Synthesis and analysis of novel glycerolipids for the treatment of metabolic syndrome

Article abstract of DOI:10.1021/jm801019s

Tetradecylthioacetic acid (TTA) 1 is a peroxisome proliferator-activated receptor (PPAR) agonist found to improve insulin sensitivity, lower blood lipid levels, enhance fatty acid oxidation, and promote anti-inflammation in vivo. In an attempt to enhance these properties, two key thioether fatty acid (Thefa) lipids, ditetradecylthioacetyl phosphatidylcholine 2 and tritetradecylthioacetyl glycerol 3, are synthesized and administered po to male Wistar rats at two different doses to study and compare metabolic outcomes relative to the administration of 1 alone after 6 days. Liposomal formulations of 1 and 2 are also prepared to evaluate acute metabolic responses (at 3 h) post iv injection. Across all metrics measured, 1-induced responses post po administration are in line with previous data. Responses induced from 3 are mostly equivalent to 1-induced responses. By contrast, 2-induced responses almost always outperform those of 1 and 3. Therefore, 2 may represent a new lead for the treatment of metabolic syndrome.

Full text of DOI:10.1021/jm801019s

1172
J. Med. Chem. 2009, 52, 1172–1179
Synthesis and Analysis of Novel Glycerolipids for the Treatment of Metabolic Syndrome
Michael R. Jorgensen,† Yushma Bhurruth-Alcor,† Therese Røst,‡ Pavol Bohov,‡ Melanie Mu¨ller,† Cristina Guisado,†
Kostas Kostarelos,† Endre Dyrøy,‡ Rolf K. Berge,‡ Andrew D. Miller,*,† and Jon Skorve*,‡
Imperial College Genetic Therapies Centre, Department of Chemistry, Flowers Building, Armstrong Road, Imperial College London, London
SW7 2AZ, United Kingdom, Institute of Medicine, UniVersity of Bergen, Haukeland UniVersity Hospital, N-5021 Bergen, Norway
ReceiVed August 13, 2008
Tetradecylthioacetic acid (TTA) 1 is a peroxisome proliferator-activated receptor (PPAR) agonist found to
improve insulin sensitivity, lower blood lipid levels, enhance fatty acid oxidation, and promote anti-
inflammation in vivo. In an attempt to enhance these properties, two key thioether fatty acid (Thefa) lipids,
ditetradecylthioacetyl phosphatidylcholine 2 and tritetradecylthioacetyl glycerol 3, are synthesized and
administered po to male Wistar rats at two different doses to study and compare metabolic outcomes relative
to the administration of 1 alone after 6 days. Liposomal formulations of 1 and 2 are also prepared to evaluate
acute metabolic responses (at 3 h) post iv injection. Across all metrics measured, 1-induced responses post
po administration are in line with previous data. Responses induced from 3 are mostly equivalent to 1-induced
responses. By contrast, 2-induced responses almost always outperform those of 1 and 3. Therefore, 2 may
represent a new lead for the treatment of metabolic syndrome.
Introduction
within promoters.8,9 There are three subtypes of PPARs: R, γ,
and δ.10-17 Natural ligands of the PPARs include a wide variety
of saturated and unsaturated fatty acids plus eicosanoid deriva-
tives.18 It has been well documented that hypolipidemic fibrates
and antidiabetic thiazolidinediones (TZDs) are synthetic ligands
for PPARR and PPARγ, respectively. The thiazolidinediones
improve insulin sensitivity, decrease hepatic glucose output, and
improve nonglycemic effects.19 However, use of thiazolidinedi-
ones is known to have attendant side effects such as weight
gain, edema, and anemia with possible liver dysfunction.20
Accordingly, there have been attempts to develop dual or even
pan PPAR agonists in the hope of achieving complementary
and synergistic actions on lipid homeostasis and insulin sensitiv-
ity with minimal attendant side effects.21
Disorders of lipid metabolism are connected to many
common, life-style related diseases. Obesity and obesity-
related disorders, often referred to as the metabolic syndrome,
affect an increasing part of the population in Europe, the
USA, and even several developing countries.1-3 The key
pathophysiological features of the metabolic syndrome
include insulin resistance (impaired glucose tolerance, insulin
insensitivity, type 2 diabetes), dyslipidemia (elevated tria-
cylglycerols and low density lipoprotein (LDLa) with reduced
high density lipoprotein (HDL) cholesterol), abdominal
obesity, hypertension, and endothelial dysfunction. Whereas
the pathogenesis of the metabolic syndrome is complex and
not well understood, obesity is acknowledged as an important
causative factor. The presence of the metabolic syndrome
predicts increased risk of developing diabetes type 2 and
cardiovascular disease with fatal consequences.4-7
Over the past 10 years, peroxisome proliferator-activated
receptors (PPARs) have been targeted in drug discovery for the
treatment of metabolic diseases because of their critical role in
the regulation of lipid metabolism and fat cell differentiation.
