

2B

LC

BL

C6

2.5

62

.5

10

01

00

92

92

B

LC

BL

C

Me

N

O

H

Ph

5

00

6

2.5

62

.5

25

0

25

0

25

0

10

01

00

>

50

0

>5

00

9

29

2

23

7.1

12

12

12

12

22

BL

CB

LC

20

02

00

10

01

00

20

02

00

BL

CB

LC

H

N

O

H

2,5

-Cl

Ph

2

50

2

00

20

0

10

01

00

2

50

5

00

2

00

20

0

25

0

>5

00

3

8

28

7.3

11

11

11

11

2B

LC

BL

C2

00

20

01

00

10

0B

LC

BL

C

H

NO

H

3

-ClP

h

50

0

25

0

20

02

00

1

00

10

0

25

0

25

0

>5

00

5

00

7

8

25

4.2

10

10

10

10

2B

LC

BL

C2

00

20

02

00

20

02

00

20

0B

LC

BL

C

H

NO

H

4

-Me

Ph

2

50

2

50

2

50

2

00

20

0

20

02

00

2

00

20

0

50

0

>5

00

7

4

26

0.3

99992

BL

CB

LC

20

02

00

12

51

25

20

02

00

91

91

B

LC

BL

C

H

NO

H

P

h

25

0

25

0

20

02

00

1

25

12

5

20

02

00

2

50

>

50

0

>5

00

9

19

1

22

0.5

88882

2B

LC

BL

C2

00

20

01

25

12

52

00

20

03

31

.73

31

.7

BL

CB

LC

M

e

H

NO

2

,5-C

lP

h

25

0

20

02

00

5

00

1

25

12

5

20

02

00

2

50

>

50

0

25

0

84

3

31

.73

31

.7

77772

BL

CB

LC

20

02

00

BL

CB

LC

M

e

H

NO

3

-ClP

h

20

02

00

2

50

5

00

5

00

5

00

2

50

>

50

0

>5

00

2

5

28

8.7

66662

BL

CB

LC

20

02

00

20

02

00

12

51

25

20

02

00

20

02

00

BL

CB

LC

M

e

H

NO

4

-Me

Ph

2

00

20

0

25

0

20

02

00

1

25

12

5

20

02

00

2

00

20

0

>5

00

5

00

8

8

20

2.4

55552

BL

CB

LC

10

01

00

20

02

00

20

02

00

12

51

25

20

02

00

BL

CB

LC

M

e

H

NO

P

h

25

0

10

01

00

2

50

2

00

20

0

20

02

00

1

25

12

5

20

02

00

>

50

0

46

2

47

.5

44442

2B

LC

BL

C2

00

20

01

00

10

02

00

20

02

00

20

01

25

12

5B

LC

BL

C

H

H

NO

2

,5-C

lP

h

20

02

00

1

00

10

0

20

02

00

2

00

20

0

25

0

12

51

25

>

50

0

>5

00

3

6

23

4.5

33332

BL

CB

LC

10

01

00

10

01

00

BL

CB

LC

H

H

N

O

3-C

lPh

2

50

1

00

10

0

25

0

25

0

25

0

10

01

00

>

50

0

>5

00

1

4

26

3.6

22222

BL

CB

LC

12

51

25

2

00

20

01

00

10

0B

LC

BL

C

H

H

NO

4

-Me

Ph

1

25

12

5

20

02

00

1

00

10

0

50

0

50

0

25

0

50

0

>5

00

1

2

21

9.1

11112

BL

CB

LC

6

2.5

62

.5

12

51

25

10

01

00

10

01

00

20

02

00

B

LC

BL

C

H

H

NO

P

h

62

.56

2.5

1

25

12

5

10

01

00

2

50

2

50

1

00

10

0

>5

00

2

00

20

0

74

2

46

.9

. .A A. .

C C. .

A A. .C C

. .F F. .

A A. .

F F. .A A

. .C C. .

V V. .

C C. .V V

. .T T. .S S

. .T T. .S S

. .C C. .

E E. .

C C. .E E

. .P P. .S S

. .P P. .S S

. .T T. .

C C. .

T T. .C C

. .S S. .B B

. .S S. .B B

1122

3311

2233

RR

RR

RR

RR

mM

/1

00

gm

M/

10

0g

m

M/

10

0g

mM

/1

00

g

oof

inh

ibit

ion

f in

hib

itio

nRR

RR

RR

RR

oo

f in

hib

itio

nf

inh

ibit

ion

% G

row

th

% G

row

th

% G

row

th

% G

row

th

Gra

m p

osi

tiv

e b

act

eri

aG

ram

po

siti

ve

ba

cte

ria

G

ram

ne

ga

tiv

e b

act

eri

aG

ram

ne

ga

tiv

e b

act

eri

a

Fu

ng

iF

un

gi

Gra

m p

osi

tiv

e b

act

eri

aG

ram

po

siti

ve

ba

cte

ria

G

ram

ne

ga

tiv

e b

act

eri

aG

ram

ne

ga

tiv

e b

act

eri

a

Fu

ng

iF

un

gi

--11--11g

μg g

μ μg

μA

nti

mic

rob

ial

Act

ivit

y (

MIC

, A

nti

mic

rob

ial

Act

ivit

y (

MIC

, m

Lm

L))

An

ti T

BA

nti

TB

A

nti

ox

ida

nt

An

tio

xid

an

t A

nti

mic

rob

ial

Act

ivit

y (

MIC

, A

nti

mic

rob

ial

Act

ivit

y (

MIC

, m

Lm

L))

An

ti T

BA

nti

TB

A

nti

ox

ida

nt

An

tio

xid

an

t S

ub

stit

uti

on

sS

ub

stit

uti

on

s S

ub

stit

uti

on

sS

ub

stit

uti

on

s C

om

p.

Co

mp

. C

om

p.

Co

mp

.

1―

25

1―

25

1―

25

1―

25

cT

ab

le

Ta

ble

55..55

BL

CB

LC

Ta

ble

T

ab

le 55

..55 B

iolo

gic

al

scre

en

ing

te

st r

esu

lts

of

Sy

nth

esi

s o

f ch

rom

en

o[4

', 3

’:4

, 5]p

yra

no

[2,3

-]p

yra

zole

s B

LC

BL

C.

