Antioxidant behaviour of thia fatty acids

Article abstract of DOI:10.1071/CH02095

Eight thia fatty acids and other sulfides have been studied as inhibitors of autoxidation of arachidonic acid. The inhibitors extend the lag phase of the oxidation, to varying degrees. A carboxyl group in the vicinity of the sulfur reduces the antioxidant activity, while unsaturated sulfides are more effective than their saturated analogues. The results are consistent with the sulfides acting to reduce fatty acid hydroperoxides, which otherwise accumulate during the early stages of reaction and propagate the free-radical oxidation process.

Full text of DOI:10.1071/CH02095

P u b l i s h i n g
AUSTRALIAN JOURNAL OF
CHEMISTRY
A N I N T E R N A T I O N A L J O U R N A L F O R C H E M I C A L S C I E N C E
publishing research papers from all fields of chemical
science, including synthesis, structure, new materials,
macromolecules, supramolecular chemistry, biological
chemistry, nanotechnology, surface chemistry, and
analytical techniques.
Volume 55, 2002
© CSIRO 2002
All enquiries and manuscripts should be directed to:
Dr Alison Green
Australian Journal of Chemistry–
an International Journal for Chemical Science
CSIRO
PUBLISHING
PO Box 1139 (150 Oxford St)
Collingwood, Vic. 3066, Australia
Telephone: +61 3 9662 7630
Fax: +61 3 9662 7611
E-mail: publishing.ajc@csiro.au
CSIRO
Published by
PUBLISHING
for CSIRO and the Australian Academy of Science
w w w. p u b l i s h . c s i r o . a u / j o u r n a l s / a j c

Aust. J. Chem. 2002, 55, 647–653
Antioxidant Behaviour of Thia Fatty Acids
Christopher J. Easton,A,B Antonio Ferrante,C Thomas A. RobertsonA and Ling XiaA
A Research School of Chemistry, Australian National University, Canberra, A.C.T. 0200, Australia.
B Author to whom correspondence should be addressed (e-mail: easton@rsc.anu.edu.au).
C Department of Immunopathology, Adelaide Women’s and Children’s Hospital, North Adelaide,
S.A. 5006, Australia.
Eight thia fatty acids and other sulfides have been studied as inhibitors of autoxidation of arachidonic acid. The
inhibitors extend the lag phase of the oxidation, to varying degrees. A carboxyl group in the vicinity of the sulfur
reduces the antioxidant activity, while unsaturated sulfides are more effective than their saturated analogues. The
results are consistent with the sulfides acting to reduce fatty acid hydroperoxides, which otherwise accumulate
during the early stages of reaction and propagate the free-radical oxidation process.
C
H
02059
A
n
t
l
o
xia
d
n
t
B
e
hva
i
o
u
o
f
T
h
a
F
t
t
A
c
d
s
Manuscript received: 6 May 2002.
Final version: 26 June 2002.
Introduction
S
COOH
Fatty acids display widespread physiological activity but
their utility to address associated plant and mammalian
conditions is limited.[1] This can be attributed to the fact that
a particular disease state often relates to only one of the many
biochemical reactions of a fatty acid. In the search for
analogues which are more specific in their biochemical
reactions, oxa-,[2,3] thia-[4–7] and nitro-substituted[8]
derivatives have been prepared, and it has been demonstrated
that their biochemical reactions are quite different to those of
the natural compounds.[9–25] Tetradecylthioacetic acid (1)
(Diagram 1) has been studied quite extensively[12–21] and it
has been found to inhibit proliferation of cancer cells,[12–15]
reduce stenosis development after balloon angioplasty injury
of arteries,[16] inhibit oxidative modification of low-density
(1)
(2)
S
COOH
S
COOH
COOH
(3)
(4)
S
COOH
COOH
S
lipoprotein[17]
and
inhibit
iron–ascorbate-induced
(5)
(6)
microsomal lipid peroxidation.[18] The antioxidant behaviour
of the thia fatty acid (1) has been attributed to its interaction
with copper or iron ions in radical scavenging.[18] To
examine the antioxidant properties in more detail, we have
prepared the series of saturated and unsaturated thia fatty
acids and other sulfides (1)–(8), and studied their effects on
thin-film autoxidation[26,27] of arachidonic acid (9). The
results are consistent with a mechanism proposed earlier for
the antioxidant activity of sulfides, involving their
conversion into sulfoxides during the course of reducing
hydroperoxides to alcohols.[28,29]
S
S
(7)
(8)
S
COOH
Results and Discussion
Compounds (1)–(8) were selected to explore the importance
of the carboxyl group, the extent of unsaturation of the
aliphatic chain, and the location of the sulfur relative to the
(9)
Diagram 1
10.1071/CH02095
© CSIRO 2002
0004-9425/02/100647

648
C. J. Easton et al.
Table 1. Percentages of PUFA (9) and sulfide (1)–(8) recovered following thin-
film autoxidation
carboxyl group and the centres of unsaturation. The
saturated β- and γ-thia fatty acids (1) and (2) were prepared
by adapting literature procedures,[5] involving reaction under
basic conditions of the corresponding alkyl bromide with
mercaptoacetic acid and 3-mercaptopropionic acid,
respectively. The unsaturated analogues (3)–(5) were
prepared as described previously.[7] The diene (6) was
synthesized as outlined in Scheme 1. Accordingly, the
alkynol (10) was converted into the tosylate (11) in 65%
yield.[30] Reaction with but-3-yn-1-ol, using the method of
Jeffery et al.[31] for the synthesis of methylene-interrupted
diynes, afforded the coupled product (12), in 62% yield,
which was reduced with hydrogen over a Lindlar catalyst to
give a 92% yield of the dienol (13). The corresponding
tosylate (14) was prepared in 57% yield and treated with 3-
mercaptopropionic acid to give a 41% yield of the thia fatty
acid (6). The thioethers (7) and (8) were prepared by
coupling propane-1,3-thiol with the corresponding alkyl
bromides (all-Z)-1-bromoeicosa-5,8,11,14-tetraene and 1-
bromotetradecane, respectively.