Indeed, these nuclear receptors are transcription factors that play
an important role in the regulation of gene expression by binding
to specific peroxisome proliferator response elements (PPREs)
In the past decade, it has been found that structurally modified
fatty acids show an enhanced potency compared to natural fatty
acids in modulating critical steps in the regulation of lipid
metabolism. One such molecule is tetradecylthioacetic acid
(TTA) 1 (Scheme 1). Fatty acid 1 is a modified 16-carbon
saturated fatty acid with a thioether bridge inserted between
carbon atoms 2 and 3. This thioether bridge is considered to
stabilize the compound with respect to ꢀ-oxidation in vivo.22
In other aspects, the chemical properties of such thioether fatty
acids align with those of normal fatty acids. The pleiotropic
effects of 1 have been revealed over the past decade. The
biological responses include increased hepatic and muscle
mitochondrial fatty acid oxidation in mammals, reduced body
fat (as seen in rats), decreased plasma lipid levels, modification
of plasma and tissue fatty acid profiles, improved insulin
sensitivity, potent antioxidant effects, and anti-inflammatory
actions.23,24 There are also effects involving increased mito-
chondrial ꢀ-oxidation rates that associate with a lowered proton
electrochemical potential and enhanced expression of uncoupling
protein 2 (UCP-2).25 This multitude of biological responses
suggests that fatty acid 1 should be a potent antiatherogenic
compound. Indeed, recent clinical trials have indicated that 1
could have significant effects on lipid metabolism in humans.26
As a pan PPAR ligand, fatty acid 1 regulates the expression of
those particular lipid-metabolizing enzymes involved in catabolic
metabolic pathways in a PPAR dependent manner. Indeed, 1
* To whom correspondence should be addressed. For J.S.: phone, +47
9186 5115; fax, +47 5597 3115; E-mail, jon.skorve@med.uib.no. For
A.D.M.: phone, +44 2059 45773; fax, +44 2059 45803; a.miller@
imperial.ac.uk.
†
Imperial College Genetic Therapies Centre, Department of Chemistry.
Institute of Medicine, University of Bergen, Haukeland University
‡
Hospital.
a Abbreviations: BSA, bovine serum albumin; CDI, N,N′-carbonyl-
diimidazole; CPT-II, carnitine palmitoyl transferase-II; DBU, 1,8-
diazabicyclo(5.4.0)undec-1-ene; DMAP, 4-dimethylaminopyridine; DMPC,
dimyristoyl-L-R-phosphatidylcholine; DMSO, dimethylsulfoxide; EI, elec-
tron ionization; FAO, fatty acyl-CoA oxidase; GPC, L-R-glycero-phospho-
choline; HBTU, O-benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-
phosphate; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; MUFA,
monounsaturated fatty acid; PPAR, peroxisome proliferator-activated recep-
tor; PUFA, polyunsaturated fatty acid; SA, stearic acid; SFA, saturated fatty
acid; Thefa, thioether fatty acid; TTA, tetradecylthioacetic acid; LDL, low-
density lipoprotein; HDL, high-density lipoprotein.
10.1021/jm801019s CCC: $40.75
2009 American Chemical Society
Published on Web 01/28/2009

Hypolipidemic Effect of Di-TTA-phosphatidylcholine
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1173
Scheme 1. Structures and Syntheses of 1, 2, and 3a
a (i) NaOH/MeOH, ambient temperature, 91%; (ii) L-R-glycerophosphocholine, CDI, DBU, CH2Cl2, DMSO, 70%; (iii) glycerol, HBTU, DMAP, CH2Cl2,
83%.
appears to activate human PPARs in the following order: δ >
1).33 The triacylglycerol analogue, lipid 3, was synthesized by
coupling 1 with glycerol using O-benzotriazole-N,N,N′,N′-
tetramethyluronium-hexafluoro-phosphate (HBTU) as coupling
agent and 4-dimethylaminopyridine (DMAP) as a nucleophilic
catalyst (Scheme 1).
R > γ.27 Alternatively, fatty acid 1 has also been found to
activate murine PPARs in cells post transfection but in the
following rank order R > δ . γ.28,29 In addition, PPAR
independent metabolic pathways appear to be important for other
metabolic effects of 1 and nonmetabolic effects such as the anti-
inflammatory activities of 1. Indeed 1, as an active lipid
metabolite, may affect specific signal transduction pathways.29
In terms of metabolism, data to date suggest that fatty acid 1
is incorporated into glycerolipids in vivo, with a preference for
phospholipids over acylglycerols.30,31 Both phospholipids and
acylglycerols are partially degraded by pancreatic lipases in the
gut releasing fatty acids from the sn-1 and sn-3 positions.
Micelles are then generated from 2-monoacylglycerols, lyso-
phospholipids, cholesterol, bile salts, and free acids prior to
absorption into enterocytes by simple diffusion across plasma
membrane. Once there, lipids are resynthesized and subsequently
assembled into large lipoprotein particles known as chylomi-
crons. These pass into the general circulation via the thoracic
duct and are transported in the bloodstream to the liver. Given
this uptake mechanism, we were curious to establish if the
properties of fatty acid 1 could be enhanced and/or indeed
modified by the substitution of 1 into phospholipid or triacylg-
lycerol structures. Here we describe the testing of this hypothesis
and observe that administration of a TTA-containing phospho-
lipid does indeed lead to fatty acid 1 like behavior that is much
enhanced in many instances. By contrast, administration of a
TTA-containing triacylglycerol structure does not appear typi-
cally to result in any enhanced fatty acid 1 effects.
Decrease in Plasma and Hepatic Lipids. Male Wistar rats
were fed fatty acid 1 or lipids 2 or 3 at doses equivalent to 85
µmol or 250 µmol of TTA/animal, resulting in a general dose
dependent decrease in plasma cholesterol and triacylglycerols
levels (Figure 1A). Administration of fatty acid 1 or lipids 2 or
3 at the lower dose all resulted in broadly similar decreased
levels of cholesterol and triacylglycerols in plasma. By contrast,
administration of lipid 2 at the higher dose resulted in a more
enhanced lipid lowering effect than was observed with either
fatty acid 1 or lipid 3. Overall, administration of 2 was
responsible for a drop of approximately 60% in both plasma
cholesterol and triacylglycerol levels relative to control (Figure
1A). Similar but less emphatic trends were also observed with
liver cholesterol and triacylglycerols levels (Figure 1B). In the
liver, administration of lipid 2 was found to be responsible for
a drop of approximately 20% and 25%, respectively, in liver
cholesterol and triacylglycerol levels relative to control (Figure
1B).
