Microwave Mediated Synthesis, Characterisation and Biological activity of Certain New Mannich Bases of Isatin and Coumarin.

N. Habeela Jainab, P. Sivachidambaram, J. Yuvaraju, K. Jesintha Beyatricks, Bincy Raj

Hillside College of Pharmacy and Research Centre, Bangalore-62.

*Corresponding Author E-mail: habi.asheesh@gmail.com

ABSTRACT:

In the present study Schiff’s bases of isatin were synthesized by condensation of the keto group of isatin with the 4-methyl coumarinyl-7-oxyacetic hydrazide. The N-mannich bases of the above Schiff’s bases were synthesized by reaction of acidic imino group of isatin with formaldehyde and various primary or secondary amines. In this current study, microwave assisted parallel synthesis along with the conventional method was opted for synthesizing Schiff’s and Mannich bases of isatin and their efficacies based on the percentage yield and consumption of time for synthesis by each method were compared. The compounds synthesized were screened against some selected gram (+)ve and gram (-)ve microorganisms and activities were compared with a standard antibiotic. Similarly the prepared compounds were also subjected for antifungal screening, where activities were compared with standard antifungal agent. The compounds synthesized were also evaluated for antioxidant activity and compared with standard drug (ascorbic acid). Antioxidant activity was done by DPPH method.

KEYWORDS: Mannich bases, coumarin, isatin, antimicrobial, antioxidant.

INTRODUCTION:

Mannich bases are important compounds owing to their wide range of biological and industrial applications. They are also employed as intermediates in chemical synthesis and polymer chemistry. Several important therapeutic compounds have been synthesized via the Mannich reaction. Schiff’s and Mannich bases of isatin possess versatile activities like antibacterial[1], antifungal[2], antiviral[3], anticancer[4], antileishmanial[5], antitubercular [6] activities etc. References show heterocyclic compounds containing coumarin to exhibit broad spectrum of activity such as antimicrobial [7], antiviral [8], antitubercular[9], antipyretic [10], antiinflammatory[11] and antioxidant[12] activities. In the light of these interesting biological activities the present work was carried out with view of synthesizing some new isatin based Mannich bases from coumarin. Nowadays microwave mediated synthesis has gained importance in organic synthesis. The use of such non-conventional reaction conditions possess salient features like short reaction time compared to the conventional heating, ease of work-up and selectivity, high percentage yield etc.

So in this current study, microwave assisted parallel synthesis along with the conventional method was opted for synthesizing Schiff’s and Mannich bases of isatin and their efficacies based on the percentage yield and consumption of time for synthesis by each method were compared. The structure of the compounds synthesised has been confirmed by IR, ¹H NMR and mass spectral data. All the compounds were screened for antimicrobial and antioxidant activity.

MATERIALS AND METHODS:

All the amines were obtained from Hi-media and Loba chem. The solvents and chemicals were procured from S.D Fine Chem. Ltd., Fischer in organics Ltd. All the compounds, procured were purified and dried, whenever necessary before use, following standard methods. Melting points were determined in open capillary tubes and were uncorrected. Purity of the compounds were routinely checked by TLC using plates coated with silica-gel-G. Iodine vapour was used as visualizing agent. UV spectras were recorded on Jasco-V-530. IR spectras were recorded on Jasco FT/IR-410 at pharmaceutical analytical laboratory, College of Pharmacy, SRIPMS, Coimbatore. Microwave synthesis was carried out using DAEWOO KOG-370A. Mass spectra were recorded at QUEST Research and Training Institute, Bangalore. PMR spectra were recorded at Sastra University, Tanjore.

Scheme-II

Microwave method

Table No. 1, Amines

Amines	Compound Code
Piperidine	V a
Morpholine	V b
Dimethylamine	V c
Diethylamine	V d
N-methylaniline	V e
N-methylpiperazine	V f
4-bromoaniline	V g
4-chloroaniline	V h
4-fluoroaniline	Vi

Scheme – I, Conventional method:

Synthesis of 4-methyl-7-hydroxycoumarin (I)[13]:

Place 1 litre of concentrated sulphuric acid in a 3-litre three necked flask fitted with a thermometer, mechanical stirrer and a dropping funnel. Immerse the flask in an ice bath. When the temperature falls below 10⁰C, add a solution of 100g (0.91mol) of resorcinol in 134g (130.5ml, 1.03mol) of redistilled ethylacetoacetate drop wise and with stirring. Maintain the temperature below 10^oC by means of an ice-salt bath during the addition. Keep the reaction mixture at room temperature for about 18 hrs, then pour it with vigorous stirring into a mixture of 2 kg of crushed ice and 3 litres of water, collect the precipitate by suction filtration and wash it with three 25 ml portion of cold water. Dissolve the solid in 1500ml of 5% sodium hydroxide solution, filter and added 2M sulphuric acid (about 550ml) with vigorous stirring until the solution is acid to litmus. Collect the crude 7-hydroxy 4-methyl coumarin by filteration at the pump, wash it with four 25ml portion of cold water and dry at 100⁰C. The product obtained was recrystallised using 95% ethanol. M.P. 185⁰C. The purity of the compounds were determined by single spot on the TLC plates. The solvent system used was Toluene: Ethyl acetate: Formic acid (5:4:1).

