International Journal of Innovative Pharmaceutical ... · Isatin (1H-indole-2, 3-dione,) was first obtained by Erdman and Laurent in 1841 as a product from the oxidation of indigo

RESEARCH ARTICLE Anusha et.al / IJIPSR / 3 (9), 2015, 1305-1318

Department of Medicinal Chemistry ISSN (online) 2347-2154

Available online: www.ijipsr.com September Issue 1305

SYNTHESIS AND BIOLOGICAL EVALUATION OF ISATIN

DERIVATIVES

Anusha Ramgalla*, Sharath Chandra R.A.S*, Harish Kumar P*, Sandeep Ankam

Department of Pharm. D, CMR College of Pharmacy, Kandlakoya (V) Hyderabad, 501401,

Telangana state, INDIA

Corresponding Author Anusha Ramgalla

Intern (Gandhi Hospital), Department of Pharm.D,

CMR College of Pharmacy,

Hyderabad, Telangana state, INDIA

E-mail: [email protected]

Phone: +91-97044 60658

International Journal of Innovative

Pharmaceutical Sciences and Research www.ijipsr.com

ABSTRACT

Isatin is a versatile lead molecule for potential bioactive agents and its derivatives were

reported to posses’ wide spectrum of activity like antibacterial, antifungal, anticonvulsant,

anti-HIV, antidepressant and anti- inflammatory etc., in view of these facts we hereby

report the synthesis of some Isatin derivatives using Schiff’s reaction. The present study

deals with the synthesis of Isatin Schiff’s bases by reacting Isatin with aromatic amines

and substituted aromatic amines (p- chloroaniline, p-nitro aniline, p- flouro aniline). All the

compounds have been screened for anti-microbial activity by standard method carried out in

laboratories. Of the five synthesized compounds III-b is found to be more potent with the

activity of III-a compared to the standard against anti-bacterial activity.

Key words: Heterocyclic compounds, Schiff’s reaction, Isatin derivatives.

mailto:[email protected]




INTRODUCTION

Heterocyclic compounds are abundant in nature and are of great significance to life because

their structural subunits exist in many natural products such a s vitamins, hormones, antibiotics,

etc, hence have attracted considerable attention in the design of biologically active molecules

and advanced organic materials [1]. Organic compounds containing a ring made up, in addition

to carbon atoms of atoms other elements (hetero atoms) most often nitrogen, oxygen, sulphur, and

less frequently phosphorous, boron and silicon [2]. Isatin (1H-indole-2, 3-dione,) was first

obtained by Erdman and Laurent in 1841 as a product from the oxidation of indigo by nitric and

chromic acids. Isatins are synthetically versatile substrates, where they can be used for the

synthesis of a large variety of heterocyclic compounds, such as indoles and quinolines, and as raw

material for drug synthesis. Isatins have also been found in mammalian tissue and their function

as a modulator of biochemical processes has been the subject of several discussions [15].

Isatin is a versatile lead molecule for potential bioactive agents and its derivatives were

reported to posses’ wide spectrum of activity likeantibacterial, [1] antifungal, [2] anticonvulsant

[3], anti-HIV [4], antidepressant [5] and anti- inflammatory [6] etc., in view of these facts we

hereby report the synthesis of some isatin derivatives using Schiff’s reaction [7]. The present

study deals with the synthesis of isatin Schiff’s bases by reacting isatin with aromatic amines

and substituted aromatic amines (p- chloroaniline, p-nitro aniline, p- flouro aniline) [13].

Fig.1: Isatin

IUPAC name - 1H-indole-2,3-dione, Molecular formula: C8H5NO2, Molar mass: 147.1308

g/mol, Appearance: Orange-red solid, Melting point: 200 °C, 473 K, 392°

The chemical structures of the synthesized compounds were confirmed by means of their IR and

1HNMR spectral data. In nature, isatin is found in plants of the genus Isatis, in Calanthe

discolor LINDL and in Couroupita guianensis Aubl., and has also been found as a component

of the secretion from the parotid gland of Bufo frogs, and in humans as it is a metabolic derivative

of adrenaline.[8] Substituted isatins are also found in plants, for example the melosatin

alkaloids (methoxy phenylpentyl isatins) obtained from the Caribbean tumorigenic plant

http://en.wikipedia.org/wiki/International_Union_of_Pure_and_Applied_Chemistry_nomenclature

http://en.wikipedia.org/wiki/Chemical_formula

http://en.wikipedia.org/wiki/Molar_mass

http://en.wikipedia.org/wiki/Melting_point




Melochia tomentosa as well as from fungi: 6-(3’-methylbuten- 2’-yl) isatin was isolated from

Streptomyces albus and 5- (3’-methylbuten-2’-yl) isatin from Chaetomium globosum Isatin has also

been found to be a component of coal tar. Endogenous compounds are often used as a basis, or

structural component, of pharmacological preparations [15].

