September 2003
1087
m/z 371 (Mϩϩ1). Anal. Calcd for C22H26O5: C, 71.33; H, 7.07. Found: C,
71.29; H, 7.01.
ity, radical scavenging, and cytotoxicity on melanocytes be-
cause of the absence of phenolic hydroxyl groups in their
molecular structure. Further studies on their mechanism of
action in melanogenesis are underway.
Cell Culture Melan-a melanocytes are a highly pigmented, immortal-
ized normal murine melanocyte cell line derived from C57BL/6 mice. The
melan-a melanocytes used in this study were obtained from Dr. Dorothy
Bennett (St. George’s Hospital, London, U.K.). Cells were grown and main-
tained at 37 °C in an atmosphere of 95% air, 5% CO2 in RPMI-1640 (Bio
Whittaker, Walkersville, MA, U.S.A.) supplemented to a final concentration
of 10% heat-inactivated fetal bovine serum, penicillin 5 units/ml, strepto-
mycin 5 mg/ml and 200 nM phorbol 12-myristate 13-acetate. Cells were pas-
saged every 3 d with a maximal passage number of 33. Confluent monolay-
ers of melanocytes were harvested with a mixture of 0.05% trypsin and 0.53
mM EDTA (Gibco BRL, Grand Island, NY, U.S.A.).
Measurements of Melanin Content and Cell Viability Melanin con-
tent and cell number were measured in melan-a melanocytes. One hundred
thousand cells were seeded into each well of 24-well plates and compounds
were added to triplicate cultures. Medium was changed daily, and after 4 d of
culture, the cells were lysed with 1 N NaOH 1 ml and pipetted repeatedly to
homogenize. For analysis, 200 ml of each crude cell extract was transferred
into 96-well plates. The relative melanin content was measured at 400 nm
with an enzyme-linked immunosorbent assay (ELISA) reader (Bio-Tex In-
struments). Cell viability was determined using the crystal violet assay. The
culture medium was removed from the 24-well culture plates and replaced
with 0.5 ml of 0.1% crystal violet in 10% ethanol per well. The plates were
stained for 5 min at room temperature and rinsed four times. The crystal vio-
let retained by adherent cells was extracted with 1 ml of 95% ethanol. Ab-
sorbance was determined at 540 nm using an ELISA reader.
Experimental
Syntheis of Thymol Ester Derivatives In the synthesis of 4a—c, the
phenolic hydroxyl groups were protected by a benzyl group and deprotected.
All acids were coupled with thymol using the mixed anhydride method19) as
follows. To a solution of 3,4,5-tribenzyloxybenzoic acid (2.0 g, 4.5 mmol) in
pyridine (30 ml), benzenesulfonyl chloride (962 mg, 5.4 mmol) was added
dropwise at 0 °C. The reaction mixture was allowed to warm up to room
temperature and stirred for 30 min. To the stirring reaction mixture, thymol
(680 mg, 4.5 mmol) in pyridine (5 ml) was added dropwise for 20 min. After
additional stirring for 1 h, the reaction mixture was evaporated in vacuo and
the residue was extracted with ethyl acetate (300 ml). The ethyl acetate layer
was dried over anhydrous magnesium sulfate and evaporated in vacuo.
The crude product was separated by SiO2 column chromatography to
afford 5-methyl-2-(methylethyl)phenyl 3,4,5-tribenzyloxybenzoate (2.2 g,
85%). Pd/C (10%, 0.5 g) was suspended in a solution of 5-methyl-2-
(methylethyl)phenyl 3,4,5-tribenzyloxybenzoate (2.2 g, 3.8 mmol) in ethyl
acetate (50 ml), and the mixture was hydrogenated at 2 atm for 3 h. The cata-
lyst was removed by filtration and washed with ethyl acetate (25 ml). The fil-
trate was dried over anhydrous magnesium sulfate and evaporated in vacuo.
The crude product was separated by SiO2 column chromatography to afford
4a (0.9 g, 78%).
5-Methyl-2-(methylethyl)phenyl 3,4,5-Trihydroxybenzoate, 4a: IR vmax
(KBr) cmϪ1: 3254, 3100, 2950, 1735. 1H-NMR (DMSO-d6) d: 9.48 (bs, 3H),
7.21 (d, 1H, Jϭ7.5 Hz), 7.08 (s, 2H), 7.01 (d, 1H, Jϭ7.5 Hz), 6.89 (s, 1H),
2.99 (m, 1H), 2.26 (s, 3H), 1.12 (d, 6H, Jϭ6.9 Hz). MS (FAB) m/z 303
(Mϩϩ1). Anal. Calcd for C17H18O5: C, 67.54; H, 6.00. Found: C, 67.50; H,
5.97.