Thin films of each of the thia fatty acids and other
sulfides (1)–(8) mixed with arachidonic acid (9) in various
ratios and containing lauric acid as an inert internal standard
were stored under oxygen at 25°C for 1–7 days, and then
analysed using reverse-phase high-performance liquid
chromatography (HPLC) to determine the quantities of the
compounds (1)–(9) that remained. Alone, under identical
conditions, (1), (2) and (4)–(8) were stable for at least a
week, but (3) showed signs of decomposition. The results are
summarized in Table 1, and examples of the more effective
inhibition of oxidation of arachidonic acid (9) are illustrated
in Figures 1–4.
Sulfide Initial ratio of PUFA
to sulfide (w/w)
Time
(days)
PUFA remaining
(%)
Sulfide
remaining (%)
None
1
2
3
5
7
90
20
15
10
A
(1)
(2)
(3)
(4)
(4)
(4)
(5)
(5)
(6)
(6)
(6)
(7)
(7)
1 : 1
1 : 1
1
2
3
5
7
50
50
15
15
15
15
10
A
A
A
1
2
3
5
7
100
95
80
30
A
100
90
85
45
35
1 : 1
1
2
3
5
7
70
30
10
5
25
5
A
A
A
5
1 : 1
1
2
3
5
7
100
100
100
100
100
100
100
100
100
100
2 : 1
1
2
3
5
7
100
100
100
100
95
100
95
95
95
90
10 : 1
1
2
3
5
7
100
100
85
40
15
95
85
40
A
A
1 : 1
1
2
3
5
7
100
100
100
100
95
100
100
95
95
85
10 : 1
1
2
3
5
7
80
35
15
5
A
25
A
In the absence of any sulfide, after a lag phase of
approximately 1 day, arachidonic acid (9) undergoes rapid
autoxidation under these relatively extreme conditions and is
A
A
A
1 : 1
1
2
3
5
7
100
100
100
100
100
100
95
100
95
(i)
100
OH
OTs
2 : 1
1
2
3
5
7
100
100
100
100
100
95
100
95
95
100
(10)
(11)
(ii)
10 : 1
1
2
3
5
7
100
100
100
80
95
85
OH
60
A
(iii)
A
OH
15
1 : 1
1
2
3
5
7
100
100
100
100
100
100
90
90
90
90
(13)
(12)
(iv)
10 : 1
1
2
3
5
7
100
100
95
70
15
80
65
(v)
(6)
40
A
OTs
A
(14)
(8)
(8)
1 : 1
3
7
100
100
100
100
Scheme 1. Reagents and conditions: (i) p-toluenesulfonyl chloride,
pyridine, 0°C, 4 h; (ii) but-3-yn-1-ol, Na2CO3, CuI, n-Bu4NCl,
–30°C, room temperature, 24 h; (iii) Pd/CaCO3/Pb, quinoline,
methanol, hydrogen, 2.5 h; (iv) p-toluenesulfonyl chloride, pyridine,
chloroform, 15°C, 24 h; (v) 3-mercaptopropionic acid, sodium
methoxide, 40°C, 48 h.
10 : 1
1
2
3
5
7
95
90
65
25
10
85
20
A
A
A
A None detected.

Antioxidant Behaviour of Thia Fatty Acids
649
Fig. 1. Percentages of PUFA (9) and sulfide (4) recovered following
Fig. 3. Percentages of PUFA (9) and sulfide (7) recovered following
thin-film autoxidation of a mixture with an initial ratio (w/w) of 10 : 1.
thin-film autoxidation of a mixture with an initial ratio (w/w) of 10 : 1.
Fig. 2. Percentages of PUFA (9) and sulfide (6) recovered following
Fig. 4. Percentages of PUFA (9) and sulfide (8) recovered following
thin-film autoxidation of a mixture with an initial ratio (w/w) of 10 : 1.
thin-film autoxidation of a mixture with an initial ratio (w/w) of 10 : 1.
mostly consumed within 2 days. The sulfides (1)–(8) vary in
their ability to prolong the lag phase and reduce the extent of
reaction of arachidonic acid (9). The saturated β-thia fatty
acid, tetradecylthioacetic acid (1), is ineffective, even when
used in a 1 : 1 (w/w) ratio with arachidonic acid (9). The
saturated γ-thia fatty acid (2) has a modest effect at the same
ratio, extending the lag phase to approximately 3 days. The
saturated sulfide (8) is more effective. With a 1 : 1 (w/w)
mixture of arachidonic acid (9) and the sulfide (8), no
autoxidation is observed after 7 days, while even with a 10 : 1
mixture the lag time for autoxidation of arachidonic acid (9)
is almost 3 days. This indicates that a carboxyl group in the
vicinity of the sulfur decreases the antioxidant activity of a
sulfide. Consistent with this observation, the unsaturated β-
thia fatty acid (3) is not an antioxidant. The unsaturated γ-
thia fatty acids (4)–(6) and the sulfide (7) are active, and
more so than the saturated analogues (2) and (8). The
comparison between the unsaturated and saturated sulfides
(7) and (8) is illustrated in Figures 3 and 4. The different
effects of the unsaturated and saturated γ-thia fatty acids
(4)–(6) and (2) are even more obvious. When used in a 1 : 1
ratio with arachidonic acid (9), the unsaturated compounds
(4)–(6) prevent autoxidation for at least 7 days, whereas
under the same conditions the saturated fatty acid (2) only
prolongs the onset of oxidation to 3 days. The activity of the
polyenes (4)–(6) appears to depend on the proximity of the
unsaturated centres to the sulfur. Compounds (6), (4) and (5)
have two, four and eight methylenes between the sulfur and
the polyene moiety, and when they are used in a 1 : 10 ratio
with arachidonic acid (9) the lag time for autoxidation is 5, 3
and 1 days, respectively.
