A ureido-thioisobutyric acid (GW9578) is a subtype-selective PPARα agonist with potent lipid-lowering activity [2]

Full text of DOI:10.1021/jm9903601

J . Med. Chem. 1999, 42, 3785-3788
3785
Ch a r t 1. Chemical Structures of Fibrate Compounds
A Ur eid o-Th ioisobu tyr ic Acid (GW9578)
Is a Su btyp e-Selective P P ARr Agon ist
w ith P oten t Lip id -Low er in g Activity
Peter J . Brown,*,† Deborah A. Winegar,‡
Kelli D. Plunket,§ Linda B. Moore,§
Michael C. Lewis,‡ J oan G. Wilson,‡
Scott S. Sundseth,‡ Cecilia S. Koble,† Zhengdong Wu,|
J ames M. Chapman,| J u¨rgen M. Lehmann,§
Steven A. Kliewer,§ and Timothy M. Willson†
Departments of Medicinal Chemistry, Metabolic Diseases,
and Molecular Endocrinology, Glaxo Wellcome
Research & Development,
Research Triangle Park, North Carolina 27709, and
College of Pharmacy, University of South Carolina,
Columbia, South Carolina 29208
Received J uly 13, 1999
Hypercholesterolemia and hypertriglyceridemia are
associated with an increased incidence of coronary heart
disease,1 the leading cause of death in the western
world. Drug therapy with fibrates, such as clofibrate
(1a ), fenofibrate (2a ), and bezafibrate (3) (Chart 1), is
effective at lowering serum triglycerides and low-density
lipoprotein (LDL) cholesterol and raising high-density
lipoprotein cholesterol in humans.2 These drugs have
been shown to slow the progression of atherosclerosis
and reduce the number of coronary events in high-risk
patients.3-5 Fibrates mediate their clinical effects pri-
marily through increased clearance of circulating tri-
glyceride-rich very low-density lipoproteins (VLDL),2
leading to a reduction in the number of atherogenic
particles. Apolipoprotein C-III (apoC-III) is a 79-amino
acid glycoprotein which resides primarily on the surface
of VLDL particles and inhibits their breakdown by
lipoprotein lipase. In humans, fibrates lower serum
levels of apoC-III,6 increasing catabolism of VLDL to
smaller particles which can be removed from the
circulation by receptor-mediated uptake into the liver.7
The clinically used fibrate drugs were developed
without knowledge of their cellular target.8 In 1990,
Issemann and Green reported the cloning of an orphan
member of the nuclear receptor superfamily, designated
the peroxisome proliferator-activated receptor R (PPARR),
which was activated by the known fibrate drugs.9
Adding to the significance of this discovery was the
identification of a PPAR binding site in the proximal
promoter of the apoC-III gene,10 through which fibrates
were shown to transrepress its expression.10,11 Rodent
pharmacology studies support the hypothesis that fi-
brates mediate their lipid-lowering activity through
PPAR-mediated repression of hepatic apoC-III expres-
sion,11,12 which in turn leads to lower circulating levels
of apoC-III and increased lipoprotein catabolism.7
Three PPAR subtypes have been identified in humans
and rodents:13 PPARR is found primarily in the liver;
PPARγ, the target for the glucose- and lipid-lowering
action of the thiazolidinedione (TZD) drugs,14,15 is found
at high levels in adipose tissue and at lower levels in
the spleen and liver; and PPARδ (also known as PPARâ,
NUCI, and FAAR) is expressed in most tissues. Al-
though all three subtypes are found in the liver, the
level of PPARR expression is generally higher, and it
has been widely assumed that this is the subtype
through which fibrates mediate their lipid-lowering
activity.2,16 However, the fibrates that are used in the
clinic have not been rigorously characterized for their
activities on the three PPAR subtypes. We profiled
