INTRODUCTION:

Though the first member was synthesized by Staudinger¹ in 1907, the b-lactams as a class acquired importance since the discovery of penicillin which contains b-lactam unit as an essential structural feature of its molecule. This interest continued unabated because of the therapeutic importance of b-lactams, being a structural unit in most widely used antibiotics. The utility of b-lactams (azetidinones), as synthons for various biologically active compounds, as well as their recognition as antibacterial^2,3, antifungal⁴, anti-inflammatory⁵, anticancer^6-8, cholesterol absorption inhibitors^9,10 and anti-hepatitis¹¹has given impetus to these studies. Recently, synthesis of b-lactams have been reviewed¹². Synthesis and biological activity of Schiff bases have also been reported by different authors^13-25.

In this paper, the synthesis of some Schiff bases and their cyclization to produce b-lactam derivatives of biological significance has been reported. The newly synthesized Schiff bases and b-lactam derivatives were evaluated for antibacterial activity. Although results comprising Schiff bases are not encouraging, all the b-lactam derivatives exhibited varied activity against two Gram +ve (Staphylococcus aureus, Bacillus subtilis) and two Gram –ve (Escherichia coli, Pseudomonas aeruginosa) bacterial strains.

These studies may serve as a basis for the chemical modifications directed towards the development of a new class of antibacterial drugs.

MATERIAL AND METHODS:

All chemicals were of analytical grade and were used without further purification. Melting points were determined using open glass capillaries and are uncorrected. ¹H-NMR spectra were recorded on NMR-Spectrometer (Bruker 300 M Hz). The ¹H-chemical shifts are expressed in ppm relative to tetramethylsilane (Me₄Si). IR spectra were recorded on FT-IR Spectrometer (Lambda Scientific). Mass spectra were obtained on a Shimadzu GCMS-QP-2000 Mass Spectrometer. Elemental analyses were performed on Perkin Elmer CHN Analyzer. Column chromatography was performed on silica gel (60–120 mesh) and TLC plates were coated with silica G.

General method for preparation of Schiff bases (1, 3 and 5):

0.1 mole of amine (aniline, phenylhydrazine, or 2,4-dinitrophenylhydrazine) and 0.1 mole of carbonyl compound (antipyrine, furfuraldehyde, p-hydroxybenzaldehyde) were taken and mixed in glacial acetic acid (25 ml). The resultant mixture was refluxed for (1.5-10 hours), cooled and poured on to crushed ice. The precipitates were filtered and washed with distilled water (2x10 ml). Recrystalization from ethanol afforded the desired Schiff bases. Compounds were further purified by column chromatography (eluent: ethylacetate/hexane :: 1-3 : 9-7). The reaction was monitored by thin layer chromatography (TLC) and spots were visualized using iodine chamber.

General method for preparation of b-lactam derivatives (2, 4 and 6):

A mixture of 0.1 mole of Schiff base and 0.1mole of chloroacetylchloride were taken in 25ml ethanol/1,4-dioxane containing 0.1 mole of triethylamine. The resultant mixture was refluxed for (1-9 hours), cooled to room temperature and poured on to crush ice. Precipitates were filtered on suction, washed with distilled water (2x10 ml) and dried in air. Recrystalization from ethanol afforded the purified b-lactam derivatives. Crude product was also purified by column chromatography (eluent: ethylacetate/hexane :: 1-3 : 9-7). The reaction was monitored by thin layer chromatography (TLC) and spots were visualized in iodine chamber.

Fig. 1 Structures of Schiff Bases (1, 3, 5) and b-lactam derivatives (2, 4, 6).

2-[(2-Phenylhydrazinylidene)methyl]phenol (1).

Yield 69%, light brown powder; m.p. 140-142 ⁰C; IR (KBr) cm^-1: 3308 (O-H str.), 3272 (N-H str.), 1660 (C=N str.), 1139 (C-NH str.). ¹H NMR (CDCl₃) : d: 3.51 (s, 1H, Ar.-NH-, exchangeable with D₂O), 5.58 (s, 1H, Ar.-OH exchangeable with D₂O) 6.07 (s, 1H, Ph-CH=N-), 6.86-7.00 (m, 5H, Ar. ring-A), 7.08-7.31 (m, 4H, Ar. ring-B), MS (m/z) 212.2 (M^.+), 211.1, 183.2, 119.1, 105.1. Anal. Calcd. for C₁₃H₁₂N₂O : C, 73.56; H, 5.70; N, 13.20. Found C, 73.48; H, 5.75; N, 13.35.

4-Chloro-2-(2-hydroxyphenyl)-1-(phenylamino)-1,2-diazetidin-3-one (2).