Chapter 5 265

41

41

41

41

22

BL

CB

LC

10

01

00

20

02

00

20

02

00

20

02

00

20

02

00

BL

CB

LC

M

e

NH

H

2

,5-C

lP

h

10

01

00

2

50

2

00

20

0

20

02

00

2

00

20

0

25

0

>5

00

2

00

20

0

35

2

18

.8

40

40

40

40

2B

LC

BL

C1

25

12

51

25

12

52

00

20

01

25

12

5B

LC

BL

C

Me

N

H

H

3-C

lPh

1

25

12

5

12

51

25

2

50

2

50

2

00

20

0

12

51

25

>

50

0

>5

00

8

4

24

3.1

39

39

39

39

2B

LC

BL

C1

25

12

52

00

20

01

00

10

01

00

10

0B

LC

BL

C

Me

N

H

H

4-M

eP

h

12

51

25

2

00

20

0

10

01

00

1

00

10

0

25

0

25

0

50

0

25

0

68

2

23

.9

38

38

38

38

2B

LC

BL

C2

00

20

06

2.5

62

.5

10

01

00

12

51

25

1

00

10

0B

LC

BL

C

Me

N

H

H

Ph

2

00

20

0

62

.56

2.5

2

50

1

00

10

0

12

51

25

1

00

10

0

>5

00

2

50

3

5

26

3.8

37

37

37

37

22

BL

CB

LC

20

02

00

20

02

00

10

01

00

20

02

00

10

01

00

BL

CB

LC

H

N

H

H

2,5

-Cl

Ph

2

00

20

0

20

02

00

1

00

10

0

20

02

00

1

00

10

0

25

0

>5

00

5

00

4

7

20

0.4

36

36

36

36

2B

LC

BL

C6

2.5

62

.5

10

01

00

12

51

25

BL

CB

LC

H

N

H

H

3-C

lPh

2

50

2

50

2

50

6

2.5

62

.5

10

01

00

1

25

12

5

>5

00

5

00

5

5

21

3.1

35

35

35

35

2B

LC

BL

C1

25

12

51

25

12

51

00

10

02

00

20

0B

LC

BL

C

H

NH

H

4

-Me

Ph

5

00

1

25

12

5

50

0

12

51

25

1

00

10

0

20

02

00

>

50

0

50

0

78

2

68

.6

34

34

34

34

2B

LC

BL

C1

25

12

5B

LC

BL

C

H

NH

H

P

h

25

0

12

51

25

2

50

2

50

2

50

2

50

5

00

>

50

0

23

2

63

.3

33

33

33

33

22

BL

CB

LC

10

01

00

12

51

25

20

02

00

20

02

00

BL

CB

LC

M

e

H

NH

2

,5-C

lP

h

50

0

10

01

00

5

00

1

25

12

5

20

02

00

2

00

20

0

>5

00

5

00

5

8

25

2.2

32

32

32

32

2B

LC

BL

C2

00

20

02

00

20

02

00

20

0B

LC

BL

C

Me

H

N

H

3-C

lPh

2

00

20

0

20

02

00

2

00

20

0

25

0

25

0

25

0

>5

00

5

00

3

6

22

4.1

31

31

31

31

2B

LC

BL

C2

00

20

01

25

12

5B

LC

BL

C

Me

H

N

H

4-M

eP

h

50

0

25

0

20

02

00

2

50

2

50

1

25

12

5

>5

00

2

50

7

7

24

3.5

30

30

30

30

2B

LC

BL

C2

00

20

01

25

12

51

00

10

01

00

10

02

00

20

0B

LC

BL

C

Me

H

N

H

Ph

2

00

20

0

25

0

25

0

12

51

25

1

00

10

0

10

01

00

2

00

20

0

>5

00

5

8

20

2.8

29

29

29

29

22

BL

CB

LC

12

51

25

10

01

00

12

51

25

10

01

00

20

02

00

BL

CB

LC

H

H

N

H

2,5

-Cl

Ph

2

50

1

25

12

5

25

0

10

01

00

1

25

12

5

10

01

00

2

00

20

0

>5

00

6

2

25

4.8

28

28

28

28

2B

LC

BL

C2

00

20

01

00

10

01

00

10

0B

LC

BL

C

H

H

NH

3

-ClP

h

20

02

00

1

00

10

0

10

01

00

2

50

5

00

2

50

5

00

>

50

0

34

2

17

.0

27

27

27

27

2B

LC

BL

C2

00

20

0B

LC

BL

C

H

H

NH

4

-Me

Ph

2

50

2

50

2

50

2

50

2

00

20

0

25

0

25

0

>5

00

4

1

25

0.2

26

26

26

26

2B

LC

BL

C2

00

20

02

00

20

02

00

20

0B

LC

BL

C

H

H

NH

P

h

25

0

25

0

25

0

20

02

00

2

00

20

0

20

02

00

>

50

0

>5

00

6

2

20

1.0

. .A A. .

C C. .

A A. .C C

. .F F. .

A A. .

F F. .A A

. .C C. .

V V. .

C C. .V V

. .T T. .S S

. .T T. .S S

. .C C. .

E E. .

C C. .E E

. .P P. .S S

. .P P. .S S

. .T T. .

C C. .

T T. .C C

. .S S. .B B

. .S S. .B B

11

2233

1122

33RR

RR

RR

RR

mM

/1

00

gm

M/

10

0g

m

M/

10

0g

mM

/1

00

g

inh

ibit

ion

inh

ibit

ion

RR

RR

RR

RRin

hib

itio

nin

hib

itio

n

% G

row

th o

f %

Gro

wth

of

% G

row

th o

f %

Gro

wth

of

Gra

m p

osi

tiv

e b

act

eri

aG

ram

po

siti

ve

ba

cte

ria

Gra

m n

eg

ati

ve

ba

cte

ria

Gra

m n

eg

ati

ve

ba

cte

ria

Fu

ng

iF

un

gi

Gra

m p

osi

tiv

e b

act

eri

aG

ram

po

siti

ve

ba

cte

ria

G

ram

ne

ga

tiv

e b

act

eri

aG

ram

ne

ga

tiv

e b

act

eri

a

Fu

ng

iF

un

gi

--11--11g

μg g

μ μg

μA

nti

mic

rob

ial

AA

nti

mic

rob

ial

Act

ivit

y (

MIC

, ct

ivit

y (

MIC

, m

Lm

L))

An

ti T

BA

nti

TB

A

nti

ox

ida

nt

An

tio

xid

an

t A

nti

mic

rob

ial

AA

nti

mic

rob

ial

Act

ivit

y (

MIC

, ct

ivit

y (

MIC

, m

Lm

L))

An

ti T

BA

nti

TB

A

nti

ox

ida

nt

An

tio

xid

an

t S

ub

stit

uti

on

sS

ub

stit

uti

on

s S

ub

stit

uti

on

sS

ub

stit

uti

on

s C

om

p.

Co

mp

. C

om

p.

Co

mp

.

2266

―4

―4

1122

66―

4―

411

Ta

ble

T

ab

le 55

..66

BB

LLCC

Ta

ble

T

ab

le 55

..66 B

iolo

gic

al

scre

en

ing

te

st r

esu

lts

of

am

ino

-ch

rom

en

-an

nu

late

d p

yra

no

–p

yra

zole

s BB

LLCC

.

Chapter 5 266

5.5.5.5.5555....1.3 1.3 1.3 1.3 Biological screening test results Biological screening test results Biological screening test results Biological screening test results of of of of chromenchromenchromenchromen––––annulated pyranoannulated pyranoannulated pyranoannulated pyrano––––fused fused fused fused

pyrazoles/ pyrimidinespyrazoles/ pyrimidinespyrazoles/ pyrimidinespyrazoles/ pyrimidines BLBLBLBLCCCC42424242――――83838383 ((((Chapter Chapter Chapter Chapter 3333).).).).

Antimicrobial screening test results of chromen―annulated pyrano-fused

pyrazoles BLCBLCBLCBLC42424242––––71717171 that have been described in Chapter 3Chapter 3Chapter 3Chapter 3 are shown in Table 5.7Table 5.7Table 5.7Table 5.7,

along with FRAP values. For claiming the biological potential of the compound,

moderate, good or excellent, MIC values of the compound were directly

compared at least with that of any of the standard reference drugs used in the

study, against respective bacterial species, refereeing Table 5.3Table 5.3Table 5.3Table 5.3 on page 260.

Considering compounds having at least Ampicillin–equivalent potency, it can be

seen that many compounds displayed good anti-bacterial potential. Among them,

resistivity of BLCBLCBLCBLC42424242, BLC, BLC, BLC, BLC49494949, BLC, BLC, BLC, BLC53,5453,5453,5453,54 and BLCBLCBLCBLC62626262 was recorded even with MIC lower

than that of standard Ampicilin against Gram +ve Bacillus subtilis and

Clostridium tetani bacteria. From MIC values, one can put all these candidates

very close to standard Ciprofloxacin (against Clostridium tetani bacteria), and

except BLCBLCBLCBLC53535353 very close to standard Norfloxacin drug (against Bacillus subtilis

bacteria), in the activity. With MIC value further lower, compound BLCBLCBLCBLC53535353

resembled more potent standard drug Chloramphenicol, against Bacillus subtilis

bacteria. Compounds showed better resistivity against only Bacillus subtilis

bacteria include BLCBLCBLCBLC47, 47, 47, 47, BLCBLCBLCBLC50,51, 50,51, 50,51, 50,51, BLCBLCBLCBLC61, 61, 61, 61, BLCBLCBLCBLC66, 66, 66, 66, BLCBLCBLCBLC69 69 69 69 and BLCBLCBLCBLC71717171.... All candidates

except BLCBLCBLCBLC69696969 and BLCBLCBLCBLC71717171 have standard Norfloxacin-like activity, and those having

resistivity against Clostridium tetani bacteria include BLCBLCBLCBLC52 52 52 52 and BLCBLCBLCBLC59, 59, 59, 59, which

have Ciprofloxacin–equivalent activity. Compounds BLCBLCBLCBLC69696969 and BLCBLCBLCBLC71717171 with MIC

value again lower resemble both the standard drugs; Chloramphenicol and

Ciprofloxacin, in activity against Bacillus subtilis bacteria. Compounds which

showed resistivity against a spectrum of more than one bacterial type were also

Chapter 5 267

found, with at least Ampicillin-equivalent potency. In this context, compound

BLCBLCBLCBLC54 54 54 54 showed excellent inhibitory action against a spectrum of maximum five

bacterial types, such as Bacillus subtilis, Clostridium tetani, Streptococcus

pneumoniae, Escherichia coli and Salmonella typhi. Next come compounds BLCBLCBLCBLC44444444

and BLCBLCBLCBLC64646464 (against Bacillus subtilis, Clostridium tetani, Streptococcus

pneumoniae and Salmonella typhi bacteria), BLCBLCBLCBLC46464646 (against Bacillus subtilis,

Clostridium tetani, Escherichia coli and Vibrio cholerae bacteria), BLCBLCBLCBLC48484848 and

BLCBLCBLCBLC71717171 (against Bacillus subtilis, Clostridium tetani, Escherichia coli and

Salmonella typhi bacteria,) BLCBLCBLCBLC51515151(against Bacillus subtilis, Clostridium tetani,

Streptococcus pneumoniae, and Escherichia coli bacteria), BLCBLCBLCBLC57575757 (against