Increased Expression of PPARr Regulated Genes. Fatty
acid 1 is an agonist for the different PPARs and is a reasonably
potent PPARR agonist.27 Hence, the activities of three important
products of PPARR-regulated genes that are all involved in fatty
acid catabolism were examined by different assays on biopsy
tissue. These three gene products were 3-hydroxy-3-methyl-
glutaryl-CoA (HMG-CoA) synthase, fatty acyl-CoA oxidase
(FAO), and carnitine palmitoyl transferase-II (CPT-II). HMG-
CoA synthase is a mitochondrial protein that typically acts as
the rate-determining enzyme for ketone body formation. FAO
is the rate-determining enzyme peroxisomal fatty acid oxidation.
Both were found to increase in a dose dependent manner at 6
days post po administration of fatty acid 1 or lipids 2 or 3
(Figure 2). However, administration of lipid 2 at the higher dose
resulted in, by far, the largest increases in mitochondrial HMG-
CoA synthase and FAO activities above control by ap-
proximately 4-, and 5- to 6-fold, respectively.
Results
Chemistry. The chosen phospholipid and triacylglycerol
targets for “incorporation” of 1 were ditetradecylthioacetyl-L-
R-phosphatidylcholine 2 and tritetradecylthioacetyl triglycerol
3 (Scheme 1).32 Fatty acid 1 was synthesized by a nucleophilic
substitution between thioglycolic acid 4 and tetradecylbromide
5 under basic conditions. Thereafter, 1 was activated with N,N′-
carbonyldiimidazole (CDI) and reacted with L-R-glycerophos-
phocholine (GPC) to give lipid 2. The final coupling reaction,
adapted from Warner et al., was optimized using a dichloro-
methane-dimethylsulfoxide (DMSO) solvent system (Scheme

1174 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Jorgensen et al.
Figure 1. Effects of low (85 µmol) and high (250 µmol) equimolar doses of TTA, fed as unesterified fatty acid 1 or lipids 2 or 3 on levels of
cholesterol (dark striped bars) and triacylglycerol (light speckled bars) in plasma (A) and liver (B). Male Wistar rats were fed the different compounds
by gastric intubation for 6 days. The values represent the means ( SD from 5 rats in each group. *: significant difference from control animals (P
< 0.05). †: significant difference from control and the situations where animals were treated at same dose level with either 1 or lipid 3 (P < 0.05).
Figure 3. Effect of low (85 µmol) and high (250 µmol) equimolar
doses of TTA fed as unesterified fatty acid 1 or lipids 2 or 3 on fatty
Figure 2. Effect of low (85 µmol) and high (250 µmol) equimolar
acid oxidation of [1-14C]-palmitoyl-CoA in liver homogenates (A) and
doses of TTA, fed as unesterified fatty acid 1 or lipids 2 or 3 on the
on the mitochondrial CPT-II enzyme activity (B). Male Wistar rats
were fed the different compounds by gastric intubation for 6 days. The
enzyme activities of HMG-CoA synthase (A) and FAO (B). Male
Wistar rats were fed the different compounds by gastric intubation for
6 days. The values represent the means ( SD from 5 rats in each group.
values represent the means ( SD from 5 rats in each group. *:
significant difference from control animals (P < 0.05). §: significant
*: significant difference from control animals (P < 0.05). §: significant
difference from control and 1 treated animals at the lower dose (P <
difference from control and 1 at the lower dose (P < 0.05). †: significant
0.05). ‡: significant difference from control, plus 1 and 2 treated animals
difference from control, plus 1 and 3 treated animals at same dose level,
at the lower dose (P < 0.05). †: significant difference from control,
and also from 1 at the lower dose (P < 0.05).
plus 1 and 3 treated animals at same dose level, also from 1, 2, and 3
treated animals at the lower dose (P < 0.05).
In a similar way, the activity of CPT-II, the protein system
responsible for the translocation of fatty acids over the inner
mitochondrial membrane was found to increase in a dose
administration of lipid 2 at the higher dose was no more effective
than administration of fatty acid 1 or lipid 3 at the same higher
dose.
