Selective peroxisome proliferator-activated receptor δ isosteric selenium agonists as potent anti-atherogenic agents in vivo

Article abstract of DOI:10.1016/j.bmcl.2010.10.103

We report the synthesis and in vivo activity of a novel anti-atherogenic agent, isosteric selenium PPARδ-selective ligand. This ligand did not cause significant body or liver weight changes and did not have obvious adverse effects on intestinal polyp formation. Our overall results clearly demonstrate that PPARδ is a viable drug candidate for targeting and treating atherosclerosis.

Full text of DOI:10.1016/j.bmcl.2010.10.103

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7239–7242
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Selective peroxisome proliferator-activated receptor d isosteric selenium
agonists as potent anti-atherogenic agents in vivo
Jungwook Chin a, , Jun Young Hong a, , Jaehwan Lee a, , Hoosang Hwang a, Hyunsil Ko a, Hyukjae Choi a,
Dongyup Hahn a, Jaeyoung Ko a, Sang-Jip Nam a, Jungae Tak a, Jungyeob Ham b, Heonjoong Kang a,c,
⇑
a Center for Marine Natural Products and Drug Discovery, School of Earth and Environmental Sciences, Seoul National University, NS-80, Seoul 151-747, Republic of Korea
b Korea Institute of Science and Technology, 290 Daejeon-dong, Gangneung 210-340, Republic of Korea
c Research Institute of Oceanography, SNU 151-741, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the synthesis and in vivo activity of a novel anti-atherogenic agent, isosteric selenium
PPARd-selective ligand. This ligand did not cause significant body or liver weight changes and did not
have obvious adverse effects on intestinal polyp formation. Our overall results clearly demonstrate that
PPARd is a viable drug candidate for targeting and treating atherosclerosis.
Received 18 August 2010
Revised 30 September 2010
Accepted 20 October 2010
Available online 26 October 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
PPARd-selective ligand
Anti-atherogenic agents
Intestinal polyp formation
Atherosclerosis is a disease characterized by enlarged arterial
blood vessels that are induced by the dysfunction of lipid metabo-
lism. This disease progresses via chronic inflammation resulting
from the interaction between lesional lipoproteins and immune
cells.1 The peroxisome proliferator-activated receptors (PPARs)
are transcription factors that are activated by their cognate ligands
and act as master regulators in mammalian physiology and devel-
In contrast, Graham et al. demonstrated the anti-atherogenic
potential of the PPARd agonist in LDLRÀ/À mice.11 However, the
inhibitory effect was only observed with a high dose regimen
(60 mg/kg) or long-term treatment (16 weeks) and was associated
with the adverse effect of increased liver weight. The compound
was reported to have no selectivity for PPARd above 1
the other isotypes.7 Considering the serum concentration of
GW0742 (21 M at 60 mg/kg) in the study by Graham et al., the
anti-atherogenic activity might have been mediated through
lM over
opment.2 Among the three different isoforms (PPAR-
a
, -
c, and -d),
l
PPARd regulates lipid homeostasis and inflammation in the human
body, and the impairment of these processes can lead to athero-
sclerosis.3 Several studies have shown that the activation of PPARd
promotes fatty acid catabolism and reverses cholesterol transport
and represses inflammatory gene expression. These effects suggest
a beneficial role of PPARd-selective agonists in the treatment of
atherosclerosis.4–6
Prior research has shown that the activation of PPARd by
GW0742,7 a PPARd agonist, increases the level of high-density
lipoprotein-cholesterol (HDL-c) in DBA/1 mice.8 Considering the
atheroprotective role of HDL-c,9 it was expected that the PPARd-
selective agonist would have anti-atherogenic potential.
PPAR
a or c activation. In this regard, it still unclear whether
PPARd-selective agonists have anti-atherogenic effects.12
We developed a series of novel selenium-containing PPARd-
selective ligands to resolve this issue (Fig. 1).13 Isosterism is a
useful strategy for molecular modification and a rational approach
in drug design.14 Isosteric analogs possess an equally well-estab-
lished biological potency in protein–receptor interaction.15 Our
O
O
OH
R1
R2 R3
S
However, recent studies have produced incompatible results
regarding this prediction. In a study by Li et al., there was no
obvious inhibitory anti-atherogenic effect of the PPARd agonist,
Se
F3C
N
R1 = H, R2 = H,
R3 = H
2f
2g
2h
2i
R1 = F, R2 = H,
R3 = H
R3 = H
1f
1g
1h
independent of the PPARa and PPARc
agonists, in LDLRÀ/À mice.10
R
2 = CH3,
R1 = H,
R
3 = H
R
1 = F, R2 = CH3,
R3 = H
R3 = CH3
R
1 = H, R2 = CH2CH3,
R1 = F, R2 = CH2CH3, R3 = H
⇑
1i
R
1 = F, R2 = CH3,
R2 = CH3,
R3 = CH3
Corresponding author. Tel.: +82 2 880 5730; fax: +82 2 883 9289.
E-mail address: hjkang@snu.ac.kr (H. Kang).
R1 = H,
These authors contributed equally to the study.
Figure 1. Structures of selenoether-containing PPARd agonists.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.103

