101

CHAPTER VI

Synthesis of Novel Sydnone Derivatives

as

Anti-Microbial Agents

102

INTRODUCTION

The chemistry of heterocycles is one of the interesting branches of

chemistry, for its theoretical implications and for the diversity of

synthetic procedures. This has resulted in virtually limitless series

of structurally novel synthetic heterocyclic compounds with a wide

range of physical, chemical and biological properties, scanning a

broad spectrum of reactivity. Heterocycles, widely distributed in

nature as alkaloids, vitamins, antibiotics are important not only in

the medicinal world, but also in the field of agriculture. The large

majority of pharmacologically active compounds are synthetic

heterocyles. Amongst the most important and interesting

heterocyclic compounds are the ones that possess aromatic

properties and many of them are five-membered rings. Of the large

number of non-benzenoid aromatic heterocycles known, the

mesoionic compounds have attracted attention, because of their

structure, chemical properties and varied pharmacological

properties. The most extensively studied class of mesoionic

compounds is the “sydnone” An anormous amount of reaserch on

synthetic and pharmacological studies of sydnones has been

reported from this laboratory during the last three decades. This

present work is directed to further synthetic utility of sydnones for

creative development of bisheterocycles of pharmacological

interest.

103

These sydnones have been prepared by obtaining the N-

arylsubstituted glycines from the corresponding primary amines,

followed by nitrosation and cyclisation with acetic anhydride.

Sydnones (III) belong to the mesoionic class of nonbenzenoid

aromatic compounds. The mesoionic compounds have attracted

much attention because of their interesting physical, chemical and

biological characteristics. Many mesoionic compounds have been

reported since 1949 when the concept of mesoionic molecules was

introduced by Baker and Ollis1. The concept of mesoionicity was

developed to explain the electronic structure of sydnone – the

cyclodehydration product which was prepared by Earl and

Mackney2 in 1935 by treating N-nitroso-N-phenylglycine (I) with

acetic anhydride.

The original structure proposed was that of a bicyclic ring (II)

which Baker et al1 found to be untenable in the light of its

properties. They described the molecule to be a resonance hybrid

of a number of dipolar and tetrapolar structures (IIIa-IIIf) and

further indicated that it would be misleading to arbitrarily select

one contributing structure among a number of canonical forms

with varying degree of importance. A general adjective “mesoionic”

104

(Mesomeric+ionic) was therefore introduced to describe molecules

having such electronic distribution.

The definition of mesoionic compounds was given by Baker1

as, “A compound may appropriately be called mesoionic if it is a five

membered heterocyclic compound which cannot be represented

satisfactorily by any one covalent or polar structure and possesses a

sextet of electrons in association with all atoms comprising the ring”.

The aromaticity of these compounds can be explained by the

classical sextet theory. There are a total of seven 2pz electrons

contributed by the five atoms forming the ring, and one 2pz

electron on the exocyclic atom (IV). A sextet of electrons will be

obtained if one of the seven 2pz electron is paired with the single

electron on the exocyclic atom. The ring would thus become

positively charged balanced by the negative charge on the exocyclic

atom (V).

This draws comparison to tropone (VIa-b), which however, is

not mesoionic since it can be represented by a covalent structure

(VIa).

The mesoionic compounds are generally classified into two

types depending on position of the two heteroatoms contributing

two electrons each to the aromatic sextet. Thus in type A, the two

105

atoms are nonadjacent while in type B they are adjacent to each

other.

Out of the 144 possible mesoionic systems of type A,

sydnone is the most widely studied because of its facile synthesis

and marked physiological activity.

Much less is known of the 84 possible systems of type B,

though the first mesoionic compound discovered, dehyrodithizone,

is of this type. The compounds of type A differ markedly in their

physical and chemical properties from that of type B members. The

important mesoionic compounds of both types are listed below:

Type A

Type B

Of all the mesoionic systems, the study of sydnones still

remains a field of interest because of their diverse chemical and

biological properties, and their utility in heterocyclic synthesis.

Comprehensive reviews on mesoionic compounds have been

published by Baker and Ollis3a, Stewart 3b, Kier and Roche3c, Ohta

and Kato3d.

CHEMISTRY OF SYDNONES

Sydnones, (III) the best known of the mesoionic compounds

were not studied intensively until 1946, even though they were

reported first in 1935. Thereafter, a number of studies directed

106

towards understanding the chemical, physical and biological

properties of sydnones have been carried out. While the initial

studies of sydnones was directed towards understanding their

physical characteristics, later studies have been directed towards

exploiting its potential as synthons in organic synthesis.

