INTRODUCTION:

Coumarin and its derivatives have extensive and diverse applications. Naturally occurring coumarins are found in many plants, notably in high concentration in tonka bean, woodruff, lavender, licorice, strawberries, apricots, cherries, cinnamon, sweet clover and bison grass. Coumarin and its derivatives possesses a wide spectrum of biological activity. ^{[1] [2] [3] [4]}Many of the compounds have proved to be active as antitumor ^[5], antibacterial ^[6], antifungal ^[7], anticoagulant ^[8] and anti-inflammatory ^{[9] [10] [11]}. In addition, these compounds are used as additives to food and cosmetics. ^[12]Coumarins can be synthesized by various methods including the Perkin, ^[13] Pechmann, ^[14] Knoevenagel, ^[15] Reformatsky ^[16] and Wittig. ^[17]

In our previous study we have utilized Pechmann condensation for synthesis of Coumarin nucleus, since it proceeds from very simple starting materials and gives good yield of variously substituted Coumarins. the Pechmann reaction has been the most widely used method since it proceeds from very simple starting materials and gives good yields of variously substituted Coumarins.^[18]Substituted phenols are condensed with β- keto esters in the presence of an acid to afford Coumarins. Various acids such as H₂SO₄, H₃PO₄, CF₃COOH, p-toulene sulphonic acid, P₂O₅, POCl₃ and metal halides have been used to carry out this reaction. ^[19] Several acid catalysts have been used in the von Pechmann reaction including sulfuric acid, aluminium chloride,^[20] phosphorus pentoxide,^[21] trifluoroacetic acid^[22] and many more.^[23] However, these catalysts have to be used in excess; for example, sulfuric acid in ten to twelve equivalents, trifluoroacetic acid in three to four equivalents^[24] and phosphorous pentoxide is required in a five-fold excess. Moreover, in some cases, mixtures of substituted phenols, β-ketoesters and the acidic catalyst were allowed to stand overnight or for a number of days (depending on their reactivity) or were heated above 150°C, and undesired side-products such as chromones, in addition to coumarin were isolated.^[25] As a result, the disposal of excess acid waste leads to environmental pollution. Consequently, there is scope for further development of milder reaction conditions, increased variation of the substituents in both components and better yields. In our study we have utilized stannous chloride dehydrate to condense 2 amino phenols with ethyl acetoacetate to obtain Coumarin derivative. Later is converted to Schiff bases^[26]by reacting with various Aromatic aldehydes in presence of ethanol. In this study same method is used for synthesis of Coumarin derivatives. Also this paper emphasis on synthesis of Coumarin derivatives by using microwave irradiation technique because of green chemistry^{[27] [28] [29]}.The synthesized compounds were evaluated for Anti-inflammatory activity.

MATERIALS AND METHODS:

Scheme for Synthesis:

Schiff’s bases of 8- amino-4- methyl-2H-chromen-2-one

Figure No. 1 Scheme for Synthesis Coumarin Derivatives

Thin layer chromatography was used to assess the course of reactions and the purity of the intermediates and final compounds, giving a single spot on TLC plate (Silica gel G), using various solvent systems. Visualization of the compounds on chromatographic plates was done by exposure to iodine vapors.

Table no.1 List of Aromatic aldehyde

Compound	Aromatic Aldehyde	Compound	Aromatic Aldehyde
II _a	C₆H₅	II _f	(CH₃)₂N-C₆H₄
II _b	3 Cl - C₆H₄	II _g	2 NO2-C₆H₄
II _c	3, 4 Cl- C₆H₃	II _h	4 CH₃O-C₆H₄
II _d	2 OH-C₁₁H₇	II _i	2 OH-C₆H₄
II _e	C₆H_5-CH₂CH=CH	II _j	4 OH-C₆H₄

Melting points of the synthesized compounds were taken on melting point apparatus and are uncorrected.

Infra-red (IR) spectra of the intermediates and final compounds were recorded on Jasco FTIR-410 spectrophotometer using KBr pellet method and ¹H NMR spectra of intermediates and final compounds were recorded in CDCl₃ solution and proton chemical shift are relative to tetramethylsilane as internal standard.

Chemicals:

All chemicals and solvents were procured from commercial sources, purified and dried using standard procedures from literature whenever required. Chemicals used for the synthesis are 2-Amino phenol, Ethyl acetoacetate, Stannous chloride dehydrate, Ethanol, Aromatic aldehydes, Silica gel G, DMSO, Chloroform, enlisted below with their manufacturer mentioned in parentheses. The chemicals were procured from various companies like Research-Lab fine Mumbai, Thomas Baker Pvt. Ltd. Mumbai, Research Lab Poona, S. D. Fine Chem. Ltd. Mumbai, Loba Chemie Pvt. Ltd., Mumbai.

