Novel phenoxyalkylcarboxylic acid derivatives as hypolipidaemic agents

Article abstract of DOI:10.3109/14756366.2011.589840

Novel phenoxyalkylcarboxylic acid derivatives based on the natural scaffolds, flavonoids, or resveratrol were designed, synthesized, and evaluated for hypolipidaemic activity. Among the compounds, 30b lowered the triglycerides by 48.5% (P<0.05) and total cholesterol by 44.2% (P<0.05), respectively, and was more effective than the reference drug fenofibric acid in a Triton WR-1339-induced hyperlipidaemic mice model orally (300mg/kg body weight). 30b also showed 59.4% triglycerides lowering in an alloxan-induced diabetic mice model orally (150mg/kg body weight). Receptor docking studies revealed that compound 30b could interact with the amino acid residues in the ligand-binding domain essential for the activation of the PPARα. The results indicate that resveratrol should be a better scaffold to derive a new class of hypolipidaemic agents in comparison with a flavonoid scaffold.

Full text of DOI:10.3109/14756366.2011.589840

Journal of Enzyme Inhibition and Medicinal Chemistry, 2012; 27(2): 311–318
© 2012 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.589840
SHORT COMMUNICATION
novel phenoxyalkylcarboxylic acid derivatives as
hypolipidaemic agents
Wei Li1,2, Hao-yan Jia1, Xin-hua He1, Wei-guo Shi1, and Bo-hua Zhong1
1Department of Pharmaceutical Chemistry, Beijing Institute of Pharmacology and Toxicology, Beijing, P.R. China and
2Present address: CAS Key Laboratory of Pathogenic Microbiology and Immunology (CASPMI), Institute of Microbiology,
Chinese Academy of Sciences, Beijing, P.R. China
Abstract
Novel phenoxyalkylcarboxylic acid derivatives based on the natural scaffolds, flavonoids, or resveratrol were designed,
synthesized, and evaluated for hypolipidaemic activity. Among the compounds, 30b lowered the triglycerides by
48.5% (P <0.05) and total cholesterol by 44.2% (P <0.05), respectively, and was more effective than the reference
drug fenofibric acid in a Triton WR-1339-induced hyperlipidaemic mice model orally (300mg/kg body weight). 30b
also showed 59.4% triglycerides lowering in an alloxan-induced diabetic mice model orally (150 mg/kg body weight).
Receptor docking studies revealed that compound 30b could interact with the amino acid residues in the ligand-
binding domain essential for the activation of the PPARα. The results indicate that resveratrol should be a better
scaffold to derive a new class of hypolipidaemic agents in comparison with a flavonoid scaffold.
Keywords: Scaffolds, flavonoids, resveratrol, hypolipidaemic, PPARα
Introduction
rhabdomyolysis, idiosyncratic destruction of muscle tis-
sue, leading to renal failure.10–12 Elevated serum transam-
inase concentrations may occur with fenofibrate.13
Fenofibrate retains some hepatotoxicity, especially in the
rodents.14 Asaconsequence,novelmolecularscaffoldsare
expected to provide a superior clinical profile compared
with that of existing fibrates for therapeutic intervention
in dyslipidaemia and other metabolic disorders.15
Flavonoids are naturally occurring phenolic com-
pounds that are widely distributed in plants fulfilling
many functions, such as antioxidant, antiallergic, anti-in-
flammatory, antimicrobial, and anticancer activities.16–18
Consumers and food manufacturers have become inter-
ested in flavonoids for their medicinal properties, espe-
cially their potential role in the prevention of cancers
and cardiovascular diseases.19 ey are relatively of low
toxicity compared with other active plant compounds
(for instance, alkaloids). e chemical structure of fla-
vonoids are based on a C15 skeleton with a chromone
Hyperlipidaemia is the major risk factor for coro-
nary heart disease (CHD), ischaemic cerebrovas-
cular diseases, and peripheral vascular diseases.1–3
Phenoxyalkylcarboxylic acid derivatives (fibrates), such
as
fenofibrate
(2-[4-(4-chlorobenzoyl)phenoxy]-2-
methylpropanoic acid isopropyl ester, 1a), gemfibrozil
(5-(2,5-dimethylphenoxy)-2,2-dimethyl-pentanoic acid,
2) (Figure 1), have been widely used in the clinic as an
important class of lipid-modifying agents by lowering
serum triglycerides and raising HDL cholesterol (HDLc)
and remain the first choice for the treatment of severe
hypertriglyceridaemia.4–6 e effects of fibrates are attrib-
uted to the activation of the peroxisome proliferator-ac-
tivated receptor alpha (PPARα), which is a known target
for the treatment of dyslipidaemia.7–9 However, most
fibrates can cause myopathy (muscle pain with creatine
phosphokinase elevations). Moreover, in combination
with statin drugs, fibrates can cause an increased risk of
Address for Correspondence: Bo-hua Zhong, Department of Pharmaceutical Chemistry, Beijing Institute of Pharmacology and Toxicology,
27 Tai-Ping Road, Haidian District, Beijing 100850, P.R. China. Tel/Fax: +86 10 66931639. E-mail: bohuazhong@yahoo.com
(Received 17 February 2011; revised 16 May 2011; accepted 16 May 2011)
311