Nomenclature:

Several methods of naming the sydnone ring are currently in

use.The original nomenclature by Baker1 is based on the unknown

1,2,3-oxadiazole (X). According to this phenyl sydnone is named as

ψ-5-keto-3-phenyl-3,5-dihydro-1-oxo-2,3-diazole. The symbol ψ

refers to the delocalization of the electrons in the ring.

a) Ollis and Ramsden4 named the sydnone ring as,

1) Mesoionic 1,2,3-oxadiazolium-5-olate based on the

dipolar structure (XI).

2) Mesoionic 1,2,3-oxadiazole-5-one, emphasizing the

double bond character of the exocyclic bond. (IIIb-c)

b) An alternate nomenclature proposed by Katritzky5, is based

on the betaine structure (IIIb). It is one of the important

contributors to the resonance hybrid and is considered as

the anhydro compound of quaternary base (XII). Thus 3-

107

phenylsydnone is named as anhydro-5-hydroxy-3-phenyl-1-

oxa--2,3-diazolium hydroxide.

c) Besides these the trivial name sydnone (suggested in honour

of University of Sydney where it was first synthesized) is also

used (ex. chemical abstracts).

Synthesis of sydnone:

The most common method of preparing sydnone is by the

cyclodehydration of N-substituted-N-nitroso-α-amino carboxylic

acids with acetic anhydride1.

Although this proceeds sluggishly at room temperature, it is

rapid when heated. Trifloroacetic anhydride rapidly promotes ring

closure even at low temperatures. The mechanism of cyclisation

suggested by Baker involves the formation of a mixed anhydride as

intermediate.

The only other alternate method6 reported for the

preparation of sydnone(XIV) is by heating N-nitrososydnoneimine

(XV) in an organic solvent. However, this method is not practically

feasible because of the difficulty in preparing the starting material.

Chemical properties:

108

Both the aromatic and dipolar nature of sydnone are

reflected in its chemical properties. While the ability of sydnone to

undergo electrophilic substitution reaction shows its aromatic

nature, the ready cleavage of the ring by acids and 1,3-dipolar

cycloaddition reaction brings forth its dipolar nature (cyclic

azomethine imine). This suggests that the degree of aromaticity of

the sydnone ring is about the same order as that of the furan ring.

Electrophilic Substitution Reactions:

4- Unsubstituted sydnones undergo the usual electrophilic

substitution reactions like chlorination7, bromination8,

sulfonation9, formylation10, acetylation11 and chlorosulfonation12.

Metallation:

The hydrogen atom at the 4-position of 3-phenylsydnone is

acidic enough to be lithiated by n-butyllithium, affording 4-

lithium-3-phenylsydnone. 4-Bromo-3-phenylsydnone could be

converted to the corresponding Grignard by treatment with

magnesium in ether in the presence of methyl iodide13.

Hydrolysis:

A synthetically very useful reaction that sydnones undergo is

the hydrolysis to substituted hydrazines1 (XIX) with hydrochloric

acid.

109

The sydnone ring thus acts as a masked hydrazine group.

This reaction has been recommended by Fugger et al14 as a

convenient method of preparing hydrazines from primary amines

via sydnone, especially when the hydrazines are inaccessible or

otherwise accessible with difficulty.

The synthetic utility of this reaction has been well exploited

in our laboratory in the one-pot synthesis of various heterocycles

using reagents such as 1,3-diones15, cyclohexanone16 and levulinic

acid17.

1,3-Dipolar Cycloadditions:

One of the interesting and novel reaction of sydnone, which

has received detailed attention is the 1,3-dipolar addition of a wide

variety of unsaturated compounds to sydnone giving rise to

heterocycles18a viz. pyrazoles, pyrazolines, oxadiazoles and other

mesoionic compounds. This is an addition-elimination reaction,

involving the loss of carbon dioxide.

Pyrazoles (XXIII) are formed by the cycloaddition of

acetylenic compounds to sydnone18b, while pyrazolines (XXIV) are

formed with olefinic compounds.

110

The synthetic utility of this reaction has been demonstrated

by Badami and Puranik17 by synthesising 5-halogenopyrazoles

from 4-halosydnones. This is the only method of preparing 5-

halopyrazoles, because halogenation of pyrazoles occur only at 4-

position.

