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INTERNATIONAL RESEARCH JOURNAL OF PHARMACY www.irjponline.com ISSN 2230 – 8407

Research Article

ANTIMICROBIAL ACTIVITY OF DIFFERENT THIOSEMICARBAZONE COMPOUNDS AGAINST MICROBIAL PATHOGENS

Negi Parul1, Nandy Subhangkar2*, Mahato Arun3 1Department of Medicinal Chemistry, Sardar Bhagwan Singh Post Graduation Institute, Balawana, UTU, Uttarakhand, India

2Department of Pharmacology, Vedica College of Pharmacy, RKDF University, Bhopal MP, India 3Department of Medicinal Chemistry, Sardar Bhagwan Singh Post Graduation Institute, Balawana, UTU, Uttarakhand, India

Article Received on: 15/03/12 Revised on: 24/04/12 Approved for publication: 19/05/12

*E-mail: subhangkarnandy@gmail.com ABSTRACT Thiosemicarbazone belongs to a large group of thiourea derivatives, whose biological activities are a function of parent aldehyde or ketone moiety. They have been evaluated over the last 50 year as antiviral, antibacterial, antifungal, antimalarial, anticancer, leprosy, rheumatism, trypanosomiasis and coccidiodis. Thiosemicarbazones were prepared by simple process in which N4-thiosemicarbazone moiety was replaced by aliphatic, arylic and cyclic amines. Present study reported the anti-microbial activity of different thiosemicarbazone compounds against certain bacterial and fungal pathogens viz. Bacillus cereus, Staphylococcus epidermis, Moraxella cattarhalis, Staph. Saprophyticus, Candida albicans and Aspergillus flavans. KEY WORDS Thiosemicarbazone derivetives, Synthesis, Characterisation, Antibacterial activity, Antifungal activity. INTRODUCTION Thiosemicarbazone exhibit various biological activities and have therefore attracted considerable pharmaceutical interest. They have been evaluated over the last 50 year as antiviral, antibacterial, antifungal, antimalarial, anticancer, leprosy, rheumatism, trypanosomiasis and coccidiodis1. In past few years thiosemicarbazones derived from 2-formylpyridine and related aldehydes have been of great interest because of their antineoplastic action. A thiosemicarbazone is a derivative of a semicarbazone which contain a sulfur atom in place of the oxygen atom. In organic chemistry semicarbazone is the derivatives of an aldehyde or ketone formed by a condensation reaction between ketone or aldehyde and semicarbazide. Nitrofurazone is the semicarbazone antibiotics2. Thiosemicarbazone belongs to a large group of thiourea derivatives, whose biological activities are a function of parent aldehyde or ketone moiety.3 Conjugated N-N-S tridentate ligand system of thiosemicarbazide (NH2-CS-NH-NH2) seem essential for anticancer activity, possibly due to the observation that structural alteration that hinders a thiosemicarbazone’s ability to function as a chelating agent tend to destroy or reduce its medicinal activity.4 Fruther more, the most active thiosemicarbazone are those which possess the trans isomer ( i.e. hydrogen bonding involving O…NH). In number of cases the transition metal complexes of TSCS showed greater biological activity than the uncomplexed ligands. This observation further encouraged detailed studies on coordination chemistry involving TSCs.5, 6 Thiosemicarbazone are known iron-chelating agents by bonding through the sulfur and azomethine nitrogen atoms. They have been found to be shown activity against extracellular protozoan such as Plasmodium falciparum, Trichomonas vaginalis, Trypanosome cruzi, and other parasites. Iron chelating agents are use to treatment for malaria. The replacement of the X (chlorine, hydroxyl, bromo atom by iodine atom led to compounds with a less toxicity and better biological activity. These promoted us to explore the biological activities of thiosemicarbazone analogs of aromatic iodine. Thiosemicarbazone could inhibit ribonucleotide reductase (RR) activity. RR catalyzes the

synthesis of deoxyribonucleotides are present in extremely low levels in mammalian cell; it is a crucial and rate-controlling step in the pathway leading to the biosynthesis of DNA. Mammalian ribonucleotide reductase (RR) is composed of two dissimilar protein, (R1), which is contain polythiol and (R2), which contain non - heme iron and a free tryosyl radical. Both the R1and R2 subunit contribute to the active site of the enzyme.8 Since thiosemicarbazone are known iron chelators and as such can destabilize or damage the non-heme iron-stabilized tryosyl free radical and thus inhibit the catalytical function of RR. Mammalian ribonucleotide reductase is located in the cytoplasm and is regulated by the cell cycle. Ribonucleotide reductase is a multi submit enzyme responsible for the reduction of ribonucleotide to their corresponding deoxyribonucleotides, which are the building blocks for DNA replication and repair in all living cell.9 Thiosemicarbazones were prepared by simple process in which N4-thiosemicarbazone moiety was replaced by aliphatic, arylic and cyclic amines. Literature survey revealed that the presence of certain bulky groups at N4 of the thiosemicarbazone moiety greatly enhances biological activity. Disease which is caused by microorganism (bacteria, fungi, viruses, protozoa and helminthes) and parasites after being transmitted from one host or reservoir to the other host is known as infection disease. These may be mild, severe or deadly to the host. Smallpox, distemper, and measles are amongst the diseases known to have entered human populations at this time.10 Bacteria are grouped in a number of different ways. They usually have a size of 0.3-2.0 micrometer. Most bacteria are of one of three typical shapes-rod-shaped (bacillus), round (coccus, e.g., streptococcus), and spiral (spirillum). An additional group, vibrios appears as incomplete spirals. The cytoplasm and plasma membrane of most bacterial cells are surrounded by a cell wall; further classification of bacteria is based on cell wall characteristics. They can also be characterized by their patterns of growth, such as the chains formed by streptococci 11 Gram negative bacteria are encased in a triple-layer. The outer most layers contain lipopolysaccharide (LPS) and are decolorized by the acetone /alcohol wash. For example: E. coli, Pseudomonas

