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Chapter - VI
6.1 Introduction & Literature review
6.2 Antibacterial activity
6.3 Antimycobacterial activity
6.4 Anticonvulsant activity
6.5 References
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Chapter - VI
6.1. Introduction & Literature review:Biological evaluation is that phenomenon which explains the
role of drugs in living organisms. Biological evaluation depends upon
the study of various bacteria present in cell membrane of living
system. In fact first life on the earth was started by bacteria called
cyanobacteria,, later to it eukaryotic life started and evolved biological
life like animals and humans as well. And one most important thing
with bacteria is its inclusion into the eukaryotic system was nothing
but the most important organelle
or
of cell called mitochondria.
Bacteria are the simplest prokaryotic organisms. They are
highly adaptable and can service extremes of temperature,
temperature pH,
oxygen tension and osmotic / atmospheric pressure and are therefore
found in almost all natural sources.
so
• Size of Bacteria:Bacteria:
Bacteria are very small size organisms. Which are barely
visible under the light microscope. The smallest bacteria are about 0.1
million in diameter while the largest may be 60 x 6 microns.
• Shape of Bacteria:
Bacteria:-Bacteria occurs in three main shape
Fig.6.1. Shape of Bacteria
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Chapter - VI
1. Spherical ⇒ Spherical bacteria are called Cocci.
2. Rod-like ⇒ Rod like bacteria is called Bacilli.
3. Spiral ⇒ Spiral bacteria are called Spirilla.
The following table gives some of disease causing bacteria in
human being.
Sr.
No.
Disease
Bacterium
01
Meningitis
Neisseria meningitides
02
Lobar pneumonia
03
Boils
04
Scarlet fever
05
Food poisoning
06
Tetanus
07
Diphtheria
08
Tuberculosis
09
Plague
10
Typhoid
Salmonella typhii
11
Cholera
Vibrio comma
12
Syphilis
Treponemapallidum
Streptococcus pneumonia
Staphylococcus sp.
Streptococcus scarlatinae
Clostridium botalinum
Clostridium tetani
Cynobacterium diphtheria
Mycobacterium tuberculosis
Pasturellapestis
Biological activity:In pharmacology, biological activity describes the beneficial or
adverse effect of a drug on living matter. When a drug is a complex
chemical mixture, this activity is exerted by the substances active
ingradient or pharmacophore but can be modified by the other
constituents. Activity is generally dosage-dependant and it is not
uncommon to have effects ranging from beneficial to adverse for one
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Chapter - VI
substance when going from low to high doses. Activity depends
critically on fulfillment of the ADME criteria.
Where as a material is considered bioactive if it has interaction
with or effect on any cell tissue in the human body. Biological
activity is usually taken to describe beneficial effect i.e. the effect of
drug candidates. The main kind of biological activity is substances
toxicity.
Antimicrobial:An anti-microbial is a substances that kills or inhibits the
growth of micro-organisms
(1)
. Such as bacteria, fungi or protozoans.
Antimicrobial drugs either kill microbes (micro-biocidal) or prevent
the
growth
of
microbes
(microbiostatic).
Disinfectants
are
antimicrobial substances used on non-living objects or outside the
body. Technically, antimicrobials are only those substances that are
produced by one microorganism, of course, in today’s common
usage; the term of antimicrobial is used to refer to almost any drug
that attempts to rid your body of a bacterial infection. Antimicrobials
include not just antibiotics, but synthetically formed compounds as
well. The discovery of antimicrobials like penicillin and tetra cycline
paved the way for better health for millions around the world. Before
penicillin became a viable medical treatment in the early 1940s, no
true cure for gonorrhea, strep throad or pneumonia existed. Patients
with infected wounds often had to have a wounded limb removed, or
face death from infection now, most of these infections can be cured
easily with a short course of antimicrobials.
However
with
the
development
of
anti-microbials,
microorganisms have adapted and become resistant to previous
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Chapter - VI
antimicrobial agents. The old antimicrobial technology was based
either on poison or heavy metals, which may not have killed the
microbe completely, allowing the microbe to survive change and
become resistant to the poisons and / or heavy metals.
6.2. Antibacterial activity:Introduction:An antibacterial is a substance or compound that kills or slows
down the growth of bacteria(2). The term “antibiotic” was coined by
Selman in 1942 to describe any substance produced by a
microorganism that in antagonistic to the growth of other
microorganisms in high dilution
(3)
. This definition excluded
substances that kill bacteria but are not produced by microorganisms
(such as gastric juices and hydrogen peroxide). It also excludes
synthetic antibacterial compounds such as Sulfonamides. With
advances in medicinal chemistry, most of today’s antibacterials
chemically are semi-synthetic modification of various natural
compounds(4). These include for example, the β-lactam antibacterials,
which include the penicillin, the cephalosporins and the carbapenems.
