Synthesis, Reactivity and Antimicrobial activity of ...

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Vol 66, No. 5;May 2013

Synthesis, Reactivity and Antimicrobial activity of Coumarinic and Chromonic Heterocycles Marwa S. Salem,* Hamed A. Y. Derbala, Mona B. Lashlam, Hassan M. F. Madkour Synthetic Organic Chemistry Laboratory, Chemistry Department, Faculty of Science, Ain Shams University, Abbasiya, Cairo, 11566, Egypt. *

Corresponding author e-mail: [email protected]

Abstract 3,5-Dichloro-2-hydroxyacetophenone has been utilized as synthon for the synthesis of 4-methyl-3cyanocoumarin and chromone-3-carboxaldehyde derivatives. The reactivity of both coumarinic and chromonic heterocyclic systems towards different electrophiles and nucleophiles have been studied. Antibacterial and antifungal activity of selected coumarin and chromone derivatives has been screened. The biological study revealed that chromonic derivatives have more inhibitory action than coumarinic ones as well as some of the former namely; chromenopyrazole has inhibitory effect more than the standard Amphorericin B. Keywords: 2-Oxo-2H-chromene, 4-Oxo-2H-chromene, Schiff bases, o-Hydroxyacetophenone, Antimicrobial activity. 1. Introduction Several natural products with the coumarinic and chromonic moieties exhibit interesting biological and pharmacological properties. They are anti-leishmanial, [1,2] antibacterial, [3-5] anti-HIV active, [6,7] antiviral, insecticidal [8] and anticoagulant, [9, 10] anticancer, [10-17]antihepatotoxic, antioxidant, [18,19] anti‐inflammatory, [20] antispasmodic, estrogenic, [21] antimicrobial, [22‐24] antifungal. [25] Additionally, coumarin derivatives have been used as food additives, perfumes, cosmetics, dyes [26, 27] fluorescent probes and triplet sensitizers, and herbicides. [28] In continuation of our previous works, [1,2,29-34]the present work aimed at utilization of the reactivity of 3-cyano-4-methylcoumarin and 3-formylchromone derivatives towards different electrophilic and nucleophilic reagents to get new fused and non-fused heterocyclic systems and evaluate them for antifungal and antibacterial activities. 2. Results and discussion 2.1 Chemistry From a synthetic point of view, 3,5-dichloro-2-hydroxyacetophenone occupies an important role in the synthesis of various heterocyclic systems such as 3-cyano-4-methylcoumarin 2 and 3-formylchromone 3 derivatives. Previously,[35] it has been reported that unexpected Claisen condensation of 3,5‐dichloro‐2‐hydroxy-acetophenone with excess of ethyl cyanoacetate in the presence of sodium metal gave 7‐amino‐2,4‐dichloro‐9‐hydroxy‐6‐oxo‐6H‐benzo[c]chromene ‐8‐carbonitrile (1). However, when 3,5‐dichloro‐2‐hydroxy-acetophenone reacted with ethyl cyanoacetate in presence of piperidine as a basic catalyst afforded 6,8-dichloro-4-methyl-2-oxo-2H-chromene-3-carbonitrile 2. [1] 6,8Dichloro-4-oxo-4H-chromene-3-carbaldehyde 3 was obtained by using Vilsmeier–Haack reaction [36] (cf. scheme 1).

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OH CN Cl

NH2

(i) [35]

Cl

O 1

O

CH3

O Cl

Cl

(ii)

CN O

OH

Cl

Cl

O

2 O

(iii)

Cl

CN

CN (i)

COOEt

CHO

Cl O 3

, Na (ii) COOEt , EtOH/ pip. (iii) DMF/ POCl3, H2O

Scheme 1 A series of 4-styrylcoumarins have been synthesized by knovenagel condensation between substituted 4-methylcoumarin derivative 2 and different aromatic aldehydes namely, 4-methylbenzaldehyde, 4chlorobenzaldehyde and benzaldehyde in presence of catalytic amount of piperidine to afford 6,8dichloro-2-oxo-4-[2-substitutedethenyl]-2H-chromene-3-carbonitriles 4a-c respectively(cf. scheme 2). It has been reported [36] that styrylcoumarins have anti–inflammatory activity against TNF-α and IL-6, antitubercular activity against Mycobacterium tuberculosis H37Rv strain. Styrylcoumarins 4a-c prepared in our laboratory, have also been found to be potent anti-leishmanial reagents. [1] Benzo[c]coumarin derivatives 5a-c have been readily obtained by two alternative routes. In the first route, 4-methylcoumarin derivative 2 was treated with arylidine-malononitrile in refluxing ethanol containing few drops of piperidine. 7-Amino-2,4-dichloro-4-aryl-6-oxo-benzo[c]chromene-8carbonitrile (5a-c) were also obtained by heating the styryl derivative 4a-c with malononitrile in boiling ethanol containing piperidine as basic catalyst. [1,30]The formation of 5a-c from the second route is a convincing chemical evidence for structures assigned to the products 5a-c. The present work presents to the art a typical example of heterocyclic systems transformations through the conversion of the starting coumarin derivative 2 to 2-oxoquinolin-1(2H)-ylthiourea 6 by treatment with thiosemicarbazide in refluxing dry pyridine. The IR of the compound 6 displayed absorption bands attributable to ʋ C=O quinolinone at 1744 & 1705 cm-1. The appearance of two absorption bands for the carbonyl functionality of quinolinone 6 is due to dipolar interaction between C=O and C=S. For illustration, conformer A (the higher value) and B (the lower value) are represented. CH3 Cl

CN N Cl HN

O S

CH3 Cl

CN N Cl HN

NH2 A

O NH2 S

B

The authors extend this work by conversion of cyano functionality of 4-methylcoumarin derivative 2 into acetyl function by treatment with Grignard reagent. The reaction of coumarin 2 with methylmagnesium iodide gave 3-acetyl-6,8-dichloro-4-methylcoumarin 7. The structure of 7 has been confirmed by all spectral data. IR spectrum showed the disappearance of ʋ C≡N and appearance of ʋ C=O coumarin & ʋ C=O acetyl at 1783 &1721 respectively recalling the similar field effect on the absorption of carbonyl function in conformer A (cf. scheme 2). We intend to study the relative reactivity of carbonyl group at position 3 and that of the coumarin nucleus at position 2. Thus the reaction with hydrazine hydrate in boiling ethanol produced hydrazone 8. The IR of compound 8 displayed absorption bands attributable to ʋ C=O coumarin at 1684 & ʋ C=N at 1635 and the disappearance of ʋ C=O acetyl. When 3-acetylcoumarin 7 was allowed to react with hydrazine hydrate 635

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in boiling glacial acetic acid, the pyrazole derivative 9 was obtained. The IR spectrum showed the disappearance of ʋ C=O coumarin. Z

(i)

Cl

CN

O O Cl 4a, Z = Me; 4b, Z = Cl; 4c, Z = H (iii) Z

CH3 Cl

CN O Cl

CN

(ii) Cl

O

NH2

2

O

O

Cl 5a, Z = Me; 5b, Z = Cl; 5c, Z = H CH3 Z

Cl

CHO

CN

(iv) N Cl HN

(v)