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aeuriginosa. Bacterial parasites that cause disease are called pathogens. Natural defense against harmful bacteria is provided by antibodies. Certain bacterial diseases, e.g., tetanus, can be prevented by injection of antitoxin or of serum containing antibodies against specific bacterial antigens; immunity to some can be induced by vaccination; and certain specific bacterial parasites are killed by antibiotics12. New strains of more virulent bacterial pathogens, many of them resistant to antibiotics, have emerged in recent years. Fungi grow as single cell, as in yeast, or as multicelluler filamentous colonies, as in mold and mushroom. They do not contain chlorophyll. So they are saprophytic (obtain food from dead organic matter) or parasites (obtain nourishment from living organisms). Most fungi are not pathogenic, and the body’s normal flora contains many fungi13. Present study reported the anti-bacterial activity of different thiosemicarbazone compounds against certain bacterial pathogens viz. Bacillus cereus, Staphylococcus epidermis, Moraxella cattarhalis, Staph. saprophyticus. EXPERIMENTAL Equipments Melting points were determined in open capillaries and were uncorrected by melting point determining apparatus (Tashniwal Bros. Pvt. Ltd). Purity of the compounds were checked by TLC. IR data were recorded in KBr disks on Hitachi 270-30 Infrared spectrophotometer and H1NMR spectra on Brucker AV of NMR spectrometer 500MHz.

Heating mantle and refluxing assembly (Rolex Ltd), Electronic balance (FEW Pvt. Ltd). MATERIALS Hydrazine hydrate, Potassium hydroxide, Methyl iodide, Diethyl ether, p-Chloro benzaldehyde, P-Hydroxy benzaldehyde, Propylamine, Isopropyl amine, Ethylamine, DMSO, (CDH), Carbon disulfide(Merck Ltd), Isopropanol, p-Bromo benzaldehyde, Nutrient Agar , Muller Hinton Agar , Sabaourds Dextrose Agar (Hi Media Pvt. Ltd), Methanol, Ethanol , Chloroform (Ranbaxy fine Chemicals) METHODS SYNTHESIS OF INTERMEDIATES AND FINAL COMPOUNDS 1) Synthesis of (2E)-2-(4-bromobenzylidene)-N-propylhydrazinecarbothioamide (A1) 2.4gm (0.02 mol) Methyl dithiocarbazinate added with 3.68gm (0.02 mol) of bromo benzaldehyde. This mixture was dissolved in 7.0ml isopropanol. Then the reaction mixture was stirred for 2 hrs and cooled overnight. Next day reaction mixture was washed with cooled isopropanol or ethanol. After the completion of reaction a crèmish yellow product obtained (Schiff base of methyl dithiocarbazinate). 0.45gm (0.002mole) Schiff base added with 0.164ml (0.002mol) propylamines. This solution then dissolved in 15ml methanol and reflux until the evaluation of methyl mercaptan completely ceased (8-24 hr). After completely ceased methyl mercaptan the reaction mixture was cooled in ice bath. Thiosemicarbazone was obtained after washing with cooled water.

NH 2 N H

S

S C H 3 NN H

S

S C H 3

Br

p-B rC 6H 5C H O

M ethyl Thiocarbazinate

( lll )

m ethyl (2E )-2-(4-brom obenzylidene)hydrazinecarbodith ioate

( lV a )

(2E)-2(4 brom obenzylidene)-N -

P ropythydrazinecarbothioam ide

NN H

Br S

N HC H 3

C 3H 9N

A 1

Yeild-33%, M.P-1490C, Rf = 0.65.

Scheme-I

2) Synthesis of (2E)-2-(4-bromobenzylidene)-N-(propan-2yl) hydrazinecarbothioamide (A2) 2.4gm (0.02 mol) Methyl dithiocarbazinate added with 3.68gm (0.02 mol) of Bromo benzaldehyde. This mixture was dissolved in 7.0ml isopropanol. Then the reaction mixture was stirred for 2 hrs and cooled overnight. Next day reaction mixture was washed with cooled Isopropanol or ethanol. After the completion of reaction a cremish yellow product obtained (Schiffbase of methyl dithiocarbazinate). 0.45gm (0.002 mol) Schiff base added with 0.164ml (0.002mol) iso-propylamines. This solution then dissolved in 15ml methanol and reflux until the evaluation of methyl mercaptan completely ceased (8-24 hr). After completely ceased methyl mercaptan the reaction mixture was cooled in ice bath. Thiosemicarbazone was obtained after washing with cooled water.

