Synthesis and pharmacological evaluation of a new class of peroxisome proliferator-activated receptor modulators.

Article abstract of DOI:10.1016/S0960-894X(02)00791-6

A series of 5-substituted 2-benzoylaminobenzoic acids has been synthesized and assayed for PPARalpha/gamma activity. Both dual activators and selective PPARgamma agonists have been identified. This class of compounds was shown to activate the PPARgamma receptor through interaction with a novel binding site.

Full text of DOI:10.1016/S0960-894X(02)00791-6

Bioorganic & Medicinal Chemistry Letters 12 (2002) 3565–3567
Synthesis and Pharmacological Evaluation of a New Class of
Peroxisome Proliferator-Activated Receptor Modulators
Markus Thor,a,* Katarina Beierlein,a Graeme Dykes,a Anna-Lena Gustavsson,a
Jessica Heidrich,a Lena Jendeberg,a Bengt Lindqvist,a Cecile Pegurier,b
Patrick Roussel,c Martin Slater,b Stefan Svensson,a Mona Sydow-Backman,a
Ulla Thornstroma and Jonas Uppenberga
aBiovitrum AB, Department of Medicinal Chemistry, Rapsgatan 7, Uppsala 751-37, Stockholm, Sweden
bBiofocus Plc., Sittingbourne, UK
cPharmacia Corporation, Nerviano, Italy
Received 1 July 2002; revised 9 September 2002; accepted 16 September 2002
Abstract—A series of 5-substituted 2-benzoylaminobenzoic acids has been synthesized and assayed for PPARa/g activity. Both dual
activators and selective PPARg agonists have been identified. This class of compounds was shown to activate the PPARg receptor
through interaction with a novel binding site.
# 2002 Elsevier Science Ltd. All rights reserved.
The peroxisome proliferator-activated receptors
(PPARa, PPARd and PPARg) belongto the nuclear
receptor superfamily and function as ligand-activated
transcription factors.1 These receptors play a key role in
various tissues controllingthe expression of egnes
involved in lipid and carbohydrate metabolism. Interest
in the PPARs has increased with the discovery of the
clinically useful thiazolidinedione (TZD) class of insulin
sensitizers (e.g., rosiglitazone and troglitazone) which
are potent and selective PPARg agonists used in the
treatment of type II diabetes. However, the success of
these drugs has been hampered by cases of liver toxicity
and side-effects such as fluid retention and weight gain.2
This has prompted the search for non-TZD ligands with
different characteristics compared to the glitazones.
further demonstrates that these targets are suitable for
therapeutic intervention.3 Accordingly, a dual action
drugwhich binds and activates the PPAR a/g receptors
is hypothesized to mechanistically target the two major
metabolic abnormalities observed in type II diabetic
patients, insulin resistance and dyslipidemia.
We herein report the synthesis and initial pharmacolo-
gical evaluation of a new series of 5-substituted 2-ben-
zoylaminobenzoic acids as PPARa/g modulators.
BVT.142 (Fig. 1) was identified in a cell based reporter
gene assay as being a dual activator of the PPARa/g
receptors. In the course of expandingthe chemistry
around BVT.142 more potent dual activators, as well
as selective PPARg agonists and antagonists, have
been identified through variations at the 5-position.
We have been able to obtain co-crystals of the
PPARg receptor and a number of analogues within
this series. X-ray experiments show that these com-
pounds activate the receptor through interactions
with a novel bindingsite.
Type II diabetes is almost invariably accompanied by a
dyslipidemia which is highly atherogenic and thus
represents a major risk factor for the development of
premature atherosclerosis and coronary artery disease.
The findingthat the established hypolipidemic fibrate
drugs (e.g., bezafibrate and clofibrate) most probably
exert their effect by activation of the PPARa receptor
The Mitsunobu reaction4 and Suzuki coupling5 were
identified as suitable synthetic methods for variations
at the 5-position of BVT.142. The synthesis of aryl
ethers utilizingthe Mitsunobu reaction is outlined in
Scheme 1.
*Correspondingauthor. Tel.:+46-8-6973844; fax:+46-8-6973867;
e-mail: markus.thor@biovitrum.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00791-6