312 W. Li et al.
ring bearing a second aromatic ring B in position 2 or
3 (Figure 2). Various subgroups of flavonoids including
flavones, isoflavonoids, chalcones, and flavanones are
classified according to the substitution patterns of ring C
(Figure 1).
ese beneficial effects of flavonoids and resveratrol
and their diverse chemical structures have attracted
much attention, and they may provide novel scaffolds as
therapeutic drugs. In this article, novel hypolipidaemic
agents based on the flavonoid and resveratrol scaffolds
were studied. e structure–activity relationship (SAR)
on the fibrates shows that they consist of an acidic head
group and a large lipophilic backbone.36 e classical
acidic head group could be 2,2-dimethyloxyvaleric acid
or oxyisobutyric acid.37 Here, we described the design
and synthesis of novel hypolipidaemic agents by the
combination of the fibrate head groups and the flavonoid
or resveratrol scaffolds, which probably would provide
synergistic pharmacological effects. e flavone, isofla-
vonoid, chalcone, flavanone, and resveratrol backbones
were selected as the scaffolds in this study.
e stilbene-based components, or stilbenoids, have
been suggested to have many health benefits including
antioxidative, anti-inflammatory, antileukemic, antibac-
terial, antifungal, antiplatelet aggregation, vasodilator,
and antitumour activities.20–27 Resveratrol (trans-3,5,4′-
trihydroxystilbene), the most widely studied stilbene,
is a phytoalexin produced naturally by several plants
when attacked by pathogens such as bacteria or fungi.
Resveratrol is found in the skin of red grapes and is a con-
stituent of red wine, and some scientists believe it is one
of the factors behind the French Paradox.28,29 In mouse
and rat experiments, anticancer, anti-inflammatory,
blood sugar-lowering and other beneficial cardiovascu-
lar effects of resveratrol have been reported.30 Resveratrol
protects our heart and blood vessels by directly scav-
enging oxidants, which could cause oxidation of lipids,
and by preventing oxidative stress-induced apoptosis
of endothelial cells.31,32 It may also help to prevent heart
damage after a cardiac arrest. Reduced platelet aggrega-
tion by resveratrol can reduce the risk of atherosclerosis.33
Resveratrol has also been demonstrated to reduce blood
lipid levels in animals.34 Some naturally occurring or syn-
thetic resveratrol analogues have shown to lower plasma
lipid levels when fed to hamsters or to significantly
activate PPARα.35
Materials and methods
Chemistry
All common laboratory chemicals were purchased from
commercial sources and used without further purifica-
tion. Melting points (uncorrected) were obtained by
using an XT4 MP apparatus (Taike Corp., Beijing, China).
1H NMR and 13C NMR were measured on a JNM-ECA-400
NMR spectrometer (at 400 MHz for 1H and 100 MHz for
13C) using TMS as an internal standard. Coupling con-
stants (J) are given in hertz. Electrospray mass spectra
(ESI-MS) were recorded on an API3000 spectrometer.
in-layer chromatography (TLC) was carried out on
Figure 1. Synthetic triglyceride-lowering drug (fenofibrate, fenofibric acid, and gemfibrozil); naturally occurring flavone (chrysin),
isoflavonoids (daidzein, formononetin, genistein), chalcone (isoliquirtigenin), flavanone (naringenin), and resveratrol.