Bacillus subtilis, Clostridium tetani, Escherichia coli and Vibrio cholerae

bacteria) and BLCBLCBLCBLC70707070(against Bacillus subtilis, Escherichia coli, Salmonella typhi

and Vibrio cholerae bacteria). Similarly, compounds BLCBLCBLCBLC49494949,50,50,50,50, BLCBLCBLCBLC52,53, 52,53, 52,53, 52,53, BLCBLCBLCBLC55,5655,5655,5655,56

and BLCBLCBLCBLC59595959 covered a maximum three bacterial types in general. Compounds with

potency more than Ampicillin but less than other standard drugs were also

found in the study. Examples include BLCBLCBLCBLC44,46, 44,46, 44,46, 44,46, BLCBLCBLCBLC52, 52, 52, 52, BLCBLCBLCBLC55,56, 55,56, 55,56, 55,56, BLCBLCBLCBLC60, 60, 60, 60, BLCBLCBLCBLC64,65,64,65,64,65,64,65, BLCBLCBLCBLC68 68 68 68

(against Bacillus subtilis bacteria) and BLCBLCBLCBLC46,47,46,47,46,47,46,47, BLCBLCBLCBLC55, 55, 55, 55, BLCBLCBLCBLC58, 58, 58, 58, BLCBLCBLCBLC60, 60, 60, 60, BLCBLCBLCBLC68 68 68 68 (against

Clostridium tetani bacteria).

Analyzing antifungal test results, it is noted that BLCBLCBLCBLC47,48,47,48,47,48,47,48, BLCBLCBLCBLC52, 52, 52, 52, BLCBLCBLCBLC59,60,59,60,59,60,59,60,

and BLCBLCBLCBLC69696969, have Griseofulvin–equivalent activity, against Candida albicans fungi.

Compounds with more potency than this standard drug include BLCBLCBLCBLC43434343,,,, BLCBLCBLCBLC53 53 53 53 and

BLCBLCBLCBLC56565656. The test results against Aspergillus fumigatus fungi are surprisingly very

poor.

All compounds were screened against M. tuberculosis H37Rv bacteria, for

their possible antitubercular activity. The inhibitory action was determined as %

Growth inhibition. Compounds BLCBLCBLCBLC46 46 46 46 and BLCBLCBLCBLC54545454 revealed more than 90 % Growth

inhibition, indicating they are potential antitubercular agents.

Ferric reducing antioxidant power (FRAP) value of all the compounds lies

in the 93–207(mM/100gm) range, indicating they posses moderate to good

antioxidant activity.

Table 5.8 Table 5.8 Table 5.8 Table 5.8 displays MIC values of all chromen–annulated pyrano–fused

pyrimidine derivatives BLCBLCBLCBLC72727272––––83838383 against various bacteria and fungi, along with

their FRAP values. It showed majority of compounds have antibacterial potential

Chapter 5 268

that is at least comparable to that of standard Ampicillin drug. It is noted that

Ampicillin has lowest antibacterial resistance in comparison with other standard

drugs used in the study. Candidates with potency more than standard Ampicillin

drug include BLCBLCBLCBLC72―76, 72―76, 72―76, 72―76, and BLCBLCBLCBLC79―8279―8279―8279―82. . . . Among them, BLCBLCBLCBLC76767676 with lower MIC value

(~100µgmL-1) showed Norfloxacin-like potential against Bacillus subtilis

bacteria, and Ciprofloxacin-like resistivity against Clostridium tetani bacteria.

Compound BLCBLCBLCBLC73737373, on the other hand, revealed Ciprofloxacin-equivalent activity

against both the bacteria. Additionally, however, it has Chloramphenicol-

equivalent activity against former Bacillus subtilis bacteria. Compounds with

activity against only Bacillus subtilis bacteria are BLCBLCBLCBLC74,7574,7574,7574,75 and BLCBLCBLCBLC82828282, all have

Norfloxacin-equivalent activity. Additionally, compound BLCBLCBLCBLC82828282 with MIC 62.5

µgmL–1 also resembles more potent standard drug Chloramphenicol in activity

against same bacteria.

When analyzed anti-fungal screening test results, it was observed that

none of the compounds has Nystatin–equivalent potency against any of the

fungal species used. It was however, at least Griseofulvin–equivalent potency

was recorded for BLCBLCBLCBLC73, 73, 73, 73, BLCBLCBLCBLC77,78, 77,78, 77,78, 77,78, BLCBLCBLCBLC81818181––––83 83 83 83 against Candida albicans fungus. Among

them BLCBLCBLCBLC82,83 82,83 82,83 82,83 have better resistivity then that of standard Griseofulvin, against

same fungal species, indicating that antifungal test results are not overall

disappointing.

As such, there has been no remarkable change in the bioactivity replacing

pyrazolone by pyrimidine in the fusion with chromenopyran unit. However,

compounds BBBBLCLCLCLC72,8372,8372,8372,83 with pyrimidine fusion, showed improved activity against

Bacillus subtilis bacteria.

Inhibitory power of all compounds was determined as % Growth of

inhibition, against M. tuberculosis H37Rv bacteria, for their antitubercular

activity study. Only one compound BLCBLCBLCBLC79 79 79 79 has registered highest 91% Growth of

inhibition, indicating moderate antitubercular activity.

FRAP value of compounds doesn’t exceed 137 mM/100gm, indicating

they have moderate antioxidant activity.

Chapter 5 269

…

……

……

……

……

.Co

nti

nu

ed

……

……

……

……

….C

on

tin

ue

d......

……

……

……

……

….C

on

tin

ue

d…

……

……

……

……

.Co

nti

nu

ed

......