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NH 2 N H

S

S C H 3 NN H

S

S C H 3

B r

p -B rC 6H 5C H O

M e th y l T h io c a rb a z in a te

( l l l )

m e th y l (2 E ) -2 -(4 -b ro m o b e n z y l id e n e )h y d ra z in e c a rb o d ith io a te

( lV a )

NN H

B r S

N H C H 3

C H 3

(2 E )-2 (4 b ro m o b e n z y l id e n e )-N -

(P ro p a n -2 -y l)h y d ra z in e c a rb o th io a mid e

(A 2 )

C 3H 9N

Yeild-25%, M.P-1400C, Rf= 0.40

Scheme-II 3) (2E)-2-(4-bromobenzylidene)-N-ethylhydrazinecarbothioamide (A3) 2.4gm (0.02 mol) Methyl dithiocarbazinate added with 3.68gm (0.02 mol) of Bromo benzaldehyde. This mixture was dissolved in 7.0ml isopropanol. Then the reaction mixture was stirred for 2 hrs and cooled overnight. Next day reaction mixture was washed with cooled Isopropanol or ethanol. After the completion of reaction a cremish yellow product obtained (Schiff base of methyl Dithiocarbazinate). 0.45gm (0.002 mol) Schiff base added with 0.112ml (0.002mol) ethylamine. This solution then dissolved in 15ml methanol and reflux until the evaluation of methyl mercaptan completely ceased (8-24 hr). After completely ceased methyl mercaptan the reaction mixture was cooled in ice bath. Thiosemicarbazone was obtained after washing with cooled water.

NH 2 N H

S

S C H 3 NN H

S

S C H 3

B r

p - B r C 6 H 5 C H O

M e t h y l T h i o c a r b a z i n a t e

( l l l )

m e t h y l ( 2 E ) - 2 - ( 4 - b r o m o b e n z y l i d e n e ) h y d r a z i n e c a r b o d i t h i o a t e

( l V a )

NN H

B r S

N H C H 3

( 2 E ) - 2 - ( 4 - B r o m o b e n y l i d e n e ) - N -

e t h y l h y d r a z i n e c a r b o t h i o a m i d e

( A 3 )

C 2 H 7 N

Yeild-32%, M.P. 1460C, Rf-= 0.60

Scheme-III 4) N'-[(E)-(4-bromophenyl) methylidene] morpholine-4-carbothiohydrazide (A4) 2.4gm (0.02 mol) Methyl dithiocarbazinate added with 3.68gm (0.02 mol) of bromo benzaldehyde. This mixture was dissolved in 7.0ml isopropanol. Then the reaction mixture was stirred for 2 hrs and cooled overnight. Next day reaction mixture was washed with cooled Isopropanol or ethanol. After the completion of reaction a cremish yellow product obtained (Schiff base of methyl dithiocarbazinate). 0.45gm (0.002 mol) Schiff base added with 0.174ml (0.002mol) morpholine. This solution then dissolved in 15ml methanol and reflux until the evaluation of methyl mercaptan completely ceased (8-24 hr). After completely ceased methyl mercaptan the reaction mixture was cooled in ice bath. Thiosemicarbazone was obtained after washing with cooled water.

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NH 2 N H

S

S C H 3 NN H

S

S C H 3

B r

p-B rC 6H 5C H O

M ethy le T h iocarbaz inate

( ll l )

m ethy l (2E )-2-(4-brom obenzy lidene)hydraz inecarbod ith ioate

( lV a )

NN H

B r S

N

O

N '-[(E )-(4-brom opheny l)m ethy lidene]m orpho line-4-carboth iohydraz ide (A 4)

C 4H 9N O

Yield 32%, M.P.-1510C, Rf = 0.76

Scheme-IV 5) (2E)-2-(4-chlorobenzylidene)-N-propylhydrazinecarbothioamide (A5) 2.4gm (0.02mol) Methyl dithiocarbazinate added with 2.9gm (0.02 mol) of p- chloro benzaldehyde. This mixture was dissolved in 7.0ml isopropanol. Then the reaction mixture was stirred for 2 hrs and cooled overnight. Next day reaction mixture was washed with cooled Isopropanol or ethanol. After the completion of reaction a cremish yellow product obtained (Schiff base of methyl dithiocarbazinate). 0.39gm (0.002 mol) Schiff base added with 0.164ml (0.002mol) Propylamines. This solution then dissolved in 15ml methanol and reflux until the evaluation of methyl mercaptan completely ceased (8-24 hr). After completely ceased methyl mercaptan the reaction mixture was cooled in ice bath. Thiosemicarbazone was obtained after washing with cooled water.

NH 2 N H

S

S C H 3

m e th y l h y d ra z in e c a rb o d i th io a te

NN H

S

S C H 3

C l

( l l l )

p - C lC 6 H 4 C H O

m e th y l ( 2 E ) - 2 - ( 4 - c h lo ro b e n z y l id e n e )h y d ra z in e c a rb o d i th io a te

(2 E ) -2 - (4 c h lo ro b e n z y l id e n e ) -N -

p ro p y lh y d ra z in e c a rb o th io a m id e

(A 5 )

NN H

S

N HC H 3

C l

( lV b )

C 3 H 9 N

Yeild-41%, M.P. – 1450C, Rf- 0.45

Scheme-V 6) (2E)-2-(4-chlorobenzylidene)-N-(propan-2yl) hydrazinecarbothioamide (A6) 2.4gm (0.02 mol) Methyl dithiocarbazinate added with 2.9gm (0.02 mol) of p- chloro benzaldehyde. This mixture was dissolved in 7.0ml isopropanol. Then the reaction mixture was stirred for 2 hrs and cooled overnight. Next day reaction mixture was washed with cooled isopropanol or ethanol. After the completion of reaction a cremish yellow product obtained (Schiff base of methyl dithiocarbazinate). 0.396gm (0.002mol) Schiff base added with 0.164ml (0.002mol) isopropylamine. This solution then dissolved in 15ml methanol and reflux until the evaluation of methyl mercaptan completely ceased (8-24 hr). After completely ceased methyl mercaptan the reaction mixture was cooled in ice bath. Thiosemicarbazone was obtained after washing with cooled water .