O S

NH2 6 CH3 O Cl

CH3 NNH2

CH3 O Cl

O

(vi)

Cl

CH3 Cl

7

O 8

O

(vii) CH3

CH3

O

N

Cl

N Cl

9 CN (i) Ar-CHO, EtOH/ piperidine, reflux; (ii) Ar CN , Ar = C6H5(CH3)-4, EtOH, piperidine, reflux; CN (iii) , EtOH, piperidine, reflux; (iv) thiosemicarbazide, dry pyridine , reflux; (v) CH3MgI; CN (vi)N2H4, EtOH, reflux; (vii) N2H4, AcOH, reflux

Scheme 2 3-Formylchromone 3 represents a very reactive system owing to the presence of three active sites which are a keto-function, a conjugated second carbonyl group at C-3 and above all, the carbon atom at position 2 of chromone nucleus. C-2 is very reactive towards Michael addition of nucleophiles with concomitant opening of the γ-pyrone ring, followed by new cyclization. The reaction of compound 3 with several nitrogen nucleophiles, as primary heterocyclic amines namely 2-aminopyrimidine and 2aminopyridine in boiling ethanol afforded Schiff bases 10a,b respectively (cf. scheme 3).

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O (i)

N Cl

CHO

(ii)

Cl

O Cl

O Cl

3 (iii)

N

O 10a, X=N 10b, X= CH O CHO

O Cl

X

Cl

NH2

11 O

Cl

[41] Cl

N NH

OH 12

N N Cl

(iii)

O Cl

13

(i) 2-aminopyrimidine and / or 2-aminopyridine, EtOH; (ii) NH2OH.HCl, EtOH; (iii) N2H4.H2O, EtOH

Scheme 3 Reaction of 3-formylchromone 3 with hydroxylamine hydrochloride is an interesting point as we obtain results which are contradictory with the previously reported ones. [37-40] All these publications have been previously reported to provide the 3-cyanochoromone; whether the reaction was carried out in 95% ethanol containing few drops of hydrochloric acid. However, the 2-amino-6,8-dichloro-4-oxo-4Hchromene-3-carbaldehyde (11) was the sole product. According to our speculation, the latter product was formed as a result of nucleophilic attack of the amino group of hydroxylamine on the electronically deficient carbon atom of formyl group to afford oxime derivative which was subjected to dehydration to give 3-cyanochromone derivative followed by Michael addition of nucleophilic hydroxyl group of water molecule on C-2 resulting in opening of the γ-pyrone ring, and followedllowed by new cyclization (cf. scheme 4). The IR showed strong absorption bands at 3327 & 3302 (NH2), 1747 (C=O formyl), 1652 (C=O chromone). O

O CHO

Cl O Cl

NH2OH

O

H

Cl

N

-H2O

OH

-H2O

O

O

Cl

Cl

3

CHO O

H2 O O

O Cl

CN

Cl

NH2

Cl

CN

Cl OH

O

Cl

11

Scheme 4 It has been claimed that [41] treatment of 3-formylchromone 3 with hydrazine hydrate in boiling ethanol lead to cleavage of the pyran ring and afforded the corresponding pyrazole (12). In our laboratory, treatment of 3-formylchromone 3 with the same reagent under the same reaction conditions gave 6,8-dichlorochromeno[4,3-c]pyrazole (13) as a sole product. This suggested that the reaction involved two successive 1,2-addition of (NH2-NH2) as a bi-dentate nucleophile with both formyl and 4oxo groups, that means no cleavage has occurred. In our investigations we could establish the behaviour of 3-formylchromone 3 towards carbon nucleophiles such as cyanoacetamide, barbaturic acid, malononitrile, malonic acid, diethyl malonate, ethyl acetoacetate and acetyl acetone in different conditions. Thus, condensation of chromone-3carboxyaldehyde 3 with cyanoacetamide in dry pyridine afforded 5-(3,5-dichloro-2-hydroxybenzoyl)637

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2-oxo-1,2-dihydropyridine-3-carboxamide (14). 3-Formylchromone 3 was easily condensed with barbituric acid to afford 15 after 10 minutes of reflux in pyridine (cf. scheme 5). It was found that, whether the reaction was carried out with one or two mole equivalents of barbituric acid, compound 15 was obtained. Formation of 15 in both cases is probably due to the high thermodynamic stability of the product acquired by the extended conjugation involving 4-hydroxy pyran moiety integrated with two segments of the utilized reagent constituting two alternative conjugated C=C. In addition, it could be also functionalized in the higher reactivity of barbituric acid as a nucleophilic carbon reagent. 3-Formylchromone 3 readily reacted with malononitrile, malonic acid and / or diethyl malonate in pyridine at boiling point under reflux to afford 2-((6,8-dichloro-4-oxo-4H-chromen-3-yl)methylene) malononitrile (16a), 3-(6,8-dichloro-4-oxo-4H-chromen-3-yl)acrylic acid (16b) and diethyl 2-((6,8dichloro-4-oxo-4H-chromen-3-yl)methylene)malonate (16c) respectively. It has been found that the structures similar to those of 16a-c strongly inhibit the secretion of histamine; therefore they can serve as suitable agents for the treatment of allergic diseases, especially bronchial asthma. [35, 42-44] Thus, we can claim that compounds 16a-c have anticipated activity in treatment of asthma and allergy. Reaction of 3-formylchromone 3 with excessive ethyl acetoacetate in piperidine-ethanol medium gave diethyl 5-(3,5-dichloro-2-hydroxybenzoyl)-2-methylisophthalate (17).The proposed mechanism involves initial condensation of 3 with ethyl acetoacetate followed by Michael addition of the second molecule of ethyl acetoacetate and subsequent rearrangement. O NH

Cl

(i)

O OH

CONH2

Cl 14

O HN

NH

O OH (ii)

O

Cl

O O Cl 15

NH N H

O

O

O Cl

X

(iii)

X

O Cl 16a, X = CN, CN 16b, X = COOH, H 16c, X = COOEt, COOEt

O CHO

Cl O Cl

O (iv)

Cl

COOEt OH

3 Cl

CH3 COOEt

17 O (v)

Cl

COCH3 COCH3

O Cl

18 CH3

(vi)

HO

Cl

Cl

COCH3

N

O 19

OH COCH3

(vii)

Cl

HO O Cl

20 (i) cyanoacetamide, Pyridine; (ii) barbituric acid, dry pyridine; (iii)malononitrile, malonic acid and/ or diethyl malonate, pyridine; (iv) ethyl acetoacetate, EtOH,pipredine; (v)2,4-pentanedione , Ac2O/ AcONa, (vi) 2,4-pentanedione , EtOH/ AcONH4; (vii) 2,4-pentanedione , AcOH/ HCl