64

64

64

64

2B

LC

BL

C

12

51

25

1

00

10

0

BL

CB

LC

3

-NO

M

e

3-C

lPh

2

00

2

50

1

25

12

5

20

0

10

01

00

2

50

>

50

0

>5

00

7

8

18

2.4

63

63

63

63

2B

LC

BL

C

25

02

50

B

LC

BL

C

3-N

O

Me

4

-Me

Ph

2

50

2

50

5

00

2

50

2

00

2

50

5

00

2

50

25

0

46

1

97

.4

62

62

62

62

2B

LC

BL

C

10

10

00

10

01

00

2

50

25

0

BL

CB

LC

3

-NO

M

e

Ph

1

01

000

1

00

10

0

25

0

25

0

25

0

20

0

>5

00

2

50

25

0

63

1

30

.6

61

61

61

61

22

BL

CB

LC

1

25

12

5

BL

CB

LC

5

-NO

M

e

2,5

-Cl

Ph

1

25

12

5

25

0

25

0

20

0

25

0

20

0

>5

00

5

00

6

9

17

9.8

60

60

60

60

2B

LC

BL

C

10

01

00

B

LC

BL

C

5-N

O

Me

2

-ClP

h

20

0

20

0

10

01

00

2

50

2

50

2

50

>

50

0

>5

00

7

6

19

1.2

59

59

59

59

2B

LC

BL

C

10

01

00

1

25

12

5

BL

CB

LC

5

-NO

M

e

3-C

lPh

2

50

1

00

10

0

20

0

25

0

25

0

12

51

25

>

50

0

50

0

54

2

05

.1

58

58

58

58

2B

LC

BL

C

BL

CB

LC

5

-NO

M

e

4-M

eP

h

25

0

20

0

25

0

25

0

50

0

20

0

>5

00

>

50

0

79

2

04

.6

57

57

57

57

2B

LC

BL

C

10

01

00

1

25

12

5

BL

CB

LC

5

-NO

M

e

Ph

2

50

2

50

2

50

1

00

10

0

25

0

12

51

25

>

50

0

>5

00

2

5

19

9.6

56

56

56

56

2B

LC

BL

C

10

01

00

1

00

10

0

25

02

50

B

LC

BL

C

Cl

Me

2

,5-C

lP

h

20

0

25

0

10

01

00

1

00

10

0

20

0

20

0

>5

00

2

50

25

0

37

1

93

.0

55

55

55

55

BL

CB

LC

1

00

10

0

BL

CB

LC

C

l M

e

2-C

lPh

2

00

2

00

1

00

10

0

20

0

25

0

25

0

>5

00

>

50

0

48

1

66

.4

54

54

54

54

BL

CB

LC

1

00

10

0

12

51

25

1

25

12

5

12

51

25

1

00

10

0

94

94

B

LC

BL

C

Cl

Me

3

-ClP

h

10

01

00

1

25

12

5

12

51

25

1

25

12

5

10

01

00

2

00

>

50

0

>5

00

9

49

4

17

7.6

53

53

53

53

BL

CB

LC

6

2.5

62

.5

10

01

00

1

25

12

5

25

02

50

B

LC

BL

C

Cl

Me

4

-Me

Ph

6

2.5

62

.5

10

01

00

2

00

2

50

2

50

1

25

12

5

50

0

25

02

50

3

8

19

3.0

52

52

52

52

BL

CB

LC

1

25

12

5

12

51

25

BL

CB

LC

C

l M

e

Ph

2

00

1

25

12

5

12

51

25

2

00

2

50

2

00

>

50

0

50

0

46

1

57

.8

51

51

51

51

2B

LC

BL

C

12

51

25

1

00

10

0

12

51

25

B

LC

BL

C

Me

M

e

2,5

-Cl

Ph

1

25

12

5

25

0

10

01

00

1

25

12

5

25

0

20

0

50

0

>5

00

1

8

93

.1

50

50

50

50

BL

CB

LC

1

25

12

5

BL

CB

LC

M

e

Me

2

-ClP

h

12

51

25

2

50

2

00

2

50

2

00

2

00

5

00

>

50

0

76

1

12

.8

49

49

49

49

BL

CB

LC

1

00

10

0

10

01

00

1

00

10

0

BL

CB

LC

M

e

Me

3

-ClP

h

10

01

00

1

00

10

0

20

0

20

0

10

01

00

2

00

>

50

0

>5

00

3

9

17

3.2

48

48

48

48

BL

CB

LC

1

25

12

5

10

01

00

BL

CB

LC

M

e

Me

4

-Me

Ph

2

50

2

50

2

00

1

25

12

5

10

01

00

2

50

>

50

0

50

0

26

2

04

.6

47

47

47

47

BL

CB

LC

1

00

10

0

10

01

00

BL

CB

LC

M

e

Me

P

h

10

01

00

2

00

2

00

2

00

2

50

1

00

10

0

>5

00

5

00

8

4

19

1.2

46

46

46

46

2B

LC

BL

C

12

51

25

1

00

10

0

93

93

B

LC

BL

C

H

Me

2

,5-C

lP

h

20

0

20

0

25

0

12

51

25

2

00

1

00

10

0

>5

00

>

50

0

93

93

1

32

.6

45

45

45

45

BL

CB

LC

B

LC

BL

C

H

Me

2

-ClP

h

25

0

25

0

20

0

25

0

25

0

20

0

>5

00

>

50

0

36

1

79

.8

44

44

44

44

BL

CB

LC

12

51

25

1

00

10

0

BL

CB

LC

H

M

e

3-C

lPh

2

00

2

50

1

25

12

5

20

0

10

01

00

2

50

>

50

0

>5

00

7

4

20

4.6

43

43

43

43

BL

CB

LC

25

02

50

B

LC

BL

C

H

Me

4

-Me

Ph

2

50

2

50

5

00

2

50

2

00

2

50

5

00

2

50

25

0

12

1

92

.6

42

42

42

42

BL

CB

LC

10

01

00

1

00

10

0

BL

CB

LC

H

M

e

Ph

1

00

10

0

10

01

00

2

50

2

50

2

50

2

00

>

50

0

>5

00

6

5

17

9.8

.A

. .A A

.A .

C. .

C C.

C . .

F F. .A A

. .F F. .

A A . .

C C. .V V

. .C C. .

V V . .

T T. .S S. .

T T. .S S . .

C C. .E E

. .C C. .

E E . .

P P. .S S. .

P P. .S S . .

T T. .C C

. .T T. .

C C . .S S. .

B B. .S S. .

B B2222

1111RR

RR

RR

RR

RR

RR

Gra

m p

osi

tiv

e b

act

eri

aG

ram

po

siti

ve

ba

cte

ria

% G

row

th o

f in

hib

itio

n

% G

row

th o

f in

hib

itio

n

Fu

ng

iF

un

gi

Gra

m p

osi

tiv

e b

act

eri

aG

ram

po

siti

ve

ba

cte

ria

%

Gro

wth

of

inh

ibit

ion

%

Gro

wth

of

inh

ibit

ion

F

un

gi

Fu

ng

i m

M/

10

0g

mM

/1

00

g

mM

/1

00

gm

M/

10

0g

An

tio

xid

an

tA

nti

ox

ida

nt

An

tio

xid

an

tA

nti

ox

ida

nt

inh

ibit

ion

inh

ibit

ion

in

hib

itio

nin

hib

itio

n

An

ti T

BA

nti

TB

A

nti

TB

An

ti T

B

--11--11g

μg g

μ μg

μS

ub

stit

uti

on

sS

ub

stit

uti

on

s A

nti

mic

rob

ial

Act

ivit

y (

MIC

, A

nti

mic

rob

ial

Act

ivit

y (

MIC

, m

Lm

L))

Su

bst

itu

tio

ns

Su

bst

itu

tio

ns

An

tim

icro

bia

l A

ctiv

ity

(M

IC,

An

tim

icro

bia

l A

ctiv

ity

(M

IC,

mL

mL

))

Co

mp

.C

om

p.

% G

row

th o

f %

Gro

wth

of

Co

mp

.C

om

p.

% G

row

th o

f %

Gro

wth

of

42

―7

14

2―

71

42

―7

14

2―

71

.T

ab

le 5

.T

ab

le 5

.77

B

LC

BL

CT

ab

le 5

.T

ab

le 5

.77 B

iolo

gic

al

scre

en

ing

te

st r

esu

lts

of

chro

me

n–

an

nu

late

d p

yra

no

–fu

sed

py

razo

les

BL

CB

LC

Chapter 5 270

83

83

83

83

BL

CB

LC

1

25

12

5

10

01

00

25

02

50

B

LC

BL

C

H

Et

Et

S

25

0

25

0

25

0

25

0

12

51

25

1

00

10

0

50

0

25

02

50

6

8

13

0.4

82

82

82

82

BL

CB

LC

6

2.5

62

.51

00

10

0

25

02

50

B

LC

BL

C

H

Et

Me

O

6

2.5

62

.5

20

0

20

0

50

0

10

01

00

2

00

5

00

2

50

25

0

56

9

7.2

81

81

81

81

2B

LC

BL

C

12

51

25

12

51

25

12

51

25

BL

CB

LC

3

-NO

M

e

Et

S

20

0

12

51

25

1

25

12

5

50

0

20

0

12

51

25

>

50

0

50

0

78

1

06

.4

80

80

80

80

2B

LC

BL

C

10

01

00

10

01

00

10

01

00

BL

CB

LC

3

-NO

M

e

Me

O

2

50

1

00

10

0

10

01

00

2

50

2

00

1

00

10

0

>5

00

>

50

0

54

9

0.2

79

79

79

79

2B

LC

BL

C

10

01

00

1

25

12

51

00

10

0B

LC

BL

C

5-N

O

Me

E

t S

5

00

1

00

10

0

25

0

12

51

25

2

00

1

00

10

0

>5

00

>

50

0

91

1

06

.6

78

78

78

78

2B

LC

BL

C

10

01

00

BL

CB

LC

5

-NO

M

e

Me

O

2

50

2

50

2

50

2

00

1

00

10

0

20

0

>5

00

5

00

4

3

93

.2

77

77

77

77

BL

CB

LC

1

25

12

5B

LC

BL

C

Cl

Me

E

t S

2

50

2

00

2

00

2

50

1

25

12

5

25

0

>5

00

5

00

2

6

11

3.2

76

76

76

76

BL

CB

LC

1

00

10

01

25

12

51

25

12

5B

LC

BL

C

Cl

Me

M

e

O

10

01

00

1

25

12

5

25

0

12

51

25

2

50

2

00

>

50

0

>5

00

8

4

10

4.6

75

75

75

75

BL

CB

LC

1

00

10

0B

LC

BL

C

Me

M

e

Et

S

10

01

00

2

00

2

50

2

00

2

00

2

50

>

50

0

>5

00

7

9

87

.0

74

74

74

74

BL

CB

LC

1

25

12

51

25

12

51

25

12

5B

LC

BL

C

Me

M

e

Me

O

1

25

12

5

20

0

20

0

12

51

25

1

25

12

5

50

0

25

0

>5

00

7

3

13

7.8

73

73

73

73

BL

CB

LC

6

2.5

62

.5

12

51

25

12

51

25

BL

CB

LC

H

M

e

Et

S

62

.56

2.5

1

25

12

5

12

51

25

2

50

2

00

2

50

5

00

5

00

3

8

11

3.2

72

72

72

72

BL

CB

LC

1

00

10

0B

LC

BL

C

H

Me

M

e

O

25

0

10

01

00

2

50

2

00

2

50

5

00

>

50

0

>5

00

4

5

91

.2

. .A A. .

C C. .

A A. .C C

. .F F. .

A A. .

F F. .A A

. .C C. .

V V. .

C C. .V V

. .T T. .S S

. .T T. .S S

. .C C. .

E E. .

C C. .E E

. .P P. .S S

. .P P. .S S

. .T T. .

C C. .