3566
M. Thor et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3565–3567
7. Palladium-catalyzed cross-couplingof 7 with an array
of aryl or heteroaryl boronic acids afforded biaryls 8a–e
or a mixture of 8a–e and the correspondingcyclized
benzoxazine equivalent. Subsequent ester hydrolysis
gave the target compounds 9a–e.
Utilizingthe intermediate 3, a number of diaryl ethers
have also been prepared as outlined in Scheme 3. Simple
nucleophilic aromatic substitution reactions of 2-chloro
nitrogen containing heterocycles yielded, after hydro-
lysis, compounds 10a–d.
Figure 1. The dual PPARa/g activator BVT.142.
Startingfrom commercially available 2-amino-5-
hydroxy benzoic acid 1 the correspondingmethyl ester 2
was formed by treatment with concentrated sulphuric
acid in methanol. Subsequent couplingof 2 with 2,4-
dichlorobenzoyl chloride provided the amide 3. The key
intermediate 3 was reacted with a set of alcohols in the
presence of 1,10 - azo - bis(N,N - dimethylformamide)
(TMAD) and polymer supported triphenylphosphine in
a mixture of dichloromethane and tetrahydrofuran to
give the adducts 4a–f. Ester hydrolysis, using1 M aqu-
eous lithium hydroxide, afforded the target compounds
5a–f as lithium salts.
The three different series of compounds have been
screened in a PPARa/g competitive bindingassay and a
cell-based reporter gene efficacy assay. The latter is
based on an expression vector containingthe liagnd
bindingdomain of the human PPAR a/g receptor and
the yeast transcription factor GAL4. Data for some
selected analogues is presented in Table 1.
The introduction of different substituents in the 5-posi-
tion of the BVT.142 scaffold allowed us to identify
compounds with an improved potency at the PPARa
and PPARg receptors. In general, it seems easier to gain
activity at PPARg within these three series although
dual activators are found in both the Mitsunobu and
Suzuki arrays. Aryl ethers 5a and 5b are significantly
more active on both receptors compared to the original
The synthesis of biaryl analogues was performed as
shown in Scheme 2. Couplingof the commercially
available methyl 2-amino-5-iodobenzoate
2,4-dichlorobenzoyl chloride provided the intermediate
6
with
Scheme 1. Preparation of aryl ethers 5a–f utilizingthe Mitsunobu reaction. Reagents: (a) H 2SO4, MeOH; (b) 2,4-dichlorobenzoyl chloride, pyridine;
(c) R-OH, TMAD, polymer supported PPh3, THF/DCM (1:1); (d) 1 M LiOH, THF.
Scheme 2. Preparation of biaryls 9a–e utilizing the Suzuki coupling. Reagents: (a) 2,4-dichlorobenzoyl chloride, diisopropylethylamine, THF;
(b) Pd(PPh3)4, Ar-B(OH)2, 2N Na2CO3, DME; (c) 1 M LiOH, dioxane.
Scheme 3. Preparation of diaryl ethers 10a–d. Reagents: (a) K2CO3, DMF, heat; (b) 1 M LiOH, THF.