Penicillin, the first natural antibiotic discovered by Alexander
Fleming in 1929 in his small lab accidentally. When he was working
with some fungus, he found that there is zone of inhibition in the
plate. Then he isolated the active ingredient and studied it found that
it is having potential to kill micro-organism called bacteria. The term
antibiosis meaning “against life”, was introduced by the French
bacteriologist Vuillemin as a descriptive name of the phenomenon
exhibited by these early antibacterial drugs
(4,5)
. Antibiosis was first
described in 1877 in bacteria when Louis Pasteus and Robert Koch
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Chapter - VI
observed that an airborne bacillus could inhibit the growth of Bacillus
anthraces
(6)
. These drugs were later renamed antibiotics by Selman
Waksman, an American microbiologist in 1942 (3, 4).
Pharmacodynamics:Testing the susceptibility of staphylococcus aurous to
antibiotics by the Kirby-Bauer disk diffusion method. Antibiotics
diffuse out from antibiotic-containing disk and inhibit growth of S.
aurous resulting in a zone of inhibition. The successful outcome of
antimicrobial therapy with antibacterial compounds depends on
several factors. These include host defense mechanisms, the location
of infection, and the pharmacokinetic and pharmacodynamic
properties of the antibacterial (7).
A bactericidal activity of antibacterials may depend on the
bacterial growth phase, and it often requires ongoing metabolic
activity and division of bacterial cells (8). These findings are based on
laboratory studies, and in clinical setting have also been shown to
eliminate bacterial infections
(7,9)
since the activity of antibacterials
depends frequently on its concentration
(10)
, in vitro characterization
of antibacterial activity commonly includes the determination of the
minimum inhibitory concentration and minimum bacteriacidal
concentration of an antibacterial (7,11). To predict clinical outcome, the
antimicrobial activity of an antibacterial is usually combined with its
pharmacokinetic profile, and several pharmacological parameters are
used as markers of drug efficiency.
Classes:Antibacterials are commonly classified based on their
mechanism of action, chemical structure or spectrum of activity. Most
antibacterial antibiotics target bacterial functions or growth processes.
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Chapter - VI
Antibiotics that target the bacterial cell wall (such as penicillin and
cephalosphorins) or cell membrane (for example, polymixins) or
interfere with essential bacterial enzymes (such as quinolones and
sulfonamides) have bactericidal activities. Those that target protein
synthesis, such as the aminoglucosides, macrolides and tetracyclines,
usually bacteriostatic. Further categorization is based on their target
specificity. “Narrow-spectrum” antibacterial antibiotics target specific
types of bacteria, such as gram-negative or gram-positive bacteria,
where as broad-spectrum antibiotics affect a wide range of bacteria.
gram-positive bacteria B.Subtilis
gram-negative bacteria such as E. Coli
There is great importance to bacteria in recent world to produce
industrial enzymes as well as biotechnologically derived human
therapeutic proteins. It has become possible by genetic engineering.
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Human genes are isolated and recombined into the bacterial genetic
system and genes are expressed inside the bacteria and therapeutic
proteins are isolated and purified and marketed. For example human
insulin expression in E. coli. Human intestinal microflora play
important role to the host by producing vitamins and
and enzymes. They
also induce some substances which keeps live the immunity of the
host. For example Lactobacillus species converts sugar into lactic
acid.
Bacterial Cell Structure:Structure:
The bacterial cell differs dramatically in structure and function
compared to mammalian cells
(12)
. The bacterial cytoplasm is
separated from the external environment by a cytoplasmic membrane
as shown in Fig. 5.2 A. The bacterial cell wall is chemically ddistinct
from mammalian cell walls and so is constructed by enzymes that
often have no direct counterpart in mammalian cell construction. In
addition, bacteria possess a crucial structure surrounding the entire
cell, the Peptidoglycan (PG), which forms a su
succulus
cculus around the
bacterial cell, is an essential cell wall polymer since interference with
its synthesis or structure leads to lose of cell shape and integrity
followed by bacterial death.
FIG.6.2.A.
FIG.
CELL STRUCTURE
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Chapter - VI
The peptidoglycan (13) layer as shown in Fig. 5.2 B consists of a
matrix of polysaccharide chains composed of alternating N
Nacetylmuramic acid (MurNAc) and N-acetylglucosamine
N acetylglucosamine (GlcNAc)
sugar moieties cross--linked
linked through pentapeptide side chains.
