Chapter X

ANTmACTERIAL AND ANTIFUNGAL ACTIVITIES OF SOME

LANTHANIDE(III) CHLORIDE AND NITRATE COMPLEXES OF

2-(N-INDOLE-2-0NE)AMINO-3-CARBOXYETHYL-4, 5, 6, 7­

TETRAHYDROBENZO[blTHIOPHENE

From the inception of civilization, human being struggles for

existence against the affliction of disease, decay and death. It is the eternal

want of human to remain healthy and cure from his surroundings. But in

the ancient era, millions of people died from various infectious diseases

like plague, cholera, diarrhea, tuberculosis etc, in epidemic form, which has

instigated the man to endeavor for remedy for their sufferings. In fact in the

way of long struggle for relief from many infectious diseases as well as

illness, physical discomforts, injuries, pain and wounds, man had achieve

the mastery over the diseases specially infectious diseases when Alexander

Fleming accidentally discovered penicillin from a microorganism.

Although antibiotics are life saving drugs but nowadays, because of

careless and promiscuous use of antibiotics, various pathogenic microbes

are gaining resistance. To overcome this problem and to treat many serious

infectious diseases like AIDS, cancer, viral fever, continuous search of new

228

more potent antibiotic is indispensable. A large number of organic

compounds have been developed as antimicrobial agents. Development of

resistance among different species of pathogenic bacteria has given new

dimension to this area.

!satin and its derivatives are unique among the organic compounds

mainly because of their biological significance. Apart from the synthetic

flexibility with which they can be transformed into other derivatives, they

form an important class of compounds which possess activity against

. 325 326 1 327 fu 1 328-331 . 1332 dtuberculosis, ' eprosy, nga , vua an bacterial333•334

, . h

· 335 · · 336 d

t' 1 t 337-339 mfectlons, r eumatlsm, trypanosom1as1s an as an 1convu san s.

Similarly thiophene and its analogs represent an important class of

compounds with diverse biological activities.340 Substituted thiophenes

h . I d. . h 341ave potentia ra 10-protectlve c aracter, 1 · 3

42ana ges1c, antitussive,

antiinflammatory, bactericidal, fungacidal etc activities. Schiff bases play

an outstanding role in many biological processes.343 Their antimicrobial

activities have been reported to increase on coordination with metal ions.

These two biologically important compounds have been incorporated

through an azomethine functionality and the resulting ligand and thus metal

chelates have been examined as antibacterial and antifungal agents. Metal

chelates of sulphur containing Schiff bases have received major attention

229

of biochemists because of their versatile use as antimicrobial agents.344

Compounds containing C=S and c=o groups possess fairly good

fungitoxicity.345,346 There are recent reports on the antimicrobial activities

of lanthanide(III) complexes.347,348 All these observations prompted us to

synthesise some lanthanide(III) Schiff base complexes of 2-(N-indole­

2-one)amino-3-carboxyethyl-4,5,6,7-tetrahydrobenzo[b]thiophene and

enticed to the evaluation of the antibacterial and antifungal activities. So

this chapter is solely concerned with antibacterial and antifungal activities

of lanthanum(III), cerium(III), praseodymium(III), neodymium(III),

samarium(III), europium(III), gadolinium(III), dysprosium(III),

ytterbium(lII) and lutetium(III) nitrate and chloride complexes of

2-(N-indole-2-one)arnino-3-carboxyethyl-4,5,6,7-tetrahydrobenzo[b]­

thiophene synthesised during this investigation.

EXPERIMENTAL

Antibacterial and antifungal activities of lanthanum(III) chloride and

nitrate complexes of 2-(N-indole-2-one)amino-3-carboxyethyl-4,5,6,7­

tetrahydrobenzo[b]thiophene were studied against the four bacteria

Escherchia coli, Vibrio cholerae, Salmonella typi and Bacillus megaterium

230

and the four fungi Penicillium notatum, Aspergillus niger, Candida

lb ' d S h .. b d'ff' h d 193 194349a leans an ace aromyces cerevlszae y agar 1 uSlOn met o. ' ,

Escherchia coli

Escherchia coli is a type of fecal coli form, Gram negative and non-

sporing bacteria commonly found in the intestines of animals and human.

The virulent strains of Escherchia coli act as specific pathogens of the gut

(enteritis) and of extra-intestinal sites (urinary tract infection, wound

infection). Most strains are mobile by peritrichalate flagella. Regarding

biochemical reaction they break down most of the sugars. Infection often

causes severe bloody diarrhea and abdominal cramps. They are usually

sensitive to many antibiotics such as ampicillin, tetracyclin,

chloramphenicol, gentamycin etc. They are aerobic and facultative

anerobes and grow in ordinary culture media (nutrient broth, nutrient agar)

at 37°C in 8 to 24 h.

Vibrio cholerae

Vibrios are gram negative, highly motile curved rods with a single

polar flangellum and appear to 'vibrate'. Some are pathogenic. Of the

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vibrios that are chemically significant to human, Vibrio cholerae, the

agent of cholera, is the most important. Most vibrios have relatively simple

growth factor requirements and will grow in synthetic media with glucose

as a sole source of carbon and energy. However, since vibrios are typically

marine organisms, most species require 2 - 3% NaCl or a sea water base for

optimal growth. In liquid media vibrios are motile by polar flagella. These

organisms are sensitive to acid pH but tolerate alkaline pH (9 - 9.6) very

well.

Salmonella typi

Salmonella typi is a Gram negative facultative rod shaped bacterium

in the same protea-bacterial family, trivially known as 'enteric' bacteria. It

causes typhoid fever and it can be transmitted by the fecal-oral route. It is

excreled by human in feces and may be transmitted by contaminated water,

food or by person - to - person contact. Growth temperature is minimum at

7 °C, growth greatly reduced at < 15 °C, maximum at 45 °C and optimum

at 35 - 37 °C. Optimum pH is 7 - 7.5.

232

Bacillus megaterium

Bacillus megaterium is a gram positive spore producing bacteria. It

is a rod shaped Eubacteria and is found in the soil. Colonies form in chains

due to sticky polysaccharides on the cell wall. It is very important in the

biotech industry due to its size and its' enzyme and cloning ablities.

Penicillium notatum

Penicillium notatum, also known as Penicillium chryogenum, is the

blue - green mold on bread, fruits and nuts. It grows at its peak during the

spring and the winter, but the mold has no seasonal variations. It is a well­

known group of brushed - shaped microorganism and is heterotropic.

Aspergillus niger

Aspergillus niger is a fungus and one of the most common species of

the genus Aspergillus. It causes black mold on certain types of fruit and

vegetables and is a common contaminent of food. It is less likely to cause

disease than some other Aspergillus species, but if large amounts of spores

are inhaled, a serious lung disease aspergillosis can occur. It is one of the

most common causes of otomycosis (fungal ear infections), which can

233

cause pain, temporary hearing loss and in severe cases damage to the ear

canal and tympanic membrane.

Candida albicans

Candida albicans is a diploid sexual fungus and a casual agent of

opportunistic oral and vaginal infections in humans. Systemic fungal

infections have emerged as important causes of morbidity and mortality in

immunocompromised patients (eg. AIDS, cancer, chemotherapy, organ or

bone marrow transplantation). It is among the many organisms that live in

the human mouth and gastrointestinal tract. Under normal circumstances, it

lives in 80 % of the human population with no harmful effects, although

overgrowth results in candidiasis.

Saccharomyces cerevisiae

Saccharomyces cerevisiae commonly known as bakers yeast' is a

single celled ascomycete. Widely used as a simple eukaryotic model,

particularly in recombinant DNA and cell cycle studies as well as in mating

type and hetrokaryon compatibility studies. It can reproduce both asexually

and sexually; and can be cultured in either the haploid or the diploid state.

234

Microorganisms are ubiquitous. They are found in soil, air, water,

food, sewage and on body surfaces. The microbiologist separates these

mixed populations into individual species for study. A culture containing a

single unadulterated species of cells is called a pure culture. The survival

· and continued growth of microorganisms depend on an adequate supply of

nutrients and a favourable growth environment. For the former, most

microbes must use soluble low - molecular weight substances that are

frequently derived from the enzymatic degradation of complex nutrients is

a culture medium. Basically, all culture media are liquid, semisolid or solid.

A liquid medium lacking a solidifying agent is called a broth medium. A

broth medium supplemented with a solidifying agent called agar results in a

solid or semisolid qiedium. Agar serves as an excellent solidifying agent

because it liquefies at 100 °C and solidifies at 40 °C. Because of these

properties, organisms, especially, pathogens can be cultivated at

temperatures of 37 .5 °C or slightly higher without fear of the medium

liquefying. A completely solid medium requires an agar concentration of

about 1.5 % to 1.8 %. A concentration of less than 1 % agar results in a

semisolid medium. A solid medium has the advantage that it presents a

hardened surface on which microorganisms can be grown using specialized

techniques for the isolation of discrete colonies. Each colony is a cluster of

235

cells that originates from the multiplication of a single cell and represents

the growth of a single species of microorganism. Such a defined and well -

isolated colony is a pure culture. Also, while in the liquefied state, solid

media can be placed in test tubes, which are then allowed to cool or harden

in a slanted position, producing agar slants. These are useful for

maintaining pure cultures.

Nutrient broth

It was prepared by dissolving peptone (1 g), meat extract (0.5 g) and

sodium chloride (0.5 g) in distilled water (100 mL). The pH of the solution

was adjusted to 7 .2 -7.4.

Nutrient agar

This medium is used for maintaining pure bacterial culture and to

lawn the bacteria for detecting antibacterial activity. It was prepared by

dissolving peptone (0.5 g), meat extract (0.2 g), sodium chloride (0.5 g)

and agar (2.5 g) in distilled water (100 mL). The pH of the medium was

maintained at 7 .2 - 7.4.

236

Subculture

One day prior to the test, the bacteria were inoculated in nutrient

broth (inoculation medium) and kept in an incubator at 37°C for 24 h.

Seed layer medium

The bacterial subculture was taken in the seed layer medium for

lawning. It consist of peptone (l g), yeast extract (0.8 g), glucose (0.2 g),

sodium chloride (0.6 g) and agar (0.2 g) in distilled water (100 mL) and

sterilized.

Compound solution

A stock solution of the ligand and the complexes were prepared by

dissolving 0.5 mg of the compounds in 1 mL of DMSO so that their

concentration is 500 ~ g / mL.

Zone of inhibition

When a filter paper disc, impregnated with the compound solution is

placed on agar the compound will diffuse from the disc to the agar. This

diffusion will place the compound in the agar only around the disc. The

237

solubility of the compound and its molecular size will determine the size of

the area of chemical infiltration around the disc. If an organism is placed on

the agar it will not grow in the area around the disc if it is susceptible to the

chemical. This area of no growth around the disc is known as a zone of

inhibition'.

Antibacterial activity

The antibacterial activity of ISAT and its chloro and

nitrato complexes of lanthanum(III), cerium(III), praseodymium(III),

neodymium(III), samarium(III), europium(III), gadolinium(III),

dysprosium(III), ytterbium(III) and lutetium(III) were evaluated by the agar

diffusion method. The hot nutrient agar solution (20 rnL) was poured into

sterilized petri dishes and allowed to attain room temperature. The seed

layer medium was melted and cooled to about 45 °C with gentle shaking.

The previously grown subculture was added to the seed layer medium

aseptically and mixed well. It was immediately lawned into petri dishes and

allowed to attain room temperature. Filter paper discs (6 mm dia.) were

soaked in solution of the test compounds in DMSO and placed after drying

off the solvent on petri dishes containing lawn culture of different bacteria.

The plates were incubated to an optimum temperature of 37 °C for 48 h. At

238

the end of incubation period, the zones of inhibition around the discs were

measured. Gentamycin was used as reference standard.

Antifungal activity

Antifungal activity of the ligand and the complexes was tested by

the agar diffusion method using Flucanazole as the standard. Instead of

agar petri dishes in antibacterial study, agar slants prepared in sterilized test

tubes were used. The test tubes were closed with cotton plugs to maintain

sterile ervironment. Incorporation of the test compounds in SDA

(sabouraud dextrose agar) inoculation of the fungal culture, the presence,

absence or degree of growth were determined.

RESULTS AND DISCUSSION

Antimicrobial activities of the ligand, ISAT and its chloro and

nitrato lanthanide(III) complexes against the bacteria and fungi mentioned

above were studied and the results have been presented in Table X. 1 to

Table X. 4 and Fig X. 1 to Fig X. 3. It is interesting to observe that

the complexes are more potent bactericide and fungicide than the ligand.

This is in consistent with the reports that antimicrobial properties of organic

239

compounds are considerably enhanced by complexation with lanthanide

(III) ions. 350-352

A possible mode of toxicity can be speculated in the light of.

chelation theory. 353 Chelation reduces the polarity of the metal ions

considerably, mainly because of the partial sharing of its positive charge

with donor groups and possible 1t-electron delocalization on the whole

chelate ring. The lipids and polysaccharides are some important

constituents of cell wall and membranes, which are preferred for metal ion

interaction. In addition to this, cell wall also contains many

aminophosphates, carbonyl and cysteinyl ligands, which maintain the

integrity of the membrane by acting as a diffusion barrier and also provides

suitable sites for binding. Chelation can reduce not only the polarity of the

metal ion, but it increases the lipophilic character of the chelate and the

interaction between metal ion and the lipid is favoured. This may lead to

the breakdown of the permeability barrier of the cell resulting in

interference with the normal cell processes.

