Phenyl Hydrazone Derivatives of Benzofuran: Synthesis and Their Antimicrobial Activities

A series of (Z)-1-benzo [b] furan-2-yl-3-(substituted phenyl) prop-2-en-1-one 1-phenylhydrazone derivatives (C1-C12) of benzofuran were synthesized with satisfactory yield and pharmacologically evaluated for their in vitro antimicrobial activity. All the synthesized compounds were in good agreement with elemental and spectral data. A majority of the tested compounds showed good to moderate antimicrobial activity against all tested pathogenic bacterial and fungal strains.


INTRODUCTION
Antimicrobials reduce or completely block the growth and multiplication of bacteria. This has made them unique for the control of deadly infectious diseases caused by a variety of pathogens.
They have transformed our ability to treat infectious diseases such as pneumonia, meningitis, tuberculosis, malaria and AIDS [1]. These agents are those inhibitory chemicals which are employed to kill microorganisms or prevent their growth. Infectious diseases account for approximately one-half of all deaths in tropical countries. Although deaths from bacterial and fungal infections have dropped in the developed world, these are still major causes of death in the developing country [2]. In addition, primary and opportunistic fungal infections continue to increase rapidly because of the increased number of immune compromised patients (AIDS, cancer and transplants) [3]. According to WHO, each year 1.4 million children died of gut infections and diarrhoea caused by gramnegative bacteria like Pseudomonas, Salmonella, Shigellae and gram positive rods like Corynebacterium diptheriae [4]. Decades of antibiotic use have resulted in the development of widespread resistance to commonly prescribed antibacterial agents [5]. Therefore, there is a need to develop new, potent, fastacting antimicrobial drugs with low toxicity. In the design of new compounds, development of hybrid molecules through the combination of different pharmacophores in one structure may lead to compounds with increased antimicrobial activity [6].
Despite numerous attempts to develop new structural prototype in the search for more effective antimicrobials, benzofuran still remain as one of the most versatile class of compounds against microbes and therefore, are useful substructures for further molecular exploration. Benzofuran's literature is enriched with progressive findings of the moiety in respect of antimicrobial activity [7]. Benzofuran and its derivatives constitute the most versatile and valuable source of antimicrobial compounds. They appear to transcend the chemotherapeutic boundaries of other antiparasitic drugs with a spectrum of activity that includes the majority of fungi, bacteria, protozoa and helminthic species. The prime objective for the current study is to develop novel derivatives of benzofuran moiety and finally screen them against different microbial strains (bacteria and fungi) at variable concentrations. The rationale for the study includes the designing of the derivatives having some common structural features that are important for the compound to exhibit an antimicrobial activity that includes the following: [8-10].
1. A lipophilic bicyclic aromatic ring system. 2. Another bulky lipophilic group (e.g. phenyl, tert-butyl)as a side chain. 3. Two lipophilic domain linked by a spacer of appropriate length with polar centre at defined position, for example, Naftifine, Butenafine, Terbinafine, Debacarb, Penicillins and Cephalosporins.
In view of the above mentioned facts and in continuation of our interest in the synthesis of heterocycles containing benzofuran moiety, to identify new candidates that may be of value in designing new, potent, selective and less toxic antimicrobial agents, we report herein the synthesis and antimicrobial evaluation of some novel structure hybrids incorporating the benzofuran moiety with phenylhydrazone through different linkages. This combination was suggested in an attempt to investigate the influence of such hybridization and structure variation on the anticipated biological activities, hoping to add some synergistic biological significance to the target molecules. The substitution pattern of benzofuran ring was carefully selected so as to confer different electronic environment to the molecules.

