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141
CHAPTER-6
ANTIMICROBIAL STUDIES
206
6.1. Introduction
6.1.1. Antimicrobials and their importance
An antimicrobial is a substance that kills or inhibits the growth of microorganisms such as bacteria,
fungi, or protozoans. This capability makes them unique for the control of deadly infectious diseases
caused by a large variety of pathogenic microorganisms.
Today, more than 15 different classes of antimicrobials are known. They differ in chemical
structure and mechanism of action. Specific antimicrobials are necessary for the treatment of specific
pathogens.
Since their discovery, antimicrobial agents have substantially reduced the threat posed by
infectious diseases. The use of these "wonder drugs", combined with improvements in sanitation,
housing, and nutrition, and the advent of widespread immunization programmes, has led to a dramatic
drop in deaths from diseases that were previously widespread, untreatable, and frequently fatal. Also,
these drugs have contributed to the major gains in life expectancy by helping to control many serious
infectious diseases.
Antimicrobials can be divided into two classifications based upon their effects on target cells.
Substances that actually kill microorganisms are termed ‘bactericidal’. Compounds that only inhibit the
growth of microorganisms are termed ‘bacteriostatic’. The decision to use a bactericidal or
bacteriostatic drug to treat infection depends entirely upon the type of infection. Some examples of
bactericidal and bacteriostatic drugs are Streptomycin, Aminoglycosides, Penicillin, Sulfonamides,
Tetracycline etc.
Also, based on their range of activity, antimicrobial drugs can be classified as (i) narrow
spectrum drugs, which are only active against a relatively small number of gram-positive organisms, (ii)
moderate spectrum drugs, which are effective against gram-positive and the most systemic, enteric and
207
urinary tract gram-negative pathogens, (iii) narrow and moderate spectrum drugs like beta-lactam
antibiotics, (iv) broad spectrum drugs which are active against all prokaryotes with two exceptions,
Mycobacteria and Pseudomonas and (v) drugs which are effective against Mycobacteria only.
6.1.2. Antimicrobial agents and their mechanism of action
In medication of microbial infections different types of AMDs are used. Before the discovery of
Penicillin, in the early 1940's, no true cure for gonorrhea, strep throat, or pneumonia existed. Patients
with infected wounds often had to have a wounded limb removed, or face death from infections. Now,
most of these infections can be cured easily with a short course of many exiting active antimicrobials.
The old antimicrobial technology was based either on poisons or heavy metals, which may not have
killed the microbe completely, allowing the microbe survive, change, and become resistant to the
poisons and/or heavy metals. However, it has been observed that with the development of new
antimicrobials, microorganisms have adapted and become resistant to previous antimicrobial agents. A
schematic representation of the history of antimicrobials has been captured in Fig-6.1.
208
Fig-6.1: History of antimicrobials
The antimicrobial agents function by attacking various cellular targets which include cell wall,
plasma membrane, nucleic acids and proteins synthesis of the microbe. The precise mechanisms of
action of antimicrobial drugs are still not clear, but the following possible views were proposed for their
mode of action.
• Inhibition of cell wall synthesis: Certain antimicrobials work by inhibiting the cell wall synthesis.
Therefore, they have little effect on host cells, which do not contain peptidoglycan. Penicillin,
Bacitracin, Cephalosporine and Vancomycin act in this way.
• Inhibition of protein synthesis: Several antimicrobial agents like Chloramphenicol,
Erythromycin, Streptomycin, Tetracyclines etc. act by inhibiting protein synthesis. As ribosomes
of prokaryotic cells are slightly different from those of eukaryotes, they can be used as a target.
• Injury to the plasma membrane: This is a mode of action for certain antibacterials and
antifungals. Antifungals are able to work mostly against fungus cell membranes because they
contain ergosterol instead of cholesterol. However, these antimicrobials are potentially quite toxic
to the host. The examples include Polymixins (antibacterial), and Amphotericin B, Miconazole,
and Ketoconazole (antifungal).
• Inhibition of nucleic acid synthesis: These drugs interfere with DNA replication and
transcription, but their selective toxicity varies. Rifampin and certain quinolone derivatives are the
examples under this mode of action.