Fig.6.2.B.PEPTIDOGLYYCAN
.2.B.PEPTIDOGLYYCAN LAYER OF E.COLI
Classification of Antibacterial Agents: -Based
Based on the severnity of
damage (14) to the bacteria they are divided into:
1. Bactericidal agenst act primarily by killing bacteria with an
efficiency of >99.9%.
2. Bacteriostatic agents act primarily by inhibiting the growth of
the bacteria.
Based on the mode of action they can be further divided into
following four main categories.
1. Cell wall synthesis inhibitors.
2. Cell Membrane Agents.
3. Protein synthesis inhibitors.
i) Impairing 50S subunit.
ii) Impairing 30S subunit.
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Chapter - VI
4. Nucleic acid synthesis inhibitors.
i) DNA replication and repair
ii) Transcription
A representative listing of the antibacterial compounds
currently in clinical practice or in development along with a
schematic overview of their targets
(15)
is shown in Fig. 5.2 C. More
recently it has been shown that the bactericidal antibiotics, having
distinct drug-target
target interactions stimulate the production of highly
harmful hydroxyl radical in gram-positive
gram
and gram-negative
negative bacteria
which finally contribute to the cell death. In contrast,
c
a Bacteriostatic
drug tested doesn’t lead to production of hydroxyl radical (16).
FIG.6.2.C.SCHEMATIC VIEW OF A BACTERIAL CELL WITH SITES
OF ACTION OF VARIOUS ANTIBIOTICS.
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Chapter - VI
6.2.1. Antibacterial Screening:Some of the synthesized derivatives from chapter three and
chapter four were then assayed for their in-vitro antibacterial activity
against a panel of pathogenic as well as standard bacterial strains such
as Staphylococcus aurous, Klebsiella pneumonia, Salmonella
typhimurium, Pseudomonas aeroginosa, Bacillus subtilis, Proteus
vulgaries, Xanthomonascampestri Pv, Citri, Xanthomonascampestri
Pv. Malvacerum, Bacilluthurengensis and Escherichia coli. Based on
previous literature and scope of the bacterial species were selected
under the different scheme. Gentamicin, Kanamycin, Streptomycin
and Cefotaxime sodium were procured from commercial sources. The
purities and potencies of the agents recovered from commercial
sources. The purities and potencies of the agents recovered from
commercial preparations were documented by showing that the MICs
of antibacterials were within acceptable limits against the known
strains.
*Determination of MIC in terms of Zone of Inhibition:The antibacterial activity was tested by agar disc diffusion
method. The killing or growth inhibition property of the agents was
scored as clear zone of inhibition surrounding the disc and is
measured in mm scale.
*Materials & Method:
•
The bacterial strains were inoculated in fresh sterile MHB
(Muller Hinton Broth) media tube (4.5 m) and were incubated
for 18-24 hrs at 370C in a B.O.D. incubator.
•
Standard antibiotic Gentamicin, Kanamycin, Streptomycin and
Cefotaxime sodium were prepared clear solutions with final
(1mg/ml).
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Chapter - VI
•
The above antibiotic solutions were poured on sterile disc at a
final concentration of 40 mcg/disc for Gentamicin, Kanamycin
while 40 mcg/disc for Streptomycin and Cefotaxime sodium.
•
All discs were dried completely by incubating into hot air oven
in sterile petri dishes.
•
On MHA (Muller Hinton Agar) plates, the bacterial suspension
was poured and spread evenly with the help of glass spreader.
•
After drying the plates completely, the antibiotic loaded discs
were kept on the plates.
•
All plates were incubated at 370C in a B.O.D. (Biological
Oxygen Demand) incubator for 24 hours.
•
Results were recorded and antibiotic activity was quantified by
measuring the zone of inhibition surrounded to the disc and it
were measured in ‘mm’ scale and presented in the respective
tables.
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Chapter - VI
*Table 6.2 (A): The antibacterial data synthesized substituted 3Methyl-7-(1-methyl-1H-indazol-3-yl-carboxamido)
8-oxo-5-thia-
azabicyclo [4.2.0] oct-2-ene-e-carboxylic acid.