EXPERIMENTAL
All the solvents were of AR grade and were obtained from SDFCL Ltd., Himedia, Central drug House (p) Ltd and Loba-chemicals. All the microorganisms used in the present screening were procured from the Department of Microbiology, Gulbarga University, Gulbarga. Melting points were determined in open capillary tubes and are uncorrected. All the compounds were subjected to elemental analysis (CHN) and the measured values agreed within ± 0.4% with the calculated ones. Thin layer chromatography was performed on silica gel G (Merck). The spots were developed in an iodine chamber and visualized with an ultraviolet lamp. The solvent systems used were benzene:acetone (8:2, v/v) and toluene: ethylacetate:formic acid (5:4:1, v/v). Ashless Whatman No. 1 filter paper was used for vacuum filtration. The IR spectra were recorded in KBr pellets on a (BIO-RAD FTS 135) WIN-IR spectrophotometer. The FAB mass spectra of all the compounds were recorded on a JEOL SX102/DA-600 mass spectrometer using argon/xenon (6 kV, 10 mA) as the FAB gas. The 1H-NMR spectra were recorded on a Bruker model DPX 300 FT-NMR spectrometer in CDCl 3 using tetramethylsilane (Me 4 Si, TMS) as an internal standard. The chemical shifts are reported in the δ ppm scale. The physicochemical &spectral data of the synthesized title compounds were listed in Tables 1 and 2.

General Procedure for the Synthesis of 1-(1-benzofuran-2-yl) ethanone (B) [7]
The mixture of salicylaldehyde (0.1 mole) (A), chloroacetone (0.1 mole) and anhydrous potassium carbonate (30 g) were gently refluxed in dry acetone (150 ml) for 13 hr. The reaction product after cooling was filtered and the filtrate on the removal of the solvent under reduced pressure furnished 2-acetyl benzofuran as dark yellow colored solid. The product obtained was recrystallized from petroleum ether.

General Procedure for the Synthesis of (Z)-1-benzo[b]furan-2-yl-3-(Substituted phenyl) prop-2-en-1-one (B 1 -B 12 )
A solution of 2-acetyl benzofuran (1.6 ml, 0.01 mole) and various aromatic aldehyde (0.01 mole) in ethanol (4.6 ml) was cooled to 5 to 10°C in an ice bath. The cooled solution was treated with drop wise addition of aqueous sodium hydroxide (4 ml, 50%). The resulting dark solution was diluted with ice water and carefully acidified using diluted hydrochloric acid. The benzofuran analogues of chalcone which were crystallized were collected by filtration after washing with sodium bicarbonate and water. It was further purified by re-crystallization from ethanol.

General Procedure for the Synthesis of (Z)-1-benzo[b]furan-2-yl-3-(Substituted phenyl) prop-2-en-1-one-1-Phenylhydrzone (C 1 -C 12 )
A mixture of phenylhydrazine hydrochloride (1.44 g, 0.01 mole), sodium acetate (0.82 g, 0.01 mole) in ethanol (10 ml) was stirred at RT for 10 min.    B 12 ) (0.01 mole) was added slowly. The resulting reaction-mixture was allowed to stir for about 2 hr and completion of the reaction was monitored by TLC. The reaction-mixture was then poured into ice water (50 ml) where upon the crude compound was precipitated as yellow solid. The residue obtained after filtration was washed with water and dried. The crude product was purified by recrystallization from absolute alcohol. Table 3. Antimicrobial activity of the synthesized title compounds (C 1 -C 12 )

Biological Activity
All the synthesized compounds were tested for their in vitro antimicrobial activity against the   benzofuran (B 1 -B 12 ).These chalcones were stirred at room temperature with phenylhydrazine hydrochloride and sodium acetate in ethanol for 2 hours to precipitate the title compounds (C 1 -C 12 ). The product was then recrystallized from absolute ethanol. The structure of synthesized compounds was confirmed by elemental analysis and spectral data (IR, 1 H NMR, MS).

CONCLUSION
Thus, various derivatives of phenylhydrazone (C 1 -C 12 ) were prepared with the objective of developing better antimicrobial agents. All the derivatives were found to have a promising class of compounds with an interesting pharmacological profile. Among these the compounds, C 8 and C 11 showed maximum antibacterial and antifungal activity, respectively. Hence, it is clear from structure activity relationship (SAR), that the compounds synthesized showed significant antimicrobial activity stating the importance of electron withdrawing substituent's on the phenyl group. In conclusion, the benzofuran incorporated hydrazone derivatives can be regarded as a newer class of antimicrobial agents. They were also found to be less toxic which indicates better tolerability of the compounds having strong future prospects.