clofibric acid (1b), fenofibric acid (2b), and bezafibrate
(3) for their PPAR agonist activity on the human and
murine receptors (Table 1). In agreement with earlier
reports,9,16 these fibric acids activated PPARR at high
micromolar concentrations. Interestingly, all three com-
pounds showed significant cross-reactivity with the
other PPAR subtypes. Clofibric acid (1b) and fenofibric
acid (2b) were dual activators of PPARR and PPARγ,
with ∼10-fold selectivity for PPARR, while bezafibrate
(3) activated all three PPAR subtypes at comparable
doses. The PPAR selectivity data is consistent with the
proposal that PPARR mediates the lipid-lowering activ-
ity of the fibrate drugs,2,16 although their cross-reactivity
with PPARγ may contribute to some of the observed
pharmacology.17
Since the fibrates (1-3) are relatively weak PPARR
agonists, we were interested in profiling compounds
with increased potency and selectivity for PPARR as
lipid-lowering drugs. We have previously described a
series of ureido-fibrates (Chart 1) with either potent
PPAR activity18,19 or potent lipid-lowering activity in
hyperlipidemic rats.20 Four of the compounds (4-7) with
good in vivo activity were assayed for their PPAR
agonist activity (Table 1). These ureido-fibrates (4-7)
were potent agonists of murine PPARR. However, like
fibrates (1-3), they showed only moderate levels of
subtype selectivity. In addition, all four ureido-fibrates
* Address correspondence to Peter J . Brown, Department of Me-
dicinal Chemistry, NTH-M1108, Glaxo Wellcome Research & Develop-
ment, P.O. Box 13398, 5 Moore Dr., Research Triangle Park, NC 27709-
3398. Tel: (919) 483-1195. Fax: (919) 315-3900. E-mail: pjb5890@
glaxowellcome.com.
† Department of Medicinal Chemistry.
‡ Department of Metabolic Diseases.
§ Department of Molecular Endocrinology.
| College of Pharmacy.
10.1021/jm9903601 CCC: $18.00 © 1999 American Chemical Society
Published on Web 09/04/1999

3786 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19
Ta ble 1. PPAR and Lipid-Lowering Activity of Fibrates
Communications to the Editor
compda
R1
murine receptor activityb EC50 (µM)
human receptor activityb EC50 (µM)
MEDc
(mg/kg)
no.
X
R2
PPARR
PPARγ
PPARδ
PPARR
PPARγ
PPARδ
1b
2b
3
4
5
6
7
8
clofibric acid
fenofibric acid
bezafibrate
cC7H13
nC7H15
(CH2)5cC6H11
nC7H15
50
18
90
0.12
0.033
0.053
0.010
0.005
>300
250
55
ia
ia
110
1.4
5.5
23
55
30
50
1.4
0.41
0.15
0.79
0.05
>300
300
60
ia
ia
20
0.79
3.1
nd
nd
1.4
nd
50
50
1.0
0.1
1.0
0.1
0.2
O
O
O
O
S
F
0.012
0.87
1.0
0.40
1.5
0.006
0.28
1.6
0.20
1.0
H
H
F
3.2
2.6
F
nC7H15
a
b
Compound structures in Chart 1. Compounds were assayed for agonist activity on PPAR-GAL4 chimeric receptors in transiently
transfected CV-1 cells as described (refs 14, 15, and 19); EC50 ) the concentration of test compound that gave 50% of the maximal reporter
activity ( 10%, n ) 4-6. All compounds were full agonists unless indicated; >300 ) agonist activity observed only at 300 µM.; ia )
inactive at 300 µM.; nd ) not determined. c Lipid-lowering activity in male Sprague-Dawley rats fed a 1% cholesterol, 0.5% cholic acid
diet. Animals (n ) 6/group) were dosed orally once daily for 3 days with either vehicle (5% bicarbonate) or doses of test compound ranging
from 0.1 to 50 mg/kg as described (ref 20). Fenofibrate (2a ) and the dicyclohexylamine salt of 8 were used for the in vivo studies; all other
compounds (3-7) were the free acids. MED ) minimum effective dose producing a 40-60% decrease in serum TLDL cholesterol relative
to vehicle-treated controls, where TLDL cholesterol ) (VLDL + LDL) cholesterol.