dependent manner at 6 days post po administration of fatty acid
Modification of Fatty Acid Composition. The metabolism
1 or lipids 2 or 3 (Figure 3B), with the largest increase of
of fatty acid 1 involves coenzyme A activation and incorporation
into esterified lipids, mostly phospholipids more than triacylg-
approximately 3-fold above control after administration of 2 at
the higher dose. By contrast, when mitochondrial ꢀ-oxidation
lycerols. Only minor amounts of free fatty acids can be detected
levels were measured as a function of [14C]-palmitoyl CoA
in plasma and liver.16,30 As a result of feeding fatty acid 1, the
isolated from liver homogenates, levels were found to increase
in a dose dependent manner at 6 days post po administration of
fatty acid 1 or lipids 2 or 3 (Figure 3A), but in this case,
fatty acid composition is known to be changed in plasma and
tissues.17,31 In our case, analysis of the fatty acid composition
in plasma and liver revealed that the levels of 1 became

Hypolipidemic Effect of Di-TTA-phosphatidylcholine
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1175
Table 1. Fatty Acid Composition of Plasma and Liver in Male Wistar Rats Fed 85 µmol or 250 µmol of 1 for 6 Days as Fatty Acid 1 or Lipids 2 or 3a
daily dose/rat
1
SFA
MUFA
PUFA, n-3
18:2, n-6
LCPUFA, n-6
LCn-6/18:2, n-6
A. Plasma FA wt %
control
1 85 µmol
3 (equiv 85 µmol of 1)
2 (equiv 85 µmol of 1)
1 250 µmol
3 (equiv 250 µmol of 1)
2 (equiv 250 µmol of 1)
0.00
35.60 ( 1.56
36.02 ( 0.38
36.21 ( 1.26
35.65 ( 1.27
35.00 ( 0.54
34.67 ( 1.05
35.25 ( 0.97
21.47 ( 3.51
23.49 ( 2.35
21.80 ( 2.96
22.53 ( 4.54
21.32 ( 2.48
21.63 ( 4.01
21.62 ( 4.55
2.85 ( 0.39
20.06 ( 2.50
18.46 ( 1.91
19.73 ( 2.32
19.52 ( 0.98
19.68 ( 1.35
18.25 ( 0.92
17.97 ( 2.06
19.38 ( 4.1
18.75 ( 2.0
19.08 ( 1.22
19.06 ( 3.66
20.82 ( 1.72
22.24 ( 2.13
22.07 ( 4.88
0.98 ( 0.26
1.03 ( 0.19
0.98 ( 0.12
0.97 ( 0.14
1.06 ( 0.11
1.22 ( 0.09
1.25 ( 0.34
0.32 ( 0.20
0.51 ( 0.26
0.90 ( 0.55
1.60 ( 0.76b
1.49 ( 0.68b
1.95 ( 0.75b
2.47 ( 0.12
2.35 ( 0.16
2.42 ( 0.26
2.24 ( 0.20b
2.29 ( 0.18b
1.86 ( 0.41b
B. Hepatic FA wt %
control
1 85 µmol
3 (equiv 85 µmol of 1)
2 (equiv 85 µmol of 1)
1 250 µmol
3 (equiv 250 µmol of 1)
2 (equiv 250 µmol of 1)
0.00
39.56 ( 1.35
39.67 ( 0.90
39.82 ( 1.12
39.67 ( 1.21
38.71 ( 0.65
38.98 ( 0.49
39.22 ( 0.56
14.68 ( 2.00
17.35 ( 1.87
17.27 ( 2.41
16.66 ( 3.20
16.86 ( 2.01
15.91 ( 1.33
16.33 ( 1.40
6.06 ( 0.29
13.11 ( 1.86
11.15 ( 1.39
11.28 ( 1.07
11.99 ( 1.31
11.02 ( 1.80
10.67 ( 0.76
26.09 ( 1.51
25.70 ( 0.64
25.81 ( 1.75
26.13 ( 1.48
27.66 ( 0.54
28.73 ( 1.09e
2.02 ( 0.26
2.34 ( 0.33
2.30 ( 0.25
2.19 ( 0.13
2.56 ( 0.41
2.70 ( 0.21b
0.36 ( 0.20
0.69 ( 0.35
1.28 ( 1.07
2.03 ( 1.02b
2.04 ( 0.8b
5.44 ( 0.40b
5.18 ( 0.42b
4.91 ( 0.52b
4.94 ( 0.48b
4.97 ( 0.34b
4.05 ( 0.44b
3.34 ( 1.55b e
8.79 ( 1.08b e
30.30 ( 1.60b e
3.49 ( 0.46b c d e
,
,
,
, , ,
a The values represent means ( SD from 5 rats in each group. b Significant difference (P < 0.05) from control animals. c Significant difference (P < 0.05)
from 1 at same dose level. d Significant difference (P < 0.05) from 3 at same dose level. e Significant difference (P < 0.05) from 1 at the low dose.
Table 2. Liposomal Formulations of Fatty Acid 1 and Lipid 2a
triacylglycerol and cholesterol levels in plasma appeared to
support the possibility that there are acute 1/DMPC and 2/SA
liposome mediated lipid lowering effects compared to the
control, but BSA-associated free fatty acid 1 administration was
at least as effective, if not more so, especially where the lowering
of cholesterol (Chol) levels were concerned (Table 3). A dose
response effect was not strongly evident on either plasma lipids
or the hepatic content of fatty acid 1. Only a few animals were
available for analysis of the hepatic content of fatty acid 1, so
these data have not been treated statistically. These data also
seem to indicate a nondose response except where the liver fatty
acid 1 content was determined after administration of the 2/SA
liposomes at the highest total lipid dose (Table 3B).
[total lipid] ꢁ potential TC
diameter
(nm)
liposome
[1] (mg/mL)
(mg/mL)
(mV)
(°C)
1/DMPC (1:1)
2/SA (2:1)
8.9
12.5
28.6
14.7
-36
-10
36 265.3 ( 114.3
44 209.1 ( 84.6
a ꢁ potentials are estimates.
substantial post po administration of fatty acid 1 or lipids 2 or
3 (Table 1). Saturated fatty acid (SFA) and monounsaturated
fatty acid (MUFA) levels were unchanged relative to control
post po administration of fatty acid 1 or lipids 2 or 3 (Table 1).
In contrast, plasma and liver levels of polyunsaturated fatty acids
(PUFA, n-3), and 18:2, n-6 fatty acids were obviously decreased
in a dose dependent manner after administration of the three
compounds (this decrease was seen for 22:6, n-3, 20:5, n-3 fatty
acids as well, data not shown). Administration of lipid 2 at the
highest dose resulted unexpectedly in the most significant
decline of both PUFA, n-3 and 18:2, n-6 fatty acid levels,
particularly in liver tissue, relative to controls. Conversely,
plasma and liver levels of long-chain PUFAs (LCPUFA, n-6;
LCn-6/18:2, n-6) were increased in a dose dependent manner
by administration of fatty acid 1 or lipids 2 or 3, and once again
administration of lipid 2 at the highest dose resulted in the most
significant increase in LCPUFA, n-6 and LCn-6/18:2, n-6 levels,
particularly in liver tissue, relative to controls (Table 1).
