International Journal of Research In Pharmaceutical ...formation. The thiazines are of great importance because they act as precursor for the synthesis of Cepha-losporins...
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Razia sultana et al., (2018) Int. J. Res. Pharm. Chem & Analy., 1(1), 25-35
International Journal of Research In Pharmaceutical Chemistry and Analysis
Synthesize, characterize and evaluation of anti-bacterial and anti-fungal activity of thiazines
Razia Sultana*1, Arsalan Sarmad2, B. Syed Salman3 1Department of Pharmaceutical chemistry, 2Department of Pharmacology, 3Department of Pharmaceutics, Global college of pharmacy, Moinabad, Hyderabad, India. ABSTRACT
1,3–Thiazineshavebeenappliedasusefulstartingmaterialsinthestereoselectivesynthesisofcompoundsofpharmacologicalinterestandtheyhaveservedaschiralligantsandauxiliariesinenantioselectivetrans-formation.ThethiazinesareofgreatimportancebecausetheyactasprecursorforthesynthesisofCepha-losporins (3,6-dihydro-2H-,1,3-thiazine) and then converted to the thiazine derivatives (an heterocycliccompounds).Therefore, itwasthoughttocombinechalconesmoietytothiazinederivativetogether inamolecularframeworktoseethebiologicalactivities,additiveeffortoftheseringstowardsbiologicalactiv-itieslikeAnti-bacterialandAnti-fungalactivities.
Thiazines a heterocyclic compound having fourcarbon atoms and one nitrogen atom and sulphuratom at varied positions in the sixmembered ringexist as 1,2- thiazine,1,3-thiazine and 1,4- thiazineand subsequently their derivatives having N-C-Slinkage have been used as anti-tubercular, anti-bacterial, anti-microbial, anti-tumor, insecticidal,fungicidal, herbicidal agents, transquilizers andvarious dyes etc[5-7], anti-parkinsonian[8], calciumchannel modulators, Anti- pyretic, anti-mycobacterialagents,CannabinoidreceptoragonistsandAnti-Oxidantagents.
Theheterocycliccompoundswhichcontainnitrogenandsulphurpossesanenormoussignificanceinthefieldofmedicinalchemistry.Manyresearchershavesynthesized different thiazines derivatives thatexhibit various biological activities such as anti-tubercular,anti-fungal,anti-bacterial,analgesic,anti-inflammatory etc. Some thiazine derivatives in thedevelopment phase due to their versatility of thethiazine skeleton, it’s chemical simplicity andaccessibility. Many compounds of thiazines wereknownasphenothiazines.Phenothiazinesareusedasvermifugeforlivestockandalsoasaninsecticide.[11]
The ability to treat bacterial infections whichchemotherapeuticagentsrepresentsoneofthemostimportant medical achievement of the lastmillennium.Unfortunately theemergenceofmicro-organismsresistanttoanti-bacterialagentshasbeencontinuedtothepresentday.Itwaswellknownthatbacteriaproducebeta–lactamasesandtheseenzymeshad the natural role of destroying beta–lactamantibiotics. The latest twist to this increasinglyalarming situation has been the recent isolation ofmethicillin resistant strains of Staphylococcusaureus.
The fungal diseases of humans are mainly dividedintothreegroups.Thefirsttypeisdermatophytosescaused by epidermaphyton species, microsporiumspeciesandtycophytonspeciesconsistofcontagioussuperficial skin infections that are limited to theepidermal region. The second type is CandidiasiscausedbyCandida species canaffect the skin,nailsandmucousmembranes and occasionally becomessystemic.Thethirdtypeissubcutaneous,pulmonary,lymphatic and systemic mycoses caused by
Aspergillus species and Candida species invade theskin, lungs, lymphatic tissue and various organs ofsusceptible hosts who have accidentally been incontact with the fungal environment. There areincreasingdetectionofsystemicmycosesinpatientssufferingfromdelilitatingdiseasessuchasneoplasiasinpersonsonlongteamparenteralnutrition.
