PART. VI

ANTIMICROBIAL ACTIVITIES OF SOME

HETEROCYCLIC COMPOUNDS

Introduction:

Treatment of diseases with chemical substances has been Icnown since the 15()()'s. The chemi­

cal substances used for the treatment of inleclious diseases and diseases caused by the

proliferation of mallgnanat cells are called as chemotherapeulic agents.

Chemical agents not only provide the structural basis and energy supply of living

oragnisms but also regulate their functional activities. The interaction between poieni chcnuciil

and living system contribute to the understanding of life processes and provide effective

methods for the treatment, prevention and diagnosis of many diseases. Chemical compounds

used for this purpose are called 'drugs' and their actions on living system are relerred to as

'drug effect'. The subject of drug is as old as disease. Sickness has been mans heritage from

the begining of his existence and search for remedies to combat is perhaps equally old.

The histroy of drugs is almost as old as the history of man, but medicine really began

with the Greeks who were well aware of the value of opium and Hippocrates {4(){) B.C.) has

justly been termed 'The Father of MetfJcine'. It is interesting thai even primitive people

could discover relationships between drug and disease. The u.se of drugs has been so preva­

lent throughout histroy that Siv Willialit Osier vSlaled (1894) with some juslillcaiion thai.

"Man has an inborn craving for medicine".

Fighting disease with drug is the timeless struggle. Us beginings echoed out of thi'

primeval jungle. Man's survival on this planet has depended uopn its success.

The concept that substances derived from one living organism may kill another

327

(antibiosis) is almost as old as the science of microbiology. Indeed, the application of anli-

bioiic therapy, without recognition of it as such, is considerably older. The Chinese were

aware, over 2500 years ago, of the therapeutic properties of moldy curd of soyabean applied

to carbuncles, boils and similar infections and used this materials standard ireaimeni in such

disorders.

Antibiotics are substances which are synlhesised by micro-organisms and are leihitl

to other micro-organisms in low concentrations. An effective antibiotic must be seleclivuly

toxic to the microbial pathogen with little toxicity for the human host.

Antibacterial agents have been used in folk remedies from early times. The earliest

source of medicine come from 'Egypt' and two kingdom of'Assyria' and 'Babylonia'. The

'papyri' were the first written account of medical experiences from Egypt and dale back lo

1900 B.C. The papyrus discovered by Fiber in 1872 was prepared in 1500 B.C. & main­

tained about 700 herbal medicines including the X)phium'. A Babylonian c|ay-iablei (700

B.C.) has been discovered which mentions about 300 drugs.

Modern medicine is considered to date from 'Hippocratus' a greek physician (450

b.C.) who for the first time interoduced the concept of disea.se as a pathologic process and

tried to organise the science of medicine on the basis of observation, analysis and deduc­

tion.

Till the begiiiing of 19lh century the treatment of disease consisted of abnoxious rem­

edies such as flesh excreta and metalic and plant preparations. James Ciregory (1753-1H211

was responsible for poplilarising heroic symptomatic treatment consisting of hlin)d leiliny,

large doses of emetics and drastic purgatives often with dislraous results. Such ireaimeni

without any rational basis was called 'Allopathy' (meaning the other suffering).

328

The concept of 'Homeopathy' was firsl introduced in the early lyth century hy

'Hanneman' who thought that 'IJke curse Like' and dilution potentiates the action of drugs.

Homoeopathy outlines the therapy lor various ailments with drugs in very high dilution.

Various animal experiments have been designed to study the effects of drugs on living

organisms and isolated tissues and these give an insight in to where and how a drug act.

Knowledge of the mode of action of drug, its effect on various body systems and the prob­

able adverse effect is important.

The work, Pasteur and Koch established thai. Microorganisms were the cause of in­

fectious disease. Paul Ehrlich was the fust lo propo.se thai infectious di.sea.se.s might hi'

curable by using chemicals that inhibit or kill the infectious agent but do not harm the hosi

at the concentration employed. He discovered the famous organoansenical compoiinil

'salvarson' which was active against the causative organisms of 'syphillus'. Ii was he who

used the term 'chemotherapy'. According to his theory of diiig action, cells po.sse.sses chemiciil

receptors to which the drug bind. He recognised the importance of quantitative measurment

to determine the drug dose, that would be effective against the causative agent and not have

toxic effect on the host. He also pioneered methods for screening a large number of com­

pounds for biological activity in relation lo chemical structure. Chemical varienis of eflec-

live compounds were then synthesized and tested to see whether they have improved anti­

microbial activity and reduced toxicity.

When a living system Is in imbalance through distortion of normal bodily proce.s,ses a

pathologic condition is present, it is then that the chemical stimulus, the pharmacologic

agent, the drug, is administered to attempt lo reestablish the organism to normal physiologic

and chemical balance. A drug is administered to alleviate symptoms and possibly cure ilie

329

diseased condition. Examples will serve luilher to show this relationship between the sci­

ences.

The irihydrlc alcohol glycerin C,H,(OH)^, As a product of digestion from edible luis

the biological chemical is coticerned with its late in the body. It", however, one replaces the

three hydroxyl groups in the glycerin molecule with three nitrate radicals, producing glyceiyl

trinitrate CjHjCNO,),, the compound serves no longer as a food. The oily glyceryl iriniiruie,

in exceedingly small doses, dilates the blood vessels, causing a fall in blood pressure. Ilu'

coronary arteries share in this dilatatiDh and hence glyceryl trinitrate serves as a drug, ii

therapeutic agent. It is used also by the physician in the treatment of hypertension and

angina pectoris.

The effect of ethyl alcohol C^H^OH on the animal organism is intriguing and illus­

trates the divisions of interest among the basic medical sciences. When ingested in small

quantities alcohol produces no symptoms. It is metabolized, producing carbon dioxide and

water and each gram gives rise to seven calories. Hence in small quantities alcohol serves

merely as a food and its fate in the body is mainly a biochemical problem. In farger amounts

alcohol profoundly affects the central nervous system, causing a depression ol the inhibi­

tory centres in the brain and produces a syndrome of inebriety.

As a product of a gland of internal secretion the physiologist studies the effect ol

epinephrine on the living organism. The action of epinephrine on the organism under stress

nad its effect on metabolism are of Prime Physiologic Importance. As a product exiracled

from the medullary portion of the adrenal galnds and available for injection as a therapeullc

agent. It is a potent drug, useful in the treatment of the asthmatic spasm bacause it dllaU's

the bronchioles upon injection. Its stimulation of the myocardium has proved useful in

330

collapse.

Indeed one can readily judge from the examples cited that there are no sharp lines ul

demarcation between the basic medical sciences. These sciences are the study ol the human

body in health and in disease, structurally, functionally and chemically. Thc ultimate pur­

pose of each is the same i.e. "knowledge of life proce.s.ses and the utilization of this knowl­

edge for the health of mankind".

Historical Development:

There are several significant events in the histroy of .science. One of the first of these

was the life and work of the Swiss physician -chemist paracelsus. Prior to the linie ol para-

celsus the purpose of chemistry had been to search for the philo.sopher's stone, a ficiionul

charm which would confer perpetual youth and convert the ba,ser metals into gold. He thun

dered his .stirring message through the ranks of the medical and chemical praciiiioners ul

his lime. "The purpose of chemislry is not to make gold but to study the fundamenial sci­

ences and turn them against diseases". " Not gold but medicine is the purpo.se ol chemistry"

declared paracelsus. It portended the synthesis of thousands of compounds for pharmacologic

testing and possible medical application.