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J. Chin et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7239–7242
Table 1
Selectivity of PPARd agonists
research interest therefore lay on the discovery of more potent and
selective analogs of any known PPARd ligand, viz GW501516 or
GW0742. As a proof-of concept, the sulfur–selenium bioisoster-
ism16 was applied to GW501516 and GW0742 series because
molecular modeling study suggested a bulkier element at the sul-
Compd
hPPAR
a
(EC50, nM)
hPPARd (EC50, nM)
hPPARc (EC50, nM)
1f
i.a.a
0.9
11.0
6.2
4.5
1.0
8.5
7.0
1.8
i.a.a
1g
1h
1i
120.0
83.1
15.2
350.3
39.4
i.a.a
i.a.a
i.a.a
fur fit the receptor better. a-Substituted phenylpropanoic acid was
100.2
a versatile template for the design of PPAR subtype-specific ago-
2f
i.a.a
nists.17 Further modification had thus been applied to the
tion of the carboxylic acid region and its alkyl substituent.
a-posi-
2g
2h
2i
i.a.a
270.6
290.3
A series of novel selenoether-containing PPARd ligands were
efficiently prepared via a simple one-pot lithium exchange reac-
tion.18 First, we prepared the key intermediate selenides 1a and
2a (86% and 85% yields, respectively) from 4-iodo-2-methylphenol
via a one-pot reaction. This reaction included the in situ protection
of phenol with isopropylmagnesium chloride, lithium–halogen ex-
change by tert-butyllithium, selenolate formation, and quenching
the resultant selenolates with the corresponding chloromethyl thi-
azole (1 or 2). In the second step, treatment of 1a or 1b with K2CO3
and bromoester in acetone produced ester intermediates 1b–2d
with quantitative yields. In the final step, ester hydrolysis with
2 M LiOH afforded the target compounds 1f–1h and 2f–2h with
greater than 90% yields. Notably, compounds 1i and 2i were syn-
thesized by refluxing procedures (Scheme 1, see Supplementary
data for detailed experimental procedures).
24.6
a
EC50 values are higher than 1 lM.
Compounds 1g and 2g (methyl group at R2) serve as
agonists. Compound 1h (ethyl group at R2) also behaved as an
/d dual agonist. Yet compounds 2h (ethyl group at R2) acts as a
a/d dual
a
c
/d dual agonist. Compounds 1i and 2i (dimethyl side chains at
R2 and R3) were pan-agonists for PPARs (Table 1).
We fed apolipoprotein E-deficient mice (ApoeÀ/À- HC/HF diet
(1.25% cholesterol, 21% fat) for 14 weeks and treated them with
one of the most potent and selective compounds (1f) at 2 mg/kg/
day for the last 4 weeks. The newly developed PPARd ligand atten-
uated atherosclerosis, resulting in an increase in HDL-c and the
suppression of pro-atherogenic molecules. The results, along with
our previous study using the well-known agonist GW501516,19
present clear evidence of the anti-atherogenic potential of PPARd.
The atherosclerosis model mice,20 apoEÀ/À mice, were treated
with 1f to examine its anti-atherogenic potential. Compound 1f
was used at a lower concentration (2 mg/kg) than in previous stud-
ies10,11 (5 mg/kg by Li et al.; 6 and 60 mg/kg by Graham et al.) to
minimize its cross-reactivity toward the other two subtype recep-
tors. The dose regimen also allowed us to directly assess the effect
of the PPARd ligand on atherosclerosis because a higher dose treat-
ment of this compound led to weight loss. The ligand decreased the
extent of lesion fatty streak throughout the aorta by 25% (vehicle:
Remarkably, compounds 1f, 2f, and 2i displayed excellent
potency for PPARd (EC50 = 0.9, 1.0, and 1.8 nM, respectively) in a
cell-based co-transfection assay. Compound 1f exhibited the most
potent and selective agonistic activity for PPARd over the other
subtypes PPARa and c (Table 1 and Fig. 2). Time-resolved fluores-
cence resonance energy transfer (TR-FRET) was conducted to
investigate the direct binding of 1f and PPARd. The dose-dependent
sigmoidal curve showed that 1f facilitated direct interaction be-
tween the fluorescein-conjugated copeptide and hPPARd protein,
indicating the direct binding of 1f to the receptor protein (Fig. 2,
see also Supplementary data).
Scheme 1. Reagents and conditions: (a) isopropylmagnesium chloride, 0 °C, 10 min, anhydrous THF; (b) tert-butyllithium, À78 °C, 0.5 h; (c) selenium power, À78 to 0 °C, 1 h;
(d) compound 1 or 2, 0.5 h; (e) K2CO3, bromoesters; (f) 2 M LiOH, 0.5 M NaHSO4; (g) Cs2CO3, DMF, ethyl 2-bromoisobutyrate; (h) 5 N NaOH, EtOH, reflux (see Supplementary
data for detailed experimental procedures and yields).