• Inhibition of the synthesis of essential metabolites: Generally sulfas and Trimethoprim functions by
this way. They interfere with the pathway on which bacteria synthesize folic acid. Since humans
produce folic acid by a different pathway, these drugs have less effect on human cells.
209
In conclusion, the schematic representation of the mechanism of action of antimicrobial agents is given
in Fig-6.2.
Fig-6.2. Schematic representation of mechanism of action on bacterial cell
During the past 60 years, antimicrobial drugs have been critical in the fight against infectious
diseases caused by bacteria and other microbes. However, in the past decade these disease-causing
microbes have become resistant to the antimicrobial drug therapy causing severe public health problem.
Wound infections, gonorrhea, tuberculosis, pneumonia, septicemia and childhood ear infections are just
a few of the diseases that have become hard to treat with antimicrobials. One part of the problem is that
bacteria and other microbes that cause infections are remarkably resilient and have developed several
ways to resist antibiotics and other antimicrobial drugs. Currently, resistance to first-line antimicrobial
agents is further aggravated. Infections caused by these resistant microbes fail to respond to treatment
resulting in prolonged illness and greater risk of death. Nowadays, the alarming rates of emerging and
re-emerging microbial threats coupled with increasing antituberculosis, antibacterial and antifungal
resistance; particularly in regard to multi drug-resistant microbes [1] are major concerns to the public
210
health as well as scientific communities worldwide. Although new and more expensive drugs have
developed, their cost is beyond the common man’s reach. As a consequence, these trends have
emphasized the pressing need for new, more effective, cheaper and safe antimicrobial agents. This has
instigated the scientific community to carry out extensive research activities on design and development
of new antimicrobials.
6.1.3. Antimicrobial screening
In general, antimicrobial activity of any substance can be investigated by various screening
methods (WHO/CDS/CSR/RMD, 2003). Both in vivo and in vitro methods are widely used for
screening of compounds for antimicrobial activity. Amongst them, in vitro methods are extensively
used for the preliminary evaluation of antifungal, antibacterial and antituberculosis activities. Further,
in vivo studies are employed on animal models of the human condition necessary to elucidate the
mechanisms of antimicrobial action and to develop drugs that can control infection caused by
pathogenic microbes. Furthermore, the toxicity (IC50) studies of these molecules are performed on a
mammalian Vero cell line in order to pass them into phase trials.
During the last decades, several experimental procedures were developed for Antimicrobial
Susceptibility Testing (AST) by CLSI (Clinical and Laboratory Standards Institute) that created
standards to perform ASTs. These methods are extensively being used to determine the molecular
potency against microbes.
Generally, in vitro antimicrobial susceptibility testing methods are divided mainly into three
types, viz. (i) Diffusion, (ii) Dilution and (iii) Diffusion and Dilution methods. Some of the important
antimicrobial testing methods have been discussed in the following sections.
6.1.3.1. Diffusion methods
211
Diffusion method involves two important techniques, viz. Stokes method and Kirby-Bauer
method. These methods are typically used for antimicrobial susceptibility testing, which are being well
recommended by the NCCLS.
(i) Stokes method
In this method a known quantity of bacteria is grown on agar plates in the presence of thin wafers
containing relevant standard antibiotics. If the bacteria are susceptible to a particular antimicrobial, an
area of clearing surrounds the wafer where bacteria are not capable of growing (called a zone of
inhibition). Also, the rates of antimicrobial diffusion are determined and these values are used to
estimate the bacteria’s sensitivity to that particular antimicrobial agent. In general, larger zones
correlate with smaller concentration of test compounds for a specific microorganism. This information
can be used to choose appropriate antimicrobials to combat a particular infection.