The formation of dibenzoylpyrazole (XXV) and dimethyl

dicarboxylate (XXVI) by the 1,3-dipolar cycloaddition of dibenzoyl

acetylene (DBA) and dimethylacetylene dicaroxylate (DMAD) to

sydnones has also been reported from this laboratory19.

Recently, intramolecular 1,3-dipolar cycloaddition of

functionalised sydnones has been used to build indazole

derivatives20(XXVII).

Benzynes also react with 3-phenylsydnone to form 2-

phenylindazole21 (XXVIII). Phenyl isocyanate reacts with 3-phenyl

sydnone in a 1,3 dipolar fashion to give mesoionic 1,2,3-triazoles22

(XXIX).

3-Phenylsydnones when treated with bromine in presence of

acetic anhydride result in the formation of oxadiazolinones23

(XXX). The formation of oxadiazolinone in this reaction was

111

explained by Stewart24 in terms of a 1,3-dipolar addition of acetic

anhydride to the intermediate product 4-bromo-3-phenylsydnone.

Badami et al, have extended this reaction to many more

sydnones25. Recently the catalytic role played by HBr, liberated in

the reaction has been highlighted by the same authors26.

All these reactions demonstrate the importance of sydnone

in heterocyclic synthesis. Since sydnones are readily obtained from

primary amines, they serve as valuable intermediates for

converting primary amines to various heterocyclic systems, which

are many a time inaccessible or prepared with difficulty by other

methods. Sydnones are the only 5-membered mesoionic

compounds, which undergo such a wide variety of synthetically

useful reactions.

BIOLOGICAL ACTIVITY:

Sydnones and mesoionic compounds in general possess

structural features which have been of considerable interest to

medicinal chemists. The common structural feature found in these

compounds is the oppositely charged four atom dipole segment

which is the hallmark of many pharmacologically active classes of

drugs27.

112

Its significance perhaps lies in its ability to electrostatically

interact with two complementary partially charged positions on

receptor macromolecules, such as a protein helix.

The biological properties of sydnone were first studied by

Brooker and Walker28 in 1957. They screened 3-methy-4-

alkylsydnones as potential amino acid antagonists, since then a

number of studies dealing with the physiological activity of

sydnone have been reported.

An extensive pharmacological study of a large number of 3-

phenylsydnones has been carried out by Oehme et. al.29. They

have reported that the 3-arylsydnones are less toxic than the 3-

alkylsydnones. Further, the 3-alkylsydnones in general exhibit

CNS stimulation effect while the 3-arlysydnones exert CNS

depression effect.

Greco, Nyberg and Cheng30 tested N-piperonylsydnone

(XXXI) for antimalarial activity and found it to be active against

Plasmodium berghei in mice at a dose of 10 mg/kg. Later, Popoff

and Singhal31 claimed antimalarial properties for a series of

sulphonylsydnones (XXXII), but the effect was mild compared to

the piperonylsydnone.

Daeniker and Druey32 have reported antitumor activity for

ethylene-bis- sydnone (XXXIII).

113

Hill et al33 prepared a series of 3-(2-arylthio) and 4-

(methylthio) sydnones (XXXIV) and tested them for

antiinflammatory activity. Several of these compounds were more

potent then hydrocortisone and phenylbutazone versus adjuvant

arthritis in Mice.

In search of antibacterial agent Naito et al34 prepared

pencillium derivatives (XXXV) of sydnones that were found to be

active against gram positive and gram negative microbes, both in

vivo and in vitro.

A large number of cephalosporin derivatives (XXXVI) of sydnones35

have been prepared, which are reported to possess

antistreptococcal and antistaphylococcal activities.

Sydnones are also reported to show antifungal36,

anticonvulsant, analgesic37, diuretic38a and hypotensive, 38b

activities.

The synthesis of a large number of sydnone derivatives

incorporating biologically important heterocyclic moieties viz,

thiazole44, indazoline39, pyrazole40, pyrazolines41, thiadiazole42,

diazepine43, isatin44, quinoline45 etc. have been reported from this

114

laboratory and various pharmacological properties have been

claimed.

REVIEW OF LITERATURE

The ability of sydnones to undergo 1,3-dipolar cycloaddition

reactions has being extensively used in heterocyclic ring

construction. It provides a facile and convenient means of

synthesizing a wide variety of five membered ring nitrogen

heterocycles.

