Discovery of potent and selective agonists for the free fatty acid receptor 1 (FFA1/GPR40), a potential target for the treatment of type II diabetes

Article abstract of DOI:10.1021/jm8010178

A series of 4-phenethynyldihydrocinnamic acid agonists of the free fatty acid receptor 1 (FFA₁) has been discovered and explored. The preferred compound 20 (TUG-424, EC₅₀ = 32 nM) significantly increased glucose-stimulated insulin secretion at 100 nM and may serve to explore the role of FFA₁ in metabolic diseases such as diabetes or obesity.

Full text of DOI:10.1021/jm8010178

J. Med. Chem. 2008, 51, 7061–7064
7061
sustained enhancement of GSIS after prolonged exposure to
FFA1 agonists.12 Furthermore, two single nucleotide polymor-
phisms of FFA1 significantly correlating to obesity and impaired
insulin secretion further validate the link between the receptor
and the disease,14,15 although another study failed to establish
a disease link for the receptor.16
Discovery of Potent and Selective Agonists
for the Free Fatty Acid Receptor 1 (FFA1/
GPR40), a Potential Target for the
Treatment of Type II Diabetes
The implication of FFA1 in insulin secretion has attracted
considerable attention to the receptor as a new potential
therapeutic target for type II diabetes. Interestingly, it is still
unclear if agonists or antagonists are the desired therapeutic
principle. Although stimulation of FFA1 may acutely promote
GSIS, extended exposure of FFA1 to fatty acids might mediate
lipotoxicity and ꢀ-cell dysfunction. Steneberg and co-workers
found support for the latter view in favor of antagonists from
studies with FFA1 deficient mice;10 however, more recent studies
have challenged these results in favor of agonist therapy.12,13,17
Agonists and antagonists of FFA1 are therefore required to
resolve this controversy and to further validate FFA1 as an
antidiabetic target.
Drug discovery efforts have already resulted in published
FFA1 ligands. Recently, a series of agonists derived from
4-(benzylamino)dihydrocinnamic acid was described by
GlaxoSmithKline,18,19 of which compound GW9508 (4-(3-
phenoxybenzylamino)phenylpropionic acid, pEC50 ) 7.32)
appears to be the preferred one, even though it lacked activity
in primary rat or mouse islets and hence is of limited value for
in vivo evaluation of the function of FFA1.9 Other FFA1 agonists
and a few antagonists have also been disclosed, most of which
only exhibit potencies in the micromolar range.5,12,20,21
Most saturated and unsaturated fatty acids of 10 or more
carbon atoms exhibit some degree of agonist activity on
FFA1.4-6 The receptor thus appears to tolerantly recognize
relatively large, elongated lipophilic carboxylic acids. With this
in mind, conformationally constrained carboxylic acids more
suitable for optimization were screened in a calcium fluorescence
assay22 using 1321N1 cells stably expressing the human FFA1.
This resulted in identification of the alkyne 1 (Table 1) as a
full agonist 10-fold more potent than oleic acid. The compound
and analogues were synthesized via ethyl 4-iodophenoxyacetate
using the Sonogashira coupling reaction to assemble the
aromatic rings around the alkyne (Scheme 1). Removing the
terminal phenyl ring (2), moving the phenethynyl to the
3-position (3), or replacing the central triple bond by a double
bond to give stilbene-4-oxyacetic acid (4) led to complete loss
of activity, clearly demonstrating that 1 is in possession of
specific receptor recognizing properties rather than just emulat-
ing the general structure of a fatty acid (Chart 1).
Elisabeth Christiansen,† Christian Urban,‡ Nicole Merten,§
Kathrin Liebscher,# Kasper K. Karlsen,†
Alexandra Hamacher,‡ Andreas Spinrath,§ Andrew D. Bond,†