In 1630 the use of peruvian bark in the treatment of malarial fever was discovered. Ii

became known as the cinchona bark, l-rom this bark two centuries later the Irench pharma­

ceutical chemists caventou and pelletier i.solated the alkaloid quinine, the first specific driij;

to be used in an infectious disease, in the treatment of malaria.

In 1760 Louis Cadet, apothecary to the l-rench army, made cacodyl oxide (CH_).As-

0-As(CH,), by heating together pota.ssium acetate and ansenious oxide. This was the lusi

lime that man had succeeded in attaching an ansenic atom to a carbon atom. The experiment

.31

portended the synthesis of the organic arsenicals. It meant that the toxicity ol this poieni

element aresenic could he modified hy combining it in a multiplicity ol'organic iiiolecules.

In 1783 the English physician William Withering published his epoch-making mono­

graph on the use of digitalis entitled, "An Account of the purple l-'oxglove and its Medicinal

Use." Withering has used digitalis in the treatment of dropsy and believed thai he was deal­

ing with a drug which had a direct action upon the kidney.

In 1807, the German apothecary Serturner isolated from opium the alkaloid moi-

phine. He showed by experiments on dogs that much of the norcotic activity ol iipium was

dependent upon morphine. Thie was the first alkaloid that was isolated IVom a plant, there

after strychnine, caffeine, emetine, atrophine and quinine were to be separated as pure ci ys

tailine alkaloids.

The "Ipecacuanha root" was used in Brazil and Asia for treting amoebic dyseniry. Its

active constituent 'emelia' was isolated in 1817.

One of the most significant events in the histroy ol" mankind was the synthesis ol' urea

by Wholer in 1828. Wohler made urea from ammonium cyanate NH^OCN-(NHJX'O by

the evaporation of an aqueous solution of the latter. He had made urea without a kidney. He

had synthesised an organic chemical compound without the ilervention of the niysienous

vital force. He had begun an era in chemistry and he had laid the basis for the changing o\

the character of civilization. Organic chemi.srly, carbon chemistry, was to ri.se I'riim this

experiment. The chemist was to become a creator. More than a half million new chemical

entities were to appear. New dyes, new plastics and new perfumes were made.

In the middle of the seventeenth century Christopher Wren and Robert Boyle at Ox­

ford University showed that a quill inserted into the vein of a dog provided a means ol

332

injecting drugs directly into the blood stream. Based upon these studies Alexander Wood ol

b^dinburgh developed the hollow hypodermic needle in 1853. The hypoderma needle niaile

it possible to iject drugs directly into the blood stream. Dependability and rapiduy oJacliim

were achieved by this development.

In 1900 Abel and his associates seprated epinephrine from the medullary portion ol

the adrenal glands. He prcpai'ed the material as a benzoyl derivative and later in 1902 Takamine

separated the ba.se epinephrine in burr-like crystals. Its synthesis was accompli.shed by Sioll/

a few years later.

In 1905, Reid Hunt, working in Abel's laboratory obtained an extract i'rom the adrenal

medulla which exhibited marked depressor respon.ses when injected into cais. later. Hum

was able to show that what he had separated from the adrenal glands was acetylcholine, iluis

within a period of live years the respective harmones of the sympathetic and para.sympaihclie

divisions of the autonomic nervous system were discovered in professor Abel's lahoraU)ry

In 1910, Paul Bhrlich announced the preparation of ar.sphenamine . his 6()6ih com

pound studied as an anti-syphilitic drug. The importance of arsenamine anil sub.seqiieiu

drugs in the cure of syphilis cannot be overestimated. It represented the linsi hactericidal

synthetic drug with a definite specificity for an invading pathogen. Later. Mapar.sen.

sulfanilamide, penicillin and streptomycin were to follow. Hhrlich's synthesis and experl

menial trial of these arsenical ircponemicidal agents were referred to by him as "chemo­

therapy", which is the use of a defmite chemical compound U) combat the causative agent ol

an infectious disea.se.

Turner in 1913 he interpeted the blood How from the renal artery of a dog hy a seiies

of lubes consisting of permeable membranes. Blood pa.ssing through these tubes was re-

333

turned to the circulation. The tubes were surrounded with soUilions ol' the various crysuil

loids of blood. Cryslalloidal substances in the circulating blood dilTused oui uuo the sur

rounding fluid, cells and colloids remained in the hlot)d. This experiment, referred ui hs

Abel as 'Vividiffusion' and often called the "artificial kidney", for the first lime, lo ideniils

amino acids unconjugaled in the blood. Recenlly this ingenious experiment has lieen UMHI

clinically in the treatment of uremia lo remove nitrogenous waste from the blood.

In 1919 Abel and his as.sociales obtained from the po.sterior lobe of the pilunary glanil

a principle which when injected produced a pre.s.sor respon.se and al.so acted upon ihe uiei us.

cau,sing its contraction. The pre.s.sor and oxyioxic activities of the pituitary principle were

due to one molecular entity.

In 1927 Kamm and his as,sociales showed that the Principle .separated by Abel con­

sisted of at least two chemical moieties, one. oxytocin, which was responsible for uieruie

contraction, and the other, pilressin which acted upon the blood pressure.

In 1920 Young, While, and Hill -synlhesized and showed the u.se of mercurochrome as

an antiseptic agent. Il was hoped that this compound would .serve as the great ilierapeiilic

sterilizing agent"MagnaTherapiaStervlisuns" which Khrlich had predicted. I'hrlich pleaded

for a drug which would sierilize the blood when infecled with streptococci or siapiiylocoeei.

ju.st as arsphenamine killed Ihe spirochaeies. mercurochrome was not this drug, yei it repie

sented a milestone on the tortuous road of progress which led U) it. In ihe blood and tissue

cells the molecule of mercurochrome underwent decomposition and the carhon-merciirs

bond ruptured, resulting in the formation of ionic mercury. This is nephrotoxic and iluis

• rendered mercurochrome unavailable for systemic therapy.

In 1927 Dohme, Leonard and Cox developed the u.se of hexylre.sorcinoi as an gerini-

cidal agent. It was shown that by the iniroduclion of alkyl groups al posiiioii "4" in ihi'

resorcinol molecule, the germicidal value ol" the molecule could be enhanced, luriher. the

germicidal capacity of the compound appeared to increa.se with the number of carbon aionis

in the alkyl side chain. A maximum was obtained al six and hence the u.se ol hexylresort i

nol.