O
H
N
S
N
N O
CH3
Bacteria
N
R1
Streptomycine
O
OR
100 µg/ml
standard
3.2.a 3.2.b 3.2.c 3.2.d 3.2.e
Zone of Inhibition
Kiebsiell pneumonia
8
-
-
-
-
-
Salmonella typhy
7
-
-
-
-
-
Psuedomanusauroginosa
9
9
10
-
7
11
Bacillus subtilis
7
11
-
12
-
-
E. coli
10
10
12
10
12
11
Proteus vulgaris
11
12
-
-
-
10
Xanthomonuscampe strip
V. Malvacerum
9
-
-
-
8
-
Bacillus thurengensis
11
11
13
15
13
7
Xanthaomonascampe
strip V. citri
6
-
-
-
-
-
Staphylococcus aureus
9
10
11
14
-
8
Summary & Conclusion:We screened 3.2 (a-e) compounds for antibacterial activity.
Concentrations of the compounds used in nutrient agar plates were
100 µg / ml each. Linear growths of the test bacteria were measured
every day and zone of inhibition of the isolates was recorded. It is
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Chapter - VI
clear that Table
able 5.5 that compound 3.2.c had maximum zone of
inhibition for Bacillus thuregensis, Staphylococus aureus, Bacillus
Subtilis and showed least zone of inhibition of E. coli. It did not show
any zone of inhibition to remaining bacteria. Compound 3.2.b showed
showe
maximum zone of inhibition of Bacillus thurengensis and E. coli.
Compound 3.2.a showed maximum zone of inhibition Proteus
vulgaris, Bacillus Subtilis and Bacillus thurengensis. Compound 3.2.d
showed maximum zone of inhibition Xanthaomonassampstrip V.
Malvacerum
vacerum and Compound 3.2.e showed maximum zone of
inhibition at E. coli.
Streptomycin
e 100 µg/ml
standard
3.2.a
3.2.b
3.2.c
3.2.d
3.2.e
Graphical representation of antibacterial activity of compound
3.2(a-e)
e) against Streptomycin as a standard
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Chapter - VI
Table 6.2 (B):-Antibacterial data of substituted 3-(acetoxymethyl7-(2-(7-methyl-2-p-tolylimidazo[1,2-a] pyridine-3yl) acetamide-8oxo-sthia-1-azabicyclo [4.2.0] oct-2-ene-2-carbaylic acid.3.3 (a –
e):
Bacteria
Standard
(Ceftaxime
sodium
N
N
N
O H
O
100 µg / ml
S
N
RI
OR
O
3.3.a
3.3.b
3.3.c
3.3.d
3.3.e
Zone of Inhibition
E. coli
30
20
17
14
13
15
S – aureus
20
-
-
-
-
-
S – typhi
25
18
15
13
13
14
B – megaterium
24
15
13
10
14
15
Summary & Conclusion:We screened all five derivatives of 3-(acetoxymethyl-7-(2-(7methyl-2-p-tolylimidazo[1, 2-a] pyridine-3yl) acetamide-8-oxo-5thia1-azabicyclo [4.2.0] oct-2-ene-2-carbaylic acid 3.3 (a-e). The
concentration of derivatives used in disc were 100 µg / ml each. The
activity of these derivatives were measured against four different
bacterial species by using cefotaxime sodium as standard drugs.
Among the tested series 3.3.a has shown maximum activity against E.
coli and S. Typhi. 3.3.b also showed good activity against E. coli and
S. Typhi. 3.3.e also show comparatively good activity against E. coli
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Chapter - VI
and B. Megaterium.The overall impact of all the derivatives against
cefotaxime sodium was inferior against all bacterial species.
35
30
E. coli
25
20
S – aureus
15
S – typhi
10
5
B – megaterium
0
Standard
3.3.a
3.3.b
3.3.c
3.3.d
3.3.e
Graphical representation of antibacterial activity of compound
3.3(a-e) against Ceftaxime sodium as a standard
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Chapter - VI
Table 6.2 (C): The antibacterial data of synthesized substituted
spiro – [2H-1, 3-benzoxazine-2, 1-cyclohexan]-4 (3H)-one. 4.2(a-g)
O
Zone of inhibition in (mm)
H N
S.
aureus
E. coli
Standard
[Nor floxacin 100 µg/ml
25
24
24
24
4.2 a
20
21
20
-
4.2 b
-
22
19
21
4.2 c
21
20
-
15
4.2 d
24
17
19
-
4.2 e
23
21
20
22
4.2 f
20
13
21
19
4.2 g
15
14
17
23
O
R
R
P.
B.
subtilis aeruginosa
Bacteria
Summary & Conclusion:We screened comp. 4.2 (a-g) for antibacterial activity. The
result indicates that all compounds showed good antibacterial activity.
Table 6.2 C compounds 4.2e showed very good activity against S.
aureus, E. coli, B. subtilis and P. aeruginosa. From the above
observation it is clear that the Benzoxazine derivatives are more
active and play a prominent role in the antimicrobial activity.
