Journal of Agricultural and Food Chemistry
Article
226 (C13H17ClO [M + 2]+·33); 224 ([M]+·100); 211(19); 209 (57);
189 (13); 185 (20); 183 (66); 167 (19); 155 (73); 143 (88); 139
(14); 119 (23); 105 (23); 91 (2); 77 (26); 65 (7); 41 (69).
1-(Allyloxy)-4-chloro-5-isopropyl-2-methylbenzene (3b). The
compound is a reddish brown liquid. 1H NMR, δH (300 MHz,
CDCl3): 1.22 (d, 6H, J = 6.9 Hz,CH(CH3)2); 2.18 (s, 3H,CH3); 3.33
(sept. 1H, J = 6.9 Hz, CH(CH3)2); 4.54 (dt, 2H, J = 5.1; 1.5 Hz,
CH2); 5.22−5.44 (m, =CH2); 5.26−5.46 (m, 2H; =CH2); 6.10 (m,
1H, CH=); 6.72 (s, 1H, Ar−H6); 7.10 (s, 1H, Ar−H3). 13C NMR, δC
(75 MHz, CDCl3): 15.58 (CH3); 22.71 (CH(CH3)2); 30.16
(CH(CH3)2); 69.82 (CH2); 109.67 (C6); 117.15 (=CH2); 124.09
(C2); 126.01 (C4); 130.97 (C3); 133.44 (CH=); 143.60 (C5); 155.63
(C1). MS (EI) m/z (%): 226 (C13H17ClO [M + 2]+·26); 224 ([M]+·
77); 211(32); 209 (100); 181 (9); 174 (48); 158 (31); 145 (19); 141
(6); 128 (10); 115 (14); 105 (4); 91 (10); 77 (8); 51 (6); 43 (7).
1-(Allyloxy)-4-chloro-2-methoxybenzene (3c). The compound is a
reddish brown liquid. 1H NMR, δH (300 MHz, CDCl3): 3.86 (s, 3H,
OCH3); 4.57 (dt, 2H, J = 5.4 Hz; 1.5 Hz; CH2); 5.26−5.42 (m, 2H,
=CH2); 5.99−6.12 (m, 1H, CH=); 6.77−7.26 (m, 3H,Ar−H). 13C
NMR, δC (75 MHz, CDCl3): 56.05 (OCH3); 70.10 (CH2); 112.34
(C3); 114.24 (C6); 118.19 (=CH2); 120.19 (C5); 126.00 (C4);
132.97 (CH=); 146.67 (C1); 150.04 (C2).MS (EI) m/z (%): 200
(C10H11ClO2 [M + 2]+·2); 198 ([M]+·6); 157 (100); 143 (5); 129
(24); 111 (14); 99 (8); 93 (50); 79 (16); 65 (30); 51 (13); 41 (16).
2-(Allyloxy)-1-isopropyl-4-methylbenzene (4a). The compound is
an uncolored liquid. 1H NMR, δH (300 MHz, CDCl3): 1.22 (d, 6H, J
= 6.9 Hz, CH(CH3)2); 2.23 (s, 3H, CH3); 3.28−3.51 (m, 1H,
CH(CH3)2); 4.52−4.56 (m, 2H, CH2−CH=CH2); 5.25−5.49 (m,
2H, CH=CH2); 5.88−6.16 (m, 1H, CH=CH2); 6.76 (d, 1H, J = 7.5
Hz, Ar−H5); 6.67 (s, 1H,Ar−H3),7.11 (d, 1H, J = 7.5 Hz, Ar−H6).
13C NMR, δC (75 MHz, CDCl3): 21.40 (CH3); 22.75 (CH(CH3)2);
26.59 (CH(CH3)2); 68.73 (CH2); 113.86 (C3); 112.65
(=CH2);116.61 (C5); 125.88 (C1); 127.10 (C6); 133.74 (CH=);
136.20 (C4); 155.64 (C2). MS (EI) m/z (%): 190 (C13H18O [M]+·
82); 175 (100); 147 (57); 133 (47); 121 (85); 105 (47); 91 (53); 77
(25); 65 (11); 41 (52).
