3990 J. Agric. Food Chem., Vol. 56, No. 11, 2008
Mastelic´ et al.
tion of 10 µg/mL and introduced into the spectrometer with the flow
of 5 µL/min. Reference for spectrometer calibration was sodium iodide
clusters.
antioxidant activity of phenols depends on the electronic and
steric effects of the ring, substituents, and the strength of
hydrogen-bonding interactions between the phenol and the
solvent (9–12).
Many essential oils exhibit antioxidant and antimicrobial
activities (13–15). Phenols, such as thymol, carvacrol, and
eugenol, and monocyclic hydrocarbons, such as terpinolene,
R-terpinene, and γ-terpinene, belong to the most active natural
antioxidants found in the essential oils (16–18). However, due
to their poor water solubility and the requirement of high
concentrations to reach a therapeutic effect, the efficiency of
these compounds in treatment is limited.
In this paper, we report on the synthesis of three hydroxy-
methyl derivatives of thymol, carvacrol, and eugenol as well
as a diarylmethane derivate of carvacrol that, to the best of our
knowledge, has not been synthesized before. Chemical modi-
fications, such as introducing polar hydroxymethyl moiety into
phenol structures, can change antioxidant activity in comparison
with the starting compounds (19). Hydroxymethylation reactions
(nucleophilic addition to the carbonyl group) can be catalyzed
by either acids or bases, and electrophilic aromatic substitution
reaction of the newly formed hydroxymethylphenol with residual
phenol can yield methylenediphenols (20–22). The antioxidant
properties of prepared derivatives were evaluated, for the first
time, by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and Rancimat
methods, as well as their antibacterial, antiyeast, and antipro-
liferative activities and compared to those of the parent
compounds.
NMR Spectroscopy. The liquid-state one- and two-dimensional 1H
and 13C NMR spectra (600.13 MHz for 1H, 150.90 MHz for 13C) were
measured in DMSO-d6 at 298 K using standard 1H, APT, COSY,
HSQC, and HMBC techniques. Chemical shifts, in parts per million,
were referred to TMS as internal standard. The resolution in 1H NMR
and 13C NMR spectra was 0.29 and 0.54 Hz per point, respectively.
Synthesis of Compounds. Hydroxymethylation of CarVacrol. To
a solution of 8 g of NaOH (200 mmol) in 100 mL of water and 15 g
of carvacrol (100 mmol) was added 10 mL of 35% methanal (ca. 125
mmol) in several portions. Reaction temperature was 50 °C and reaction
time, 24 h. The resulting mixture was neutralized with 20% acetic acid
(to pH 5) and extracted with ether. The combined ether extracts were
concentrated by removal of ether on a rotating evaporator. The residue
was submitted to steam distillation to remove residual carvacrol. Water-
non-soluble residue was purified by column chromatography on silica
gel with petroleum ether/ethyl acetate, Ψ ) 3:2 (v/v). Two derivatives
of carvacrol, 1 and 2, were obtained with yields of 15 and 10%.
4-(Hydroxymethyl)-5-isopropyl-2-methylphenol: colorless oil; purity,
96.4% by GC; MS (EI), m/z (%) 180 (M+; 27), 162 (81), 147 (100),
119 (36), 109 (17), 91 (51), 77 (32), 65 (17), 51 (16), 39 (27); 1H
NMR (DMSO-d6) δ 1.13 (d, 6H, J ) 6.7 Hz, H-5′′), 2.07 (s, 3H, H-2′),
3.13 ppm (m, 1H, J ) 6.7 Hz, H-5′), 4.39 (d, 2H, J ) 5.3 Hz, H-4′),
4.77 (t, 1H, J ) 5.3 Hz, H-4′′), 6.68 (s, 1H, ar-H-6), 6.95 (s, 1H, ar-
H-3), 9.00 (s, 1H, H-1′); 13C NMR (DMSO-d6) δ 15.5 (C-2′), 23.9
(C-5′′), 27.6 (C-5′), 60.7 (C-4′), 111.2 (ar-C-6), 120.1 (ar-C-2), 129.0
(ar-C-4), 131.0 (ar-C-3), 144.9 (ar-C-5), 154.5 (ar-C-1).
4,4′-Methylenebis(5-isopropyl-2-methyl)phenol: white crystals, mp
173 ( 0.5 °C; purity, 95.7% by GC; MS (EI), m/z (%) 312 (M+; 13),
269 (2), 162 (100), 161 (18), 147 (12), 121 (6), 91 (5), 77 (3); HRMS
(negative ESI), calcd for C21H28O2 [M - H]- 311.2011, found
MATERIALS AND METHODS
1
311.1996; H NMR (DMSO-d6) δ 1.09 (d, 12H, J ) 7.0 Hz, H-5′′),
General. Synthesized compounds were purified on a silica gel 60
(Kieselgel 60, 0.040–0.063 mm, Merck) column or by recrystallization
and analyzed by TLC, mass spectrometry, and NMR analysis. TLC
was performed on silica gel 60 precoated plates (Kieselgel 60, thickness
) 0.2 mm, Merck). Melting points were obtained using an Kofler
Mikroheitztisch apparatus (Reichert) and were uncorrected. Gas chro-
matograph model 5890 with mass selective detector model 5971A
(Hewlett-Packard) was used for purity determination and mass spectral
analysis of prepared compounds. High-resolution mass spectra was
measured on Waters Micromass Q-ToF micro electrospray ionization
spectrometer. NMR experiments were performed on a Bruker AV600
spectrometer equipped with a 5 mm TBI probe with z-gradient.