M. Thor et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3565–3567
Table 1. In vitro bindingaffinity ( Ki) and potency (EC50) for selected
compounds
3567
Ki (mM)a,c
EC50 (mM)b,c,d
Compd
5-Substituent
PPARa PPARg PPARa PPARg
BVT.142
5a
5b
5c
5d
5e
5f
9a
9b
9c
9d
9e
10a
10b
10c
Methyl
30
25
20
>100
19
>100
>100
28
2.2
0.4
0.6
1
9.5
5
3.8
>100
2.5
>100
n.d.
12
10
>100
>100
1.6
0.3
0.8
0.7
0.6
0.7
3.2
0.7
1
2-Thienylmethoxy
2-(3-Thienyl)ethoxy
2-(2-Thienyl)ethoxy
OCH2CH2SCH3
4-Ethoxybenzyloxy
2-(2-Pyridinyl)ethoxy
3-Thienyl
1.6
3.6
1.5
0.9
0.5
1.2
0.7
1.4
0.18
0.17
0.1
0.16
2-Furyl
22
3-Ethoxyphenyl
8-Quinolinyl
3-Carboxyphenyl
5-Nitro-2-pyridinyloxy >100
3-Nitro-2-pyridinyloxy >100
>100
>100
67
0.3
0.3
>100 >100
>100
>100
>100
>100
0.4
0.4
1.3
0.45
2-Pyrimidinyloxy
6-Chloro-2-pyrazinyloxy
>100
21
10d
Figure 2. The bindingsite of 5a in the PPARg receptor.
aLigand binding affinities were determined by displacement of a tri-
tiated tracer by the unlabeled compound to a GST-PPAR fusion-pro-
tein, for PPARa, containingresidues 167–468 and 3H-GW2331 as
tracer, and for PPARg, containingresidues 204–477 and 3H-Rosigli-
tazone as tracer.
ligands such as rosiglitazone, which appear to activate
the PPARg receptor through interactions with amino
acids (e.g., His323, His449 and Tyr473) located deep
into the ligand binding pocket, compound 5a occupies a
region in proximity with helix 3. In Figure 2 the key
interaction between the carboxylic acid group of 5a and
the backbone nitrogen of Ser342, situated near the
entrance of the PPARg pocket, is shown.
bTransactivation potency was measured by luciferase activity in Caco-
2/TC7 cells transiently co-transfected with an expression vector for the
fusion-protein Gal4-PPARa (167–468) or Gal4-PPARg (204–477),
respectively, and a reporter vector containing4xGAL4RE-luciferase,
after 24 h of incubation with compounds.
cValues are the mean of at least two experiments, where nꢀ2, and
Xlfit version 2.0 (Business Solutions Limited) was used for curve fit-
tinganalysis.
dn.d.=not determined.
Other variations around BVT.142, such as carboxylic
acid isosters and various modifications of the
2,4-dichloro moiety, have also been investigated but are
not within the scope of this paper.
hit. Interestingly, compound 5c, which only differs from
5b in the position of the sulfur in the thiophene ring,
does not bind or activate the PPARa receptor but still
retains PPARg receptor activity. In general, analogues
containingan aromatic portion in the 5-substituent are
more active, however compound 5d shows that aliphatic
substituents could also be of interest. In the Suzuki
biaryl series, compounds 9a and 9b are dual PPARa/g
agonists while compounds such as 9c and 9d are potent
and highly selective PPARg agonists. From the same
series the PPARg antagonist 9e was also identified. The
analogues showing the highest binding affinities are the
diaryl ethers 10a–d. Interestingly, all four compounds,
prepared accordingto Scheme 3, are highly selective and
potent PPARg agonists. The data demonstrates the pos-
sibility to modify the effect on the PPARa and PPARg
receptors by variations in the 5-position of BVT.142.
References and Notes
1. Mangelsdorf, D. J.; Evans, R. M. Cell 1995, 83, 841.
2. King, A. B. Diabetes Care 2000, 23, 557.
3. Vu-Dac, N.; Chopin-Delannoy, S.; Gervois, P.; Bonnelye,
E.; Martin, G.; Fruchart, J. C.; Laudet, V.; Staels, B. J. Biol.
Chem. 1998, 273, 25713.
4. Hughes, D. L. In Organic Reactions; Paquette, L. A., Ed.;
John Wiley & Sons: New York, 1992; Vol. 42, pp 335–656.
5. Miyaura, N.; Suzuki, A. Chem. Rev. 1995, 95, 2457.
6. Nolte, R. T.; Wisely, G. B.; Westin, S.; Cobb, J. E.; Lam-
bert, M. H.; Kurokawa, R.; Rosenfeld, M. G.; Willson, T. M.;
Glass, C. K.; Milburn, M. V. Nature 1998, 395, 137.
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8. The ligand binding domain of hPPARg was produced in E.
coli and the protein was purified to homogeneity and con-
centrated to a final concentration of 10 mg/mL.9 Crystals of
PPARg-LBD complexes were grown by the hanging drop dif-
fusion method. A GRIP-1 derived co-activator peptide was
co-crystallized with the receptor. All data were collected at
room temperature usinga Rigaku RU300 rotatinganode and
an Raxis4 image plate detector. The crystals diffracted to 2.9
A resolution. A detailed description of the structure will be
published elsewhere.
It has been suggested that PPARg and other nuclear
receptors are activated by a common mechanism in which
the ligand stabilizes helix 12.6,7 This in turn allows for the
recruitment of co-activator proteins and subsequent
stimulation of transcriptional activity. We have been
able to obtain the structure of human PPARg LBD in
complex with the PPARg agonists 5a–b and 9a.8
The X-ray crystallographic data shows that these com-
pounds bind to a site different from the reported bind-
ingsite of the thiazolidinediones. In contrast to TZD
9. Uppenberg, J.; Svensson, C.; Jaki, M.; Bertilsson, G.; Jen-
deberg, L.; Berkenstam, A. J. Biol. Chem. 1998, 273, 31108.