Journal of Enzyme Inhibition and Medicinal Chemistry

Novel phenoxyalkylcarboxylic acid derivatives as hypolipidaemic agents 313
(10 g, 39.4 mmol) with anhydrous K2CO3 (4.35 g, 31.5
mmol) in anhydrous acetone (250 mL), and the solu-
tion was stirred and refluxed for 12 h. e reaction mix-
ture was filtered and the filtrate was concentrated. e
residue was purified by silica gel chromatography (PE/
EtOAc 85:15) to afford 14a (3.2 g, 27.4% yield) as a white
solid. 1H NMR (400 MHz, DMSO-d6) δ 3.81 (3H, s, OCH3),
5.27 (2H, s, OCH2O), 6.80–6.84 (3H, m, Ar-H), 6.95 (1H,
dd, J= 9.0, 2.2 Hz, Ar-H), 7.36 (2H, d, J= 8.4 Hz, Ar-H), 7.98
Figure 2. Chemical structures of flavonoids.
(1H, d, J= 8.6 Hz, Ar-H), 8.32 (1H, s, Ar-H), 9.50 (1H, s,
4′-OH); C17H14O5, MW calcd. 298.29; ESI-MS (MeOH): 299
[M+H]+.
silica gel plates with a fluorescence indicator of F254
(0.2 mm, Qingdao Haiyang), and the spots were visual-
ized in UV light. TOF-HRMS spectra were determined on
an Agilent 1100 instrument.
4′,5-Dihydroxy-7-methoxymethoxyisoflavone (14b)
e titled compound was prepared from 4′,5,7-
trihydroxyisoflavone and ethyl 2-bromo-2-methylpro-
panate according to the method of compound 14a with
the following yield: 44.8 %; yellow powder; 1H NMR
(400 MHz, DMSO-d6) δ 3.78 (3H, s, OCH3), 5.23 (2H, s,
OCH2O), 6.21 (1H, d, J = 2 Hz, Ar-H), 6.39 (1H, d, J = 2
Hz, Ar-H), 6.85 (2H, m, Ar-H), 7.49 (2H, m, Ar-H), 8.41
(1H, s, Ar-H), 9.54 (1H, s, 4′-OH), 12.89 (1H, s, 5-OH);
C17H14O6, MW calcd. 314.29; ESI-MS (MeOH): 315
[M+H]+.
e following illustrated the synthesis and character-
istics of three representative target compounds (11, 17a,
17b), and the details of the synthesis of the other com-
pounds, compound characterization data are available
in the Supporting Materials.
5-Hydroxy-7-(1-methyl-1-ethoxycarboxylethoxy)flavone (10)
Ethyl 2-bromo-2-methylpropanate (2.21 mL, 15 mmol)
was added to a solution of 5,7-dihydroxyflavone (1.90 g,
7.5 mmol) with anhydrous K2CO3 (3.14 g, 22.5 mmol) and
KI (0.25 g, 1 mmol) in anhydrous dimethylformamide
(DMF) (40 mL), and stirring was continued for 12 h at
120°C. e reaction mixture was cooled and filtered. e
filtered solvent was removed under reduced pressure and
the residue was purified by silica gel chromatography
(PE/EtOAc 85:15) to afford 10 (2.07 g, 75% yield) as a yel-
low solid. Mp. 231–233°C; 1H NMR (DMSO-d6) δ1.18 (3H,
m, CH3), 1.66 (6H, m, 2CH3), 4.22 (2H, m, CH2), 6.14 (1H,
d, J= 2 Hz, Ar-H), 6.55 (1H, d, J= 2 Hz, Ar-H), 7.04 (1H, s,
Ar-H), 7.60 (3H, m, Ar-H), 8.10 (2H, m, Ar-H), 12.73 (1H,
br.s, 5-OH); C21H20O6, MW calcd. 368.38; ESI-MS (MeOH):
369 [M+H]+.