Autoxidation of arachidonic acid (9) gives a range of
products. From the results shown in Table 1 and Figures 1–4,
it is clear that the thia fatty acids and other sulfides (1)–(8)
also react in the presence of arachidonic acid (9). The
reactions involving the unsaturated compounds (3)–(7) gave
complex mixtures, presumably as a result of polyene
oxidation, and it was not practical to isolate discrete
products. The saturated sulfide (8) only reacted in the
presence of a 10-fold excess of arachidonic acid (9) and it
was then not feasible to separate the product or products of
reaction of the sulfide (8) from those formed from the fatty

650
C. J. Easton et al.
National University. HPLC was performed using a Waters HPLC
system with ultraviolet (UV) and refractive index (RI) detection, and an
Alltech Spherisorb octadecylsilane (ODS) (4.6 mm by 250 mm, 3 µm)
column. The mobile phase comprised acetonitrile, methanol and
phosphoric acid (30 mM). Column chromatography was carried out
using Merck Silicagel 60.
O
S
COOH
(15)
O
S
COOH
Arachidonic acid (9) was purchased from Nu–Chek Prep. Inc.,
Elysian, Minnesota, U.S.A. The thia fatty acids (3)–(5) and (all-Z)-1-
(16)
bromoeicosa-5,8,11,14-tetraene
were
prepared
as
reported
previously.[7] Other chemicals were commercially available from
Aldrich Chemical Company.
Diagram 2
Tetradecylthioacetic Acid (1)
acid (9). By contrast, reactions of the thia fatty acids (1) and
(2), each as a 1 : 1 (w/w) mixture with arachidonic acid (9),
resulted in their smooth conversion into the sulfoxides (15)
and (16) (Diagram 2), which were isolated in yields of 59 and
50%, respectively.
The above results are consistent with earlier mechanistic
studies, by Bateman and Hargrave,[28,29] of the effect of
sulfides as antioxidants. The lag phase of autoxidation of
Mercaptoacetic acid (288 mg, 3.13 mmol) was added under an
atmosphere of dry nitrogen to a stirred solution of sodium methoxide,
prepared from sodium (180 mg, 7.82 mmol) and methanol (20 mL).
After the initial white precipitate had dissolved, a solution of 1-
bromotetradecane (725 mg, 2.61 mmol) in diethyl ether (2 mL) was
added and the mixture was stirred for 16 h at room temperature. The
crude reaction mixture was poured into an equal volume of
hydrochloric acid (10% v/v), and the organic phase was separated and
washed with water and brine, and dried over sodium sulfate. After
removal of the solvent, the residue was subjected to flash column
chromatography, using diethyl ether–hexanes (20 : 80) to diethyl
ether–hexanes–acetic acid (60 : 40 : 2) for elution, to afford tetradecyl-
thioacetic acid (1) (580 mg, 77%) as a white solid after recrystallization
from diethyl ether–hexanes, m.p. 68°C (Found: C, 66.5; H, 10.9; S,
10.8. Calc. for C16H32SO2: C, 66.6; H, 11.2; S, 11.1%). IR νmax (Nujol)
3200–2600(br), 2950s, 2910s, 2840s, 1680s, 1460s, 1425w, 1375s,
1265m, 1140w, 908w, 725w cm–1. 1H NMR δ (300 MHz, CDCl3) 0.88,
3H, t, J 6.6 Hz, C14´ CH3; 1.26–1.40, 22H, m, C3´–C13´ CH2;
1.56–1.64, 2H, m, C2´ CH2; 2.66, 2H, t, J 7.4 Hz, C1´ CH2; 3.26, 2H,
s, C2 CH2. 13C NMR δ (75.5 MHz, CDCl3) 14.68, 23.26, 29.30, 29.46,
29.75, 29.93, 30.06, 30.15, 30.22, 32.49, 33.36, 34.05, 177.57. Mass
spectrum (EI) m/z 288 (M+, 12%), 230 (21), 229 (100), 111 (6), 97 (17),
83 (27), 69 (30), 55 (34). HRMS (M+), found: 288.2121; C16H32SO2
requires 288.2123.
polyunsaturated fatty acids involves
a build-up of
hydroperoxides, which initiate free-radical chain processes.
The sulfides are thought to act as antioxidants by reducing
the hydroperoxides to the corresponding alcohols, extending
the lag phase, and in the process the sulfides are oxidized to
sulfoxides. When the sulfides are consumed, the normal
autoxidation process resumes. As noted above, a carboxyl
group in the vicinity of the sulfur decreases the antioxidant
effect. This can be attributed to the inductive electron-
withdrawing property of a carboxyl group, to reduce the
nucleophilicity of the sulfur and thereby decrease the rate of
reaction with the hydroperoxides. In preliminary studies we
have found that the relative rates of reaction of the β- and γ-
thia fatty acids (1) and (2) and the sulfide (8), with tert-
butylhydroperoxide are ca. 1 : 4 : 25. The observation that
unsaturated sulfides are more effective than their saturated
analogues as antioxidants presumably reflects more efficient
intramolecular reduction of hydroperoxides with the former
compounds. Somewhat anomalously, among the compounds
tested the β-thia fatty acids (1) and (3) are the weakest
antioxidants and yet they are the most readily consumed
(Table 1). It appears that the reactivity of the sulfides (1) and
(3) is too low to prevent a build-up in the concentration of
hydroperoxides, which therefore reach sufficiently high
concentrations to consume the sulfides (1) and (3) despite
their low reactivity. Under less extreme conditions the
sulfides (1) and (3) would be expected to show greater
antioxidant activity, where their relatively low reactivity
towards hydroperoxides would still be sufficient to consume
the hydroperoxides as they are formed.
