Design and synthesis of indane-ureido-thioisobutyric acids: A novel class of PPARα agonists

Article abstract of DOI:10.1016/j.bmcl.2007.10.041

A series of aminoindane derivatives were synthesized and shown to be potent PPARα agonists. The compounds were obtained as racemates in 12 steps, and tested for PPARα activation and PPARα mediated induction of the HD gene. SAR was developed by variation to the core structure as shown within. Oral bioavailability was demonstrated in a Sprague-Dawley rat, while efficacy to reduce plasma triglycerides and plasma glucose was demonstrated in db/db mice.

Full text of DOI:10.1016/j.bmcl.2007.10.041

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6773–6778
Design and synthesis of indane-ureido-thioisobutyric acids:
A novel class of PPARa agonists
Jay M. Matthews,* Xiaoli Chen, Ellen Cryan,à Dennis J. Hlasta,
Philip J. Rybczynski,§ Kim Strauss, Yuting Tang,
June Z. Xu, Maria Yang,– Lubing Zhouk and Keith T. Demarest
Drug Discovery, Johnson & Johnson, East Coast RED, Spring House, PA 19477-0776, USA
Received 3 August 2007; revised 12 October 2007; accepted 12 October 2007
Available online 17 October 2007
Abstract—A series of aminoindane derivatives were synthesized and shown to be potent PPARa agonists. The compounds were
obtained as racemates in 12 steps, and tested for PPARa activation and PPARa mediated induction of the HD gene. SAR was
developed by variation to the core structure as shown within. Oral bioavailability was demonstrated in a Sprague–Dawley rat, while
efficacy to reduce plasma triglycerides and plasma glucose was demonstrated in db/db mice.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Peroxisome proliferators-activated receptors (PPARs)
are members of a nuclear hormone receptor superfamily
that are activated by endogenous saturated and unsatu-
rated fatty acids, as well as synthetic ligands. To date,
three nuclear receptor isoforms, PPARa, PPARd, and
PPARc have been isolated in various species, with the
PPARa-subtype being the first isoform cloned in multi-
ple species, including rodent and humans. The PPARs
as a whole play a central role in regulating the storage
and catabolism of dietary fats,1 with each PPAR recep-
tor differentially expressed in a tissue-specific manner,
subsequently playing a crucial role in lipid and lipopro-
tein homeostasis.2,3 The PPARs have also been hypoth-
esized to treat obesity-related syndromes, such as type 2
diabetes.4
sion in the liver, kidney, heart, and brown adipose
tissue, with lower levels of expression in muscle, testis,
spleen, and the lung. The PPARa receptor is specifically
involved in the regulation and expression of target genes
responsible for lipid and lipoprotein metabolism. Upon
ligand activation, the PPARa isoform forms a heterodi-
mer with retinoid X receptor (RXR), with the heterodi-
mer causing a downstream cascade effect that leads to
the expression of target genes responsible for fatty acid
degradation, such as fatty acid binding protein (FABP)
and b-oxidation enzymes (acyl-CoA oxidase and 3-en-
oyl-hydroxyacyl CoA dehydrogenase (HD)). PPARa
also regulates high-density lipoprotein (HDL) levels,
which are involved in reverse cholesterol transport, by
inducing the transcription of the major HDL apolipo-
proteins, apolipoprotein A-I and apolipoprotein A-II.5
PPARa is predominantly expressed in various metabol-
ically active tissues, which include high levels of expres-
The currently prescribed fibrates, such as clofibrate and
fenofibrate (Fig. 1), are hypolipidemic agents effective at
lowering elevated serum triglyceride levels. The triglyc-
eride lowering activity of the fibrates is a result of in-
creased lipoprotein lipolysis followed by b-oxidative
degradation of fatty acids. The latter action combined
with a reduction of fatty acid and triglyceride synthesis
results in a decrease of VLDL production by the liver.