Mushroom Tyrosinase Assay Mushroom tyrosinase, L-tyrosine, and L-
DOPA were purchased from Sigma Chemical (St. Louis, MO, U.S.A.). Ty-
rosinase activity was determined using the a method of Pomerantz20) with
minor modification. Twenty-five microliters of 0.5 mM L-DOPA, 25 ml of 10
mM L-tyrosine, 875 ml of 50 mM phosphate buffer (pH 6.5), and 25 ml of test
sample solution were mixed. Then 50 ml of mushroom tyrosinase (1600
U/ml) was added. The amount of dopachrome produced in the reaction mix-
ture was determined against a blank (solution without enzyme) at 475 nm
(OD475) using a spectrophotometer (Shimadzu Corporation, Kyoto, Japan).
DPPH Assay DPPH reagent was prepared at a DPPH concentration of
80 mg/ml in MeOH. A test sample (50 ml) was dissolved in DMSO and
mixed with 100 mM Tris–HCl buffer (pH 7.4, 50 ml), distilled water, and 400
ml of DPPH ethanolic solution (50 ml). The mixture was shaken well and al-
lowed to stand for 20 min in the dark. The absorbance was measured at 515
nm using an Elx800 microtiter plate reader (Bio-Tek Instruments, Vermont,
U.S.A.).
5-Methyl-2-(methylethyl)phenyl 4,5-Dihydroxy-3-methoxybenzoate, 4b:
1
IR vmax (KBr) cmϪ1: 3250, 3100, 2949, 1734. H-NMR (DMSO-d6) d: 9.56
(bs, 2H), 7.29 (s, 1H), 7.25 (d, 1H, Jϭ7.5 Hz), 7.19 (s, 1H), 7.10 (d, 1H,
Jϭ7.5 Hz), 6.94 (s, 1H), 3.84 (s, 3H), 2.98 (m, 1H), 2.29 (s, 3H), 1.16 (d,
6H, Jϭ6.9 Hz). MS (FAB) m/z 317 (Mϩϩ1). Anal. Calcd for C18H20O5: C,
68.34; H, 6.37. Found: C, 68.29; H, 6.31.
5-Methyl-2-(methylethyl)phenyl 4-Hydroxy-3,5-dimethoxybenzoate, 4c:
1
IR vmax (KBr) cmϪ1: 3252, 3100, 2951, 1732. H-NMR (DMSO-d6) d: 9.45
(bs, 1H), 7.36 (s, 2H), 7.25 (d, 1H, Jϭ7.5 Hz), 7.01 (d, 1H, Jϭ7.5 Hz), 6.93
(s, 1H), 3.83 (s, 6H), 2.97 (m, 1H), 2.29 (s, 3H), 1.16 (d, 6H, Jϭ6.9 Hz). MS
(FAB) m/z 331 (Mϩϩ1). Anal. Calcd for C19H22O5: C, 69.07; H, 6.71.
Found: C, 68.99; H, 6.70.
References and Notes
5-Methyl-2-(methylethyl)phenyl 3,4,5-Trimethoxybenzoate, 4d: IR vmax
1
(KBr) cmϪ1: 3100, 2950, 1730. H-NMR (CDCl3) d: 7.47 (s, 2H), 7.25 (d,
1) Sturm R. A., Mutat. Res., 422, 69—76 (1998).
2) Marmol V. D., Ito S., Jackson I., Vachtenheim J., Berr P., Ghanem G.,
Morandini R., Wakamatsu K., Huez G., FEBS Lett., 327, 307—310
(1993).
3) Amae S., Yasumoto K., Takeda K., Udono T., Takahashi K., Shibahara
S., Biochim. Biophys. Acta, 1492, 505—508 (2000).
4) Marmol V. D., Beermann F., FEBS Lett., 381, 165—168 (1996).
5) Jimbow K., Obata H., Pathak M. A., Fitzpatrick T. B., J. Invest. Der-
matol., 62, 436—449 (1974).
1H, Jϭ7.5 Hz), 7.07 (d, 1H, Jϭ7.5 Hz), 6.93 (s, 1H), 3.95 (s, 3H), 3.94 (2,
6H), 3.01 (m, 1H), 2.34 (s, 3H), 1.23 (d, 6H, Jϭ6.9 Hz). MS (FAB) m/z 345
(Mϩϩ1). Anal. Calcd for C20H24O5: C, 69.75; H, 7.02. Found: C, 69.71; H,
6.98.
