Iraqi J Pharm Sci, Vol.23(1) 2014 Antimicrobial evaluation of new derivatives of coumarin 35 Synthesis and Antimicrobial Evaluation of New 6 and7 Substituted Derivatives of Coumarin Rouaa A. Muften * , Kawkab Y.Saour ** and Ammar A.Razzak Mahmood **,1 * Ibn-Zuhr Hospital, Ministry of Health. Baghdad, Iraq. ** Department of Pharmaceutical Chemistry, College of Pharmacy, University of Baghdad Baghdad,Iraq. Abstract A series of benzohydrazide derivatives attached to coumarin moiety at position 6 and 7 have been synthesized. The reaction of coumarin derivatives (coumarin I and II) with p-nitrophenyl hydrazine yield Schiff bases (compound1a and IIa).These Schiff bases were refluxed with benzoyl chloride to give benzohydrazide derivatives of coumarin substituted at its 6 or 7 nucleus position (Ia1 and IIa1).The reaction and the purity of the products were checked by thin layer chromatography (TLC). The structures of the final compounds and their intermediates were confirmed by their melting points, infra red spectroscopy, and elemental microanalysis(CHN). Compounds (Ia1 and IIa1) were evaluated for their preliminary antibacterial and antifungal activities. Compound (IIa1) has good antibacterial activity against Staphylococcus aureus other than bacterial species, and was equivalent to ofloxacin as (standard drug). Key word: Coumarin, Schiff base, benzohydrazide derivatives, antimicrobial activity. كمضاداخ 7و6تحضير والتقييم الثايولوجي لمشتقاخ الكومارين المعوضح في الموقعين للمايكروتاخ ،رؤى علي مفتن * كوكة يعقوب ساعور ** وعمارعثذ الرزاق محمود **،1 * . انعراق، بغذاد ،َزارة انصحت ،مسخشفى ابه زٌر ** . انعراق،بغذاد، جبمعت بغذاد ،كهيت انصيذنت، ميبء انصيذالويتيفرع انك الخالصح . ان حفبعم مشخمبث انكُمبريه )مركب 6َ7حم حخهيك سهسهت مه مركببث انبىسٌَبيذرازايذ انمخصهت بىُاة انكُمبريه في انمُلعيه ( (I ( َII( مع )p-Nitrophenyl hydrazine اعطج لُاعذ شف, مركب )((1a(َ )IIa نمذ اسخعمهج ٌذي انمُاعذ مع كهُريذ.)) (. نمذ جرث مرالبت جميع IIa1 ,Ia1) 6َ7انبىسَيم نيعطي مشخمبث انبىسٌَبيذرازايذ نهكُمبريه انمعُض عهى وُاحً في انمُلعيه ركببث انُسطيت َانمركببث انىٍبئيت انخفبعالث َانخبكذ مه ومبَة انمركببث بُاسطت كرَمُحغرافيب انطبمت انرليمت, كمب حم مخببعت سير انم َاثببث ٌُيخٍب مه خالل ليبش درجبث االوصٍبر َانخحهيم انطيفي نالشعت ححج انحمراء, َانخحهيم انذليك نهعىبصر.نمذ حم حمييم انفعبنيت Staohylococcus( يمخهك فعبنيت جيذة ضذ (IIa1( ضذ انجراثيم َانفطريبث,حيث َجذ ان انمركب IIa1,Ia1انمبذئيت نهمركبيه) aureus .دَن غيرٌب مه اصىبف انبكخريب َكبن مكبفئب نفعبيت االَفهُكسبسيه كذَاء ليبسي جراثيم .لل المضادجالفعاليح ، الكومارين، قواعذ شف ، مشتقاخ الثنسوهايذرازايذ الكلماخ المفتاحيح: Introduction Coumarins (2H-1benzopyran-2ones) are important oxygen containing fused heterocycles used in drugs and dyes. Coumarins bound their class name to "coumarou" the vernacular name of Tonaka bean, from which coumarin itself was isolated in 1820.Coumarin and its derivatives represent one of the most active classes of compounds possessing a wide spectrum of biological activity (1-3) .Novobiocin and chlorobiocin are established antimicrobials containing a coumarin skeleton (4) .Many of these compounds have been proven to be active as,antibacterial (5-7) , antifungal (8) ,anti-Inflamat- ory (9) ,anticoagulant (10) and anti tumor agents (11) . Azomethine group (C=N) containing compounds typically known as Schiff bases have been synthesized by condensation of primary amines with active carbonyl form. Schiff bases from a significant class of compounds in medicinal and pharmaceutical chemistry with several biological applications that induce antibacterial and antifungal activities (12-16) . It's known that Schiff bases react smoothly with acid chloride to give the corresponding addition products (17-21) . Experimental section: 1. Chemicals: The specific chemicals used in this work are supplied from HIMEDIA, Fluka and SIGMA companies.All the solvents were of annular grade and used without further purification. 