Though the fibrates have been effective ligands of
PPAR, their mechanism of action was not realized until
the early 1990s. Consequently, the fibrates have weak
affinities (>20 lM) in both rodent and human cloned
PPARa receptors, with extremely poor subtype-selectiv-
Keywords: Aminoindane; PPARa agonist; Hypolipidemic; Hypogly-
cemic; Dyslipidemia.
*
Corresponding author. Tel.: +1 215 628 5214; fax: +1 215 628
3297; e-mail: jmatthew@prdus.jnj.com
Present address: Merck & Co., Rahway, NJ 07065, USA.
Present address: BD Diagnostics, One Becton Drive, Franklin Lakes,
à
NJ 07417, USA.
Present address: Amicus Therapeutics, 6 Cedar Brook Drive,
§
Cranbury, NJ 08512, USA.
Present address: Palatin Technologies, Cranbury, NJ 08512, USA.
–
k
Present address: Lifescan, 199 Grandview Road, Skillman, NJ 08558,
USA.
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.10.041

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J. M. Matthews et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6773–6778
F
tribromide at reduced temperatures affords phenol 6,
O
EtO2C
X
R
HO2C
O
which was further acylated with dimethylthiocarbamoyl
chloride in the presence of 1,4-diazabicyclo[2.2.2]octane
(DABCO) to provide thiocarbamate 7. Newman–Kwart
rearrangement of thiocarbamate 7 to carbamate 8 was
achieved at 340 ꢁC in excellent yields.
N
H
Cl
N
F
Clofibrate
C7H15
GW2331; X = O, R = Et
GW9578; X = S, R = Me
Cl
PriO2C
O
R1
O
O
N
NH
The synthesis of structure 1 was completed using the
chemistry outlined in Scheme 2. Deprotection of phthal-
imide 8 to afford 2-aminoindane 9 was achieved using
hydrazine at elevated temperatures. In one-pot, depro-
tection of the thiophenol (not shown) was accomplished
using standard base hydrolysis conditions, followed by
immediate treatment with tert-butyl 2-bromoisobutyrate
to afford thiobutyrate 10. Acylation of amine 10 with
acid chlorides followed by reduction of the resultant
amide with boron–THF complex provided secondary
amine 11, which was further acylated with arylisocya-
nates and deprotected under acidic conditions to afford
analogs of 1.
Fenofibrate
R
HO2C
S
1
Figure 1. Structures of clofibrate, fenofibrate, GW2331, GW-9578,
and thioisobutyric acid 1.
ity. Despite the low affinity for the PPARs, clofibrate
has been shown to improve glucose tolerance in type 2
diabetes patients, with obesity a risk factor in the devel-
opment of diabetes. With the uncertainty of whether the
a- or c-component (or the combination thereof) of clo-
fibrate is responsible for the therapeutic effect, the need
for a PPARa-selective agonist has been a large thrust
amongst several groups in the last 10 years.6–13 Re-
cently, GlaxoSmithKline (GSK) reported the discovery
of GW2331, a ureidofibrate, found to be a potent, dual
PPARa/c agonist.14 Subsequently, a similar motif was
revealed by GSK in the form of GW-9578, a ureidobu-
tyric acid, which was disclosed to be a potent and selec-
tive PPARa agonist with favorable hypolipidemic
activity in vivo compared to fenofibrate.15,16 Herein,
we report the design and synthesis of novel indane-ure-
ido-thioisobutyric acids as PPARa agonists.
Alternatively, intermediate amine 10 was reductively
aminated with aldehydes or ketones, using sodium tri-
acetoxyborohydride as the reducing agent, to afford sec-
ondary amines 11, which were subsequently converted
to final products 1.
In the event that alkylation of the second urea nitrogen
is desired, the chemistry shown in Scheme 3 was utilized.