In 1935 'Gerhard Domagk's showed the u.se ol'certain sulfonated dyes in the ireui

meni of streptococcal septicemias. The dyes prontosil and Neoproniosil were broken down

in the body and excreted in the utine partially as sulfanilamide, a simple, non-U)xic. organic

molecule, used in the treatment of infectious di.sea.se as an anti-infective drug. However, m

the development of u.seful anti-infective drugs, the sulfonamides played an eijualiy impor­

tant role. These drugs pointed the way to the u.se of .structures similar to bacterial mctabi)liU's

as anti-infcciive agents. The.se .substances, which resemble in chemical consiiiuiion .some

essential metabolite of the invading organism, are nor u.sed extensively in medicine. The

.similarity between the genuine and spurious metabolite is .so .striking that (he organism

accepts the sub.stitute in place of its genuine metabolite. The phenomenon is illustruied In

the competitive action of sulfanilamide and para-aminoben/.oic acid for certain pathogenii.

bacteria. Their structural similarity may be .seen in the accompanying formulas

SO,NH, COOH

^ ' ( | )

Sulfanilamide Para-aminobenzoic acid

In 1929 Fleming's discovered the Penicillin, active again.si gram-positive organi.snis

and against certain gram-negative organism. Other drugs frimi molds an4 bacteria have

335

followed and the discovery of sireplo inycin in 1943, aureomycin and ehloromyceiin u\

1948 are outstanding among them.

In 1922 Luckhardt introduced the unsaturated hydrocarbon ethylene into general

anesthesia. This was followed by the brilliant suggestion of Leake to combine ihe esseniiul

features of the molecules of ethyl ether and ethylene. This resulted in the synihesis ol ihe

hybrid molecule divinyl oxide CH^.CH-O-CTLCH,. This volatile anesthetic lias heconie ii

valuable agent especinlly for anesthesias of short duration.

Lucas and Henderson in 1929 demiin.strated the value of the hydrocarbon cyclopropani'

as a general anesthetic.

The use of the barbituric acid drugs, such as Amytal and Lvipol Sodium, us inuave

nous general anesthetics led to the introduction of penloihal sodium as an aneslhelic agcni

by Lundy in 1934.

Chen's discovery of the use of alkaloid ephedrine in 1922 heralded the synthesis and

subsequent therapeutic use of Amphetamine, Propadrine, Neosynephrine and oihei

sympathomimetic amines, used in the treatment of na.sal congestion and other conditions.

In 1952 morphine, the principal alkaloid of opium, was .synthe.si/ed by (iaies and

Tschudi.

In last decade three new interesting types of agents have been intioduced. these tue

represented by Dicumarol Tridione and Banadryl. Dicumarol is u.sed to prolong ihe cloiiiniJ

time ol blood. Tridione appears to be a specific in the treatment of petit mal epilepsy. Henadi yl

is a specific antagonist to histamine and used in treatment of various disturbances produceil

by excessive histamine release.

During World war II. the use of British Anli-Lewisile (BAL) as a specific drug in

336

heavy metal poisoning. Besides, liie release of atomic energy/compounds coniaining radiin

active atoms available for the treatment of polycythemia, hyperthyroidism c other diseases.

[mediately, following World War II, among the substances available are Vitamin B|, in the

treatment of pernicious anemia and corti.sone and AcTH in the treatmem of arthritis and

other diseases.

337

Review of literature

The world's oldest pharniacoligicul wriuings come Irom 'India' and "China". The

great herbal or Chinese materia medica. 'Pan TSaO' was probably written in 27 ' H.C llu'

earliest Indian record are the 'Vedas'Although there are medical discnptions MI 'kigvedu'

(25()()-30()() B.C.) it was 'charka' a renowned ancient Indian physician and later "Siisluitra'

and "Vagbhat' who discovered various medical preparations included in "Ayurveda', the

science of life'. Initially these consisted mostly nonpoi.sonous vegetable drugs and miner­

als. Thus charka described about 3{){) vegetable dcugs and.cla.ssified them according to iheir

effects, mostly on symloms into fifty groups.

Development of modern pharmacology as a.science is fairly recent and probably siaiU'd

taking shape following the introduction of experimental procedure by Iranciiis magendie

(1783-1855) and was expanded by claude Berhard (1813-1878). The name of osvvald

.schmiedeberg (1838-1921) is commonly a,s.sociated with the development olexperinieniil

pharmacology in Germany and John, .lacob Abel (1857-1938) palycd a .similar role in the

United stales.

Spectacular developments in physiology biochemistry and organic chemistry during

the recent years have greatly accelerated the advance in pharmacology.

Literature show that much work have been done over many heterocyclic compountis

for their antibacterial activities including both gram-positive and gram-negative bacteria, us

well as antifungal activity. Chalcone and their substituted derivatives are repoited to have

antibacterial', antifungal, antiparasitic , antitubercular, antiinflammatory and insect repel-

leni.properties-\ Bhattet al syrithesized quinoline derivatives of chalcones amis screened

the products forantibacterial activities, while Ahluwalia and co-workers' screened in viirn

338

dihydrochalcones and their derivatives against some microbial organisms.

Flavones play vital role in plant lil'e'' and are antibacterial'^ Pyrimidine dcrivaiiVi's

are reported to possess various biological" and antibacterial properiics'". It was ihereliiif

thought to synthesize some new members and test them for their antibacirial aciiviiy.

Pyrazolines and isoxazolines""* revealed diat these compounds are not only useed in

textile and cinematographic films but they also show widely dilTerening bacteriological

activity. Ozawa and co-workers''^ worked on pyrazolines and found them elTeciive in killing

house Hies on contact. The insecticidal properiics of pyrazolines was studied MI 19X2 hy

Van-Hes and co-workers-" pyrazolines and lluorinated heteroaryl pyrazolines also po.ssess

antibacterial activity-'-- Roda and co-worker,s-' synthesised some new 2-pyrazoHne denvii

tives and tested them for antimicrobial activity.

Like pyrazolines and isoxazolines. pyrazoles and isoxazoics were also synihcsiMul

and" tested Tor antibacterial activity. Sharma and co-workers-' reported ih.ii

hydroxyarylpyrazoles were found to be effective antimicrobials, the antifungal nature of I

substituted 3-(2-hydroxyphenyl)-3-(4-nltrophenyl) pyrazoles was studied by (liri-\ While

trifluromethyl-1-arylpyrazole were reported to be analgesic, antipyretic and anliinllammuloiy

agents-*. Anderson and i'aolella-' showed thai l-phenylpyrazo!e derivatives are effeciive

antidiabetic. While Faucher-** reported that (phosphanadilhioacetamido)-phenyl pyrazoles

are good insecticides. Chlorosubslituted isoxazole.s-"" also possess anlibaclerial activity.

Sharma et dV' synlhesLsed chlorosubslituted i.soxazole derivatives as very effeciive aniihuc-

terial agents. Recently Den Uma and Ashok Rao" reported 1.4-bis (5-aryl-3-is'oxazolyl)

benzenes to be antibacterial agents. .lolly V. S. el al""* reported .synthesis and biological

activities of .some new aryl pyrazoles the lillle compounds shows maximum ami IIIV actlv

339

ity 38.60% and moderate activity at 50 g.nil when tested against several strains of bacteria

and fungi Muller et aP' reported 4-aminoiT>ethyidiphenyl-2,4-dipyiiydro-3H-pyra/.oline-V

ones as herbicides. Salmon and Rogar*° prepared -(4-penlaniiorosulphynylphenyl )-pyra/.oles

as insecticides and acaricides. The tittle compounds showed 80-100 killing ot S pesis. i.e.

Mussca domestica at 500 ppm. Hartllel el aH' reported suhstiiuled pyra/.oles as broad leal

weed herbicides against a wide variety of weed species.

