Design, synthesis, and evaluation of a new class of noncyclic 1,3-dicarbonyl compounds as PPARα selective activators

Article abstract of DOI:10.1016/j.bmcl.2004.04.053

Lipid accumulation in nonadipose tissues is increasingly linked to the development of type 2 diabetes in obese individuals. We report here the design, synthesis, and evaluation of a series of novel PPARα selective activators containing 1,3-dicarbonyl moie

Full text of DOI:10.1016/j.bmcl.2004.04.053

Bioorganic & Medicinal Chemistry Letters 14 (2004) 3507–3511
Design, synthesis, and evaluation of a new class of noncyclic
1,3-dicarbonyl compounds as PPARa selective activators
Zhibin Li,a, Chenzhong Liao,a,b, Ben C. B. Ko,c,d Song Shan,a Edith H. Y. Tong,c,d
Zihui Yin,a Desi Pan,a Vincent K. W. Wong,c Leming Shi,a Zhi-Qiang Ning,a
Weiming Hu,a Jiaju Zhou,b Stephen S. M. Chunge and Xian-Ping Lua,*
aChipscreen Biosciences, Ltd, Research Institute of Tsinghua University, Suite C301, PO Box 28, Shenzhen 518057, China
bInstitute of Process Engineering, Chinese Academy of Sciences, PO Box 353, Beijing 100080, China
cDepartment of Chemistry, The University of Hong Kong, Hong Kong SAR, China
dOpen Laboratory of Chemical Biology of the Institute of Molecular Technology for Drug Discovery and Synthesis,
The University of Hong Kong, Hong Kong SAR, China
eInsitute of Molecular Biology, The University of Hong Kong, Hong Kong SAR, China
Received 7 March 2004; revised 19 April 2004; accepted 19 April 2004
Available online
Abstract—Lipid accumulation in nonadipose tissues is increasingly linked to the development of type 2 diabetes in obese individuals.
We report here the design, synthesis, and evaluation of a series of novel PPARa selective activators containing 1,3-dicarbonyl
moieties. Structure–activity relationship studies led to the identification of PPARa selective activators (compounds 10, 14, 17, 18,
and 21) with stronger potency and efficacy to activate PPARa over PPARc and PPARd. Experiments in vivo showed that com-
pounds 10, 14, and 17 had blood glucose lowering effect in diabetic db/db mouse model after two weeks oral dosing. The data
strongly support further testing of these lead compounds in other relevant disease animal models to evaluate their potential thera-
peutic benefits.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
O
Increasing evidence suggests that lipid accumulation in
nonadipose tissues, such as pancreatic islet cells and
skeletal muscle, is causally related to the development of
type 2 diabetes in obese individuals.1 Peroxisome pro-
liferator activated receptor-a (PPARa), PPARc and
PPARd are ligand-activated transcriptional factors that
belong to the nuclear hormone receptor superfamily,
which are essential in controlling lipid, glucose, and
energy homeostasis.2;3 At present, PPARc agonists
rosiglitazone4 (1, Fig. 1) has been successfully prescribed
for patients having type 2 diabetes. The fibrate agents,
such as fenofibrate and Wy-146432 (2), are low affinity
PPARa agonists; fenofibrate and beazfibrate have been
COOH
N
N
S
NH
O
S
N
N
N
O
Cl
1
2
O
O
COOH
NH
O
Br
N
O
O
4
3
Figure 1. Reference compounds.
widely used clinically as antihyperlipidemic drugs. These
drugs increase HDL cholesterol levels and lower LDL
and VLDL cholesterol levels with stronger triglyceride-
lowering effect than statins, the HMG-CoA reductase
inhibitors. Recently published data showed that PPARd
activators could also be used in the treatment of
Keywords: PPARa activators; Type 2 diabetes; Noncyclic 1,3-dicar-
bonyl compounds; Metabolic diseases.
* Corresponding author. Tel.: +86-755-26711889; fax: +86-755-2695-
7291; e-mail: xplu@chipscreen.com
Contributed equally to this work.
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.04.053