(ii) Kirby method
The Kirby-Bauer test, known as the disk-diffusion method, is the most widely used antibacterial
susceptibility test in determining the precise antibiotics used to treat the exact infection. This method
relies on the inhibition of bacterial growth measured under standard conditions. For this test, a culture
medium, specifically the Mueller-Hinton agar, is uniformly and aseptically inoculated with the test
organism and then filter paper discs, which are impregnated with a specific concentration of a particular
antimicrobial, is placed on the medium. The organism will grow on the agar plate while the
antimicrobial ‘works’ to inhibit the growth. If the organism is susceptible to a specific antimicrobial
drug, there will be no growth around the disc containing the antibiotic. Thus, a ‘zone of inhibition’ can
be observed and measured to determine the susceptibility to an antimicrobial for that particular
organism.
6.1.3.2. Dilution Methods
212
Dilution technique mainly includes minimum inhibition concentration (MIC) method, which can be
further classified as broth dilution and agar dilution methods.
Minimum Inhibitory Concentration (MIC) method
MIC method is generally used to determine the minimal concentration of antimicrobial to inhibit
or kill the microorganisms completely. This can be achieved by dilution of antimicrobial solution in
either agar or broth media. The dilutions are normally expressed in log2 serial dilutions (i.e. two fold).
In this method, a pure culture of a single microorganism is grown in appropriate broth. The culture is
standardized using standard microbiological techniques (nearly 1 million cells per milliliter). The
compound under screening is diluted a number of times, 1:1, using a sterile diluent. After dilution, a
volume of the standardized inoculum equal to the volume of the diluted compound is added to each
dilution vessel bringing the microbial concentration to approximately 500,000 cells per milliliter. The
inoculated, serially diluted antimicrobial agent is incubated. After incubation, the dilution vessels are
observed for microbial growth, the results of which are usually indicated by turbidity or colour change.
The last tube in the dilution series that does not demonstrate growth corresponds to the minimum
inhibitory concentration (MIC) of the antimicrobial agent.
(i) Broth dilution method
The Broth dilution method is a simple technique for testing a small number of isolates, even
single isolate. It involves serial dilution of the antimicrobial agent in a liquid medium, which is then
inoculated with a standardized number of organisms and incubated for a prescribed time. The lowest
concentration of antibiotic preventing appearance of turbidity is considered to be the minimal inhibitory
concentration. It has the added advantage that the same tubes can be taken for Minimum Bactericidal
Concentrations (MBC) tests also.
213
(ii) Agar dilution method
In this method, the compounds under screening are diluted on log2 dilution intervals where each
petri dish contains 50 per cent of the concentration of the given compound in the previous dilution. The
diluted solution is incorporated into the agar medium and mixed by gentle rotation and poured into petri
dish. A control plate without any antimicrobial agent incorporated into the medium is also used along
with each compound tested, to check for growth of the test and control strains. Readings are recorded
after the petri dishes have been incubated. The main advantage of the method is that it is possible to test
several organisms on the same plate.
6.1.3.3. Dilution and Diffusion method
Dilution and diffusion method is a convenient method to screen the antimicrobial susceptibility
of any substance. It is also known as Epsilometer test (E test). This ‘exponential gradient’ testing
methodology is generally used for the quantitative antimicrobial screening wherein both dilution of
antimicrobial and diffusion of antimicrobial into the medium involve. In this method, a thin inert carrier
strip containing a predefined stable antimicrobial gradient is used. It is then applied onto an inoculated
agar plate. Then, there is an immediate release of the drug. On incubation for 24 hours, a symmetrical
inhibition ellipse is produced. The intersection of the inhibitory zone edge and the calibrated carrier
strip indicates the MIC value over a wide concentration range (>10 dilutions) with inherent precision
and accuracy. E test is simple, easy to perform and is a reliable method for determination of MIC.
Also, it has been shown to be a good alternative to the agar and broth dilution tests, particularly for the
strains such as Haemophilus. influenza. However its cost and limited availability is a concern.
The latest ‘genotypic’ technique for detection of antimicrobial resistance genes has also been
promoted as a way to increase the speed and accuracy of susceptibility testing. Numerous DNA based
assays are being developed to detect bacterial antibiotic resistance at the genetic level. These methods,
214
when used in conjunction with phenotypic analysis, offer the promise of increased sensitivity,
specificity, and speed in the detection of specific known resistance genes and can be used in tandem
with traditional laboratory AST methods.