3-Tetradecylthiopropionic Acid (2)
3-Mercaptopropionic acid (261 mg, 2.46 mmol) was added under an
atmosphere of nitrogen to a stirred solution of sodium methoxide,
prepared from sodium (142 mg, 6.17 mmol) and methanol (20 mL).
After the initial white precipitate had dissolved, a solution of 1-
bromotetradecane (568 mg, 2.05 mmol) in diethyl ether (2 mL) was
added. The reaction mixture was stirred for 16 h at room temperature.
After workup and purification by flash column chromatography, using
diethyl ether–hexanes (20 : 80) to diethyl ether–hexanes–acetic acid
(60 : 40 : 1) for elution, the title compound (2) (450 mg, 73%) was
obtained as a white solid, m.p. 67°C (Found: C, 67.3; H, 11.3; S, 10.4.
Calc. for C17H34SO2: C, 67.5; H, 11.3; S, 10.6%). IR νmax (Nujol)
3100–2600(br), 2965s, 2910s, 2840s, 1680s, 1460s, 1405w, 1375m,
1265m, 1255w, 1231w, 1210w, 1200m, 1080w, 915m, 725m cm–1. 1H
NMR δ (500 MHz, CDCl3) 0.88, 3H, t, J 6.7 Hz, C14´ CH3; 1.25–1.38,
22H, m, C3´–C13´ CH2; 1.56–1.61, 2H, m, C2´ CH2; 2.54, 2H, br, C1´
CH2; 2.66, 2H, t, J 6.6 Hz, C3 CH2; 2.79, 2H, br, C2 CH2. 13C NMR δ
(75.5 MHz, CDCl3) 14.69, 23.26, 27.16, 29.44, 29.80, 29.93, 30.02,
30.10, 30.17, 30.23, 32.49, 32.78, 35.25, 178.50. Mass spectrum (EI)
m/z 302 (M+, 21%), 230 (24), 229 (100), 185 (2), 161 (4), 119 (8), 106
(24), 97 (15), 89 (21), 83 (22), 69 (25), 55 (32). HRMS (M+), found:
302.2272; C17H34SO2 requires 302.2279.
Experimental
Melting points were determined using a Reichert microscope with a
Köfler heating stage and are uncorrected. 1H and 13C nuclear magnetic
resonance (NMR) spectra were recorded on Gemini 300 and Unity
Inova 500 spectrometers with tetramethylsilane as the internal standard
(δ 0.00). Infrared (IR) spectra were recorded on Perkin–Elmer 683 and
7700 spectrophotometers. Low- and high-resolution electron ionization
(EI) mass spectra and chemical ionization (CI) mass spectra were run
on a Fisons VG Autospec. Microanalyses were carried out by the
Microanalytical Laboratory, Research School of Chemistry, Australian
Pent-2-ynyl p-Toluenesulfonate (11)
Pent-2-yn-1-ol (10) (1.03 g, 12 mmol) was dissolved in chloroform
(10 mL) and the solution was cooled in an ice bath. Pyridine (1.90 g,
24 mmol) was then added, followed by p-toluenesulfonyl chloride
(3.43 g, 18 mmol) in small portions, with constant stirring. After 4 h,
diethyl ether (30 mL) and water (7 mL) were added and the organic
layer was separated and washed successively with 1 N HCl (7 mL), 5%
aqueous NaHCO3, water (7 mL) and brine (20 mL), and then dried with

Antioxidant Behaviour of Thia Fatty Acids
651
Na2SO4. The solvent was removed under reduced pressure and the
residue was chromatographed on silica gel, using diethyl ether–hexanes
(20 : 80) as eluent, to yield the title compound (11) (1.85 g, 65%) as a
colourless oil (Found: C, 60.2; H, 5.9; S, 13.2. Calc. for C12H14SO3: C,
60.5; H, 5.9; S, 13.5%). IR νmax (film) 2980m, 2940w, 2878w, 2240m,
1598s, 1495w, 1450m, 1360s, 1180s, 1175s, 1095s, 1020m, 1000m,
960s, 940s, 840s, 815s, 735s, 662s cm–1. 1H NMR δ (300 MHz, CDCl3)
0.98–1.03, 3H, m, C5 CH3; 2.04–2.10, 2H, m, C4 CH2; 2.44, 3H, s,
ArCH3; 4.69, 2H, m, C1 CH2; 7.35 and 7.82, 4H, d and d, J 8.7 Hz, ArH.
13C NMR δ (75.5 MHz, CDCl3) 12.91, 13.72, 22.22, 59.35, 71.72,
92.33, 128.69, 130.30, 133.90, 145.47. Mass spectrum (EI) m/z 238
(M+, < 0.1%), 209 (1), 155 (24), 139 (100), 129 (6), 117 (18), 107 (10),
92 (42), 91 (87), 83 (29), 66 (50), 65 (48).
57%) as a colourless oil (Found: C, 65.2; H, 7.4; S, 11.3. C16H22SO3
requires C, 65.3; H, 7.5; S, 10.9%). IR νmax (film) 3005m, 2960s,
2930m, 2870m, 1599m, 1462m, 1377s, 1310w, 1290w, 1189s, 1178s,
1
1100s, 1020w, 973s, 815s, 770m, 660s cm–1. H NMR δ (300 MHz,
CDCl3) 0.95, 3H, t, J 7.6 Hz, C9 CH3; 2.00–2.05, 2H, m, C8 CH2;
2.38–2.44, 2H, m, C2 CH2; 2.45, 3H, s, ArCH3; 2.72, 2H, t, J 7.0, C5
CH2; 3.99–4.04, 2H, m, C1 CH2; 5.20–5.28, 2H, m; 5.34–5.50, 2H, m;
7.33, 7.80, 4H, d and d, J 8.5 Hz, ArH. 13C NMR δ (75.5 MHz, CDCl3)
14.78, 21.09, 22.20, 26.12, 27.64, 70.20, 123.53, 126.94, 128.47,
130.37, 132.61, 132.92, 145.28. Mass spectrum (EI) m/z [277
(M – OH)+, 1%], 155 (25), 139 (2), 122 (67), 107 (47), 93 (100), 91
(77), 79 (66), 67 (47), 55 (32); (CI) m/z 312 (M + NH4)+.