Discussion
The pleiotropic effects of fatty acid 1 are well documented,
and significant benefits are conferred on lipid metabolism,
immune response, and oxidative stress post administration of
1.23,24 In this study, it has now been demonstrated that oral
feeding with esterified or unesterified 1 or intravenous injection
of the fatty acid will differentially affect the absorption and
distribution of this potent fatty acid. Across all metrics measured,
the responses to fatty acid 1 and lipid 3 were mostly equivalent.
By contrast, the responses to lipid 2 almost always outperformed
the responses to 1 and 3. Compared to administration of fatty
acid 1 or lipid 3, administration of lipid 2 appeared to result in
significant increases in the enzyme activities of PPARR
regulated gene products and mitochondrial ꢀ-oxidation in
addition to having the largest impact on plasma and hepatic
fatty acid composition (Figures 1, 2, and 3). Most of these effects
were dose dependent, although not to the same extent. Especially
with regard to enzyme activities, the changes observed with the
highest dose used were substantially greater (4-6 fold) than
the decrease in plasma and hepatic lipid levels (Figures 1 and
2). This indicates that the changes in lipid levels are not simply
a reflection of the upregulation of the PPAR regulated genes,
but genes of other regulatory pathways involved in lipid
metabolism are also affected. Regarding the incorporation of
fatty acid 1 into lipids, the highest level of incorporation of 1
was attained in plasma and liver after feeding lipid 2 in a dose
dependent manner (Table 1). The hepatic and plasma fatty acid
composition was to a large extent also changed in a dose
dependent manner in line with changes to the hepatic content
of fatty acid 1 across all the different formulations tested. These
modifications have been observed previously in studies with
Liposomal Delivery of 1 and 2. To examine the potential
role of intestinal absorption in the enhanced effects of lipid 2
compared to the effects of fatty acid 1 after po administration,
fatty acid 1 and lipid 2 were formulated into liposomes with
dimyristoyl-L-R-phosphatidylcholine (DMPC) (1:1, m/m) or
stearic acid (SA) (2:1, m/m), respectively (Table 2). Free fatty
acid 1 was also prepared in bovine serum albumin (BSA)
solution to ensure solubility. The effects of iv administration
of liposomes 1/DMPC (1:1, m/m), 2/SA (2:1, m/m), or DMPC
control liposomes in PBS to male Wistar rats (tail vein injection)
were compared with the effects of the iv administration of BSA-
associated free fatty acid 1 in the same animal model. The 2/SA
liposomes (liposomal 2) were administered at a total lipid 2
dose of 9 or 46 µmol (that were equivalent with fatty acid 1
doses of 12 or 62 µmol), 1/DMPC liposome formulations
(liposomal 1) were administered with total fatty acid 1 doses
of 12 or 62 µmol. Our expectation was that iv administration
of these liposomes might reveal evidence for an acute fatty acid
1 response in a matter of 2-4 h as opposed to the 6 day effects
studied post po administration. Indeed, the data concerning

1176 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Jorgensen et al.
Table 3. Plasma Triacylglycerol (TAG) or Cholesterol (Chol) Levels (A) and Hepatic Content of Fatty Acid 1 (B) in Rats Injected Intravenously with
Either DMPC Control Liposomes (DMPC), a BSA-Associated Solution of Free Fatty Acid 1 (Free 1), 1/DMPC (1:1) Liposomes (Liposomal 1), or 2/SA
(2:1) Liposomes (Liposomal 2) (see Table 2)a
DMPC
free 1
liposomal 1 [1 /DMPC (1:1)]
[1] 12 µmol [1] 62 µmol
A. Plasma Lipid Levels (mmol/L)
liposomal 2 [2/SA (2:1)]
at 3 h
liposomes
[1] 12 µmol
[total] 9 µmol
[total] 46 µmol
TAG
Chol
1.97 ( 0.28
1.80 ( 0.24
1.51 ( 0.27b
1.43 ( 0.34b
1.67 ( 0.32
1.64 ( 0.16
1.38 ( 0.28b
1.65 ( 0.63
1.73 ( 0.25
1.69 ( 0.6
1.52 ( 0.29
1.72 ( 0.24b
B. Hepatic Content wt % of Fatty Acid 1
wt % of 1
0.0
0.24
0.18 0.30
0.13
0.55
a In (A), the values represent means ( SD from 4-7 rats in each group. In (B), the values are representative average data from 3 rats in each group.
b Significantly different (P < 0.05) from control animals.
1,23 the main features being the decrease in PUFA, n-3 fatty
acids and an apparent increase in the long chain (20C and more)
n-6 fatty acids.
iv compliant formulations to accelerate the bioavailability of 1
to the liver. Indeed iv administration of liposomal 1 leads to an
equivalent level of 1 in the liver compared to the amount
observed post po administration even though the dose of iv
administered liposomal 1 is almost an order of magnitude lower
(Tables 1B and 3B). Having said this, some caution is necessary
because there is the possibility that the metabolic effects of fatty
acid 1 may also vary according to whether or not 1 is
administered in free form or in a liposome associated form,
owing to potential differences in physical properties between
liposomal 1 and free fatty acid 1 (Table 3). Nevertheless, iv
administration of liposomal 1 may represent a useful alternative
to po administration for specific purposes such as the targeted
delivery of modified fatty acids and similar compounds. Overall,
less 1 seems to be required for a biological responses post
administration by iv injection compared with the amount of 1
required to provoke responses post po administration, a char-
acteristic which is characterized by the fact that the lowest ratio
between the dose administered and the hepatic content of 1 is
obtained after iv injection (compare Tables 1 and 3).