Synthesis of 7-carbethoxymethoxy-4-methylcoumarin (II)[14]:

A mixture of I (4.75g, 27 mmoles), ethylchloroacetate (4.1ml, 33 mmoles), potassium carbonate (4.50g, 33 mmoles) and dimethyl formamide (28 ml) was stirred for 20 hr. The reaction mixture was poured on to ice water. The solid thus separated was filtered, dried and crystallized from methanol to give II. M.P. 88 - 90°C. The purity of the compounds were determined by single spot on the TLC plates. The solvent system used was Toluene: Ethyl acetate: Formic acid (5:4:1).

Synthesis of 4-methylcoumarinyl-7-oxyacetichydrazide (III)[15]:

To a solution of compound II (2.62g, 0.01 mole) in ethanol (20ml) was added hydrazine hydrate (0.9ml, 0.014 mole) and the reaction mixture refluxed for 3 hrs. The solvent was concentrated to a small volume, on cooling it gave a solid mass which was filtered and crystallized from methanol to gave a buff coloured compound (III). M.P. 198-200°C. The purity of the compounds were determined by single spot on the TLC plates. The solvent system used was Toluene: Ethyl acetate : Formic acid (5:4:1).

Synthesis of Schiff’s Base (IV)[16]:

A mixture of III (12.4g, 0.05mole) and isatin (7.35g, 0.05 mole) in 40ml of dioxan and a few drops of glacial acetic acid was refluxed for about 2 hours and then cooled. Yellow solid separated was filtered, dried and recrystallised from dimethyl formamide to give (IV). M.P.258-262⁰C. The purity of the compounds were determined by single spot on the TLC plates. The solvent system used was Toluene: Ethyl acetate: Formic acid (5:4:1).

Synthesis of Mannich Bases V (a-i)[16]:

The compound IV (7.54g, 0.02 mole) was suspended in minimum quantity of dimethyl formamide. To that solution, slightly more (1.41ml, 0.02mole) formaldehyde and (0.025 mole) either primary or secondary amine was added with vigorous stirring. The reaction mixture was heated on a water bath for one hour, and then cooled. The solid thus separated was filtered, dried and recrystallised from dioxan to give V (a-i). The purity of the compounds were determined by single spot on the TLC plates. The solvent system used was Toluene: Ethyl acetate: Formic acid (5:4:1).

Microwave method:

Synthesis of Schiff’s Base (IV)[17]:

A suspension of III (1.245g, 0.005 mol) and Isatin (0.7356g, 0.005 mol) in dioxan and glacial acetic acid (one drop) was irradiated at 80% power for 3 minutes. The solid so obtained was recrystallised from dimethyl formamide to give IV.

Synthesis of Mannich Bases V (a-i)[17]:

A suspension of IV (0.9425g, 0.0025 mol) in dimethyl Formamide, Formaldehyde (0.5ml) and either primary or secondary amine (0.0025 mol) was irradiated at 80% power for 2 minutes. The solid product separated was recrystallised using dioxan to give V (a-i). The characterization data of synthesized compounds are given in Table 2.

Table No:2, Characterization data of isatin based mannich bases of coumarin

S. No	Comp code	R	Molecular Formula	Mol wt.	% Yield		Reaction Time		M.P ^0C	Rf Value
					Conventional	Micro wave	Conventional hr	Micro Wave min
1	V a		C26 H26 N4 O5	475	72.26	88.58	1	2	268	0.514
2	V b		C25 H24 N4 O6	477	68.72	85.51	1	2	170	0.500
3	Vc		C23 H22 N4 O5	434	67.18	81.91	1	2	160	0.476
4	V d		C25 H26 N4 O5	462	65.39	79.56	1	2	182	0.461
5	V e		C28 H24 N4 O5	497	62.57	76.43	1	2	260	0.449
6	V f		C27 H25 N5 O5	490	69.79	83.38	1	2	243	0.450
7	V g		C27 H21 Br N4O5	561	64.83	77.03	1	2	262	0.437
8	V h		C27 H21 Cl N4O5	517	64.24	76.85	1	2	258	0.419
9	V i		C27 H21 FN4 O5	500	60.75	74.30	1	2	264	0.427

