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
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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
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rom
en
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late
d p
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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
…
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Chapter 5 270
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5
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4
3
93
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77
77
77
77
BL
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1
25
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5B
LC
BL
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E
t S
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50
2
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2
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2
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25
12
5
25
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2
6
11
3.2
76
76
76
76
BL
CB
LC
1
00
10
01
25
12
51
25
12
5B
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BL
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M
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10
01
00
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25
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5
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51
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50
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50
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8
4
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75
75
75
75
BL
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M
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10
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7
9
87
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74
74
74
74
BL
CB
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1
25
12
51
25
12
51
25
12
5B
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BL
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Me
M
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Me
O
1
25
12
5
20
0
20
0
12
51
25
1
25
12
5
50
0
25
0
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7
3
13
7.8
73
73
73
73
BL
CB
LC
6
2.5
62
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12
51
25
12
51
25
BL
CB
LC
H
M
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Et
S
62
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2.5
1
25
12
5
12
51
25
2
50
2
00
2
50
5
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5
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3
8
11
3.2
72
72
72
72
BL
CB
LC
1
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10
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LC
BL
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Me
M
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25
0
10
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2
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2
50
5
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50
0
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4
5
91
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. .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
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eria
Gra
m p
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e b
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G
ram
ne
ga
tiv
e b
act
eri
aG
ram
ne
ga
tiv
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act
eri
a
Fu
ng
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un
gi
mM
/1
00
gm
M/
10
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m
M/
10
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mM
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00
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An
tio
xid
an
tA
nti
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da
nt
An
tio
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an
tA
nti
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da
nt
inh
ibit
ion
inh
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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
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mic
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ial
Act
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MIC
, m
Lm
L))
An
tim
icro
bia
l A
ctiv
ity
(M
IC,
An
tim
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(M
IC,
mL
mL
)) S
ub
stit
uti
on
sS
ub
stit
uti
on
s S
ub
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on
sS
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stit
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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
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Ta
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5.
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5.88
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log
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62
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0
84
1
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70
70
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70
BL
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1
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0
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51
25
1
25
12
5
BL
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0
50
0
25
0
10
01
00
1
25
12
5
12
51
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50
0
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8
1
20
6.6
69
69
69
69
BL
CB
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6
2.5
62
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10
01
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74
2
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68
68
68
68
BL
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BL
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Et
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46
1
78
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67
67
67
67
BL
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1
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5
BL
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E
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0
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6
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66
66
66
66
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3-N
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Me
10
01
00
2
50
2
00
2
50
2
50
2
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50
0
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00
8
9
15
7.2
65
65
65
65
2B
LC
BL
C
12
51
25
1
00
10
0
BL
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3
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2
00
2
50
1
25
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5
20
0
10
01
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2
50
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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
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%
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
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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
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MIC
, A
nti
mic
rob
ial
Act
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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
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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
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l M
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–
20
0
20
0
62
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2.5
1
25
12
5
12
51
25
2
00
>
50
0
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00
3
3
19
1.2
1
10
11
01
10
11
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62
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1
00
10
0
12
51
25
1
00
10
0
BL
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–
2
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6
2.5
62
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0
10
01
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1
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5
10
01
00
5
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5
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8
7
19
6.0
1
09
10
91
09
10
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LC
BL
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01
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1
25
12
5
12
51
25
B
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BL
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–
2
00
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50
2
00
1
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10
0
12
51
25
1
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12
5
20
0
>5
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6
3
18
2.4
1
08
10
81
08
10
8B
LC
BL
C
10
01
00
B
LC
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H
–
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01
00
2
00
2
50
2
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2
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0
38
1
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10
71
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71
07
BL
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10
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1
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12
5
92
92
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51
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2
00
1
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51
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2
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9
29
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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.