A well known and one of the earliest reported cycloaddition

reaction of sydnone is with acetylenic compounds. This reaction,

first reported by Huisgen and Grashey 46a, provides a powerful

method for the construction of the pyrazole ring system. The

presence of electron-withdrawing substituents on the dipolarophile

greatly facilitates this reaction. The addition of

dimethylacetylenedicarboxylate (DMAD), for example, proceeds

rapidly (< 2 hrs) while acetylene takes 40 hours, almost 20 times

more. This one-pot ring transformation by reaction of (DMAD) with

some 3-arylsydnones leading to the formation of the pyrazole 3,4-

dicarboxylates has been reported from our laboratory46b.

Such pyrazole dicarboxylates have been obtained by a

cumbersome method from the reaction of α-halogenated

phenylhydrazines with β-ketoesters and β –diketones. While, these

115

compounds are obtained in a single step from sydnones in

excellent yield (~90-98 %) and purity. Hence, this route provides a

convenient and simple method using easily accessible and

inexpensive chemicals. In view of this, we thought of extending this

reaction to other derivatives of 3-arylsydnones. In the present

work, we were interested in using this reaction for functionalised

3-arylsydnones with an aim of utilizing these cycloadducts for the

preparation of pyrazolo [3,4-d]pyridazines. The functional group

also would be utilized to build a heterocyclic ring resulting in the

formation of a bisheterocyclic system. One such sydnone

derivatives we selected for this work was the 3-[4-(1-oxo-3-aryl-

prop-2-en-1-yl)] phenyl sydnones, which can be used as

bifunctional compounds.

Compounds with this enone moiety – the chalcone residue

are known to possess marked biological properties. Apart form

this; functional group is a useful building block for the

heterocycles like pyrazolines, thiazepines, diazepines etc. Making

use of the enone residue and the latent functionaliy of sydnone

ring of 3-[4-(1-oxo-3-aryl-prop-2-en-1-yl)] phenyl sydnones, we

could prepare some bis heterocyles containing the pyrazolo[3,4-d]-

pyridazine in combination with the pyrazoline and the

benzothiazepine rings. It may be mentioned here that only a few

aryl pyrazolopyridazines are reported in the literature whereas

116

such heterocyclyl phenyl pyrazolopyridazines are not found in the

literature.

Sydnones (III) are the heterocycles, which belong to the

mesoionic class of non-benzenoid aromatic compounds, and serve

as valuable precursors in heterocyclic synthesis. They undergo

one-step ring conversion to a variety of heterocycles by 1, 3-dipolar

cycloaddition reactions. Some of these reactions, using the latent

functionality of sydnones, have been reported.

The major part of this thesis includes use of 1,3-dipolar

cycloaddition reactions to sydnones as well as its azide derivatives,

resulting in the formation of a variety of biologically active

heterocyclic ring systems.

Some chalcone derivatives of sydnones (X) and (XI) were

prepared in laboratory and screened for their antibacterial activity

47.

The 3-[p-(5‟-aryl-2‟pyrazolin-3‟-yl) phenyl] sydnones (XV)

synthesised by Dambal and coworkers48 have been reported to

possess good antibacterial activity.

Satyanarayana and coworkers49 have reported

benzothiazepine derivatives of sydnones of this type (XXVI) and

screened them for their anti-inflammatory activity.

117

Hiremath and et al.,50 from our laboratory have reported the

synthesis and antimicrobial activity of some pyrazole derivatives

(XL)

Valenta and et al.51 have reported the potential neuroleptic,

antipsychotic drugs as followsbelow. (XL-1) and (XL-2)

(AMISULPRIDE)

In view of the above mentioned important biological and

pharmacological properties of sydnone, other drugs like sulprides

particularly amisulpride, repglinide and other benzamide

derivatives and use them for building new heterocycles by

coupling sydnone ring with side chains of well known drugs like

sulprides particularly amisulpride, repglinide and other benzamide

derivatives resulting in the formation of some new heterocyclic

systems, in anticipation that they might possess pronounced

biological activity.

The present work involves the exploitation of the

functionality of the sydnone ring in the synthesis of some

heterocyclic systems, which are difficult or in some cases not

accessible by routine methods. The important reaction is the

amidation of heterocyclic / aromatic amines with 3-(4-carboxy

118

phenyl) sydnone. All the compounds have been characterised by

elemental analysis and spectral data viz., IR, 1H-NMR, 13C-NMR

and Mass. We have also carried out in vitro antimicrobial testing

has also been carried out to study the structure-activity

relationship (SAR).