In conclusion, we are of the opinion that the biological
responses of 1, 3, and 2 are mediated by effects on gene
expression and by direct interference in the metabolism of
hepatic and plasma lipoproteins. Particularly at the higher dose,
2 is the most profound agent of these changes. Overall, po
administration of 2 gave better results than 1 and 3. Given this
promising data, other novel lipid analogues can now be
developed as potential therapeutics for metabolic syndrome
related conditions such as hyperlipidaemia and atherosclerosis.
The simplest explanation for the better performance of lipid
2 is that this compound has a higher intestinal absorption than
fatty acid 1, thereby facilitating incorporation into plasma and
hepatic lipids post absorption. This is supported by the fact that
the quantity of 1 recovered from liver post intravenous
administration of free 1 is at least equivalent to if not more
than the quantity of 1 recovered post intravenous administration
of liposomal 2 (administered in such an amount as to correspond
to an approximately equimolar dose of 1) (see Table 3). In fact,
it has been demonstrated that approximately 90% of 1 absorbed
actually becomes incorporated into phospholipids post po
administration,30,31 and this rather uncommon distribution for
a fatty acid appears to be a central component of effects
mediated by 1 on metabolic responses. Therefore, the promo-
tional influence on metabolic responses of feeding 2 (Figures
1, 2, and 3) may well be consistent with these metabolic
observations. Accordingly, lipid 2 could just be regarded as a
more suitable “means of carriage” to enhance the bioavailability
of 1 to influence metabolic responses. However, we do not
necessarily believe that the enhancing effects of lipid 2 after
po administration can be due to such proposed fatty acid
1-prodrug-like behavior alone. Indeed, the observed capacity
of lipid 2 to enhance metabolic responses, over and above the
effects of 1 and 3 post po administration, may also be associated
with other unique properties and characteristics that are not
necessarily linked with the release of free 1 in a timely fashion
such as membrane active functions. We shall be carrying out
further investigations into this area of research in order to
determine if lipid 2 has such potentially unique properties that
could contribute to the control of metabolic responses in a
manner independent of fatty acid 1.
The liposome data are also interesting. A comparison can be
made between the extent of accumulation of 1 into liver post
po administration of 1 or lipid 2, and the contrasituation post
iv administration of either free 1, injected in association with
BSA, liposomal 1, or liposomal 2. In the first instance, po
administration of lipid 2 leads to the largest increase in 1 found
in the liver (Table 1A). In the second instance, administration
of free 1 leads to the highest levels of accumulation of 1 in
liver, followed by the administration of liposomal 1 (Table 3B).
The administration of free 1 also resulted in the most significant
decrease in plasma triacylglycerols and cholesterol levels at 3 h
post injection. Certainly, such data could be considered con-
sistent with the argument above that lipid 2 is primarily a “means
of carriage” for fatty acid 1 post po administration to enhance
the bioavailability of this fatty acid 1 to influence metabolic
responses. However, these data more properly demonstrate that
acute effects of fatty acid 1 might be able to take advantage of
Experimental Section
Synthesis. All reactions were carried out under an atmosphere
of nitrogen or argon, in oven-dried glassware, unless otherwise
stated. Flash column chromatography was performed with Merck
silica gel 60 (230-240 mesh) according to the method of W. C.
Still.34 TLC refers to thin layer chromatography performed on
precoated Merck silica gel 60 F254 aluminum-backed plates and
visualized with a UV lamp (254 nm) and/or stained with acidic
ammonium molybdate(IV), basic potassium manganate(VII; KM-
nO4), iodine, or phosphomolybdic acid. CH2Cl2 was distilled over
P2O5, and other solvents were bought and predried as required.
Hipersolv CHCl3 was used for some reactions. All chemicals were
purchased from Sigma-Aldrich, Lancaster and Merck Biosciences.
Special solvent mixtures are: solvent A, CH2Cl2/MeOH/H2O 345:
90:10; solvent B, CH2Cl2/MeOH/H2O 65:25:4; solvent C, CH2Cl2/
1
MeOH/AcOH 92:7:1. H NMR spectra were recorded on either a
Bruker DRX300 or Avance 400 using residual isotopic solvent
(CDCl3, δH ) 7.27 ppm; CD3OD, δH ) 3.84 ppm) as an internal
reference. Data is reported as follows: (br ) broad; s ) singlet, d
) doublet, t ) triplet, q ) quartet, m ) multiplet; coupling
constant(s) in Hz; integration; assignment). Mass spectra were
recorded using VG-070B, Joel SX-10,2 or Bruker Esquire 3000
ESI instruments.

Hypolipidemic Effect of Di-TTA-phosphatidylcholine
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1177
Tetradecylthioacetic Acid 1. Tetradecyl bromide (5.36 mL, 18.0
mmol) was added to a solution of thioglycolic acid (1.25 mL, 18.0
mmol) in 25% NaOH in MeOH (6.03 g in 24.0 mL), and the
mixture was stirred vigorously at room temperature for 72 h. After
dilution with water, the mixture was acidified to pH 1 with
concentrated HCl and the aqueous phase was extracted with diethyl
ether. After drying over MgSO4 and concentration in vacuo, this
residue was then purified by flash column chromatography on silica
gel (hexane/EtOAc 3:1) to yield 4.73 g (91%) of 1 as a hygroscopic
white gum. δH (CDCl3) 3.27 (s, 2H, SCH2COOH), 2.68-2.65 (t, J
) 7.6 Hz, 2H, SCH2), 1.66-1.58 (m, 2H), 1.41-1.36 (m, 2H),
1.27 (s, 20H), 0.908-0.874 (t, J ) 6.8 Hz, 3H); m/z (EI+) 288;
found [M]+, 288.2121, C16H32O2S requires [M]+, 288.2123.
