The first total synthesis of the marine fatty acid (±)-2-methoxy-13- methyltetradecanoic acid: A cytotoxic fatty acid to leukemia cells

Article abstract of DOI:10.1016/S0009-3084(03)00110-5

The recently discovered marine fatty acid (±)-2-methoxy-13- methyltetradecanoic acid was synthesized for the first time in six steps (26% overall yield) starting from commercially available methyl 12- methyltridecanoate. The synthetic approach provided enough material to corroborate the structure of the acid, which was recently identified in the sponge Amphimedon complanata from Aguadilla, Puerto Rico, and to test its cytotoxicity to three leukemia cell lines. The key step in the synthesis was the addition of trimethylsilyl cyanide to 12-methyltridecanal under triethylamine catalysis. Nuclear magnetic resonance data are provided for the first time for this methoxylated fatty acid and the synthetic approach utilized is of general applicability since it can be used in the synthesis of other methyl-branched 2-methoxylated fatty acids. We also report that the acid (±)-2-methoxy-13-methyltetradecanoic acid is cytotoxic to human chronic myelogenous leukemia K-562 (EC₅₀=238μM), histiocytic lymphoma U-937 (EC₅₀=250μM), and promielocytic leukemia HL-60 (EC ₅₀=476μM) in RPMI 1640 medium.

Full text of DOI:10.1016/S0009-3084(03)00110-5

Chemistry and Physics of Lipids 126 (2003) 149–153
The first total synthesis of the marine fatty acid
( )-2-methoxy-13-methyltetradecanoic acid:
a cytotoxic fatty acid to leukemia cells
Néstor M. Carballeira∗, Heidyleen Cruz,
Elsie A. Orellano, Fernando A. González
Department of Chemistry, University of Puerto Rico, P.O. Box 23346, San Juan 00931-3346, Puerto Rico
Received 26 June 2003; received in revised form 31 July 2003; accepted 31 July 2003
Abstract
The recently discovered marine fatty acid ( )-2-methoxy-13-methyltetradecanoic acid was synthesized for the first time in six
steps (26% overall yield) starting from commercially available methyl 12-methyltridecanoate. The synthetic approach provided
enough material to corroborate the structure of the acid, which was recently identified in the sponge Amphimedon complanata
from Aguadilla, Puerto Rico, and to test its cytotoxicity to three leukemia cell lines. The key step in the synthesis was the addition
of trimethylsilyl cyanide to 12-methyltridecanal under triethylamine catalysis. Nuclear magnetic resonance data are provided
for the first time for this methoxylated fatty acid and the synthetic approach utilized is of general applicability since it can
be used in the synthesis of other methyl-branched 2-methoxylated fatty acids. We also report that the acid ( )-2-methoxy-13-
methyltetradecanoic acid is cytotoxic to human chronic myelogenous leukemia K-562 (EC50 = 238 ␮M), histiocytic lymphoma
U-937 (EC50 = 250 ␮M), and promielocytic leukemia HL-60 (EC50 = 476 ␮M) in RPMI 1640 medium.
© 2003 Elsevier Ireland Ltd. All rights reserved.
Keywords: Sponges; Fatty acids; 2-Methoxy-13-methyltetradecanoic acid; Synthesis; Leukemia
1. Introduction
hydroxy-13-methyltetradecanoic acid, a component
of gliding bacteria such as Flexibacter elegans and
of the Actinomycetales such as Arthrobacter simplex
(Fautz et al., 1979; Yano et al., 1969). The biosyn-
thesis of 1 is unusual and could be the result of an
hitherto unrecognized biosynthetic pathway in nature
leading to these rare 2-methoxylated iso and anteiso
C15–C16 fatty acids (Carballeira, 2002). Of equal im-
portance could be the study of the biological potential
of these novel methoxylated fatty acids. Therefore, a
good synthesis and a more complete characterization
of acids such as 1 is important, if we are to fully
understand its biochemistry and/or medical potential.
The ( )-2-methoxy-13-methyltetradecanoic acid
(1) is a novel fatty acid which was recently iden-
tified, together with other 2-methoxylated iso and
anteiso C15–C16 fatty acids, in the sponge Amphime-
don complanata collected in Aguadilla, Puerto Rico
(Carballeira and Alicea, 2001). Fatty acid 1 is the
methoxylated analog of the bacterial fatty acid 2-
∗
Corresponding author. Tel.: +1-787-764-0000x4791;
fax: +1-787-756-8242.
E-mail address: ncarball@upracd.upr.clu.edu (N.M. Carballeira).
0009-3084/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/S0009-3084(03)00110-5