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Chapter - VI
25
S. aureus
20
E. coli
15
10
B. subtilis
5
P. aeruginosa
0
Standard
4.2 a
4.2 b
4.2 c
4.2 d
4.2 e
4.2 f
4.2 g
Graphical representation of antibacterial activity of compound
4.2(a-g)
g) against Norfloxacin as a standard
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Chapter - VI
Table 6.2 (D): The
he antibacterial data of synthesized substituted 33
(2-Bromopropionyl)
Bromopropionyl) spiro [2H - 1, 3 – benzoxazine - 2, 1 cyclohexane] – 4 (3H) – one. 4.3 (a-g)
O
O
Zone of inhibition in (mm)
Br
N
R
R
S.
B.
P.
E. coli
aureus
subtilis aeruginosa
O
Bacteria
Standard
[Nor floxacin 100 µg/ml
4.3 a
4.3 b
4.3 c
4.3 d
4.3 e
4.3 f
4.3 g
25
24
24
24
24
26
24
25
23
24
21
20
24
23
19
20
20
21
19
22
24
22
19
20
22
25
22
-
Summary & Conclusion:Conclusion:
We screened comp. 4.3(a
4.3(a-g) for antibacterial activity. All the
synthesized compounds have shown mild to good activity against
pathogenic bacteria.. The synthesized compounds 4.3(e)
4.3(e) have been
shown to be more potent than other synthesize compou
compounds.
The overall impact of all the derivatives against Norfloxacin
was inferior against all bacterial species.
30
S. aureus
25
20
E. coli
15
B. subtilis
10
P.
aeruginosa
5
0
Standard
4.3 a
4.3 b
4.3 c
4.3 d
4.3 e
4.3 f
4.3 g
Graphical representation of antibacterial
antibacterial activity of compound
4.3(a-g)
g) against Norfloxacin as a standard
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Chapter - VI
Table 6.2 (E):-The
The antibacterial data of synthesized substituted
substitu
Bromo spiro base. 5.3(a-i):
5.3
Zone of inhibition in (mm)
O
Br
O
R
2
N
R
R1 O
O
CH2
R
4
CH3
S.
aureus
E.
coli
B.
P.
subtilis aeruginosa
3
Bacteria
Standard
24
25
23
24
[Vancomycine 25 µg/ml
5.3a
21
24
22
23
5.3b
20
21
19
19
5.3c
18
23
21
18
5.3d
22
19
16
18
5.3e
16
15
17
19
5.3f
21
18
18
18
5.3g
20
17
19
21
5.3h
19
21
20
22
5.3i
22
18
18
21
Summary and conclusion: We screened 5.3(a-i)
i) for antibacterial
activity. The result indicates that all compounds showed good
antibacterial activity. 5.3a showed very significant activity against S.
aureus, E. coli, B. subtilis and P. aeruginosa. From the above
observation it is clear
lear that the Bromo spiro base auxillaries are more
active and play a prominent role in the antimicrobial activity which
helpful to improve the antibacterial activity of Carbapenems.
25
S. aureus
20
15
E. coli
10
B. subtilis
5
P.
aeruginosa
0
Standard
5.3a 5.3b
5.3c
5.3d
5.3e
5.3f
5.3g
5.3h
5.3i
Graphical representation of antibacterial activity of compound
5.3(a-i)
i) against Vancomycineas a standard
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Chapter - VI
6.3. Anti-mycobacterial activity:Anti-mycobacterial agents are generally used in combination
with other antimicrobials since treatment is prolonged and resistance
develops readily to individual agents.
1.
Para-amino salicylic acid (PSA) (Bacteriostatic) PSA is
specific for mycobacterium tuberculosis.
2.
DAPSONE (Bacteriostatic) : DAPSONE is used in treatment of
leprosy.
3.
Isoniazid (INZ) (Bacteriostatic). :Isoniazid inhibitsysthesis of
mycolic acids. It is used in treatment of tuberculosis. Isoniazid
(INZ) is an organic compounds that is the first-line antituberculosis medication in prevention and treatment. It was
first discovered in 1912 and later in 1951 it was found to be
effective against tuberculosis. Isoniazid never used on its own
to treat active tuberculosis because resistance quickly develops.
Isoniazid is prodrug and must be activated by a bacterial
catalase-peroxidase
enzyme
that
in
mycobacterium
tuberculosis. Isoniazid is bactericidal to rapidly-dividing
mycobacteria but is bacteriostatic if the mycobacterium is
slow-growing.