General Procedure for synthesis of Schiff’s bases:

1. Synthesis of 8-amino-4-methyl-2H- chromen-2-one (I)

2. Synthesis of Schiff’s bases of 8-amino-4-methyl-2H- chromen-2-one (II _a-j)

STEP I: Synthesis of 8-amino-4-methyl-2H- chromen-2-one (I) ^{[31] [32]}:

A mixture of 2-amino phenol (10mmol), β-keto esters i. e. ethyl acetoacetate (10mmol) and stannous chloride dehydrate (10 mol %) was refluxed for 2 hrs at 45 ⁰ C on Heating mental. Then, the reaction mixture was cooled to room temperature, and it was poured in ice cold water and stirred for 10min. The precipitated product was collected by ﬁltration, washed with water and dried. The product obtained was recrystallized from appropriate solvent (i. e. ethanol) to afford corresponding pure Coumarin product.

Table No. 2 Physicochemical data for the compound I:

Compd. No.	M. P.	Molecular Formula	% Yield	R_f value
I	172 ⁰C	C₁₀H₉NO₂	90%	0.76

STEP II: Synthesis of Schiff’s bases of 8-amino-4-methyl-2H- chromen-2-one (II _a-j):

Conventional Synthesis:

In this method, compound (I) 8-amino-4-methyl-2H- chromen-2-one (0.01mol) and substituted aromatic aldehydes (a-j) (0.01mol) were taken in absolute alcohol (30 ml) in round bottom flask. The reaction mixture was refluxed for 5 h. Completion of reaction was monitored by using TLC. After completion of reaction, mixture was cooled and poured into crushed ice. The precipitate obtained was recrystallized using ethanol.

Microwave Synthesis:

Compound (I) 8-amino-4-methyl-2H- chromen-2-one (0.01 mol) and substituted aromatic aldehyde (a-j) (0.01 mol) were taken in two necked flasks, absolute alcohol (20 ml) was added to it. The reaction mixture was stirred for some time and irradiated with microwaves at 40 %, 280 W. After completion of reaction, after specific time given Table 3 depending on the derivative, the mixture was cooled and poured onto crushed ice. The precipitate obtained was recrystallized using ethanol.

Table No. 3 Parameters for Microwave synthesis

Compound	Power level	Output in Watts	MW % power	Time (min)
2a	4	280	40	4
2b	4	280	40	7
2c	4	280	40	5
2d	4	280	40	9
2e	4	280	40	8
2f	4	280	40	7
2g	4	280	40	8
2h	4	280	40	9
2i	4	280	40	5
2j	4	280	40	5

Table No. 4 Physicochemical data for the compound II _(a-j):

Compd. No.	M. P.	Molecular Weight	% Yield		R_f value
Compd. No.	M. P.	Molecular Weight	Conventional	Microwave	R_f value
II _a	102 ⁰C	263.29	65	75	0.73
II _b	167 ⁰C	297.73	79	86	0.68
II _c	132 ⁰C	332.18	71	82	0.75
II _d	70 ⁰C	331.36	64	72	0.7
II _e	142 ⁰C	289.32	77	83	0.88
II _f	68 ⁰C	306.35	57	69	0.67
II _g	136 ⁰C	308.28	69	76	0.64
II _h	182 ⁰C	293.31	73	81	0.84
II _i	242 ⁰C	279.29	60	70	0.87
II _j	220 ⁰C	279.29	76	87	0.8

(Mobile phase: Tolune: Acetone, 5:4)

Spectral Data:

4-methyl-8-{[(Z)-phenylmethylidene] amino}-2H-chromen-2-one (2a), IR ν: 3065, 3033, 2831, 1721, 1600, 1495, 1383, 1272, 1111, 746, 713.1H-NMR (CDCl3, 300 MHz): δ 2.45 (s, 3H, CH3), δ 8.517 (s, 1H, CH), δ 7.431 (d, 1H, CH), δ 7.513 (t, 1H, CH), δ 7.545 (s, 1H, CH), δ 7.368 (d, 1H, CH), δ 7.260 (s, 1H, CH), δ 7.66 (s, 1H, CH).

8-{[(Z)-(3-chlorophenyl) methylidene] amino}-4-methyl-2H-chromen-2-one (2b) IR ν: 3073, 2923, 1396, 1596, 1574, 1483, 1417, 1384, 1303, 1263, 1138, 808, 750, 720. 1H-NMR δ 2.079 (s, 3H, CH₃), δ 9.986 (s, 1H, CH), δ 7.647 (s, 1H, CH), δ 3.961 (d, 1H, CH), δ 7.431 (t, 1H, CH), δ 7.609 (d, 1H, CH), δ 8.014 (d, 1H, CH), δ 8.095 (s, 1H, CH), δ 7.867 (t, 1H, CH), δ 7.582 (d, 1H, CH).

