INTRODUCTION:

Coumarin moiety has an easy accessibility in the biological systems and is reported to have varied useful biological properties¹. The literature survey also reveals the medicinal importance of coumarins derivatives, which covers the whole gamut of chemotherapeutic agents. Many biologically active synthetic coumarins derivatives have been patented. A collective bibliography of the relevant literature till 1936 is available in E. Merck’s, Jahresbericht². The natural coumarin Daphnin in the plant, protects the plant from harmful effect of short wave radiaitions³. The phytocidal action of coumarins to inhibit germination and root growth was reported⁴ in nature in 1947.

Some coumarins are found to be anthelmintic⁵ agents. Calophyllolids, a complex of coumarins isolated from calophyllum was found to show anti tubercular⁶ activity. Coumarins also acts as a growth regulator⁷ in the number of plants. Some polycyclic coumarins shows anticarcinogenic properties^8,9. Nitrogen mustards were synthesized¹⁰ from 6-substituted coumarins as a potential anticancer agent. Some coumarins like 3-aminocoumarins; methoxy and hydroxyl derivatives of coumarins show antibacterial, antifungal activities^11-16. Recently, it was found that, coumarin dyes¹⁷ were used in optical recording medium.

The coumarin dyes¹⁸ at 0.0001-1% in lubricating oils intended for use in hydraulic fluids or coolents are extremely well suited for indicating or detecting leaks in hydraulic system and cooling system.

Coumarins also show cytostatic activity¹⁹, antitumour²⁰, antioxidative and pro-oxidative effects²¹, antimicrobial²², antidiabetics²³, antileukemic²⁴, antiviral²⁵, antifungal^26,27 properties. Interest in the study of hydrazones has been growing because of their antimicrobial, antituberculosis and antitumour activities^28-35. Pyrazoles are well documented to posses antihypertensive³⁶, antibacterial³⁷, antiinflammatory³⁸, and antitumour³⁹ properties. Certain pyrazoline compounds have been reported to posses biological activities⁴⁰. Some of the pyrazoline derivatives have hypoglycemic⁴¹ and anesthetic properties⁴².

In the present work, novel Hydrazones, Pyrazoles and Pyrazolones are synthesized and check their antibacterial activities.

Scheme I: Synthesis of 8-amino-6-chlorocoumarine from salicyladehyde.

Scheme II: Synthesis of hydrazones from β-keto esters and β-diketone compounds.

Scheme III: Synthesis of pyrazoles and hydrazoles.

Comp ound	R	R’	Comp ound	R	R’
7a	OH	NH₂	8a	OH	CH₃
7b	OH	CH₃	8b	CH₃	CH₃
7c	CH₃	CH₃	8c	CH(CH₃)₂	CH₃
7d	CH(CH₃)₂	CH₃	------	--------	---------

Experimental Work:

The 8-amino-6-chlorocuomarin has been synthesized by known literature methods. Salicylaldehyde (1) was chlorinated by using sulphuryl chloride give compound 2. The compound 2 was nitrated by using fuming nitric acid and acetic anhydride gives compound 3. Compound 3 was heated with sodium acetate and acetic anhydride at 175⁰C for about 8 hrs forming compound 4 which was reduced further by using calcium chloride and zinc powder gives compound 5 (8-amino-6-chlorocoumarin).

1. Synthesis of hydrazones:

a. Diazotization:

Placed 0.01 mole of 8-amino-6-chlorocoumarin in 250 ml three necked round bottom flask fitted with a mechanical stirrer, thermometer and surrounded by an ice-bath. It was dissolve in 25 ml of hydrochloric acid and cooled to 0 – 5⁰C. In another beaker dissolve 0.011 moles of sodium nitrite in 5 ml of distilled water and cooled to 0 – 5⁰C. Add the sodium nitrite solution slowly with stirring in round bottom flask containing amine solution. The stirring was continuous for further 10 minutes.

b. Coupling:

Placed 0.01 mole of β-ketoester or β-diketone in another 250 ml three necked round bottom flask fitted with mechanical stirrer, thermometer and surrounded by ice salt bath. It was dissolved in 150 ml of absolute ethanol and add 5 g of sodium acetate. The temperature was maintained between 0 – 5ºC. The above diazotized mixture of coumarin is added slowly with stirring. After completion of addition, the stirring was continue for another 1 hour at 0 – 5ºC. The resulting reaction mass is then poured into crushed ice and adjusted the pH = 6 by adding dilute NaOH solution. The solid obtained was filtered and dried at 50⁰C till constant weight. Record their yield and m.p (Table-1).

2. Synthesis of pyrazoles and pyrazolones:

a. From Hydrazine hydrate:

Placed 0.01 mole of hydrazone (6a-e) in 150 ml three neck round bottom flask fitted with a mechanical stirrer, thermometer and reflux condenser. It was dissolve in 60 ml methanol and added 0.015 moles of hydrazine hydrate slowly with stirring. The reaction mixture was refluxed for 4 hours in water bath. The resulting reaction mixture was cooled to room temperature and quenched in crushed ice. The separated solid was filtered, washed with water and dried. The crude solid was recrystallized in 50% methanol. Record yield and m.p (Table-2).