6.3.1: Antimycobacterial Screening:In vitro anti-mycobacterial screening was done for the
synthesized compounds 2.2.2C, 2.2.2e, 2.2.2f, 2.2.2r. Screened
against standard strain H37Rv and two human strain [Human strain-I
and Human strain-II]. Isolated from patients suffering from
pulmonary tuberculosis in different concentration from 12.5, 25, 50,
100, 200, 400 µg/ml and the isoniazid used as standard at a cone of 50
µg/ml.
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Chapter - VI
Material Required:1. Lowenstein-Jensen (LJ) medium slopes containing various
concentration of the compound.
2. Control strain H37Rv.
3. Two strains of Mycobacterium tuberculosis isolated from
patients suffering from pulmonary tuberculosis.
Preparation of drug containing slopes:The synthesized compounds were dissolved in DMSO and
added to the egg fluid salt solution in such a way that to give final
concentration of 12.5, 25, 50, 100, 200, 400 µg of the compounds per
ml of the medium. The above was insisted at 900C for 50 minutes
only once.
Preparation of the bacterial suspension:The bacterial culture of a control strain and 2 test strain about
2/3 loop full (3mm internal diameter) is mixed with 1ml sterile
distilled water in BIJOU bottle containing 3-5, glass beads, shaken in
a vertexed bottle for about 1mm to get a uniform suspension.
Susceptibility test procedure:1 loop full of bacterial suspension was inoculated on L. J.
medium slopes containing the test compounds. A drug free slope is
also included as a control. All the slopes were incubated at 370C for
15 days. The result and data are given in table V and VI.
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Chapter - VI
Table 6.3.A: - Antimycobacterial activity of compound 2.2.2 (c, e, f, i):H37Rv
Conc.
µg/ml
Human Strain I
Human Strain II
2.2.2c
2.2.2e
2.2.2f
2.2.2i
2.2.2c
2.2.2e
2.2.2f
2.2.2i
2.2.2c
2.2.2e
2.2.2f
2.2.2i
12.5
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
25
++
+++
+++
+++
++
+++
+++
+++
++
+++
+++
+++
50
–
++
++
+
+
++
++
+
+
++
++
++
100
–
–
–
–
–
–
+
+
+
+
+
+
200
–
–
–
–
–
–
–
–
–
–
–
–
400
–
–
–
–
–
–
–
–
–
–
–
–
–: No growth of mycobacterium tuberculosis
+: Growth of mycobacterium tuberculosis below 100 colonies
++: Growth of mycobacterium tuberculosis between 100-200 colonies
+++: Growth of mycobacterium tuberculosis above 200 colonies
Table 6.3.B: Minimum Inhibitory Concentration
Minimum Inhibitory Concentration
Compounds
H37Rv
Human Strain
I
Human Strain
II
2.2.2c
50
100
200
2.2.2e
100
100
200
2.2.2f
100
200
200
2.2.2i
100
200
200
Summary & Conclusion:In vitro anti-mycobacterial screening was done for the
synthesized compounds 2.2.2c, 2.2.2e, 2.2.2f, 2.2.2i. Synthesized
compounds screened against standard strain H37Rv and two human
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Chapter - VI
strain isolated from patients suffering from pulmonary tuberculosis in
different concentration from 12.5, 50, 100, 200, 400 µg/ml and the
Isoniazid used as standard at a cone of 50 µg/ml.
Synthesized compounds 2.2.2c, 2.2.2e, 2.2.2f & 2.2.2i showed
significant antitubercular activity.
Minimum inhibitory concentration
600
500
Axis Title
400
Human Strain II
300
Human Strain I
200
H37Rv
100
0
2.2.2c
2.2.2e
2.2.2f
2.2.2i
Graphical representation of MIC of compound 2.2.2(c,e,f,i))
211
Chapter - VI
6.4. Anticonvulsant Activity:Anticonvulsant evaluation by Maximal Electro Shock (MES)
Method:
The anticonvulsant activities of the compounds were evaluated
by maximal electro shock method using rats,where the electro shock
is applied through the corneal electrode producing optic stimulation
cortical excitations. The MES convulsions are divided into five
phases such as (a) Toxic flexion (b) Tonic extension (c) Clonic
Convulsion (d) Stupor and (e) Recovery of death. A drug is known to
possess anticonvulsant properties. If it reduces or abolishes the
extensor phase or MES convulsions, for the evaluation anticonvulsant
activity the total 16 groups of animals were kept fasting for 10 – 14
hrs. The animals were divided into 16 groups each containing 6
animals. In the 14 groups were served for testing the synthesized
compounds, one as control and one as standard (phenytoin 25 mg/kg
of body weight) was used as a standard drug.