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NH 2 N H

S

S C H 3

m e th y l h yd ra z in e ca rb o d ith io a te

NN H

S

S C H 3

C l

( l l l) ( lV b )

NN H

S

N H C H 3

C l C H 3

p -C lC 6H 4C H O

m e th y l (2 E )-2 -(4 -ch lo ro b e n zy lid e n e )h yd ra z in e ca rb o d ith io a te

(2 E )-2 -(4 ch lo ro b e n zy lid e n e )-N -

(p ro p a n -2 -y l)h yd ra z in e ca rb o th io a m id e

(A 6)

C 3H 9N

Yield – 35%, M.P. – 1520C, Rf- 0.43

Scheme-VI 7) (2E)-2-(4-chlorobenzylidene)-N-ethylhydrazinecarbothioamide (A7) 2.4gm (0.02mol) Methyl dithiocarbazinate added with 2.9gm (0.02 mol) of p- chloro benzaldehyde. This mixture was dissolved in 7.0ml isopropanol. Then the reaction mixture was stirred for 2 hrs and cooled overnight. Next day reaction mixture was washed with cooled isopropanol or ethanol. After the completion of reaction a cremish yellow product obtained (Schiff base of methyl dithiocarbazinate). 0.396gm (0.002 mol) Schiff base added with 0.112ml (0.002mol) ethylamine. This solution then dissolved in 15ml methanol and reflux until the evaluation of methyl mercaptan completely ceased (8-24 hr). After completely ceased methyl mercaptan the reaction mixture was cooled in ice bath. Thiosemicarbazone was obtained after washing with cooled water .

NH2 NH

S

S CH3

methyl hydrazinecarbodithioate

NNH

S

S CH3

Cl

(lll) (lV b)

NNH

S

NH CH3

Cl

p-ClC6H4CHO

methyl (2E )-2-(4-chlorobenzylidene)hydrazinecarbodithioate

(2E)-2-(4chlorobenzylidene)-N-

ethylhydrazine carbothioamide

(A7)

C2H7N

Yeild-38%, M.P.- 1460C, Rf = 0.86

Scheme-VII 8) (2E)-2-(4-hydroxybenzylidene)-N-propylhydrazinecarbothioamide (A8) 2.4gm (0.02mol) Methyl dithiocarbazinate added with 2.5gm (0.02mol) of p- hydroxyl benzaldehyde. This mixture was dissolved in 7.0ml isopropanol. Then the reaction mixture was stirred for 2 hrs and cooled overnight. Next day reaction mixture was washed with cooled isopropanol or ethanol. After the completion of reaction a cremish yellow product obtained (Schiff base of methyl dithiocarbazinate). 0.46gm (0.002 mol) Schiff base added with 0.164ml (0.002mol) propylamines. This solution then dissolved in 15ml methanol and reflux until the evaluations of methyl mercaptan completely ceased (8-24 hr). After completely ceased methyl mercaptan the reaction mixture was cooled in ice bath. Thiosemicarbazone was obtained after washing with cooled water.

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NH 2 N H

S

S C H 3

m ethyl hydrazinecarbodith ioate

NN H

S

S C H 3

OH

(lll) (lV c)

NN H

S

N H

OH

C H 3

p -O H C 6H 4C H O

(2E )-2-(4chlorobenzylidene)-N -

ethylhydrazine carboth ioam ide

(A 8)

m ethyl (2E )-2-(4-hydroxybenzylidene)hydrazinecarbodith ioate

C 3H 9N

Yeild-25%, M.P- 1390C, Rf= 0.64

Scheme-VIII 9) (2E)-2-(4-hydroxybenzylidene)-N-(propan2yl) hydrazinecarbothio amide (A9) 2.4gm (0.02mol) Methyl dithiocarbazinate added with 2.5gm (0.002mol) of p- hydroxy benzaldehyde. This mixture was dissolved in 7.0ml isopropanol. Then the reaction mixture was stirred for 2 hrs and cooled overnight. Next day reaction mixture was washed with cooled Isopropanol or ethanol. After the completion of reaction a cremish yellow product obtained (Schiff base of methyl dithiocarbazinate). 0.46gm (0.002mol) Schiff base added with 0.164ml (0.002mol) isopropylamines. This solution then dissolved in 15ml methanol and reflux until the evaluation of methyl mercaptan completely ceased (8-24 hr).After completely ceased methyl mercaptan the reaction mixture was cooled in ice bath. Thiosemicarbazone was obtained after washing with cooled water .