Christel Drewke,§ Susanne Ullrich,# Matthias U. Kassack,‡
Evi Kostenis,§ and Trond Ulven*,†
Department of Physics and Chemistry, UniVersity of Southern
Denmark, CampusVej 55, DK-5230 Odense M, Denmark,
Pharmaceutical Biochemistry, Institute of Pharmaceutical and
Medicinal Chemistry, UniVersity of Du¨sseldorf, UniVersitaetsstrasse
1, D-40225 Du¨sseldorf, Germany, Department of Molecular,
Cellular and Pharmacobiology Institute for Pharmaceutical Biology,
UniVersity of Bonn, Nussallee 6, 53115 Bonn, Germany, and
Department of Internal Medicine, DiVision of Endocrinology,
Diabetology, Vascular Medicine, Nephrology, and Clinical
Chemistry, UniVersity of Tuebingen, Otfried-Mu¨ller-Strasse 10,
D-72076 Tu¨bingen, Germany
ReceiVed August 12, 2008
Abstract: A series of 4-phenethynyldihydrocinnamic acid agonists of
the free fatty acid receptor 1 (FFA1) has been discovered and explored.
The preferred compound 20 (TUG-424, EC50 ) 32 nM) significantly
increased glucose-stimulated insulin secretion at 100 nM and may serve
to explore the role of FFA1 in metabolic diseases such as diabetes or
obesity.
Insulin is secreted from pancreatic ꢀ-cells in response to
elevated plasma glucose concentrations and restores the optimal
level by eliciting glucose uptake into cells and storage as
glycogen. Dysfunction in this mechanism leads to abnormal
plasma glucose levels, which is the hallmark of diabetes, a
disease currently afflicting 6% of the adult world population
and with increasing prevalence.1 Type II diabetes, accounting
for 85-95% of cases, is linked to obesity and is characterized
by improper insulin secretion and insulin resistance, often
culminating in the metabolic syndrome, a cluster of diseases
including diabetes, obesity, and hyperlipidaemia. Nutrients, like
fatty acids, have long been known for their capacity to amplify
insulin secretion, although the underlying mechanism has been
unclear. Importantly, amplification of GSISa by fatty acids is
operative in situations of ꢀ-cell compensation for insulin
resistance.2
The seven-transmembrane receptor FFA1 (GPR40/FFAR1)3
is highly expressed in pancreatic ꢀ-cells and is activated by
physiological concentrations of free fatty acids.4-6 Activation
of FFA1 enhances GSIS but does not affect insulin secretion at
low glucose concentrations.6-9 Enhancement of GSIS by FFA1
has been confirmed in vivo,10-13 and one study also observed
To investigate the influence of substituents on the terminal
phenyl ring, analogues with methyl groups introduced in each
position were synthesized (Scheme 1). Whereas a methyl
substituent in the ortho position (5) led to a moderate loss of
activity and a methyl in the para position (6) gave a dramatic
loss of activity, the introduction of a methyl in the meta position
(7) resulted in 2-fold increase in agonistic activity. The
introduction of polar substituents on the terminal phenyl ring
in all cases yielded compounds with deteriorated activity;
however, the meta substitution (8) was again favored over para
substitutions (9, 10).
* To whom correspondence should be addressed. Phone: +45 6550 2568.
Fax: +45 6615 8780. E-mail: ulven@ifk.sdu.dk.
†
University of Southern Denmark.
University of Du¨sseldorf.
University of Bonn.
University of Tuebingen.
‡
§
Propionate analogues were synthesized from dihydrocinnamic
acid by iodination and coupling to the arylethynyl moiety
directly or via introduction of the ethyne followed by coupling
#
a Abbreviations: FFA1/2/3, free fatty acid receptor 1/2/3; GSIS, glucose-
stimulated insulin secretion; DMR, dynamic mass redistribution.
10.1021/jm8010178 CCC: $40.75
2008 American Chemical Society
Published on Web 10/24/2008