The activities of each group were measured after the intervals
of half-an-hour are compounds administering including control and
standard.
Results and data are given in table 6.4.A.
212
Chapter - VI
Table 6.4.A:- Anticonvulsant activity of some synthesized
compounds by MES method:
Compound
Mean time in various phases
of convulsions (seconds) ±
standard error mean
Recovery
MES
Test
Percentage protection
(% abolition of tonic
extensor phase)
Flexion
Extensor
Control
6.833 ± 0.40
10.00 ± 0.73
√
0/6
--
2.2.2a
3.67 ± 0.21
1.83 ± 0.83
√
3/6
50%
2.2.2b
3.33 ± 0.21
2.16 ± 0.74
√
2/6
33.33%
2.2.2c
4.66 ± 0.33
2.00 ± 0.69
√
2/6
33.33%
2.2.2d
4.33 ± 0.49
4.66 ± 0.49
√
0/6
--
2.2.2e
5.33 ± 0.61
5.00 ± 0.57
√
0/6
--
2.2.2f
4.33 ± 0.42
1.00 ± 0.63
√
4/6
66.66%
2.2.2g
6.66 ± 0.66
7.30 ± 0.61
√
0/6
--
2.2.2h
4.66 ± 0.49
1.00 ± 0.63
√
5/6
83.33%
2.2.2i
5.66 ± 0.33
9.16 ± 0.74
√
0/6
--
2.2.2j
6.66 ± 0.33
9.83 ± 0.60
√
0/6
--
2.2.2k
6.16 ± 0.47
8.66 ± 0.49
√
0/6
--
2.2.2l
6.50 ± 0.42
9.66 ± 0.61
√
0/6
--
2.2.2m
4.66 ± 0.22
3.66 ± 0.33
√
0/6
--
2.2.2n
4.83 ± 0.47
5.33 ± 0.88
√
0/6
--
Standard
(Phenytoin)
4.00 ± 0.36
0.00 ± 0.00
√
6/6
100 %
Summary & Conclusion:All the synthesized compounds exhibit significant to moderate
anticonvulsant activity. Compounds 2.2.2a, 2.2.2b, 2.2.2c, 2.2.2d,
2.2.2e, 2.2.2h, 2.2.2m, 2.2.2n showed significant anticonvulsant
activity in the flexion phase, other compounds showed moderate
activity. In the extensor phase 2g showed most significant activity
when compared with 2.2.2a, 2.2.2b, 2.2.2c, 2.2.2d, 2.2.2e, 2.2.2h,
2.2.2m, 2.2.2n. Compounds 2.2.2i, 2.2.2j, 2.2.2k, 2.2.2l showed
reduction in the duration of the flexion and extensor phase. 2.2.2e and
2.2.2g least active in the flexion and showed significant activity in
extensor phase.
213
Chapter - VI
Activity zone shown by synthesized indazole comp.(Table-6.2A)
Activity zone shown by synthesized Imidazole comp. (Table-6.2B)
Activity zone shown by synthesized Benzoxazine comp. (Table-6.2C)
214
Chapter - VI
Activity zone shown by Bromoxazine comp. (Table-6.2D)
Activity zone shown by Bromo spiro base comp. (Table-6.2E)
215
Chapter - VI
6.5.References:
1.
M.R. Shiradkar, K.K. Murahari, H.R. Gangadasu, T. Suresh,
C.C. Kalyan, D. Panchal, R. Kaur, et al. Bioorg. Med.
Chem.2007, 15, 3997.
2.
Y. Tanin, Bioorg. Med. Chem.2007, 15, 2479.
3.
E. Gursoy, N. Guzeldemirci- Ulusoy, Eur. J. Med. Chem. 2007,
42, 320.
4.
A. Nayyar, R. Jain, Curr. Med. Chem. 2005, 12, 1873.
5.
R.M. Fikry, N.A. Ismael, A.A. EI-Bahnasawy, A.A. Sayyed,
El-Ahl., Phos. Sulf and Silicon, 2004, 179, 1227.
6.
A. Walcourt, M. Loyevsky, D.B. Lovejoy, V.R. Gordeuk, D.R.
Richardson. Int. J. Biochem. Cell Biol. 2004, 36, 401.
7.
H.S. Patel, H. J. Mistry., Phos. Sulf and Silicon, 2004, 179,
1085.
8.
H.S. Patel, V.K. Patel, Ind. J. Het. Chem. 2003, 12, 253.