Alkylation of urea 1 using sodium hydride and the
appropriate alkyl halide afforded tert-butyl protected
The synthetic approach utilized toward the synthesis of
the indane-ureido-thioisobutyric acids is shown in
Scheme 1. Treatment of 5-methoxy-1-indanone 2 with
butyl nitrite at elevated temperature afforded oxime 3,
which was subsequently reduced to aminoindane 4 via
catalytic hydrogenation, using carbon-activated palla-
dium. Protection of aminoindane 4 was achieved using
phthalic anhydride at elevated temperature to afford
intermediate 5. Demethylation of ether 5 using boron
O
a
O
O
NH
N
2
(100 %)
N
S
N
S
9
O
8
d or e
b, c
NH
2
O
S
(70 %)
O
10
f, g
NH
R
O
S
(60 %)
O
11
O
O
a
b
1
R
O
NOH
(70 %)
MeO
MeO
NH
MeO
(33 %)
2
3
N
R
HO
S
1
O
O
c
NH
N
2
Scheme 2. (a) EtOH, H2NNH2, 78 ꢁC, 30 min; (b) MeOH, KOH,
65 ꢁC, 5 h; (c) tert-butyl 2-bromoisobutyrate, RT, 18 h; (d) i—CH2Cl2,
TEA, RC(O)Cl, 0 ꢁC, 16 h; ii—THF, borane–THF (1.0 M), RT, 5 h;
(e) DCE, RCHO, NaBH(OAc)3, RT, 18 h; (f) CH2Cl2, R1PhNCO, RT,
16 h; (g) CH2Cl2, TFA, RT, 1 h.
(68 %)
MeO
O
4
5
O
d
e
N
(95 %)
(70 %)
HO
O
6
O
O
f
O
S
1
N
1
N
R
O
N
R
O
N
N
S
(75 %)
N
O
NH
a, b
N
R
O
8
7
O
2
O
HO
R
S
R
S
1
Scheme 1. (a) MeOH, HCl, CH3(CH2)3ONO, 45 ꢁC; (b) AcOH,
H2SO4, Pd–C (10%), H2, 60 psi, 18 h; (c) DMF, NaH (60%), 125 ꢁC,
96 h; (d) CH2Cl2, BBr3, ꢀ50 ꢁC, 5 h; (e) DMF, DABCO, RT, 16 h; (f)
neat, 340 ꢁC, 12 min.
12
O
O
Scheme 3. (a) DMF, NaH (60%), R2-Br, RT, 5 h; (b) CH2Cl2, TFA,
RT, 1 h.

J. M. Matthews et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6773–6778
6775
H
N
To determine the role of stereochemistry on gene activa-
tion, compounds 32, 33, and 35 were resolved by chiral
HPLC to yield each enantiomer in >98% ee. The abso-
lute configuration of the (S)-(+) enantiomer of 35 was
initially characterized by vibrational circular dichroism
(VCD).17 The results in Tables 1 and 2 indicate that
(R)-(ꢀ) enantiomer of 35 is the preferred absolute con-
figuration in all three cases.
1
1
N
R
R
N
b, c
NH
2
a
O
14
13
1
R
O
N
NH
HO
R
S
1
O
Compounds 15–32 were evaluated for their agonist
activity by using a rat hepatoma cell line for the induc-
tion of PPARa target gene, 3-enoyl-hydroxyacyl CoA
dehydrogenase (HD).18 The results obtained in Table 1
were compared with corresponding data for GW-9578
(PPARa agonist) as a reference compound. The direct
analogue of GSK-9578, 15, exhibited no activity in the
HD gene induction assay. However, introduction of o-
substitution, such as a 2-trifluoromethyl group, as in
19, enhanced receptor affinity (EC50 = 6.4 lM). Im-
proved HD induction was realized through the introduc-
tion of a p-trifluoromethyl, as in 27 (EC50 = 1.2 lM).
Methylthio analogue 28 demonstrated an additional 3-
fold increase in potency over 27, while trifluoromethyl
thio analogue 29, isopropyl analogue 30, dimethylamino
analogue 31, and trifluoromethoxy analogue 32 show a
further 5-fold enhancement in potency in the HD induc-
tion assay. Each of the enantiomers of 32, (R)-(ꢀ)-
32and (S)-(+)-32, was tested, with only enantiomer
(R)-(ꢀ)-32 demonstrating notable PPARa receptor
activity (EC50 = 0.172 lM).
