SYNTHESIS AND ANTI MICROBIAL EVALUATION OF SOME OXAZINE DERIVATIVES SEMINAR BY G.VIJENDHAR (08GD1R0007) M HIMA BINDU (08GD1R0014) K. RACHANASREE (08GD1R0023) M.PRADEEP KUMAR (08GD1R0024) M.MANISHA (08GD1R0047) CH. MAHENDAR REDDY (08GD1R0050) UNDER THE GUIDENCE OF V.KALYAN VARMA, M.Pharm. Department of Pharmaceutical Chemistry CHILKUR BALAJI COLLEGE OF PHARMACY (SPONSORED BY SRINIVASA EDUCATIONAL ACADEMY, CHITTOOR) (APPROVED BY AICTE,PCI, NEW DELHI) (Affiliated to Jawaharlal Nehru Technological University, Hyderabad, A.P).
INTRODUCTION• 1,3-Oxazines are a well-known structural modify for biologically active substances. They exhibit antifungal, antibiotic, antiviral, and antitumor properties and are also used as acetylcholinesterase (AChE) inhibitors; potential targets for Alzheimer’s drugs.• The biological activity of oxazine derivatives was reported as early as 1937 (Novelli& Adams, 1937).Later, several workers reported the fungistatic and bacteriostatic-including tuberculostatic-activity of these compounds (Urbanski& Slopek, 1951; Lane, 1953; Kay &Lane, 1953; Shono&Takahashi, 1954).
OBJECTIVES• 1,3 oxazines derivatives are an interesting class of heterocyclic compounds being studied in recent years and are reported to posses a wide spectrum of biological activities such as antibacterial, antifungal, anti-inflammatory, anti-helminthec, antitumor and anti HIV activities.• Many heterocyclics have shown antifungal activity due to N- C=O-N group and this group is also present in the structure of 1,3- Oxazine nucleus.• The above observations stimulated us to synthasise more number of useful therapeutic derivatives having Oxazine nucleus .• 1.synthesis and evaluation of Oxazine derivatives.• 2.evaluation of the antimicrobial activities like antibacterial and antifungal activity of some Oxazine derivatives.
METHODOLOGY A mixture of 30ml of 10% sodium hydroxide , 50ml of ethanol and 0.01mol of cyclohexanone placed in a 250 ml beaker ,which was immersed in ice bath as well as equipped with a mechanical stirrer ,The stirrer was started 0.02ml of benzaldehyde was added to the mixture and stirring continued After 2 hours, the stirrer was removed and the reaction mixture was kept in an ice chest over night. The product was filtered, washed with ice cold water , followed by ice cold ethanol , dried and recrystallized from DMF
METHODOLOGY-2 • A mixture of 0.01mol 2,6 –dibenzylidene cyclohexanone of 0.015mol of urea and 0.01ml of potassium hydroxide dissolved in 10ml of water was reflexed in ethanol for 10 hours. Later ethanol was removed under reduced pressure and the residue was treated with ice cold water .The precipitate thus obtained was filtered washed with water,dried and recrystallized from ethanol.
ANTIMICROBIAL ACTIVITY• Microorganisms used: gram positive bacteria viz:Staphylo cocus aureus and Bacillus subtilus and S.N Ingredients Weight(g) O gram negative bacteria viz: Escherichia coli• Preparation Of Inoculum: 1. Beef extract 4.0 The suspensions of all the organisms were prepared as per Mac- 2. Peptone 5.0 Farland Nephelometer standard(Baily and Scott 1990). A 24hr old culture was 3. Agar 20.0 used for the preparation of bacterial suspension. Suspensions of organisms 4. Distilled water q.s 1000ml were made in sterile isotonic solution of sodium chloride (0.9 %W/V) and the 5. Ph 5.4 turbidity was adjusted.• udomonus aeruginosa species.
PROCEDURE:• Inoculum was added to 30ml of sterile nutrient agar medium and was poured into sterile petridishes for solidifying.• 0.1ml of test solution was added to the petridishes, 0.1ml of the ampicillin at a concentration of 100μg/ml was taken as standard reference.• The petridishes were kept in the refrigerator at 4C for 15 minutes for diffusion to take place. After diffusion , the petridishes were incubated at 37c for 24hr and zone of inhibition were observed and measured using a scale.• Antibacterial activity of all the compounds was carried out against all four microorganisms. The same media was used for both sub culturing and for estimating antibacterial activity.