The amidation by heating the mixture of compound (SD-III)

and (RS)-2-(aminomethyl)-1-ethyl pyrrolidine (R-I) to 100 0C

proceeded very sluggishly, the addition of small amount of sodium

methoxide had not much influenced the progress of reaction and

compound (SDD-I) was obtained after 15 hrs in the yield of 5%

only. For this reason, the mixed anhydride of the 3-(4-carboxy

phenyl)sydnone with triethylamine and ethyl chloroformate in

dichloromethane was prepared in situ at 0-5 0C, and was

subjected to the action of (RS)-2-(aminomethyl)-1-ethyl pyrrolidine

(R-ii); under these conditions the benzamide derivative of sydnone

(SDD-II) was obtained in the yield of 95%.

A series of benzamide derivative of sydnones (SDD II-IX) were

prepared by using different heterocyclic chiral and aromatic

primary amines (R II-IX) under above optimized conditions which

afforded good yields (75-95 %).

119

Scheme-IV

N

NO O

HO

HO

NH2

HO

ONH

HO

O

COOH

Ac2O /N

HO

O

COOH

NO

HCl / NaNo2

0-5 0C

N

NO O

HO

O _

Triethylamine /

Ethyl chloroformate / 0-50C

R NH2

R =

CH3

N

H3C

CH3

H

I) II) III)

IV) V) H

Cl-CH2COOH

NaOH

4-Amino benzoic acid

VI)

Cl

Cl

ClVII)

H3CO

H3CVIII)

CH3

IX)

(SD-I)

(SD-II)(SD-III)

OCH3

N

NO O

HO

NHR _

(SDD I-IX)

O

O

H3C

Mixed Anhydride

0-50C

N

H3C

H

120

Preparation of Compound SD-I:

To a well stirred boiling suspension of 4- amino benzoic acid

(0.1mol) in water (1lit) was added a neutralized solution of mono

chloro acetic acid (57.0g;0.6 mol) and the mixture was refluxed for

40 hrs. The solid obtained after cooling was dissolved in sodium

carbonate (10%) and filtered. The filtered was acidified with

hydrochloric acid (pH 5-6) and the precipitated solid was filtered

and washed with cold water and crystallized from boiling water.

Preparation of Compound SD-II:

To a well stirred mixture of SD-I (0.01mol) in water (2ml) and

Conc.HCl (25ml) was cooled to 0-2 0C and added sodium nitrite

(0.012mol) in water (10ml) drop wise during 30 min by maintaining

the temperature 0-2 0C (temperature should not allowed to the

above 50C).The reaction mixture was allowed to stand overnight

and the separated solid was filtered, washed thoroughly with ice

cold water , dried in air, and recrystallized from methanol.

Preparation of Compound SD-III:

The compound SD-II (0.01mol) and acetic anhydride ((0.02 mol))

were introduced in to a clean round bottomed flask and heated to

95-100 0C on water bath for 3hrs and cooled the reaction mass.

The thick mass was then poured on to crushed ice with constant

stirring. The separated solid product was collected by filtration.

121

Preparation of Compound SDD-I-IX:

The compound SD-III was dissolved (0.01mol) in MDC (50ml).

The triethyl amine (0.01mol) was added slowly with stirring. The

reaction mixture was cooled to 0-5 0C and slowly in drop wise

added ethylchloro formate (0.01ml) in MDC(10ml) with continues

stirring over the period of 30min. The substituted amine (0.01mol)

in MDC (15ml) was added slowly to the above reaction mixture over

a period of 15 min at 0-5 0C with stirring. Further the reaction

mixture was stirred for 30min at room temperature. Then the

solvent was evaporated and the crude mass was added to ice cold

water with stirring, the separated solid was filtered and washed

with water and recrystallized from suitable solvent.

122

BIOLOGICAL ACTIVITY

ANTI-BACTERIAL ACTIVITY

All the compounds synthesized in the present investigation

were screened for their anti-bacterial activity by subjecting the

compounds to standard procedures. Anti-bacterial activities were

tested on nutrient agar medium against Bacillus Pumilus, Bacillus

substilis, Escheria coli and Pseudomonas aeruginosa. Which are

representative type of gram positive and gram negative organisms

respectively. The anti-bacterial activity of the compounds was

assessed by disc-diffusion method.

Preparation of nutrient agar media:

Media composition and procedure:

The nutrient agar media was prepared by using the following

ingredients.

