Antifungal activity of 4-hydroxy-3-(3-methyl-2-butenyl)acetophenone against Candida albicans. Evidences for the antifungal mode of action.

José R. Soberóna,b,c,*, Emilio F. Lizarragaa,c,d, Melina A. Sgarigliaa,b,c, María B. Carrasco Juáreza,b, Diego A. Sampietroa,b,c, Aida Ben Altabefa,c,d, César A.N. Catalána,c,d, Marta A. Vattuoneb,c

SUPPLEMENTARY MATERIAL

FIGURE 1: Chemical structures of sample compounds

Semi-synthesis of 6-acetyl-2,2-dimethyl-chromane (CHR).

4HMBA (1.5 g) was dissolved in 40 mL of ethanol containing 4.5 mL of concentrated HCl and refluxed for 5 h. After cooling the reaction mixture was processed as previously described (Lizarragaet al., 2014),to yield crystalline CHR.Mp, UV-vis, MS, 1H-NMR spectra and X-Ray structure were reported elsewhere (Lizarragaet al., 2012).

Semi-synthesis of 4-acetyloxy-3-(3-methyl-2-butenyl)acetophenone (AcA).

4HMBA (1 g) was mixed with 1.5 mL of acetic anhydride dissolved in 7 mL of anhydrous pyridine and stirred for 16 h (Villeccoet al., 2008); then, 15 mL of concentrated HCl diluted in 40 mL of distilled water was added, stirred for 15 min and extracted with CH2Cl2 (3 x 70 mL). The organic extracts were pooled, washed with 10% CuSO4 (2 x 30 mL) and water (1 x 15 mL). After drying (anh. Na2SO4) and solvent evaporation, 993 mg (yield 82.3%) of crude AcA was obtained and purified by column chromatography on silica gel (230-400 mesh) using a mixture of n-hexane-ethyl acetate 85:15 as elution solvent to yield 752.2 mg of AcA(oil), 99.3% pure by GC.

Semi-synthesis of 4-acetyloxy-3-(2,3-epoxy-3-methylbutyl)acetophenone (EPX).

To AcA (600 mg) dissolved in 30 mL of CHCl3 and cooled in ice was added in small portions 600 mg of 77% m-chloroperbenzoic acid (de Heluaniet al., 1989). The mixture was stirred overnight, washed with 10% Na2SO3 (2 x 10 mL), 5% NaHCO3 (2 x 10 mL) and water (1 x 10 mL). After drying (Na2SO4) and solvent evaporation, 458.7 mg (71.6%) of crude EPX was obtained which was purified by column chromatography on Silica gel 230-400 mesh using a mixture of n-hexane-ethyl acetate 80:20 as elution solvent, to yield 146.6 mg of EPX (oil), 97.7% pure by GC.

SPECTROSCOPIC DATA

4HMBA.Needles.Mp, UV, MS, 1H- and 13C-NMRspectra were identical to those reported (Tomás-Barberán et al., 1990, Marchese et al., 2007).

CHR.Needles. Mp 94-95ºC.UV, MS, IR, 1H- RMN spectra and X-Ray structure were previously reported (Lizarragaet al., 2014).

AcA.Pale-yellow oil.EIMS: m/z (rel. int.%) [M]+ 246 (1); 203 (54); 189 (86), 175 (5), 161 (42), 149 (68), 133 (25), 91 (11), 77 (10), 43 (100).1H NMR (200 MHz, CDCl3) δ 7.78-7.89 m (2H, H-2 and H-6), 7.12 br d (1H, 8.1 Hz, H-5), 3.28 br d (2H, 7.1 Hz, H-1’), 2.58 s (3H, Me-6’), 2.33 s (3H, Ac), 1.75 br s and 1.71 br s (3H each, Me-4’ and Me-5’).