Solvent system used for TLC: Toluene: Ethylacetate: Formic acid (5:4:1)

Solvent used for recrystallization: Dioxan

Va: IR(KBr)cm^-1: 3266.82 (N-Hstr), 2814.60 (-CH₂ str), 1729.83(C=O str of coumarin), 1693.19 (C=O str of indole), 1623.77 (C=O str of amide), 1589.06 (C=N str), 1524.45 (C=C str), 1151.29 (C-N str), 749.20 (Arm C-H bending), ¹H NMR (DMSO-d₆) δ ppm: 2.41 (3H, -CH3), 2.67 (4H, -CH2-N-CH2-), 4.51 (2H, -OCH2-), 6.26 (1H, =CH-), 7-7.8 (8H, Aromatic protons & NH protons), Mass(m/z): 474(M-1)^-1, 376, 175, 161, 132.

Vb: IR(KBr)cm^-1: 3284.18 (N-H str), 2809.78 (-CH₂ str), 1728.87 (C=O str of coumarin), 1698.02 (C=O str of indole), 1614.13 (C=O str of amide), 1582.31 (C=N str), 1519.63(C=C str), 1150.33 (C-N str), 764.63 (Arm C-H bending), ¹H NMR (DMSO-d₆) δ ppm: 2.42 (3H, -CH₃), 2.52 (4H, -CH2-N-CH2-), 4.52 (2H, -OCH2-), 6.27 (1H, =CH-), 7-7.8 (8H, Aromatic protons & NH protons).

Antimicrobial studies[18,19]:

Antimicrobial activity was determined using disc diffusion method by measuring the inhibition zone in mm. All the compounds v (a-i) were screened in vitro for antibacterial activity against gram positive organisms Staphylococcus aureus, Bacillus subtilis, gram negative organisms Escherichia coli, Pseudomonas aeruginosa and antifungal activity against Candida albicans and Aspergillus niger at 500 μg/disc and 250 μg/disc. Standard antibacterial drug ciprofloxacin (5 μg/disc) and antifungal drug fluconazole (25μg/disc) were also tested under similar conditions and these compounds showed highest activity against all these organisms tested. The data of compounds with their activity are given in Table 3.

Table No: 3, Data of Antibacterial Screening.

S. No	Compound	Gram(+) ve bacteria				Gram (-) ve bacteria
		Staph aureus NCIM 5021		Bacillus subtilis NCIM 2010		Eischerichia Coli NCIM 2911		Pseudomonas aeruginosa NCIM 5029
		500 mg/ disc	250 mg/ disc	500 mg/ disc	250 mg/ disc	500 mg/ disc	250 mg/ disc	500 mg/ disc	250 mg/ disc
1	V a	-	-	12	10	-	-	-	-
2	V b	-	-	15	12	14	11	-	-
3	V c	-	-	-	-	-	-	-	-
4	V d	-	-	-	-	-	-	-	-
5	V e	-	-	-	-	-	-	-	-
6	V f	-	-	-	-	-	-	-	-
7	V g	-	-	12	<10	-	-	-	-
8	V h	-	-	13	<10	11	<10	-	-
9	V i	-	-	15	13	15	12	-	-
10	Std Ciprofloxacin 5 mg/disc	32 mm	-	36 mm	-	38 mm	-	33 mm	-

Anti-oxidant activity [20,21]:

The compounds synthesized were evaluated for antioxidant activity and compared with standard drug (ascorbic acid). The activity was evaluated using the DPPH method. Stock solution of (1.0 mg/ml) test compounds prepared using 10% methanolic solution of DMSO were diluted to get various concentrations of 0.5mM, 0.2mM, 0.1mM using methanol. After this 1.5ml of various concentration of test compound were added to 1.5ml of 0.2mm of DPPH radical in methanol (final concentration of DPPH was 0.1mm and final concentration of test compounds were 0.25mM, 0.1mM, 0.05Mm). The mixture was shaken vigorously and allowed to stand for 30min; absorbance at 517nm was determined and the percentage activity was calculated. Scavenging activity (AA%)={[(Ab+As)-Am]/Ab}X100%, Ab : absorbance of 0.1mM DPPH methanol solution at 517nm, As : absorbance of various concentration solution of test compounds at 517nm, Am : absorbance of mixture methanol solution at 517nm.