Table No 1: m.p., yield and Spectral data of the product of scheme II.

Structure	M.p.⁰C	Yield	NMR data δ (ppm)	IR data ν (cm^-1) and mass
8-amino-6-chlorocoumarin (5) (straw yellow solid)	236	60%	5.77 (2H, bs, 2H); 6.47-6.50 (1H, d); 6.87-6.90 (2H, d), 7.88-7.91 (1H, d)	3485, 3381, 1716, 1562, 1229, 1154, 1083. m/z = 195.4
Pentane-2,3,4-trione-3-[(6-chloro-2-oxo-2H-chromen-8-yl)]hydrazone (6a)	160	70%	2.47 (6H, d); 6.65-6.69 (1H, d); 7.64 (1H, d); 7.84 (1H, d); 8.04-8.07 (1H, d); 14.38 (1H, bs)	3436, 1761, 1740, 1602, 1571, 1170, 1114 m/z = 307.5 (M + 1)
Ethyl(2Z)-2-[(6-chloro-2-oxo-2H-chromen-8-yl)hydrazono]-3-oxobutanoate (6b)	145	50%	1.31 (3H, s); 2.45 (3H, m); 4.32 (2H, m); 6.62-6.65 (1H, d); 7.56-7.72 (2H, m); 8.01-8.04 (1H, d); 12.30 (1H, bs)	3285, 1739, 1530, 1114, 1138 (weak), 836 m/z = 337.6 (M+1)
Diethyl-2-[(6-chloro-2-oxo-2H-chromen-8-yl)hydrazono]-3-malonate (6c)	210	65%	1.28-1.35 (6H, q); 4.25-4.39 (4H, m); 6.63-6.66 (1H, d); 7.54 (2H, s); 8.02-8.05 (1H, d); 12.52 (1H, bs).	3420, 3058, 2985, 2925, 1757, 1712, 1522, 1116, 1097(weak), 863. m/z = 367.8 (M+1)
Ethyl(2Z)-2-[(6-chloro-2-oxo-2H-chromen-8-yl)hydrazono]-3-cyano-3-oxopropanoate (6d)	178	50%	1.35 (3H, t); 4.38-4.40 (2H, q); 6.67-6.70 (1H, d); 7.67-7.73 (2H, dd); 8.05-8.08 (1H, d); 13.22 (1H, bs)	3443, 1753, 1534, 1172, 1111 (weak), 855. m/z = 348.8 (M+1)
(3Z)-5-methylhexane-2,3,4-trione 3-[(6-chloro-2-oxo-2H-chromen-8-yl) hydrazone] (6e)	165	60%	1.19 (6H, d); 2.24 (3H, s); 4.39 (1H, m); 6.65-6.69 (1H, d); 7.64 (1H, d); 7.84 (1H, d); 8.03-8.06 (1H, d); 12.32 (1H, bs)	3432, 1767, 1682, 1520, 1177, 1118 (weak), 864. m/z = 335.9 (M+1)

Table No 2: M.p., yield and Spectral data of the product of scheme III.