Although a variety of methods exist, the goal of in vitro antimicrobial susceptibility testing is to
provide a reliable predictor of how an organism is likely to respond to antimicrobial therapy in the
infected host. This type of information aids the clinician in selecting the appropriate antimicrobial
agent, aids in developing antimicrobial use policy, and provides data for epidemiological surveillance.
Such epidemiological surveillance data provide a base to choose the appropriate empirical treatment
(first-line therapy) and to detect the emergence and/or the dissemination of resistant bacterial strains or
resistance determinants in different bacterial species. The selection of a particular AST method is based
on many factors such as validation data, practicality, flexibility, automation, cost, reproducibility,
accuracy, and individual preference.
In our present study, serial dilutions method has been followed for the investigation of
antimicrobial properties of newly synthesized compounds, since this method is popular in laboratory
due to low cost, reproducibility in results, convenient to perform and accuracy. The detailed
experimental procedures along with screening results have been discussed in this chapter.
6.2. Experimental protocol
All the newly synthesized compounds were screened for their antibacterial activity. For this,
Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa
microorganisms were employed. Antimicrobial study was assessed by Minimum Inhibitory
Concentration (MIC) by serial dilution method [2]. Several colonies of Staphylococcus aureus, Bacillus
subtilis, Escherichia coli and Pseudomonas aeruginosa were picked off a fresh isolation plate and
inoculated in corresponding tubes containing 5 ml of trypticase soya broth. The broth was incubated for
215
6 hrs at 37 oC until there was visible growth. Mc Farland No.5 standard was prepared by adding 0.05 ml
of 1% w/v BaCl2 .2H2O in Phosphate Buffered saline (PBS) to 9.95 ml of 1% v/v H2SO4 in PBS. The
growth of all the four cultures was adjusted to Mc Farland No.5 turbidity standard using sterile PBS.
This gives a 108 cfu/ml suspension.
The working inoculums of above mentioned four different microorganisms containing 105 cfu/ml
suspension was prepared by diluting the 108
cfu/ml suspension, 103 times in trypticase soya broth
Preparation of Anti-microbial Suspension (50µg/ml).
Dissolved 0.5 mg of each compound in 10 mL of trypticase soya broth to get 50µg/mL. This
suspension was filter sterilized in syringe filters.
Preparation of dilutions.
In all, for each of the anti-microbial compounds and standard antimicrobial i, e Ceftriaxone, 24
tubes of 5 ml capacity were arranged in 4 rows with each row containing 6 tubes. Then 1.9 mL of
trypticase soya broth was added in the first tube in each row and then 1 ml in the remaining tubes. Now,
100µl of filtered anti microbial suspension was added to the first tube in each row and then after mixing
the content, 1 ml was serially transferred from these tubes to the second tube in each of the rows. Then
the contents in the second tube of each of the rows were mixed and transferred to the third tube in each
of the rows. This serial dilution was repeated till the sixth tube in each of the rows. This provided anti
microbial concentrations of 50, 25, 12.5, 6.25, 3.125, 1.6125 µg /mL in the first to sixth tube
respectively in each row. Finally, 1 ml of 105 cfu /ml of Staphylococcus aureus, Bacillus subtilis,
Escherichia coli and Pseudomonas aeruginosa suspension were added to the first, second, third and
fourth rows of tubes respectively. Along with the test samples and Ceftriaxone (standard), the inoculums
control (without antimicrobial compound) and broth control (without antimicrobial compound and
inoculum) were maintained. All the test sample and control tubes were then incubated for 16 hrs at
216
37oC. After incubation, the tubes showing no visible growth were considered to be representing the
MIC. The results of each series of compounds are discussed below.