4′-(1-Methyl-1-ethoxycarboxylethoxy)-7-
methoxymethoxyisoflavone (15a)
e titled compound was prepared from 14a and ethyl
2-bromo-2-methylpropanate according to the method
of compound 10 with the following yield: 60%; white
solid; 1H NMR (400 MHz, DMSO-d6) δ 1.16 (3H, m,
CH3), 1.64 (6H, s, 2CH3), 3.83 (3H, s, OCH3), 4.25 (2H,
m, CH2), 5.33 (2H, s, OCH2O), 6.81–6.84 (3H, m, Ar-H),
6.97 (1H, dd, J = 9.0, 2.2 Hz, Ar-H), 7.37 (2H, d, J = 8.4
Hz, Ar-H), 8.02 (1H, d, J = 8.6 Hz, Ar-H), 8.34 (1H, s,
Ar-H); C23H24O7, MW calcd. 412.43; ESI-MS (MeOH):
413 [M+H]+.
5-Hydroxy-7-(1-methyl-1-carboxylethoxy) flavone (11)
K2CO3 (0.41 g, 3.00 mmol) was added to a solution of 10
(0.37 g, 1.00 mmol) in MeOH/H2O (1:1, 20 mL) and the
solution was stirred and refluxed for 12 h. e mixture
was concentrated under reduced pressure. e residue
was poured into ice water (10 mL), acidified with a 3N
HCl aqueous solution to pH 3–4 and extracted with EtOAc
(3 × 20 mL). e organic extracts were washed with brine,
dried over Na2SO4, and filtered. e solvent was removed
under reduced pressure and the residue was purified by
silica gel chromatography (PE/EtOAc 7:3) to afford 11
4′-(1-Methyl-1-ethoxycarboxylethoxy)-5-hydroxy-7-
methoxymethoxyisoflavone (15b)
e titled compound was prepared from 14b and ethyl
2-bromo-2-methylpropanate according to the method
of compound 10 with the following yield: 50.3%; yellow
1
powder; H NMR (400 MHz, DMSO-d6) δ 1.17 (3H, m,
CH3), 1.63 (6H, s, 2CH3), 4.24 (2H, m, CH2), 3.78 (3H, s,
OCH3), 5.24 (2H, s, OCH2O), 6.22 (1H, d, J= 2 Hz, Ar-H),
6.40 (1H, d, J= 2 Hz, Ar-H), 6.84 (2H, m, Ar-H), 7.45 (2H,
m, Ar-H), 8.42 (1H, s, Ar-H), 12.90 (1H, s, 5-OH); C23H24O8,
MW calcd. 428.43; ESI-MS (MeOH): 429 [M+H]+.
1
(0.22 g, 65% yield) as a white solid. Mp. 264–266°C; H
NMR (400 MHz, DMSO-d6) δ 1.65(6H, m, 2CH3), 6.17(1H,
d, J= 2 Hz, Ar-H), 6.51 (1H, d, J= 2 Hz, Ar-H), 7.02 (1H, s,
Ar-H), 7.57–7.63 (3H, m, Ar-H), 8.09 (2H, m, Ar-H), 12.71
(1H, br.s, 5-OH), 13.21 (1H, br.s, COOH); C19H16O6, MW
calcd. 340.33; ESI-MS (MeOH): 341[M+H]+; TOF-HRMS
m/z= 341.1023 [M+H]+ (C19H17O6 requires 341.1025).
4′-(1-Methyl-1-ethoxycarboxylethoxy)-7-hydroxyisoflavone
(16a)
Six millilitres of 10% aqueous HCl was added to a solu-
tion of 15a (1 g, 2.4 mmol) in MeOH (30mL) and the mix-
ture was stirred and refluxed for 30 min. e solution was
poured onto water (40 mL), filtered, washed with water
(100 mL), and dried in vacuum to give 16a as a white solid
(0.75 g, 85% yield) without further purification.
4′-Hydroxy-7-methoxymethoxyisoflavone (14a)
Chloromethyl methyl ether (3.17 mL, 39.4 mmol) was
added dropwise to a solution of 4′,7-dihydroxyisoflavone
© 2012 Informa UK, Ltd.

314 W. Li et al.
diabetic plus drug-treated groups containing 10 mice in
each. Mice of experimental groups were administered
suspension of the desired test samples orally (made in
0.5% CMC-Na) at 150 mg/kg dose daily from Day 1 to 3.