J. Chin et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7239–7242
7241
Figure 2. Co-transfection assay was conducted to confirm the selectivity and potency of 1f against mouse PPARs for the following in vivo experiments. The 1f compound
showed more potent agonistic activity to mouse PPARd than GW501516, currently known as the most potent and selective PPARd agonist (EC50 = 2.3 and 5.8 nM for 1f and
GW501516, respectively). The other PPAR subtypes were barely activated by 1f, in contrast to GW501516, which robustly activated these receptors at micro-molar
concentrations.
17.07 2.20: 1f: 12.87 2.32%, p = 0.004, Fig. 3). Plasma HDL-c le-
vel, a key protective factor against atherosclerosis,9 was signifi-
cantly increased by 1f, and this effect accounted for the reduced
formation of lesions (Fig. 4).
Quantitative real-time PCR analysis showed that endothelial
cell adhesion molecules, such as intercellular adhesion molecule-
1 (ICAM-1), were down-regulated by 1f in the aortic lesion. The
expression level of the pro-atherogenic cytokine Interleukin-6
(IL-6) was also greatly reduced by 1f. The results indicate that
the PPARd agonist alleviated the inflammatory response in the le-
sion. However, the expression of genes involving cholesterol
homeostasis remained unchanged by 1f (see supporting Informa-
tion). During experimental periods, ApoeÀ/À mice were weighed
every week. There were no significant changes in body weight be-
tween the treated and the control groups, suggesting that the anti-
atherogenic effect of 1f was not caused by ligand-induced weight
reduction (see Supplementary data).21 In addition, liver weight
was not adversely increased, a finding in contrast to the study done
by Graham et al. (see Supplementary data).11
Several studies have suggested that PPARd has carcinogenic po-
tential in colonic tumorigenesis.22 Many groups have reported
mixed results, indicating that the issue has yet to be resolved.23
Thus, 1f was given to the spontaneous intestinal adenoma model,
adenomatous polyposis coli multiple intestinal neoplasia mice24
(APCMin/+ mice), to assess its carcinogenicity. The results demon-
strated that the ligand did not have any significant detrimental
effect on intestinal polyp formation (Fig. 5). In addition, the plasma
HDL-c level was also significantly increased by 1f in APCMin/+ mice,
which is the mechanism by which PPARd agonists protect mice
from atherosclerosis (see Supplementary data).
Figure 3. Effect of 1f on atherosclerotic lesion progression. Eight-week-old apoEÀ/À
mice were fed an atherogenic diet for 14 weeks and treated with either 1f (n = 7) or
vehicle (n = 6) for the last 4 weeks. The extent of atherosclerotic lesion was
Figure 4. HDL-c-increasing effect of 1f in apoEÀ/À mice. Data are expressed as
**
determined by the en face staining method (see Supplementary data). p <0.005.
*
means SEMs. p <0.05.

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J. Chin et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7239–7242
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Transport and Maritime Affairs (MLTM) and in part the BK21 pro-
gram from Ministry of Education, Science and Technology (MEST),
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.10.103.
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