T T. .C C

. .S S. .B B

. .S S. .B B

1122

1122

RR

RR

RR

XX

Gra

m p

osi

tiv

e b

act

eria

Gra

m p

osi

tiv

e b

act

eria

G

ram

ne

ga

tiv

e b

act

eri

aG

ram

ne

ga

tiv

e b

act

eri

a

Fu

ng

iF

un

gi

Gra

m p

osi

tiv

e b

act

eria

Gra

m p

osi

tiv

e b

act

eria

G

ram

ne

ga

tiv

e b

act

eri

aG

ram

ne

ga

tiv

e b

act

eri

a

Fu

ng

iF

un

gi

mM

/1

00

gm

M/

10

0g

m

M/

10

0g

mM

/1

00

g

An

tio

xid

an

tA

nti

oxi

da

nt

An

tio

xid

an

tA

nti

oxi

da

nt

inh

ibit

ion

inh

ibit

ion

RR

RR

RR

XX

in

hib

itio

nin

hib

itio

n

An

ti T

BA

nti

TB

A

nti

TB

An

ti T

B

--11--11g

μg g

μ μg

μA

nti

mic

rob

ial

Act

ivit

y (

MIC

, A

nti

mic

rob

ial

Act

ivit

y (

MIC

, m

Lm

L))

An

tim

icro

bia

l A

ctiv

ity

(M

IC,

An

tim

icro

bia

l A

ctiv

ity

(M

IC,

mL

mL

)) S

ub

stit

uti

on

sS

ub

stit

uti

on

s S

ub

stit

uti

on

sS

ub

stit

uti

on

s C

om

p.

Co

mp

. %

Gro

wth

of

% G

row

th o

f C

om

p.

Co

mp

. %

Gro

wth

of

% G

row

th o

f

72

―8

37

2―

83

72

―8

37

2―

83

Ta

ble

5.

Ta

ble

5.88

BL

CB

LC

Ta

ble

5.

Ta

ble

5.88

Bio

log

ica

l sc

ree

nin

g t

est

re

sult

s o

f ch

rom

en

–a

nn

ula

ted

py

ran

o–

fuse

d p

yri

mid

ine

s B

LC

BL

C.

71

71

71

71

2B

LC

BL

C

62

.56

2.5

1

00

10

0

BL

CB

LC

H

E

t 2

,5-C

lP

h

62

.56

2.5

2

50

2

00

1

00

10

0

25

0

10

0

>5

00

>

50

0

84

1

93

.2

70

70

70

70

BL

CB

LC

1

00

10

0

12

51

25

1

25

12

5

BL

CB

LC

H

E

t 2

-ClP

h

25

0

50

0

25

0

10

01

00

1

25

12

5

12

51

25

>

50

0

>5

00

8

1

20

6.6

69

69

69

69

BL

CB

LC

6

2.5

62

.5

10

01

00

B

LC

BL

C

H

Et

3-C

lPh

6

2.5

62

.5

25

0

10

01

00

2

50

2

50

2

00

>

50

0

50

0

74

2

00

.2

68

68

68

68

BL

CB

LC

B

LC

BL

C

H

Et

4-M

eP

h

20

0

20

0

25

0

25

0

25

0

20

0

>5

00

>

50

0

46

1

78

.6

67

67

67

67

BL

CB

LC

1

25

12

5

BL

CB

LC

H

E

t P

h

25

0

25

0

20

0

12

51

25

2

50

2

50

>

50

0

>5

00

6

9

20

4.6

66

66

66

66

2B

LC

BL

C

10

01

00

B

LC

BL

C

3-N

O

Me

10

01

00

2

50

2

00

2

50

2

50

2

00

>

50

0

>5

00

8

9

15

7.2

65

65

65

65

2B

LC

BL

C

12

51

25

1

00

10

0

BL

CB

LC

3

-NO

M

e

2

00

2

50

1

25

12

5

20

0

10

01

00

2

50

>

50

0

>5

00

5

6

13

0.8

. .A A. .

C C. .

A A. .C C

. .F F. .

A A. .

F F. .A A

. .C C. .

V V. .

C C. .V V

. .T T. .S S

. .T T. .S S

. .C C. .

E E. .

C C. .E E

. .P P. .S S

. .P P. .S S

. .T T. .

C C. .

T T. .C C

. .S S. .B B

. .S S. .B B

22221111

RR

RR

RR

RR

RR

RR

Gra

m p

osi

tiv

e b

act

eri

aG

ram

po

siti

ve

ba

cte

ria

%

Gro

wth

of

inh

ibit

ion

%

Gro

wth

of

inh

ibit

ion

F

un

gi

Fu

ng

i G

ram

po

siti

ve

ba

cte

ria

Gra

m p

osi

tiv

e b

act

eri

a

% G

row

th o

f in

hib

itio

n

% G

row

th o

f in

hib

itio

n

Fu

ng

iF

un

gi

mM

/1

00

gm

M/

10

0g

m

M/

10

0g

mM

/1

00

g

An

tio

xid

an

tA

nti

ox

ida

nt

An

tio

xid

an

tA

nti

ox

ida

nt

inh

ibit

ion

inh

ibit

ion

in

hib

itio

nin

hib

itio

n

An

ti T

BA

nti

TB

A

nti

TB

An

ti T

B

--11--11g

μg g

μ μg

μS

ub

stit

uti

on

sS

ub

stit

uti

on

s A

nti

mic

rob

ial

Act

ivit

y (

MIC

, A

nti

mic

rob

ial

Act

ivit

y (

MIC

, m

Lm

L))

Su

bst

itu

tio

ns

Su

bst

itu

tio

ns

An

tim

icro

bia

l A

ctiv

ity

(M

IC,

An

tim

icro

bia

l A

ctiv

ity

(M

IC,

mL

mL

))

Co

mp

.C

om

p.

% G

row

th o

f %

Gro

wth

of

Co

mp

.C

om

p.

% G

row

th o

f %

Gro

wth

of

Ta

Tab

le 5

.b

le 5

.77 (

Co

nti

nu

ed

)(C

on

tin

ue

d)

Ta

Ta

ble

5.

ble

5.77

(C

on

tin

ue

d)

(Co

nti

nu

ed

)

Chapter 5 271

5.5.5.5.5555....1.41.41.41.4 Biological screening test results of Biological screening test results of Biological screening test results of Biological screening test results of thiochromeno[2,3thiochromeno[2,3thiochromeno[2,3thiochromeno[2,3----bbbb]quinoline]quinoline]quinoline]quinoline----

heterocycles BLCheterocycles BLCheterocycles BLCheterocycles BLC88884444––––111111111111 ((((Chapter 4Chapter 4Chapter 4Chapter 4).).).).

The in vitro biological screening test results of all thiochromeno[2,3-

b]quinoline-heterocycles BLCBLCBLCBLC84848484––––111111111111,,,, described in Chapter 4,Chapter 4,Chapter 4,Chapter 4, have been shown in

Table 5.9Table 5.9Table 5.9Table 5.9, along with results on their antioxidant activity study. For claiming the

biological potential of the compound, moderate, good or excellent, MIC values of

the compound were directly compared at least with that of any of the standard

reference drugs used in the study, against respective bacterial species, refereeing

Table 5.3Table 5.3Table 5.3Table 5.3 on page 260.

As can be seen in TableTableTableTable 5.95.95.95.9, a compound has minimum

Ampicillin‒equivalent resistivity, at least, against one of the bacterial species

used in the study, based on MIC values. It means that none of the compounds one

can find, with activity which is not comparable to that of standard drugs used. In

this context, maximum six bacterial species and one minimum species could be

controlled and inhibited effectively by a compound. Among the compounds

Chapter 5 272

tested, BLCBLCBLCBLC91919191 and BLCBLCBLCBLC98989898 showed good activity against all six bacteria. It has

Norfloxacin-equivalent potential against Bacillus subtilis bacteria and Ampicillin-

equivalent power against others. Next is BLCBLCBLCBLC101101101101, which can inhibit five different

bacterial species, i.e. all except Streptococcus pneumoniae bacteria, with

bioactivity comparable to those standards used in the study. It showed

Norfloxacin-like potency against Bacillus subtilis bacteria, Ciprofloxacin-like

resistivity against Clostridium tetani bacteria, Chloramphenicol-like activity

against Escherichia coli bacteria and Ampicillin-like behavior against both

Salmonella typhi and Vibrio cholerae bacteria. Compounds having significant

activity against maximum three or four different types of bacterial species

include, BLCBLCBLCBLC85858585, BLCBLCBLCBLC90909090, BLCBLCBLCBLC95959595,96969696, BLCBLCBLCBLC99999999, BLCBLCBLCBLC103103103103, and BLCBLCBLCBLC109,110109,110109,110109,110 (inhibited four

different bacterial species) BLCBLCBLCBLC86868686,87878787, BBBBLCLCLCLC100100100100, BLCBLCBLCBLC105105105105, BLCBLCBLCBLC109109109109 and BLCBLCBLCBLC111111111111 (inhibited

three bacterial species,) and revealed Norfloxacin-equivalent, Ciprofloxacin-

equivalent, Chloramphenicol-equivalent and Ampicillin-equivalent bioactivities.