Animals of control group were given an equal amount
of 0.5% CMC-Na. On the second day, after 30 min of
administration of overnight fasted mice, alloxan was
administered (110 mg/kg body weight) by injecting via
tail vein. e blood was collected on Day 4, 48 h after
alloxan administration and serum was separated by
centrifugation at low speed and assayed for triglycerides
and glucose. e analysis of serum biochemical param-
eters was performed on a Semi-automatic Vital Microlab
300 Biochemistry Analyzer by standard enzymatic
methods. e assay kits were purchased from BioSino
Biotechnology and Science Inc.. Differences between
two groups were analysed using unpaired Student’s t-test
and one-way ANOVA for multiple comparisons. P< 0.05
was considered significant.
4′-(1-Methyl-1-ethoxycarboxylethoxy)-5,7-
dihydroxyisoflavone (16b)
e titled compound was prepared from 15b according
to the method of compound 16a with 90% yield as a yel-
low powder without further purification.
4′-(1-Methyl-1-carboxylethoxy)-7-hydroxyisoflavone (17a)
e titled compound was prepared from 16a according
to the method of compound 11 with the following yield:
1
72 %; white solid; Mp. 246–248°C; H NMR (400 MHz,
DMSO-d6) δ 1.54 (6H, m, 2CH3), 6.85–6.88 (3H, m, Ar-H),
6.94 (1H, dd, J= 8.8, 2.4 Hz, Ar-H), 7.48 (2H, m, Ar-H),
7.97 (1H, d, J= 8.8 Hz, Ar-H), 8.35 (1H, s, Ar-H), 10.80 (1H,
s, 7-OH), 13.02 (1H, br.s, COOH); C19H16O6, MW calcd.
340.33; ESI-MS (MeOH): 341 [M+H]+, 363 [M+Na]+; TOF-
HRMS m/z= 363.0843 [M+Na]+ (C19H16NaO6 requires
363.0845).
4′-(1-Methyl-1-carboxylethoxy)-5,7-dihydroxy
isoflavone (17b)
Molecular modelling
e titled compound was prepared from 16b according
to the method of compound 11 with the following yield:
72%; yellow powder; Mp. 222–224°C; 1H NMR (400 MHz,
DMSO-d6) δ 1.54 (6H, m, 2CH3), 6.23 (1H, d, J= 2 Hz,
Ar-H), 6.40 (1H, d, J= 2 Hz, Ar-H), 6.87 (2H, m, Ar-H), 7.46
(2H, m, Ar-H), 8.39 (1H, s, Ar-H), 10.94 (1H, s, 7-OH),
12.91 (1H, s, 5-OH), 13.14 (1H, s, COOH); C19H16O7, MW
calcd. 356.33; ESI-MS (MeOH):357 [M+H]+, 379 [M+Na]+;
TOF-HRMS m/z= 379.0792 [M+Na]+ (C19H16NaO7 requires
379.0794).
ecrystalstructureofPPARαincomplexwithGW409544
(1K7L) recovered from the Brookhaven Protein Data
Bank was used as the target for molecular docking. e
docking calculations of compounds with PPARα were
performed with the AutoDock Vina1.0 program (Oleg
Trott et al., e Scripps Research Institute, La Jolla, CA).
ese images were generated using the Discovery Studio
2.5 Visualizer program (Accelrys Software Inc.).
Results and discussion
Biology
Chemistry
In vivo hypolipidaemic activity evaluation in Triton WR-1339-
induced hyperlipidaemic mice
e flavone and isoflavonoid derivatives were synthe-
sized starting with the natural scaffolds, chrysin (5,7-di-
hydroxyflavone, 3), daidzein (4′,7-dihydroxyisoflavone,
4), formononetin (7-hydroxy-4′-methoxyisoflavone, 5),
genistein (4′,5,7-trihydroxyisoflavone, 6), one common
natural flavone and three common natural isoflavonoids.
e target compounds were prepared by the alkylation of
the phenols with ethyl 2-bromo-2-methylpropanate, and
then hydrolysis of the esters as shown in Schemes 1 and
2 and S1 (in Supplementary Materials). As the 7-hydroxyl
group is more reactive than 4′-hydroxyl group, the latter
could be selectively alkylated following MOM protection
of the 7-hydroxyl group. MOM deprotection and then
hydrolysis of the esters provided the isoflavonoid deriva-
tives 17a and 17b.
e method for the synthesis of chalcone derivatives
is shown in S2 (in Supplementary Materials). After aldol
condensation and MOM deprotection, the chalcone
scaffolds were obtained and the phenols were alky-
lated with ethyl 2-bromo-2-methylpropanate. Alkaline
hydrolysis of the esters gave the target compounds 23a
and 23b.