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N.M. Carballeira et al. / Chemistry and Physics of Lipids 126 (2003) 149–153
Due to small amounts of material (0.9%) in a com-
methyltridecanoate (0.30 g, 1.24 mmol) dissolved in
diethyl ether (8 ml). The resulting gray mixture was
stirred for 2 h and then carefully quenched with a sat-
urated aqueous ammonium chloride solution (8 ml).
Diethyl ether (10 ml) was then added and the organic
layer was separated and dried over Na2SO4, filtered,
and concentrated in vacuo, affording 12-methyl-1-
tridecanol (0.28 g, 1.30 mmol) in a 93% yield.
plex fatty acid methyl ester mixture of 72 methyl
esters, the 2-methoxy-13-methyltetradecanoic acid
(1) was only identified by gas chromatography–mass
spectrometry and nuclear magnetic resonance spec-
tral data for 1 is still lacking in the lipid literature
(Carballeira and Alicea, 2001). Despite the fact that
the stereochemistry at the ␣-carbon of 1 was also not
determined, earlier studies with other longer-chain
2-methoxylated fatty acids have established the stere-
ochemistry at the ␣-carbon in these methoxylated
fatty acids as R (Ayanoglu et al., 1983). In addition,
there are no reported syntheses for 1, but a methodol-
ogy is needed if we are to obtain sufficient quantities
to study the biological activity of 1 or related analogs,
in particular their cytotoxicity to leukemia cell lines.
Such a study becomes more evident due to a recent
report that the 13-methyltetradecanoic acid (iso-15:0)
effectively inhibits both in vitro and in vivo tumor
growth by inducing apoptosis in seven carcinoma cell
lines, including the K-562 leukemia cell line with an
ID50 of 11 ␮g/ml (Yang et al., 2000). Therefore, in
this contribution we describe the first total synthesis
of racemic 1 and report that it is cytotoxic to the
three leukemia cell lines K-562, HL-60, and U-937.
In addition, 1 is more cytotoxic to K-562 than the
previously reported 13-methyltetradecanoic acid.
2.3. 12-Methyltridecanal
To a stirred solution of pyridinium chlorochromate
(0.28 g, 1.30 mmol) in 8 ml of CH2Cl2 was added
dropwise 12-methyl-1-tridecanol (0.28 g, 1.30 mmol)
in 8 ml of CH2Cl2 at room temperature. After 24 h
the reaction mixture was filtered through Florisil and
washed with diethyl ether (25 ml). Evaporation of
the solvent afforded the known 12-methyltridecanal
(0.28 g, 1.29 mmol) in a 98% yield (Guth and Grosch,
1995; Kerscher et al., 2000).
2.4. ( )-2-Trimethylsilyloxy-13-
methyltetradecanonitrile
To 12-methyltridecanal (0.24 g, 1.14 mmol) in 10 ml
of anhydrous CH2Cl2 were added dropwise (0.11 g,
1.14 mmol) of trimethylsilyl cyanide and catalytic
amounts of Et3N (10%) at 0 ◦C. The reaction mixture
was stirred for 2 h at 25 ◦C. After this time the solvent
was removed in vacuo affording 0.34 g (1.09 mmol)
the 2-trimethylsilyloxy-13-methyltetradecanonitrile
for a 96% yield. The nitrile was used as such for the
next step without further purification. GC-MS (70 eV)
m/z (relative intensity) 311 (M+, 1), 296 (98), 284
(17), 269 (65), 241 (5), 227 (3), 213 (5), 194 (7), 185
(12), 171 (23), 157 (6), 155 (3), 151 (2), 143 (16),
140 (11), 130 (7), 129 (55), 123 (7), 115 (17), 114
(17), 109 (12), 101 (24), 97 (24), 95 (24), 91 (24), 84
(30), 81 (23), 75 (100), 73 (97), 69 (42), 67 (21), 59
(16), 57 (38), 55 (50).
2. Materials and methods
2.1. Instrumentation
1H NMR (300 MHz) and 13C NMR (75 MHz)
were recorded on a Bruker DPX-300 spectrometer.
1H NMR chemical shifts are reported with respect
to internal (CH3)4Si, 13C NMR chemical shifts
are reported in parts per million relative to CDCl3
(77.0 ppm). GC/MS analyses were recorded at 70 eV
using a Hewlett Packard 5972A MS ChemStation
equipped with a 30 m ×0.25 mm special performance
capillary column (HP-5MS) of polymethyl siloxane
crosslinked with 5% phenyl methylpolysiloxane.
2.5. ( )-2-Hydroxy-13-methyltetradecanoic acid
Into a 25-ml round-bottomed flask was placed
the 2-trimethylsilyloxy-13-methyltetradecanonitrile
(0.34 g, 1.09 mmol) in 7 ml of dimethoxyethane
(DME). Concentrated HCl (7 ml) was added and the
reaction mixture was heated at 90 ◦C for 24 h. The
2.2. 12-Methyl-1-tridecanol
To a stirred solution of LiAlH4 in dry diethyl ether
(8 ml) at 0 ◦C was added a solution of methyl 12-

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
extracted with ether (2 × 8 ml). The organic layer 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-
ganic phase was dried over Na2SO4, filtered, and
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,