6.3 Results and discussions
6.3.1. Antimicrobial studies of 5H-Chromeno [2,3-d] pyrimidine derivatives
O N
N
HNR
160 (a-l)
a: R = H
b: R = 2,4-Dichlorophenyl
c: R = 2-Methyl,4-triflouromethylphenyl
d: R = 4-(1-Benzylpiperidine)
e: R = Benzyl
f: R = 4-Chlorophenyl
g: R = 1-Napthyl
h: R = 2,4-Dimethylphenyl
i: R = Adamantyl
j: R = 3-Fluoro-5-methylphenyl
k: R = 5-Methyl-2-thiazolyl
l: R = 2-Chloro-4-fluorophenyl
Fig-6.3: Chromenopyrimidine derivatives
In this series twelve newly synthesized compounds (160 a-l) were screened for antibacterial
activity against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa
strains using Ceftriaxone as a standard. The results are summarised in Table-6.1. The antibacterial
screening revealed that some of the tested compounds showed good inhibition against various tested
microbial strains. Among the screened samples 160a, 160b and 160c have not showed any antibacterial
property against all bacterial strains. However compounds 160d, 160e, 160f, 160g, 160h which contains
benzylpiperdine, benzyl, 4-chlorobenzyl, 1-naphthyl and 2,5-dimethylphenyl moieties respectively have
showed excellent antibacterial activity at 1.6125 µg/mL concentration against Staphylococcus aureus,
bacteria as compared to the standard drug Ceftriaxone which is active at 3.125 µg/ml concentration.
Similarly compounds 160d, 160e, 160f, 160g and 160h have showed same activity as that of the
217
standard which is active at1.6125 µg/ml against Bacillus subtilis. However none of the compounds were
active against bacterial strains Escherichia coli and Pseudomonas aeruginosa.
Table - 6.1: Antibacterial activity data in MIC (µg/ml)
Compound
No. S. aureus B.subtilis E.coli P.aeruginosa
160a * * * * * * * * * * * *
160b * * * * * * * * * * * *
160c * * * * * * * * * * * *
160d 1.6125 1.6125 3.125 * * *
160e 1.6125 1.6125 25.00 * * *
160f 1.6125 1.6125 50.00 * * *
160g 1.6125 1.6125 * * * * * *
160h 1.6125 1.6125 * * * * * *
160i * * * * * * 50.00 50.00
160j 50.00 * * * * * * * * *
160k * * * * * * * * * 25.00
160l * * * * * * * * * 25.00
Ceftriaxone
(Standard) 3.125 1.6125 1.6125 1.6125
Inoculum
control
* * * * * * * * * * * *
Broth
control No growth No growth No growth No growth
* * * Indicates Growth in all concentrations
218
6.3.2 Antimicrobial studies of Chromeno Oxadiazole derivatives
O N
N
ONR
R
210 (a-n)
S
NO
O OH
H NH
N
HN
NO
O
O
NOS
N NH2
Ceftriaxone (Standard )
a: R = 2-Chlorophenyl g: R= 3-Nitrophenylb: R = 4-Chlorophenyl h: R = 5-Bromo-2-fluorophenylc: R = 2- Hydroxyphenyl i: R = Phenyld: R = 3,4-Dimethoxyphenyl j: R = 2-Thiophenyle: R = 4-Fluorophenyl k: R = 4-Hydroxy-3-methoxyphenylf: R = Cinnamyl l: R = 4-Bromophenylm:R = 2- Thiazolyl n: R = 4-Fluoro-3-phenoxyphenyl
Fig-6.4: Chromeno-Oxadiazole derivatives
The in vitro antibacterial activity of newly synthesized compounds (210 a-n) were determined by
serial dilution method as explained in experimental protocol by measuring MIC values. In this work,
Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa were used to
investigate the antibacterial activity. Ceftriaxone was used as standards for the comparison of
antibacterial activity. Results of antimicrobial studies have been presented in Table 6.2.
219
Table-6.2. Antimicrobial activity data of the compounds (210 a-n) in MIC (µg/ml)
Antibacterial activity
Comp.