Analyzing the activity results further, it follows that highest 10 compounds

revealed Norfloxacin-equivalent potency. BLCBLCBLCBLC84,85, 84,85, 84,85, 84,85, BLCBLCBLCBLC88, 88, 88, 88, BLCBLCBLCBLC90,91, 90,91, 90,91, 90,91, BLCBLCBLCBLC96,98, 96,98, 96,98, 96,98,

BLCBLCBLCBLC101,102, 101,102, 101,102, 101,102, and BLCBLCBLCBLC107,108107,108107,108107,108 among are active against Bacillus subtilis bacteria, while

BLCBLCBLCBLC110110110110 against Clostridium tetani bacteria. Compound BLCBLCBLCBLC110110110110 showed

additionally Chloramphenicol‒equivalent potency also, against same bacteria.

Both Chloramphenicol‒ and Ciprofloxacin‒equivalent potencies were revealed

by compounds BLCBLCBLCBLC85858585, BLC, BLC, BLC, BLC96969696 and BLCBLCBLCBLC111111111111 against Streptococcus pneumoniae

bacteria. BLCBLCBLCBLC101010101111, and BLCBLCBLCBLC105105105105, on the other hand, showed the Chloramphenicol-

equivalent potency against Escherichia coli bacteria. Finally Compound BLCBLCBLCBLC99999999

registered excellent potency against Salmonella typhi bacteria, in the line with

that of standard Chloramphenicol.

Although the anti-fungal activities of compounds are so poor, some of

them, BLCBLCBLCBLC89, 89, 89, 89, BLCBLCBLCBLC95,96, 95,96, 95,96, 95,96, BLCBLCBLCBLC106, 106, 106, 106, and BLCBLCBLCBLC110110110110 have revealed noticeable anti-fungal

activity, comparable directly to that of standard drug Griseofulvin, against

Candida albicans fungi. Moreover, compounds BLCBLCBLCBLC86,87, 86,87, 86,87, 86,87, BLCBLCBLCBLC91, 91, 91, 91, BLCBLCBLCBLC100,101, 100,101, 100,101, 100,101, and

BLCBLCBLCBLC103103103103 have shown even better resistivity power, in comparison with

Griseofulvin, against identical fungal species.

Chapter 5 273

Analyzing all the compounds structurally for bioactivity, it was concluded

that introduction of chloro-functionality in the polyheterocycles provided with

better bioactivity, in general.

The inhibitory effect of compounds towards M. tuberculosis H37Rv

bacteria has been expressed in terms of % Growth of Inhibition (Table 5.9Table 5.9Table 5.9Table 5.9)....

Among the compounds tested, at least three compounds were found with %

Growth of Inhibition more than 90%. For examples, BLCBLCBLCBLC106106106106, BLC, BLC, BLC, BLC95959595 and BLCBLCBLCBLC107, 107, 107, 107,

with 94 %, 91% and 92% Growth inhibition values respectively, revealed good

antitubercular activity.

Data on antioxidant activity study of the compounds are good to

moderate. However, with highest FRAP value (398), compound BLCBLCBLCBLC87878787 appeared

as potential antioxidant agent. Avoiding few compounds, majority of the

compounds revealed FRAP value around 200.