Scheme 5 638

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Condensation of 3-formylchromone 3 and 2,4-pentanedione in acetic anhydride-sodium acetate medium afforded 3-(2-acetyl-3-oxobut-1-enyl)-6,8-dichloro-4H-chromen-4-one (18). On the other hand when the reaction was conducted in boiling ethanol catalyzed by ammonium acetate, the product was 3-acetyl-7,9-dichloro-10b-hydroxy-2-methyl-10bH-chromeno[4,3-b]pyridine (19). The proposed mechanism of this reaction involves the initial base-catalyzed condensation of 3 with 2,4-pentanedione to obtain 3-(2-acetyl-3-oxobut-1-enyl)-6,8-dichloro-4H-chromen-4-one (18), followed by conversion of hydroxyl group in the enolic form of compound 18 into amino group under the effect of ammonia and then cyclization by the attack of the amino group on the electronically deficient carbon of carbonyl group to give chromeno[4,3-b]pyridine derivative (19) (cf. scheme 6). O

O

O Cl

COCH3

EtOH/ AcONH4

Cl

H3COC O COCH3

-H2O

Cl

O

18

CH2

Cl

COCH3

HN HO

Cl O

CH3

Cl

CH2

COCH3

O

COCH3

Cl

O

H2N

CH2

HO O

Cl

NH3

COCH3

Cl

COCH3 OH

O

COCH3

Cl

3

H

COCH3

OH

COCH3

O

H C O

Cl

CHO +

Cl

HO

Cl

O

O

Cl

COCH3

N

Cl

Cl

19

Scheme 6 2,4-Dichloro-8-acetyl-9-hydroxy-10a-hydroxy-10aH-benzo[c]chromene 20 has been obtained by the addition of the solution of 3 in acetic acid to a preheated (70–80 ºC) solution of 2,4-pentanedione in acetic acid containing a catalytic amount of hydrochloric acid. The formation of 20 can be explained by a plausible mechanism (cf. scheme 7). O

O

O Cl

COCH3

AcOH/ HCl OH

COCH3

H

Cl O

H3COC O COCH3

-H2O

Cl

OH

Cl O

Cl

OH COCH3

COCH3 HO

O Cl

O Cl

O Cl

Cl

COCH3

Cl

18

O H COCH3

OH

H2C O

O

Cl

H

COCH3

Cl

COCH3 OH

H2C

COCH3 COCH3

Cl

3

O

H C O

Cl

CHO +

Cl

HO O Cl 20

Scheme 7 2.2 Antimicrobial Evaluation: Antibacterial and antifungal activities of selected synthesized compounds were screened using the Diffusion Disc Method. The experiments were performed using test bacterial organisms belonging to the Gram-positive and Gram-negative groups namely 639

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Staphylococcus aureus, Neisseria gonorrhoeae, Pseudomonas aeruginosa and Escherichia coli respectively as well as Candida albicans and Aspergillus flavus as tested fungi. 2.2.1 Antibacterial Activity: Table 1 and Fig. 1 revealed the inhibitory activity of twenty five selected compounds, 4- 9 and 10-20 derived from the starting compounds 4-methylcoumarin derivative 2 and 3formylcoromone derivative 3 respectively. The following points have been noticed: (1) All compounds derived from 3-formylchromone derivative 3 have more inhibitory action than compounds derived from 4-methylcoumarin derivative 2. (2) All compounds under investigation have more inhibitory action than the starting compounds except compounds, 4a-c, 5b, 7 and 8. (3) The tested compounds could be classified, according to their activities into four groups: a) Group 1: Compounds (13, 15, and 18) have good inhibitory action against the growth of bacteria strands of the tested microorganisms. b) Group 2: Compounds 3, 5a, 5c, 6, 9, 10a,b, 16a-c and 20 have moderate inhibitory action against the growth of bacteria strands of the tested microorganisms. c) Group 3: Compounds 11, 14 and 19 don't have any inhibitory effect except against Escherichia coli (G-), Pseudomonas aeruginosa (G-) and Staphylococcus aureus (G+) respectively. d) Group 4: Compound 17 has inhibitory effect in all tested microorganisms except against Escherichia coli (G-). 2.2.2 Antifungal Activity: Table 2 and Fig. 2 revealed the inhibitory activity of ten selected compounds, 2, 4b, 4c, 5b, 7, 8 and 13-15, 19 derived from the starting compounds 4-methylcoumarin derivative 2 and 3-formylcoromone derivative 3 respectively. The following points have been noticed: The tested compounds could be classified, according to their activities into three groups: a) Group 1: Compound 13 has inhibitory effect more than Amphotericin B against Aspergillus flavus. b) Group 2: The two compounds (7 and 15) have good inhibitory action against the growth of bacteria strands of the tested microorganisms. c) Group 3: Compounds 4b, 4c, 5b, 8, 14, and 19 don't have inhibitory action against the tested microorganisms. 3. Experimental 3.1 Instrumentation All melting points are uncorrected and were measured on a Gallenkamp electric melting point apparatus. The infrared spectra were recorded using potassium bromide disks on a Pye Unicam SP-3300 infrared spectrophotometer. 1H NMR were run at 300 MHz on a Varian Mercury VX-300 NMR spectrometer using tetramethylsilane (TMS) as internal standard in deuterated dimethylsulphoxide (DMSO-d6) or chloroform (CDCl3). Chemical shifts are quoted as δ. The mass spectra were recorded on Shimadzu GCMS-QP-1000EX mass spectrometer at 70 e.V. All the spectral measurements as well as elemental analyses were carried out at the Micro analytical Center of Cairo University. 3.2 Synthesis General procedure for synthesis of (5b,c). Method A: To a mixture of 4-styrylcoumarin derivative 4a-c (10 mmol) and malononitrile (12 mmol) was added few drops of piperidine in ethanol (30 mL). The reaction mixture was heated under reflux for 2h.The solid obtained after concentration and cooling was collected by filtration and recrystallized to afford benzo[c]chromene 5b,c. Method B: A solution of cyanocoumarin 2 (5 mmol, 1.27 g) and 4-substituted-benzylidenemalono-nitrile derivatives (10 mmol) in ethanol (30 ml) containing few drops of piperidine was heated at boiling point under reflux for 4 h. The solid that separated out after distilling off was filtered off and recystallized from the proper solvent to yield benzo[c]coumarin 5b,c.