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Z. Li et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3507–3511
dyslipidemia.5 Fatty acids, for example, 2-bromo-hexa-
decanoic acid6;7 (3) are known as weak activators of
PPARd. Because of the side effects of PPARc agonists,
such as weight gain and water retention,8 and the
potential beneficial effects of the PPARa and PPARd,9
balanced triple agonists for PPARa=c=d or selective
agonists for PPARa or d are of great interest for drug
discovery efforts worldwide.10
A
C
B
O
O
more
lipophilic
noncyclic
1,3-dicarbonyl
NH
O
N
O
O
Figure 2. Chemical structure of the lead compound JTT-501.
The framework of JTT-501, like most of PPAR activa-
tors, can be divided into three key regions: (A) the acidic
head part, (B) the linker part, and (C) the hydrophobic
tail part (Fig. 2). Thus, chemical modifications of the
acidic head part and the lipophilic part13 might change
their selectivity to PPARa, PPARc, or PPARd recep-
tors.
Here, we describe the design, synthesis, and evaluation
of a new class of PPARa selective activators containing
1,3-dicarbonyl moieties, leading to the identification of
PPARa selective activators as candidate leads for the
potential treatment of metabolic disorders such as dia-
betes, obesity, and hyperlipidemia.
We first modified the acidic head part into a series
of 1,3-dicarbonyl compounds, and the lipophilic part
into 2,2,5,5-tetramethyl-2,3,4,5-tetrahydronaphthalene.
These two modifications generated compounds 5–9 that
have no PPARa and PPARd activities while remaining
as partial PPARc agonists (Table 1). Substitution of the
four methyl groups with hydrogen atoms from this
lipophilic tail formed compounds 10–13 that had
activities to all three PPAR subtypes, suggesting that
2. Result and discussion
JTT-50111 (4), a dual agonist of PPARa=c, is an isox-
azolidine-3,5-dione. Its activity is likely to be mediated
through a malonic amide metabolite that is generated by
hydrolysis of the heterocyclic ring.12 We therefore took
this moiety as template to develop structurally new
human PPARa selective activators.
Table 1. Cell-based in vitro transcription activation of test compounds on hPPARa, hPPARc, and hPPARda
R1
R2
O
O
R3
No.
R1
R2
R3
PPARa EC50 lM
(%maxb)
PPARc EC50 lM
(%maxb)
PPARd EC50 lM
(%maxb)
5
–COOCH3
–COOCH3
–COOH
–COOCH3
–COOH
–COOH
–CONH2
–CONH2
–COOCH3
–COOH
–COOH
–CONH2
–COOCH3
–COOH
–COOH
–CONH2
–COOCH3
–COOH
–COOH
–CONH2
2,2,5,5-Tetramethylcyclohexane
2,2,5,5-Tetramethylcyclohexane
2,2,5,5-Tetramethylcyclohexane
2,2,5,5-Tetramethylcyclohexane
2,2,5,5-Tetramethylcyclohexane
Cyclohexane
iac (–)
ia (–)
0.15 (66)
0.09 (64)
2.05 (9)
0.83 (15)
ia (–)
ia (–)
ia (–)
6
7d
ia (–)
ia (–)
ia (–)
ia (–)
ia (–)
ia (–)
8
–COOCH3
–COOH
9
10
11
12d
13
14
15d
16
17
18
19
20d
21
1
–COOCH3
–COOCH3
–COOH
0.03 (210)
ia (–)
ia (–)
1.5 (55)
0.69 (60)
2.11 (39)
3.43 (22)
0.53 (78)
ia (–)
3.31 (134)
2.45 (100)
4.68 (80)
ia (–)
Cyclohexane
Cyclohexane
–COOCH3
–COOCH3
–COOH
Cyclohexane
Benzene
ia (–)
0.07 (264)
ia (–)
1.27 (144)
ia (–)
Benzene
Benzene
Benzene
–COOCH3
–COOH
0.044 (264)
0.25 (213)
0.17 (215)
0.09 (207)
ia (–)
1.12 (59)
3.02 (8)
3.16 (27)
3.80 (14)
ia (–)
1.05 (125)
2.63 (111)
3.16 (67)
3.09 (56)
ia (–)
–COOCH3
–COOCH3
–COOH
Hexahydropyridine
Hexahydropyridine
Hexahydropyridine
Hexahydropyridine
–COOH
0.14 (210)
7.3 (16)
15.5 (100)
nde
2.88 (14)
0.04 (100)
ia (–)
3.62 (83)
9.3 (42)
1.46 (20)
3.72 (100)
ia
Rosiglitazone
Wy-14643
2-Bro
2
3
nd
0.083 (NA)
4
JTT-501f
1.9 (NA)
a Data represent the mean values from at least three independent experiments each in triplicate.
b %max responses were calculated as Wy-14643 at 5 lM as 100% for PPARa; Rosiglitazone at 1 lM as 100% for PPARc; and 2-bromohexadecanoic
acid at 5 lM as 100% for PPARd.
c ia ¼ inactive.
d Compounds 7, 12, 15, and 20 are chemically instable and decomposed to CO2 and monoacid at different rates when activity measured in vitro.
e nd ¼ not determined.
f The EC50 originated from a same type of cell-based transcription activation assay reported for JTT-501 was adapted from reference 3; NA: not
available.