Process for the preparation of substituted pyra/.oline has been reported by (iallen

Kamp et al"-. The title compounds are proves to he in.secticide intermediaie. Chene el al''

reported the preparation of phenyl pyrazoles as agrochemical fungicides. Suhhunwad (i.K

and Vibhute Y. B. ** synihesised some new pyra/olines and N-phenyipyra/olines ami sereeiu'd

for antibacterial and antifungal activity. Trivedi et ai"'' reported pyra/.oline derivatives ol

phenothiazine. These compounds when as.sayed shows effective aniunicrobial activity uiul

tuberculostatic activity. 3,5-diaryl substituted pyrazoles were reported by Has. Chandra el

al"** as antibacterial agents.

Similarly 1-3-thiazines are also reported to po.ssess antimicrobial^'' and pesticidal '*

properties Khadse"^ has synthesised a series of 4-(5-nitro-2-furyl)-2-(subsliiuied

phenylamino)thiazoles. 4-(2,4.5-trichlorophenyl)-2-(substituted phenylamino)thia/.oles and

4-(di and trihydroxyphenyI)-2-(subslilUled phenylamino) thiazoles and studied iheir

antitubercular activity. Many of the.se compounds showed antituhercular aciiviiy ai \.SU

mg/ml. Concentration 6-amino-2-alkylthiohenzothia/.oIes'"' were found U) possess

bacteriostatic activity against mycobaclerium tuberculosis, /awadzka ei aP synlhesi.sed

and studied pharmacological action of ihiazole derivatives. Ciudadhe'-^ has reporled

antimicrobical activity of iodosubstituled-4.6-diaryl-2-imino-6H-2,3-dihydro-1. ^Ihia/ a/nu'.H

340

and 5-(2-hydroxy-3-iodo-5-melhylphenyl)-4-anis()yl-2-ainino-l,3-lhia/,()le. Kamekar" ru

ported antimicrobial activity of ;^,5-diaryl-4-aroyl isoxa/olines pyra/olines and 4.fi-dJaryl

5-aroy}-2-amino-6H-2,3-dihydro-1,3-lhia/.incs.

Raghuwanshi'*" reported the synthesiscd and aniimicrohial as well as aniiltnigal iic

tivity of 1,3-thiazines obtained from nitoCchitlcones. Modi and Nailv'' have reporied synllu'

sis of some new l-H-3(2"-hydroxy-4"-melhoxy-5"-bromophen-l"-yl)-5-subsiiliiicd phenyl

2-pyrazolines and related compounds and atheir antibacterial activity. Ankhiwala"" has syii

ihesised some l-phenyl-3,5-diaryI-!.2-pyrazolines and 3.5-diaryl-l.2-isoxa/olines and re

ported their antibactrial activity similarly. Thore et al" have reported synthesis of some

new pyrazolines and isoxazolines and their antibacterial activity. Ahmad Roshan'' reporied

synthesis and antimicrobial activity of 3-{2-hydroxyphenyl)-.'i-(4-substiiiiied phenyl)-1-

pyrazoleacetic acids. Newly synthesised 1.3-thiazines'*'' from chloro and

chlorobromosubslituted chalcones have been reported to show antimicrobial activity,

Shingare et al* " have reported antimicrobial activity of some dihydropyridino pyra/ole.s.

Raut and Doshi "' have reported synthesis and antimicrobial activity ol I -H-3-(2-hydroxy- V

brQmo-.S-chlorophenyO-.S-substiluted phenyl-2-pyra/olines and their acetyl and txMV/.oyl dc

rivatives. Recently Deshmukh" reported synthesis and antimicrobial activity oi

dichlorosubstituted 3.5-diarylisoxazolines, isoxazoles, pyrazolines. pyrazoles and 4.ri-diaryl

1.3-thiazines.

Antimicrobial susceptibility testing is carried out on a large scale in clinical laboralu

ries as a guide to antibiotic therapy when one adds to these clinical tests those done m

pharmaceutical and research laboratories in screening and evaluation ol' new compounds,

"They are carried out by many dllTerent methods with variable degrees of accuracy and lel-

341

evance. In the managemejit of a specific inrcclion in a palieni the icsi is being pciTormed to

cieiermine whether or not the infecting organism is susceptible lo a series ol aniibiiiiic ihal

might be relevant in treatment. Antibiotic sensitivity tests are also often used as an aiti iii

the identification of infecting organisms. All are helpful in indicating the ideniiis ol isiiiah's

from clinical material.

The disk -diffusion method provides a simple , convenient, and reliable lesi specially

appUcable in routine clinical bacteriology laboratory. Itcon.sistsof inipregnaiing small disks

.of a standard filler paper with given amounts of a chosen range of antihioiics. These are

placed on plates of culture medium previously spread uniformly with an inoculum of ihe

bacterial isolated to be tested. After incubation, the degree of .sensitivity is determined by

measuring the easily visible areas of inhibition of growth produced by the difiusion of the

antibiotic from the disks into the surrounding medium.

Paper di.scs as reserviors of antibiotics for sensitivity testing were introduced in the

1940's (Vincent and Vincent, 1944, Morley, 1945, Bondi et al 1947) and are now more

commonly used than others such as cups (Ro.se and Miller. 1939) tablets (I .und et al 19511

or wells cut in the medium. Although it is po.ssible to produce dry filler-paper discs in llu'

laboratory (I-air-brother and Sherris, 1959; I .each and Willis, 1971) most laboratories ctin

sider this to be too-time consljming and con.sequently rely on commercial st)uices.

Problem

From the review of literature it is quite evident that heterocyclic compounds have

antibactrial, anUfungal. antiparasitic and in.secticidal activity. The work presented in this

chapter deals with heterocyclic compounds synlhesi.sed by us have been .studied for antibac­

terial activity again.si .some common pathogenic bacteria .such as Escherichia coli. Klebsiella

.142

pneumoniae, Pseudomonasaeruginosa, Proieus niirabilis. Salmonella typhi. Vibrio ciiolerue,

Shigella dysentery. Staphylococcus aureus (coagulase +ve) and staphylococcus alhiis

(coagula.se -ve) as well as antifungal activity against some common pathogenic lungi such

as. Candida albicans, cryptococcus neoformans, A.spergillus lumigalus. Aspergillus ingei.

Aspergillus flavus, Mucor .spp. and Rhizopus spp.

The following compounds were tested.

List of Compounds

Parti

A) Synth0sis of isoxazoUnes

1) 2-Benzoyloxy-5-methylacetophenone

2) 2-Benzoyloxyaceiophenone

3) 1 -(2-Hydroxy-5-melhylphenyl)-3-phenyl-1.3-propanedione.

4) 1 -(2-Hydroxyphenyl)-3-phenyl-1,3-Propanedione.

5) 3-Benzoyl-6-methylflavttnone

6)3-Benzoyl-4'-melhoxy-6-melhyinavanone

7)3-Benzoyl-2-(2'-furyl)-6-melhylchromanone

8) 3-Benzoylflavanone

9) 3-Benzoyl-4'-melhoxynavanone

lU) 3-Benzoyl-2-(2'-fui'yl) chromanone

11) 3-(2-Hydroxy-5-methylphenyl)-4-benzoyl-5-phenyli.soxa/.oline

12)3-(2-Hydroxy-5-nielhylphenyl)-4-benzoyl-.')-(4'-methoxyphenyl)i,soxa/.oliiic

13)3-(2-Hydroxy-5-melhylphenyl)-4-benzoyl-5-(2'-furyl)i,soxa/.oline

14)3-(2-Hydroxyphenyl)-4-benzoyl-5-phenyli.soxazoIine

343

15)3-(2-Hydr()xyphenyl)-4-benzoyl-(4'-melhoxyphenyl)isoxa/,i)linc

16) 3-(2-Hydroxyphenyl)-4-benzoyl-5-(2'-ruryl)isoxa/oIine.