3-[(3´Z,6´Z)-Nona-3´,6´-dienylthio]propionic Acid (6)
Nona-3,6-diyn-1-ol (12)
3-Mercaptopropionic acid (150 mg, 1.41 mmol) was added, under an
atmosphere of nitrogen, to a stirred solution of sodium methoxide,
prepared from sodium (64 mg, 2.78 mmol) and methanol (20 mL). After
the initial white precipitate had dissolved, a solution of (3Z,6Z)-nona-
3,6-dienyl p-toluenesulfonate (14) (276 mg, 0.94 mmol) in diethyl ether
was added. The mixture was stirred at 40°C for 2 days under nitrogen,
and then hydrochloric acid (10% v/v, 20 mL) and diethyl ether (20 mL)
were added. The organic phase was separated and washed with water and
brine, and dried over sodium sulfate. After removal of the solvent, the
residue was chromatographed, using diethyl ether–hexanes–acetic acid
(60 : 40 : 2) as eluent, to afford 3-[(3´Z,6´Z)-nona-3´,6´-
dienylthio]propionic acid (6) (88 mg, 41%) as a colourless oil (Found:
C, 62.9; H, 8.7; S, 14.0. C12H20SO2 requires C, 63.1; H, 8.8; S, 14.0%).
IR νmax (film) 3400–2500(br), 3005m, 2960m, 2910m, 2870w, 1713s,
Pent-2-ynyl p-toluenesulfonate (11) (1.37 g, 5.78 mmol) was added at
–30°C under nitrogen to a well stirred suspension in dimethyl-
formamide (15 mL) of but-3-yn-1-ol (368 mg, 5.25 mmol), sodium
carbonate (834 mg, 7.87 mmol), tetrabutylammomium chloride (1.46 g,
5.25 mmol) and copper(I) iodide (1.00 g, 5.25 mmol). The mixture was
stirred at room temperature for 24 h. Diethyl ether (30 mL) and 1 M HCl
(30 mL) were then added. After filtration through Celite, the organic
phase was washed with brine and dried over sodium sulfate, and the
solvent was removed under reduced pressure. Chromatography of the
residue on silica gel, with diethyl ether–hexanes (40 : 60) as eluent, gave
the product (12) (442 mg, 62%) as a colourless oil (Found: C, 79.6; H,
8.8. C9H12O requires C, 79.4; H, 8.9%). IR νmax (film) 3650–3100(br),
2975s, 2938s, 2905s, 2880s, 2500m, 1415m, 1375w, 1320s, 1180w,
1120w, 1040s, 900m, 735w cm–1. 1H NMR δ (300 MHz, CDCl3) 1.10,
3H, t, J 7.4 Hz, C9 CH3; 1.96, 1H, br, OH; 2.13–2.20, 2H, m, C8 CH2;
2.41–2.45, 2H, m, C2 CH2; 3.11–3.13, 2H, m, C5 CH2; 3.69, 2H, t, J
6.1 Hz, C1 CH2. 13C NMR δ [75.5 MHz, (D6)acetone] 10.14, 13.07,
14.72, 24.03, 61.95, 75.08, 76.83, 78.46, 82.42. Mass spectrum (EI) m/z
135 [(M – H)+, 12%], 121 (44), 107 (30), 105 (51), 103 (29), 93 (44), 91
(100), 79 (58), 77 (80), 65 (41), 63 (29), 57 (14), 53 (27), 51 (37). HRMS
(M – H)+, found: 135.0811; C9H11O requires 135.0810.
1
1459m, 1377w, 1264m, 1195w, 1140w, 940w cm–1. H NMR δ (500
MHz, CDCl3) 0.97, 3H, t, J 7.8 Hz, C9´ CH3; 2.05–2.08, 2H, m, C8´ CH2;
2.34–2.39, 2H, m, C2´ CH2; 2.57–2.60, 2H, t, J 7.4 Hz, C1´ CH2; 2.67,
2H, t, J 7.3 Hz, C3 CH2; 2.78–2.82, 4H, m, C5´ CH2, C2 CH2; 5.27–5.32,
1H, m; 5.37–5.47, 3H, m; 5.50–6.10, 1H, br s, COOH. 13C NMR δ (75.5
MHz, CDCl3) 14.83, 21.14, 26.20, 27.19, 27.95, 32.62, 35.21, 127.37,
127.97, 130.53, 132.72, 178.66. Mass spectrum (EI) m/z 228 (M+, 34%),
169 (14), 159 (18), 155 (45), 133 (8), 122 (54), 119 (42), 113 (12), 107
(44), 93 (100), 89 (66), 79 (57), 77 (53), 67 (52), 61 (33), 55 (43). HRMS
(M+), found: 228.1182; C12H20SO2 requires 228.1184.