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7-Amino-2,4-dichloro-9-(4-chlorophenyl)-6-oxo-6H-benzo[c]chromene-8-carbonitrile (5b). Recrystallised from dioxane as pale green crystals, Yield 79 %, mp over 320 °C. IR (KBr) ν, cm-1: 3410 & 3298 (NH2), 2217(C≡N) , 1710 (C=O coumarin). 1H NMR spectrum (DMSO‐d6, δ ppm): 8.32 & 8.18 (2d, 2H, Ar-H, Jm= 3Hz, 2.4Hz), 7.53-7.42 (m, 5H, Ar-H), 6.60 (br.s, 2H, exchangeable, NH2). Ms: m/z (418, M+4 , 36.6 %), (417, M+3 , 23.8 %), (416, M+2 , 100.0 %), (415, M+1 , 31.3 %), (414, M , 93.1%), 399(6.5 %), 388 (8.6 %), 379 (11.8 %), 353 (14.0 %). 7-Amino-2,4-dichloro-6-oxo-9-phenyl-6H-benzo[c]chromene-8-carbonitrile (5c).Recrystallised from dioxane as yellow crystals, Yield 72%, mp over 300°C. IR (KBr) ν, cm-1: 3441& 3336 (NH2), 2199 (C≡N), 1681(C=O coumarin). 1H NMR spectrum (DMSO‐d6, δ ppm): 8.14 & 7.96 (2d, 2H, Ar-H, Jm= 3Hz, 2.4Hz), 7.31-7.20 (m, 6H, Ar-H), 6.38 (br.s, 2H, exchangeable, NH2). Ms: m/z (383, M+3 , 90.8 %), (382, M+2 , 74.4 %), (381, M+1 , 53.0 %), 354 (64.0 %), 339 (63.0 %), 320 (84 %), 304(66 %), 295(64 %), 261 (100 %), 260(71 %). 1-(6,8-Dichloro-3-cyano-4-methyl-2-oxoquinolin-1(2H)-yl)thiourea (6). A mixture of cyanocoumarin 2 (5 mmol, 1.27 g) and thiosemicarbazide (5 mmol, 0.45 g) in dry pyridine (20 mL) was refluxed for 3h., left to cool, acidified with cold dilute hydrochloric acid. The crude solid product that deposited was collected by suction,washed with cold water, dried and then recrystallised from ethanol to give 6 as brown crystals. Yield 89%, mp 201-202 °C. IR (KBr) ν, cm-1: 3439 & 3341(NH2 & NH), 2233 (C≡N), 1744& 1705 (C=O), 1377 (C=S). 1H NMR spectrum (DMSO‐d6, δ ppm): 8.78 (s, 2H, exchangeable, NH2), 8.08 & 8.03 (2d, 2H, Ar-H, Jm = 3 Hz), 3.73(br.s, 1H, exchangeable, NH), 2.71 (s, 3H, CH3). Ms: m/z (329, M+3 , 12.0 %), (328, M+2 , 17.3%), (327, M+1 , 11.4%), (326, M , 12.2%), 325 (14.0%), 310 (13.4%), 295 (11.4%), 253 (23.5%), 227 (18.4%), 129 (24.5%), 69 (100.0%). 3-Acetyl-6,8-dichloro-4-methyl-2H-chromen-2-one (7). A solution of coumarin derivative 2 (10 mmol, 2.53 g) in tetrahydrofuran (50 mL) was added dropwise to methylmagnesium iodide (10 mmol, 1.66 g) in dry ether (100 mL) within 15 min. The reaction mixture was refluxed on a water bath for 4h and left overnight at room temperature, poured into saturated solution of ammonium chloride, extracted by diethyl ether and dried using anhydrous magnesium sulfate, filtered and then recrystallised from ethyl acetate to give 7 as yellow crystals. Yield 41 %, mp 155-156 °C. IR (KBr) ν, cm-1: 1784(C=O coumarin), 1721(C=O acetyl). 1H NMR spectrum (DMSO‐d6, δ ppm): 8.13 & 7.83 (2d, 2H, Ar-H, Jm= 3Hz, 2.4Hz), 2.73 (s, 3H, -COCH3), 2.09 (s, 3H, -CH3). Ms: m/z (274, M+4 , 0.76 %), (273, M+3 , 5.6%), (272, M+2 , 33.9%), (271, M+1 , 39.0%), (270, M , 100.0%), 235 (41.1%), 178 (41.9%), 177 (23.9%), 89 (36.0%). 6,8-Dichloro-3-[1-ethanehydrazonoyl]-4-methyl-2H-chromen-2-one(8). To a solution of acetylcoumarin derivative 7 (5 mmol, 1.35 g) in ethanol (30 mL) hydrazine hydrate (10 mmol, 0.32 g) was added. The reaction mixture was refluxed for 4h, left to cool, and then poured onto cold dilute hydrochloric acid. The solid that separated out was collected by filtration, washed with water, dried and recrystallised from ethanol to give the hydrazon 8 as yellow crystals. Yield 72%, mp 218-219 °C. IR (KBr) ν, cm-1: 1684(C=O coumarin), 1636 (C=N). 1H NMR spectrum (DMSO‐d6, δ ppm): 8.06 & 7.95 (2d, 2H, Ar-H, Jm = 2.4 Hz), 7.03 (br.s, 2H, exchangeable, NH2), 2.87 (s, 3H, NH2N=C-CH3), 2.71 (s, 3H, CH3). Ms: m/z (286, M+2 , 7.65%), (285, M+1 , 8.38%), (284, M , 8.09%), 270 (10.7%), 214 (10.0%), 135 (8.8 %), 83 (34.4%), 69 (100.0%). 6,8-Dichloro-3,4-dimethylchromeno[2,3-c]pyrazole (9). A solution of acetylcoumarin derivative 7 (5 mmol, 1.35g) and hydrazine hydrate (5 mmol, 0.96 g) in glacial acetic acid (20 mL) was refluxed for 6h. The reaction mixture was allowed to cool, and then poured into water. The solid that deposited was collected, dried and finally recrystallised from dioxane to afford 9 as brown crystals. Yield 81 %, mp 283-284 °C. IR (KBr) ν, cm-1: 1632 (C=N). 1H NMR spectrum (DMSO‐d6, δ ppm): 8.16 & 7.96 (2d, 2H, Ar-H, Jm = 2.7 Hz), 3.56 & 2.99 (2s, 6H, CH3). Ms: m/z (269, M+3 , 7.5 %), (268, M+2 , 49.6%), (267, M+1 , 100.0%), (266, M , 49.2%), 252 (6.5%), 237 (5.0%), 202 (5.8%), 77 (26.2%). General procedure for synthesis of (10 a,b). To a solution of 3-formylchromone derivative 3 (5 mmol, 1.22 g) in absolute ethanol (20 mL), the appropriate amine namely, 2-amino pyrimidine and / or 2-amino pyridine (5 mmol) was added. The 641