Male Kunming mice (18–22 g) bred in the animal house
of the institute were divided into Triton, Triton plus drug-
treated groups containing 10 mice in each. Overnight
fasted mice of experimental groups were administered
suspension of the desired test samples (made in 0.5%
CMC-Na) at 300 mg/kg (i.g.) or 100 mg/kg (i.p.) dose.
After 30 min, Triton WR-1339 (Sigma, St. Louis, MO)
was administered (400 mg/kg body weight) by injecting
via tail vein. Blood was withdrawn 22 h later from retro-
orbital plexus and serum was separated by centrifugation
at low speed and assayed for total cholesterol and trig-
lycerides. e analysis of serum biochemical parameters
was performed on a Semi-automatic Vital Microlab 300
Biochemistry Analyzer by standard enzymatic methods.
eassaykitswerepurchasedfromBioSinoBiotechnology
and Science Inc., Changping, China. Differences between
two groups were analysed using unpaired Student’s t-test
and one-way ANOVA for multiple comparisons. P< 0.05
was considered significant.
In vivo hypolipidaemic activity evaluation in alloxan-induced
diabetic mice
Male Kunming mice (18–22 g) bred in the animal house
of the institute were divided into diabetic control,
Cyclization of 21b by treatment with sodium acetate
and then MOM deprotection gave the flavanone scaffold.
e subsequent alkylation and hydrolysis afforded the
Journal of Enzyme Inhibition and Medicinal Chemistry

Novel phenoxyalkylcarboxylic acid derivatives as hypolipidaemic agents 315
target compound 26 as shown in S3 (in Supplementary
Materials).
In previous studies, simple groups were introduced at
reference drug fenofibric acid (2-[4-(4-chlorobenzoyl)
phenoxy]-2-methylpropanoic acid, 1b), the active
metabolite of fenofibrate.
4′ position and the methoxy groups were kept at the 3 and
5 positions of resveratrol.38 In our target compounds, the
classical fibrate head groups were introduced at the 3, 5,
and/or 4′ positions of resveratrol by the alkylation of the
phenols with either ethyl 2-bromo-2-methylpropanate or
isobutyl 5-chloro-2,2-dimethylvalerate, and the remain-
ing phenolic hydroxyl groups could be methylated by
iodomethane or not. e compounds with two or three
fibrate head groups were also obtained by the one-pot
synthesis method in order to better understand the SAR.
Furthermore, as the reactivity of the 4′-hydroxyl group of
resveratrol was comparable with the 3-hydroxyl group,
the resveratrol-4′-O- and 3-O-monosubstitutes were
synthesized by the one-pot synthetic approach. e syn-
thesis routes are shown in S4 and S5 (in Supplementary
Materials). All the target compounds above were con-
firmed by spectroscopy. Like resveratrol, the trans con-
figuration of the target compounds were confirmed as
the relatively large coupling constant (~16 Hz) between
H-7 and H-8.