Peptone (Bacteriological) 20gm

Beef extract(Bacteriological) 05gm

Sodium chloride 05gm

Agar agar 20gm

Distilled water up to 1000ml

Weighed quantities of peptone and beef extract were

dissolved in distilled water by gentle warming and then specified

amount of agar was dissolved by heating on water bath. Then the

123

PH of the solution was adjusted to 7.2 to 7.4 by adding the sodium

chloride and the volume of the final solution was made up to

1000ml with distilled water. Then it was transferred in to a

suitable container, plugged with non-adsorbent cotton and the

media was sterilized by in autoclave at 121oC for 20minutes at 15

lbs pressure.

Preparation of test solution:

Directly taken 40μg of the compound was dissolved in 1 ml

of DMSO, now the concentration of the test solution was 40μg/ml.

Preparation of standard antibiotic solution:

Ciprofloxacin was used as a standard anti-biotic for

comparison and solution was prepared by using sterile water, the

solution was diluted by using sterile water, so that the

concentration of the solution were 40μm/ml.

Preparation of discs:

Discs of 6-7mm in diameter were punched from NO:1

Whattmann filter paper with sterile cork borer of same size. These

discs were sterilized by keeping in oven at 1300C for 60 minutes.

The standard and test solutions were added to each disc and discs

were air-dried.

Method of testing:

The sterilized media was cooled to 450C with gentle shaking

to bring about uniform cooling and then inoculated with 18-24hrs

124

old culture under aseptic conditions, mixed well by gentle shaking.

This was poured in to sterile Petri dishes and allowed the medium

to set. After solidification all the Petri dishes were transferred to

laminar air flow unit. Then the discs which were previously

prepared were carefully kept on the solidified media by using

sterilized forceps. These Petri dishes were kept as it is for one hour

for diffusion at room temperature and then for incubation at 370C

for 24 hours in an incubator.

The extent diameter of inhibition after 24 hours was

measured as the zone of inhibition in millimeters and the results

were shown in table 13.

TABLE-13: Antibacterial Activity of compounds SDD 1-9

Compound

code

Inhibition zone

diameter in mm

S.No B.

pumilus

B.

substilis

E.

coli

P.

Aureginosa

1 SDD-1 18 14 17 18

2 SDD-2 18 16 16 16

3 SDD-3 15 11 18 16

4 SDD-4 17 14 19 15

5 SDD-5 18 15 10 14

6 SDD-6 18 18 17 16

7 SDD-7 14 16 16 17

8 SDD-8 16 17 15 16

9 SDD-9 17 18 18 17

10 SDD-10 16 14 15 17

11 Standard

Ciprofloxacin 20 17 19 20

Average of triplicate ± Standard deviation

125

Note:-08 -12 mm poor activity, 13 – 16 mm moderate activity, 17 –

20 mm good activity.

ANTIFUNGAL ACTIVITY: Procedure:

The anti-fungal activity of all compounds was determined on

potato dextrose agar medium against Aspergillus Niger,

Colletotrichum and Fussarium verticilloids and Ketoconazole

40μg/ml was used as a standard and solvent DMSO was used as

control.

The sterile molten potato dextrose medium was cooled to 450C

and inoculated with test organism and mixed the contents

thoroughly and poured in to the sterile Petri dishes under aseptic

conditions. Then the above procedure was repeated. All the

inoculated Petri dishes were incubated at 280C for 4 days in case

of fungi and in case of yeast. The results were showed in the table

14.

PREPARATION OF SUB-CULTURE:

Peeled potato 200-300 gm

Dextrose 5 gm

Distilled water up to 1000 ml

Peeled potato were cut in to pieces and boiled for 30 min to get

extract. The extract is filtered through muslin cloth. The dextrose

was added to the filtrate and final volume is adjusted to 1000ml

126

with distilled water. Then it was sterilized by autoclave at 1210C

for 20 min.

Note: Two days before testing the culture is prepared by

inoculating the fungus from master culture in to potato dextrose

medium and incubated for 48 hr at room temperature.

Preparation of fungal medium:

The potato dextrose medium is prepared by dissolving.

Peeled potato 200-300gm

Dextrose 5gm

Agar 20gm

Distilled water up to 1000ml

Peeled potato were cut in to pieces and boiled for 30min to

get the extract. The extract is filtered through muslin cloth. The

dextrose and agar were added to the filtrate and the final volume is

adjusted to 1000ml with distilled water. Then it was sterilized by

autoclave at 1210C for 20 min.

Preparation of solution of test compounds:

Test solutions were prepared by accurately weighting 40μg of

the test compound and dissolving in 1 ml of DMSO to give

40μg/ml concentration of test compound solution.