Part II

B) Synthesis of A^'PyrazoUnes :

17) 3-(2-Hydroxy-5-methylphenyl)-4-ben/.oyl-5-phenyl-A--pyr{)/.oline

18)3-(2-Hydroxy-5-mtJthylphenyl)-4-beil/:oyl-5-(4'-melhoxyphenyl)-A-pyra/oline

19)3-(2-Hydroxy-5-methylphenyl)-4-benzoyl-5-(2'-rui7l)A--pym/olinc

2U)3-(2-Hydroxyphenyl)-4-benzoyl-5-phenyl-A--pymzoline

21) 3-(2-Hydroxyphenyl)-4-benzoyl-5-(4-mclhoxyphenyl)A--pyrazoline

22)3-(2-Hydroxyphenyl)-4-benzoyl-5-(2'-ruryl)-A--pyia/.()line

23) l-carboxamido-3-(2-hydroxy-5-melhyIphciiyI)-4-benz()yl-5-phcnylpyra/i)liiie

24) l-carboxamido-3-(2-hydroxy-5-meihylphcnyl)-4-bcn/.()yl-5-{4'nielhi)xyplu'nyl)-

pyrazoline

25) l-carboxamido-3-(2-hydi"oxy-5-melhylphenyl)-4-bcnzoyl-5-(2'-ruiyl)pyra/olinc

26) l-carboxamido-3-(2-hydroxyphenyl)-4-benzoyl-5-phenylpyrazoIine

27) l-carboxamido-3-(2-hydroxyphenyl)-4-benzoyl-5-{4'-mclhoxyphenyl)pyra/oline

28) l-carboxamido-3-(2-hydroxyphenyl)4-benzoyI-5-(2'-riiryl)pyra/.olinc.

Part III

C) Synthesis of Isoxazoles

29) 3-Benzoyl-6-methylflavone

30) 3-Benzoyl-4'-melhoxy-6-niclhoxynavone

31) 3-Benzoyl-2-(2'-furyl)chromone

32) 3-Benzoylflavone

344

33) 3-Benzoyl-4'-melhoxynavone

34) 3-Ben/.()yl-2-(2'-turyl) chromone

35)3-(2-Hydroxy-5-mclhylphcnyl)-4-bi;n/.()yl-5-phcnyli.si)xa/olc

36)3-(2-hydroxy-5-methylphcnyl)-4-bcnzoyl-5-(4'-nielhoxyphenyl)isoxa/(ilc

37)3-(2-Hydroxy-5-melhylphenyl)-4-hen/oyl-5-(2'-ruryl)isi)xa/,olc

38) 3-(2-Hydioxyphenyl)-4-hen/.oyl-5-phenylisi)xa/.()le

39)3-(2-Hydroxyphenyl)-4-bcnzoyl-5-(4'-melhoxyphcnyl)i,st)xa/.ole

40)3-(2-Hydroxyplienyl)-4-ben?.oyl-5-(2'-ruryl)isoxazole

Part IV

D) SyMhesis ^tf Pyrazoles

41) 3-(2-Hydroxy-5-mclhylphenyI)-4-bciizoyl-5-phenylpyra/.()le

42)3-(2-Hydroxy-5-melhylphenyl)-4-benzoyl-5-(4'-niclhoxyphcnyl)pyrazA)k'

43)3-(2-Hydroxy-5-melhylphenyl)-4-benzi)yl-5-(2'-ruryl)pyrazole

44) 3-(2-Hydroxyphenyl)-4-benzoyi-5-Phenylpyrazoles

45)3-(2-Hydroxyphenyl)-4-benzoyI-5-(4'-nielhoxyplienyI)pyrazole

46)3-(2-Hydroxyphenyl)-4-benzoyl-5-(2'-rLiryl)pyrazolc

47) l-Carboxamido-3-(2-Hydroxy-5-melhylphenyl)-4-bcnzoyl-5-phcnylpyia/i)le

48) l-Carboxamido-3-(2-Hydroxy-5-methylphenyl)-4-benzoyl-5-(4'-melh()xyphcnyl

pyrazole

49) l-Carboxamido-3-(2-Hydroxy-5-mclhylphenyI)-4-benzoyl-5-(2'-l'iiryl)pyni/ole

50) l-Cai"boxaiTiido-3-(2-Hydroxyphenyl)-4-henzi)yl-5-phenylpyra/.olc

51) l-Carboxamido-3-(2-Hydroxyphcnyl)-4-bcn/.i)yl-5-(4'-nielhoxyphcnyl)pyra/ole

52) l-Carboxaniido-3-(2-Hydr()xyphenyl)-4-bcnzoyl-5-(2'-ruryl)pyia/.i)lc

345

The bacteria used are known human pathogens :

1. Escherichia coli:

It lives only in human or animal intestine as commensal in gastroinlesiinal iraci. I )e

teciion of H. coli in drinking water is taken as evidence of recent polUilion wiili human or

animal excreta. They are Gram-negative, with micro capsule, non-sporing motile wilh

peritrichous flagella, producing lactose feiiTienting colonies on Mac-conkey medium, oxidiisi*

negative, catalase-positive, Indole-positive, MR. positive, V.P. - negative citrate ulili/.aiion.

Negative urease- negative , growth in KCn, liquelles gelatin responsible lor causing.

a) Urinary Tract Infections : Commonest lesion

Lesions are : cystitis, pyelitis, pyelonephritis. Perinephric abscess and asymp­

tomatic bacteriuria. li coli .serotypes commnly responsible lor urinary iraci inleciion

are 0,1,2,4,6 and 7.

b) Diarrhoea or gastroenteritis :

Certain serotypes produce fatal type of ga.stroenterilis in inlanis e.g. 4,26. 46.

55, 86, 111, 112, 119, 127 and 129. Lactic acid pri)duced from lacU).se rermenialitio

may cause irritation of the colon. Result is violent nau.sea, vomitling and diarrhoeii

c) Peritoneal and biliary infections :

Peritonitis. Acute cholecystitis. Acute cholangitis and pyelephelebiiis.

d) Neonatal infections :

E. coli bacteriaemia (Mostly in premature neonates) Neonatal menmgills,

pyelonephritis.

e) Bacteriaemia and Septicaemia :

Serious most manilestalion ofH. coli infection. Gram negative endotoxic shock

346

Colonies of Klebsiella pneumoniae on Macconkey agar (Lactose fermenter, lirge, mucoid, oil paint like)

Colonies of Staphylococcus aureus on P.P. agar (Bright pink colour)

may result from bacleriaemia.

f) Pyogenic infections or ohscesses anvnherc in the body :

Subcutaneous ilitections following injeclions. I'eriunal abscess, isochioieciiil

abscess, appendicular abscess, metastatic infections in liver, lungs, brani and bomv

2) Klebsiella :

It is found in the mucosa of upper respiratory tracl, intestine and genilourinni \

tract. It is non-motile, capsulate (both in ti.ssues & in in culture) gram-negaiive. plump,

.short rods surrounded by clear halo (capsule). Producing lacto.se fernieniing (pmk) large,

convex, vLscous. confluent, mucoid colonies with oil paint like appearance (due to abun­

dant production of Extracellular slinie). On mac-conkey medium, responsible for caus­

ing Friedlander's Pneumonia (destructive Pneumonia), uninary iraci infeciions,

suppurative lesions elsewhere in body (biliary tract, .PeriU)neum. mastoids, paranasal

sinuses, menings.)

3) Pseudomonas aeniginosa :

It produces large, opaque, irregular colonies of buiyrous consistency. It gives

musty or earthy smell and.is mainly a.s.socialed with formatit)n t)f "Blue pus". Ii cau.ses

suppurative otitis media. Wound infections, eye infections and urinary iraci uUeciions li

is commonest and most serious cause of infection in exten.sive burns and responsible lor

causing bacteriaemia /.septicaemia in jmmunocompromi.sed hosts. Two special leaiiiivs

of pseudomonas septicaemia

a) Ecthyma gangrenosum : purple -black ulcerations in ano-genilal region and

b) pas.sage of green urine It is al.so one of the mo.st troublesome agents causing no.soconiiiil/

hospital infections. It also cau.se infantile diarrhoea.

:M7

4) Staphylococcus aureus :

Gram-positive cocci arranged in grapc-Iil<e irregular clusters. non-mi)iili\

nonsporing, non capsulaicd coagulase-posiiive, toxigenic, niannitol ferincniing. phos

phales -positive, MR & VP-posilive. I^athogenic staphylococci producing golden yellow

colonies on nutrient agar. Staphylococcus may cause the majority o\ acute pyogenic

lesions in man. Staphylococcal lesions are characteristically localized.

Pathogenic lesions :

a) Cutaneous lesions :

Furuncles. styes, boils, ab.scess, carbuncles, I'oUiculitis, Hidradradenites suppurativa,

impetigo, pemphigus neonatoniupi.

b) Deep infection :

Acute osleomyelilis, Brodies abscess, tonsillitis, pharyngitis, sinusitis. I'neumonui.

pulmonary abscess, breast abscess, renal absces.ses, meningitis, endocardills.

staphylococci, scaled'skin syndrome, staphylococcal bacteriaemia and sepiicaemiu.

Toxic shock syndrome usually begins within 5 dayson.sei of men.ses in young women

who use tampons (with positive vaginal culture of staphylococcus aureus)

c) Staphylococcal food poisoning :

It results when food coniaminled with enieroioxin, produced by siaphylocticciis

is consumed e.g. meal, fish, milk and milk products. Diarrhea and vomiiiing set in

within 6 hours of taking contaminated food.

d) Nosocomial (hospital) infection :

Staphylococci are one of the most notorious agents for cross infection in

hospitals.

348

5) Staphylococcus alhus :

li is coagulase negative slaphylococcus which are normal comiiiensals ol skui. mis

irils, conjunclive mouth, throat, prtiducing porcelain white colonies on nuincni agar li

may act as opportunist pathogens causing ache, pustules and stitch abscess, iirmary uacl

infection with pyuria.

6) Salmonella Typhi:

Gram negative rods, motile with I'eritrichous riagella, non capsulaie. mostly

fimbriate showing non-lactose fermenier and colourless colonies on Mac-conkey's

medium. Lesion produced by salmonella

a. Enteric Fever (Typhoid or paratyphoid fever)

b. Acute gastroenteritis

c. Food poisoning

d. Septicaemia lesion any where in body (octeomyeliiis, endocarditis, abscess)

f. Asymptomatic intestinal infection.

g. Carrier stales : I) Intestinal 2) Biliary 3) Urinary.

7) Vibrio Cholerae :

They are thin, curved, comma -shaped, (iram negative, rigid and actively monk'

bacilli showing darling type of motility. They are non-laclo.se fermenting, growiiij.' ;ii

alkaline pH, Producing transluscent. rai.sed, yellowi.sh, mucoid colonies on TC'Bs agar.

causing an acute disease cholera resulting from colonisation of small uuesinu' hy

strains of Vibrio cholerae (cla.ssical/FI-lar), charateri/ed by

a) Profuse watery (Rice water stool) diarrhoea

. b) Effortless vomitling

349

c) Muscle cramps

d) Oliguria

e) Features of oligaemic shock

8) Shigella dysentery :

Organisms of genus shigella are parasites of human intestine and cause hacillary

dysentry (vShigellosis). A genus oi" Family enlerobacteriaceae, slender, gram -negalive

rods, non motile, non Hagellate. non capsulate. non lactose I'ermenting pale colonies on

Mac-conkey medium, Anaerogenic, lermentalive, oxidase negative, grow best in pres­

ence of bile salts, marked antigenic sharing among themselves and with other enierii

bacilli.

I-esion : Baclllary dy.sentry .

There is lYequenl passage of loose motion containing blood and mucous vvilh

griping pain and tenesmus. Shigellil dysenteriae type I u.saully causes complicaiios liki'

arthritis, toxic neuritis, conjunctivitis, paretilis, intus.sceplion and myocardiiis.

9) Proteus tnirahilis:

'Proteus' is named so because of its

a) variable morphology (tendency to vary from almost coccal forms to king filamenunis

fornis)

b) variable biochemical reactions.

'Proteus' was a Greek -God who often changed his appearance.

Gram -negative enlerobacteria, highly motile with peritrichous flagclla. non-

lactose fermenting, showing spreading growth (Swarming) on .solid media. Ii gives fishy

of seminal smell.

350

Growth is KCN -positive. All strains arc indole positive except I'roieus-mirahihs

Rapid hydrolysis of urea with liberation ol ammonia (urease - positive) All sirainn

deaminaie phenylalanine to phcnylpyruvic acid (positive IM'A lest)

Lesions :

Mostly secondary invaders (attacks tissues already damged by other pathogens) op­

portunist pathogen, cause urinary tract infection, pyogenic lesions like abscess, inleciion ol

wound, ear or respiratory tract, bacteremia/septicaemia. Endotoxic shock. Summer diar­

rhoea in infants.

Super infection :

Proteus is extremely rtislslant to most of the commonly u.sed antibiotics and may

cause superinfection during lopg term antibiotic therapy.

Relation of Urease activity of Proteus to renal calculi/infections :

Proteus (Urease) • Splits urea to ammonia :

1) Urine becomes highly alkaline -helps formation of ammonium -magnesium phosphaio

stones &

2) Ammonia inactivates C4 (Fourth componani of complement) causing further infection.

The fungi used are known human pathogens

I. Candida albicans :

Yeast like fungus, opporturiist pathogen. It acts as a bridge in between superficial and

deep seated mycosis. In human beings, Candida albicans is an endosaprophyte of the

gastrointestinal tract and vagina. The thalkis of Candida consists of yeast cells and

pseudohyphae. They reproduce by budding, ferment a number {){' sugars and a.ssimilaie

nitrogen. Microscopic examination of pathological material shows round i>r oval yeast cells

35:

Colonies of Candida albicans on Sabouraud's dextrose agar (Creamy white, mucoid, yeasty, pasty, oilpaint like)

/!^SpPJiCill-L-0.