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reaction mixture was refluxed for 9h, left to cool, collected by filtration, dried and then recrystallised to give (10a,b). 6,8-Dichloro-3-((pyrimidin-2-ylimino)methyl)-4H-chromen-4-one (10a). Recrystallised from dioxane as brown crystals, Yield 87 %, mp 129-130 °C. IR (KBr) ν, cm-1: 1658 (C=O chromone), 1601 (C=N). 1H NMR spectrum (CDCl3, δ ppm): 8.60-8.55 (m, 3H, pyrimidine), 8.31 (s, 1H, -CH=N-), 7.90 & 7.54 (2d, 2H, Ar-H, Jm= 3Hz, 2.7Hz), 7.01 (s, 1H, C2-H). Ms: m/z (323, M+4 , 2.8 %), (322, M+3 , 2.8 %), (321, M+2 , 3.5 %), (320, M+1 , 3.0 %), (319, M , 8.0%), 240 (6.3%), 209 (3.6%), 188 (3.0%), 122 (3.3%), 97 (4.7%), 80 (100.0%). 6,8-Dichloro-3-((pyridin-2-ylimino)methyl)-4H-chromen-4-one (10b). Recrystallised from dioxane as brown crystals, Yield 76 %, mp 180-181 °C. IR (KBr) ν, cm-1: 1661 (C=O chromone), 1605 (C=N). 1H NMR spectrum (DMSO‐d6, δ ppm): 8.83-8.35 (m, 3H, pyridine), 7.90 &7.89 (2d, 2H, Ar-H, Jm= 2.4Hz), 7.44 (s, 1H, -CH=N-), 7.18- 7.11 (m, 1H, C2-Hpyran, 1H, C6-Hpyridine). Ms: m/z (319, M , 00.0 %), (271, M-CH≡C-C≡CH , 11.1%), 214 (60.0%), 188 (51.1%), 150 (13.3%), 111 (31.1%), 94 (95.6%), 66 (100.0%). 2-Amino-6,8-dichloro-4-oxo-4H-chromene-3-carbaldehyde (11). A mixture of 3-formylchromone derivative 3 (5 mmol, 1.22 g) and hydroxylamine hydrochloride (5 mmol, 0.37g) in absolute ethanol (25 mL) was heated under reflux for 48 h. The solid product that formed was collected by filtration, dried and recrystallised from dioxane to give 11 as yellow crystals. Yield 86 %, mp 296-297 °C. IR (KBr) ν, cm-1: 3356 & 3302 (NH2), 1747 (C=O formyl), 1652 (C=O chromone). 1H NMR spectrum (DMSO‐d6, δ ppm): 10.60 (br.s, 2H, exchangeable, NH2), 9.38 (s, 1H, CHO), 7.99 & 7.88 (2d, 2H, Ar-H, Jm = 2.4 Hz). Ms: m/z (259, M+2 , 5.6 %), (258, M+1 , 12.5%), (257, M , 12.5%), 227 (12.5%), 188 (100.0%), 152 (13.9%), 69 (9.7 %), 68 (47.2%). 6,8-Dichlorochromeno[4,3-c]pyrazole (13). A mixture of 3-formylchromone derivative 3 (5 mmol, 1.22 g) and hydrazine hydrate (5 mmol, 0.16 g) in ethanol (25 mL) was heated under reflux for 24h, left to cool. The solid product that formed was collected by filtration, dried and recrystallised from ethanol to give 13 as white crystals. Yield 85 %, mp 249-251 °C. IR (KBr) ν, cm-1: 1626 (C=N). 1H NMR spectrum (DMSO‐d6, δ ppm): 8.48 & 8.36 (2s, 2H, ArH), 7.57 (s, 1H, pyrazole-H), 6.30 (s, 1H, C2-Hpyran moiety). Ms: m/z (242, M+4 , 11.4 %), (241, M+3 , 60.5%), (240, M+2 , 32.4%), (239, M+1 , 100.0%), (238, M , 38.0%), 147 (18.4%), 97 (10.9%), 73 (19.3%). 5-(3,5-Dichloro-2-hydroxybenzoyl)-2-oxo-1,2-dihydropyridine-3-carboxamide(14). A mixture of 3-formylchromone derivative 3 (5 mmol, 1.22 g) and cyanoacetamide (5 mmol, 0.47 g) in dry pyridine (20 mL) was heated under reflux for 1h. Left to cool, collected by filtration, dried and recrystallised from dimethyl formamide to give 14 as white crystals. Yield 78 %, mp over 320 °C. IR (KBr) ν, cm-1: 3408(OH), 3281, 3142 (NH & NH2). 1H NMR spectrum (DMSO‐d6, δ ppm): 12.98 (br.s, 1H exchangeable, OH), 10.41(s, 2H, exchangeable, NH2), 8.73(s, 1H, exchangeable, NH), 8.63 (d, 1H, pyridineH, Jm= 2.4Hz), 8.08 (d, 1H, Ar-H, Jm = 3Hz), 7.78 (d, 1H, Ar-H, Jm = 3Hz), 7.40 (d, 1H, pyridine-H, Jm = 2.4Hz). Ms: m/z (326, M , 00.0%), (310, M-OH, +H , 61.5%). 5-(6,8-Dichloro-3-((2,4,6-trioxo-dihydropyrimidin-5(6H)-ylidene)methyl)-4-hydroxy-2Hchromen-2-ylidene)pyrimidine-2,4,6(1H,3H,5H)-trione (15). To a solution of 3-formylchromone derivative 3 (5 mmol, 1.22 g) in 10 mL of pyridine, barbituric acid (5 mmol or 10 mmol, 0.64 g or 1.28 g) in (10 mL) pyridine was added. The reaction mixture was refluxed for 10 minute, left to cool at room temperature. The solid product that formed was collected by filtration, washed with light petroleum ether (bp 40-60°C), dried and then recrystallised from dimethyl formamide to furnish 15 as yellow crystals. Yield s 58 % and 95 % respectively, mp over 300 °C. IR (KBr) ν, cm-1: 3385 (OH), 3203 (NH), 1729 (C=O), 1620 (C=C). 1H NMR spectrum (DMSO‐d6, δ ppm): 9.94 (br s, 4H, exchangeable, 4NH), 8.64 (s, 1H exchangeable, OH), 8.40 (s, 1H, CH olefinic), 7.727.53(m, 2H, Ar-H). Ms: m/z (481, M+2 , 82.9 %), (480, M+1 , 42.9 %), (479, M , 100.0%), 396 (14.3%), 395 (28.6%), 261 (20.0%), 185 (20.0%), 85 (28.6%), 84 (14.3%). 2-((6,8-Dichloro-4-oxo-4H-chromen-3-yl)methylene)malononitrile (16a). A mixture of 3-formylchromone derivative 3 (5 mmol, 1.22 g) and malononitrile (5 mmol, 0.33 g) in dry pyridine (20 mL) was heated under reflux for 12 h, left to cool. Acidification of the reaction 642