S. aureus B.subtilis E.coli P.aeruginosa
210a 3.1250 6.2500 3.1250 3.1250
210b 6.2500 3.1250 12.5000 1.6125
210c 1.6125 1.6125 1.6125 1.6125
210d 3.1250 3.1250 1.6125 1.6125
210e 1.6125 3.1250 3.1250 1.6125
210f 3.1250 1.6125 1.6125 1.6125
210g 6.2500 3.1250 3.1250 3.1250
210i 12.5000 6.2500 6.2500 6.2500
210j 6.2500 6.2500 3.1250 3.1250
210k 6.2500 3.1250 3.1250 3.1250
210l 12.5000 3.1250 6.2500 3.1250
210m 6.2500 6.2500 3.1250 3.1250
210n 6.2500 6.2500 3.1250 3.1250
Standard 3.1250 1.6125 1.6125 1.6125
Inoculum
control
* * * * * * * * * * * *
Broth control No growth No growth No growth No growth
220
* * * Indicates Growth in all concentrations
The antibacterial screening revealed that some of the tested compounds showed good inhibition
against various tested microbial strains. The result indicated that among the tested compounds, 210c and
210f which contains hydroxyl and cinnamyl functional groups showed excellent activity against all the
tested bacterial strains compared to standard drug Ceftriaxone. 210e showed excellent activity as that of
standard, against Staphylococcus aureus and Pseudomonas aeruginosa. Compound 210d showed
similar anti-microbial activity against Staphylococcus aureus, Escherichia coli and Pseudomonas
aeruginosa as compared to the standard drug. Compounds 210a and 210b showed moderately good
anti-microbial activity against all the tested microbial strains. The remaining compounds have showed
less activity against all of the four tested bacterial strains compared to standard, Ceftriaxone.
6.3.3 Antimicrobial studies of new (1H-Pyrazol-3-yl)-1, 2, 4-oxadiazole derivatives
NH
N
R O
NN
R1
263(a-l)
a: R= 4-Me, R1= H; e: R= 4-Me, R
1= 4-OMe; i: R= 4-Me, R
1= 3-OMe;
b: R= 4-Me, R1= 4- Cl; f: R= Me, R
1= 2-Br,4-Cl; j: R= CF3, R
1= 4-F;
c: R= 3-OMe, R1= 4- Cl; g: R= 4- Cl, R
1= 2,4- Cl k: R= CF3, R
1= 4-N(CH3)2
d: R= 3-OMe, R1= 4- F; h; R= 3-OMe, R
1= 4-NO2 l: R= Me, R
1= Morpholine
Fig-6.5: (1H-Pyrazol-3-yl)-1, 2, 4-oxadiazole derivatives
The in vitro antimicrobial activity of newly synthesized compounds (263 a-l) was determined by
serial dilution method as explained in experimental protocol. In this work, Staphylococcus aureus,
Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa were used to investigate the
antibacterial activity. Antimicrobial study was assessed by Minimum Inhibitory Concentration (MIC) by
221
serial dilution method. Newly synthesized compounds (263a-l) were also screened for their antifungal
activity against Candida albicans. Antifungal activity was compared with the standard drug
Fluconazole. Among the screened samples, 263c and 263d which contains methoxy, chloro and
methoxy fluoro at 3rd and 4th positions respectively emerged as potent antimicrobial agents. Results of
antimicrobial studies have been presented in Table 6.4. Compound 263c showed excellent activity
against Staphylococcus aureus compared to standard. 263c showed similar activity against Bacillus
subtilis and Pseudomonas aeruginosa compared to standard drug Ceftriaxone. 263b and 263c showed
similar activity as that of standard, against Pseudomonas aeruginosa and 263h had shown similar
activity against Staphylococcus aureus compared to Ceftriaxone. The remaining compounds showed
moderately good activity against all of the four tested bacterial strains compared to standard,
Ceftriaxone.
Antifungal activity data revealed that compounds 263c and 263d had shown similar activity against the
fungal stain Candida. albicans compared to the standard drug Fluconazole. Other derivatives were less
active compared to standard.
Table 6.3: Antimicrobial activity data of the compounds (263 a-l) in MIC (µg/ml)
Antibacterial activity Antifungal
activity Comp.
