Synthesis of Multi-substituted 4 , 5-Dihydrofuran Derivatives from ( S )-Limonene and 1 , 3-Dicarbonyl Compounds and their Biological Activities

Multi-substituted 4,5-dihydrofuran derivatives were regioselectively synthesized by the reaction of α-carbo radical produced from 1,3-dicarbonyl compounds by oxidation with Mn(OAc)3 in Acetic acid and S-(-)-limonene. All the compounds were tested for their antibacterial and antifungal activities by the disc-diffusion technique.


Introduction
Monoterpenes are widely used in the pharmaceutical, cosmetic and food industries as active components of drugs and ingredients of artificial flavours and fragrances 1,2 .Monoterpenes and limonene in particular, are an important class of naturally occurring chiral compounds 3 widely used in organic synthesis 4 as starting materials for optically pure molecules 5 , as a core of chiral auxiliaries 6 and as asymmetric ligands employed in enantioselective transformations 7 .In nature, limonene is abundant in both enantiomeric forms, (R)-limonene and (S)-limonene 8 .From a chemical point of view, these terpenes are very versatile since they present two double bonds that can be selectively converted to several functional groups [9][10][11] .The biological activities of (R)-limonene and (S)-limonene have been studied 12 against Staphylococcus aureus ATCC 12600, Bacillus cereus ATCC 11778, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 11775, Pseudomonas aeruginosa ATCC 9027, Klebsiella pneumoniae ATCC 13883 and Cryptococcus neoformans ATCC 90112 bacteria.They have shown considerable activity.
In addition, furans and their derivatives are available in nature 3 and one of the most important heterocyclic compounds 14 for the total synthesis of the complicated naturally occurring metabolites in organic chemistry 15 .Furans and their derivatives have a broad spectrum of biological activity such as antiprotozal 16 , antiviral 17 , antimicrobial 17 , anticancer 17 , HIV-1 activity 18 and antimalarial 19 .
Therefore, this study focuses on the synthesis of 4,5-dihydrofuran derivatives 3 and 4 with a limonene and dihydrofuran ring and the determination of their biological activities toward 12 different human pathogen microorganisms.

Experimental
Mn(OAc) 3 , (S)-limonene, Acetic acid and 1,3-dicarbonyls were commercial products with highest reagent grade.IR spectra (neat or in CHCl 3 ) were recorded on a Jasco FT/IR-430 spectrometer. 1 H and 13 C NMR spectra were recorded on a Bruker Avance III 400 instrument.As internal standards served TMS (δ 0.00) for 1 H NMR and CDCl 3 (δ 77.0) for 13 C NMR spectroscopy; J values are given in Hz.The multiplicities of the signals in the 1 H NMR spectra are abbreviated by s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad) and combinations thereof.Elemental analyses were obtained by a LECO CHNS 932 Elemental Analyzer.

General procedure for the synthesis of benzofuran and 4,5-dihydrofuran derivatives
A solution of Mn(OAc) 3 (2.97 g, 11.1 mmol) in 15 mL of glacial Acetic acid was heated under N 2 atmosphere at 80 0 C until it dissolved.After Mn(OAc) 3 was dissolved completely, the solution was cooled to 60 0 C and a solution of 1,3 dicarbonyl compounds variable (1.04 g, 7.4 mmol) and S-(-)-limonene (0.5 g, 3.7 mmol) in 5 mL acetic acid was added to this mixture.The reaction was finished when the dark brown colour of the solution disappeared.Acetic acid was evaporated under reduced pressure; H 2 O was added to the residue and extracted with ethyl acetate (3 x 20 mL).The combined organic phases were neutralized with saturated NaHCO 3 solution, dried (Na 2 SO 4 ), and evaporated.Crude products were purified by column chromatography on silica gel using n-hexane/ethyl acetate (9:1) as eluent.

Microbiology Preparation of microorganisms
A total of 12 microbial cultures belonging to ten bacterial and two fungal species were used in this study (Table 1).The cultures were grown in Mueller-Hinton Broth (Merck) for all the bacterial strains by 24 h of incubation at 36 o C. C. albicans and C. utilis were grown in Sabouraud Dextrose Broth (Merck) by incubation for 24 h at 25 o C.

Disc-diffusion assay
Antimicrobial tests were carried out by disc-diffusion method [21][22] using 100 µL of suspension containing 10 8 CFU/mL of bacteria and 10 6 CFU/mL of yeast spread on Nutrient Agar (NA), Sabouraud Dextrose Agar (SDA) and Potato Dextrose Agar (PDA) medium, respectively.The blank discs (Oxoid = 6 mm in diameter) were impregnated with 20 µL of the each substances (105 µg/disc) and placed on the inoculated agar.Negative controls were prepared using the same solvents (Methanol) employed to dissolve each substances.Sulbactam (30 µg) + Cefoperazona (75 µg) (105 µg/disc) was used as positive reference standard to determine the sensitivity of a strain of each microbial species tested.The inoculated plates were incubated at 36 o C for 24 h for clinical bacterial strains, 48 h for yeast and 72 h for fungi strains.Antimicrobial activity was evaluated by measuring the zone of inhibition against the test organisms.
AcOH, 80 0 C to 60 0 C Scheme 1 The structures of the compounds were determined on the basis of spectroscopic data ( 1 H NMR, 13 C NMR, IR and Elemental analysis) and comparison with literature data 20 .All spectroscopic data were in accord with the proposed structures.

Antibacterial and antifungal activity
The newly synthesized compounds (3a-g and 4a-g) were screened for their antibacterial activity against two yeasts bacteria viz., Candida albicans ATCC 1213, Candida utilis KUEN 1031), five gram positive bacteria viz., Streptococcus homilis, Bacillus cereus DSM 4312, Bacillus subtilis ATCC 6633, Staphylococcus Aureus ATCC 29213, Streptococcus Pyogenez ATCC 176 and five gram negative bacteria viz., Proteus Vulgaris KUEN 1329, Escherichia coli 111, Pseudomonas aeruginosa ATCC 9027, Salmonella enteridis ATCC 13076, Enterococous fecaelis ATCC 29122 by using disc-diffusion method.A reference standard drug SCF (Sulbactam (30 µg) + Cefoperazona (75 µg)) and methanol were used as positive and negative control, respectively.The experiments were performed in triplicate in order to minimize the errors.Zone of inhibition produced by each compound was measured in mm.The results of antibacterial studies were given in Table 2.