Additionally, conditions that depress cell mediatedimmunity, such as the latter half of pregnancy,excessive use of corticosteroids andimmunosuppressive and defects in thymus glandtissue, results in marked enhancement ofsusceptibility to serious fungal diseases. Manyremedies have been used against fungal infectionsbut the ideal Anti-fungal agents has not yet beenfound.
The 1,3-thiazine nucleus is active core ofCephalosporinwhichareamongthewidelyusebeta-lactam anti- biotics. A large group of dyes hasphenothiazine structure, including methylene bluethiazine are used for dyes, transquilizers. Thiazinecanhelp toreducesomeof thatextrawaterweightyoumaybeholdingon to in stomach.Thiazine isafairlybasicdiureticsupplement,itreduceswaterandincreasevascularity,soitisuseasanabolicagentinmedicine.[12]
Themechanisminvolvedinthepreparationofchal-conesisbyClaisenSchmidtreaction.Thereactionbe-tweenanaldehyde(R-CHO)orketone(R=O)andanaromatic carbonyl compound lacking an Alpha-hy-drogeniscalledtheClaisenSchmidtreaction.Amix-tureof30mlof10%sodiumhydroxide,50mlofeth-anoland0.01molofcyclohexanoneplacedina250mlbeaker,whichwasimmerseinicebathaswellasequippedwithamechanicalstirrer.Thestirrerwasstarted,0.02molofbenzaldehydewasaddedtothemixture and stirring continued. After 2 hours, thestirrerwas removed and the reactionmixturewaskept in an ice cold overnight. The product was fil-tered, washedwith ice cold water, followed by icecold ethanol, dried and recrystallised fromDMF orethanol.
Figure 1: Preparation of 2, 6-di Benzelidene Cyclohex-
anone
Recrystallizationforsynthesizedchalcone
Theproductorsample is taken inaconical flasktothisaddquantitysufficientofethanoluntilitgetsdis-solveonheating,filtertheproducttoremovetheim-puritiesinthepresenceofheating,andcoolthesolu-tion, yellow colour needle shape crystals are ob-tained.[38]
Preparation of 2- Imino-8-benzylidene-4-phenyl5,6-dihydro-4H,7H-(3,1)benzothiazine
Figure 2: Preparation of 2- Imino-8-benzylidene-4-
phenyl 5,6-dihydro- 4H, 7H- (3,1) benzothiazine
Recrystallizationforsynthesizedthiazine
Theproductorsample is taken inaconical flasktothisaddquantitysufficientofethanoluntilitgetsdis-solveonheating,filtertheproducttoremovetheim-puritiesinthepresenceofheating,andcoolthesolu-tion yellow colour needle shape crystals are ob-tained.[38]
Anti–bacterialActivity
Methodfollowed-Agardiffusionmethod.
WorkingProcedure-Stocksolutionsofthesynthe-sizedcompoundsandstandarddrugsusedwerepre-pareddimethylsulfoxide taken in the concentrationofµg/0.1ml.
Cyclohexanone Benzaldehyde 2,6-dibenzelidene
cyclohexanone
Razia sultana et al., (2018) Int. J. Res. Pharm. Chem & Analy., 1(1), 25-35
Microorganisms used-Standard cultures of grampositivebacteriaStaphylococcusaureusandBacillussubtilusandgramnegativebacteriaEscherichiacoliand Pseudomonas aeruginosa species were taken.Themicroorganismswereidentifiedbyvariousstain-ing techniques and bio-chemical reactions. Themi-croorganismsweremaintainedbysub-culturingandusedatregularintervalsinnutrientagarmedium.
Preparationofinoculum-Thesuspensionofalltheorganisms were prepared as per Mac-FarlandNephlometerstandard(BailyandScott1990).A24houroldculturewasusedforthepreparationofbac-terial suspension. Suspensions of organisms weremade insterile isotonicsolutionofsodiumchloride(0.9%w/v)andtheturbiditywasadjusted.
Preparationofassaymedium
Beefextract4.0,Peptone5.0,Agar20.0,Distilledwa-ter1000ml,mentionedquantitiesofdifferentingre-dientswereaccuratelyweighedanddissolvedinap-propriate amount of distilled water. Media so pre-paredwas sterilizedbyautoclavingat120°c for15minutes.ThepHshouldbemaintainedat5.4.