Yield 65%, light brown powder; m.p. 130-132 ⁰C; IR (KBr) cm^-1: 3301 (O-H str.), 3275 (N-H str.), 1741 (C=O str.), 1135 (C-NH str.), ¹H NMR (CDCl₃ : d: 3.35 (s, 1H, Cl-CH), 3.61 (s, 1H, Ar.-NH-exchangeable with D₂O), 5.50 (s, 1H, Ar.-OH, exchangeable with D₂O) 6.87-7.01 (m, 4H, Ar. ring-A), 7.13-7.32 (m, 5H, Ar. ring-B). MS (m/z) 289.7 (M^.+), 290.5, 274.6, 261.7, 214.6, 212.6, 196.6. Anal. Calcd. for C₁₄H₁₂N₃O₂Cl : C, 58.04; H, 4.18; N, 14.51. Found C, 58.46; H, 4.25; N, 14.44.

1,5-Dimethyl-2-phenyl-3-[(phenylimino)methyl]-2,3-dihydro-1H-pyrazol-4-amine (3).

Yield 59%, light brown powder; m.p. 95-97ºC; IR (KBr) cm^-1: 3270 (N-H str.), 1669 (C=N str.), ¹H NMR (CDCl₃) : d: 1.25 (s, 3H, C-CH₃), 2.14 (s, 2H, -NH_2,exchangeable with D₂O), 2.51 (s, 1H, N-CH-C=N-), 2.64 (s, 3H, -N-CH₃), 6.08 (s, 1H, -CH=N-), 6.82-7.06 (m, 5H, C₆H₅-N=CH-), 7.09-7.27 (m, 5H, C₆H₅-N<). MS (m/z) 292.4 (M^.+), 201.3, 200.3, 158.1, 104.1, 103.1. Anal. Calcd. for C₁₈H₂₀N₄ : C, 73.94; H, 6.90; N, 19.17. Found C, 73.82; H, 6.97; N, 19.38.

1-(4-Amino-1,5-dimethyl-2-phenyl-2,3-dihydro-1H-pyrazol-3-yl)-4-chloro-2-(furan-2-yl)-1,2-diazetidin-3-one (4).

Yield 35%, brownish-yellow powder; m.p.170-172ºC; IR (KBr) cm^-1 : 3279(N-H str.), 1740 (C=O str.), ¹H NMR (CDCl₃) : d: 1.29 (s, 3H, CH₃-C<), 2.53 (s, 3H, CH₃-N<), 2.62 (s, 1H, N-CH-), 3.17 (s, 1H, Cl-CH<), 3.57 (s, 2H, NH₂-C=C<,exchangeable with D₂O), 6.58 (dd, 1H, Furyl C₄-H), 6.68 (d, 1H, Furyl C₃-H), 6.89 (d, 1H, Furyl C₅-H), 6.98-7.36 (m, 5H, C₆H₅-N<). MS (m/z) 359.8(M^.+), 315.7, 313.7, 203.6, 201.6, 106.5. Anal. Calcd. for C₁₇H₁₈N₅O₂Cl : C, 56.75; H, 5.04; N, 19.47. Found C, 56.60; H, 5.11; N, 19.35.

3-{[2-(2,4Dinitrophenyl)hydrazinylidene]methyl}-1,5-dimethyl-2-phenyl-2,3-dihydro-1H-pyrazol-4-amine (5).

Yield 70%, brown powder; m.p. 187-189ºC; IR (KBr) cm^-1: 1665 (C=N str.), 1539 [(Ar-NO₂) asym. str.], 1340 [(Ar-NO₂) sym. str.]. ¹H NMR (CDCl₃) : d: 1.29 (s, 3H, CH₃-C=), 2.45 (s, 3H, CH₃-N<), 2.52 (s, 2H, NH₂-C, pyrazole), 2.59 (s, 1H, N-CH-C=), 4.21(s, 1H, C₆H₅-NH, exchangeable with D₂O),), 6.11 (s, 1H,-CH=N-), 6.55-7.08 (m, 5H, ring-A), 7.19-8.45 (m, 3H, ring-B). MS (m/z) 397.4 (M^.+), 367.3, 366.3, 337.4, 320.3, 304.2, 274.3. Anal. Calcd. for C₁₈H₁₉N₇O₄ : C, 54.40; H, 4.82; N, 24.68 Found C, 54.05; H, 4.89; N, 24.35.

1-{4-Amino-2-[(2,4dinitrophenyl)amino]-1,5-dimethyl-2,3-dihydro-1H-pyrazol -3-yl}-4-chloro-2-(furan-2-yl)-1,2-diazetidin-3-one (6).