S.
aureus B.subtilis E.coli P.aeruginosa C. albicans
263a 3.1250 3.1250 3.1250 6.2500 12.5000
263b 6.2500 3.1250 3.1250 1.6125 12.5000
263c 1.6125 1.6125 3.125 1.6125 6.25000
263d 3.1250 1.6125 3.1250 1.6125 6.25000
263e 12.5000 6.2500 3.125 6.2500 12.5000
263f 6.2500 12.500 6.2500 6.2500 12.5000
263g 6.2500 3.1250 3.1250 6.2500 25.0000
263h 3.1250 6.2500 3.1250 3.1250 12.5000
263i 12.5000 6.2500 12.5000 3.1250 12.5000
263j 6.2500 12.5000 12.5000 12.5000 25.0000
222
263k 12.5000 12.5000 25.0000 12.5000 25.0000
263l 12.5000 25.0000 12.5000 12.5000 25.0000
Standard 3.1250 1.6125 1.6125 1.6125 6.2500
Inoculum control
* * * * * * * * * * * * * * *
Broth control
No growth
No growth No
growth No growth No growth
* * * Indicates Growth in all concentrations
6.3.4 Antimicrobial studies of Homoallylamines and β-amino-ketones
Antimicrobial studies of Homoallylamines
R NH
R1
(331 a-q)
Fig-6.6: Homoallylamine derivatives
Table-6.4: Homoallylamine derivatives
S. No R R1
331a Phenyl Napthyl
331b 2-Benzofuran 3,4-Diflurobenzyl
331c Cyclopropyl 4-t-Butylphenyl
331d 2,4-Difluorophenyl 2,4,5-Trifluorophenyl
331e 2-Benzofuran Napthyl
331f 2,4-Difluorophenyl 4-t-Butylaniine
331g 2-Fluoro-5-methoxyphenyl 3-Fluorophenyl
331h Cyclopropane carboxaldehyde 4-Fluoro-3-trifluoromethyl- Phenyl
331i Cyclohexane carboxaldehyde 4-Morpholinophenyl
331j Cyclohexane carboxaldehyde 2,5-Dimemethylphenyl
331k 2-Allyloxyphenyl 4-(4-Chlorophenoxy) Phenyl
331l 5-(2-Chlorophenyl)furan-2-carbaldehyde 4-(4-Chlorophenoxy) phenyl
223
331m 1-Acetyl-1H-3-indolyl Benzo[d]thiazol-7-amine
331n 2,4-Difluorophenyl 2,4-Difluorophenyl
331o 2,6-Difluorophenyl 4-Chloro-3-fluorophenyl
331p 3-Thiophenyl 4-Cyanophenyl
331q 3-Ethoxyphenyl 4-Fluorophenyl
Seventeen newly synthesized compounds were screened for antibacterial activity by MIC
method. Among the screened samples, 331a, 331i, 331j, 331k, 331m and 331p have showed very poor
antibacterial property against all bacterial strains. Compounds 331d, 331g, 331n, 331o have showed
excellent antibacterial activity at 1.6125 µg/ml concentration against all microorganisms as compared to
the standard drug Ceftriaxone. Interestingly all the above four biologically active molecules are halogen
substituted, which is accounted for their significant antibacterial activity. Compound 331h which is also
trifluoro substituted is active against Staphylococcus aureus and Bacillus subtilis however which has not
showed any activity against other two bacterial strains. Remaining compounds showed moderate
antibacterial activity. Reults of this antimicrobial study is summarised in Table-6.5.