8-{[(Z)-(3, 4-dichlorophenyl) methylidene] amino}-4-methyl-2H-chromen-2-one (2c) IR ν: 2974, 2925, 1701, 1588, 1384, 1192, 1132, 818, 752. 1H-NMR δ 2.230 (s, 3H, CH₃), δ 9.953 (s, 1H, CH) δ 7.476 (t, 1H, CH) δ 7.413 (s, 3H, CH₃) δ 7.438 (d, 1H, CH), δ 7.584 (d, 1H, CH), δ 7.962 (s, 1H, CH), δ 7.737 (d, 1H, CH), δ 7.962 (d, 1H, CH), δ 7.650 (d, 1H, CH).

4-methyl-8-{[(1Z,2E)-3-phenylprop-2-en-1-ylidene]amino}-2H-chromen-2-one (2d); IR ν: 3059, 3026, 2924, 2852, 1676, 1624, 1596, 1575, 1450, 1384, 1159, 1124, 972, 749, 688. 1H-NMR δ 2.363 (s, 3H, CH₃), δ 9.890 (s, 1H, CH) δ 7.454 (m, 5H, CH), δ 7.517 (s, 1H, CH), δ 7.024 (d, 1H, CH), δ 7.574 (t, 1H, CH), δ 7.860 (d, 1H, CH), δ 7.316 (t, 1H, CH), δ 6.670 (d, 1H, CH).

8-({(Z)-[4-(dimethylamino) phenyl] methylidene} amino)-4-methyl-2H-chromen-2-one (2e) IR ν: 3048, 2917, 2821, 1660, 1597, 1548, 1533, 1483, 1432, 1371, 1313, 1231, 1064, 864, 812. 1H-NMR δ 3.168 (s, 3H, CH₃), δ 9.740 (s, 1H, CH), δ 7.179 (s, 1H, CH), δ 6.724 (d, 1H, CH), δ 6.945 (t, 1H, CH), δ 7.040 (d, 1H, CH), δ 7.584 (d, 1H, CH), δ 7.962 (d, 1H, CH).

(8-({(Z)-[4-(dimethylamino) phenyl] methylidene} amino)-4-methyl-2H-chromen-2-one)

(2f) IR ν: 3048, 2917, 2821, 1660, 1597, 1548, 1483, 1432, 1371, 1231, 1164, 824, 812. 1H-NMR δ 3.168 (s, 3H, CH₃), 6.724 (d, 1H, CH), 6.945 (t, 1H, CH), 7.040 (d, 1H, CH), 7.179 (s, 1H, CH), 7.584 (d, 1H, CH), 7.962 (d, 1H, CH), 9.740 (s, 1H, CH).

(4-methyl-8-{[(Z)-(3-nitrophenyl) methylidene] amino}-2H-chromen-2-one) (2g) IR ν:

3072, 2925, 2854, 1702, 1614, 1531, 1483, 1453, 1355, 1201, 1135, 808, 731. 1H-NMR δ 2.227, 7.536 (d, 1H, CH), 7.695 (s, 1H, CH), 7.743 (t, 1H, CH), 8.166 (d, 1H, CH), 8.224 (d, 1H, CH),8.349 (s, 1H, CH).

(8-{[(Z)-(4-methoxyphenyl) methylidene] amino}-4-methyl-2H-chromen-2-one) (2_h) IR ν: 2930, 2840, 1682, 1599, 1577, 1509, 1384, 1262, 1216, 1160, 834, 757. 1H-NMR δ 3.895 (s, 3H, CH₃), 6.745 (d, 1H, CH), 7.023 (d, 1H, CH), 7.516 (s, 1H, CH), 7.575(t, 1H, CH), 7.858 (d, 1H, CH), 9.980 (s, 1H, CH).

(8-{[(Z)-(2-hydroxyphenyl) methylidene] amino}-4-methyl-2H-chromen-2-one)(2 _i)IR ν: 3417, 1607, 1588, 1541, 1464, 1440, 1385, 1298, 1259, 1151, 1127, 838, 757. 1H-NMR δ 1.661 (s, 3H, CH₃), 6.815 (d, H, CH), 6.886 (t, H, CH), 6.997(t, H, CH), 7.206 (d, H, CH), 7.425 (t, H, CH), 7.562 (d, H, CH), 7.609 (s, H, CH), 10.595 (s, H, CH), 11.02 (s, H, OH).