Solubility: Conc Hcl/H2SO4 & NaOH/ KOH

It has the ability to form salts of silver, perchloric acid and mercury.

Exhibits Tautomerism.

CO CO

CO COH

N N

H Tautomerism H

Fig. 2

Chemically Isatin may be characterized as the lactum of o-amino-benzoyl formic acid. It

possesses both amide and keto carbonyl groups, active H attached to nitrogen or ( oxygen) an

aromatic ring which should substituted in 5,7 positions.[9]

MECHANISM OF ACTION: ISATIN

In 1988, isatin was identified as a major constituent of tribulin, a low-molecular-weight

inhibitor of MAO type B (MAO-B) and furthermore, urinary concentrations of isatin in

patients with Parkinsons disease tend to increase according to the severity of disease.

These results suggest that urinary Isatin may become a diagnostic marker for the clinical

severity of Parkinsons disease and that endogenous isatin, a new biological modulator, may play

a role in the regulation of the brain levels of ACh by increasing the level of DA under stress,

identified isatin in the urine and the brain of stroke-prone spontaneously hypertensive rats

(SHRSP). [10] Tribulin may contain metabolites of isatin or related endogenous compounds. The

physiological and pathological roles of isatin and tribulin are not yet clear. In humans, tribulin

levels increase as a result of exercise and old age. Tribulin excretion is significantly higher in

females than males. [11] Tribulin output is transiently raised following alcohol withdrawal,

benzodiazepine withdrawal, lactate- induced panic attacks, and migraine attacks. Tribulin output

thus appears to be raised in a variety of different conditions related to stress, agitation, or

anxiety. These observations suggest that during stress, activated catecholamine-synthesizing cells




and corticotropin- releasing factor cells, both of which play central roles in stress

responses, may be involved in isatin production. Cold immobilization stress, for

example, has been associated with the serotonergic system [1]. Cold restraint stress increases

tribulin in the rat heart and kidney. Tribulin acts on central benzodiazepine receptors, and has

been proposed to be an anxiety-promoting agent [12]. The potency of the MAO inhibitory and

benzodiazepine-receptor-binding inhibitory components of tribulin is roughly equal. It is a

selective MAO-B inhibitor. At much higher concentrations, it inhibits a variety of other enzymes,

such as alkaline phosphatase. Tribulin can be extracted from tissue and body fluids with ethyl

acetate [1]. Isatin is believed to be a component of tribulin and a selective inhibitor of MAO-B.

Other components of tribulin, the ethyl and methyl esters of indoleacetic and 4-

hydroxyphenylacetic acid selectively inhibit [14]. The synthetic and metabolic pathways of isatin

have not been established. It has been suggested that dietary tryptophan may be converted into an

indole by the gut flora and then transported to the liver where it is oxidized. Urinary isatin

concentration is significantly reduced in germ-free rats (0.22 mg/mL) compared to control rats

(0.66 mg/mL). This suggests that isatin is derived, at least in part, from the interaction of gut

flora with tryptophan- containing food [1].

STRUCTURE ACTIVITY RELATIONSHIP [1]

NH

O

NH

O

O

2-Indolidone 2, 3, indole dione

Fig.3 Fig.4

1.Bond acceptor at the position (3)

2. Free rotation bond OH

3. Bond donor (1)

4. Polar surface area-37.38

5. C5, C6 & C 7 substitution generally enhanced CNS activity some di and trihalogenanted

isatins.




METABOLISM [2]

Isatin was originally identified as one component of the endogenous monoamine oxidase (MAO)

inhibitory activity. The isatin concentration in blood can exceed 1 µM. Based on tissue isatin and

water content calculations; basal isatin tissue concentrations are different in various organs

(Table 1). Highest concentrations in the brain are in hippocampus, cerebellum and striatum

(1–1.3 µM), whereas in peripheral organs the highest concentrations are in seminal vesicles and

vas deferens: 47.4–79 µM. In the heart the maximal basal concentration approaches to 3 µM. In

other rat organs basal isatin concentrations vary from 0.3 µM (spleen) to 1.5 µM (liver). In the

rat, stress causes the 2–3 fold increase of isatin content in the brain and heart, 2.5–6 fold increase

of isatin output in urine and its concentration in serum reaches 2.9 ± 0.29 µM. This suggests that

under certain conditions isatin concentrations may be as high as 10 µM and even more (if we take

into consideration exceptionally high concentrations in seminal vesicles and vas deferens).