Structure	M.p.⁰C	Yield	NMR data δ (ppm)	IR data ν (cm^-1) and mass
(4E)-4-amino-1H-pyrazole-4,5-dione 4-[(6-chloro-2-oxo-2H-chromen-8-yl) hydrazone] (7a)	160	70%	6.16 (2H, s); 6.64-6.67 (1H, d), 7.56 (1H, s); 8.04 (2H, m); 10.78 (1H, s); 13.2 (1H, bs)	3413, 3319, 1737, 1622, 1562, 1177, 1112 (weak), 842. m/z = 306.8 (M+1)
(4Z)-3-methyl-1H-pyrazole-4,5-dione 4-[(6-chloro-2-oxo-2H-chromen-8-yl) hydrazone] (7b)	210	65%	2.67 (3H, s); 6.64-6.72 (1H, m); 7.44-7.96 (3H, m), 10.91 (1H, bs), 13.80 (1H, bs)	3420, 3233, 1731, 1672, 1545, 1119, 1084 (weak), 858. m/z = 305.9 (M+1)
6-chloro-8-[(E)-3,5-dimethyl-1H-pyrazol-4-yl)diazenyl]-2H-chromen-2-one (7c)	178	50%	2.50 (6H, m); 6.64-6.69 (1H, d); 7.72-7.76 (1H, d); 7.90 (1H, d); 8.08-8.11 (1H, d); 13.06 (1H, bs).	3338, 1721, 1579, 1179, 1141, 914. m/z = 303.60 (M+1)
6-chloro-8-[(E)-(5-isopropyl-3-methyl-1H-pyrazol-4-yl)diazenyl]-2H-chromen-2-one (7d)	190	60%	1.45-1.64 (6H, m); 2.41 (3H, s); 3.23 (1H, m); 6.63-6.72 (1H, d); 7.74 (1H, d); 7.89 (1H, d); 8.07-8.14 (1H, d); 13.12 (1H, bs)	3218, 1664, 1520, 1102 (weak), 857. m/z = 331.6 (M+1)
(4Z)-3-methyl-1-phenyl-1H-pyrazole-4,5-dione 4-[(6-chloro-2-oxo-2H-chromen-8-yl) hydrazone] (8a)	145	50%	2.33 (3H, s); 6.65-6.68 (1H, d); 7.21-7.26 (1H, t); 7.44-7.63 (3H, m); 7.83-8.06 (4H, d); 13.51 (1H, bs)	3435, 1741, 1552, 1156, 1116 (weak), 877. m/z = 381.6 (M+1)
6-chloro-8-[(E)-3,5-dimethyl-1-phenyl-1H-pyrazol-4-yl)diazenyl]-2H-chromen-2-one (8b)	165	60%	2.73 (6H, m); 6.31-6.33 (1H, d); 7.35 (5H, m); 7.76-7.78 (1H, d); 7.95 (1H, m); 8.23-8.29 (1H, d)	3433, 1721, 1595, 1176, 1120 (weak), 831. m/z = 379.6 (M+1)
6-chloro-8-[(E)-(5-isopropyl-3-methyl-1-phenyl-1H-pyrazol-4-yl)diazenyl]-2H-chromen-2-one (8c)	138	60%	1.51-1.66 (6H, m); 2.45 (3H, s); 3.42 (1H, m); 6.63-6.72 (1H, d); 7.11-7.56 (5H, m); 7.74 (1H, d); 7.89 (1H, d); 8.07-8.14 (1H, d).	3312, 1749, 1562, 1119 (weak), 859. m/z = 407.6 (M+1)

b. From phenyl hydrazine:

Placed 10 mmol of hydrazone in 150 ml three neck round bottom flask fitted with a mechanical stirrer and reflux condenser. Dissolve it in glycial acetic acid and add 10 mmol of sodium acetate. To this solution add 15 mmol of phenyl hydrazine and the resulting mixture was refluxed for 4 hours in boiling water bath. It was the cooled to room temperature and quenched over crushed ice. The solid separates was filtered, washed with water and dried. The crude solid was recrystallized in methanol.

Biological Studies:

Some derivatives of amino coumarins¹ shows anti microbial activities such as 2-amino benzopyranothiazoles² and pyrazoles^3,4. Hence in the present work, we synthesized these compounds and checked for their antimicrobial activities (Table-3).

The antimicrobial activity of these compounds can be studied by paper disc diffusion method. The paper disc is impregnated with desired compounds; usually 1% of the solution is prepared. The standard Wattmans filter paper disc is dipped in the solution and placed on the medium with the test organisms preferably one gram positive and one gram negative, so that the antibacterial spectrum of the given compound can be found out. The plates are incubated for 24 hours and zone of inhibition, if any is observed and recorded. The compound diffuses in the medium and inhibits the test organism on the medium producing a visual zone of inhibition.

Table-3-

Sr. No.	Name of the test organism	Compound
Sr. No.	Name of the test organism	05	6a	6b	6c	6d	7b	7c	8b
01	S. aureus.	-	+	+	-	-	+	-	+
02	S. typhi.	-	-	-	-	-	-	-	-

Key : + zone of inhibition observed; - zone of inhibition not observed.

The compounds 6a, 6b, 7b and 8b were found to be effective against the given test organism S. aureus, whereas S. typhi was completely resistant towards the given compounds. Of these compounds 6a was found to be the most effective.

RESULT AND DISCUSSION:

Salicylaldehyde (1) was chlorinated by using sulphuryl chloride give compound 2. The compound 2 was nitrated by using fuming nitric acid and acetic anhydride gives compound 3. Compound 3 was heated with sodium acetate and acetic anhydride at 175⁰C for about 8 hrs forming compound 4 which was reduced further by using calcium chloride and zinc powder gives compound 5 (8-amino-6-chlorocoumarin). The amino coumarine can be diazotized and then treated with active methylene compounds such as (1,3-diketone, 3-ketoester, etc) forming series of hydrazones (6a-e) with moderate yield. The NH proton of hydrazones is in conjugation with carbonyl groups so can be easily condensed with hydrazine hydrate and phenyl hydrazine hydrochloride forming pyrazoles (7a-d) and hydrazoles (8a-c).

The compounds are then tested for their antimicrobial activity, some of them are effective (6 is most effective) against the given test organism S. aureus, whereas S. typhi was completely resistant towards the given compounds.

ACKNOWLEDGMENT:

The authors are grateful to the Principal, Patkar-Varde College, Goregaon (W), Mumbai 60, India, for his constant encouragement and the spectral analysis. We are also thankful to HOD, Life Science Laboratory, University of Mumbai, Mumbai 01 for the antimicrobial analysis.

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Received on 10.08.2011 Modified on 17.08.2011

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