Z. Li et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3507–3511
3509
Table 2. In vivo anti-diabetes activity of tested compounds in db/db
mice
the 2,2,5,5-tetramethyl-2,3,4,5-tetrahydronaphthalene
group might be too big to interact with PPARa and d
favorably. Changing the lipophilic part to a planar
group (naphthalene), such as compounds 14–17, led to
stabilization of the interaction between the compounds
and PPARa as evidenced by strong transcription acti-
vation of PPARa, although the substitutions were made
in the acidic head part of the dimethyl malonate group.
The substitution of the dimethyl malonate group in
compound 14 with 2-methoxycarbonyl propionic acid
(compound 16) resulted in weakening of PPARc agonist
activity while marginally enhancing PPARa and d
activities; the substitution in compound 14 with 2-carb-
amoyl propionic acid (compound 17) caused significant
decrease in PPARc activity, as indicated in Table 1,
where the maximal response of the PPARc activity to
the compound was only 8% of that of rosiglitazone, but
the activation of PPARa by the compound remained
strong. Replacement of a carbon atom with a nitrogen
atom in the 2,3,4,5-tetrahydronaphthalene ring in com-
pounds 18–21 further decreased their ability to activate
PPARc and d, while relatively strong PPARa activation
by these compounds still remained. The activity profiles
in Table 1 indicate that the compounds 10 and 19 are
potent activators with good selectivity for PPARa while
the compounds 17, 18, and 21 also exhibit good selec-
tivity for PPARa since the efficacies for PPARc were
significantly decreased; the compounds 14 and 16 show
only modest selectivity for PPARa.
Compound
Dosage (mg/kg)
Reduction in BG (%) after
two weeks of oral dosinga
Vehicle
10
0
0
30
10
30
20
5
38.15
35.08
46.76
42.22
62.96
14
14
17
Rosiglitazone
a Data represent the mean of the percent reduction in blood glucose
(BG) level measured using blood samples from mice (N ¼ 4 to 6
mice).
lowering effect of compound 17 is interesting since it is
almost inactive for PPARc stimulation, suggesting that
the glucose-lowering effect may indirectly result from its
lipid modulation. It should be noted that the com-
pounds 10 and 14 are pro-drug forms that are converted
to the acid form quickly in vivo. It is speculated that
these two compounds may have short half-lives that
could generate a favorable side-effect profile. Efforts are
currently underway to test the efficacy of these com-
pounds in other animal models of type 2 diabetes and
dyslipidemia.
In conclusion, the in vitro and in vivo results show that
by modifying the acidic head part and the lipophilic part
of the template compound JTT-501, we obtained
PPARa selective activators that are potentially useful
for the development of drugs against type 2 diabetic or
other metabolic diseases.
AutoDock14–17 was used to dock compound 17
into PPARa co-crystallized with AZ 242,18 PPARc co-
crystallized with rosiglitazone,19 and PPARd co-crys-
tallized with GW2433,20 respectively. Figure 3 shows
that compound 17 docks into the active sites with the
carboxylic acid in the same position as the co-crystal-
lized ligands in PPARa and PPARd, but not in PPARc.
The calculated pKi by AutoDock were 8.14, 5.89 and
7.06 to PPARa, PPARc, and PPARd. This data is in
good agreement with the in vitro activities measured.
3. Experimental Section
3.1. Chemistry
Compounds 5–21 were synthesized as outlined in
Scheme 1. The commercially available phenols upon
reaction with p-bromoethoxy benzaldehyde gave benz-
aldehyde derivatives a. Knoevenagel condensation be-
tween the aldehydes a and dimethyl malonate gave the
benzylidene b. Catalytic hydrogenation of b with 5%
palladium on carbon gave the dimethyl malonate c.
Partial hydrolysis of c with 1 equiv of sodium hydroxide
gave the half-ester d. The Schotten–Baumann reaction
Compounds 10, 14, and 17 were tested in preliminary
experiments using diabetic db/db mouse model. As
shown in Table 2, all three compounds were able to
lower the increased blood glucose levels as compared to
the vehicle treatment after 12-days oral dosing, but they
were less effective than rosiglitazone. The blood glucose-
Figure 3. From left panel to right panel: compound 17 (purple) docked into the PPARa receptor co-crystallized with AZ 242 (green), the PPARc
receptor co-crystallized with rosiglitazone (green) and the PPARd receptor co-crystallized with GW 2433 (green).