Yield 55%, yellow powder; m.p.195-197ºC; IR (KBr) cm^-1: 1738 (C=O str.), 1549 [(Ar-NO₂) asym. str.], 1348 [(Ar-NO₂) sym. str.]. ¹H NMR (CDCl₃) : d: 1.20 (s, 3H, CH₃-C=C<), 2.59 (s, 1H, N-CH-C=), 2.70 (s, 3H, CH₃-N-N<), 3.21 (s, 1H, Cl-CH<), 3.61 (s, 2H, NH₂-C=C<, exchangeable with D₂O),), 4.19 (s, 1H, Ar.-NH-N<, exchangeable with D₂O),), 6.45 (dd, 1H, Furyl C₄-H), 6.95 (d, 1H,Furyl C₃-H), 7.07 (d, 1H, Furyl C₅-H), 7.19-8.38 (m, 3H, -C₆H₃-NO₂). MS (m/z) 464.8(M^.+), 362.3, 337.7, 277.2, 201.3, 200.3. Anal. Calcd. for C₁₇H₁₇N₈O₆Cl : C, 43.93; H, 3.69; N, 24.12 Found C, 42.01; H, 3.75; N, 24.14.

RESULTS AND DISCUSSION:

All the test compounds (Schiff bases and β-lactam derivatives) were screened for their in vitro antibacterial activity by agar-well diffusion method against Gram +ve (Staphylococcus aureus, Bacillus subtilis) and Gram –ve (Escherichia coli, Pseudomonas aeruginosa) microorganisms by preparing 200 µg/ml of test solution of each compound. Zone of inhibitions in mm were noted. The zone of inhibition for Schiff Bases (1, 3 and 5) and β-lactam derivatives (2, 4 and 6) varied from 0 to 25 mm. The results have been documented in Table 1. Those compounds which showed significant antibacterial activity were selected for minimum inhibitory concentration (MIC) studies by making dilutions of different concentrations varying from 1 to 50 µg/ml, and the results of minimum inhibitory concentrations have been shown in Table 2. The activity of control dimethyl sulphoxide was also checked for its toxicity. Ampicillin was used as standard antibacterial agent for comparing the activity of test compounds.

Table 1. Screening of Schiff bases and Beta-lactam derivatives for anti-bacterial activit (zone of inhibition in mm.)

Comp. No.	MICROBIAL SPECIES
Comp. No.	*E. coli*	*B. subtilis*	*P.aeruginosa*	*S. aureus*
MTCC code	1687	441	424	737
1	10	14	16	15
2	17	22	18	25
3	00	09	00	13
4	15	17	08	08
5	00	20	00	13
6	14	18	08	15

00-09: Weak activity; 10-16: Moderate Activity; 17-25: Significant activity

Table 2. Minimum inhibitory concentration (µg/ml.) of the following compounds against selected bacteria

Comp. No.	1	2	Ampicillin
*E. coli*	25	15	04
*B. subtilis*	20	10	03
*p. aeruginosa*	30	15	05
*S. aureus*	20	07	03

The present investigations suggest that:

Schiff bases (1, 3 and 5) were found less affective against Gram +ve (Staphylococcus aureus, Bacillus subtilis) and Gram –ve (Escherichia coli, Pseudomonas aeruginosa) microorganisms but β-lactam derivatives (2, 4 and 6) showed moderate to significant activity. It has been found that amongst all the test compounds explored for antibacterial evaluation, Comp. 2 showed maximum activity against all microbes. The activity of control dimethyl sulphoxide was also checked for its toxicity and it has been found that it has no effect on the growth of any microorganisms taken.

CONCLUSION:

The Schiff bases were found to exhibit either no or low to moderate activity against one or more bacterial species. On the contrary all the β-lactam derivatives exhibited varied activity against different bacteria. The inactive and less active Schiff bases became active and more active after cyclization, respectively. These studies may serve as a basis for the chemical modifications directed towards the development of a new class of antibacterial agents.

ACKNOWLEDGEMENTS:

The authors feel a tremendous paucity of words to express gratitude and indebtedness to thank the Head, Sophisticated Instrumentation Facility, Punjab University Chandigarh for ¹H-NMR spectra and Mass spectra. We also wish to acknowledge the help rendered by Prof. G. S. Roy for carrying out the anti-microbial studies for bringing this manuscript in the present form.

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Received on 08.11.2011 Modified on 22.11.2011

Research J. Pharm. and Tech. 5(1): Jan. 2012; Page 129-132

General method for preparation of Schiff bases (1, 3 and 5):

General method for preparation of b-lactam derivatives (2, 4 and 6):

MTCC code

CONCLUSION:

REFERENCES: