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CHAPTER-6

ANTIMICROBIAL STUDIES

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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

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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.

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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.

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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

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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.

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(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,

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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

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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.

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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

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* * * 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

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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

1. Desselberger, U. “Emerging and re-emerging infectious diseases.” J. Infect. 2000, 40(1), 3-15.

2. Mackie, Mc. Cartney. “Practical Medical Microbiology,” 1989, Vol. 13.

3. Mouilleron, S., Badet-Denisot M. A., Golinelli-Pimpaneau B. “Ordering of C-terminal loop and

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.

4. Thomas, S., Joel, F., Modest, V. K., Jacqueline, R. R., Christian, R. “OSIRIS, an entirely in-House

Developed Drug Discovery Informatics System” J. Chem. Inf. Model. 2009, 49 (2), 232–246.

5. Schüttelkopf, A. W., Van Aalten, D. M. F. “PRODRG: a tool for high-throughput crystallography of

protein-ligand complexes” Acta Cryst. 2004, D60, 1355-1363.

6. Morris, G. M., Goodsell, G. M., Halliday, R. S., Huey, R., Hart, W. E., Belew, R. K., Olson, A. J.

“Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy

function” Journal of Computational Chemistry, 1998, 19 (14), 1639 – 1662.