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 (Lizarraga et al., 2014), to yield crystalline CHR.
Mp, UV-vis, MS, 1H-NMR spectra and X-Ray structure were reported elsewhere
(Lizarraga et 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 (Villecco et 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 Heluani et 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-NMR spectra 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 (Lizarraga et 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-yellow oil. 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. albicans ATCC 10231 is a FLU sensitive strain, while C.
albicans 12-99 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-0 and FIC values generated by 4HMBA, FLU and CAS in
combinatory experiments (checkerboard assay) against C. albicans ATCC 10231
and 12-99.
MIC-0 (mg L-1)
Combined
Strain
FIC
drugs
4HMBA
FLU
12-99
4HMBA + FLU
40
32
ATCC 10231
4HMBA + CAS
10
0.06
0.490
12-99
4HMBA + CAS
40
0.12
0.980
CAS
0.625
TABLE S2: MIC values generated by 4HMBA and FLU alone and in combinatory
experiments (checkerboard assay) against C. albicans mutant strains.
Drugs alone
Drugs in combination
MIC-2 (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
Strain
MIC-0 (mg L-1)
REFERENCES (for supplementary material)
de Heluani CS, de Lampasona MP, Catalán CAN, Goedken VL, Gutierrez AN,
Herz W. Guaianolides, heliangolides and other constituents from Stevia 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 Xenophyllum
incisum. Biochem Syst Ecol 35 (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-acetyl2,2-dimethyl-chromane. Spectrochim. Acta Mol. Biomol. Spectros. 127 (2014) 7484.
Lizarraga E, Romano E, Rudyk R, Catalán CAN, Brandán SA. Structural study,
coordinated normal analysis and vibrational spectra of 4-hydroxy-3-(3-methyl-2butenyl)acetophenone. Spectrochim Acta A 97 (2012) 399-406.
Tomás-Barberán F, Iniesta-San Martín E, Tomás-Lorente F, Romero A.
Antimicrobial phenolic compounds from three spanish Helicrysum species.
Phytochem 29 (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.