Synthesis and Investigation of Antimicrobial and Antioxidant Activity of 3-Nitro-N-( 3-chloro-2-oxo-substituted-phenyl-azetidin-1-yl ) naphtho [ 2 , 1-b ] furan-2-carboxamides

Ethyl-3-nitronaphtho[2,1-b]furan-2-carboxylate (2) has been synthesized from ethyl naphtha [2,1-b]furan-2-carboxylate (1). This nitro product on reaction with hydrazine hydrate formed 3-nitronaphtho [2,1-b] furan-2-carbohydrazide (3). Various Schiff bases 3-nitro-N(aryl-methylene)-substituted-naphtho [2,1-b]furan2-carbohydrazides 4(a-g) were obtained by treating hydrazide (3) with different aldehydes. These Schiff bases on reaction with chloro acetyl chloride in presence of triethylamine in dioxane yielded 3-nitro-N-(3-chloro-2-oxo-substituted-phenylazetidine-1-yl) naphtha [2,1-b]furan-2-carboxamides 5(a-g). All the newly synthesized compounds have been characterized by spectral and analytical studies. The Schiff bases, 4a-g and azetidione derivatives, 5a-g have been studied for antioxidant and antimicrobial activities.

Hence, with a view to further assess the pharmacological profile of this class of compounds, it was thought worthwhile to synthesize some new congeners of β-lactam heterocycles by incorporating the naphtho[2,1-b]furan nucleus bearing nitro group and azetidinone moieties in a single molecular framework.The present work deals with the synthesis of the title compounds starting from ethyl naphtho[2,1-b]furan-2-carboxylate, followed by their antioxidant and antimicrobial screening.

Experimental
Melting points were recorded in open capillary tubes and are uncorrected.IR (cm -1 ) were recorded in KBr on FT-IR (Research spectrometer) Shimadzu 8201 PC (4000-400cm -1 ) and NMR on Bruker DRX-300 (300MHz-FT-NMR with low and high temperature facility -90 o to + 80 o C).Standard chemical shifts are given in δ ppm values.Compounds were checked for their purity by TLC on silica gel plates using chloroform and ethyl acetate of varying polarities and spots were located by iodine vapour.

Synthesis of ethyl 3 -nitronaphtho[2,1-b]furan-2-carboxylate (2)
A cooled nitrating mixture of con HNO 3 and con H 2 SO 4 (1:2, 15 mL) was added very slowly to a cooled solution of ester 1 (2.4 g, 0.01 mol) in glacial acetic acid (4 mL) and the mixture was stirred for about 30 minutes at 0 to 15 o C. The stirring was continued for 2 h and the reaction mixture was poured on to crushed ice.The product, that separated as solid, was collected, dried and recrysatllised from aqueous ethanol.
The carbohydrazide (3) was treated with various substituted aromatic aldehydes in refluxing ethanol to obtain corresponding Schiff bases, 3-nitro-N 1 (aryl-methylene)substituted-naphtho[2,1-b]furan-2-carbohydrazides (4a-g) in good yield.The structures of the Schiff bases (4a-g) were established by IR spectral data, showing carbonyl absorption band in between 1680-1695 cm -1 and C=N stretching frequency at 1620-1621 cm -1 .The structure assigned, was supported by 1 H NMR spectra in which a multiplet at δ 8.3 to 8.5 due to aromatic protons was observed.
Synthesis of 3-nitro-N-(3-chloro-2-oxo-substituted-phenyl-azetidine-1-yl)naphtho[2,1-b] furan-2-carboxamide 5(a-g) was accomplished by the reaction between 3-nitro-N 1 (arylmethylene)-substituted-naphtho[2,1-b]furan-2-carbohydrazides 4a-g with chloro acetyl chloride in presence of triethyl amine in dioxane.The structure of 5a was confirmed by elemental analysis and spectral studies.The IR spectrum of 5a exhibited the sharp adsorption band at 1709 cm -1 due to amide carbonyl group and at 1657 cm -1 due to carbonyl group of β-lactam ring.The 1 H NMR spectrum showed a singlet integrating for three protons at δ 3.8 due to OCH 3 protons.A multiplet at δ 7.0-8.5,two doublets one at δ 7.4 and another at δ 7.6 were attributed to ten aromatic protons, one CHPh and one CHCl protons respectively.The CONH proton appeared as a singlet downfield at δ12.1.The physical and analytical data of the newly synthesised compounds is presented in Table 1.

Antioxidant activity
The antioxidant activity of azetidinones and Schiff bases was determined by DPPH radical method.The compounds were taken at 100 µg concentration by their dry weight and mixed with 5 mL of 0.1 mM solution of DPPH radical in methanol and incubated at 20 0 C for 20 min in darkness.The control was prepared as above without the compound and methanol was used for the baseline correction.The absorption of each solution was measured at 517 nm using "Shimadzu 175" spectrophotometer.The % of radical scavenging activity was expressed as percentage activity using the following formula.
(Control absorbance-sample absorbance) % of Radical scavenging activity = Control absorbance 100 Where control absorbance is the measurement of DPPH radical solution without compound and sample absorbance is the measurement of DPPH radical solution with the compound.Ascorbic acid (5 mg/mL) was used as standard for the comparison of antioxidant activity.The results revealed that, the compounds 4g and 5c exhibit moderate activity when compared to the standard ascorbic acid whose concentration was 5 mg/mL.

Antimicrobial activity
The antimicrobial activity of newly synthesized compounds was determined by cup-plate method.The in vitro antimicrobial activity was carried out against 24 h old culture of four bacteria and three fungii.The bacteria used were Bacillus subtilis, Escherichia coli, Micrococus luteus and Staphylococcus aureus and fungii used were Aspergillus niger, Trichoderma spp and Penicillum spp.The compounds were tested at a concentration of 500 µg/mL in DMF against both the organisms.The chloroamphenicol and fluconazole were used as standard for comparison of antibacterial and antifungal activities respectively.The zone of inhibition was compared with the standard drug after 24 h and incubation at 37 o C for antibacterial activity and 72 h at 25 o C for antifungal activity.
The compound 5f showed promising activity against Bacillus subtilis, Micrococus luteus and Staphylococcus aureus.The compounds 5d and 5e exhibited moderate activity against E. coli.For antifungal activity, there was no zone of inhibition.The compounds did not exhibit activity against Aspergillus niger, Trichoderma spp and Penicillum spp organisms.

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The physical and analytical data of the newly synthesised compounds