Chelation is not the only criterion for antibacterial activity.354 Some

important factors which determine the activity are, nature of the metal ion,

nature of the ligand, coordinating sites, geometry of the complex,

concentration, hydrophilicity, lipophilicity and presence of co-ligand. Steric

240

Table X. 1 Antibacterial activity of ISAT and [Ln(ISAT)(N03)3]

Inhibition zone (mm)

Compound Escherchia Vibrio Salmonella Bacillus

coli. cholerae typhi megaterium

!SAT 6 7 8 7

[La(ISA T)(N03)3] 8 10 12 11

[Ce(ISA T)(N03)3] 9 11 14 12

[Pr(ISA T)(N03)3] 10 12 13 14

[Nd(ISAT)(N03)3) 11 11 15 13

[Sm(ISAT)(N03)3) 10 12 17 15

[Eu(ISAT)(N03)3) 12 14 18 14

[Gd(ISAT)(N03)3) 11 15 16 13

[Dy(ISA T)(N03)3) 10 14 15 12

[Yb(ISAT)(N03)3) 12 16 14 15

[Lu(ISA T)(N03)3) 11 15 13 14

241

Table X. 2 Antibacterial activity of ISAT and [Ln(ISAT)Cb]

Inhibition zone (mm)

Compound Escherchia Vibrio Salmonella Bacillus

coli. cholerae typhi megaterium

ISAT 6 7 8 7

[La(ISAT)Ch] 10 12 14 13

[Ce(ISAT)Ch] 11 13 15 14

[Pr(ISAT)Ch] 12 14 14 13

[Nd(ISAT)Ch] 12 15 16 14

[Sm(ISA T)Cl3] 13 14 15 13

[Eu(ISA T)Ch] 14 16 18 15

[Gd(ISAT)Ch] 12 15 16 14

[Dy(ISAT)Ch] 11 16 17 15

[Yb(ISA T)Ch] 13 16 17 14

[Lu(ISAT)Ch] 12 15 16 13

242

243

Table X. 4 Antifungal activity of ISAT and [Ln(ISAT)ChJ

% Activity

CompoundP. notatum A. niger C. albicans S. cerevisiae

ISAT +++ +++ +++ +++

[La(ISAT)ChJ + + + +

[Ce(ISAT)CI3J + + + +

[Pr(ISAT)CI3J +

[Nd(ISAT)CI3J + +

[Sm(ISAT)ChJ + + + +

[Eu(ISAT)CI3J + + + +

[Gd(ISAT)ChJ +

[Dy(ISAT) ChJ +

[Yb(ISAT)CI3J + +

[Lu(I AT)ChJ + +

25% inhibition: +++ 50% inhibition: ++ 75% inhibition: + 100% inhibition:

244

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246

Fig X.l

Antibacterial activity

247

Fig X.2

Full growth of fungi

248

Fig X.3

Antifungal activity

249

and pharmacokinetic factors also play important roles in deciding the

potency of an antimicrobial agent. The interaction of metal ions with

cellular compounds is due to the fact that all these structures contain a

variety of functional groups that can act as metal-binding sites. The

difficulty is how to obtain these interactions in cells and organisms where

non-polar membranes exist to hinder the movement of charged metal ions

in to the cell, where a myriad of metal-binding sites exist to complete for

the metal ion. In order to achieve this, the complex should possess

sufficient lipid solubility to permit transport of the metal across the

membranes and it must have high thermodynamic stability to reach the site

without being dissociated. Finally, it can act as metal complex with cells

to produce a selective effect in order to obtain therapeutic value.355

Role of co-ligands on antimicrobial activity was also examined. On

comparing the activities of lanthanide nitrate and chloride complexes it has

been observed that the chloride complexes exhibited more inhibition. The

bonding capacity of nitrate ion towards lanthanide ion is greater than that of

the chloride ion. As a result of this the extent of metal ion available in

lessened for the display of antimicrobial activity. Besides this mode of

action, the compounds may indulge in the formation of hydrogen bonded

interaction throu ah the coordinated anion, azomethine group etc. with theb

250

active centres of the cell constituents resulting in the interference with the

normal cell processes. Hence it can be concluded that antimicrobial activity

possessed by metal complexes is an intricate blend of several contributions.