5-Methyl-2-(methylethyl)phenyl 3,4,5-Triethoxybenzoate, 4e: IR vmax
1
(neat) cmϪ1: 3101, 2953, 1735. H-NMR (CDCl3) d: 7.36 (s, 2H), 7.24 (d,
1H, Jϭ7.5 Hz), 7.04 (d, 1H, Jϭ7.5 Hz), 6.92 (s, 1H), 4.12 (m, 6H), 2.92 (m,
1H), 2.29 (s, 3H), 1.35 (t, 6H, Jϭ5.4 Hz), 1.27 (t, 3H, Jϭ5.4 Hz), 1.10 (d,
6H, Jϭ6.9 Hz). MS (FAB) m/z 387 (Mϩϩ1). Anal. Calcd for C23H30O5: C,
71.48; H, 7.82. Found: C, 71.41; H, 7.77.
6) Patick E., Juberg D. R., O’Donoghue J., Maibach H. I., Food Chem.
Toxicol., 37, 169—175 (1999).
7) Coupvray A. L., Sevran H., U.S. Patent 5468472 (1995).
8) Katagiri T., Okubo T., Oyobikawa M., Futaki K., Sagaku M., “Pro-
ceedings of the 20th IFSCC Congress,” Cannes, volume 1, 1998, pp.
93—102.
9) Ito S., Kato T., Ishikawa K., Kasuga T., Jimbow K., Biochem. Pharma-
col., 36, 2007—2011 (1987).
5-Methyl-2-(methylethyl)phenyl 3,4,5-Tripropoxybenzoate, 4f: IR vmax
1
(neat) cmϪ1: 3100, 2950, 1735. H-NMR (CDCl3) d: 7.43 (s, 2H), 7.20 (d,
1H, Jϭ7.5 Hz), 7.02 (d, 1H, Jϭ7.5 Hz), 6.87 (s, 1H), 4.02 (m, 6H), 3.01 (m,
1H), 2.34 (s, 3H), 1.90 (m, 6H), 1.22 (d, 6H, Jϭ6.9 Hz), 1.06 (m, 9H). MS
(FAB) m/z 429 (Mϩϩ1). Anal. Calcd for C26H36O5: C, 72.87; H, 8.47.
Found: C, 72.81; H, 8.42.
10) Dooley T. P., Gadwood R. C., Kilgore K., Thomasco L. M., Skin Phar-
macol., 7, 188—200 (1994).
11) Curto E. V., Kwong C., Hermersdorfer H., Glatt H., Santis C., Virador
V., Hearing V. J., Dooley T. P., Biochem. Pharmacol., 57, 663—672
(1999).
12) Hase T., Murase T., Shibuya J., Tokimitsu I., Nishizawa Y., Jpn. Patent
08283137 (1996).
13) Riley P. A., Sawyer B., Wolff M. A., J. Invest. Dermatol., 64, 86—89
(1975).
5-Methyl-2-(methylethyl)phenyl 3,4,5-Tributoxybenzoate, 4g: IR vmax
1
(neat) cmϪ1: 3100, 2952, 1734. H-NMR (CDCl3) d: 7.43 (s, 2H), 7.20 (d,
1H, Jϭ7.5 Hz), 7.02 (d, 1H, Jϭ7.5 Hz), 6.90 (s, 1H), 4.05 (m, 6H), 3.01 (m,
1H), 2.34 (s, 3H), 1.80 (m, 6H), 1.48 (m, 6H), 1.23 (d, 6H, Jϭ6.9 Hz), 0.97
(m, 9H). MS (FAB) m/z 471 (Mϩϩ1). Anal. Calcd for C26H36O5: C, 74.01;
H, 8.99. Found: C, 73.97; H, 8.93.
5-Methyl-2-(methylethyl)phenyl (2E)-3-(3,4,5-Trimethoxyphenyl)prop-2-
enoate, 4h: IR vmax (KBr) cmϪ1: 3100, 2950, 1707, 1620. 1H-NMR (CDCl3)
d: 7.80 (1H, d, Jϭ15.9 Hz), 7.25 (s, 1H), 7.20 (1H, d, Jϭ7.5 Hz), 7.01 (1H,
Jϭ7.5 Hz), 6.90 (s, 1H), 6.82 (s, 2H), 6.60 (1H, d, Jϭ15.9 Hz), 3.91 (s, 3H),
2.99 (s, 6H), 3.01 (m, 1H), 2.33 (s, 3H), 1.22 (d, 6H, Jϭ6.9 Hz). MS (FAB)
14) Alena F., Jimbow K., Ito S., Cancer Res., 50, 3743—3747 (1990).
15) Passi S., Picardo M., Nazzaro-Porro N., Biochem. J., 245, 537—542