NH 2 N H

S

S C H 3

m e t h y l h y d r a z in e c a r b o d i t h io a t e

NN H

S

S C H 3

OH

( l l l ) ( lV c )

NN H

S

N H C H 3

OH C H 3

p - O H C 6 H 4 C H O

( 2 E ) - 2 - ( 4 c h lo r o b e n z y l id e n e ) - N -

( p r o p a n - 2 - y l ) h y d r a z in e c a r b o t h io a m id e

( A 9 )

m e t h y l ( 2 E ) - 2 - ( 4 - h y d r o x y b e n z y l id e n e ) h y d r a z in e c a r b o d i t h io a t e

C 3 H 9 N

Yeild-25%, M.P. – 1450C, Rf =0.45

Scheme-IX 10) (2E)-N-ethyl-2-(4-hydroxybenzylidene) hydrazinecarbothioamide (A10) 2.4gm (0.02mol) Methyl dithiocarbazinate added with 2.5gm (0.02mol) of p- hydroxy benzaldehyde. This mixture was dissolved in 7.0ml isopropanol. Then the reaction mixture was stirred for 2 hrs and cooled overnight. Next day reaction mixture was washed with cooled Isopropanol or ethanol. After the completion of reaction a cremish yellow product obtained (Schiff base of methyl dithiocarbazinate). 0.46gm (0.002 mol) Schiff base added with 0.112ml (0,002mol) ethylamines. This solution then dissolved in 15ml methanol and reflux until the evaluation of methyl mercaptan completely ceased (8-24 hr).After completely ceased methyl mercaptan the reaction mixture was cooled in ice bath. Thiosemicarbazone was obtained after washing with cooled water.

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NH 2 NH

S

S CH 3

m ethyl hydrazinecarbodithioate

NNH

S

S CH 3

OH

(lll) (lV c)

NNH

S

NH CH 3

OH

p-O HC 6H 4CHO

(2E)-2-(4hydroxybenzylidene)-N-

ethylhydrazine carbothioam ide

(A 10)

m ethyl (2E )-2-(4-hydroxybenzylidene)hydrazinecarbodithioate

C 2H 7N

Yeild-28%, M.P. – 1450C, Rf = 0.78.

Scheme-X ANTIBACTERIAL ACTIVITY Preparation of standard bacterial suspensions The cultures of Bacillus cereus, Staphylococcus epidermis, Moraxella cattarhalis, Staph. Saprophyticus organisms were obtained from MTCC and Microbiology department. These cultures were maintained on nutrient agar slants at first being incubated at 370C for about 18-24 hour and then stored at 40C. Fresh cultures obtained by transferring a loopful of culture into Nutrient Broth and then incubated at 370C over night. To test antibacterial activity, disc diffusion method was used. Preparations of Media for Bacterial strain Muller Hinton agar media (gm/liter) was used as a media preparation. For 1000 ml Muller Hinton agar preparation, Peptone-5gm, Sodium Chloride-8gm, Beef infusion-3gm, and Agar-16gm were weighed and dissolved in 1000 ml of distilled water and adjusted to pH 7.3-7.4 which was sterilized by autoclaving at 1210C for 15 minutes at 15 psi pressure and was used for sensitivity tests. Disc diffusion method 1) 20-25ml of pre-autoclaved Muller Hinton agar medium

was poured into 90 mm diameter pre sterilized petriplates under aseptic conditions and was allowed to solidify at room temperature.

2) 0.1ml of fresh pure cultures of Bacteria spreaderd on MHA plates with the help of Spreader under aseptic condition.

3) For the preparation of disc whatman filter paper were used to prepare discs. Paper discs (dia. 6mm) are sterilized by auto calving each disc was impregnated with 10µl of (100ml/mg) stock solution of respective synthetic compound. The petriplates were incubated at 370C for 24 hours. The inhibition zone diameter were observed and recorded.14-21

ANTIFUNGAL ACTIVITY Preparation of standard bacterial suspensions The following cultures were used for anti-fungal activity viz. Aspergillus niger, Tricoderma viride, Fusarium oxysporium, candidia albicans were obtained from MTCC and Microbiology department. These cultures were maintained on Sabaourds dextrose agar slants (SDA) at first being incubated at 270C for about 5-7 days and then stored at 40C. Fresh culture were obtained by transferring a loopful of culture into Potato dextrose broth and then incubated at 270C for 3 days.

Preparation of Media Sabourand Dextrose Agar (Hi-media) media (SDA) was used for cultivation of fungi and particularly pathogenic fungi associated with skin infections6. Formula gm/litre Peptone 10g, dextrose 40g and agar 15g; pH5.6 ± 0.2. 65 gm of SDA was dissolved in 1000ml of distilled water. The medium was sterilized by autoclaving at 121oC for 15 minutes at 15psi pressure. Disc diffusion method 1) 25ml of pre autocalved SDA media was poured into pre

sterilized Petriplates and allowed to solidify 0.1ml of fresh pure cultures of fungi spreaded on SDA plates with the help of Spreader under aseptic condition.

2) For the preparation of disc what man filter paper were used to prepare discs. Paper discs (diameter 6mm) are sterilized by auto calving each disc was impregnated with 10µl of (100ml/mg) stock solution of respective synthetic compound. The petriplates were incubated at 370C for 24 hours. The inhibition zone diameter were observed and recorded.14-21

3) The solution of drugs was prepared in that solutions in which the synthetic compounds are completely dissolve (DMSO). It is a colorless liquid with boiling point 1890C. It is miscible with H2O, chloroform, alcohol, ethyl acetate and ethanol.

4) DMSO is a very good solvent for experimental purpose with high polarity. DMSO is an ideal solvent at 1 to 2% a perhaps the least toxic for assay system.

5) Accurately weigh (50mg) compound were transferred to different 10ml volumetric flask. These were dissolved in 1ml DMSO than make up the volume upto mark with DMSO, these solution (each having 10000µg/ml)used as stock solution from these stock solution further dilution were made to get concentration in the range to 5000µg/ml to 78.125ml (bacteria) 1000µg/ml to 3.91µg/ml(fungal).