Scheme 4. (a) THF, CDI, 50 ꢁC, 15 min; (b) THF, 11, 50 ꢁC, 2 h; (c)
CH2Cl2, TFA, RT, 1 h.
intermediates (isolated), which were subsequently
deprotected to afford compounds 12.
In instances when the required isocyanates are not com-
mercially available, the requisite analogs were synthe-
sized using the chemistry shown in Scheme 4. Anilines
13 were treated with carbonyldiimidazole, at elevated
temperatures, to afford intermediate 14, to which was
added a solution of compound 11, to afford the tert-bu-
tyl protected esters, which were deprotected to afford
compounds 1.
Table 1. Biological data for aminoindane derivatives
1
R
O
NH
N
Using analogue 32 as a starting point, the length of the
urea side chains (R and R2) as well as the substitution pat-
tern on the urea-phenyl moiety (R1), as shown in Table 2,
was explored. Des-heptyl analogue 33 demonstrated com-
parable affinity to 32 in the HD gene induction assay.
Varying the chain length indicated that the smaller, ali-
phatic chains were favored. For example, ethyl analogues
35 (4-fold), 36 (7-fold), and 37 (9-fold) demonstrated a
significant enhancement in potency compared to 32, while
octyl analogue 54 showed a 6-fold loss of activity. Branch-
ing of the aliphatic side chain was tolerated when the
branched portion was several carbon atoms removed
away from the urea, as in 57 (EC50 = 0.479 lM), albeit
branching was not an optimal substitution. Substitution
of the second urea nitrogen (R2), demonstrated by com-
pounds 39 and 46, with aliphatic chains afforded inactive
compounds. Further exploration of the structure–activity
relationship of the trifluoromethoxyphenyl moiety affor-
ded analogues 60–63. Additional substitution on the
urea-phenyl ring led to either less potent or inactive com-
pounds. Each enantiomer of 35, (R)-(ꢀ)-35 and (S)-(+)-
35, was studied, and both enantiomers furnished notable
PPARa receptor potency (EC50 = 0.014 and 0.171 lM,
respectively). However, (R)-(ꢀ) was 10-fold more potent
than the (S)-(+).
HO
C H
S
7
15
1
O
Compounda
R1
PPARa-HD18 EC50 (lM)
15
16
2,4-diF
H
2-F
>10
>10
17
18
>10
7.54
2-CH3
2-CF3
2-OCF3
2-SCF3
3-F
19
20
6.39
4.99
21
22
1.96
2.67
23
24
3-Cl
2.09
2.73
3-CF3
3-OCH3
4-OCH3
4-CF3
4-SCH3
4-SCF3
4-i-Pr
25
26
1.11
0.910
1.27
27
28
0.419
0.210
0.229
0.252 (0.700)
0.294
0.169
>13
29
30
31
4-N(CH3)2
4-OCF3
4-OCF3
4-OCF3
32
(R)-(ꢀ)-32b
(S)-(+)-32b
GSK-9578
0.166
a Target compounds were purified by reverse-phase semi-prep HPLC,
isolated, and tested as carboxylic acids unless otherwise noted.
Purities were judged by reverse-phase HPLC/MS at 215 and 254 nm
(YMC J’Sphere C-18 column, 0.4 · 5 cm; mobile phase: MeCN–
H2O). All compounds were characterized by ESI-MS and 400-MHz
1H NMR.
b >98% enantiomeric purity by chiral HPLC (Chiralcel OD column,
0.46 · 25 cm; mobile phase: 95:5: hexanes/i-PrOH, with 0.1% TFA;
flow rate: 1 mL/min).