1 Corresponding author E. mail: kubbaammar1963@gmail.com Received:13 /4/2013 Accepted:5 /2/2014 Iraqi J Pharm Sci, Vol.23(1) 2014 Antimicrobial evaluation of new derivatives of coumarin 36 2. Detection Equipments: Melting points were determined by capillary tube method on Banested (UK). The FTIR spectra were recorded on FTIR spectrophotometer, Shimadzu (Japan) at the college of Science(Al-Mustansaryia University).The CHN microanalysis were carried out using Euro A(Elemental Analyzer), (Italy) performed in the college of Science-Al- Mustansaryia University. Thin layer chromatography (TLC) was run on silica gel GF254 (60), Merck (Germany) to check both purity of compounds and their reactions progress. The antimicrobial study of the synthesized final products was done in Al- Kindy college of Medicine-University of Baghdad. Synthesis: A. Synthesis of 4-methyl -2 – oxo -2H- chromene-6-carbaldehyde (compound I): Methyl acetoacetate (0.28 mole, 30ml)was mixed with re-distilled trimethyl orthoformate (0.27mole,30 ml) in dry methanol (25)ml and then conc. HCl (0.3ml) was added. The mixture was distilled immediately to obtain methyl-β-methoxy crotonate. Later polyphosphoric acid 100gm was added to a mixture of p-hydroxy benzaldehyde (0.05mole ,6.2 gm) and methyl- β- methoxy – crotonate (0.055 mole, 8 gm).The mixture was stirred for 90 minutes on steam bath at 80-85 °C and then poured into ice and water. The solid product was collected, washed and dried. Re-crystallization was from ethanol (22) . Percent yield, physical appearance, m.p., Rf values are listed in table (1), While the IR characteristic absorption bands are listed in table (2). B. Synthesis of 4- methyl -6 - ( 2 - ( 4- nitrophenyl) hydrazano) methyl) -2H- chromene-2-one (compound Ia) Equimoles of compound (I) (0.01mole ,1.88gm) and p-nitrophenyl hydrazine (0.01mole,1.53gm) were dissolved in 25 ml of methanol, to the resulting mixture 4-5 drops of piperidine was added and the mixture was refluxed for 10 hours. After completing the reaction, the mixture was poured into crushed ice. The product obtained was filtered and purified from methanol (23) . Percent yield, physical appearance, m.p., and Rf values are listed in table (1) and the IR characteristic absorption bands are listed in table (2) and the elemental micro analysis data are listed in table (3). C. Synthesis of N- (chloro (4- methyl -2 - oxo-2H- chromene - 6 -yl) methyl)-N'-(4- nitrophenyl) benzohydrazide (compound Ia1). Benzoyl chloride (0.01 mole, 1,2ml) was added drop wise to a solution of equimolar quantity of compound (Ia), (0,01 mole,3.23gm),dissolved in 1,4 dioxane(15 ml) and the mixture was refluxed for 6-8 hrs. Later the mixture was poured into ice with stirring. The product was filtered and purified from ethanol (24) , Percent yield, physical appearance, m.p.,and Rf values are listed in table (1),IR characteristic absorption bands are listed in table (2) and the elemental microanalysis data are listed in table (3). D. Synthesis of 4-methyl-2-oxo-2H- chromene-7-carbaldehyd (compound II). A flask containing (45) ml conc. sulfuric acid was immersed in an ice bath and the temperature was kept below 10°C. A solution of (0,1 mole,12.2 gm) of 3-hydroxy benzaldehyde in (0.1 mole, 1.2 ml) of methyl acetoacetate was then added, and the temperature should be kept below 10°C during the time of addition. Later the reaction mixture was kept at R.T for 48 hrs. after which, it was poured into a flask containing a mixture of ice and water, and the formed solid product was filtered off, washed with water, and re- crystallized from ethanol (25) . Percent yield, physical appearance, m.p., and Rf values are listed in table(1), while the IR characteristic absorption bands are listed in table (2). E. Synthesis of 4-methyl-7-((2-(4-nitrophenyl) hydrazono) methyl)-2H-chromen-2-one (compound IIa). To a solution of compound (II), (0.01 mole,1.88gm) in absolute ethanol (25 ml), an equimolecular amount of p-nitropheny hydrazine was added(0.01 mole,1.53 gm), to the above solution and a catalytic amount of piperidine (4-5) drops. The reaction mixture was heated under reflux for 10 hrs. It was then cooled at R.T, poured into crushed ice, filtered and purified from methanol (26) . Percent yield, physical appearance, m.p., and Rf values are listed in table (1), While the IR characteristic absorption bands are listed in table (2). F. Synthesis of N-(Chloro(4-methyl-2-oxo- 2H-chromene-7-yl)methyl)-N'-(4-nitro phenyl) benzohydrazide ( compound II a1): Compound (IIa), (0.01 mole,3.23 gm) was dissolved in 1,4-dioxane (15 ml) and to which (0.01 mole,1.2 ml) of benzoyl chloride was added dropwise, and the mixture was refluxed for 6-8 hrs. Afterwhich, the mixture was poured into ice with stirring. The product Iraqi J Pharm Sci, Vol.23(1) 2014 Antimicrobial evaluation of new derivatives of coumarin 37 obtained was filtered and purified from ethanol (24) . Percent yield, physical appearance ,m.p., and the Rf values are listed in table (2).The elemental microanalysis data are listed in table (3). Table (1) The physical