Determination of Minimum inhibitory Concentration of synthesized drugs (Thiosemicarbazone derivatives) Minimum inhibitory concentration of any antimicrobial substance is the lowest concentration of the drug which inhibits the visible growth of microorganism. To access MIC, different variable concentration of the drug (1000µg/ml, 500µg/ml, 250µg/ml,125µl/ml, 62.5µg/ml, 31.25µg/ml, 15.63µg/ml,7.81µg/ml3.91µl/ml)were used by disc diffusion method. Positive controls were equally set up using DMSO

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as control solvent. The plate with least concentration of drug showing growth inhibition was recorded as MIC. Reading was expressed as (-) -if inhibition of growth is seen and (+) - If inhibition of growth is not seen. The result of each plate were observed and recorded after 24-36 hour in case of bacteria by measuring the inhibition zone (diameter after suitable incubation in mm).Data showing zones of inhibition caused by synthesized Thiosemicarbazone derivatives are already described in the below, while the plate showing zones in the photograph. RESULTS The synthetic evaluation was carried out by Krishnan et al22 showed that compounds with a prototype structure (A) with p-hydroxy substitution in the aromatic ring and substitution of amines groups were thought to provide a starting point for design and synthesis of newer therapeutic anticancer molecules. Based on these observations 10 molecules (A1,A2.A3,A4,A5,A6,A7,A8,A9, A10 ) were designed and synthesized using different aromatic amines by changing (X) in the aromatic ring23-27. The molecules were characterized by means of spectral data (IR, NMR). (Table 1) ANTIMICROBIAL ACTIVITY The different thiosemicarbazone derivatives synthesized were found to possesses antimicrobial activity against the various bacterial culture that is gram (+) and gram (-) bacterial culture namely Staphylococcus epidermidis, Bacillus cereus, Moraxella cattarhalis, Staph. Saprophyticus and various fungal cultures that is Candida albicans, Aspergillus flavans. The results of antibacterial activity data of compounds are as given in Table 2-5 and Figure 1-7. The Results of MIC of compounds Antibacterial screening of the synthesized compounds A2, A3, A5. A6, A7, A8, A9,A10 was conducted using Bacillus cereus, Strephylococcus epidermidis, Moraxella cattarhalis, Staph. saprophyticus. Compound A10 had minimum inhibitory concentration, equal to that of the standard drug (15µg/ml) for Bacillus cereus. A10 was found to be a potent antibacterial agent against Bacillus cereus. Compounds A3 and A9 has shown minimum inhibitory concentration equal to that of the standard drug (15µg/ml) were found to be potent antibacterial agent against Staphylococcus epidermidis .The MIC value of the Compounds A5 and A9 for Moraxella cattarhalis was found to be 15.63µg/ml which was equal to standard drug. Compounds A5 and A9 has shown minimum inhibitory concentration of Staph. Saprophyticus which was equal of to standard drug (31.25µg/ml). Compounds A5 and A9 are found to be relatively more active than rest of the molecules which suggest that the presence of OH and Cl group in the aromatic ring is favorable for antibacterial activity.( Table 6) All the compounds synthesized were subjected to antifungal screening against Candida albicans and Aspergillus flavens. All the compounds were found to be active at μg/ml concentration. Amongst all the compound A3 and A10 (IC=15.63µg/ml) was found to inhibited the growth of Candida albican and A10 (IC=15.63µg/ml) against Aspergillus flavan. Compounds A3 and A10 are found to be relatively more active than rest of the molecules which suggest that the presence of OH group in the aromatic ring is favorable for antifungal activity. DISCUSSION All Thiosemicarbazone derivatives (A1 – A10) were prepared according to the modified procedure described in the literature 28, 29, starting from hydrazine hydrate, potassium