Procedure: The Petri dishes were thoroughlywashed and sterilized in hot air oven at 160°c for1hour.Inoculumwasaddedto30mlofsterilenutri-entagarmediumandwaspouredintopetridishesforsolidifying. Boresweremade on themediumusingsterilebore.0.1mloftestsolutionwasaddedtotherespectivebore,0.1mloftheampicillinataconcen-trationof100µg/0.1mlwastakenasstandardrefer-ence. A control having only DMSO in the cup wasmaintainedineachplate.ThePetridisheswerekeptintherefrigeratorat4°cfor15minutesfordiffusiontotakeplace.Afterdiffusion,thepetridisheswerein-cubatedat37°c for24hoursandzoneof inhibitionwereobservedandmeasuredusingascale.Antibac-terial Activity of the compound was carried outagainstallfourmicroorganisms.Thesamemediawasusedforsub-culturingandforestimatinganti-bacte-rialactivity.
Anti-fungalActivity
Workingprocedure-Stock solutionsof the synthe-sizedthiazinesandstandarddrugwerepreparedinDMSOintheconcentrationof100µg/0.1ml.
Fungi used-Standard cultures of Candida albicansand Aspergillus niger were taken. The fungi weremaintainedbysubculturingandusedatregularinter-vals.
Sabourd’sagarmedium:40mgofSucrose,10mgofPeptone, 20mg of Agar, 1000ml ofDistilledwater,Thismediumwas found forboth sub-culturingandalsoforestimatingtheantifungalactivity.ThepHofthemediumplaysanimportantroleforthegrowthoffungi.Acidicmediumfavoursthegrowthbutexcessofacidmaynotcomeagartosolidify.HencethepHof
Preparation of assay medium: The mentionedquantitiesofadifferentingredientswereaccuratelyweighed anddissolved inwater. Thepreparedme-dium was sterilized in autoclave at 1210c for 15minutes.
Workingprocedure:Aninoculumwaspreparedbysuspendingasingleisolatedcolonyinabout5mlofnormal saline This is mixed slowly to achieve asmoothsuspension.Lateronedropoftween20wasaddedforfilamentousfungiandthemouldwasbro-kenbyshaking.Asterilecottonswabwasmoistenedintheinoculumssuspensionandexcessofmoisturewasremovedbyrollingthecottonswabontheinsideof the tube, above fluid level 30 ml of sterile hotSabouraud’sagarmediumwaspouredineachplateandallowtohardenonalevelsurface.ThesurfaceofSabouraud’s agarmedium plate was streakedwiththehelpofmoistenedcottonswabinallthedirectionions.ThesurfaceofSabouraud’sagarplatewasdriedout 350C. Later 5 bores per plateweremadeusingsterilecorkborer.Theaboveoperationwascarriedoutinasepticconditionand0.1mltestsolutionwasaddedtotherespectiveboreand0.1mlAmphotercinwas taken as standard reference. A control havingonlyDMSOwasmaintainedineachplate.Theplatesareincubatedat350cfor48hours.Laterthevaluesofzonesofinhibitionwererecorded.[41-50][13]
TheantibacterialactivityofnewlysynthesizedChal-cone or 2,6-dibenzylidene cyclohexanone and thia-zine derivativeswere evaluated against gram posi-tivebacteriathatisStaphylococcusaureusandBacil-lussubtilisandgram-negativebacteriathatisEscher-chiacoliandPseudomonasaeruginosa.ThestandarddrugusedasAmpicillin.
The zone of inhibition exhibited by the compoundagainststaphylococcusaureus is15mm.AmongallthecompoundsonederivativethatishadshowntheactivityagainstStaphylococcusaureusindicatingtheintroductionofnitrogenandsulphurinenhancestheactivity.
The zones of inhibition shown by the compoundagainstEscherchiacoliandPseudomonasaeruginosa
are12and13mmrespectively.Amongallthecom-pounds exhibited maximum zones of inhibitionagainstEscherchiacoliandPseudomonasaeruginosaby15and17mmrespectively, indicatingthe intro-ductionofnitrogenandsulphurinenhancestheac-tivityagainstgramnegativebacteria.