(3Z,6Z)-Nona-3,6-dien-1-ol (13)
Nona-3,6-diyn-1-ol (12) (198 mg, 1.45 mmol) was hydrogenated at
atmospheric pressure, in the presence of a mixture of quinoline (44 mg)
and palladium (5%) on calcium carbonate (100 mg), poisoned with
lead, in methanol (25 mL). The reaction was stopped after 2.5 h when
the uptake of hydrogen was 61 mL. Removal of the solvent under
vacuum, followed by silica gel column chromatography, using diethyl
ether–hexanes (35 : 65) as eluent, gave (3Z,6Z)-nona-3,6-dien-1-ol (13)
(187 mg, 92%) as a colourless oil (Found: C, 77.4; H, 11.8. C9H16O
requires C, 77.1; H, 11.5%). IR νmax (film) 3500–3160(br), 3011s,
(all-Z)-Eicosa-5,8,11,14-tetraenyl Propyl Sulfide (7)
Using the procedure described above for the preparation of 3-
tetradecylthiopropionic acid (2), propane-1,3-thiol (26 mg, 0.34 mmol)
was added under an atmosphere of nitrogen to a stirred solution of
sodium methoxide, prepared from sodium (20 mg, 0.87 mmol) and
methanol (10 mL). After the initial white precipitate had dissolved, a
solution of (all-Z)-1-bromoeicosa-5,8,11,14-tetrane (101 mg, 0.29
mmol) in diethyl ether (1 mL) was added. The mixture was stirred for
15 h at room temperature. After workup, flash column chromatography,
using hexanes for elution, gave the title compound (7) (75 mg, 75%) as
a colourless oil (Found: C, 78.9; H, 11.4; S, 9.0. C23H40S requires C,
79.2; H, 11.6; S, 9.2%). IR νmax (film) 3005s, 2950s, 2920s, 2850s,
1650w, 1450m, 1390w, 1375w, 1290w, 1260w, 1230w, 910w, 720m
cm–1. 1H NMR δ (300 MHz, CDCl3) 0.89, 3H, t, J 6.8 Hz, C20 CH3;
0.99, 3H, t, J 7.2 Hz, C3´ CH3; 1.26–1.35, 6H, m, C17 CH2, C18 CH2,
C19 CH2; 1.43–1.48, 2H, C3 CH2; 1.57–1.64, 4H, m, C2 CH2, C2´
CH2; 2.05–2.13, 4H, m, C4 CH2, C16 CH2; 2.50–2.51, 4H, m, C1 CH2,
C1´ CH2; 2.80–2.86, 6H, m, C7 CH2, C10 CH2, C13 CH2; 5.32–5.43,
8H, m, C5 CH, C6 CH, C8 CH, C9 CH, C11 CH, C12 CH, C14 CH,
C15 CH. 13C NMR δ (75.5 MHz, CDCl3) 14.13, 14.67, 23.17, 23.60,
26.22, 27.41, 27.81, 29.44, 29.91, 32.11, 32.54, 34.79, 128.12, 128.48,
128.64 (2C), 128.90, 129.11, 130.40, 131.06. Mass spectrum (EI) m/z
348 (M+, 44%), 305 (38), 273 (4), 251 (6), 237 (14), 205 (17), 177 (19),
161 (36), 150 (27), 131 (29), 119 (40), 105 (48), 93 (77), 91 (76), 81
(79), 79 (95), 67 (100), 55 (69). HRMS (M+), found: 348.2854;
C23H40S requires 348.2851.
1
2960s, 2930s, 2870s, 1462m, 1377m, 1050m, 722m cm–1. H NMR δ
(300 MHz, CDCl3) 0.97, 3H, t, J 7.6 Hz, C9 CH3; 2.01–2.12, 2H, m,
C8 CH; 2.32–2.40, 2H, m, C2 CH2; 2.82, 2H, t, J 7.1 Hz, C5 CH2; 3.64,
2H, m, C1 CH2; 5.27–5.43, 3H, m; 5.49–5.56, 1H, m. 13C NMR δ (75.5
MHz, CDCl3) 14.82, 21.14, 26.20, 31.33, 62.77, 125.90, 127.40,
132.04, 132.74. Mass spectrum (EI) m/z 140 (M+, 2%), 122 (15), 111
(7), 109 (12), 107 (22), 98 (12), 96 (19), 95 (21), 93 (72), 91 (33), 81
(39), 79 (56), 68 (31), 67 (100), 55 (59), 54 (21), 53 (21). HRMS (M+),
found: 140.1203; C9H16O requires 140.1201.
(3Z,6Z)-Nona-3,6-dienyl p-Toluenesulfonate (14)
(3Z,6Z)-Nona-3,6-dien-1-ol (13) (167 mg, 1.19 mmol) was dissolved in
chloroform (5 mL) and the solution was cooled in an ice bath. Pyridine
(376 mg, 4.76 mmol) was then added, followed by p-toluenesulfonyl
chloride (340 mg, 1.78 mmol) in small portions with stirring. The
mixture was stirred for 24 h at 15°C. Diethyl ether (15 mL) and water
(5 mL) were added and the organic layer was separated and washed
successively with 1 N HCl (10 mL), 5% aqueous NaHCO3, water (10
mL), and brine (10 mL), and then dried over Na2SO4. The solvent was
removed under reduced pressure and the residue was chromatographed
on silica gel, with diethyl ether–hexanes (20 : 80) as eluent, to yield
starting material (13) (15 mg, 9%) and the title compound (14) (201 mg,
Propyl Tetradecyl Sulfide (8)
Using the procedure described above for the synthesis of (all-Z)-eicosa-
5,8,11,14-tetraenyl propyl sulfide (7), propane-1,3-thiol (165 mg, 2.16

652
C. J. Easton et al.
mmol) was added under an atmosphere of nitrogen to a stirred solution
of sodium methoxide, prepared from sodium (82 mg, 3.56 mmol) and
methanol (10 mL). After the initial white precipitate had dissolved, a
solution of 1-bromotetradecane (500 mg, 1.80 mmol) in diethyl ether (2
mL) was added. The mixture was stirred for 15 h at room temperature.
After workup, flash column chromatography, using hexanes for elution,
gave the title compound (8) (435 mg, 89%) as a colourless oil (Found:
C, 75.1; H, 13.3; S, 11.5. C17H36S requires C, 74.9; H, 13.3; S, 11.8%).