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mixture with cold dilute hydrochloric acid gave the crude solid product which was collected by filtration, dried and then recrystallised from ethanol to give 16a as brown crystals. Yield 86 %, mp 200202 °C. IR (KBr) ν, cm-1: 2227(C≡N), 1677 (C=O chromone). 1H NMR spectrum (DMSO‐d6, δ ppm): 8.29 (s, 1H, -CH=C-), 8.00 (s, 1H, C2-H), 7.54 & 7.40 (2d, 2H, Ar-H, Jm = 2.7Hz). Ms: m/z (293, M+2 , 20.8 %), (292, M+1 , 29.2 %), (291, M , 00.0%), 97(25.0%), 86 (20.8%), 72(50.0%), 71 (58.3%), 60 (100.0%). General procedure for synthesis of (16 b,c). A mixture of 3-formylchromone derivative 3 (10 mmol, 2.43 g), malonic acid and / or diethyl malonate (10 mmol) in dry pyridine (15 mL) was heated under reflux for 1.5h and / or 12h respectively, left to cool, the resulting precipitate was recrystallised from the proper solvent to afford 16b,c respectively. 3-(6,8-Dichloro-4-oxo-4H-chromen-3-yl)acrylic acid (16b). Recrystallised from ethanol as brown crystals. Yield 85 %, mp 265-266 °C. IR (KBr) ν, cm-1: 3450 (OH), 1690 (C=O acid), 1660 (C=O chromone). 1H NMR spectrum (DMSO‐d6, δ ppm): 12.47 (br.s, 1H exchangeable, OH), 8.98(s, 1H, C2-H), 8.23& 8.03 (2d, 2H, Ar-H, Jm= 2.4Hz), 7.44 &7.12 (2d, 2H, -CH=CH-, J= 15.9 Hz). Ms: m/z (288, M+4 , 0.8 %), (287, M+3 , 1.0 %), (286, M+2 , 3.8 %), (285, M+1 , 3.1 %), (284, M , 4.9%), 241(65.5%), 240 (27.6%), 239 (100.0%), 238 (79.3%), 97 (16.2%), 74 (10.7%). Diethyl-2-((6,8-dichloro-4-oxo-4H-chromen-3-yl)methylene)malonate (16c).Recrystallised from dimethyl formamide as white crystals. Yield 85 %, mp over 300 °C. IR (KBr) ν, cm-1: 1734 (C=O ester), 1683 (C=O chromone), 1653 (C=C). 1H NMR spectrum (DMSO‐d6, δ ppm): 8.96 (s, 1H, -CH=C-), 8.29 & 8.13(m, 3H, Ar-H), 4.62 (q, 4H, 2(-CH2-CH3), J= 6.9 Hz), 1.47(t, 6H, 2(-CH2-CH3), J= 6.9 Hz). Ms: m/z (384, M , 0.0), (161, 31.3%), (153, 100 %), 152 (47.5%), 97(12.5%). Diethyl-5-(3,5-dichloro-2-hydroxybenzoyl)-2-methylisophthalate (17). To stirred solution of 3-formylchromone derivative 3 (5 mmol, 1.22 g) and ethyl acetoacetate (15 mmol, 1.98 g) in ethanol (30 mL) at 0-20 ºC was added drops of piperidine. The resulting red solution was allowed to warm at room temperature for several hours, neutralized with dilute acetic acid and extracted with diethyl ether. The ether layer was then washed with sodium carbonate, and dried by using anhydrous magnesium sulfate. Concentration in vacuum gave yellow crystals which recrystallised from ethanol to afford 17 as yellow crystals. Yield 30 %, mp 77-79 °C. IR (KBr) ν, cm−1: 3426 (OH), 1741 (C=O ester), 1637 (C=O ketone).1H NMR spectrum (CDCl3, δ ppm): 12.19 (s, 1H exchangeable, OH), 8.17(s, 2H, Ar-H), 7.65 & 7.47 (2d, 2H, Ar-H, Jm = 2.7HZ), 4.43 (q, 4H, 2(CH2-CH3), J= 7.2 Hz), 2.82 (s, 3H, CH3), 1.41(t, 6H, 2(CH2-CH3), J= 7.2 Hz). Ms: m/z (428, M+4 , 3.4 %), (427, M+3 , 3.6 %), (426, M+2 , 11.6 %), (425, M+1 , 6.2 %), (424, M , 18.4%), 382 ( 14.6%), 380 (79.7%), 379 (49.7%), 378 (100.0%), 351 (16.1%), 349 (15.6%), 304 (39.4). 3-(2-Acetyl-3-oxobut-1-enyl)-6,8-dichloro-4H-chromen-4-one (18). A mixture of 3-formylchromone derivative 3 (5 mmol, 1.22 g), 2,4-pentanedion (5 mmol, 0.5 mL) and fused sodium acetate (5 mmol, 0.44 g) in acetic anhydride (10 mL) were reflux with stirring for 3h on the water bath. The mixture was cooled neutralized by saturated solution of sodium carbonate and extracted by using dichloromethane. The ether layer was dried by using anhydrous magnesium sulfate; filtered, concentrated then recrystallised from ethanol to give 18 as brown crystals. Yield 35 %, mp 186187 °C. IR (KBr) ν, cm-1: 1774 (C=O ketone), 1670 (C=O chromone).1H NMR spectrum (DMSO‐d6, δ ppm): 8.28 (s, 1H, CH=C-), 7.98 &7.77 & 7.39 (3s, 3H, Ar-H), 2.33 & 2.13 (2s, 6H, CH3). Ms: m/z (325, M+1 , 62.0 %), (324, M , 70.0%), 307 (8.9), 297 (9.4%), 97 (23.0%), 55 (100.0%). 3-Acetyl-7,9-dichloro-10b-hydroxy-2-methyl-10bH-chromeno[4,3-b]pyridine (19). To a solution of 3-formylchromone derivative 3 (5 mmol, 1.22 g) in ethanol (20 mL), acetyl acetone (5 mmol, 0.5 mL) and ammonium acetate (5 mmol, 0.39 g) was added. The reaction mixture was heated at reflux for 24h, left to cool at room temperature. The crude solid product that deposited was collected by filtration, dried and then recrystallised from dimethyl formamide to give 19 as white crystals. Yield 85 %, mp over 300 °C. IR (KBr) ν, cm-1: 3358 (OH), 1687 (C=O).1H NMR spectrum (DMSO‐d6, δ ppm): 8.32 & 8.18 (2s, 2H, pyridine-H), 8.05 & 7.74 (2s, 2H, Ar-H), 6.70 (2s, 1H exchangeable, OH), 2.74 (s, 3H, COCH3), 2.63 (s, 3H, CH3). Ms: m/z (326, M+3 , 16.1 %), (325, M+2 , 61.3 %), (324, M+1 , 48.4 %), (323, M , 54.8%). 2,4-Dichloro-8-acetyl-9-hydroxy-10ahydroxy-10aH-benzo[c]chromene (20).