Fig.5

Although the pathways of endogenous isatin formation (Figure) still require better experimental

support there is in vitro evidence that isatin can be formed from indole, typically produced by

tryptophan catabolism in the gut, and that this involves microsomal cytochrome P450. Besides

excretion with urine, the catabolic pathways of isatin include further, possibly spontaneous,

oxidation and dimerization yielding the indigoid pigments, indigo and indirubin, hydrogen

peroxide-dependent conversion into anthranilic acid and NADPH-dependent reduction to 3-

hydroxy- 2-oxoindole. All these compounds have been found in urine. However, it remains

unclear whether indigoid formation precedes urinary excretion and aerobic transformation of the

urinary components. Interestingly, during in vitro oxidation of indole by cytochrome P450

enzymes, isatin was an intermediate, which was then transformed into the indigoid pigment major

end products.




Binding sites

The distribution of isatin-binding sites in the particulate fraction reduced in the following order:

brainstem brain hemispheres = cerebellum heart, kidneys liver. In the soluble fraction there was

a different rank of isatin-binding [2]. There are biological targets that specifically bind isatin and

also those sensitive to inhibition by physiologically relevant concentrations of isatin. The former

group includes MAO, the cytosolic enzymes glycerol-3-phosphate dehydrogenase,

glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase and ubiquitin. Other isatin binding

proteins detected in both soluble and particulate fractions of various tissues using optical

biosensor technique require detailed investigation and identification [16]. Isatin and its analogues

act on a large number of biological targets and have a wide variety of actual and potential

pharmacological actions. There is some evidence that isatin may attenuate the effects of certain

pharmaceutical agents on specific biological targets, such as monoamine oxidase inhibitors and/or

neuro protective drugs. Isatin can be present in blood in the micromolar range and increases in

response to stress. This suggests that endogenous isatin, and possibly its precursors and

metabolites, may affect the sensitivity of biological targets to therapeutics employed a range of

diseases and disorders [2].

MATERIALS & METHODS

Chemicals utilised in the study were of analytical grade and the sources of chemicals are listed

below:

Table 1: Chemicals used

Sl.no. Name of Chemicals Supplier

1 P-Chloro aniline SD Fine

2 4-flouro aniline SD Fine

3 O-Chloro aniline SD Fine

4 Chloral Hydrate SD Fine

5 4-Nitroaniline SD Fine

6 Hydroxyl amine Hcl SD Fine

7 Sodium sulfate SD Fine

8 Hcl concentrated SD Fine

9 H2SO4 concentrated SD Fine

10 Glacial acetic acid SD Fine

11 Ethanol SD Fine

12 Glycine SD Fine

13 2,4,dinitro phenyl hydrazine SD Fine




General requirements for isolation of compounds

Thin Layer chromatography:

Thin Layer chromatography based on UV absorption: The principal involved inTLC is almost

same as that of coloumn chromatography.

Elution:

1. The sample is dissolved in an appropriate solvent and applied as spots along one side

of the layer approximately one cm from the edge.

2. The plate is placed in the chromatography chamber where the eluent is lying. The

initial spots must not bath in the eluent. The whole is covered with lid to prevent the

eluent fro-vaporising.

3. As the eluent front migrates through the sorbent, the compounds of the sample also

migrate but at different rates (depending on their polarity), resulting in separation.

4. When the solvent front has reached a point near the top of the plate, the plate is

removed and dried. The spots are visualised with UV light. Consequently, only UV

active compounds appear.

Thus, the compounds that migrates the most are less polar. A good eluent enables

to get a maximal distance between the spots.

-------------------------- Eluent

TLC Plate

Compounds

Deposit line --------------------------- Initial spot

Fig.6: TLC Plate




EXPERIMENT

Present Work

In view of the biological prominence of the isatin derivatives, it was planned to synthesize some

novel isatin derivatives and schiff’s bases. And the synthesized compounds are screened for

antimicrobial activity.

The compounds have been synthesized as per the scheme presented below.