Chapter 5 274

11

11

11

11

11

11

BL

CB

LC

6

2.5

62

.5

12

51

25

1

25

12

5

BL

CB

LC

C

l M

e

–

20

0

20

0

62

.56

2.5

1

25

12

5

12

51

25

2

00

>

50

0

>5

00

3

3

19

1.2

1

10

11

01

10

11

0B

LC

BL

C

62

.56

2.5

1

00

10

0

12

51

25

1

00

10

0

BL

CB

LC

H

H

–

2

00

6

2.5

62

.5

20

0

10

01

00

1

25

12

5

10

01

00

5

00

5

00

8

7

19

6.0

1

09

10

91

09

10

9B

LC

BL

C

10

01

00

1

25

12

5

12

51

25

B

LC

BL

C

Cl

Me

–

2

00

2

50

2

00

1

00

10

0

12

51

25

1

25

12

5

20

0

>5

00

6

3

18

2.4

1

08

10

81

08

10

8B

LC

BL

C

10

01

00

B

LC

BL

C

H

H

–

10

01

00

2

00

2

50

2

50

2

00

2

50

2

50

>

50

0

38

1

69

.3

10

71

07

10

71

07

BL

CB

LC

1

25

12

5

10

01

00

1

25

12

5

92

92

B

LC

BL

C

Cl

–

N-P

h

12

51

25

2

00

1

00

10

0

12

51

25

2

00

2

50

>

50

0

>5

00

9

29

2

20

8.6

1

06

10

61

06

10

6B

LC

BL

C

10

01

00

9

49

4

BL

CB

LC

H

–

N

-Ph

2

50

2

50

5

00

1

00

10

0

25

0

50

0

50

0

50

0

94

94

2

06

.0

10

51

05

10

51

05

BL

CB

LC

6

2.5

62

.5

10

01

00

B

LC

BL

C

Cl

–

O

25

0

25

0

25

0

62

.56

2.5

2

00

1

00

10

0

>5

00

>

50

0

27

2

16

.6

10

41

04

10

41

04

BL

CB

LC

1

25

12

5

BL

CB

LC

H

–

O

2

50

5

00

2

00

1

25

12

5

20

0

25

0

>5

00

>

50

0

30

2

23

.9

10

31

03

10

31

03

BL

CB

LC

1

25

12

5

12

51

25

B

LC

BL

C

Cl

–

N-P

h

20

0

25

0

12

51

25

2

00

1

25

12

5

10

0

>5

00

2

50

5

6

22

5.5

1

02

10

21

02

10

2B

LC

BL

C

12

51

25

1

00

10

0

10

01

00

2

50

25

0

BL

CB

LC

H

–

N

-Ph

1

25

12

5

20

0

10

01

00

1

00

10

0

25

02

50

2

50

>

50

0

>5

00

8

7

20

3.8

1

01

10

11

01

10

1B

LC

BL

C

10

01

00

1

00

10

0

62

.56

2.5

1

25

12

5

12

51

25

B

LC

BL

C

Cl

–

O

10

01

00

1

00

10

0

20

0

62

.56

2.5

1

25

12

5

12

51

25

>

50

0

25

0

52

2

04

.2

10

01

00

10

01

00

BL

CB

LC

1

00

10

0

10

01

00

B

LC

BL

C

H

–

O

20

0

25

0

25

0

10

01

00

1

00

10

0

25

0

>5

00

2

50

1

6

24

3.5

9

99

99

99

9B

LC

BL

C

10

01

00

1

00

10

0

10

01

00

6

2.5

62

.5

10

01

00

B

LC

BL

C

Cl

–

- 2

00

1

00

10

0

10

01

00

1

00

10

0

62

.56

2.5

1

00

10

0

50

0

>5

00

4

4

21

5.8

9

89

89

89

8B

LC

BL

C

10

01

00

1

25

12

5

10

01

00

1

00

10

0

12

51

25

B

LC

BL

C

H

–

- 1

00

10

0

25

0

12

51

25

1

00

10

0

10

01

00

1

25

12

5

>5

00

>

50

0

76

2

07

.0

97

97

97

97

BL

CB

LC

1

00

10

0

10

01

00

B

LC

BL

C

Cl

H

S

20

0

10

01

00

2

50

2

50

2

50

1

00

10

0

>5

00

>

50

0

14

2

06

.4

96

96

96

96

BL

CB

LC

1

00

10

0

12

51

25

6

2.5

62

.5

12

51

25

B

LC

BL

C

H

H

S

10

01

00

1

25

12

5

62

.56

2.5

5

00

2

50

1

25

12

5

>5

00

5

00

6

3

19

9.8

9

59

59

59

5B

LC

BL

C

10

01

00

1

25

12

5

20

02

00

1

25

12

5

91

91

B

LC

BL

C

Cl

Me

O

2

50

1

00

10

0

25

0

12

51

25

2

00

20

0

12

51

25

>

50

0

50

0

91

91

2

01

.4

94

94

94

94

BL

CB

LC

5

00

50

0

20

02

00

1

25

12

5

BL

CB

LC

H

M

e

O

50

0

50

05

00

2

00

20

0

12

51

25

2

00

5

00

5

00

>

50

0

78

2

13

.4

93

93

93

93

BL

CB

LC

2

00

20

0

12

51

25

2

50

25

0

20

02

00

B

LC

BL

C

Cl

H

O

20

0

20

02

00

1

25

12

5

25

0

25

02

50

2

00

20

0

>5

00

>

50

0

23

2

02

.4

92

92

92

92

BL

CB

LC

B

LC

BL

C

H

H

O

25

0

20

0

25

0

20

0

20

0

25

0

>5

00

>

50

0

78

2

56

.8

91

91

91

91

BL

CB

LC

1

00

10

0

12

51

25

1

00

10

0

12

51

25

B

LC

BL

C

Cl

–

–

10

01

00

2

50

1

25

12

5

10

01

00

1

00

1

25

12

5

>5

00

2

50

5

2

25

8.8

9

09

09

09

0B

LC

BL

C

10

01

00

1

25

12

5

12

51

25

B

LC

BL

C

H

–

–

10

01

00

1

25

12

5

50

0

12

51

25

1

00

2

00

>

50

0

>5

00

3

0

20

0.6

8

98

98

98

9B

LC

BL

C

12

51

25

1

00

10

0

BL

CB

LC

C

l H

–

2

00

5

00

2

50

1

25

12

5

10

01

00

5

00

2

50

5

00

6

5

20

1.0

8

88

88

88

8B

LC

BL

C

12

51

25

1

25

12

5

BL

CB

LC

H

H

–

1

25

12

5

20

0

12

51

25

2

00

2

00

2

00

>

50

0

>5

00

8

7

20

4.8

8

78

78

78

7B

LC

BL

C

12

51

25

3

97

.63

97

.6

BL

CB

LC

C

l M

e

–

25

0

25

0

50

0

12

51

25

2

00

2

50

>

50

0

25

0

16

3

97

.63

97

.6

86

86

86

86

BL

CB

LC

1

25

12

5

10

01

00

B

LC

BL

C

H

Me

–

2

00

2

50

1

25

12

5

10

01

00

2

50

5

00

>

50

0

25

0

50

2

17

.0

10

01

00

62

.56

2.5

1

25

12

51

00

10

01

00

10

0

50

0

62

.56

2.5

1

25

12

5

20

0

10

01

00

5

00

4

1

10

4.8

8

58

58

58

5B

LC

BL

C

BL

CB

LC

C

l –

–

>

50

0

12

51

25

12

51

25

5

00

2

50

2

50

2

50

2

50

>

50

0

36

9

7.8

8

48

48

48

4B

LC

BL

C

BL

CB

LC

H

–

–

>

50

0 . .

A A. .C C

. .A A. .

C C . .

F F. .A A

. .F F. .

A A . .

C C. .V V

. .C C. .

V V . .

T T. .S S. .

T T. .S S . .

C C. .E E

. .C C. .

E E . .

P P. .S S. .

P P. .S S . .

T T. .C C

. .T T. .

C C . .S S. .

B B. .S S. .

B B1111

RR

RR

XX

mM

/10

0g

mM

/10

0g

mM

/10

0g

mM

/10

0g

G

ram

po

siti

ve

ba

cte

ria

Gra

m p

osi

tiv

e b

act

eri

a

Gra

m n

eg

ati

ve

ba

cte

ria

Gra

m n

eg

ati

ve

ba

cte

ria

F

un

gi

Fu

ng

i G

ram

po

siti

ve

ba

cte

ria

Gra

m p

osi

tiv

e b

act

eri

a

Gra

m n

eg

ati

ve

ba

cte

ria

Gra

m n

eg

ati

ve

ba

cte

ria

F

un

gi

Fu

ng

i

inh

ibit

ion

inh

ibit

ion

RR

RR

XX

in

hib

itio

nin

hib

itio

n

% G

row

th o

f %

Gro

wth

of

% G

row

th o

f %

Gro

wth

of

An

ti T

BA

nti

TB

A

nti

TB

An

ti T

B

--11--11g

μg g

μ μg

μS

ub

stit

uti

on

sS

ub

stit

uti

on

s A

nti

mic

rob

ial

Act

ivit

y (

MIC

, A

nti

mic

rob

ial

Act

ivit

y (

MIC

, m

Lm

L))

An

tio

xid

an

tA

nti

ox

ida

nt

Su

bst

itu

tio

ns

Su

bst

itu

tio

ns

An

tim

icro

bia

l A

ctiv

ity

(M

IC,

An

tim

icro

bia

l A

ctiv

ity

(M

IC,

mL

mL

)) A

nti

ox

ida

nt

An

tio

xid

an

t C

om

p.

Co

mp

. C

om

p.

Co

mp

.

8844

––1

11

11

188

44––

11

11

11

bT

ab

le

Ta

ble

55..99

BL

CB

LC

Ta

ble

T

ab

le 55

..99 B

iolo

gic

al

scre

en

ing

te

st r

esu

lts

of

thio

chro

me

no

[2,3

-]q

uin

oli

ne

-he

tero

cycl

es

BL

CB

LC

.

Chapter 5 275

5.5.5.5.5555.2. Anti.2. Anti.2. Anti.2. Anti----proliferative activity of some representative compoundsproliferative activity of some representative compoundsproliferative activity of some representative compoundsproliferative activity of some representative compounds

Results on anti-proliferative activity study of some selected and

representative compounds are given in TableTableTableTable 5.105.105.105.10. For comparison, we have

included the antiproliferative data of the standard anticancer drugs cisplatin,

etoposide and camptothecin against the same panel of solid tumor cell lines.

When compared to the standard anticancer drugs, the lead drug BLCBLCBLCBLC14141414 showed

similar activity profile. This is of relevant interest in the drug-resistant cell lines

T-47D and WiDr. From the set of tested compounds, only compound BLCBLCBLCBLC3333 was

inactive against all cell lines tested. The most active compound of the series was

BLCBLCBLCBLC11114444, which showed GI50 values in the 3.3-6.2 µM range against all cell lines

tested. The results on the antiproliferative activity do not provide clear

structure-activity relationships. Overall, the results show that nitro derivatives

BLCBLCBLCBLC1111––––16161616 are more active than the corresponding amino analogs BLCBLCBLCBLC26262626----41414141.

However, the activity is not correlated to the location (R2 or R3) of the nitro

group attached to the aromatic ring. Additionally, the effect of substituent R1 on

the modulation of the antiproliferative activity is not clear.

Table 5.Table 5.Table 5.Table 5.10101010: Antiproliferative activity of candidates of representative compoundsAntiproliferative activity of candidates of representative compoundsAntiproliferative activity of candidates of representative compoundsAntiproliferative activity of candidates of representative compounds

Cell line Cell line Cell line Cell line (origin)

CompoundCompoundCompoundCompound A549A549A549A549

(lung)

HBLHBLHBLHBL----100100100100

(breast)

HeLaHeLaHeLaHeLa

(cervix)

SW1573SW1573SW1573SW1573

(lung)

TTTT----47D47D47D47D

(breast)

WiDrWiDrWiDrWiDr

(colon)

BLCBLCBLCBLC1111 7.6 (±1.8)7.6 (±1.8)7.6 (±1.8)7.6 (±1.8) 31 (±9.9) 7.4 (±3.0)7.4 (±3.0)7.4 (±3.0)7.4 (±3.0) 16 16 16 16 (±0.6)(±0.6)(±0.6)(±0.6) 5.9 (±4.0)5.9 (±4.0)5.9 (±4.0)5.9 (±4.0) 6.0 (±0.5)6.0 (±0.5)6.0 (±0.5)6.0 (±0.5)

BLCBLCBLCBLC3333 >100 >100 >100 >100 >100 >100

BLCBLCBLCBLC8888 7.9 (±0.9)7.9 (±0.9)7.9 (±0.9)7.9 (±0.9) 7.47.47.47.4bbbb 11 (±2.7)11 (±2.7)11 (±2.7)11 (±2.7) 9.6 (±0.7)9.6 (±0.7)9.6 (±0.7)9.6 (±0.7) 20 (±0.9)20 (±0.9)20 (±0.9)20 (±0.9) 13 (±5.8)13 (±5.8)13 (±5.8)13 (±5.8)

BLCBLCBLCBLC14141414 4.5 (±0.7)4.5 (±0.7)4.5 (±0.7)4.5 (±0.7) 3.93.93.93.9bbbb 3.5 (±0.5)3.5 (±0.5)3.5 (±0.5)3.5 (±0.5) 6.2 (±0.8)6.2 (±0.8)6.2 (±0.8)6.2 (±0.8) 3.3 (±0.3)3.3 (±0.3)3.3 (±0.3)3.3 (±0.3) 4.5 (±0.9)4.5 (±0.9)4.5 (±0.9)4.5 (±0.9)

BLCBLCBLCBLC15151515 7.9 (±2.0)7.9 (±2.0)7.9 (±2.0)7.9 (±2.0) 8.38.38.38.3bbbb 4.8 (±1.7)4.8 (±1.7)4.8 (±1.7)4.8 (±1.7) 36 (±16)36 (±16)36 (±16)36 (±16) 3.7 (±0.7)3.7 (±0.7)3.7 (±0.7)3.7 (±0.7) 4.0 (±04.0 (±04.0 (±04.0 (±0.4).4).4).4)