2-(Allyloxy)-4-isopropyl-1-methylbenzene (4b). The compound is
an uncolored liquid. 1H NMR, δH (300 MHz, CDCl3): 1.24 (d, 6H, J
= 6.9 Hz, CH(CH3)2); 2.22 (s, 3H, CH3); 2.86 (sept. 1H, J = 6.9 Hz,
CH(CH3)2); 4.84 (dt, 2H, J = 5.1 Hz; 1.5 Hz; CH2-CH=); 5.25−5.48
(m, 2H, CH=CH2); 6.00 (m, 1H, CH=CH2); 6.70 (d, 1H, J = 1.0 Hz,
Ar−H3); 6.75 (dd, 1H,J = 7.5; 1.5 Hz, Ar−H5), 7.07 (d, 1H, J = 7.5
Hz, Ar- H6). 13C NMR, δC (75 MHz, CDCl3): 15.85 (CH3); 24.10
(CH(CH3)2); 34.09 (CH(CH3)2); 68.73 (CH2); 109.87 (C3); 116.80
(=CH2); 118.21 (C5); 124.25 (C1); 130.44 (C6); 133.81 (CH=);
147.79 (C4); 156.65 (C2). MS (EI) m/z (%): 190 (C13H18O [M]+·
100); 175 (82); 133 (38); 121 (35); 105 (59); 79 (26); 77 (26); 65
(10); 55 (13); 41 (56).
1-(Allyloxy)-2-methoxybenzene (4c). The compound is an
uncolored liquid. 1H NMR, δH (300 MHz, CDCl3): 3.88 (s, 3H,
OCH3); 4.61 (dt, 2H, J = 5.4; 1.5 Hz, CH2); 5.26−5.44 (m, = CH2);
6.03−6.16 (m, 1H, CH=); 6.91 (m, 3H, Ar−H). 13C NMR, δC (75
MHz, CDCl3): 55.85 (OCH3); 69.82 (CH2); 111.72 (C3 or C6);
113.55 (C3 or C6); 117.84 (=CH2); 120.69 (C4 or C5)*; 121.19 (C4
or C5)*; 133.39 (CH=); 147.47 (C1or C2)*; 149.47 (C1 or C2)*. ∗,
attributions may be inverted. MS (EI) m/z (%): 164 (C10H12O2
[M]+·70); 123 (100); 95 (69); 77 (66); 67 (11); 65 (22); 52 (20);
41 (35).
(75 MHz, CDCl3): 33.81 (CH2); 55.99 (OCH3); 108.64 (C5);
115.38 (=CH2); 119.36 (C3); 122.22 (C4); 125.84 (C2); 136.64
(CH=); 143.37 (C1); 146.36 (C6). MS (EI) m/z (%): 164
(C10H12O2[M]+·100); 149 (35); 131 (27); 121 (16); 103 (22); 91
(18); 77 (22); (8); 55 (18); 39 (8).
Antimicrobial Activity. Antimicrobial activity was evaluated by
the agar diffusion test using solutions of all components in DMSO at
200 μg mL−1 according to the methodology described by the National
Committee for Clinical Laboratory Standards.30 Gram-positive
bacterium Staphylococcus aureus ATCC 6538 and Gram-negative
bacterium Escherichia coli ATCC 11229 were used. Suspensions of the
microorganisms were activated twice in BHI broth (Himedia, India)
and incubated at 35 °C for 18−24 h. To obtain isolated colonies,
streaking was done in Petri dishes containing count agar plates (PCA,
Himedia, India) and incubated at 35 °C for 18−24 h. Isolated
colonies were selected to prepare the bacterial inocula in saline
solution at 0.85% (w/v). The optical density of bacterial inocula was
spectrophotometrically adjusted at 600 nm (Thermo Scientific
Multiskan Go, USA) to give approximately 1.0 × 108 CFU mL−1
(Abs600 nm = 0.1). An aliquot of 0.1 mL of bacterial suspension
(Abs600 nm = 0.1) was spread onto Muller-Hinton agar plate (Sigma-
Aldrich, Sao Paulo, Brazil). Discs (6 mm) impregnated with 5 μL of
̃
each tested compound were placed on the surface of the agar.