IR νmax (film) 2960s, 2910s, 2850s, 1460s, 1375w, 1290w, 1270w,
890w, 720w cm–1. 1H NMR δ (300 MHz, CDCl3) 0.87, 3H, t, J 6.5 Hz,
C14´ CH3; 0.99, 3H, t, J 7.4 Hz, C3 CH3; 1.25, 22H, m, C3´–C13´ CH2;
1.54–1.63, 4H, m, C2 CH2, C2´ CH2; 2.47–2.51, 4H, m, C1 CH2, C1´
CH2. 13C NMR δ (75.5 MHz, CDCl3) 14.13, 14.71, 23.28, 23.59, 29.55,
29.85, 29.94, 30.12, 30.18, 30.23, 30.33, 32.50, 32.69, 34.78. Mass
spectrum (EI) m/z 272 (M+, 52%), 243 (18), 229 (100), 196 (8), 187 (2),
168 (5), 145 (6), 131 (15), 111 (14), 97 (22), 89 (34), 83 (27), 76 (33),
69 (32), 57 (30), 55 (44).
29.70, 29.89, 29.93, 30.09, 30.18, 30.22, 32.49, 52.27, 53.47, 166.93.
Mass spectrum (EI) m/z 305 [(M+1)+, 1%], 287 (50), 243 (60), 229
(94), 196 (12), 168 (6), 149 (6), 125 (10), 111 (21), 97 (45), 83 (63), 69
(74), 57 (100), 55 (91). HRMS (M+1)+, found: 305.2153; C16H33SO3
requires 305.2150.
3-Tetradecylsulfinylpropionic Acid (16)
Reaction of 3-tetradecylthiopropionic acid (2) (30 mg, 0.10 mmol), as
described above for the synthesis of the sulfoxide (15), afforded the title
compound (2) (16 mg, 50%) as a colourless solid, m.p. 166–167°C
(Found: C, 64.3; H, 10.5. C17H34SO3 requires C, 64.1; H, 10.8%). IR
νmax (Nujol) 3600–2500(br), 2965s, 2910s, 2840s, 1695m, 1460s,
1375s, 1330w, 1305w, 1125w, 1040w, 1025w, 920w, 720w cm–1 1H
.
NMR δ (500 MHz, CDCl3) 0.81, 3H, t, J 7.0 Hz, C14´ CH3; 1.19–1.26,
20H, m, C4´–C13´ CH2; 1.34–1.37, 2H, m, C3´ CH2; 1.68–1.72, 2H, m,
C2´ CH2; 2.70–2.76, 1H, m; 2.82–2.89, 3H, m; 2.88–3.03, 1H, m;
3.05–3.10, 1H, m; 7.96, 1H, br s, COOH. 13C NMR δ (75.5 MHz,
CDCl3) 14.67, 23.19, 23.24, 27.78, 29.29, 29.72, 29.91, 30.09, 30.17,
30.20, 32.47, 46.66, 52.53, 174.37. Mass spectrum (EI) m/z 301
[(M – OH)+, 27%], 246 (21), 245 (16), 229 (100), 196 (5), 121 (15), 94
(22), 97 (22), 83 (29), 71 (32), 70 (34), 57 (51); (CI) m/z 319 (MH+).
HRMS (M – OH)+, found: 301.2197; C17H33SO2 requires 301.2201.
Antioxidant Behaviour of Thia Fatty Acids and Sulfides (1)–(8)
Solutions in dichloromethane (2 mL) containing arachidonic acid (9)
(18 mg) and one of the sulfides (1)–(8) (1.8–18 mg) were prepared with
lauric acid (18 mg) as an inert internal reference standard to aid
monitoring consumption of the other species. Samples of the solutions
(100 µL) were added to glass Petri-dishes (80 mm diameter) followed
by ethanol (400 µL). After evaporation of the solvent, a well distributed
thin film was formed on each Petri-dish. The Petri-dishes were placed
in a desiccator, which was then evacuated, filled with oxygen and stored
at 25°C in the dark. Dishes were removed from the desiccator after 1, 2,
3, 5 and 7 days. The mixture on each dish was dissolved in diethyl ether
and transferred to a 2 mL vial. After evaporation of the solvent, the
residue was dissolved in the HPLC mobile phase (100 µL) and 10% of
the solution was analysed by HPLC. Table 2 shows the mobile phases
used for the various thia fatty acids and sulfides (1)–(8), and their
retention times on HPLC. The results shown in Table 1 are the average
of triplicate experiments analysed in duplicate and the results of repeat
experiments and assays varied by less that 10%.
Acknowledgment
This work was made possible through the generous support
of Peptech (Australia) Ltd.
References
[1] A. Sinclair, R. Gibson (Eds), Essential Fatty Acids and
Eicosanoids 1992 (American Oil Chemists Society: Champaign,
Illinois).
[2] M. J. Pitt, C. J. Easton, C. J. Moody, A. Ferrante, A. Poulos, D. A.
Rathjen, Synthesis 1997, 1240.
[3] M. J. Pitt, C. J. Easton, T. A. Robertson, L. M. Kumaratilake, A.
Ferrante, A. Poulos, D. A. Rathjen, Tetrahedron Lett. 1998, 39,
4401.
[4] S. M. Lau, R. K. Brantley, C. Thorpe, Biochemistry 1988, 27,
5089.
[5] O. Spydevold, J. Bremer, Biochim. Biophys. Acta 1989, 1003, 72.
[6] S. Skrede, H. N. Sorensen, L. N. Larsen, H. H. Steineger, K.
Hovik, O. S. Spydevold, R. Horn, J. Bremer, Biochim. Biophys.
Acta 1997, 1344, 115.
[7] M. J. Pitt, C. J. Easton, A. Ferrante, A. Poulos, D. A. Rathjen,
Chem. Phys. Lipids 1998, 92, 63.
[8] C. J. Easton, L. Xia, M. J. Pitt, A. Ferrante, A. Poulos, D. A.
Rathjen, Synthesis 2001, 451.
[9] M. Costabile, C. S. T. Hii, B. S. Robinson, D. A. Rathjen, M. Pitt,
C. Easton, R. C. Miller, A. Poulos, A. W. Murray, A. Ferrante, J.
Immunol. 2001, 167, 3980.