NH2

RCL3CCH(OH)2

NA2SO4 NH2OH.HCL

NHO

N OHR

CONC.H2SO4

N

H

O

O

R

2-(hydroxyimino)-N-phenylacet

amide

1H-indole-2,3-di

one

N

H

O

OR

1H-indole-2,3-di

one

NH2CH2COOH

C2H4O2 N

H

O

N NH

COOH

R

[(2Z)-2-(2-oxo-1,2-dihydro-3H-indol-3-ylidene)hydrazino]

acetic acid

SCHEME-I

SCHEME- 2

70-800

II

aniline

III II - substituted Isatins

III- isatin schiff’s bases

GENERAL PROCEDURE [4,12,15]

Synthesis of Isonitrosoacetanilide (II): Isonitrosoacetanilides have been synthesised from

substituted anilines (1) on reaction with chloral hydrate and hydroxyl amine hydrochloride.

Synthesis of Isatins (III): Substituted Isonitrosocetanilde on subsequent reaction with sulphuric

acid at 800C yielded corresponding indolin2,3-diones.

These compounds were characterised by physical data available in literature.




Synthesis of Isatin Schiff’s bases: [11]

Equimolar quantities of substituted isatins and aromatic amine were added into 10 mL of absolute

ethanol containing a few10 ml of glacial acetic acid in a 250-mL round bottom flask. The reaction

mixture was refluxed for 3 hours hour and then checked for completion by TLC. The solvent was

stripped off and the product was re-crystallized from ethanol (99.5%) and characterized by

elemental analysis, IR.

The physical and spectral data of the synthesized compounds are listed in Table 5.1 respectively.

EXPERIMENT [12]

A) Synthesis of Isonitrosoacetanilide: A mixture of chloral hydrate (7 gms) and sodium

sulphate (20 gms) is taken in a Round bottomed flask and add 150 ml of water. To this

solution then add , in order a solution of 5 grams of substituted aniline in 50 ml. of

water to which concentrated hydrochloric acid has been added to dissolve the

substituted aniline and finally a solution of 10 gms of hydroxylamine hydrochloride, in 50

cc of water. The flask is then heated in a mantle so that vigourous boiling begins in about

40- 45 minutes. After one to two minutes of vigourous boiling the reaction is complete.

During the heating period, some crystals of substituted isonnitroso acetanilide get

separate. On cooling the solution in running water the reminder crystallises, is filtered

with solution, and air dried. The yield is 4.5 to 5 grams. (80-91 % of the theoretical

amount) of a product melting at 1750C.

B) Synthesis of Isatin: 30 ml of concentrated Sulphuric acid is warmed to 500C in a 50

ml RBF fitted with an efficient mechanical stirrer, to this 4 grams of dry isonitroso

acetanilide is added at such a rate as to keep the temperature between 600

to 700 but not

higher. External cooling should be applied so that the reaction can be carried out more

rapidly .After completion of the isoniroso compound is finished , the solution is heated to

800

and kept at this temperature for about 10 minutes to complete the reaction. Then the

reaction mixture is cooled to room temperature and poured upon ten to twelve times of its

volume of crushed ice. After standing for about one and half hour, the substituted isatin is

filtered with suction, washed several times with cold water to remove the sulphuric acid ,

and then dried in air. The yield of crude substituted isatin which melts at 189-1920, is 45-

50 g, (71-78 % of the theoretical amount).




C) SYNTHESIS OF ISATIN SCHIFF’S BASES:[11]

Equi-molar quantities of substituted isatins and aromatic amine were added into 10 mL of

absolute ethanol containing a few 10 ml of glacial acetic acid in a 250-mL round bottom

flask. The reaction mixture was refluxed for 3 hours hour and then checked for completion

by TLC. The solvent was stripped off and the product was recrystallized from ethanol

(99.5%) and dried.

ANTIBACTERIAL ACTIVITY SCREENING [3,14]

Antibacterial activity:

Agar Diffusion Test:

a. Preparation of Test Solution:

Stock solution of synthetic compounds and standard drugs were prepared in DMSO. The

test compounds were used at a concentration of 1µM. Ampicillin was used as a

antibacterial standard at a conc. of 0.002 µM/0.1ml.

b. Micro-organisms used:-

1. Staphylococcous aureus( Gram Positive)

2. Escherisia Coli( Gram Negative Bacteria)

The microorganisms were maintained by sub-culturing and used at regular intervals in

nutrient agar medium.

c. Preparation of Mueller –Hinton agar medium: The ingredients of the medium are as

follows:

Table 2: Mueller –Hinton agar medium

Ingredients Concentrations (g/l)

Beef Infusion 300

Casein hydrolysate 17.5

Starch 1.5

Agar 17

Final PH

at 250C 7.4±0.3

About 3.8gm of the above medium was suspended in 100ml of distilled water in a conical flask,

corked with cotton and sterilized by autoclaving at 15 lbs pressure, 1210c for 15 min.

d. Procedure:

The petri dishes were thoroughly washed and sterilised in a hot air oven at 160 0c for one hour.