Inoculated plates were incubated at 35 °C for 24 h. At the end of
incubation, the diameter of inhibition zones was measured with a
ruler and recorded in mm. DMSO was used as negative control and
ampicillin (10 μg disc, Bio-Rad, France) as positive control. The
experiment was conducted under completely randomized design
(CRD) with three repetitions in duplicate. Data from the agar
diffusion test were analyzed using the R software31 and were subjected
to analysis of variance (ANOVA) and Duncan test at 5% of
probability.
Determination of Minimum Inhibitory Concentration (MIC).
The minimum inhibitory concentration (MIC) and the minimum
bactericidal concentration (MBC) of the natural phenol derivatives
were tested against strains of: Staphylococcus aureus (ATCC 6538),
Escherichia coli (ATCC 11229), Pseudomonas aeruginosa (ATCC
15442), Salmonella enterica Typhimurium (ATCC 13076), Bacillus
cereus (ATCC 14579), Listeria innocua (ATCC 33090), and
Salmonella enterica ssp. enterica isolated from vegetables commercial-
́
ized in Alegre city, Espirito Santo. The MIC was carried out using the
in vitro broth microdilution method. Broth BHI (Himedia, India)
supplemented with the compounds was added to 96-well titration
microplates. A two-fold dilution factor was used ranging from 220 to
3.44 μg mL−1. The concentrations tested were: 220 μg mL−1, 110 μg
mL−1, 55 μg mL−1, 27.5 μg mL−1, 13.75 μg mL−1, 6.87 μg mL−1, 3.44
μg mL−1. Microplates were separately, inoculated with 5 × 105 ufc
mL−1 of each strain of bacteria in exponential growth phase. After the
inoculations, the microplates were aerobically incubated at 35 °C for
24 h. MIC was considered the lower concentration of the tested
compounds able to inhibit microorganism’s visual growth.32
Determination of Minimum Bactericidal Concentration
(MBC). The minimum bactericidal concentration (MBC) was
determined from the inoculation by spreading BHI of 0.1 mL of
the microplate wells content that did not present growth in the MIC
test in agar. Plates were aerobically incubated at 35 °C for 24 h. MBC
was defined as the lower concentration that did not present bacterial
multiplication.32
Experimental Planning and Statistical Data Analysis. The
experiment was conducted under completely randomized design
(CRD) with three repetitions. For the diffusion in agar test, the results
were analyzed with the R software submitted to analysis of variance
(ANOVA). When the different semisynthetic compounds presented
significant effect, they were compared by the Duncan test at 5% of
probability. For the comparison of the two tested bacteria, results
were submitted to t test for the averages comparison (p > 0.05)31.
Molecular Properties and Statistical Analysis. The molecular
properties of the natural phenols and their semisynthetic derivatives
were computed with the Spartan 6.033 software using the semi-
empirical PM6 method.34 Geometries were fully optimized with the
Synthesis of Compound 5c. Synthesis of 5c was exemplified by
the synthesis of the 2-allyl-6-methoxyphenol (5c), known as ortho-
eugenol. The allyl ether obtained, 4c (3g), was introduced in a sealed
glass tube, which was heated between 200 and 240° for 6 h in a sand
bath. After this period, the tube was submitted to a cooling bath until
it reached room temperature, when the tube could be opened and the
product purified by column chromatography using the mixture
hexane/dichloromethane (5:1) as eluent.29
2-Allyl-6-methoxyphenol (5c). The compound is a yellowish
1
liquid. H NMR, δH (300 MHz, CDCl3): 3.42−3.44 (m, 2H, CH2);
3.89 (s, 3H, OCH3); 5.04−5.13 (m, 2H, =CH2); 5.72 (s, 1H, OH);
5.96−6.10 (m, 1H, CH=); 6.74−6.84 (m, 3H, Ar−H). 13C NMR, δC
C
J. Agric. Food Chem. XXXX, XXX, XXX−XXX