hydroxide and carbon disulfide. Compounds were white or yellow powdered and crystal easily soluble in DMSO, acetone, methanol, chloroform and water. Purity of the compounds was confirmed by TLC analysis. Solvents were used for TLC analysis of all compounds were same, in which ratio of the solvent system was 9.8: 0.2. The IR spectra of the derivatives are in the accordance to the literature data for the same type of the compounds. Characteristic intense bands in the range of (3500 cm-1) str are associated with C=N. The (OH) band of phenolic oxygen occurs almost at the same frequency for all Thiosemicarbazone at 3600 cm-1. The intense C=S band is observed in the range 1160 cm-1. The compounds were also characterized by 1H NMR. A multiplet at δ = 6.5-7.7 [4H] shows the presence of aromatic ring and gradual shifting of these peaks in different derivatives shows that there is an electronegative group attached to the ring. Peak at δ = 2.0 indicates the presence of -NH group. The biological activities of the compounds were evaluated against antibacterial and antifungal by used various described strains.The results of the zone inhibition were summarized in.The concentration of test compound used was 12.5mg/ml and Penicillin G, Erythromycin, Amphicilline, Amoxicillin were taken as the standard drug for bacteria and Ketoconazole and fluconazole as standard for fungi. DMSO was used as a solvent control. CONCLUSION The result of antibacterial screening reveals that all the synthesized compounds were active against gram (+) ve and gram (-) ve bacteria and fungi. Compound A10 was found to be highly active against Bacillus cereus, while A3 and A9 showed the good activity against Streptococcus epidermidis. Compounds A5 and A9 was active against Morexella cattrihelis and staph. saprophyticus. The result of antifungal activity reveals that compounds A3 and A10 have significant activity against Candida albicans, and compound A10 also have a inhibitory activity against Aspergillus flavans. So it can be concluded that the presence of 4-OH in aromatic ring have shown better activity against bacteria and fungi. It was also concluded that the carbon side chain increases the antimicrobial and antifungal activity. REFERENCES (1) Jose MV, Teresa P, Adriana A, Maria G, Javier M, Margarita L T, Fernandez. The key role of sulfur in thiosemicarbazone compounds crystal and molecular structure. J. Organ Chem. 2001; 623: 176-184. (2) Dimmock JR, Vashishta SC, Stables JP. Biological properties of semicarbazone. Eur. J. Med. Chem. 2000; 35: 241-242. (3) Du X, Guo C, Hansall E, Doyle PS, Caffrey C R, Holler TP, Mc Kerrov JH, Cohen FE. Thiourea derivatives. J. Med. Chem. 2002; 45: 2695-2696. (4) Liberta AE, West DX. Thiosemicarbazone derivatives as a potent anticancer activity. Biometal 1992; 5: 121-122 (5) Pandeya SN, Dimmock JR, Pharmazie 1993; 48: 659-670. (6) Quiroga AG, Ranninger CN. The transition metal complexes of thiosemicarbazone, Coordinate. Chem. Rev. 2004; 248: 119-120 (7) Azuolas JK, Caple IW. Novel thiosemisemicarbazone derivatives bearing aromati6c iodine moiety. Aust J. Vet 1994; 61: 223-224 (8) Adoracion GQ, Carmen N R. Contribution to the SAR field of metallated and coordination complexes studies of the palladium and platinum derivatives with selected thiosemicarbazone as antitumor drugs. Coordn. Chem. Rev. 2004; 248: 119-133. (9) Shao JB, Zhou BC, Yen Y. Ribonucleotide Reductase inhibitors and Future drug design. Curren. Can. Drg. Targ. 2006; 6: 409- 431. (10) Bhawsar SB, Mane DV, Sinde DB, Shingare MS, Deokate AS, and Congwane, LV. Synthesis of some 8-[6-substituted-1, 3-benzothiazol-2-yl) amino methyl] substituted hydroxyl coumarins and Evaluation of their antibacterial activity. Indian J Het Chem. 1996; 8: 23-24. (11) http://www.answers.com/topic/Bacteria 2, Jun, 2011. (12) Latrofa A, Franco M, Lopedota A, Rosato A, Carone D, and Vitali C. Structural modification and antimicrobial activity of N-cycloalkylidene-2, 3-

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dihydro-1, 3-benzothiazoles, N-cycloalkyl-2-acylalkylidene-2, 3-dihydro-1, 3-benzothiazoles. IL Farmaco 2005; 60: 291-297. (13) http://www.answers.com/topic/Fungi. Jun 2011; 2. 14) Cowan MM. Plant products as antimicrobial agents. Clin Microbiol Rev 1999; 12(4): 564-582. 15) Cushnie TPT, Lamb AJ. Antimicrobial activity of flavonoids. Int J Antimicrob Agents 2005; 26: 343-56 16) Dr. S. S. Kadam, K. R. Mahadik, K. G. Bothara. Principles of Medicinal Chemistry 18th edition. Nirali Prakashan 2007; volume-1: 67. 17) Frobisher M, Hinsdill RD, Crabtree KT, Goodheart CR. Fundamentals of Microbiology. 9th ed. Saunders Company: Philadelphia, PA, W.B. 1974. p. 319- 330. 18) Gerard J. Tortora, Berdell R. Funke and Christine L. Microbiology An introduction, Pearson education. 8th edition; 591-592. 19) Kokoska L, Polesny Z, Rada V, Nepovim A, Vanek T. Screening of some Siberian Medicinal plants for antimicrobial activity. J Ethnopharmacol 2002; 82: 51-53. 20) Lehmann PF. Immunology of fungal infections in animals. Vet immunol Immunopatholo 1985; 10: 33-69. 21) Pelczar M. J. and Reid J. D., Jr. E. C. S. Chan. Microbiology. 5th ed. New Delhi: Tata Mcgraw Hill 1974; 473: 491. 22) Hu W, Zhou W, Xia C, Wen X. Synthesis and anticancer activity of thiosemicarbazone. Bioorg. Med. Chem. Lett. 2006. 23) Wang Y, Liu MC, Lin TS, Sartorelli AC. Synthesis and antitumor activity of 3-and 5-Hydroxy 4-methyl

pyridine-2carboxaldehyde thiosemicarbazone. J. Med. Chem. 1992; 35: 3667-3671. 24) Klayman D L, Bartosevich J F, Griffin T S, Manson C J, Scovill JP. 2-Acetylpyridine thiosemicarbazones. A new class of potential antimalarial agents, J. Med. Chem. 1979; 22: 855-862. 25) Krishnan K, Design. Synthesis and antimalarial activity of thiosemicarbazone. Thesis submitted for partial fulfillment for the degree of M. Pharm, Birla Institute of Technology, Mesra, Ranchi. 2007; 42: 1-43. 26) Audrieth LF, Scott E S, Kippur P S. Hydrazine derivatives of the carbonic and thiocarbonic acids. I. The preparation and properties of thiocarbohydrazine. J. Org. Chem. 1953; 24: 733-741. 27)Klayman DL, Bartosevich JF, Griffin TS, Manson CJ, Scovill JP 2-Acetylpyridine thiosemicarbazones. 1. A new class of potential antimalarial agents. J. Med.Chem. 1979; 22: 855-862. 28) Du X, Guo C, Hansell E, Doyle PS, Caffrey CR, Holler TP, Mc Kerrow JH. Semicarbazone and thiosemicarbazone derivatives were prepared and tested in vitro against a chloroquine resistant strain of Plasmodium falciparum. J. Med. Chem. 2002; 45: 2695-2707. 29) Santos IG, Abram Alberto R, Vázquez-López E, Sanchez A. Isatin b-thiosemicarbazone derivatives was synthesized and evaluated for their anti-HIV activity in HTLV-IIIB strain in the CEM cell line. Inorg. Chem. 2004; 43: 1834-1835.

