INTRODUCTION:

A large number of medicinal compounds which have been discovered belong to a major class of heterocycles containing Sulphur. Among the variety of compounds studied thiophene derivatives form an important class. Thiophene has exhibited an array of biological activities ranging from anti-inflammatory,^1-2 antimicrobial,^3-5 antioxidant,⁶ anticancer,⁷ HIV-1 reverse transcriptase inhibitors,⁸ anti-tubercular⁹ and they are also active against rheumatoid diseases.¹⁰ Cephalothin, Cephalorodine, Cefoxitin, Ticarcillin, Ticonazole, Suprofen acid are examples of some clinically useful drugs having thiophene ring.

In the current literature survey, it has been observed that drug designed by molecular modification is more rational and productive foundation of new drug. So, an attempt was made to synthesize new substituted thiophenes as anti-inflammatory agents adapting Gewald reaction.^11-12 Hence the synthesis of “2-amino-N-(3-chloro-4-fluorophenyl) thiophene-3-carboxamide” was carried out. A new series of compounds were synthesized from 2-amino-N-(3-chloro-4-fluorophenyl)thiophene-3-carboxamide and the required aryl aldehydes to yield seven new Schiff bases. All the synthesized compounds were characterized on the basis of their physical and spectral data.

All The compounds were screened for their anti-inflammatory activity (in vitro) by using Diclofenac Sodium at the concentration of 100µg/0.1ml as standard.¹³

v These compounds were also screened for antibacterial activity against a G+ve and G–ve bacteria, using Ampicillin as the standard. All the test compounds as well as the standard were used at the concentration of 50µg/0.1ml

v . Five compounds were screened for anti-oxidant activity by using ferric ion reduction method.

MATERIAL AND METHODS:

The melting point of the compounds was taken in open capillaries and is uncorrected. The infrared spectrum was recorded using KBr as the medium, utilizing SHIMADZU Infrared spectrophotometer. ¹H NMR were recorded from Astra-Zeneca Pharma India Ltd. Bangalore. All the reactions were monitored using thin layer chromatography (TLC) using a glass plate coated with Silica Gel G or GF₂₅₄ and spots were visualized either by iodine vapour or by irradiation with ultraviolet light (254 nm).

Synthesis of N-(3-chloro-4-fluorophenyl)-2-cyanoacetamide, [A]

A mixture of 4-fluoro-3-Chloro aniline (7.53 g; 0.05 M) and ethylcyano acetate (5.7 ml; 0.05 M) was taken in a round bottom flask and refluxed at 180ºC for 2 hrs. The reaction mixture was left at room temperature overnight. The solid obtained was collected, washed with ethanol and dried. Recrystallized from ethanol:water mixture. Yield

8.58 g (78.25%); mp 140ºC. IR (KBr) V_max 3279.1 (N-H), 3107.43 (ArC-H), 2926.11(AliC-H), 2262.58 (CN), 1666.55 (C=O) cm^-1.

Scheme-1

Synthesis of 2-amino-N-(3-chloro-4-fluorophenyl) thiophene-3-carboxamide, [B]

To a mixture of 2-amino-N-(3-chloro-4-fluorophenyl)-2-cyanoacetamide(2.12 g; 0.01 M) and acetaldehyde (0.54 ml; 0.01 M) in alcohol (30 ml) was added sulphur (0.32 g; 0.01 M) in portions followed by the addition of tri ethyl amine (1.01 ml; 0.01 M) drop wise with stirring. The reaction mixture was stirred for 15-20 min at 40-45⁰C and chilled over night. The solid obtained was filtered, washed with ethanol and crystallized from chloroform. Yield 2.07 g (76.87%), mp 92ºC, IR (KBr) V_max 3402.54 (N-H), 3336.96 (NH₂), 3128.64 (ArC-H), 1629.9 (C=O), 804.34 (C-S) cm^-1.

General method for the preparation of 2-substituted-N-(3-chloro-4-fluorophenyl)thiophene-3-carboxamide, [B1-7]

A mixture of the starting compound B (1.3 g; 0.005 M) and the required aryl aldehydes (0.005 M) in ethanol (30 ml) was taken in a round bottom flask and catalytic amount of glacial acetic acid (2-5 drops) was added. Refluxed it for 2 hrs. The mixture was cooled to room temperature. The solid separated was filtered, washed with ethanol and recrystallized from DMF:Water mixture.

N-(3-chloro-4-fluorophenyl)-2-{[(1E)-phenylmethylene] amino}thiophene-3-carboxamide, [B1]

Yield 1.11 g (61.8%); mp 110ºC. IR (KBr) V_max 3290.37 (N-H), 3010.45 (ArC-H), 2985.95 (AliC-H), 1641.62 (C=O), 782.49 (C-S) cm^-1.