ANTIFUNGAL ACTIVITYFUNGI USED: S.no Ingrediants Weight in g• Standard cultures of Candida albicans and Aspergillus niger. 1. Dextrose 40NUTRIENT MEDIUM• Dextrose,Peptone,Agar 2. Peptone 10 Distilled water, These ingredients 3. Agar 20 were accurately weighed and dissolved water.The medium Is 4. Distilled water q.s 1000ml prepared was sterilized by autoclaving at 121C for 15 minutes
• WORKING PROCEDURE:• An inoculum was prepared by suspending a single isolated colony in above 5ml of normal saline. Later one drop of tween 20 was added for filamentous fungi and the mouid was broken by shaking.• A sterile cotton swab was moistened in the inoculum suspension and excess of moisture was removed by rolling the cotton swab on the inside of the tube, above fluid level 30ml of sterile hot sabauads agar medium was poured in each plate and allowed to harden on a level surface of sabourauds agar medium plate was streaked with the help of moistened cotton swab in all the direction ions.• The surface of plate was dried out 35°C. Later 5 bores per plate were made using sterile cork bores.• The operation was carried out in asceptic condition and 0.1ml test solution was added to the respective bore and 0.1ml Amphotercin was taken as standard reference. A control having only DMSO was maintained in each plate. The plates are incubated at 35°C for 48hrs. Later the values of zones of inhibition were recorded
RESULTS Sl. NO STRUCTURE ZONE OF INHIBITION IN MM S.a B.s E.c P.a 1. 2–imino-8–benzylidene-4– phenyl5,6,7–trihydroxy-4H,7H– 21 18 15 17 (3,1)benzoxazineCONTROL DMSO - - CONTROL DMSO STD Ampicillin 19 17 STD Ampicillin
6. DISCUSSION• The structure of new compounds prepared during the present investigation have been authentically established by their UV,IR spectral studies.In the following the spectral studies of some selected compounds have been detailed.• The compound 2,6-dibenzylidine-cyclohexanone[I(1)]has been prepared by condensing 1mol of cyclohexanone with 2moles of benzaldehyde the for I(1)as been indicated by its uv spectrum.the starting material cyclohexanone exhibited λmax at 287nm.the compoundsI(1) exhibited λmax at 328nm.this clearly indicates that the bathochromic shift may be attributed because of =CH-Ar chromophore.• The formation of I(1)as been indicated by its IR spectrum the starting material exhibited γmax at 1661cm-1 due to C=O group.the compoundsI(1) exhibited γmax at 1607cm-1 due to C=Ogroup.the appearance of a characteristic band at C=O is mainly due to α,β,and α-1 β-1.this clearly indicates the formation of I(1).
ANTIBACTERIAL ACTIVITY: STRUCTURE ACTIVITYRELATIONSHIPS• The zone of inhibition shown by the compound 2-imino-8-benzylidene- 4-phenyl 5,6,7-trihydro-4H-7H-(3,1)benzoOxazine[II(1)] against Staphylococcus aureus is 21nm.• Among all the compounds, two derivatives viz.2-imino-8(4- methyl benzylidene)-4[methyl phenyl]-5,6,7-trihydro-4H,7H- (3,1)benzoxazine[II(2)] and 2-imino-8-(4-chioro benzylidene)-4-[4- chloro]-5,6,7-trihydro-4H,7H-(3,1)benzoxazine II(4) has shown highest zone of inhibition indicating that introduction of groups such as –CH3,-Cl in II (1) enhances the activity against Staphylococcus aureus.
ANTIFUNGAL ACTIVITY: STRUCTURE ACTIVITY RELATIONSHIPS• The standard reference drug was Amphotericin B• The zone of inhibition shown by the compound 2-imino-8- benzylidene-4-phenyl-5,6,7-trihydro-4H,7H-(3,1)benzoxazine. II(1) aganist Candida albicans is 13mm.• Among all the derivatives the compound 2-imino-8-(4- chiorobenzylidene)-4-(4-chlorophenyl)-5,6,7-trihydro-4H,7H- (3,1)benzoxazine II(4) had shown highes zone of inhibition indicating that introduction of chloro substituent in II(1) enhances the activity.
CONCLUSSION & SUMMARY• The aim of our present study was to synthesize,characterize and to evaluate antibacterial and antifungal activity of some oxazines was completed successfully.• The intermediate compounds, 2,6-diarylidene-cyclohexanone derivatives I(1-4) have been prepared by condensation of one mole of cyclohexanone with two moles of aromatic aldehyde.• The final compounds,2-imino-8-arylidene-4-aryl-5,6,7-trihydro-4H,7H-(3,1)benzoxazine derivatives II(1-4) have been prepared by cyclo condensation of I(1-4) with urea.• The structure of some synthesized compounds have been authentically established by their,UV,IR spectral studies.• The antibacterial activity of II (1-4)has been evaluated. The anti fungal activity of II (1-4) has been evaluated.