BLCBLCBLCBLC26262626 28 (±1.8) 21 (±0.9) 12 (±1.5) 16 (±2.1) 23 (±7.9) 31 (±6.3)

BLCBLCBLCBLC28282828 34 (±0.1) 37 (±4.8) 21 (±5.7) 35 (±1.4) 30 (±2.8) 48 (±8.7)

BLCBLCBLCBLC29292929 27 (±3.0) 19 (±2.6) 19 (±3.2) 28 (±6.3) 26 (±6.6) 28 (±3.8)

BLCBLCBLCBLC30303030 34 (±2.1) 52 (±8.4) 25 (±15) 36 (±0.9) 31 (±3.8) 42 (±23)

BLCBLCBLCBLC31313131 33 (±0.2) 39 (±5.5) 27 (±20) 43 (±4.1) 32 (±14) 37 (±2.9)

BLCBLCBLCBLC32323232 30 (±0.3) 31 (±5.8) 19 (±9.5) 29 (±0.5) 23 (±4.1) 26 (±3.2)

BLCBLCBLCBLC33333333 60 (±0.1) 35 (±1.8) 27 (±5.1) 45 (±1.1) 38 (±5.6) 57 (±25)

BLCBLCBLCBLC34343434 35 (±3.6) 36 (±0.2) 27 (±7.5) 35 (±0.7) 33 (±4.8) 47 (±17)

[A] - 1.9 (±0.2) 2.0 (±0.3) 3.0 (±0.4) 15 (±2.3) 26 (±5.3)

[B] - 1.4 (±0.1) 3.3 (±1.6) 14 (±1.5) 22 (±5.5) 23 (±3.1)

[C] - 0.23

(±0.05) 0.6 (±0.4)

0.25

(±0.12) 2.0 ±(0.5) 1.8 (±0.7)

[A]:Cisplatin, [B]: Etoposide, [C]: Camptothecin. a Values are given in μM and are means of two to

four experiments; standard deviation is given in parentheses.b Only was experiment was done

Chapter 5 276

ReferencesReferencesReferencesReferences

1. Ehrenberg's Symbolae physioe. Animalia evertebrata. Decas prima. Berlin, 1828.

2. Gram, H. C. Fortschr. Med. 1884188418841884, 2, 185.

3. Murray, P. R.; Pfaller, M. A.; Rosenthal, K. S. Medical Microbiology. 5th Ed.; 2009.

4. URL: http://www.textbookofbacteriology.net/normalflora.html

(16/12/2011).

5. URL: http://en.wikipedia.org/wiki/Streptococcus_pneumoniae

(16/12/2011).

http://www.textbookofbacteriology.net/S.pneumoniae.html

(16/12/2011).

6. URL: http://en.wikipedia.org/wiki/Clostridium_tetani (16/12/2011).

http://www.textbookofbacteriology.net/clostridia.html (16/12/2011).

7. URL: http://en.wikipedia.org/wiki/Bacillus_b vsubtilis (16/12/2011).

http://www.textbookofbacteriology.net/Bacillus.html (17/12/2011).

8. URL: http://en.wikipedia.org/wiki/Salmonella (17/12/2011).

http://www.sanger.ac.uk/Projects/S_typhi (17/12/2011).

http://www.textbookofbacteriology.net/salmonella.html (17/12/2011).

9. URL: http://en.wikipedia.org/wiki/Vibrio_cholerae (17/12/2011).

http://www.textbookofbacteriology.net/cholera.html (17/12/2011).

10. URL: http://en.wikipedia.org/wiki/Escherichia_coli (17/12/2011).

http://emedicine.medscape.com/article/217485-overview

(17/12/2011).

11. URL: http://en.wikipedia.org/wiki/Candida_albicans (17/12/2011).

12. URL: http://en.wikipedia.org/wiki/Aspergillus_fumigatus (17/12/2011).

http://www.aspergillus.org.uk (17/12/2011).

13 . National Committee for Clinical Laboratory Standards (NCCLS), 940, West Valley

Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA. Performance Standards

for Antimicrobial Susceptibility Testing; Twelfth Informational Supplement.

NCCLS document (2002) M100-S12 (M7) [ISBN 1-56238-454-6].

14 Cruickshank, R. Medical Microbiology; 12th Ed.; Chaurchi-Livingstone, Deinburgh,

London, 1975; Vol. II.

15. (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 Medical

Microbiology; 4th Ed.; McGraw Hill, 2004.

16. Cole, S. T.; Brosch, R.; Parkhill, J. Nature 1998199819981998, 393, 537.

17. (a) Seth, S. D.; Seth, V. Text Book of Pharmacology; 3rd Ed.; Elsevier, 2009; p X.74;

(b) Janin, Y. L.; Bioorg. Med. Chem. 2007200720072007, 15, 2479.

18. Rattan, A. Antimicrobials in Laboratory Medicine; Churchill, B. I.; Livingstone, New

Delhi, 2000; 85.

19. Halliwell, B.; Gutteridge, J. M. C. Free Radicals Biol. Med. 1995199519951995, 18, 125.

20. Halliwell, B.; Gutteridge, J. M. C. Methods in Enzymology 1990199019901990, 186, 1.

21. Halliwell, B. Nutr. Rev. 1994199419941994, 53, 253.

22. Wayner, D. D. M.; Burton, G. W.; Ingold, K. U. FEBC Lett. 1985198519851985, 187, 33.

23. Benzie, I. F. F.; Strain, J. J. Anal. Biochem. 1996199619961996, 239, 70.

24. Glazer, A. N. Methods Enzymol. 1990199019901990, 186, 161.

Chapter 5 277

25. Lewis, S. E.; Boyle, P. M. Fertil. Steril. 1995199519951995, 64, 868.

26. Cao, G.; Prior, R. L. Clin. Chem. 1998199819981998, 44, 1309.

27. Miller, N. J.; Davies, M. J. Milner, A. Clin. Sci. 1993199319931993, 84, 407.

28. Mensor, L.L.; Fabio, S. M.; Alexandre, S. R. Phytother. Res. 2001200120012001, 15, 127.

29. Re, R.; Pellegrini, N.; Pannala, A. Free Radical Biol. Med. 1999199919991999, 26, 1231.

30. (a) Benzie, I. F. F.; Strain, J. J. Redox Rep. 1997199719971997, 3, 233; (b) Benzie, I. F. F.; Strain, J. J.

Methods in Enzymology: Oxidants and Antioxidants Part A. 1st Ed.; London:

Academic Press Limited, 1999; Vol. 299, p 15; (c) Choy, C. K. M.; Benzie, I. F. F.;

Cho. P. IOVS, 2000200020002000, 41, 3293.

31. Williams, D. A.; Lemke, T.L. Foye’s Principles of Medicinal chemisty 5th Ed;

Lippincott Williams & wilkins 2002;

32 . URL: http://en.wikipedia.org/wiki/Cancer#cite_note-NCI2014-2(6/11/2014)

33. URL: http://en.wikipedia.org/wiki/Cancer#cite_note-NHS2012-

3(6/11/2014)

34 . URL: http://en.wikipedia.org/wiki/Cancer#cite_note-WHO2014-

1(6/11/2014)

35. URL: http://en.wikipedia.org/wiki/Cancer#cite_note-Enviro2008-

4(6/11/2014)

36. URL: http://en.wikipedia.org/wiki/Cancer#cite_note-WCR2014Bio-

5(6/11/2014)

37. URL: http://en.wikipedia.org/wiki/Cancer#cite_note-ACS-heredity-

6(6/11/2014)

38. URL: http://en.wikipedia.org/wiki/Cancer#cite_note-7(6/11/2014)

39. URL: http://en.wikipedia.org/wiki/Cancer#cite_note-14(6/11/2014)

40. (a) URL: http://en.wikipedia.org/wiki/Cancer#cite_note-15(6/11/2014);(b)

URL: http://en.wikipedia.org/wiki/Cancer#cite_note-Cak2012-

16(6/11/2014)

41. URL: http://en.wikipedia.org/wiki/Cancer#cite_note-WCR2014Peads-

12(6/11/2014)

42. (a)Wamidh, H. T.; Adel, M. M. Sci Pharm 2010201020102010, 78, 33; (b) Richardson, D. R.; Tran,

E.; Ponka, P. Blood, 1995199519951995, 86, 4295.

43. www.promega.com

44 . Goto, M; Holgersson, J; Kumagai-Braesch, M. Am J Transplant. 2006200620062006, 6, 2483.

45. Skehan, P.; Storeng, P.; Scudeiro, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren, J.

T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990199019901990, 82, 1107.

46. Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Paull, K.; Vistica, D.; Hose, C.;

Langley, J.; Cronise, P.; Vaigro-Wolff, A.; Gray-Goodrich, M.; Campbell, H.; Mayo, J.;

Boyd, M. J. Natl. Cancer Inst. 1991199119911991, 83, 757.