EPX. Pale-yellowoil. EIMS: m/z (rel. int.%) [M]+ 262 (1); 220 (3); 202 (10), 163 (11), 149 (38), 147 (24), 133 (6), 119 (8), 107 (9), 91 (9), 77 (17), 43 (100).1H NMR (200 MHz, CDCl3) δ 7.95 d (1H, 2.1 Hz, H-2), 7.89 dd (1H, 8.4 and 2.2 Hz, H-6), 7.19 d (1H, 8.4 Hz, H-5), 2.94 m (1H, H-2’), 2.84 m (2H, H-1’), 2.61 s (3H, Me-6’), 2.37 s (3H, Ac) 1.39 s and 1.35 s (3H each, Me-4’ and Me-5’).

FIGURE S2: Disk diffusion assay.

Legend of Fig. 1:C. albicansATCC 10231 is a FLU sensitive strain, whileC. albicans12-99 is a FLU resistant strain. A) C. albicans ATCC 10231 + FLU (6 mg/L). B)C. albicans ATCC 10231 (without FLU). C) C. albicans 12-99 + FLU (6 mg/L). D) C. albicans 12-99 (without FLU). Tested compounds: 2: CHR, 3: AcA, 4: EPX.

TABLE S1: MIC-0and FIC values generated by4HMBA, FLU and CASin combinatory experiments (checkerboard assay) against C. albicansATCC 10231 and 12-99.

Strain / Combined drugs / MIC-0 (mg L-1) / FIC
4HMBA / FLU / CAS
12-99 / 4HMBA + FLU / 40 / 32 / 0.625
ATCC 10231 / 4HMBA + CAS / 10 / 0.06 / 0.490
12-99 / 4HMBA + CAS / 40 / 0.12 / 0.980

TABLE S2:MIC values generated by 4HMBA and FLU alone and in combinatory experiments (checkerboard assay) against C. albicans mutant strains.

Strain / Drugs alone / Drugs in combination
MIC-2 (mg L-1) / MIC-0 (mg L-1) / MIC-0 (mg L-1)
FLU / 4HMBA / FLU / 4HMBA
Day 364 / 0.06 / 40 / 0.03 / 5
MCC 85 / 1.00 / 40 / 0.50 / 5
OCC 1.1 / 0.30 / 40 / 0.15 / 5
OCC 7 / 0.75 / 40 / 0.50 / 5

REFERENCES (for supplementary material)

deHeluani CS, de Lampasona MP, Catalán CAN, Goedken VL, Gutierrez AN, Herz W. Guaianolides, heliangolides and other constituents fromStevia alpine. Phytochem 28 (1989) 1931-1935.

Marchese MJA de, Heluani CS de, Catalán CAN, Griffin CA, Vaughn JB, Herz W. Incisol, an alcohol with a novel sesquiterpene skeleton from Xenophyllumincisum. BiochemSyst Ecol35 (2007) 169-175.

Lizarraga E, Gil DM, Echeverría GA, Piro OE, Catalán CAN, Ben Altabef A.Synthesis, crystal structure, conformational and vibrational properties of 6-acetyl-2,2-dimethyl-chromane. Spectrochim. Acta Mol. Biomol. Spectros.127 (2014) 74-84.

Lizarraga E, Romano E, Rudyk R, Catalán CAN, Brandán SA. Structural study, coordinated normal analysis and vibrational spectra of 4-hydroxy-3-(3-methyl-2-butenyl)acetophenone. SpectrochimActaA 97 (2012) 399-406.

Tomás-Barberán F, Iniesta-San Martín E, Tomás-Lorente F, Romero A. Antimicrobial phenolic compounds from three spanishHelicrysum species. Phytochem29 (1990) 1093-1095.

Villecco MB, Catalán JV, Vega MI, Garibotto FM, Enriz RD, Catalán CAN.Synthesis and antibacterial activity of highly oxygenated 1,8-cineole derivatives. Nat. Prod. Commun. 3 (2008) 303-312.