Synthesis and characterization of compounds
The synthesis of all compounds was performed under an argon atmosphere, using standard Schlenk techniques. Anhydrous THF (tetrahydrofurane) was obtained by distillation from sodium/benzophenone. Thin layer chromatography was performed on silica gel 60 GF254. Infrared spectra were obtained on FT/IR-4100 JASCO spectrometers (http://www.jascofrance.fr). 1H and 13C NMR spectra were recorded on a 300 MHz Bruker spectrometer (http://www.bruker.com). Mass spectrometry was performed with a Nermag R 10-10C spectrometer. Elemental analyses were performed by the microanalysis service of CNRS at Gif – sur – Yvette (https://www.imagif.cnrs.fr). The preparative HPLC (high performance liquid chromatography) separations were performed on a Shimadzu apparatus (http://www.shimadzu.fr) with a Nucleodur C18 column (length of 25 cm, diameter of 2.5 cm, and particle size of 10 μm).
1-[Bis(4-hydroxyphenyl)methylidenyl]indan 10
Titanium chloride (15.177 g, 8.79 mL, 80 mmoles) was added dropwise to a suspension of zinc powder (7.844 g, 120 mmoles) in dry THF (200 mL) at 0-20°C. The mixture was heated at reflux for 2 hours. A second solution was prepared by dissolving 1-indanone (2.643 g, 20 mmoles) and 4,4'-dihydroxybenzophenone (4.284 g, 20 mmoles) in dry THF (50 mL). This latter solution was added dropwise and the reflux was continued overnight. After cooling, the mixture was poured in water and dichloromethane was added. The mixture was acidified with diluted hydrochloric acid until dark color disappeared and was decanted. The aqueous layer was extracted with dichloromethane and the combination of organic layers was dried over magnesium sulphate. After concentration under reduced pressure, the crude product was chromatographed on silica gel column with a mixture of cyclohexane and ethyle acetate 50/50 as the eluent to give 10 as a white solid with a 79% yield. The characteristics of the product were identical to that of the literature (Kim and Katzenellenbogen 2000).
1-[Bis(4-{3-dimethylaminopropoxy}phenyl)methylidenyl]indan 4
Compound 10 (1.886 g, 6mmol), potassium carbonate(8.29 g, 60 mmol) and cesium carbonate (3.91 g, 12 mmol) were stirred in 120 mL of acetone, then 3-dimethylamino-1-propyl chloride hydrochloride (5.69 g, 36 mmol) was added. The mixture was refluxed overnight, cooled and concentrated under reduced pressure. The residue was extracted with a mixture of dichloromethane and water, then was decanted. The organic layer was washed twice with a diluted aqueous solution of sodium hydroxide followed with water, dried over magnesium sulphate then was concentrated under reduced pressure. The mixture was purified by HPLC using a 10% trietylamine solution in methanol to give 4 as oil with a 67% yield.
1H NMR (CDCl3) : δ 1.87-2.06 (m, 4 H, CH2), 2.27 (s, 6 H, NMe2), 2.28 (s, 6 H, NMe2), 2.41-2.55 (m, 4 H, CH2N), 2.93 (s, 4 H, Hindane), 3.95-4.09 (m, 4 H, CH2O), 6.50 (d, J = 7.7 Hz, 1 H, Harom), 6.78-6.92 (m, 5 H, Harom), 7.01-7.25 (m, 6 H, Harom). 13C NMR (CDCl3) : δ 27.9 (2 CH2), 31.6 (CH2 indane), 34.8 (CH2 indane), 45.8 (2 NMe2), 56.7 (CH2N), 56.8 (CH2N), 66.5 (2 CH2O), 114.1 (2 CH C6H4), 114.9 (2 CH C6H4), 125.2 (CHindane), 125.3 (CHindane), 125.9 (CHindane), 127.3 (CHindane), 130.7 (2 CH C6H4), 131.4 (2 CH C6H4), 134.6 (C), 135.6 (C), 136.7 (C), 139.6 (C), 142.0 (C), 147.9 (C), 157.9 (C), 158.4 (C). IR (KBr, ν cm-1): 3063, 3033, 2940, 2854, 2813, 2763 (CH2, CH3). MS (EI, 70 eV) m/z : 484 [M]+., 439, 86 [CH2CH2CH2NMe2]+, 58 [CH2NMe2]+.