Primarily using PPARa activity, human liver micro-
somal stability,19 and structural diversity to triage com-
pounds, several compounds were selected as potential
candidates for in vivo profile. Table 3 shows the
in vitro metabolism (human liver microsomal stability)
and pharmacokinetic profiles in rats at doses of

6776
J. M. Matthews et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6773–6778
Table 2. PPARa-mediated HD gene induction and PPARa-Gal4 co-transfection data
3
R
1
R
O
N
2
N
R
R
HO
S
1
O
Compounda
R
R1
R2
R3
PPARa-HD18 EC50 (lM)
33
(R)-(ꢀ)-33b
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
i-Pr
0.096
0.146
1.11
H
(S)-(+)-33b
34
H
CH3
2.85
35
(R )-(ꢀ)-35b
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
(CH2)2CH3
(CH2)3CH3
(CH2)3CH3
(CH2)3CH3
(CH2)3CH3
(CH2)4CH3
H
0.033
0.002
0.165
0.032
0.023
0.117
>1.0
(S)-(+)-35b
36
37
38
SCF3
N(CH3)2
OCF3
OCF3
OCF3
i-Pr
39
40
CH3
H
0.158
0.195
0.260
0.160
0.561
0.105
>5.0
41
42
H
H
43
44
H
SCF3
H
N(CH3)2
OCF3
OCF3
i-Pr
45
46
H
(CH2)4CH3
47
48
(CH2)4CH3
(CH2)4CH3
(CH2)4CH3
(CH2)5CH3
(CH2)5CH3
(CH2)5CH3
(CH2)5CH3
(CH2)7CH3
CH(CH3)2
CH2CH(CH3)2
(CH2)2CH(CH3)2
(CH2)2OCH3
(CH2)3CF3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0.159
0.046
0.141
0.166
0.180
0.249
0.219
1.03
SCF3
49
50
N(CH3)2
OCF3
i-Pr
51
52
SCF3
53
54
N(CH3)2
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
55
56
>5.0
2.34
57
58
0.386
0.425
0.707
0.598
>1.0
59
60
2-CH3
2-Br
3-Cl
61
62
>3.0
>3.0
63
3-Br
GSK-9578
Fenofibrate
Ciprofibrate
0.084
>10
>10
a Target compounds were purified by reverse-phase semi-prep HPLC, isolated, and tested as carboxylic acids unless otherwise noted. Purities were
judged by reverse-phase HPLC/MS at 215 and 254 nm (YMC J’Sphere C-18 column, 0.4 · 5 cm; mobile phase: MeCN–H2O). All compounds were
characterized by ESI-MS and 400-MHz 1H NMR. Compounds are racemates unless otherwise noted by stereochemical assignment.
b >98% enantiomeric purity by chiral HPLC (Chiralcel OD column, 0.46 · 25 cm; mobile phase: 95:5: hexanes/i-PrOH, with 0.1% TFA; flow rate:
1 mL/min).
3.0 mg/kg (iv) and 30.0 mg/kg (po). The results shown in
Table 3 suggest the compounds have reasonable oral
bioavailability, though the highest exposures of drug
were seen with 35 and 38. Both 35 and 38 exhibited oral
bioavailability in rats (59% and 58%, respectively), with
acceptable oral t1/2 (4.0 and 4.4 h, respectively), and
relatively low clearance (0.1 and 0.11 mL/min/kg,
respectively). The pharmacokinetic and in vitro pharma-
cological properties of 35 warranted assessment in phar-
macological models in mice.