3510
Z. Li et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3507–3511
COOMe
COOMe
CHO
O
OH
O
i
ii
O
O
R3
R3
R3
R3
R3
a
b
COOMe
COOH
COOMe
COOMe
O
O
O
iv
vi
iii
O
R3
c
d
COOH
CONH2
COOMe
CONH2
O
O
v
O
O
R3
e
f
COOH
COOH
COOMe
COOMe
O
O
vii
O
O
R3
R3
g
c
Scheme 1. (i) p-Bromoethoxy benzaldehyde, KOH, EtOH, refluxing; (ii) dimethyl malonate, toluene, piperidinium acetate, refluxing; (iii) 5% Pd/C,
H2, rt; (iv) 1 equiv of NaOH, rt; (v) SOCl2, 28% ammonia; (vi) NaOH, CH3 OH, rt; (vii) 2 equiv of NaOH, rt.
between an acid chloride of d and ammonia gave the
amide ester e. Hydrolysis of e with sodium hydroxide
gave the amide acid f. Hydrolysis of c with more than
2 equiv of sodium hydroxide gave the malonic acid g.
Cells were treated with compound for 24 h, and then
collected with Cell Culture Lysis buffer. Luciferase
activity was monitored using the luciferase assay kit
according to the manufacturers instructions. Light
emission was read in a Labsystems Ascent Fluoroskan
reader. To measure galactosidase activity to normalize
the luciferase data, 50 lL of supernatant from each
transfection lysate was transferred to a new microplate.
Galactosidase assays were performed in the microwell
plates using a kit from Promega and read in a micro-
plate reader.
3.2. Modeling
The docking program used in this study was AutoDock
3.0.14–17 Polar hydrogens were added and Kollman
partial atomic charges were assigned to the macromol-
ecule. Gasteiger charges were assigned to the ligands. All
molecular modeling works except docking were done in
Sybyl 6.81 package.21
3.4. In vivo animal study
Six-week old male db/db mice purchased from Jackson
Lab (USA) were given a standard diet and kept at 12 h
light/darkness cycle, a temperature of 21 °C, and a rel-
ative humidity of 50% throughout the accommodation
(two weeks) and dosing periods. All tested compounds
were suspended in water containing 0.2% of methylcel-
lulose and 0.1% Tween-80 (w/v). Mice were given tested
compounds by gavage once daily for 12 days. Blood was
collected from tail vein after 3 h fasting in the morning
for blood glucose measurement using Roche’s Accu-
Chek advantage II Glucose strip together with Accu-
Chek Advantage glucose meter.
3.3. In vitro transactivation
cDNAs for Human RXR, PPAR were obtained by RT-
PCR from the human liver or adipose tissues. Amplified
cDNAs were cloned into pcDNA3.1 expression vector
and the inserts were confirmed by sequencing. U2OS
cells were cultured in McCoy’s 5A with 10% heat-inac-
tivated fetal bovine serum in a humidified 5% CO2
atmosphere at 37 °C. Cells were seeded in 96-well plates
the day before transfection to give a confluence of 50–
80% at transfection. A total of 60 ng of DNA containing
10 ng of hRXR, 10 ng of pCMV Gal, 10 ng of nuclear
receptor expression vectors and 30 ng of the corre-
sponding reporters were cotransfected per well using
FuGene6 transfection reagent according to the manu-
facturer’s instructions. Following 24 h after transfection,
cells were incubated with 10% charcoal-stripped FBS
DMEM and were treated with the individual compound
dissolved in DMSO. The final concentration of DMSO
in culture medium was 0.1%.
Acknowledgements
The authors gratefully acknowledge financial supports
from the national ‘863’ project of People’s Republic of
China and project of ‘Most Important Biopharmaceu-
tical Projects’ by Guangdong provincial and Shenzhen

Z. Li et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3507–3511
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