The innoculum was added to the medium and was poured into sterile petridishes for solidifying.

Well (bores) were made on the medium, Using sterile borer after solidification. 0.1ml test and




standard solutions were added to the respective bores. A control having only DMSO in the bore

was maintained in each plate as control.

The petri dishes were kept at RT for 30 min, for diffusion to take place and then incubated at

370C for 24 hrs; the zone of inhibition was observed and was measured using a scale. The anti-

bacterial of al the test compounds was carried against two micro organisms.

INNOCULUM PREPARTION AND STANDRDISATION

Preparation of the inoculum of vital importance

Quantification of the number of colony forming units (CFU) is an important feature of the

inoculums to assess the efficacy of the antimicrobial agent. Of the variety methods for

standardization of the medium of the inoculums, turbidometric method using McFarland solution

is routinely used.

TURBIDITY STANDARD FOR INNOCULUM PREPARATION

To standardise the inoculum density for a susceptibility test, a BaSo4 turbidity standard,

equivalent to a 0.5 McFarland standard or its optical equivalent, (Ex: Latex suspension) should be

used. A BaSo4 0.5 McFarland standards were prepared as follows:

A 0.5 ml aliqut of 0.048 moles/lit Barium Chloide(1.175% w/v BaCl2.

2H2O) was added to 99.5 ml of 0.18 moles /lit H2SO4 (1% w/v) with constant stirring

to maintain a suspension.

The correct density of the turbidity standard was verified by using a spectrophotometer

with a 1cm light path and matched cuvette to determine the absorbance. The absorbance

at 625nm should be 0.008 to 0.10 for the 0.5 Mc Farland standards.

The BaSO4 suspension was transferred to 5 to 6 ml aliquots into screw cap bottles of the

same size, as those used in growing or diluting the bacterial inoculum.

These tubes were tightly sealed and stored in dark at RT.

The barium sulphate solution was vigourously agitated on mechanical vortex before each

use and inspected for a uniformly turbid appearance. If large particles appear, the

standard should be replaced. Latex particle suspension should be mixed by inverting

gently, not on vortex mixture.

Growth method

This method is used for non-fastidious organisms (Ex: Enterobacteriaceae, Pseudomonos species

and staphylococci). Four morphologically similar colonies were touched with a sterile loop and

were transferred into Mueller-Hinton Broth media.




The inoculated broth were incubated with shaking at 35-370c for 24 hours, until the visible

turbidity is equal to or greater than the 0.5 Mcfarland standard. After incubation the turbidity of

the actively growing broth culture is adjusted. With the sterile broth or sterile saline to obtain

turbidity optically comparable to that of 0.5 McFarland standards. The suspension is containing

approx. 1 to 2 x108 CFU/ml of desired bacteria.

STANDARDISATION WITH MCFARLAND STANDARD

The 0.5 Mc Farland solutions provides an inoculums density of approx. 105 CFU/ml. the

comparison between 0.5 McFarland solution and culture is achieved by viewing the tubes against

a sheet of white paper on which sharp black lines /stripes were drawn. If the bacterial suspension

does not appear to be the same density as the Mcfarland 0.5 then the turbidity can be reduced by

adding sterile saline or broth or increased by adding more bacterial growth. The inoculums

adjusted using turbidity standard was used within 15 minutes of preparation.

RESULTS AND DISCUSSION

Antibacterial activity:

All the synthesised compounds were tested for in-vitro anti-bacterial activity by agar diffusion

method. The MIC’s of the compounds against two pathogenic bacteria are presented in table also

included is the activity of reference compound Ampicillin. It has been observed that all the

compounds tested showed activity against tested bacteria. All the compounds showed (more

activity, less MIC), than standard Ampicillin.

Table 3: Antibacterial activity of the compounds mic’s in µg/ml

Bacteria/drug IIIa IIIb IIIc IIId IIIe Ampicillin

E.coli 625 312.5 312.5 156.2 312.5 312.5

S.Aureus 625 625 312.5 312.5 312.5 312.5

CONCLUSION

All the compounds have been synthesized and expected compound could alone be obtained. The

compounds are further characterized by Spectral and analytical data. All the compounds have

been screened for anti-microbial activity by standard method carried out in laboratories. Of the

five synthesized compounds IIIb is found to be more potent with the activity of IIIa compared to

the standard against anti-bacterial activity.