Preparation of standard anti-fungal solution

127

Ketoconazole were used as standard anti-fungal for comparison

and solution were prepared by using sterile water, so that the

concentrations of the solution were 40μg/ml.

Method of testing:

The method of testing for fungicidal activity is the same as

that of antibacterial testing. DMSO was used as a solvent control.

TABLE-14: Antifungal activity of compounds SCD 1-10

Compound

code

Inhibition zone diameter in mm

S.No

A.niger Colletotrichum

Fussarium

verticilloids

1 SDD-1 11 10 09

2 SDD-2 12 12 12

3 SDD-3 11 11 10

4 SDD-4 10 10 11

5 SDD-5 11 11 12

6 SDD-6 11 10 11

7 SDD-7 12 10 10

8 SDD-8 12 12 12

9 SDD-9 11 12 11

10 SDD-10 10 10 11

11 Ketoconazole

40µg/ ml 14 13 13

12 DMSO - - -

*Average triplicate ± Standard deviation

128

Note:- 06 – 07mm poor activity, 08 – 10mm moderate activity, 11-

12mm good activity.

Result and Discussions:

Antibacterial results of sydnone derivative of SDD-1 to SDD-

10 clearly suggest that all the compounds posses significant

activity almost equipotent with the standard Ciprofloxacin against

both Gram +ve and Gram –ve pathogenic organism like B.

pumilus, B. substilis, E. coli and P. Aureginosa. The potent

activity of this class may be due to the nature of the amino

substituents. Thus the substituents place a vital role in imparting

enhanced antibacterial activity to the compounds.

The antifungal activity of sydnone series SDD-1 to SDD-10

reveals that the compounds posses moderate to very significant

activity in comparison with standard antifungal agent Ketoconazole

at the concentration 40µ/ml. However the majority of the

compounds of the series like SDD-1, SDD-2, SDD-3, SDD-5, SDD-

7, SDD-8 and SDD-9 showed activity almost equal to that of the

standard. Hence the synthesised compounds are proved to be

better antifungal agents than antibacterial agents. The presence of

sydnone moiety as it is and the amino substituents the same

moiety which results the presence of –CONH group, is responsible

129

for enhancing the antifungal activity of the compounds. As all

these derivatives do contain –CONH group, and have shown the

significant antifungal activity comparable with that of the

standard.

REFERENCES

1. Baker W, Ollis WD and Poole VD. J Chem Soc 1949; 307.

2. Earl JC and Mackney AW. J Chem Soc 1935; 899.

3. a) Baker W and Ollis WD. Quarter Rev 1957; 11: 15: b) Stewart

FHC. Chem Rev 1957; 64: 129: c) Kier LB and Roche EB. J Pharm

Sci 1967; 56: 149: d) Ohta M and Kato H. „Nonbenzenoid

Aromatics‟, ed. Snyder, J P. Academic Press, New York, 1969;117.

4. Ollis WD and Ramsden CA. Adv Heterocyclic Chem 1976; 19: 1.

5. Katritzky AR. Chem Ind 1955; 521.

6. Baker W, Ollis WD and Poole VD. J Chem Soc 1950; 1542.

7. Badami BV and Puranik GS. Indian J Chem 1974; 12: 671.

8. Greco CV, Pesce M and Franco JM. J Heterocyclic Chem 1966; 3:

391.

9. Yashunskii VG, Vasil‟eva VF and Sheinker Yu N. Zh Obschch Khim

1959; 29: 2712: Chem Abstr 1960; 54: 10999.

10. Thoman CJ, Voaden DJ and Hunsberger IM. J Org Chem 1964;

29: 2044.

130

11. a) Greco CV, Tobias J and Kier LB. J Heterocyclic Chem 1967; 4:

160: b) Tien HJ and Ohta M. Bull Chem Soc Japan 1972; 45:

2944.

12. Ugarkar BG, Badami BV and Puranik GS. Arch der Pharmazie

1979; 312(12): 977.

13. Ohta M and Kato H. Nippon Kagaku Zasshi 1957; 78: 1653.

14. Fugger JC and Mackney AW, J Am Chem Soc 1955; 77: 1483.

15. Havanur SB and Puranik GS. Indian J Chem 1985; 24B: 864.

16. Badami BV and Puranik GS. Indian J Chem 1974; 12: 671.

17. Badami BV and Puranik GS. Rev Roumaine de Chimie 1982; 27:

281.