Colonies of Asperigillus niger on Sabouraud's dextrose agar (Powdery, sugary, sporulated, blackish)

in the process of budding and often exhibiting pseudohyphae. Cultures on sabouraud's agar

medium Produce moist, glistering, creamy colonics ol a dull white or greyish while coloui.

smooth, pasty and have a yeasty odour. The species cannot be identified either by direii

microscopy or by Uie macroscopic appearence of cultures. The species idenlificalion is buM'd

upon.

a) Rapid formation of pseudohyphae and blasU) spores

b) Production of chlamydospores un corn meal agar

c) Germ tube formation {n mammalian serum at 37"C.

d) Fermentation and assimilation of sugars.

e) Nitrogen utilization.

t) Abscence of carotenoid pigment

g) Abscence of Arthrospores

h) Type of growth on SDA, BA, SD broth.

i) Viability test for yeast with 0.1% aqueous solution of methylene blue.

j) Pathogenicity in experimental animals- Rabbit and mice-renal absces.ses and

death in 2 weeks.

Lesions :

a) Superficial candidosis:

1) Mucosal infections : Oral thrush, Vaginal thrush

2) Skin and Nail infections : Cutaneous candidosis onychia & paronychia.

3) Chronic mucocutaneous candidosis & candid granuloma.

h) Deep (systemic) candidosis :

1. Intestinal candidosis

352

2. Bronchopulmonary cancJidosis

3. Meningitis

• 4. Endocarditis

5. Septicaemia

6. Systemic infections ;

In drug addicts, alter open iiearl surgeiy, prolonged antibiotic use. immunosuppression

and in leukaemia.

2) Cryptocuccus neofarntans :

True yeast 4.2()ni in diuiiu'ier with unicellular thick gelatinous capsule,

natural source : dry pigeon excreta

Characterised by :

a) Lack of carotenoid pigment

b) Prescence of encapsulated budding cells.

c) Failure to produce any pseudomycelium

d) Do not produce a.scospores

e) Lack of fermentative ability

f) fast - growing yeast. Growth at 37"C

g) Colines smooth, tnttcoid, creamy white and pa.sty on SDA, culture may he so mucoiil

as to flow down on agar slope. .

h) No germ tube develop in mammalian serum at 37"C.

i) The only metabolic activity is the .splitting of urea,

j) confirmatory test are

I. Rapid .selective urea.se test

353

2. Rapid Nitrate reductase test

3. L-dopa Terric citrate lest

4) Niger -seed agar test.

k) Animal pathogenicity in white mice causing meningitis on intracerebral

inoculation.

1) Hislopathology

Lesions : Human inteclion (cryptococcosis) may present as meningitis, pulmonary le­

sions, skin ulcers and bone lesions.

3} Opportunistic fun^i :

Opportunistic is the name given lo the fungi that are normally saprophytic but

may become pathogenic under special conditions. The term 'asthenomycosis' is some

limes used to describe diseases caused by such lungi. Strictly speaking most lungi are

opportunistic bul we will restrict the term to those organisms that become pathogenic

when the host has an identifiable , preexisting disease which may be local i»r general

spontaneous or iatrogenic.

Aspergillus

Filamentous fungus, septate conittminanls having hyaline light coloured hypluie

Aspergillus species are rapidly growing moulds. The most important of ihse are A. lumigaliis,

A. niger, A. tlavus, A. terrus, A. Galucus and A. nidulans on sabouraud dextrose agar al

room temperaiui"e the rapid growing colony is rugose and velvety various colours are due lo

dense Production of conidia blue, greeh, yellow, black and while. On microsct>pic examinii-

tion, the mycelium is septate, unbranched , rough or smooth conidiophores with a loot citl

at their base support a large Vesicle at their tip. The vesicle in turn supports short, llask

354

shaped phialides in a single or double row, wliich produce chains of sniooilror roui'li

phialoconidia.

Aspergilli species are dilTerenliaied from each other.

1) based on dilTerences in colony pigmeniaiion

2) The size and length of the conidiophitres.

3) The shape ol" the vesicles

4) The presence of metufae

5) The position of the phiitlides

6) The size and length of chains of the spores.

1) Aspergillus fumigatus

1. Green or Green gray, sugary colonies.

2. Uniseriate phialides over uppel" one half of a henii.sperical vesicle.

?). Chain of globose conidia lending to sweep inwardly.

2) Aspergillus Niger

1. colonies black , granular

2. vesicles spherical

3. Biseriate large melulae and smaller phialides

4. Conida black and roughened.

3) Aspergillus flavus :

1. Colonies usually yellow or yellow -green

2. Vesicles round, sporulution over entire surlace

3. Phialides alone (Uniseriate) or with melulae (Bi.seriale ) may he preseni

4) Aspergillus Terrus

1. colonies cinnamon colored

355

2. Vesicles dome-like

3. Meiulae and phialides both present (Biseriaie)

4. Aleuriospores produced on submerged hyphae.

Lesions :

1. various forms ol' pulmonary disease

a) 1-ungus ball int'ecUon

b) Aspergilloma

c) Allergic bronchopulmonary and invasive pneumonitis

2. Metastatic disseminated aspergillosis involving various organs.

3. Otomycosis

4. Mycotic Keratitis

5. Onychomycosis

6. Endocarditis

7. Mycetomatous skin diseases

5) Mucorspp.:

1. cause mucormycosis

2. Colony is rapidly growing, colourless, yellowish brown.

3. Wet mount showing sporangiophol-e, s.spoiangiinn and related sporangiosptm-.s ami m

rhizoids.

4. 1-ungus is characleri.sed by broad lion-septale hyphae which are cylindrkal. irret'iil.n

or distorted in shape.

6) Rhizopus spp. :

1. Rhizopus has unbranched sporangiophores arising in griuips above the rhi/oids.

,556

EXPERIMENTAL AND DISCUSSION OF THE RESULTS.

All ihe above compounds have been charaeierised on ihe basis i)l chemical properiu's

elemental analysis and spectral data. The mehing points were recorded on "Tempo" meiim i

point apparatus and are uncorrected. The procedure for the preparation olsiarimg maieriiil

i.e. 3-aroylflavanones (compound No. 1-10) has been described in .section A ot chapter II in

part I of the thesis. The details for preparation o| 3-aroyl-llavones (Comptiund No. \\-\U)

has been described in .section A oCchapter 11 in pari III of the thesis. Procedure tor prepani

tion of 3-(2-hydroxypheilyl)-4-ben/.oyI-5-phenylisoxa/.olines (compound No. 17-22) has

been described in .section B of Chapter 11 in part 1 of thesis. The method for preparaitnn V

(2-hydroxyphenyl)-4-ben/oyl-5-phenyl---pyra/,olines (compound No. 29-34) has been tic

.scribed in Section B of Chapter 11 in part tl of the thesis. The preparation methuti tor Vi j

hydroxyphenyl)-4-ben/.oyl-5-phcnylisoxa/.oles (Compound No. 3.'S-4()) has been de.scrihiHl

in Section B of chapter I in pan 111 of the thesis. Theexperimenlal details for tlu- preparaiioii

of 3-(2-hydroxy-phenyl-4-benzoyl-5-phenylpyra/.oles (compound No. 41-46) as well as I

Carboxamido-3-(2-hydroxyphenyl)-4-ben/oyl-.*5-phenylpyra/.oles has been ilescribed in

section B of chapter II in pait IV of the thesis.

