N.M. Carballeira et al. / Chemistry and Physics of Lipids 126 (2003) 149–153
151
m, CH2), 0.85 (6H, d, J = 6.6 Hz, CH(CH3)2); 13C
NMR (75.4 MHz) δ: 176.9 (s, C-1), 80.2 (d, C-2), 58.3
(q, -OCH3), 39.0 (t, C-12), 32.3 (t, C-3), 29.9 (t), 29.7
(t), 29.6 (t), 29.5 (t), 29.4 (t), 29.3 (t), 28.0 (d, C-13),
27.4 (t), 24.8 (t, C-4), 22.6 [q, CH3]; GC-MS (70 eV)
m/z (relative intensity) 272 (M+, 4), 227 (100), 211
(1), 194 (2), 168 (1), 166 (1), 152 (1), 138 (4), 128 (1),
125 (13), 123 (4), 113 (6), 111 (34), 103 (3), 97 (75),
95 (19), 90 (24), 85 (22), 83 (96), 81 (23), 73 (10),
71 (87), 69 (94), 67 (24), 61 (3), 57 (82), 55 (82).
reaction mixture was then cooled (ice bath), made al-
kaline by the slow addition of 50% NaOH (8 ml), and
heated again for 2 h at 90 ◦C. The mixture was then
acidified with 6 M HCl (8 ml), and the product was
dried over Na2SO4, filtered, and evaporated in vacuo
affording (0.2 g, 0.735 mmol) of the known ( )-2-
hydroxy-13-methyltetradecanoic acid for a 67% yield
(Fautz et al., 1979; Yano et al., 1969).
2.6. Methyl ( )-2-methoxy-13-methyltetradecanoate
2.8. Cell culture
Into a 15-ml round-bottomed flask provided with a
magnetic stirrer and under a nitrogen atmosphere was
placed the ( )-2-hydroxy-13-methyltetradecanoic
acid (0.084 g, 0.31 mmol) in 5 ml of DMSO. The
reaction mixture was stirred for 10 min followed by
the dropwise addition of two equivalents of MeI,
further stirred for 20 min, and after this time di-
luted with hexane/ether (1:1) and washed with H2O
(2 × 8 ml) to remove the remaining DMSO. The or-
evaporated in vacuo affording 0.071 g (0.26 mmol)
of the previously reported methyl ( )-2-methoxy-
13-methyltetradecanoate for a 84% isolated yield
(Carballeira and Alicea, 2001). GC-MS (70 eV) m/z
(relative intensity) 286 (M+, 1), 227 (100), 211 (1),
194 (1), 179 (1), 139 (4), 125 (12), 111 (29), 109 (5),
104 (7), 98 (6), 97 (62), 95 (14), 87 (6), 85 (16), 83
(75), 81 (18), 79 (3), 75 (6), 71 (73), 69 (72), 67 (19),
57 (58), 55 (61).
Three nonadherent cell lines were used in this
study, namely, human histiocytic lymphoma U-937
(ATCC CRL-1593), human promielocytic leukemia
HL-60 (ATCC CCL-240), and human chronic myel-
ogenous leukemia K-562 (ATCC CCL-243). These
cells were cultured in RPMI 1640 medium contain-
ing 10% Fetalclone III serum (HL-60 and K-562) or
Cosmic calf serum (U-937) (Invitrogen, Inc.), sup-
plemented with 1% penicillin-streptomycin antibiotic
solution (Sigma Chemical Co., St. Louis, MO, USA).
Cultures were maintained at 37 ◦C in a humidified
atmosphere of 95% air/5% CO2.
2.9. Cytotoxicity assay
Cells were inoculated into 96-well microplates at
5.0×105 cells/ml (100 l/well) and incubated at 37 ◦C
for 24 h before treatment. Various concentrations (in
DMSO) of test agents were added to the cells fol-
lowed by further incubation at 37 ◦C for 48 h. Then,
cells growing in suspension were fixed in situ with
50 l/well of ice cold 80% (w/v) trichloroacetic acid
(TCA) aqueous solution to produce a final concentra-
tion of 16% TCA (w/v). The plates were incubated
for 90 min at 4 ◦C after which they were washed five
times with water and air-dried. A solution of 0.4%
(w/v) sulforhodamine B in 1% (v/v) acetic acid was
added (50 l) to each well and incubated for 15 min at
room temperature. The plates were washed five times
with 1% (v/v) acetic acid, air dried, and incubated with
10 mM Tris-base (pH 10.4) with shaking for 15 min at
room temperature. The absorbance at 490 nm of solu-
bilized stain was measured using a microplate reader
(Dynex Technologies, MRX II). The concentration of
2.7. ( )-2-Methoxy-13-methyltetradecanoic acid (1)
The methyl ( )-2-methoxy-13-methyltetradecano-
ate (0.034 g, 0.12 mmol) was placed into a 25-ml
round-bottomed flask together with 15 ml of 1 M
KOH/ethanol and the reaction mixture was refluxed
for 2 h. After this time the reaction was cooled to
25 ◦C and the solvent evaporated, hexane (8 ml) was
added, and the mixture acidified with 6 M HCl (5 ml).
The final acid was extracted with ether (2 × 15 ml),
dried over Na2SO4, filtered and evaporated in vacuo
affording 0.018 g (0.07 mmol, 52% yield) of ( )-2-
methoxy-13-methyltetradecanoic acid (1). 1H NMR
δ: 3.80 (1H, x part of ABX system, H-2), 3.44 (3H,
s, -OCH3), 1.77 (2H, m, 3-H), 1.51 (1H, m, 13-H),
1.39 (2H, m, 4-H), 1.25 (14H, brs, CH2), 1.15 (2H,