N-(3-chloro-4-fluorophenyl)-2-{[(1E)-(4-hydroxyphenyl) methylene]amino}thiophene-3-carboxamide-4(3H)-one, [B2]

Yield 1.13 g (60.42%); mp 110ºC. IR (KBr) V_max 3451.87 (OH), 3262.41 (N-H), 2920.47 (AliC-H), 1690.42 (C=O), 784.63 (C-S) cm^-1

N-(3-chloro-4-fluorophenyl)-2-{[(1E)-(4-methoxyphenyl) methylene]amino} thiophene-3-carboxamide, [B3]

Yield 0.87 g (46.87%); mp 184ºC. IR (KBr) V_max 3303.58 (N-H), 2864.26 (AliC-H), 1686.39 (C=O), 1289.74 (O-CH₃), 797.16 (C-S) cm^-1.

N-(3-chloro-4-fluorophenyl)-2-{[(1E)-(3,4,5-trimethoxy phenyl)methylene]amino} thiophene-3-carboxamide, [B4]

Yield 1.02 g (59.22%); mp 200ºC. IR (KBr) V_max 3205.8 (N-H), 3078.49 (ArC-H), 2991.69 (AliC-H), 1649.19 (C=O), 1263.42 (O-CH₃), 731.05(C-S) cm^-1.

N-(3-chloro-4-fluorophenyl)-2-{[(1E)-(4-hydroxy-3-methoxy phenyl)methylene] amino}thiophene-3-carboxamide, [B5]

Yield 1.10 g (54.58%); mp 180ºC. IR (KBr) V_max3433.41 (OH), 3315.74 (N-H), 2939.61 (AliC-H), 1666.56 (C=O), 1232.55 (O-CH₃), 763.84(C-S) cm^-1.

N-(3-chloro-4-fluorophenyl)-2-({(1E)-[4-(dimethylamino )phenyl] methylene}amino)thiophene-3-carboxamide, [B6]

Yield 0.84 g (43.80%); mp 170ºC. IR (KBr) V_max3234.73 (N-H), 3095.85 (ArC-H), 2908.75 (AliC-H), 1672.34 (C=O), 802.41 (C-S) cm^-1.

N-(3-chloro-4-fluorophenyl)-2-{[(1E)-(2-hydroxy phenyl)methylene]amino} thiophene-3-carboxamide-4(3H)-one, [B7]

Yield 1.3 g (69.32%); mp 84ºC. IR (KBr) V_max 3396.76 (OH), 3249.98 (N-H), 2886.84 (AliC-H), 1687.67 (C=O), 761.57(C-S) cm^-1.

ANTI-INFLAMMATORY ACTIVITY¹³

Bovine serum albumin (Merck Limited), Ibuprofen and all other chemicals are of analytical grade. The test compounds were dissolved in minimum amount of DMF and diluted with phosphate buffer (0.2 M, pH 7.4). Final concentration of DMF in all solution was less than 2.5%. Test solution (1 ml) containing different concentrations of drug was mixed with 1 ml of 1 mM albumin solution in phosphate buffer and incubated at (27± 1)⁰C for 15 min. Denaturation was induced by keeping the reaction mixture at (60± 1)⁰C in a water bath for 10 min. After cooling the turbidity was measured at 660 nm. Percentage inhibition of denaturation was calculated from control where no drug was added. Each experiment was done in triplicate and the average was taken. The percentage of inhibition is calculated from the following formula,

% Inhibition =100 (1- Vt/Vc)

Where, Vt – Absorbance of test solution

Vc – Absorbance of control

Table-1

Compound	R₁
B1	-H
B2	4’-OH
B3	4’-OCH₃
B4	3’, 4’, 5’-OCH₃
B5	3’-OCH₃, 4’-OH
B6	4’-N(CH₃)₂
B7	2’-OH

Table-2

Compound	Anti-inflammatory activity (% Inhibition)	Antibacterial activity Zone of Inhibition (mm.)
Compound	Anti-inflammatory activity (% Inhibition)	*S. aureus* (G –ve)	*B. subtilis* (G +ve)
B1	57.31	14	15
B2	73.65	17	18
B3	68.17	17	17
B4	53.84	16	15
B5	62.98	14	13
B6	46.33	NA	13
B7	64.26	15	16
Diclofenac Na	78.67	-	-
Ampicillin	-	20	19

NA : No Activity

Table-3

Conc in µg/ml	B4	B3	B7	B2	B5
50	9.82	7.63	19..9	20.14	7.91
100	12.00	9.81	22.35	32.46	9.00
150	16.65	11.72	32.73	49.07	14.18
200	22.89	15.26	38.44	64.03	15.28
250	27.53	17.20	50.74	86.45	17.98
IC₅₀^*	335	455	249	152	425

*IC₅₀ value not detected at the highest concentration tested. So it was determined by extrapolating the graph.