18. a) Huisgen R and Grashey R. Angew Chem 1962; 74: 29: b)

Vasil‟eva VF, Yashunkii VG and Shchukina MN. Zh Obshch Khim

1960; 30: 698: Chem Abstr 1960; 54: 24674: c) Huisgen R,

Grashey R, Gotthardt H and Schmidt R. Angew Chem Int Ed Engl

1962; 1: 48.

19. Hiremath US, Yelamaggad CV and Badami BV. Indian J

Heterocyclic Chem 1996; 519.

20. Jeng-Ping Y, Jia-Jzong Y, Teng-Yueh C and Bling U. Synthesis

2002; 12: 1775.

21. Lazaris AY. Zh Organ Khim 1966; 2: 1322.

22. Kato H, Sato S and Ohta M. Tetrahedron Lett 1967; 8(43): 4261-

4262.

131

23. Stansfield F. J Chem Soc 1958; 4781.

24. Stewart FHC. Chem Rev 1964; 64: 129.

25. Mallur SG and Badami BV. IL Farmaco 2000; 55: 65-67.

26. Kamble RR and Badami BV. J Indian Chem Soc 2002; 79: 629.

27. Kier LB and Roche EB. J Pharm Sci 1967; 56: 149.

28. Brooker P and Walker J. J Chem Soc 1957; 4409.

29. Oehme P, Goeres E, Schwarz K, Pelsch G, Faulhaber HD and

Lange P Acta. Biol Med German 1965; 14: 369: Chem Abstr 1965;

63: 6191.

30. Nyberg WH and Cheng CC. J Med Chem 1965; 8: 531.

31. Popoff IC and Singhal GH. J Med Chem 1968; 11: 613 and 886.

32. Daeniker HV and Druey J. Helv Chim Acta 1957; 40: 918.

33. a) Hill JB and Wagner H. J Med Chem 1974; 17: 1337: b) Hill JB,

Ray RE, Wagner H and Aspinall RZ. J Med Chem 1975; 18: 50.

34. Naito T, Nukagawa S, Takahashi K, Fujisawa K and Kawaguchi H.

J Antibiotics 1968; 21: 300.

35. Takano T. Chem Abstr 1972; 76: 14557t.

36. a) Smith AE and Riddell JA. Chem Abstr 1965; 56: 15868. b)

Rogers EF and Davis D. US Patent 3,189,520; Chem Abstr 1965;

63: 7605.

37. Bruzzese T, Casadio S, Marazzi-Uberti E and Turba C. J Pharm Sci

1964; 54: 677.

132

38. a) Kier LB, Dhawan D and Fregly M. J Pharm Sci 1964; 53: 677: b)

Freggly MJ, Kier LB and Dhawan D. Toxicol Appl Pharmacol 1965;

6: 529.

39. Upadhya KG, Badami BV and Puranik GS. Arch Pharm (Weinheim)

1980; 313: 684.

40. Hosamani CK and Badami BV. Indian J Heterocyclic Chem 1998;

7: 245-248.

41. Havanur SB, Badami BV and Puranik GS. J Heterocyclic Chem

1980; 17: 1049.

42. Dambal DB, Pattanshetti PP, Tikare RK, Badami BV and Puranik

GS. Indian J Chem 1984; 23B: 186.

43. Patil BM, Badami BV and Puranik GS. Indian Drugs 1995; 32(10):

439-441.

44. Kavali JR and Badami BV. IL Farmaco 2000; 55(5): 406-409.

45. Kavali JR and Badami BV. J Chem Res (S) 2000; 548-500; J Chem

Res (M) 2000; 1326-1326.

46. a) Huisgen R and Grashey R. Angew. Chem. 1962; (74):29. b)

Vasil‟eva VF, Yashunkii VG and Shchukina MN. Zh. Obshch.

Khim. 1960; 30, 698 Chem. Abstr. 1960;(54):24674.

47. Dambal DB, Badami BV and Puranik GS. Indian J.

Chem.1982;(21B):865.

48. Dambal DB, Pattanshetti PP, Tikare RK, Badami BV and Puranik

GS. Indian J. Chem.1984;(21B):186-190.

133

49. Satyanarayan K and Rao MN. Indian J. Pharm. Sci.

1993;55(6):230-233.

50. Hiremath US, Yalamaggad CV and Badami BV. J. Chem. Res (s).

502-503 (1994).

51. Michal V, Phominet, Jacquesacher and Gean-Claude Monier. US

4,401,822.