Studies on Antimicrobial Activity of Formazans containing Coumarinyl Moiety

Abstract

Compounds bearing Formazan moiety are endowed with a variety of pharmacological  activities such as insecticidal(1), bactericidal(2), antiviral(3), antiinflamatory(4); Antiparkinsonian(5) acivity. Again the Coumarinyl  moiety have been reported to have excellent physiological activity few of which are anthelmintic, antiallergic, antiartheritic, anticancerous, antimalerial antinaphylactic,antiproliferative, and more large number of activities. Looking over to these properties it was contemplated to synthesis some new Formazans having Coumarinyl moiety which may enhance the biological activity with least side effect. The structures have been characterized by the elemental analysis and the spectral data. The compounds were screened for their antimicrobial activity using different strains of Bacteria’s and Fungi.

[1] Introduction :

[2] Experiment : al

All the melting points are taken in an open capillary tube and all uncorrected I.R spectra (KBr) were recorded on Perkin Elmer spectrometer and 1H NMR spectrometer at 300MHz. the purity of compound was checked by TLC using Silica gel-G.

(I) Preparation of the Schiff base:-

(A) Preparation of 4-methyl-7-hydrazino-carbonylmethoxycoumarin²⁷

The 7-Hydroxy-4-methyl coumarin was esterified stirring for 12 hrs. with ethylchloroacetate in acetone and refluxed. The ester formed was then taken in rectified spirit to which hydrazine hydrate was added and further refluxed for 8 Hrs., then after it is cooled and poured in ice to give crystalline product with (m.p. -115ºC).yield 75%

(Found C–58.06%, H-4.83% and N-11.29%; Calculated C–58.10%, H-4.78% and N-11.33%)  For C₁₂H₁₂N_2O4

(B) Preparation of Schiff’s Base
[4-methyl-7-(substituted benzylhydrazinocarbonylmethoxy) coumarin]²⁷

A mixture of hydrazine (0.01M, 2.48 gm) was dissolved in alcohol then p-Anisaldehyde
(0.01 M, 1.36 gm) was added to it, refluxed for four hours. The reaction mixture was cooled and the product was isolated as well as crystallized in DMF to give shinning white crystal (m.p 248ºC) Yield 75%, (Found  C–65.52%, H-4.9%  and N-7.62%; Calculated  C–65.57% , H-4.9%  and N-7.65%) For C₂₀H₁₈N₂O₅.

Similarly other Schiff bases were prepared.The physical constants are recorded in Table no-1 :

TABLE NO. 1 :

[A]  PHYSICAL CONSTANT OF 4-METHYL-7-[(4’-METHOXYBENZYLHYDRAZINOCARBONYLMETHOXY] COUMARIN

SPECTRAL DATA
[B]  N.M.R SPECTRAL DATA.

[C]  IR  SPECTRAL STUDY ( SHIMADZU-2245)

[D] MASS SPECTRA

II–PREPARATION OF: -4-METHYL-7-[α-(p-CHLOROPHENYLAZO)p-METHOXY BENZALHYDRAZINO CARBONYLMETHOXY]COUMARIN.

p-chloroaniline (2.5gm0.02M) in glacial acetic acid (2 ml) and HCl (1.5ml) was diazotized  with NaNO₂ (2gm in 2ml water) in cold 0-5,^oC. The resultant diazonium chloride solution was added to the schiff’s base (3.66gms 0.01M) in pyridine at low temperature (0-5^oC) and the reaction mixture was stirred gently and left overnight at ambient temperature. Thereafter it was poured into cold water. The product was isolated and crystallized from chloroform m.p. 130^oC decomposes yield 40% ( C-61.82%; H-4.12 %; N-11.10% and Calculated  C –61.84% , H-4.16 %  and N-11.10%) for  C₂₆H₂₁N₄O₅Cl
The  formazans were characterized by elemental analysis as well as supported by its various spectroscopic data as shown in Table-II.

TABLE No. – 2
[A]   PHYSICAL CONSTANTS

4-METHYL-7-[-(P-CHLOROPHENYLAZO)SUBSTITUTEDBENZALHYDRAZINO CARBONYLMETHOXY]COUMARIN.

[B]     IR SPECTRAL STUDY ( SHIMADZU-2245)

[C] MASS SPECTRA

ANTIMICROBIALACTIVITY4-METHYL-7-[α-(p-CHLOROPHENYLAZO)p METHOXYBENZALHYDRAZINO CARBONYLMETHOXY]COUMARIN.

The nutrient agar broth & sterilized sabouraud’s agar  prepared by the  usual method, was inocculated aseptically with  0.5ml of 24 hour old subculture of  various bacteria in separate conical flasks at 40 -50 ºC and mixed well by gentle shaking . About 25 ml of agar broth  was poured and evenly spread over sterilized Petri dish (13 cm in diameter) and allowed to set for 2 hours. The cups (10mm in diameter) were formed by help of the cork borer in agar medium and inoculated with various bacteria and fungi separately the cups were filled with 0.05ml (1mg/ml) of all the test samples of azetidinones in DMF solution the plates were incubated at 37ºC for 24 hours and the control was also maintained with 0.05 ml of DMF in same way the   Zones of inhibition were measured in mm. and recorded in table no-3

TABLE No. – 3

ANTI MICROBIAL ACTIVITY OF 4-METHYL-7-[α-(p-CHLOROPHENYLAZO)p-METHOXY BENZALHYDRAZINO CARBONYLMETHOXY]COUMARIN.

[4] Result and Discussion : s

The compounds were screened for both gram positive and gram negative bacterias and  Fungus by the Cup-plate method (29,30).
The compound no-2,4 and 6 were moderately active against S.Typhosa as compared with the standard drugs, for E.COLI compound no. 10 are moderately active and compound no- 3 and 4
shows similar activity as compared to standard drug. For gram positive bacteria S.Citrus bacteria compound 4showed good activity. And for B.Mageterium compound 7 showed moderate activity as compared to standard drug.

In case of antifungal activity against A.Niger almost all the compounds showed low activity other than compound no-1,4 and 18 showed  moderate activity report comparing with the standard drug.

Acknowledgement

The authors are grateful to Department of Chemistry Saurashtra University Rajkot for providing total research facility and CDRI Lucknow for the various spectral analysis. Special thanks to the Trustees And Principals Of Institutes Shri M.D.SCIENCE COLLEGE PORBANDAR and  Shri M.M.SCIENCE COLLEGE MORBI

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