RESULTS:

All compounds were in conformity with the structures envisaged. The structures were proved on the basis of spectral data. Mannich bases of Isatin are known to possess a wide variety of biological activities. Hence the newly synthesized compounds were screened for antimicrobial and antioxidant activities. Among the newly synthesisedcompounds, B.subtilis was found to be moderately sensitive to the compounds V a (piperidinyl derivative), V b (morpholinyl derivative), V g (4- bromophenyl amino derivative), V h (4- chlorophenyl amino derivative) and V i (4- fluorophenyl amino derivative) at 500μg/disc concentration. B.subtilis was moderately sensitive to compounds V b (morpholinyl derivative) and V i (4- fluorophenyl amino derivative) at 250μg/disc concentration of the test solutions. E.coli was found to be moderately sensitive to the compound V b (morpholinyl derivative) at 500μg/disc concentration and was moderately sensitive to the compound V i (4- fluorophenyl amino derivative) even at 250μg/disc concentration. None of the compounds showed activity against S.aureus and P.aeruginosa. All the newly synthesised compounds were found to be inactive against both the antifungal organisms (Candida albicans and Aspergillus niger) at the concentrations of 500 mg/ disc and 250 mg/ disc. In antioxiadant studies, compounds V a (piperidinyl derivative), V b (morpholinyl derivative), V f (N-methyl piperazinyl derivative), V g (4-bromophenyl amino derivative), V h (4-chlorophenyl amino derivative) showed free radical scavenging activity in the range 40-70%, while rest of the compounds showed less than 40% activity. Compound V i (4- fluorophenyl amino derivative) exhibited the maximum free radical scavenging activity at final concentrations of 0.25mM, 0.1mM and 0.05mM.

Table No:4, DPPH Free Radical Scavenging Activity Of Test Compounds.

Compound Code	Concentration mM	Absorbance			AA%= {[(Ab+As)-Am]/Ab}X100%
		As	Am	Ab
V a	0.25	0.312	0.564	DPPH (0.1mM) = 1.2566	65.53
	0.1	0.1120	0.5997		61.74
	0.05	0.0986	0.6375		57.14
V b	0.25	0.1180	0.6037		61.34
	0.1	0.1105	0.6754		54.13
	0.05	0.1085	0.7183		51.47
V c	0.25	-0.0094	0.6382		48.46
	0.1	-0.0082	0.6926		44.23
	0.05	-0.0107	0.7540		39.48
V d	0.25	0.0062	0.8698		31.27
	0.1	0.0033	0.9154		27.41
	0.05	0.0018	0.9657		23.29
V e	0.25	0.0502	0.9018		32.22
	0.1	0.0482	0.9761		26.18
	0.05	0.0423	1.0617		22.37
V f	0.25	0.0673	0.6566		53.10
	0.1	0.0423	0.6913		48.37
	0.05	0.0286	0.7345		43.58
V g	0.25	0.0302	0.4675		65.19
	0.1	0.0192	0.5033		61.47
	0.05	0.0092	0.5691		55.47
V h	0.25	0.0256	0.4378		67.22
	0.1	0.0154	0.4775		63.26
	0.05	0.0064	0.5365		57.81
V i	0.25	0.0310	0.4025		70.15
	0.1	0.0198	0.4561		65.27
	0.05	0.0065	0.5123		59.67

Free Radical Scavenging activity of standard, Ascorbic acid = 96.4%

CONCLUSION:

Based on the results of the synthetic work, characterization data, antimicrobial screening and screening of free radical scavenging activity, following conclusions were made. Microwave assisted method of synthesis proved to be an efficacious synthetic method in respect of its low reaction time, increased percentage yield and ease of work up procedures when compared with conventional method of synthesis. The use of piperidine as secondary amine in the synthesis of Mannich bases increased the yield of the products as observed from the highest percentage yield of compound V a. From the results of antibacterial screening, it was observed that, presence of morpholinyl sustituent in Mannich bases of Coumarin derivative increased the sensitivity of B.subtilis and E.coli to the synthesized compounds as evident from antimicrobial activity of compound V b. Mannich base derivatives of Coumarin having 4-fluorophenyl amino substitutent showed highest free radical scavenging capacity for DPPH free radicals, then rest of the compounds in respective series as seen with compounds V i. Of the 9 compounds synthesized, compound V b can be chosen as lead moiety for the development of an effective antibacterial agent. Similarly compounds V i can be taken up as lead moiety for the development of an ideal antioxidant agent.

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Received on 17.12.2013 Modified on 11.01.2014

Accepted on 17.01.2014 © AJRC All right reserved

Asian J. Research Chem. 7(2): February 2014; Page 176-181