The synthesized compound 2-imino-8-benzylidene-4-phenyl-5,6-dihydro-4H,7H-(3,1)benzothiazine
Figure 10: fragmentation pattern of 2-imino-8-benzylidene-4-phenyl-5,6-dihydro-4H,7H-(3,1) benzothiazine
Figure 11: Preparation of 2-imino-4-benzylidene-4-phenyl-5,6-dihydro-4H,7H-(3,1) benzothiazinefrom cyclo-
hexanone
Razia sultana et al., (2018) Int. J. Res. Pharm. Chem & Analy., 1(1), 25-35
Thezoneofinhibitionshownbythecompound2,6-dibenzylidene cyclohexanone against Candida albi-cans is 13mm. The compound 2-imino-8-benzyli-dene-4-phenyl-5,6-dihydro-4H,7H-(3,1) benzothia-zineis15mmandhadshownthehighestzoneofin-hibition indicating that the introductionofnitrogenandsulphurenhancestheactivity.
Thezoneof inhibitionshownby thecompoundAs-pergillusniger is 15mm.The compound2-imino-8-benzylidene-4-phenyl-5,6-dihydro-4H,7H(3,1) ben-zothiazineis18mmandshownthemaximumactivityagainstAspergillusnigerindicatingtheintroductionofnitrogenandsulphurindicatingthatintroductionofnitrogenandsulphurenhancestheactivity.
The thiazine had shown the antifungal activityagainst Candida albicans and Aspergillus niger butnoneofthecompoundshadgreaterpotencythanthestandardreference,AmphotericinB
DISCUSSION
Thecompound2,6-dibenzylidenecyclohexanonehasbeenpreparedbycondensingonemoleofcyclohexa-nonewithtwomolesofbenzaldehyde.Theformationof 2,6-dibenzylidene cyclohexanone has been indi-catedbyitsUVspectrum.Thestartingmaterialcyclo-hexanone exhibited λmax at 287 nm. The compound2,6-dibenzylidene cyclohexanone exhibited λmax at328.5nm.Thisclearlyindicatethatthebathochromicshiftmaybeattributedbecauseof=CH-ARchromo-phore.
Thecompound2,6-dibenzylidenecyclohexanonehasbeenindicatedbyitsinfraredspectraspectrum.Thestartingmaterial exhibit γmaxat1680 cm-1due toC=Ogroup.Theappearanceof2,6-dibenzylidenecy-clohexanoneexhibitedγmaxat1660cm-1duetoC=Ogroup. The appearance of a characteristic bond atC=Oismainlyduetoα,β,andα’,β’unsaturation.Thisclearly indicate the formation of 2,6-dibenzylidenecyclohexanone.
The formation of 2,6-dibenzylidene cyclohexanonehasalsobeenindicatedbyitsnuclearmagneticreso-nancespectrum.Thepresenceofsignalsatδ7.2-7.5(10H,aromaticprotons,δ7.6(1H,protonofbenzyli-denegroup),δ7.8(1H,protonofbenzylidenegroup),δ3.5(1H,protonofC-3),δ2.9-3.1(2H,protonsofC-5)andδ1.6-1.9(2H,protonsofC-4)clearlyindicatetheformationof2,6-dibenzylidenecyclohexanone.
The compound 2- imino-8-benzylidene-4-phenyl-5,6-dihydro-4H,7H-(3,1)benzothiazinehasbeenpre-paredbycyclocondensationof2,6-dibenzylidenecy-clohexanonewiththioureainthepresenceofpotas-siumhydroxide.Theformationof2-imino-8-benzyl-idene-4-phenyl-5,6-dihydro-4H,7H-(3,1) benzothia-zinehasbeenindicatedbyitsUVspectrum.Thecom-pound 2,6-dibenzylidene cyclohexanone exhibitedλmaxat328.5nm.Thecompound2-imino-8-benzyli-dene-4-phenyl-5,6-dihydro-4H,7H-(3,1) benzothia-zineexhibitedλmax284nm.Thisclearlyindicatethatthehypsochromicshiftmaybeattributedbecauseofcyclocondensation.