(8-{[(Z)-(4-hydroxyphenyl) methylidene] amino}-4-methyl-2H-chromen-2-one) (2 _j) IR ν: 3402, 2924, 1705, 1644, 1599, 1531, 1512, 1455, 1485, 1384, 1282, 1257, 1157, 1109, 836, 752. 1H-NMR δ 2.464 (s, 3H, CH₃), 6.266 (d, H, CH), 6.715 (d, H, CH), 6.736 (d, H, CH), 6.841 (t, H, CH), 6.942 (s, H, CH), 7.631 (d, H, CH), 9.764 (s, H, CH), 11.020(s, H, OH).

Pharmacological Screening:

Method utilized:

Carrageenan induced Rat paw edema method.

No. of animals used in each Group: - 6

Gender: - Male/Female

Wt. of Animals: - 120gm – 260gm.

Standard used: - Diclofenac sodium 5mg/ml

Preparation of Drug solution:

All the compounds were dissolved in DMSO to get concentration of 10mg/kg and 20mg/kg. Diclofenac sodium was dissolved in DMSO to get concentration of 5 mg/kg, this was used as a standard for determination of anti-inflammatory activity.

Procedure:

Animals were divided into twelve groups (n=6) starved overnight with water ad libitum prior to the day of experiment. The control group had given vehicle orally, while other group given test drug and standard drug respectively. The test drug was given at dose 10 mg/kg and 20 mg/kg. Left paw was marked with ink at the level of lateral malleolus; basal paw volume was measured plethysmographically by volume displacement method using Plethysmometer (UGO Basile 7140) by immersing the paw till the level of lateral malleolus. The animals were given drug treatment. One hour after dosing, the rats are challenged by a subcutaneous injection of 0.1ml of 1percentage solution of carrageenan into the sub-plantar side of the left hind paw. The paw volume was measured again at 1, 2, 3, 4 & 5 hours after challenge. The increase in paw volume was calculated as percentage compared with the basal volume. The difference of average values between treated animals and control group is calculated for each time interval and evaluated statistically. The percent Inhibition was calculated using the formula as follows.

percentage edema inhibition = [1- (Vt / Vc)] X 100

V_tand V_care edema volume in the drug treated and control groups respectively.

Observations:

Observations were taken as a paw volume and percentage inhibition was calculated and recorded in Table No. 5

Table No. 5 Percentage Inhibition of Compounds at Dose 10 and 20 mg/kg.

Compound	Percentage Inhibition at 10 mg/kg				Percentage Inhibition at 20 mg/kg
Compound	2 hr	3 hr	4 hr	5 hr	2' hr	3' hr	4' hr	5' hr
2a	28.78	43.83	36.02	34.3	30.3	45.89	40.99	32.55
2b	18.18	36.3	32.29	31.39	28.78	43.83	36.02	34.3
2c	15.9	28.08	24.22	23.25	16.66	30.82	33.54	31.39
2d	14.39	30.82	29.19	27.9	14.39	28.08	28.57	27.32
2e	32.75	41.78	44.09	40.01	39.39	43.15	45.96	37.79
2f	3.78	6.84	9.93	9.88	7.57	10.27	11.18	10.46
2g	9.84	15.75	15.52	15.11	11.36	23.28	17.39	15.69
2h	12.12	14.38	17.39	16.86	13.36	17.8	17.39	16.27
2i	20.45	35.61	36.02	34.88	21.96	35.61	36.64	35.46
2j	21.21	34.93	30.43	23.06	32.57	41.78	34.78	32.55

RESULT AND DISCUSSION:

All the Coumarin derivatives are synthesized as per the designed scheme by using conventional as well as microwave irradiation technique. All the compounds were synthesized with satisfied yield, and characterized by IR and H¹NMR. Derivatives were screened for anti-inflammatory activity by using carrageenan induced paw edema method. Observed results were tested by using one way ANNOVA and all the results were found statistically significant with P=0.005.

CONCLUSION:

All the Coumarin derivatives were synthesized as per scheme shown in figure All the novel compounds were evaluated for anti-inflammatory activity by using Carrageenan induced rat paw edema model. Compound 2a showed maximum anti-inflammatoryactivity at both doseswhen compared with standard Diclofenac Sodium.

ACKNOWLEDGMENT:

The authors wish to thank Sophisticated Analytical Instrument Facility Division, CDRI, Lucknow; for providing ¹H-NMR spectral data. Authors also wish to extend their gratitude to Dean, Krishna Institute of Pharmacy, Karad; for providing necessary facilities.

CONFLICT OF INTEREST STATEMENT:

The authors report noconflict of interest.

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Received on 28.12.2019 Modified on 30.01.2020

Research J. Pharm. and Tech 2020; 13(6): 2906-2911.

DOI: 10.5958/0974-360X.2020.00518.1