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Concentrated hydrochloric acid (2-3 drops) was added to a solution of acetyl acetone (5 mmol, 0.5 mL) in acetic acid (5 mL) at 70-80 ºC. The reaction mixture was stirred at that temperature for 15 min. A solution of 3-formylchromone derivative 3 (5 mmol, 1.22 g) in acetic acid was added dropwise. The resultant solution was stirred for 2h at that temperature. The dark red reaction mixture was cooled and poured onto crushed ice. The solid deposited was filtered, washed with water, dried and recrystallised from ethanol to give 20 as pale brown crystals. Yield 46 %, mp 146-147 °C. IR (KBr) ν, cm−1: 3409 (OH), 1654 (C=O ketone).1H NMR spectrum (DMSO‐d6, δ ppm): 8.82-7.09 (m, 5H, Ar-H), 6.17 (br. s, 2H exchangeable, 2OH), 2.73 (s, 3H, CH3). Ms: m/z (326, M+2 , 55.0 %), (325, M+1 , 79.0 %). Measurement of antimicrobial activity using the Diffusion Disc Method: Antimicrobial activity of the tested samples was determined using a modified Kirby-Bauer disc diffusion method. [45] Briefly, 100 µl of the test bacteria/fungi were grown in 10 ml of fresh media until they reached a count of approximately 108 cells/ml for bacteria or 105 cells/ml for fungi. [46]100 µl of microbial suspension was spread onto agar plates corresponding to the broth in which they were maintained. Isolated colonies of each organism that might be playing a pathogenic role should be selected from primary agar plates and tested for susceptibility by disc diffusion method. [47]Of the many media available, NCCLS recommends Mueller-Hinton agar due to: it results in good batch-to-batch reproducibility. Disc diffusion method for filamentous fungi tested by using approved standard method (M38-A) developed by the [48] for evaluating the susceptibilities of filamentous fungi to antifungal agents. Disc diffusion method for yeasts developed by using approved standard method (M44-P) by the. [49] Plates inoculated with filamentous fungi as Aspergillus flavus at 25 0C for 48 hours; Gram (+) bacteria as staphylococcus aureus, Bacillus subtilis; Gram (-) bacteria as Escherichia coli, pseudomonas aeuroginosa they were incubated at 35-37oC for 24-48 hours and, yeast as Candida albicans incubated at 30 oC for 24-48 hours and, then the diameters of the inhibition zones were measured in millimeters. [45] Standard discs of Tetracycline (Antibacterial agent), Amphotericin B (Antifungal agent) served as positive controls for antimicrobial activity but filter discs impregnated with 10 µl of solvent (distilled water, chloroform, DMSO) were used as a negative control. The agar used is Meuller-Hinton agar that is rigorously tested for composition and pH. Further the depth of the agar in the plate is a factor to be considered in the disc diffusion method. This method is well documented and standard zones of inhibition have been determined for susceptible and resistant values. Blank paper disks (Schleicher & Schuell, Spain) with a diameter of 8.0 mm were impregnated 10µ of tested concentration of the stock solutions. When a filter paper disc impregnated with a tested chemical is placed on agar the chemical will diffuse from the disc into the agar. This diffusion will place the chemical in the agar only around the disc. The solubility of the chemical and its molecular size will determine the size of the area of chemical infiltration around the disc. If an organism is placed on the agar it will not grow in the area around the disc if it susceptible to the chemical. This area of no growth around the disc is known as (Zone of inhibition) or (Clear zone). For the disc diffusion, the zone diameters were measured with slipping calipers of the National Committee for Clinical Laboratory Standards. [50]Agar–based methods such as Etest ad disk diffusion can be good alternatives because they are simpler and faster than broth-based methods. [51, 52] Conclusion: 3,5-Dichloro-2-hydroxyacetophenone could be utilized to construct a variety of heterocyclic systems such as 4-methyl-3-cyanocoumarin, chromone-3-carboxaldehyde, chromeno[4,3-b]pyridine, benzo[c] chromene, chromeno[4,3-c]pyrazole and chromeno[2,3-c]pyrazole derivatives. Some of the synthesized heterocycles have appreciable promising antimicrobial activities. References [1] Madkour, H. M. F.; Baloch, N.; Salem, M. S.; Al-kahraman, Y. M. S. A.; Lashlam, M. B. Archives Des Sciences 2012, 65(12), 349-356. [2] Baloch, N.; Alkahraman,Y. M. S. A.; Zaidi, M. A.; Madkour, H. M. F. GJSFR (B) (Global Journal of Science Frontier Research) 2012, 12(1), 27-32. 644

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[3] Harborne, J. B.; Williams, C. A. Phytochemistry 2000, 55, 481-504. [4] Djemgou, P. C.; Gatsing, D.; Kenmogne, M.; Ngamga, D.; Aliyu, R.; Adebayo, A. H.; Tane, P.; Ngadjui, B. T.; Seguin, E.; Adoga, G. I. Res. J. Med. Plant 2007, 1(2), 65‐71. [5] Diwakar, S. D.; Bhagwat, S. S.; Shingare, M. S.; Gill, C. H. Bioorg. Med. Chem. Lett. 2008, 18, 4678‐4681. [6] Hesse, S.; Kirsch, G. Tetrahedron Lett. 2002, 43, 1213-1215. [7] Groweiss, A.; Cardellins, J. H.; Boyd, M. R. J. Nat. Prod. 2000, 63, 1537-1539. [8] Lee, B. H.; Clothier, M. F.; Dutton, F. E.; Conder, G. A.; Johnson, S. S. Bioorg Med. Chem. Lett. 1998, 8, 3317-3320. [9] Jung, J.-C.; Kim, J.-C.; Park, O.-S. Synth. Commun. 1999, 29, 3587-3595. [10] Jung, J.-C.; Jung, Y.-J.; Park, O.-S. Synth. Commun. 2001, 31, 1195-1200. [11] Huang, W.; Ding, Y.; Miao, Y.; Liu, M. Z.; Li Y.; Yang, G. F. Eur. J. Med. Chem. 2009, 44, 3687‐3696. [12] Lee, K. Y.; Nam, D. H.; Moon, C. S.; Seo, S. H.; Lee, J. Y.; Lee, Y. S. Eur. J. Med. Chem. 2006, 41, 991‐996. [13] McClure, J. W.; Harborne, J. B.; Mabry, T. J.; Mabry, H. The Flavonoids, Ed.; Chapman and Hall: London, 1975; pp 970‐1055. [14] Gamal, E. A. M.; Djemgou, P. C.; Tchuendem, M.; Ngadjui, B. T.; Tane, P.; Toshifumi, H. Z. Naturforsch 2007, 62c, 331‐338. [15] Valenti, P.; Bisi, A.; Rampa, A.; Belluti, F.; Gobbi, S.; Zampiron, A.; Carrara, M. Bioorg. Med. Chem. 2000, 8, 239-246. [16] Lim, L. ‐C.; Kuo, Y. ‐C.; Chou, C. ‐J. J. Nat. Prod. 2000, 63, 627-630.

[17] Shi, Y. Q.; Fukai, T.; Sakagami, H.; Chang, W. ‐J.; Yang, P. ‐Q.; Wang, F. ‐ P.; Nomura, T. J. Nat. Prod. 2001, 64, 181-188.

[18] Atassi, G.; Briet, P.; Berthelon, J. P.; Collonges, F. J. Med. Chem. 1985, 20, 393-402. [19] Pietta, P. J. J. Nat. Prod. 2000, 63, 1035-1042.