Tetradecylsulfinylacetic Acid (15)
A solution of tetradecylthioacetic acid (1) (30 mg, 0.104 mmol) and
arachadonic acid (9) (30 mg) in dichloromethane (2 mL) in a 500 mL
round-bottomed flask was concentrated under reduced pressure to leave
a thin film on the walls of the flask, which was filled with oxygen and
left in the dark for 7 days. The material was then chromatographed, with
diethyl ether–hexanes–acetic acid (60 : 40 : 2) to methanol as eluent, to
obtain the colourless product (15) (19 mg, 59%). 1H NMR δ (300 MHz,
CDCl3) 0.88, 3H, t, J 6.4 Hz, C14´ CH3; 1.20–1.29, 20H, m, C4´–C13´
CH2; 1.44–1.50, 2H, m, C3´ CH2; 1.77–1.82, 2H, m, C2´ CH2;
2.88–2.95, 1H, m, C1´ CH; 3.02–3.07, 1H, m, C1´ CH´; 3.63–3.68, 1H,
d, J 14 Hz, C2 CH; 3.81–3.86, 1H, d, J 14 Hz, C2 CH´; 7.92, 1H, br s,
COOH. 13C NMR δ (75.5 MHz, CDCl3) 14.69, 23.20, 23.26, 29.18,
[10] B. S. Robinson, Z. H. Huang, G. Parashakis, C. S. T. Hii, J. V.
Ferrante, A. Poulos, D. A. Rathjen, M. J. Pitt, C. J. Easton, A.
Ferrante, Lipids 1999, 34, S341.
[11] C. J. Easton, T. A. Robertson, M. J. Pitt, D. A. Rathjen, A.
Ferrante, A. Poulos, Bioorg. Med. Chem. 2001, 9, 317.
[12] F. Abdi-Dezfuli, L. Froyland, T. Thorsen, A. Aakvaag, R. K.
Berge, Breast Cancer Res. Treat. 1997, 45, 229.
[13] K. Berge, K. J. Tronstad, F. Abdi-Dezfuli, T. Ranheim, R.
Mahesparan, R. Bjerkvig, R. K. Berge, Adv. Exp. Med. Biol.
1999, 466, 205.
[14] K. J. Tronstad, K. Berge, E. Dyroy, L. Madsen, R. K. Berge,
Biochem. Pharmacol. 2001, 61, 639.
[15] K. Berge, K. J. Tronstad, E. N. Flindt, T. H. Rasmussen, L.
Madsen, K. Kristiansen, R. K. Berge, Carcinogenesis 2001, 22,
1747.
Table 2. HPLC mobile phases and retention times of thia fatty
acids and sulfides (1)–(8)
Cpd Mobile phaseA
No.
Retention time (min)
Arachidonic
Lauric
acid
Cpd
acid (9)
(1) MeOH–buffer (90 : 10)
(2) MeOH–buffer (90 : 10)
(3) CH3CN–buffer (80 : 20)
(4) CH3CN–buffer (80 : 20)
(5) CH3CN–buffer (90 : 10)
(6) CH3CN–buffer (70 : 30)
(7) CH3CN–buffer (95 : 5)
(8) CH3CN–buffer (95 : 5)
6.8
6.8
6.7
6.7
5.2
14.7
3.4
3.4
4.0
4.0
4.3
4.3
3.8
7.1
3.1
3.1
9.4
10.7
8.8
10.9
7.0
3.2
14.1
21.6
[16] K. K. J. Kuiper, Z. A. Muna, K. S. Erga, E. Dyroy, E. Svendsen,
R. K. Berge, J. E. Nordrehaug, Atherosclerosis 2001, 58, 269.
[17] Z. A. Muna, K. Doudin, J. Songstad, R. J. Ulvik, R. K. Berge,
Arterioscler., Thromb., Vasc. Biol. 1997, 17, 3255.
A The buffer used was 30 mM H3PO4.

Antioxidant Behaviour of Thia Fatty Acids
653
[18] Z. A. Muna, B. J. Bolann, X. Chen, J. Songstad, R. K. Berge, Free
Radical Biol. Med. 2000, 28, 1068.
[19] E. Raspe, L. Madsen, A.-M. Lefebvre, I. Leitersdorf, L. Gelman,
J. Peinado-Onsurbe, J. Dallongeville, J.-C. Fruchart, R. Berge, B.
Staels, J. Lipid Res. 1999, 40, 2099.
[20] M. R. Wieckowski, D. Brdiczka, L. Wojtczak, FEBS Lett. 2000,
484, 61.
[21] L. Wojtczak, M. R. Wieckowski, P. Schönfeld, Arch. Biochem.
Biophys. 1998, 357, 76.
[22] A. Ferrante, A. Poulos, C. J. Easton, M. J. Pitt, T. A. Robertson,
D. A. Rathjen, Int. Pat. WO96/11908 1996.
[23] A. Ferrante, A. Poulos, M. J. Pitt, C. J. Easton, M. J. Sleigh, D. A.
Rathjen, F. Widmer, Int. Pat. WO97/38688 1997.
[24] A. Ferrante, C. J. Easton, L. Xia, Int. Pat. WO01/21575 2001.
[25] A. Ferrante, C. J. Easton, C. S. T. Hii, Int. Pat. WO01/21172
2001.
[26] N. A. Porter, L. S. Lehman, B. A. Weber, K. J. Smith, J. Am.
Chem. Soc. 1981, 103, 6447.
[27] J. Terao, S. Matsushita, Lipids 1986, 21, 255.
[28] L. Bateman, K. R. Hargrave, Proc. R. Soc. London, Ser. A 1954,
224, 389.
[29] L. Bateman, K. R. Hargrave, Proc. R. Soc. London, Ser. A 1954,
224, 399.
[30] M. Stiles, H. L. Finkbeiner, J. Am. Chem. Soc. 1959, 81, 505.
[31] T. Jeffery, S. Gueugnot, G. Linstrumelle, Tetrahedron Lett. 1992,
33, 5757.