rodent model of type 2 diabetes characterized by severe
hyperglycemia, hypertriglyceridemia, and insulin resis-
tance.20 The 6- to 7-week-old female db/db mice were
treated with compound 35 by oral gavage at 1.0 mg/kg
for 11 days. Compound 35 demonstrated a significant
hypotriglyceridemic and hypoglycemic effect, with a
reduction in plasma triglycerides (56%), plasma glucose
(60%), and plasma insulin (53%) levels suggesting that
the compound may improve insulin sensitivity. In the
same study, 35 reduced the body weight of the mice by
59%. Similarly, (R)-(ꢀ)-35 was dosed by oral gavage
at 0.1 mg/kg in db/db mice (6- to 7-week-old female
mice; 11-day dosing regimen). Even at this low dose,
The hypolipidemic and hypoglycemic effects of com-
pound 35 were demonstrated in db/db mice, an obese

J. M. Matthews et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6773–6778
Table 3. Pharmacokinetic profiles of 35–38, 45, 48, and 49a
6777
Compound
HLM stabilityb (t1/2, min)
Cmax SE (lg/mL)
AUC SE (lg h/mL)
t1/2b (h)
CLtot (mL/min/kg)
BAc (%)
35
36
37
38
45
48
49
100
12
39.27 7.87
32.36 19.48
5.67 1.29
72.3 32.17
16.63 8.62
8.02 3.36
20.79 7.00
290.28 93.49
37.13 16.68
101.02 50.3
273.46 82.4
121.36 59.85
54.72 28.76
63.58 24.07
4.0 1.27
2.92 1.01
3.84 1.84
4.46 1.12
7.19 1.35
4.05 0.55
2.60 0.56
0.10
0.81
0.30
0.11
0.25
0.55
0.47
59
34
100
80
100
58
100
76
24
59
94
62
a Compounds were dosed in fasted rats, n = 6, po @ 30 mg/kg, iv @ 3 mg/kg.
b HLM, human liver microsome. Compounds were incubated at 37 ꢁC at 5 lM and 1 mg protein/mL microsomal prep.19
c BA = Oral bioavailability.
Table 4. Selectivity of (R)-(ꢀ)-35 for PPAR subtypes
Compound
PPARa-HD18
ED50 (lM)
PPARa-Gal421
EC50 (lM)
PPARd-Gal421
EC50 (lM)
PPARc-Gal421
EC50 (lM)
PPARc-aP222
(% BRL-49653)
(R)-(ꢀ)-35
0.002
0.084
0.170
0.45
2.1
0.15
>10
0.5
3.8%
0.46%
0.12 lM
GSK-9578
Rosiglitazone
0.19
idi, G.; Fruchart, J.-C.; Staels, B. Curr. Opin. Pharmacol.
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In addition, (R)-(ꢀ)-35 was assessed for its ability to
stimulate various human PPAR subtypes (a, c, d; Table
4) with receptor activation assessed in co-transfection
assays. For this purpose, Gal4 PPAR chimeric receptors
were expressed in transiently transfected HEK293
cells.21 Further selectivity of (R)-(ꢀ)-35 was determined
by evaluating the induction of aP2 (PPARc-aP2)
mRNA in human preadipocytes (Table 3).22 As demon-
strated in Table 4, (R)-(ꢀ)-35 is potent toward the acti-
vation of the PPARa co-transfection assay
(EC50 = 0.170 lM) and demonstrates high selectivity
over the d- and c-receptors in the PPARd and PPARc-
Gal4 co-transfection assays, as well as the aP2 induction
assay. PPAR agonists GSK-9578 (PPARa) and Rosig-
litazone (PPARc-selective) were assessed in these assays
as well.
In conclusion, we have investigated a series of indane-
ureido-thioisobutyric acids as potent and efficacious
PPARa agonists. We identified (R)-(ꢀ)-35 as a highly
selective PPARa receptor agonist and demonstrated
that this compound has excellent oral efficacy in db/db
mice, with the ability to significantly decrease hypotri-
glyceridemic and hypoglycemic effects, at a low dose
(1.0 mg/kg). On the basis of an assortment of preclinical
data, (R)-(ꢀ)-35 was advanced into development as a
selective PPARa agonist that could be useful for the
treatment of several clinical indications for which the
marketed fibrates are used.
13. Sauerberg, P.; Bury, P. S.; Mogensen, J. P.; Deussen, H.-
J.; Pettersson, I.; Fleckner, J.; Nehlin, J.; Frederiksen, K.
S.; Albrektsen, T.; Din, N.; Svensson, A.; Ynddal, L.;
Wulff, E. M.; Jeppesen, L. J. Med. Chem. 2003, 46, 4883.