Mateml and Method

The compounds enlisted above were tested against pathogenic bacteria aiul lungi Im

their antibacterial & antifungal activity using Disk diffu.sion lest method. The organisms

tested were f.scherichia coli. Klebsiella pneumonia. I'.seudonnmas aeruginosa. I'roieiis

mirabilis, Salhionella typhi. Vibrio cholerae. Shigella dy.sentry. Staphylococcus umviis

(coagulase +ve) .Staphylococcus alhus (coagulase -ve). fungi. Candida albicans

Cryplococcus neoformans, Aspergillus funiigaius. Aspergillus niger. Aspeigilltis \'\;\\\\s

357

Mucor spp. & Rhizopus spp.

Preparation of wet disks for antibiotic sensitivity tests :

Method :

Punch disks 6.25 nun In diamuler from No. I whul man liliei- paper, dispense halclu'H

• of 100 in screw-capped hollies and sierili/e hy dry heal al 14()"C lor 60 miniues. Prepare ihc

solution of compound in a Dimelhylforrnamide (DMP) solvent. So ihai 1 ml eoniaiiis Ion

limes the amouni of compoi|!id required in the disk. Add I ml solution of compound lo eiich

bottle of 100 disks and as the whole of this volume will be absorbed, assume ihai each disk

will contain approximately O.Ol ml. Suire ihe disk in wei conditions.

Culture medium :

The medium used throughout the experiment was Hl-media India (makei nuirii'Hi

agar/sabauraud's dextrose ugarand having following composition

Composition of nutrient ogol*;

Peptone 5 gm/litre

Sodium chloride 5.()gm/lltre

Beef extrijci • 1.5 gm/lilre

Yeast exti'acl 1.5 gm/litle

Agar 15 gm/lilre

pH(Approxi.) 7.4 + 0.2

The media was prepared by suspending 28 gnis ingredients in 1000 ml disiilled wuiei

Boil to di.s.solve the medium completely, and was sterili/.ed hy autoclave.ai 15 Ibs/lncli,

pressure al 121"C temperature for 15 minutes. After slerili/ation it was cooled down to

about 50" C and poured into sterile petriplates and allowed to solidify.

358

Composition of Sabouraud's dextrose agar:

Glucose 40 gm

Papione 10 gm

Agar 20 gm

Water I Hire

pH - 5.4

The media was prepared by suspending above ingredients in 1000 ml disiilled water.

Boil to dissolve the medium completely and was sterilized hy autoclave al 15 Ihs/lnch \'\vs

sure at 115"C temperature lor 15 minutes and adjust to pH 5.4. After sierili/.mion il was

cooled down to above 50"C and poured into sterile peiriplales and allowed to solidily.

The low pH and high sugar content ol" these media make them particularly selective

for fungi and inhibitory to bacteria. Sabouraud's dextrose agar is the commonesi culiiiiv

medium used for isolation, identification as well as drug sensitivity iil lungi.

Medium used : Nutrient agar/ Sabouraud dextrose agar

Size : plate 8.5 cm in diameter

Debth of agar : 14 mm

Distance between 2 disk '• 2 cm away from each other

Diameter of the antibiotic disc : 6.25 mm in diameter

Not more than 6 disks Used.

. Test Procedure:

Inoculate the culture muierial in a nutrient broth/Sabauraud dextrose broth, Kept hrulh

at 37"C for 4 hrs. incubation . Dry the culture plale nutrient agar/Sabauraud dextrose agar

until its surface is free from visible moisture. Mood the inoculating material on the surlaii'

.159

of agar unilormly in presence of taking with all aseplic precautions, supernaiani cliscardeil

Dry the plate , again lor up to 30 minutes wiihout further delay, apply the com|UHind disk.

at adequate spacing (2 cm of more apai'l) to the surface of the plate with sterile fine poinii'il

forceps and press gently to ehsUfe full contact with tlie medium and moisieninjj ul the disf

Cdiitrol was run using plane DMI ' solvent for' aseplic conditions. Incuhaie al M' (Mor IH 'I

hrs/96hrs. After incubation degree of sensitivity to drugs is determined by nuasuring ihi

visible clear areas ofgrowlh free zones (zones of inhibition) Produced by diffusion of mill

biotics into the media from the disks

Width of zones of inhibitions depends on :

a) size of inoculum

b) Nature of culture medium

c) Prescence of inhibitors

d) Concentration of agar In the medium

e) thickness of the medium in the plate

f) Condition and tirne of incubation

g) Composition of atltibiotic disk.

Zones of inhibition are measured (including fmim of disk diameter) by calipers iind

reported in mm.

The results are cited in Table No. I and Table No. 2 and illustrated in pholographli

plate.

360

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Discussion of tile Results

All the organisms sludipd, bacterial as well as fungal are human pathogens, liom itU'

results it is clear that the following compounds are effective against the said organisms.

The antibacterial activity of all the compounds was determined against \i. Coli, K.

pneumoniae, Pseu. aerugindsa. Staph albus. Slitm. typhi, staph, aureus, Vihrio cholenii'

Shigella dysenlry, Proteus nilrahilis. Almost all the compounds showed signillcani aniihui

terial activity. However ihp antibacterial activity was highest against \i. coli, SUiph, m\w\h.

Proteus mirabilis in colnparlsoti to Salm. typhi. Kl- pneumoniae, V. Cholerae & ShigcHii

dysentery, while in Pseu. aeruginosa ar|d staph alhus showed lesser activity or founil In

active.

In general the presence of methoxy and furyl groups invariably increased the antihia

terial activity of the compound.

In view of the structui'e activity relationship the appreciable inhibition was observed

by the heterocycles against all the test organisms.

The antifungal activity of the titled compounds was evoluated on cry. Neoformani*,

Candida albicans, trichophyton mentagrophytes, Microsporum Gypseuin, Mucor spp..

Rhizopus spp.,A8pergiUus niger, Aspergillus Havus & Aspergillus fumigaius.

On the basis of average percentage inhibitions all the compounds were found to d|»

play modrate to good level of toxicity against all fungi. They were , however more loMc

aganist cryp. neoforman. Candida albicans, trichophyton mentagrophytes, Microspoiiiiti

Gypseum, Mucor spp. & Rhizopus spp. than against Aspergillus Niger, Aspergillus IliiviiN

& Aspergillus fUmlgatus.

Further, the presence of methoxy and furyl groups invariably enhiuiced the fungiioxiil||

375

Photographs showing zorle of inhibition around the filter paper dis)< filled with test coiripoimds.

fcpmpounds ort nutrient agar

Photograph of Aspergillus niget miijbition by the test compounds Qi\ Sabouraud's dextrose agar ^ ^

of the compounds.

In view of Hie slriictuittl complexlcily reUllionshlp ruiiglcidul aclivily invuiiuhly In

creased against all the test organisms.

Thus from the above results, it is observed that most of the lieterocyclic minpountl»

were found more or less effective against Ihe said (bacterial & I'ungal) organisms. So iht'Hi'

compounds can easily be Used for the treytmenl of diseases caused by test palhugens. oiili

when they do not have toxic wnd other sidt; effects.

376

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