TABLE 1: CHARACTERIZATION OF THE MOLECULES

Sample code Melting point IR(cm-1) NMR(δ=ppm) A1 149°C

3373 1517 1283 1157 602

_

A2

140°C

3338 1508 1258 1163 623

8.45 7.45 2.78 1.34

A3

146°C

3483 1509 1262 1166 657

8.43 7.92 2.34 1.42

A5

145°C

3382 1594 1287 1166 819

8.45 7.34 2.45 1.45

A6

152°C

3448 1590 1295 1171 818

8.39 7.48 2.78 1.34

A7

146°C

3515 1509 1287 1166 826

8.35 7.87 2.65 1.43

A8

139°C

_

8.87 7.83 2.04 5.27

A9

145°C

3509 1509 1385 1262 1166

8.73 7.39 2.83 5.38

A10

145°C

3483 1508 1337 1288 1163

8.48 7.81 2.81 5.61

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TABLE 2: MEASUREMENT OF ZONE DIAMETER (MM) IN BACTERIA Microorganism/comp(mm) A1 A2 A3 A4 A5 A6 A7 A8 A9 A10

Bacillus cereus 10 12 14 11 12 14 14 13 13 12

Staphylococcus epidermidis 11 12 14 11 14 17 11 9 10 11

Moraxella cattarhalis - 9 9 8 8 12 7 8 10 7

Staph. Saprophytics 11 10 12 9 11 15 9 7 8 8

TABLE 3: ZONE OF STANDARD DRUGS FOR BACTERIA

M. organism/std Zone diameter (mm)

Erythromycin Amoxicillin

Bacillus cereus 19 14

Staphylococcus epidermidis 19 22

Moraxella cattarhalis 13 15

Staph .saprophytics 13 20

TABLE 4: MEASUREMENT OF ZONE DIAMETER (MM) IN FUNGI

Microorganisms/comp(mm) A1 A2 A3 A4 A5 A6 A7 A8 A9 A10

Candida albicans 12 12 13 10 15 14 12 10 13 10 Aspergillus flavans 10 14 15 11 12 15 12 11 14 12

TABLE 5: ZONE OF STANDARD DRUGS FOR FUNGAL

M. organism/std Zone diameter(mm)

Ketoconazole Fluconazole

Candida albicans 16 14

Aspergillus flavans 19 12

TABLE 6: COMPARISON OF THE CONCENTRATION (µG/ML) IN CASE OF BACTERIA

S.No Test compounds Bacillus cereus Streph epidermidis Moraxella cattarhalis Staph. saprophyticus

1. A1 10 11 - 11

2. A2 62.5 125 31.25 62.5

3. A3 31.25 31.25 62.5 125

4. A4 11 11 8 9

5. A5 31.25 31.25 15.63 31.25

6. A6 62.5 31.25 62.5 125

7. A7 250 250 250 62.5

8. A8 62.5 125 62.5 250

9. A9 31.25 31.25 15.63 31.25

10. A10 15.63 31.25 125 62.5

11. Erythromycin 15.63 15.63 31.25 31.25

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TABLE 7: COMPARISON OF THE CONCENTRATION (µG/ML) IN CASE OF FUNGI

S.No. Test compounds Candida albicans(IC) Aspergillus flavans(IC)

1. A1 12 10

2. A2 250 125

3. A3 15.63 15.63

4 A4 10 11

5. A5 31.25 31.25

6. A6 62.5 62.5

7. A7 250 125

8. A8 62.5 62.5

9. A9 62.5 62.5

10. A10 15.63 31.25

11. Fluconazole 15.63 15.63

Figure 1: Comparison of zone of inhibition in case of Bacillus cereus with synthesized compounds

Figure 2: Comparison of zone of inhibition in case of the Staphylococcus epidermidis with synthesized compounds

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Figure 3: Comparison of zone of inhibition in case of the Moraxella cattarhalis with synthesized compounds

Figure 4: Comparison of zone of inhibition in case of the Staph. Saprophyticus with synthesized compounds

Figure 5: Comparison of zone of inhibition in case of the Candida albicans with synthesized compounds

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Figure 6: Comparison of zone of inhibition in case of the Aspergillus flavans with synthesized compounds

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Figure 7: a) Zone inhibition of standard drugs in case of Bacillus cereus. b) Zone inhibition of standard drugs in case of Staph saprophyticus

c) Zone inhibition of Synthesized compounds A1-A5 in case of Bacillus cereus d) Zone inhibition of Synthesized compounds A6-A10 in case of Bacillus cereus

e) Zone inhibition of Synthesized compounds A1-A5 in case of Staph saprophyticus f) Zone inhibition of Synthesized compounds A6-A10 in case of Staph. saprophyticus

g) Zone inhibition of Synthesized compounds A1-A5 in case of Candida albicans h) Zone inhibition of Synthesized compounds A6-A10 in case of Staph saprophyticus

Source of support: Nil, Conflict of interest: None Declared