1,1-bis-[4-(3-Dimethylamoniumpropoxy)phenyl]-2-ferrocenyl-but-1-ene dichloride 9
Diamino compound 5 (2.2 g, 3.7 mmol) was dissolved into 200 mL of diethyl ether. A 2 M solution of hydrochloric acid in diethyl ether (3.7 mL, 7.4 mmol) was added dropwise into the solution. An orange precipitate was immediately formed. After stirring for 20 min, the mixture was filtered under argon and the obtained orange solid was washed with 3 × 5 mL of diethyl ether and was dried under vacuum giving compound 9 in 59% yield. Crystals contain traces of diethyl ether.
1H NMR (DMSO-d6) : δ 1.02 (t, J = 7.4 Hz, 3 H, CH3), 2.04-2.27 (m, 4 H, 2 CH2), 2.44-2.63 (m, 2 H, CH2), 2.79 (s, 12 H, NMe2H+), 3.10-3.30 (m, 4 H, CH2N), 3.85 (s, 2 H, C5H4), 3.97-4.25 (m, 11 H, C5H4 + C5H5 + CH2O), 6.86 (d, J = 8.0 Hz, 2 H, C6H4), 6.90-7.01 (m, 4 H, C6H4), 7.14 (d, J = 8.0 Hz, 2 H, C6H4). 13C NMR (DMSO-d6) : δ 16.3 (CH3), 24.8 (2 CH2), 28.1 (CH2), 42.9 (2 NMe2H+), 54.9 (2 CH2N), 65.6 (CH2O), 65.8 (CH2O), 68.8 (2 CH C5H4), 69.6 (2 CH C5H4), 69.9 (5 CH C5H5), 86.8 (C C5H4), 115.1 (2 x 2 CH C6H4), 130.8 (2 CH C6H4), 131.3 (2 CH C6H4), 137.1 (C), 137.4 (C), 137.9 (C), 138.2 (C), 157.5 (2 C) IR (KBr, ν cm-1): 3425 (NH), 3029, 2960, 2685, 2510, 2470 (CH2, CH3).
Microbial strains
Known and newly synthesized compounds were tested against Escherichia coli (ATCC 10536), Pseudomonas aeruginosa (ATCC 15442), Staphylococcus aureus (ATCC 6538) and Enterococcus hirae (ATCC 10541).
All strains were cultured in liquid LB (1% Bactotryptone, 0.5% Yeast extract, 0.5% NaCl).
Estimation of the antimicrobial effect
The estimation of the antimicrobial effect against microbial strains was performed by the method of micro-dilution in ELISA plates. A 2.54 10-3 M stock solutions of the tested products were prepared in DMSO or water, depending on their solubility. In Elisa plates and for each product a series of eight wells containing 100 μl of culture medium with decreasing concentration of the product were prepared by the successive ½ dilution.
A 100 μl of overnight shaking microbial culture, incubated at adequate temperature, depending on bacterial strains, were used to inoculate the plate wells containing different concentrations of compounds. The final concentration of each product, for a series of eight wells was 2.54 10-4 M, 1.27 10-4 M, 6.35 10-5 M, 3.18 10-5 M, 1.59 10-5 M, 7.94 10-6 M, 3.97 10-6 M and 1.98 10-6 M. The plates were incubated with shaking overnight at the same temperature, depending on bacterial strains, and their OD was measured at 620 nm.
A negative control (uninoculated wells), a positive control (seeded and without antimicrobial compound wells) were prepared under the same experimental conditions.
The inhibitory activity of the tested compounds was calculated according to the formula:
where (x) is the microbial culture containing the inhibitor and (i) is the microbial culture without inhibitor.