Ditetradecylthioacetyl-sn-glycero-3-phosphocholine 2. To a
solution of 1 (261 mg, 0.905 mmol) in CH2Cl2 (5.00 mL) was added
CDI (190 mg, 1.17 mmol) and the solution stirred for 2 h 45 min
at RT under argon. In the meantime, DBU (135 µL, 0.905 mmol)
was added to a solution of L-R-GPC (133 mg, 0.301 mmol) in
DMSO (5.00 mL) and stirred at RT under argon for 2 h 45 min.
The first solution was then transferred to the second and the mixture
stirred at RT for 16 h. The mixture was acidified with acetic acid
0.1 M (12.0 mL) and washed with CHCl3/MeOH 2:1 (5 × 10.0
mL). The organic phase was then washed with H2O/MeOH 1:1 (5
× 50 mL) and the aqueous phase extracted with CHCl3/MeOH 2:1
(3 × 100 mL). The combined phases were concentrated in vacuo
to give a brownish oil (2.78 g), which was purified by flash column
chromatography on silica gel (CH2Cl2, solvent A in CH2Cl2: 10%,
20%, 80%, 90%, solvent A, solvent B, methanol) to yield 168 mg
(70%) of 2 as a hygroscopic gum; δH (CDCl3) 5.25 (br, 1H,
OCH2CHOCH2), 4.45-4.41 (dd, J ) 12 Hz, J ) 2.8 Hz, 2H,
COOCH2CH), 4.28-4.23 (m, 2H, CHCH2OP), 4.00-3.97 (t, J )
6.2 Hz, 2H, POCH2), 3.85-3.74 (m, 2H, CH2N(CH3)3), 3.31-3.28
(br, 13H, 3 × NCH3, 2 × SCH2COOH), 2.63-2.58 (m, 4H, Hb),
1.61-1.54 (m, 4H), 1.38-1.35 (m, 2H), 1.26 (s, 40H), 0.85-0.82
(t, J ) 6.8 Hz, 6H); m/z (FAB+) 798 ([M + H]+; found [M + H]+,
798.5156, C40H81NO8PS2 requires [M + H]+, 798.5141.
before the start of the experiments.35 They had free access to tap
water and a grower rat maintenance chow: Standard Laboratory
Rat Chow R-34-EWOS-ALAB (EWOS Sweden). Two rats were
housed per cage. The animals were acclimatized for at least 5 days
before the start of the experiment. Weight gain and food intake
were recorded daily. Fatty acid 1 and lipids 2 and 3 were suspended
in 0.5% (w/v) carboxymethylcellulose (CMC) and administered by
gastric intubation in a final volume of 0.8-1.2 mL once a day for
6 days. Control rats received palmitic acid suspended in CMC. At
the end of the experiment, there was no difference in weight gain
between the groups (data not shown). At the end of the feeding
period, the animals were fasted for 12 h and anaesthetized
subcutaneously with a 1:1 mixture of Hypnorm (fentanyl citrate
0.315 mg/mL and fluanisone 10 mg/mL, Janssen Animal Health)
and Dormicum (midazolam 5 mg/mL, Hoffmann-La Roche), 0.2
mL/100 g body weight. Cardiac puncture was performed to collect
blood samples, and livers and hearts were dissected. Plasma was
prepared, and triacylglycerol and cholesterol levels were measured
using the Monotest enzymatic kit (Boehringer Mannheim, Germany)
at a dose of 300 mg/day/kg body weight. Parts of the liver were
immediately frozen in liquid nitrogen, while the rest of the liver
was chilled on ice for homogenization. The protocol was approved
by the Norwegian State Board of Biological Experiments with
Living Animals.
Homogenization and Preparation of Subcellular Fractions.
Livers and hearts from the rats were homogenized individually in
ice-cold sucrose solution (0.25 M sucrose in 10 mM HEPES buffer
pH 7.4 and 1 mM EDTA) using a Potter-Elvehjem homogenizer.
Preparation of subcellular fractions was performed as previously
described.36 The procedure was performed at 0-4 °C, and the
fractions were stored at -80 °C. Protein was assayed using the
BioRad protein assay kit (BioRad, Heraules, CA) and bovine serum
albumin as standard. To measure the hepatic fatty acid composition,
fatty acids were extracted from subcellular fractions with 2:1
chloroform:methanol.37 To the lipid extracts were added hene-
icosanoic acid (21:0) as internal standard and transesterified in 12%
BF3 in methanol (v/v). To remove neutral sterols and nonsaponi-
fiable material, extracts of fatty methyl esters were heated in 0.5
mol/L of KOH in ethanol/water (9:1, v/v). Recovered fatty acids
were re-esterified using BF3-methanol. The methyl esters were
quantified as previously described.38
Enzyme Assays. Palmitoyl-CoA oxidation was measured in the
postnuclear fraction as acid-soluble products, using [1-14C]-palmi-
toyl-CoA (Radiochemical Centre, Amersham, England) as substrate
as described previously.39 Carnitine palmitoyl transferase-II activi-
ties were measured in the mitochondrial fraction as described, and
3-hydroxy-3-methylglutaryl-CoA synthase activities were measured
according to the method of Clinkenbeard et al. in the postnuclear
fraction.40,41 Finally, fatty acyl-CoA oxidase was measured in the
postnuclear fraction as well by the coupled assay according to the
protocols of Small et al.42 The production of hydrogen peroxide
was measured by monitoring the increase in dichlorofluorescein
absorbance in the presence of palmitoyl-CoA.
Preparation, Characterization, and Effectiveness of Liposo-
mal Formulations. General Procedures. 1,2-Dimyristoyl-sn-glyc-
ero-3-phosphocholine (DMPC) and stearic acid (SA) were pur-
chased from Sigma-Aldrich. Stock solutions of the lipids (5 mg/
mL) were prepared in CH2Cl2 and stored at -20 °C. Freshly
distilled CH2Cl2 and PBS (10 mM phosphate buffer, 150 mM NaCl,
pH 7.4) were used for liposome hydration.