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N.M. Carballeira et al. / Chemistry and Physics of Lipids 126 (2003) 149–153
fatty acid that inhibited growth in 50% (EC50) was
calculated using the Prism Software (Graphpad, San
Diego, CA) from titration curves generated from at
least three independent experiments, each performed
in triplicate.
methylating the ␣-hydroxy acid with two equivalents
of methyl iodide and sodium hydride in DMSO and
then saponification of the methyl ester with KOH in
ethanol. The overall yield for the synthesis of 1 was
26%. The methyl ester of 1 co-eluted in capillary GC
with the naturally occurring methyl 2-methoxy-13-
methyltetradecanoate from the sponge A. complanata,
thus confirming the structural assignment of the nat-
ural iso methoxylated fatty acid (Carballeira and
Alicea, 2001).
3. Results and discussion
Due to the trace amounts of the iso methoxylated
fatty acids in the original sponge it was not possible
to corroborate the R stereochemistry at the ␣-carbon
of 1 (Carballeira and Alicea, 2001). Therefore, we
undertook a racemic synthesis for 1. The synthesis of
1 started with commercially available (Sigma) methyl
12-methyltridecanoate, which was converted to 12-
methyltridecanal by first reducing the methyl ester
to 12-methyl-1-tridecanol with lithium aluminum hy-
dride in ether and then oxidizing the alcohol to the
aldehyde with pyridinium chlorochromate in CH2Cl2
in very high yield reactions (Scheme 1). Interest-
ingly enough, the 12-methyltridecanal is a natural
product on its own arising from plasmalogens asso-
ciated with bacteria and protozoa in beef (Kerscher
et al., 2000). The aldehyde was then reacted with
trimethylsilyl cyanide and triethylamine affording the
2-trimethylsilyloxy-13-methyltetradecanonitrile in a
96% yield. The silyloxynitrile was then transformed
into the also naturally occurring ( )-2-hydroxy-13-
methyltetradecanoic acid (Fautz et al., 1979; Yano
et al., 1969) by first reacting the nitrile with concen-
trated HCl in DME followed by saponification with
50% NaOH. Final conversion into ( )-2-methoxy-
13-methyltetradecanoic acid (1) was achieved by first
The most significant absorption in the NMR spec-
trum of the ( )-2-methoxy-13-methyltetradecanoic
acid (1) was observed for the carbons and hydrogens
bearing the methoxy functionality. For example, the
methoxy protons resonated at δ 3.44 ppm and the
methoxy carbon was observed at δ 58.3 ppm, while
the methine ␣-hydrogen resonated at δ 3.80 ppm, the
methine ␣-carbon at δ 80.2 ppm, and the carbonyl at
176.9 ppm. These 1H and 13C NMR displacements
seem to be characteristic for saturated 2-methoxylated
fatty acids and useful as a future reference for other
similar analogs.
Using the methodology described in materials
and methods acid 1 was screened against the three
leukemia cell lines K-562 (ATCC CCL-243), HL-
60 (ATCC CCL-240), and U-937 (ATCC CRL-
1593). It displayed an EC50 of 238 ␮M (log EC50
=
−3.62 0.08) against K-562, an EC50 of 250 ␮M
(log EC50 = −3.60 0.07) against HL-60, and an
EC50 of 476 ␮M (log EC50 = −3.1 0.1) against
U-937. Therefore, acid 1 displays weak cytotoxicity
against the studied cell lines. While the mechanism
of cytotoxicity of 1 on leukemia cells remains to be
elucidated one likely possibility is for 1 to trigger
Scheme 1. Synthesis of ( )-2-methoxy-13-methyltetradecanoic acid (1).

N.M. Carballeira et al. / Chemistry and Physics of Lipids 126 (2003) 149–153
153
tumor cell death through the induction of apoptosis,
similar to the reported case of 13-methyltetradecanoic
acid where the iso functionality seems to be essen-
tial for the cytotoxicity (Yang et al., 2000). In fact,
it is important to mention that in our assay the 13-
methyltetradecanoic acid only exhibited and EC50
(Grant No. S06GM08102). H. Cruz thanks the NIH-
´
RISE program and Compañıa de Fomento Industrial
(PR) for financial assistance.
References
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K-562 cell line. This means that the 2-methoxy func-
tionality increased the cytotoxicity of the iso-15:0
acid. Moreover, just recently a novel 2-methoxy-
12-methyloctadeca-17-ene-5-ynoic acid was also
reported to have weak cytotoxicity against the hu-
man leukemia cell-line K-562 at a LC50 value of
51.3 ␮g/ml (Lee et al., 2003).
In summary, we have described the first total
synthesis (six steps) for the ( )-2-methoxy-13-
methyltetradecanoic acid (1). The synthesis provided
1
us with enough material to fully report the H NMR
and 13C NMR spectral data for 1. This information
will be useful in future identifications of other methyl-
branched 2-methoxylated fatty acids and in the syn-
thesis of related analogs. Finally, we report that 1 is
weakly cytotoxic to the K-562, HL-60, and U-937
leukemia cell lines, but more cytotoxic to K-562 than
the previously reported 13-methyltetradecanoic acid.
Yang, Z., Liu, S., Chen, X., Chen, H., Huang, M., Zheng, J., 2000.
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Acknowledgements
This work was supported by a grant from the
SCORE program of the National Institutes of Health