(4-7) were less potent as activators of human PPARR
compared to murine PPARR, further eroding their
PPARR selectivity on the human receptors. Ureido-
fibrate 6 was the only analogue that showed moderate
PPARR selectivity on both the murine and human
receptors. Ureido-fibrates 5 and 7 were moderately
PPARR-selective on the murine receptors, but dual
PPARR/PPARγ agonists on the human receptors. Ure-
ido-fibrate 4 was more potent on PPARγ than PPARR,
especially on the human receptors where its profile is
comparable to that of the TZD antidiabetic drugs.13
Previous studies19 have shown that ureido-fibrate
analogues (Chart 1, X ) O), with modified urea sub-
stituents generated by solid-phase parallel synthesis,21
do not show increased PPARR selectivity. However,
through conventional analogue synthesis we discovered
that modification of the fibrate headgroup to a thio-
isobutyric acid (TiBA; Chart 1, X ) S) increased PPARR
activity relative to PPARγ and PPARδ (Table 1, 7 vs
8). Ureido-TiBA 8 (GW9578) is a potent PPARR agonist
with 300-fold selectivity on the murine receptors and
20-fold selectivity on the human receptors. Scheme 1
shows the synthesis of the ureido-TiBA 8. Briefly,
4-bromothiophenol was alkylated with tert-butyl bro-
moisobutyrate to give the bromo ester 9 in good yield.
Heck reaction with vinylphthalimide and hydrogenation
of the olefin gave intermediate 10. Following phthal-
imide deprotection, the resulting primary amine was
coupled with heptanoic acid to afford the amide 11.
Borane reduction furnished the secondary amine which
was treated with 2,4-difluorophenyl isocyanate to give
the ureido-TiBA 8 following TFA cleavage of the tert-
butyl ester.
Sch em e 1a
a
Reagents: (i) BrC(Me)2CO2tBu, KOH, EtOH (80%); (ii) vi-
nylphthalimide, Pd(OAc)2, DIEA, (o-Tol)3P, MeCN (84%); (iii)
Wilkinson’s catalyst, EtOH, H2 (91%); (iv) hydrazine, EtOH (86%);
(v) heptanoic acid, DIC, HOBT (66%); (vi) 1 M BH3‚THF (95%);
(vii) 2,4-difluorophenyl isocyanate, CH2Cl2 (83%); (viii) 50% TFA/
CH2Cl2 (90%).
were active at doses as low as 0.1-0.2 mg/kg. Second,
the ureido-fibrate 4, which possesses potent activity on
PPARγ in addition to PPARR, did not perform signifi-
cantly better than analogues 5-8. Third, comparison
of the in vitro murine PPARR activity with the in vivo
potency of 2-8 provided a strong correlation (Figure 1).
Similar associations could not be drawn with either
PPARγ or PPARδ (Table 1).
Finally, to confirm that the observed lipid-lowering
activity in the cholesterol/cholic acid-fed rat was medi-
ated through a clinically relevant mechanism,2 the
effects of fenofibrate (2a ) and ureido-TiBA 8 on serum
apoC-III and TLDL cholesterol were determined (Figure
2). A dose-dependent reduction in TLDL cholesterol was
observed with both compounds, reaching 50% lowering
at 30 mg/kg fenofibrate (2a ) (Figure 2a) and 60%
To assess the potential utility of 8 as a lipid-lowering
drug, its activity was compared to that of fibrates 2-7
in the cholesterol/cholic acid-fed rat.20 Reduction of total
low-density lipoprotein (TLDL) cholesterol in this model
has been shown to correlate with the antihyperlipidemic
activity of fibrates in humans.22 In this model,20 fenofi-
brate (2a ) and bezafibrate (3) produced a 40-60%
decrease in TLDL cholesterol only at the highest dose
tested, while the ureido-fibrates 4-7 and ureido-TiBA
8 were active at 50-500-fold lower doses (Table 1).
Several lines of evidence suggest that the lipid-lowering
activity of 8 is due to its potent PPARR activity. First,
the analogues 5, 7, and 8 with the best PPARR activity

Communications to the Editor
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19 3787
Ack n ow led gm en t . We thank Roy Hawke and J o
Salisbury for in vivo data on 2-7, Peter Dolphin
(Dalhousie University, Canada) for the polyclonal rat
apoC-III antibody, and Dr. Bart Staels (Institute Pas-
teur de Lille) for communicating data prior to publica-
tion.
Su p p or tin g In for m a tion Ava ila ble: Detailed experi-
mental procedures for the synthesis of 8. This material is
available free of charge via the Internet at http://pubs.acs.org.
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In summary, the ureido-TiBA 8 is the most potent
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the development of hyperinsulinemia in insulin-resis-
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resistance are independent risk factors for coronary
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improved drugs for primary prevention of cardiovascular
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J M9903601