[20] Middleton, Jr. E.; Kandaswami, C.; Arborne, J. B. The Flavonoids Advances in Research since 1986 Ed.; Chapman and Hall: London, 1994; pp 619. [21] Bruneton, J. Pharmacognosy, Phytochemistry and Medicinal Plants; English Translation by Hatton, C. K.; Lavoisier Publishing: Paris. 1995; pp 265. [22] Albrecht, U.; Lalk, M.; Langer, P. Bioorg. Med. Chem. 2005, 13, 1531-1536. [23] Deng, Y.; Lee, J. P.; Ramamonjy, M. T.; Synder, J. K.; Des Etages, S. A.; Kanada, D.; Synder, M. P.; Turner, C. J. J. Nat. Prod. 2000, 63, 1082-1089. [24] Khan, I. A.; Avery, M. A.; Burandt, C. L.; Goins, D. K.; Mikell, J. R.; Nash, T. E.; Azadega, A.; Walker, L. A. J. Nat. Prod. 2000, 63, 1414-1416. [25] Mori, K.; Audran, G.; Monti, H. Synlett. 1998, 259-260. [26] Singer, L. A.; Kong, N. P. J. Am. Chem. Soc. 1966, 88, 5213-5219. [27] Zahradnik, M. The Production and Application of Fluorescent Brightening Agents; Wiley: New York, 1992. [28] Song, A.; Wang, X.; Lam, K. S. Tetrahedron Lett. 2003, 44, 1755-1758. [29] Salem, M. S.; Marzouk, M. I.; Ali, S. N.; Madkour, H. M. F. Eur. J. Chem. 2012, 3(2), 220-227. [30] Madkour, H. M. F. Heterocycles 1993, 36 (5), 947-959. [31] Al- Kahraman,Y. M. S. A.; Madkour, H. M. F.; Ali; D.; Yasinzai M. Molecules 2010, 15, 660-671. [32] Hamed, A. A.; Madkour, H. M. F.; Al-Nuaimi, I. S.; Hussain, B. A. Anales De Quimica de la Sociedad Espanola de Quimica (An. Quim.)1994, 90 (5-6), 359-364. 645

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[33] Al-Kahraman, Y. M .S.; Madkour, H. M. F.; Sajid, M.; Azim, M. K.; Bukhari, I.; Yasinzai, M. World J. Chem. 2011, 6(1), 19-24. [34] Madkour, H. M. F.; El-Shiekh, Y. W.; Ahmed A. F. A.; Farag, A. A. Middle- East Journal of Scientific Research 2011, 9(4), 520-526. [35] El-Shaaer, H. M. Eur. J. of Chem. 2012, 3(1), 51-56. [36] Kuen-Chien, K.; Kuo-Liang, H.; Shiu-Yu, K.; Tsai-Fang, T.; Hung-Liang, C. Hua Hsueh Hsueh Pao 1976, 34 (2), 123-128; Chem Abstr. 1977, 87,184319m. [37] Upadhyay, K.; Bavishi, A.; Thakrar, S.; Radadiya, A.; Vola, H.; Parekh, S.; Bahavsar, D.; Savant, M.; Parmar, M.; Adlakha, P.; Shah, A. Bioorg. Med. Chem. Lett. 2011, 21 (8), 2547-2549. [38] Nohara, A., Umetani, T., Sanno, Y. Japan Kokai 7, 552, 067, 1975; Chem Abstr 1976, 84,105400t. [39] Nohara, A.; Kuriki, H.; Saijo, T.; Sugihara, H.; Kanno, M.; Sanno, Y. J. Med. Chem. 1977, 20,141-145. [40] Nohara, A.; Ishiguto, T.; Sanno, Y. Tetrahedron Lett. 1974, 13, 1183-1186. [41] Basinski, W.; Jerzmanowska, Z. Pol. J. Chem. 1983, 57, 471-481. [42] Gosh, K.; Mukhopadhyay, K. K. J. Ind. Chem. Soc. 1978, 55, 386. [43] Nohara, A.; Umetani, T.; Miyata, Y.; Sanno, Y. Ger Often. 2, 253, 914, 1973; Chem Abstr 1973, 79, 31 874y. [44] Nohara, A., Kuriki, H., Saijo, T., Ukawa, K.; Murata, T.; Sanno, Y. J. Med. Chem. 1975, 18, 3437. [45] Bauer, A. W.; Kirby, W. M.; Sherris, C.; Turck, M. American Journal of clinical pathology 1966, 45,493-496. [46] Pfaller, M. A.; Burmeister, L.; Bartlett, M. A.; Rinaldi. M. G. J. Clin. Microbiol 1988, 26, 14371441. [47] National Committee for clinical Laboratory Standards. 1993. Performance VOL. 41, 1997 antimicrobial susceptibility of Flavobacteria. [48] National Committee for clinical Laboratory Standards. 2002. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Conidium-Forming Filamentous Fungi: Proposed Standard M38-A. NCCLS, Wayne, PA.USA. [49] National Committee for clinical Laboratory Standards. 2003. Method for Antifungal Disk Diffusion Susceptibility Testing of Yeast: Proposed Guideline M44-P. NCCLS, Wayne, PA, USA. [50] National Committee for clinical Laboratory Standards. 1993. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard M7A3.NationalCommittee for Clinical Laboratory Standards, Villanova, Pa. [51] Liebowitz, L. D.; Ashbee, H. R.; Evans, E. G. V.; Chong, Y.; Mallatova, N.; Zaidi, M. D. Gibbs Global Antifungal Surveillance Group 2001, 4, 27-33. [52] Matar, M. J.; Ostrosky-Zeichner, L.; Paetznick,V. L.; Rodriguez. J. R.; Chen, E. J.; Rex. H. Antimicrob Agents Chemother 2003, 47, 1647-1651.

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Table 1: The inhibition zone diameters of some selected compounds against tested organisms:

Sample/ Standard Control:

Inhibition zone diameter (mm/mg sample) Esche Neisseria richia Pseudomonas gonorrhoeae a coli a aeruginosa (G-) a (G-) (G- )

b 0.0 DMSO Tetracycline Antibacterial 30 X(agent) 2 0.0 3 10 4a 0.0 4b 0.0 4c 0.0 5a 17 5b 0.0 5c 15 6 15 7 0.0 8 0.0 9 9 10a 13 10b 16 11 10 13 20 14 0.0 15 19 16a 12 16b 12 16c 10 17 0.0 18 17 19 0.0 20 15 a) G : Gram bacteria

0.0 30 0 .0 10 0 .0 0 .0 0 .0 16 0 .0 16 15 0 .0 0 .0 9 13 15 0 .0 16 0 .0 18 13 11 9 11 16 0 .0 16

Staphyloc occus aureus

0.0 31 0.0 12 0.0 0.0 0.0 15 0.0 15 15 0.0 0.0 11 14 15 0.0 16 11 20 12 12 9 12 17 0.0 15 b) DMSO: Solvent

a

(G+) 0.0 28 0.0 10 0.0 0.0 0.0 14 0.0 14 14 0.0 0.0 10 15 15 0.0 23 0.0 24 11 13 10 15 20 9 18

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Table 2: The inhibition zone diameters of some selected compounds against tested organisms:

Sample

Inhibition zone diameter (mm / mg sample) Aspergillus flavus Candida albicans (Fungus) (Fungus)

Control: DMSOa

0.0

0.0

AmphotericinB Antifungal agent ( Y) 2

31

28

0.0

0.0

4b

0.0

0.0

a)

4c

0.0

0.0

5b

0.0

0.0

12 0.0 32 0.0 12 0.0

16 0.0 9 0.0 17 0.0

7 8 13 14 15 19 DMSO: Solvent

Figure 2

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