Microwell plate reader (Easy-Reader 400 FW, SLT Laboratory Instru-
ments GmbH) was used for antiproliferative activity assay. A Lambda
EZ 201 UV–vis spectophotometer from Perkin-Elmer Inc. was used in
the DPPH assays. A Rancimat 743 apparatus from Metrohm A.G. was
used to measure the induction time of lard with and without
additives.
Reagents and Solvents. Thymol (99%), carvacrol (97%), eugenol
(99%), and methanal (formaline) were purchased from Fluka Chemie.
Butylated hydroxytoluene (BHT), vitamin C, Na2HPO4 ·12H2O, and
KH2PO4 were purchased from Sigma. Solvents were obtained from
Merck. Petroleum ether with bp 40–70 °C was used. All solvents were
purified prior to use in the experiments.
Gas Chromatography–Mass Spectrometry (GC-MS) Analysis.
Analyses were performed on a GC-MS using column HP-101 (dim-
ethylpolysiloxane, Hewlett-Packard): 25 m × 0.2 mm i.d., film thickness
) 0.2 µm; column temperature programmed from 70 °C isothermal
for 2 min, to 220 °C at a rate of 4 °C min-1; carrier gas, helium; flow
rate, 1 mL min-1; injector temperature, 250 °C; volume injected: 1 µL
of 1% ether solution of each compound 1:50. MS conditions were as
follows: ionization voltage, 70 eV; ion source temperature, 280 °C;
mass range, 35–350 mass units.
1.98 (s, 6H, H-2′), 2.96 (m, 2H, J ) 7.00 Hz, H-5′), 3.73 (s, 2H, H-4′),
6.50 (s, 2H, ar-H-3), 6.70 (s, 2H, ar-H-6), 8.94 (s, 2H, H-1′); 13C NMR
(DMSO-d6) δ 15.5 (C-2′), 23.6 (C-5′′), 28.0 (C-5′), 32.9 (C-4′), 111.4
(ar-C-6), 120.5 (ar-C-2), 127.7 (ar-C-4), 131.4 (ar-C-3), 144.4 (ar-C-
5), 153.6 (ar-C-1).
Hydroxymethylation of Eugenol. The same procedure as mentioned
above was used. Into a solution of 8 g of NaOH (200 mmol) in 100
mL of water and 15 g of eugenol (100 mmol) was added 10 mL of
35% methanal (ca. 125 mmol) in portions. Reaction temperature was
50 °C and reaction time, 24 h. Thereafter, the resulting mixture was
neutralized with 20% acetic acid (until pH 5) and extracted with ether.
The combined ether extracts were dried with sodium sulfate, and ether
was removed on a rotating evaporator. Derivative was purified by
column chromatography on silica gel with petroleum ether/ethyl acetate,
Ψ ) 3:2 (v/v). The yield was 51%.
4-Allyl-6-(hydroxymethyl)-2-methoxyphenol: colorless oil; purity,
94.5% by GC; MS (EI), m/z (%) 194 (M+; 48), 176 (100), 147 (51),
1
133 (30), 117 (38), 104 (38), 103 (36), 91 (39), 77 (61), 51 (30); H
NMR (DMSO-d6) δ 3.28 (d, 2H, J ) 6.1 Hz, H-4′), 3.77 (s, 3H, H-2′),
4.48 (d, 2H, J ) 5.3 Hz, H-6′), 4.98 (t, 1H, J ) 5.3 Hz, H-6′′), 5.02
(d, 1H, J ) 9.9 Hz, H-4′′′b), 5.08 (d, 1H, J ) 17.0 Hz, H-4′′′a), 5.94
(m, 1H, H-4′′), 6.66 (s, 1H, ar-H-3), 6.76 (s, 1H, ar-H-5), 8.38 (s, 1H,
H-1′); 13C NMR (DMSO-d6) δ 39.5 (C-4′), 55.8 (C-2′), 58.2 (C-6′),
110.4 (ar-C-3), 115.2 (C-4′′′), 119.3 (ar-C-5), 128.8 (ar-C-6), 129.7
(ar-C-4), 138.2 (C-4′′), 141.2 (ar-C-1), 146.9 (ar-C-2).
Hydroxymethylation of Thymol. The same procedure was followed
as previously described. Ten milliliters of 35% formaldehyde (ca. 125
mmol) in several portions was added to a solution of 8 g of NaOH
(200 mmol) in 100 mL of water and 15 g of thymol (100 mmol).
Reaction temperature was 50 °C and reaction time, 24 h. The resulting
mixture was neutralized with 20% acetic acid (pH 5) and extracted
with ether. The combined ether extracts were dried with sodium sulfate,
and ether was removed on a rotating evaporator. The residue was
purified by two crystallizations from water/ethanol, Ψ ) 2:1 (v/v), and
from petroleum ether/toluene, Ψ ) 1:1 (v/v). The yield was 75%.
4-(Hydroxymethyl)-2-isopropyl-5-methylphenol: white crystals, mp
119.0 ( 0.5 °C; purity, 97.2% by GC; MS (EI), m/z (%) 180 (M+;
High-Resolution Mass Spectrometry (HRMS). High-resolution
mass spectra were measured in negative ion mode (ESI-): capillary
voltage, 3.0 kV; cone voltage, 60 V; collision energy, 6.0 V; desolvation
temperature, 200 °C; source temperature, 100 °C; nitrogen flow, 300
L/h. Sample was prepared by dissolving in methanol to the concentra-