14. Brown, P. J.; Smith-Oliver, T. A.; Charifson, P. S.;
Tomkinson, N.; Fivush, A. M.; Sternbach, D. D.; Wade,
L. E.; Orband-Miller, L.; Parks, D. J.; Blanchard, S. G.;
Kliewer, S. A.; Lehmann, J. M.; Willson, T. M. Chem.
Biol. 1997, 4, 909.
References and notes
1. (a) Berger, J.; Moller, D. E. Annu. Rev. Med. 2002, 53,
409; (b) Berger, J. P.; Akiyama, T. E.; Meinke, P. T.
Trends Pharmcol. Sci. 2005, 26, 244; (c) Chinetti-Gbagu-

6778
J. M. Matthews et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6773–6778
15. Bown, P. J.; Winegar, D. A.; Plunket, K. D.; Moore, L. S.;
Wu, Z.; Chapman, J. M.; Kliewer, S. A.; Wilson, T. M.
J. Med. Chem. 1999, 42, 3788.
16. Brown, P. J.; Stuart, L. W.; Hurley, K. P.; Lewis, M. C.;
Winegar, D. A.; Wilson, J. G. Bioorg. Med. Chem. Lett.
2001, 11 9, 1225.
17. BioTools, 17546 Bee Line Hwy, Jupiter, FL 33458. X-ray
crystallography of an advanced intermediate of R-(ꢀ)-35
was obtained to verify the absolute configuration and will
be published in a separate manuscript.
18. HD bDNA assay. H4IIE rat hepatoma cell line was
obtained from ATCC. Cells were cultured in a tissue
culture rack with culture medium, maintained at 37 ꢁC
and 5% CO2. Twenty-four hours after initial seeding, the
HD gene induction assay was initiated. The media was
removed and replaced with low serum culture media
containing vehicle or test compounds. The cells were
incubated for 24 h, to which was added lysis buffer with
HD gene specific CE, LE, BL prbles to initiate the bDNA
HD mRNA assay. At the end of the assay, the lumines-
cence was quantitated in a Dynex MLX microtiter plate
luminometer. EC50 full dose–response data were obtained
using a 15-pt curve (10ꢀ12 M to 10ꢀ5 M).
gavaged once a day for 11 days with either 0.5%
methylcellulose in dH2O (vehicle) or test compound
(0.5% hydroxypropyl-methylcellulose) at doses of 0.03,
0.1, 0.3, 1, 3, 10 mg/kg/day. The body weight was
measured in the mornings on day 1, prior to dosing, and
on day 12 before bleeding (18–24 h after the final dose for
each group, the mice were anesthetized with CO2/O2
(70:30) and bled by retro-orbital sinus puncture into
microtubes containing clog activator and put on ice. The
serum samples were prepared by centrifugation). Serum
glucose and triglycerides were determined using COBAS
Mira Plus blood chemistry analyzer (Roche Diagnostics,
NJ). Serum insulin was measured using ALPCO insulin
ELISA kit.
21. General transfection assay and method for PPARa, d, c
receptors. HEK293 cells were grown in DMEM/F-12
media supplemented with 10% FBS and glutamine. The
cells were co-transfected with DNA for PPAR-Gal4
receptor and Gal4-Luciferase Reporter using DMRIE-C
reagent. After 18 h, the DNA-containing medium was
replaced with 5% charcoal treated FBS growth medium,
which, after 6 h, was seeded in a 96-well plate and
incubated at 37 ꢁC in a CO2 incubator overnight. The
cells were challenged with test compounds, incubated for
24 h at 37 ꢁC. The luciferase activity was assayed using the
Steady-Glo Luciferase Assay Kit from Promega.
19. Compounds were tested by Absorption Systems, in Exton,
PA.
20. db/db mouse female db/db mice (C57 BLK S/J-m+/
+Leprdb, Jackson Labs, Bar Harbor, ME), 6–7 weeks of
age used. Female db/db diabetic mice (8/group) were orally
22. Burris, T. P.; Pelton, P. D.; Zhou, L. Mol. Endocinol. 1999,
13, 410 (aP2 assay).