The formation of 2-imino-8-benzylidene-4-phenyl-5,6-dihydro-4H,7H-(3,1)benzothiazinehasbeen in-dicatedbyitsinfraredspectrum.Thecompound2,6-dibenzylidenecyclohexanoneexhibitedacharacter-isticbondat1660cm-1(C=O).Theabsenceof1660cm-1 and presence of 3411 (=NH) and 3203 (=NH)clearly indicate the formationof 2-imino-8-benzyli-dene-4-phenyl-5,6-dihydro-4H,7H-(3,1) benzothia-zine.Theformationof2-imino-8-benzylidene-4-phe-nyl-5,6-dihydro-4H,7H-(3,1)benzothiazine has alsobeen indicated by its nuclear magnetic resonancespectrum.Thepresenceof signalsatδ7.3-7.5 (10H,aromaticprotonofbenzylidenegroup),δ6.6(1H,pro-tonofiminogroup),δ6.8(1H,protonof=NHgroup),δ5.0(1H,protonofS-CHgroup),δ3.3(1H,protonofC-7),δ1.8-2.8(2H,protonsofC-5),δ1.4-1.5(2H,pro-tonsofC-6)clerlyshowtheformationof2-imino-8-benzylidene-4-phenyl-5,6-dihydro-4H,7H-(3,1) ben-zothiazine.
The formation of 2-imino-8-benzylidene-4phenyl-5,6-dihydro-4H,7H-(3,1)benzothiazinehasalsobeenconfirmedby itsmassspectrum.Themolecular ionpeak of 2-imino-8-benzylidene-4-phenyl-5,6-dihy-dro-4H,7H-(3,1) benzothiazine has been at 331,whichisingoodagreementwiththecalculatedmo-lecularweightof thecompound.Theprobable frag-mentation pattern of 2-imino-8-benzylidene-4-phe-nyl-5,6-dihydro-4H,7H-(3,1)benzothiazinehasbeendepictedasshownbelow.
CONCLUSION
ThemethodfollowedforthesynthesisofChalcone/2,6-dibezylidene cyclohexanone is by ClaisenSchmidtreactionandtheyieldwasfoundtobe2.67grams.ThemethodfollowedforthesynthesisofThi-azine/2-Imino8-benzylidene-4-phenyl-5,6-dihydro-4H,7H-(3,1) benzothiazine is by Michael additionmechanismandtheyieldwasfoundtobe3.43grams.TheAnti-microbialactivitysuchasAnti-bacterialac-tivity and Anti- fungal activity of Chalcone/ 2,6-dibenzylidene cyclohexanone was evaluated. TheAnti-microbialactivitysuchasAnti-bacterialactivityandAnti- fungal activity of Thiazine or 2- Imino-8-benzylidene-4-phenyl-5,6-dihydro-4H,7H-(3,1) ben-zothiazinewasevaluated.
Razia sultana et al., (2018) Int. J. Res. Pharm. Chem & Analy., 1(1), 25-35
12. Simerpreet and S. D Cannoo, Pharmacophore .4(3),70-88(2013).13.
13. IbadurRSiddiquiandPravinKsingh,reportedNovel one – pot synthesis of 1,3- thiazinesand1,3-ditheinsundermicro-waveirradiation.In-dian Journal of Chemistry, section- B, volume-46B,March–2007,pagenumber-499-504.
14. A.K.Pandey,C.R.Singhetal,reportedSynthesisand fungitoxicity of some S –triazolo [3,4-b][1,3]-thiazine–4–ones.IndianJournalofheter-ocyclic chemistry, volume – 17, October – De-cember,2007,pagenumber–193-194.
20. Beena KP, Sooraj TV et al, reported Synthesis,characterizationandevaluationofsome1,3-thi-azine derivatives as possible Anti –microbialagents.AM.J.PharmTechRes2013:3(4).