REFERENCES

1. Vinit Raj. A review on CNS Activity of Isatin Derivatives. International Journal of

Current Pharmaceutical Research, 2012; 4(4): 1-9.

2. Alexei M, Olga B and Vivette G. Biological targets for isatin and its analogues:

Implications for therapy Biologics. Biologics: Targets & Therapy, 2007; 1(2): 151-62.

3. Bhavesh R, Kishor S, Manish M, Mayur R. “Synthesis and antimicrobial activity of some

new isatins derivatives”. Scholars Research Library, Der Pharma Chemica, 2011; 3(4):

367-72.

4. RJH Clark. Sandmeyer isonitrosoacetanilide isatin synthesis - ChemSpider , Synthetic

Page, 2010; p-464.

5. D.Sriram ,T.Bal, P.Yogeshwari. Medicinal Chemistry Research Laboratory, Pharmacy

Group, Birla Institute of Technology and science. 2010.

6. Jan Bergman, Robert Engqvist, Claes Stalhandskec and Hans Wallberg. “Studies of the

reactions between indole-2,3-diones (isatins) and 2-aminobenzylamine Tetrahedron. 2003;

59: 1033-48.

7. Gang Chen1, Ye Wang, Xiaojiang Hao, Shuzhen Mu and Qianyun Sun Simple isatin

derivatives as free radical scavengers: Synthesis, biological evaluation and structure-

activity relationship. Chemistry Central Journal, 2011; 5:37.

8. Jiro Tatsugi, Tong Zhiwei, and Yasuji Izawa “An improved preparation of isatins from

indoles” ARKIVOC 2001; 67-73.

9. Md. Rabiul Islam and Mohammad Mohsin “Synthesis of isatin, 5- chloroisatin and

their Δ2

-1,3,4 oxadiazoline derivatives for comparative cytotoxicity study on brine

shrimp” Bangladesh J Pharmacol 2007; 2: 7-12.

10. Md. Mahbubul Hoque and Md. Rabiul Islam “ Cytotoxicity study of some indophenines

and isatin derivatives” Bangladesh J Pharmacol 2008; 3: 21-26.

11. Manjusha Verma , Surendra nath pandeya, krishnanand singh, james p. stables

“Anticonvulsant activity of Schiff basesof isatin derivatives” Acta Pharm. 54 (2004) 49-

56.

12. Organic Synthesis, Coll. Vol. 1, p.327 (1941); Vol. 5, p.71 (1925).

13. Bhanupriya Brighu, Devender Pathak, Nadeem Siddiqui, M.Shamsher Alam, Wakaur

Ashan. Search for biological activities of Isatin: A short review. 2008; 7(2): 122-25.

http://www.ncbi.nlm.nih.gov/pubmed/?term=Medvedev%20A%5Bauth%5D

http://www.ncbi.nlm.nih.gov/pubmed/?term=Medvedev%20A%5Bauth%5D

http://www.ncbi.nlm.nih.gov/pubmed/?term=Glover%20V%5Bauth%5D

https://www.google.co.in/url?sa=t&rct=j&q=&esrc=s&source=web&cd=7&ved=0CGsQFjAG&url=http%3A%2F%2Fcssp.chemspider.com%2F464&ei=z4dlUfHTLoymkwXFroCYBw&usg=AFQjCNEU-sZvviEZCdZ-N_1M_RGwFxLhkg&sig2=Rt52KCMCwYlWWCaAnRQC6g




14. Cleidson Valgas, Simone Machado de Souza, Elza F A Smânia, Artur Smânia Jr.

Screening methods to determine antibacterial activity of natural products. Braz. J.

Microbiol. 2007; 38(2).

15. Joaquim F. M. da Silva, Simon J. Garden and Angelo C. Pinto. The Chemistry of Isatins: a

Review. J. Braz. Chem. Soc., 2001; 12(3): 273-324.

16. S.N.Pandeyaa, D.Srirama, G.Nathb, E De clercqc. Biological activities of isatin and its

derivatives. Acta Pharm, 2005; 55 27-46.

Documents

International Journal of Innovative Pharmaceutical ... · Isatin (1H-indole-2, 3-dione,) was first obtained by Erdman and Laurent in 1841 as a product from the oxidation of indigo