e in vivo hypolipidaemic activity of resveratrol
derivatives in a Triton WR-1339-induced hyperlipidae-
mic mice model is shown in Table 2. e synthesized
compounds, employing the 2,2-dimethyloxyvaleric
acid group, or the oxyisobutyric acid group, showed
more potent than the parent compound, resveratrol (9)
by intraperitoneal injection (100 mg/kg body weight)
or orally (300 mg/kg body weight). e reference drug
fenofibric acid was the most potent followed by 30b at
the dose of 100 mg/kg when administration by intrap-
eritoneal injection. But at the dose of 300 mg/kg orally,
30b was identified as the most potent and lowered the
triglycerides by 48.5% (P < 0.05), total cholesterol by
44.2% (P < 0.05), whereas fenofibric acid lowered the
triglycerides by 38.2% (P < 0.05), total cholesterol by
30.8% (P < 0.05). Moreover, interestingly, methylation of
the remaining phenolic hydroxyl groups led to increased
activities: 30a was more effective than 28a, and 34 was
more effective than 28c, and 33b was more effective than
28g. e compounds, employing the 2,2-dimethyloxy-
valeric acid group, showed more potent than the com-
pounds employing the oxyisobutyric acid group at the
corresponding positions of the compounds (for instance,
28e > 28b, 28g > 28c). Furthermore, 34 exhibited more
effective than 30a suggesting the substitution position of
the fibrate head group could influence the activity. 28b
bearing two oxyisobutyric acid moieties showed more
potent than the compounds 28a (i.g., 300 mg/kg) and
28c (i.v., 100 mg/kg) which have only one oxyisobutyric
acid moiety in their molecules.
Biological activity
e ability of the compounds to affect lipid/cholesterol
homeostasis was first examined on Triton WR-1339-
induced hyperlipidaemia in mice. Table 1 shows the activ-
ity of flavonoid derivatives at the dose of 300 mg/kg body
weight orally. Out of these eight derivatives, isoflavonoid
derivative 17a and chalcone derivative 23a were the two
most potent compounds. 17a lowered the triglycerides
by 20.2% (P< 0.05), total cholesterol by 16.6% (P< 0.05),
and 23a lowered 19.8% (P< 0.05) in triglycerides, 16.3%
(P< 0.05) in total cholesterol quantity. Moreover, the iso-
flavonoid derivatives 17a and 17b showed more potent
than their parent compounds daidzein and genistein,
respectively. ese results suggested that the enhanced
hypolipidaemic activities of 17a and 17b can be attrib-
uted to the presence of the fibrate head moieties and the
modification can lead to increased activity. But none of
the synthesized compounds was more potent than the
Compounds 28e, 28f, 30a, and 30b were further tested
for their hypolipidaemic activity in an alloxan-induced
diabetic mice model at a dose of 150 mg/kg body weight
orally. In this experiment, the model mice exhibited
hypertriglyceridaemia and hyperglycaemic. ese com-
pounds showed good lowering in triglycerides, and 30b
was the most potent among them. 30b lowered 59.4%
(P< 0.01) in triglycerides, which was slightly more active
than fenofibric acid (Table 3). In addition, 28e (53.5%),
Table 1. In vivo hypolipidaemic activity of flavonoid derivatives in Triton WR-1339-induced hyperlipidaemic mice.
Compound
Dose (mg/kg)
TG (mM)
15.0 2.0
14.4 2.6
14.2 3.5
13.2 2.8*
13.6 2.1*
13.2 2.8*
13.3 2.6*
15.4 1.9
16.0 2.6
16.1 1.7
10.9 2.2*
16.5 1.2
% Reduction, TG
TC (mM)
16.4 0.9
17.0 2.3
16.2 3.2
15.8 1.1*
15.6 1.3*
15.7 1.2*
16.6 1.4
17.8 1.5
18.1 0.4
18.6 0.3
14.7 2.0*
18.8 2.0
% Reduction, TC
11
12
13
17a
300
300
300
300
300
300
300
300
300
300
300
—
8.8
12.3
13.7
20.2*
17.6*
19.8*
19.0*
6.2
12.3
9.4
13.4
15.6*
16.6*
16.3*
11.7
4.9
17b
23a
23b
26
4
6
1b
3.0
3.7
2.4
1.1
33.7*
—
21.6*
—
Triton model
*P<0.05, compared with model group.
© 2012 Informa UK, Ltd.

316 W. Li et al.
30a (52.6%), and fenofibric acid (54.1%) showed similar
triglyceride-lowering activity. Like fenofibric acid, none
of the compounds exhibited any blood glucose-lowering
activity in this model.
OH. In in vivo studies, 30b showed good hypolipidae-
mic activity, so it can be predicted that the activity may
be due to the activation of the PPARα. However, unlike
compound 30b, docking of fenofibric acid, the marketed
activator of PPARα, showed the two phenyl rings were
docked into the hydrophobic pocket formed by the heli-
ces 3, 6, and 10 adjacent to the AF-2 helix, also known
as “benzophenone” pocket (Figure 3B). ese docking
models revealed that 30b may bind to the receptor in a
different mode from fenofibric acid.