Table 6.5: Antimicrobial activity data of the compounds (331 a-q) in MIC (µg/ml)
Compound No. S. aureus B.subtilis E.coli P.aeruginosa
331a 25 25 25 25
331b 1.6125 3.125 3.125 3.125
331c * * * * * * * * * * * *
331d 1.6125 1.6125 1.6125 1.6125
331e 1.6125 1.6125 25.00 * * *
331f 3.125 3.125 3.125 3.125
331g 1.6125 1.6125 1.6125 1.6125
331h 1.6125 1.6125 * * * * * *
331i 6.250 6.250 6.250 25
331j 6.250 12.5 12.5 12.5
331k 3.125 3.125 6.250 25.00
331l 3.125 3.125 3.125 3.125
331m 12.5 12.5 12.5 12.5
331n 1.6125 1.6125 1.6125 1.6125
331o 3.125 1.6125 1.6125 1.6125
331p 6.250 6.250 3.125 6.250
331q 3.125 3.125 3.125 3.125
224
Ceftriaxone (Standard) 3.125 1.6125 1.6125 1.6125
Inoculum control * * * * * * * * * * * *
Broth control No growth No growth No growth No growth
* * * Indicates Growth in all concentrations
Antimicrobial studies of β-amino-ketones
O HN
R
R1
(335 a-k)
Fig-6.7: β-Amino-ketones derivatives
Table-6.6: β-Amino-ketones derivatives
S.No R R1
335a Phenyl t-Butylphenyl
335b 4-Fluorophenyl 2,4-Difluorophenyl
335c 2-Chlorophenyl 4-Cyanophenyl
335d 2-Fluoro-5-methoxy
phenyl 2,4-Difluorophenyl
335e 2-Fluorophenyl 3,4-Difluorophenyl
335f 2-Allyloxyphenyl 3-Fluorophenyl
335g 2-Hydroxy-3-methyl
phenyl 3-Methoxyphenyl
335h 4-Ethylphenyl
3,4,5-Trifluoro-methyl
phenyl
335i 4-Ethylphenyl
4-Methyl-3 nitro
phenyl
335j 2-Benzofuran Napthyl
335k 4-Pyridyl
2-Methyl-5-
aminoindole
Eleven new β-Amino-ketones with different substituent (Table-6.7) are synthesised. All these
compounds are screened for their in vitro antimicrobial activity using by serial dilution method as
explained in experimental protocol. In this work, Staphylococcus aureus, Bacillus subtilis, Escherichia
coli and Pseudomonas aeruginosa were used to investigate the antibacterial activity. Antimicrobial
225
study was assessed by Minimum Inhibitory Concentration (MIC) by serial dilution method. The results
are summarised in Table-6.7.
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Table-6.7: Antimicrobial activity data of the compounds (335a-k) in MIC (µg/ml)
Compound
No.
S. aureus B. subtilis E. coli P. aeruginosa
335a 6.250 6.250 12.5 6.250
335b 1.6125 1.6125 1.6125 1.6125
335c 3.125 1.6125 1.6125 1.6125
335d 3.125 1.6125 1.6125 1.6125
335e 3.125 1.6125 1.6125 1.6125
335f 6.250 3.125 3.125 6.250
335g 1.6125 1.6125 1.6125 1.6125
335h 12.5 12.5 12.5 12.5
335i 6.250 6.250 6.250 25
335j 3.125 3.125 1.6125 1.6125
335k 3.125 1.6125 1.6125 1.6125
Ceftriaxone
(Standard)
3.125 1.6125 1.6125 1.6125
Inoculum
control
* * * * * * * * * * * *
Broth control No growth No growth No growth No growth
* * * Indicates Growth in all concentrations
Antimicrobial screening showed that, most of the compounds showed significant antibacterial
activity as compared to the standard drug Ceftriaxone, Staphylococcus aureus, Bacillus subtilis,
Escherichia coli and Pseudomonas aeruginosa. Compounds 335b, 335c, 335d, 335e, 335g and 335k
have showed excellent antimicrobial activity as that of the standard drug at the same concentration
against all bacterial strains. Compounds 335b, 335c, 335d and 335e have halogens substitutions, while
compound 335k has pyridine and indole substitutions, which is accounted for the biological activity.
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Compound 335g has methoxy substitution has also showed excellent activity. Compound 335a, which
has t-butyl substitution has showed poor antibacterial activity. Remaining compounds have showed
moderate antimicrobial activity.
6.4. References
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2. Mackie, Mc. Cartney. “Practical Medical Microbiology,” 1989, Vol. 13.
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glutaminase domains of glucosamine-6-phosphate synthase promotes sugar ring opening and
formation of the ammonia channel” J Mol Biol. 2008, 377(4), 1174-1185.
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protein-ligand complexes” Acta Cryst. 2004, D60, 1355-1363.
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