ANTI-BACTERIAL ACTIVITY:

Agar diffusion method:

In our current study, the antimicrobial activity was carried out by the agar diffusion method. The micro organisms used were Staphylococcus aureus (G +ve) and Bacillus subtillis (G -ve). Here responses of organisms to the synthesized compounds were measured and compared with the response of the standard reference drug. Each test compound was dissolved in DMF to get a concentration of 50mg/ml. The standard reference drug used in the present work was Ampicillin.

ANTIOXIDANT ACTIVITY:

In Vitro Free Radical Scavenging activity (Reduction of Ferric Ions):

The reaction mixture contaning O-phenanthroline (0.5 m), ferric chloride (0.2 mM), and test compounds (different concentrations) in a final volume of 5 ml was incubated for 15-20 min at ambient temperature. The absorbance at 510 nm was measured. In another set, Sodium dithionite (0.3 mM) was added instead of the test compound and the absorbance was taken as equivalent to 100% reduction of all the ferric ions present.

Scavenging activity was expressed as Percentage Ferric ion reduction using the following formula,

Standard Abs. – Sample Abs.

% Anti radical activity =----------------------------------------x 100

Standard Abs

RESULTS AND DISCUSSION:

Chemistry:

4’-fluoro-3’-Chloro aniline when refluxed with ethyl cyanoacetate gave N-(3-chloro-4-fluorophenyl)-2-cyanoacetamide A. This cyanoacetamide undergo Gewald reaction with acetaldehyde, triethyl amine and elemental sulphur to give 2-amino-N-(3-chloro-4-fluorophenyl) thiophene-3-carboxamide B (Scheme 1). Then ethanolic solution of thiophene B refluxed with aryl aldehydes in presence of glacial acetic acid as a catalyst to yield seven new Schiff bases B1-7. The structures of the newly synthesized compounds have been established on the basis of elemental analysis, IR and ¹H NMR spectral studies.

Biological activity:

The antibacterial screenings revealed that some of the tested compounds showed good inhibition at 50µg/0.1ml concentration. The antibacterial screening indicated that among the tested compounds B2 and B3 showed excellent activity against the tested bacterial strains namely S. aureus and B. subtilis (Table 2). The compounds B6 showed least activity in the series. The remaining compounds were found to be moderately active.

Pharmacological Activity:

In-vitro anti-inflammatory screening revealed that some of the tested compounds showed good activity at 100µg/0.1ml concentration. The results indicated that among the tested compounds B2 showed excellent activity (Table 2). The compounds B6 showed least activity in the series. The remaining compounds were found to be moderately active.

Five compounds were also screened for anti-oxidant activity (reduction of Ferric Ions). Among them compound B2 showed good inhibition at lower concentration (Table 3).

CONCLUSION:

Two series of substituted thiophene have been synthesized and screened for their anti-inflammatory, anti-bacterial and anti-oxidant activities. The results suggest that among the compounds tested B2 have exhibited higher activity. It can be inferred from the above results that the new synthesized compounds possessing -OCH₃ group exhibit better antibacterial, anti-inflammatory and anti-oxidant activity.

REFERENCES:

1. Ajay D Pillai et al., Bioorg. Med. Chem. Lett. 2005; 13: 6685-92.

2. Issa M.I. Fakhr et al., European Journal of Medicinal Chemistry. 2008; 20: 1-8.

3. Isabel C.F.R. Ferreira et al., Bioorg. Med.Chem. Lettr. 2006;10: 4825-4828.

4. Eugenia Pinto et al., Bioorganic & Medicinal Chemistry. 2008; 16: 8172–8177

5. Thankamony M and Mohanan K. Asian J Chem. 2007; 19(4): 2877-2884

6. Isabel C F R Ferreira et al., Bioorg. Med. Chem. Letters. 2006;16: 1384-87.

7. Kristina Starcevic et al., Eur. J. Med. Chem. 2006; 41:1-15.

8. Krzysztof Krajewski et al., Bioorganic & Medicinal Chemistry Letters. 2006; 16: 3034-3038.

9. Pilho Kim et al., J. Med. Chem. 2006; 49:159-71.

10. The Merck Index. 13^th Edition.NJ:Merck and Co. Inc; 2001.p.1680; Monograph No. 9498.

11. Gernot A Eller and Wolfgang Holzer. Molecules. 2006; 11:371-376

12. K Gewald, E Schinke and H Bohcher. Chem. Ber. 1966; 99: 94-100.

13. G Elias, & M.N.A Rao. Indian Journal of Experimental Biology. 1988; 26, July, 540-542.

Received on 15.07.2010 Modified on 1.08.2010

Asian J. Research Chem. 3(4): Oct. - Dec. 2010; Page 1065-1068

INTRODUCTION:

200

22.89

15.26

38.44

64.03

15.28

250

27.53

17.20

50.74

86.45

17.98

IC50*

335

455

249

152

425

IC₅₀^*