Tritetradecylthioacetyl Glycerol 3. HBTU (1.16 g, 3.05 mmol)
and DMAP (1.09 g, 8.95 mmol) were added to a solution of 1
(834.0 mg, 2.89 mmol) and glycerol (85.5 mg, 0.928 mmol) in dry
CH2Cl2 under argon. The mixture was stirred at room temperature
for 19 h. The reaction had not gone to completion, so some more
TTA (93.1 mg, 0.323 mmol) was added and the mixture stirred for
a further 4 h. Citric acid solution (7%, 15.0 mL) was added to the
reaction mixture. The phases were separated and extracted with
CH2Cl2 (4 × 25.0 mL). The organic phases were combined, dried
over MgSO4, and concentrated in vacuo before purification by flash
column chromatography on silica gel (hexane, hexane/EtOAc 9:1,
8:2, 6:4, EtOAc) to yield 698 mg (83%) of 3 as a hygroscopic
white gum;
δ
(CDCl3/MeOD 2:1) 5.32-5.26 (m, 1H,
H
OCH2CHOCH2O), 4.39-4.35 (dd, J ) 12.0 Hz, J ) 4.0 Hz, 2H,
OCH2CHOCH2O), 4.26-4.21 (dd, J ) 11.8 Hz, J ) 6.2 Hz, 2H,
OCH2CHOCH2O), 3.20 (s, 6H, 3 × COCH2S), 2.61-2.57 (m, 6H,
3 × COCH2SCH2), 1.59-1.52 (m, 6H), 1.36-1.31 (m, 6H), 1.22
(br, 60H), 0.86-0.82 (t, J ) 6.8 Hz, 9H); m/z (FAB+) 901 ([M]+);
found [M + H]+, 901.5948, C51H98O6S3 requires [M + H]+,
901.5906.
Purity Evaluation. Analytical HPLC was conducted on a
Hitachi-LaChrom L-7150 pump system equipped with a Polymer
Laboratories PL-ELS 1000 evaporative light scattering detector and
a C-4 peptide column. HPLC solvent mixes assigned as follows:
mix A ) H2O/0.1% TFA; mix B ) MeCN/0.1% TFA; mix C )
MeOH. The solvent gradient used was: 0.0 min [100% A], 15-25.0
min [100% B], 25.1-45.0 min [100% C], 45.1-55.0 min [100%
A]; at a flow rate of 1 mL/min. Purities were estimated from peak
areas as 1, 99.7 ( 0.1%; 2, 99.6 ( 0.1%; and 3, 95.3 ( 0.1%.
Animals and Treatments. Male Wistar rats (Mol:Wist) from
Møllegard Breeding Laboratorium (Ejby, Denmark), weighing
170-210 g, were housed in pairs, both receiving the same treatment,
in metal wire cages in a room maintained at 12 h light-dark cycles,
at a temperature of 20 ( 3 °C and relative humidity of 65 ( 15%.
Rats were acclimatized for at least a week under these conditions
Preparation of Liposomes. The relevant lipid mixture in CH2Cl2
was dried as a thin layer in a 50 mL round-bottomed flask. PBS
was added to the lipid film and the mixture was then shaken for 10
min in a water bath at 40-50 °C. After this, the liposome
suspension obtained was sonicated for 5 min.
Differential Scanning Calorimetry (DSC). Thermograms were
obtained using a VP-DSC microcalorimeter (MicroCal Incorporated)
at a heating rate of 1 °C/min from 5 to 60 °C. The phase transition
temperature (Tc) was determined based on the peak top temperature.
Photon Correlation Spectroscopy (PCS). Particle sizes were
measured on a N4 plus MD submicron particle analyzer (Beckman

1178 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Jorgensen et al.
Response Element in the C-Promoter. J. Biol. Chem. 1995, 270,
19269–19276.
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1999, 402, 860–861.
Coulter, High Wycombe, Buckinghamshire, UK). All measurements
were performed at 20 °C and recorded at 90 °C, with an equilibrium
time of 1 min and individual run times of 300 s. The refractive
index of the buffer was set to 1.333. Unimodal analysis was used
throughout to calculate the mean particle size and standard
distribution.
Zeta Potential (ZP). Zeta potentials were measured using surface
charge electrophoresis on a Delsa 440 SX (Beckman Coulter, High
Wycombe UK).
Liposomal 1 Formulation. Fatty acid 1 was formulated by
combining 1 with DMPC. Different molar ratios of 1 to DMPC
liposomes were analyzed and characterized. The final formulation
used for the iv injection of 1 was 1/DMPC (1:1, m/m) in PBS.
Liposomal 2 Formulation. Liposomal 2 was initially formulated
from 2 alone, but it was found that the highest concentration that
could be achieved was only 2.5 mg/mL of lipid. For higher
concentrations, liposomal 2 formulations were prepared with SA.
The final formulation used for the iv injection of 2 was 2/SA (2:1,
m/m) in PBS. The doses injected were calculated in relation to the
amount of latent 1 present in 2. The liposomes were suspended in
phosphate buffered saline in the concentrations indicated and the
solution sonicated before use. Nonesterified 1 (12 mM) was
dissolved in 200 mg/mL albumin injection solution (“Octapharma”)
after dissolution in equimolar amounts of NaOH. Liposomes (Table
3) or free 1 in solution with BSA (1 mL) were injected at pH 7.0
through the tail vein. Following this, rats were anaesthetized with
Fluorane (0.4 mL/100 g body weight), and sacrificed as described
above.
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