Docking studies
Molecular modelling (docking) studies were carried out
to gain an insight into the likely binding interactions
between the most potent compound 30b and PPARα.
Our proposed binding pose of 30b superimposed on
the co-crystal structure of GW409544 inside the PPARα
ligand-binding domain (PDB:1K7L) is illustrated in
Figure 3A. GW409544 is a full PPARα and PPARγ ago-
nist.39 Docking studies revealed that one of the carboxy-
late oxygens of 30b could form a bifurcated H-bond with
the Tyr464 OH and the Tyr314 OH groups of the PPARα.
e other carboxylate oxygen of 30b was at H-bonding
distance from Ser280 OH. Ser280, Tyr314, Tyr464 are
known to be the key interaction residues for the acti-
vation of the PPARα.39 e gem-dimethyl substituents
of 30b were directed into a lipophilic pocket bounded
by Phe273, Gln277, Val444, and Leu456, a region at the
top end of the so-called “benzophenone” pocket. e
remaining two phenyl rings were docked into the hydro-
phobic pocket formed by the helices 2′, 3, and β sheet,
and one methoxy group at the 3 or 5 position of 30b
could produce a hydrogen bond interaction with Tyr334
Table 2. In vivo hypolipidaemic activity of resveratrol derivatives
in Triton WR-1339-induced hyperlipidaemic mice.
i.g., 300mg/kg
i.p., 100mg/kg
%
%
%
Reduction, Reduction, % Reduction, Reduction,
Compound
28a
28b
28c
28e
28f
TG
TC
TG
TC
2.7
ND
ND
21.2*
31.3*
ND
17.8
43.4*
13.9
35.8*
15.5
ND
35.2*
23.6*
23.0*
26.4*
48.5*
21.8*
35.8*
33.9*
38.2*
13.9
21.3*
19.2*
22.9*
22.9*
44.2*
24.5*
25.0*
33.0*
30.8*
8.3
49.7*
26.5*
23.8*
29.1*
50.3*
21.8*
38.4*
35.1*
59.6*
16.1
38.7*
14.3*
17.9*
29.8*
50.0*
21.4*
25.6*
32.1*
46.4*
10.4
28g
30a
30b
33a
33b
34
1b
9
ND: not detected. *P<0.05, compared with model group.
Figure3. (A)Proposedbindingposeof30b(purple)superimposed
on the co-crystal structure of GW409544 (golden) inside PPARα
ligand-binding domain. Blue dotted line represents hydrogen
bonding. Hydrogen atoms are not shown for clarity. e protein
backbone is shown as cyan ribbon. Important residues are shown
in yellow. (B) Proposed binding pose of fenofibric acid (blue)
superimposed on the co-crystal structure of GW409544 (golden)
inside PPARα ligand-binding domain. Blue dotted line represents
hydrogen bonding. Hydrogen atoms are not shown for clarity. e
protein backbone is shown as cyan ribbon. Important residues
are shown in yellow.
Table 3. In vivo hypolipidaemic activity of compounds in
alloxan-induced diabetic mice.
Compound Dose (mg/kg) % Reduction, TG % Reduction, GLU
28e
28f
30a
30b
1b
150
150
150
150
150
14.3
8.1
53.5**
35.9*
8.9
52.6**
59.4**
54.1**
10.2
17.2
*P<0.05, or **P<0.01, compared with model group.
Journal of Enzyme Inhibition and Medicinal Chemistry

Novel phenoxyalkylcarboxylic acid derivatives as hypolipidaemic agents 317
Scheme 1. (a) BrC(CH3)2COOC2H5, K2CO3, DMF and (b) (i) K2CO3, MeOH/H2O, reflux, (ii) aqueous HCl.
Scheme 2. (a) MOMCl, acetone, K2CO3, reflux; (b) BrC(CH3)2COOC2H5, K2CO3, DMF; (c) 10% HCl/MeOH, reflux, 0.5h; and (d) (i) K2CO3,
MeOH/H2O, reflux, (ii) aqueous HCl.
Conclusions
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