21. MukhtarHussainKhan,reportedSynthesisandantimicrobialactivityof5-amino–2,7–diaryl–6–cyano–3–isonicotianamidothiazolo[4,5-b]–2,3,4,7–tetrahydropyridines,2,7-diaryl–6-cyano–3–isonicotianamidothiazolo[4,5–b]–2,3,4,5,6,7–hexahydropyridi–5–ones,2,7–di-aryl – [4,5 – d ] [1,3 ] Thiazines and 2,6 – di-aryl[4,5-d][1,3]thiazinesand2,6-diaryl–3–isonicotianamidothiazolo[4,5–c]pyrazolines.IndianJournalofChemistryVolume46B,Janu-ary2007,pagenumber148-153.
22. Shamal K. Dolfode, M. P.Wadekar and SureshRewatkar,reportedSynthesisof1,3–Thiazinesfrom Aurone . Oriental Journal of Chemistry,2011, volume 27, no (3), page number 1265-1267.
25. SrikanthJupudi, Sandeep Talari et al, reportedScreeningofin–vitroanti-inflammatoryactiv-ityofsomenewlysynthesized1,3–thiazinede-rivatives . International Journal of Research inPharmacyandChemistry ( IJRPC )2013,3 (2)ISSN:2231–2781.
28. FlaviePeudru,Jean–FrancoisLohier,etal,re-portedSynthesisof1,3–thiazinesbyathree–component reaction and their transformationintobeta–lactam–condensed1,3–Thiazinesand 1,4 – thiazepine derivatives . Phosphorus,sulphur and silicon 2015, volume – 191, No –2,220–229.
29. GirlyTony,MeenaChandran,etal,reportedMo-leculardockingstudies:1,3–thiazineand1,3–oxazine derivatives . Journal of Pharmacy re-search2014,8(2),136–138.
30. Sindhu T . J. Meena Chandran et al, re-ported,Comparative Anti tubercular activity ofsulfa drug substituted 1,4 -thiazines and 1,3 –Thiazines.InternationalJournalofPharmaceu-ticalresearchscholars(IJPRS)volume-3, I-1,2014.
31. Pravin B. Raghuwanshi and A. G . Doshi, re-ported Synthesis and antimicrobial activity of1,3–Thiazines.AsianJournalofChemistry,Vol-ume–6,number.2(1994),291–294
32. MotumuMuraoka,MasatakaYokoyamaetal,re-ported Synthesis of 1,3 – thiazine derivativesfrom2– imino– cyclopentanedithiocarboxylicacid.BulletinofthechemistrysocietyofJapan,volume–43,number–7,2134–2137(1970).
33. Ravindar B, Srinivasa Murthy M, Afzal BashaShaik,reportedDesign,FacileSynthesis,andbi-ologicalevaluationofnovel1,3–thiazinederiv-ativesaspotentialanticonvulsantagents.
34. Lait,S.M.,Rankic,D.A.,Keay,B.A.1,3-Aminoal-cohols and theirderivatives in asymmetricor-ganicsynthesis.chem.Rev.2007,107,767-796.
39. Text book of Pharmaceutical Analysis, 4th edi-tion,Dr. S. Ravi Shankar, page number 6.1-6.9and8.1-8.5.
40. TextbookofInstrumentalmethodsofChemicalAnalysis, Gurdeep R. Chatwal and Sham K.Anand,pagenumber2.112-2.131and2.51-2.55.
41. Koketsu, M; K wong, CD, Tanaka, K, andtakenaka,Yetal.2002,Synthesisof1,3-thiazinederivatives and their evaluation as potentialAnti-microbialagent,EurJPharmaScienceVol-ume15pagenumber307-310.
43. WernerSeebacher,FerdinandBelaj, et al,New1,3-thiazoles and 1,3-thiazine from 1-thiocar-bamoyl pyrazoles, monotsheftefiirchemie 134,1623-1628(2003).
44. Guanyinheng Qui, Yi Hu, et al, Synthesis of 4-methylene-4H-benzo[d][1,3] Thiazine Via aTandem reaction of 1-(2-Alkynyl phenyl) keyooximeswithLawesson’s reagent,Royal societyofchemistry2011.
45. ArtKruithof,Marten.L.Ploeger et al,Multicom-ponent Synthesis of 3,6- dihydro-2H-1,3-Thia-zine-2-thiones,Molecules2012,17,1675-1685.