9.
K.R. Desai, Bhanvesh Naik, Ind. J. Chem. 2006, 45 (B) 267.
10.
V.V. Mulwad, B.P. Choudhari, Ind. J. Het. Chem. 2003, 12, 197.
11.
K. Akiba, M. Wad, Chem. Abstr. 1989, 111, 96964b.
12.
William Foye, Principles of Med. Chemistry, IVth edition,
Waverly, 831.
13.
Cooper G., The cell of molecular approach, 2nd edition, ASM,
500.
14.
Pankey G.A., Sabath, L.D., Clin. Infect. Dis. 2004, 38, 864.
15.
Chu, D.T.W., Plattner, J.J., Katz, L., J. Med. Chem. 1998, 39,
3853.
216
Chapter - VI
16.
Kohanski, M.A., cell. 2007, 130, 797.
17.
S.K. Kulkarni, Handbook of expt. Pharmacology, 68-72.
18.
E. Joseph & E.A. Adellburg, Textbook of Microbiology, 1978,
212-214.
19.
M.R. Rao, K. Hart, N. Devanna and K.B. Chandrasekhar,
Asian. Jou. Chem. 2008, 20, 1402.
20.
K.B. Kaymakciogln, E.E. Oruc, S. Unsalan, F. Kandemirli, N.
Shuets, S. Rollas, D. Anatholy, Eur. J. Med. Chem. 2006, 41,
1253.
21.
R. Kalsi, M. Shrimali, T.N. Bhalla, J.P. Bhartwal, Indian. J.
Pharm. Sci. 2006, 41, 353.
22.
S. Gemma, G. Kukreja, C. Fattorussa, M. Persico, M. Romano,
M. Altarelli, L. Savini, G. Campiani, E. Fattoruoso, N.
Basilico, Bioorg. Med. Chem. Lett. 2006, 16, 5384.
23.
D. Sriram, P. Yogeeswari, K. Madhu, Bioorg. Med. Chem.
Lett. 2005, 15, 4502.
24.
M.G. Mamolo, V. Falagiani, D. Zampieri, L. Vio, E. Banfi, G.
Scialino, Farmaco. 2003, 58, 637.
25.
N. Terzioglu, A. Gursoy. Eur. Jou. Med. Chem. 2003, 38, 781.
26.
S.G. Kucukguzel, E.E. Oruc, S. Rollas, F. Sahin, A. Ozbek,
Eur. Jou. Med. Chem. 2002, 37, 197.
27.
S. Rollas, N. Gulerman. Farmaco. 2002, 57, 171.
28.
L.Q. Al-Mawsawi, R. Dayam, L. Taheri, M. Witurouw, Z.
Debyser, N. Neamati, Bio-org. Med. Chem. Lett. 2007, 17
(23), 6472.
217
Chapter - VI
29.
C. Plasenicia, R. Daym, Q. Wang, J. Pinski, T.R. Jr. Burke,
D.I. Quinn and N. Neamati, Mol. Cancer. Ther. 2005, 4(7),
1105-1113.
30.
H. Zhao, N. Neamati, S. Sunder, H. Hong, S. Wang, G.W.
Milne, Y. Pommier, T.R. Jr. Burke, J. Med. Chem. 1997, 40
(6), 937.
31.
Austen K.F. Drugs, 1987, 33 (1), 18.
32.
William Foye, Principles of Med. Chem., Vth Edition, 751.
33.
Bhargava K.P, Gupta M.B, Ind. Jou. Pharm. Education, 1977,
42, 52.
34.
Caroline Charlier, Catherine Michaux. Eur. Jou. Med. Chem.
2003, 38, 645.
35.
Funk C.D, Science. 2001, 394, 1871.
36.
Vane J.R, Botting R.M, Am. Jou. Med. 1998, 104 (3A), 2S.
37.
Changhai Ding, CicuttiniFlavia, Drugs. 2003, 6, 802.
38.
Donald T, Abrahan, NityaAnand, William A, Remers, Burger’s
Medicinal Chemistry, 6th edition, 5, 537.
39.
Dannhardt G, Kiefer W, Eur. Jou. Med. Chem. 2001, 36, 109.
40.
Textbook of General Microbiology, Volume I, Pawar and
Daginawala.
41.
Textbook of Medicinal Chemistry, Alka Gupta.
42.
Textbook of Medicinal Chemistry, Parimoo.
43.
Textbook of Medicinal Chemistry, Ashutosh Kar.
44.
Introduction to Microbioloty, 2nd edition, John, L. Ingraham,
Catherine Ingraham.
218