appearance, percent yield, melting points and Rf values of the synthesized compounds and their intermediates. Solvent system Rf value Observed melting point °C Yield % Physical appearance Compd. Ethanol 8: Benzene 2 0.8 185-188 63% Dark brown crystal I Petroleum ether(40- 60°C) 5: Ethyl acetate 5 0.88 207-210 77% Light brown crystal Ia Ethanol 8: Benzene 2 0.84 110-112 89% Light walnut powder Ia1 Petroleum ether(40- 60°C) 5: Ethyl acetate 5 0.89 194-196 58% Dark brown crystal II Ethanol 8: Benzene 2 0.86 203-205 77% 93 Light brown powde IIa Ethanol 8: Benzene 2 0.83 115-118 Light walnut powder IIa1 Table (2) The IR characteristic absorption bands of the synthesized compounds IR characteristic absorption bands v` (cm-1) Compd. (1724) cm -1 for C=O of pyran ring and (1710)cm -1 for C=O stretching of aldehyde group I (1600) cm -1 for C=N stretching, (3400)cm -1 for N-H stretching and (1446) cm -1 and (1370) cm -1 for NO2 stretching Ia (1687) cm -1 C=O of amide group and (781) cm -1 for C-Cl Ia1 (1707)cm -1 for C=O of pyran ring and (1695)cm -1 for C=O stretching of aldehyde group II (1579) cm -1 for C=N stretching,(3392)cm -1 for NH-stretching and (1516) cm -1 and (1385) cm -1 for NO2 stretching IIa 1687) cm -1 for C=O of amide group and (808) cm -1 for C-Cl) IIa1 Table (3) The elemental microanalysis of the products Ia1 and IIaI Antimicrobial Activity A preliminary antibacterial and antifungal activity have been carried out according to Well Diffusion method. The prepared compounds have been studied for their antimicrobial activity in vitro against three tested bacteria ( Staphylcoccus aureus, Streptococcus spp., as Gram positive bacteria and Proteus spp., As Gram negative bacteria, and fungi (Candida spp.), were clinically activated and maintained on nutrient agar medium for testing antibacterial activity and sabouraud dextreose agar medium for antifungal activity. Ofloxacin was used a standard drug for antifungal activity. The plates were incubated at 30°C for 72 hrs., for M.wt. N% H% C% Value type Compd. 463.5 9.07 9.11 3.89 3.84 62.1 59.3 Calculated Observed Ia1 463.5 9.07 9.34 3.89 3.87 62.1 59.72 Calculated Observed IIa1 Iraqi J Pharm Sci, Vol.23(1) 2014 Antimicrobial evaluation of new derivatives of coumarin 38 the (fungal spp.), or 37°C for 24 hrs., for the bacterial spp.) (27) . The antimicrobial activity was evaluated by measuring the diameter of inhibition zone around the disk in (mm), as shown in table (4) and (5) respectively. Table ( 4) The antibacterial activity of the tested compounds. Zone of Inhibition in mm. Zone of inhibition in mm Zone of inhibition in mm Concentration µg/ml Compd. Proteus spp. Streptococcus spp. Staphylococcus aureus spp. No activity No activity No activity 2 4 8 1 5 9 3 µg/ml 25 µg/ml 60 µg/ml Ia1 No activity No activity No activity 2 6 12 4 8 14 3 µg/ml 25 µg/ml 60 µg/ml IIa1 6 11 17 7 13 18 8 10 16 3 µg/ml 26 µg/ml 60 µg/ml Ofloxacin Table (5) The antifungal activity of tested compounds Zone of inhibition in mm Candida spp. Concentration µg/ml Compd. 7 3 µg/ml Ia1 9 25 µg/ml 16 60 µg/ml 6 3 µg/ml IIa1 4 25 µg/ml 12 60 µg/ml 11 3 Ketoconazole 27 25 30 60 Results and Discussion The present investigation involves the synthesis of new series of compounds involving the coumarin moiety as shown in scheme 1 and scheme 2; the pathway of synthesis of coumarin derivatives that have a characteristic aldehyde group substituted at the 6 or 7 positions of the coumarin nucleus as shown in compound (I) and (II). Schiff bases (1a,IIa) were prepared by condensation of compound (I) and (II) with p- nitrophenyl hydrazine respectively using piperidine as catalyst; piperidine will increase the nucleophilicty of the amine group, and the reaction when followed by IR, showed the appearance of a characteristic absorption bands for (C=N) and (N-H), and disappearance of the band for (C=O) of aldehyde group as shown in table 2. The reactions between benzoyl chloride and Schiff bases are type from addition to the (C=N) moiety, and the reactions when followed by IR; illustrated the disappearance of (C=N) band, and the appearance of (C=O) of amide band and the appearance of (C-Cl) band as shown in table 2. The antimicrobial activities ( antibacterial and antifungal activities) have been carried out according to Well Diffusion methodology using ofloxacin and ketonazole as a standards respectively. All the results are fixed in table 4 and table 5. 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