7062 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22
Table 1. Activity of Alkyne Carboxylic Acids at hFFA1
Letters
Scheme 2a
pEC50
compd
X
R
(% max response)a
1
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
O
O
O
O
O
O
O
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
H
5.90 ( 0.04 (104)
5.43 ( 0.03 (96)
nab
2-Me
4-Me
3-Me
3-OH
4-OH
4-CH2OH
H
3-NH2
3-CH2OH
3-CHO
3-NO2
3-CN
3-CCH
3-CF3
3-Me
2-Me
4-Me
a Reagents and conditions: (a) I2, KIO3, H2SO4, AcOH, H2O, reflux, 5 h;
(b) MeOH, HCl (cat.), room temp, 2 h (33% over two steps after
recrystalization); (c) Me3SiCCH, Pd(PPh3)2Cl2, CuI, Et3N, DMF, 90 °C,
12 h; (d) K2CO3, MeOH, room temp, 21/2 h (90% over two steps); (e) aryl
halide, Pd(PPh3)2Cl2, CuI, Et3N, DMF, 50 °C, 12 h; (f) LiOH, H2O, dioxane,
room temp, 12 h; (g) ArCCH, CuI, Et3N, DMF, 50 °C, 12 h.
6.29 ( 0.03 (113)
5.25 ( 0.03 (86)
nab
nab
7.02 ( 0.02 (106)
5.96 ( 0.03 (96)
6.05 ( 0.03 (103)
6.45 ( 0.03 (94)
7.23 ( 0.02 (94)
7.04 ( 0.03 (92)
6.81 ( 0.05 (100)
7.05 ( 0.05 (98)
7.36 ( 0.13 (109)
7.49 ( 0.05 (105)
6.97 ( 0.03 (99)
7.06 ( 0.03 (108)
7.07 ( 0.03 (100)
7.13 ( 0.04 (101)
potent as the unsubstituted 11. Interestingly, the 3-nitrile
substituent (16) was tolerated with no change in potency,
whereas the more lipophilic 3-ethyne analogue 17 was somewhat
less potent. Likewise, the hydrophobic and electron withdrawing
3-trifluoromethyl substituent 18 displayed conserved potency
relative to 11, whereas the electron donating 3-methyl substituent
19 resulted in doubled potency. After exclusion of the sterically
slightly more demanding 17 and 18, an almost perfect correlation
between potency and lipophilicity of the meta substituted
compounds was observed.24
Moving the methyl to the ortho position (20) surprisingly
resulted in even higher potency (EC50 ) 32 nM), indicating
that structure-activity relationships are not directly transferable
from the phenoxyacetates to the dihydrocinnamates. The
4-methyl substituent led to a compound (21) equipotent with
11, compared with abolishment of activity for the corresponding
phenoxyacetate 6. Although the 2- and 3-methyl substituents
separately resulted in substantially increased potency, combining
both substituents in the same molecule (22) led to a compound
only equipotent with 11. The same was true for the sterically
similar 2-naphthyl compound (23), whereas the 3,5-dimethyl
modification (24) exhibited a modestly increased potency.
Altogether, the dihydrocinnamates appeared not only generally
more potent than the phenoxyacetates but also more tolerant to
diverse substituents on the terminal benzene ring. None of the
compounds exhibited any activity on the related receptors FFA2
(GPR43) and FFA3 (GPR41) or on nontransfected 1321N1 cells
(data not shown).
2,3-dimethyl
2,3-CHdCHCHdCH
3,5-dimethyl
a Maximal response was determined relative to the full FFA1 agonist
3-(4-(benzyloxy)phenyl)propanoic acid (TUG-20).23 b na: no activity up to
31.6 µM.
Scheme 1a
a Reagents and conditions: (a) K2CO3, acetone, room temp, 12-96 h;
(b) PhCCH, Pd(PPh3)4, CuI, DIPEA, DMF, room temp, 2 h; (c) (i)
Me3SiCCH, Pd(PPh3)4, CuI, DIPEA, DMF, room temp, 2 h; (ii) TBAF,
THF, room temp, 1 h; (d) aryl halide, Pd(PPh3)4, CuI, Et3N, DMF, 50 °C,
12 h; (e) LiOH, H2O, THF, room temp, 12 h.
Compound 20 was studied in a dynamic mass redistribution
(DMR) assay. Notably, this technology is capable of visualizing
all major G protein pathways in a single assay platform in real
time, yielding signaling pathway-specific optical signatures.25,26
In agreement with its reported coupling profile, the positive
DMR signals are well compatible with Gi- and Gq-signaling
pathway activation by FFA1.4,6 Biosensor recordings show a
clear concentration-response relationship with a significant
response occurring already at 1 nM (Figure 1). Treatment of
nontransfected cells with 10 µM 20 produced no response,
demonstrating that no signaling pathways or endogenously
expressed receptors are affected by the compound.
The insulinotropic effect of 20 was tested in the rat insulin
secreting cell line INS-1E.8,27 Increasing concentrations (100
nM to 10 µM) of 20 enhanced glucose-stimulated insulin
secretion significantly already at 100 nM and with a maximal
effect at 3 µM (Figure 2, top). The approximately 2-fold
stimulation of secretion by 20 in the presence of 12 mM glucose
is comparable to that induced by palmitate (400 µM in 0.4%
Chart 1. Structures Devoid of FFA1 Activity at 10 µM
to aryl halides (Scheme 2). The unsubstituted 4-phenethynyl-
cinnamic acid 11 exhibited a 14-fold increase in potency to give
a full agonist with an EC50 of 96 nM. Assuming structure-activity
relationships analogous to those of the phenoxyacetic acid
compounds, the initial focus was directed toward the meta
position of the terminal benzene ring. Introduction of polar
groups in general led to significant loss of potency (12-14),
whereas the more lipophilic 3-nitro analogue 15 was twice as

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22 7063
Figure 3. Crystal structure of 20.
represented by GW9508,9,18 and overlapping receptor interac-
tions at least for this part of the compound series may be
hypothesized. Using computational modeling and site-directed
mutagenesis, Tikhonova and co-workers suggested a model of
the interactions of GW9508 with FFA1.28 It is, however, not
possible to fit more rigid diphenylethynes described here in this
receptor model, which requires a folded conformation of
GW9508. As the binding mode of GW9508 has been validated
by site-directed mutagenesis, it appears natural to hypothesize
a distinct binding mode for 20, although it still seems reasonable
to presume a similar interaction for the carboxylic acid. The
crystal structure of 20 was obtained and shows a 50° twist
between planes of the two aromatic rings and the propionic acid
extended in the plane of the central ring (Figure 3). Other
conformations accessible by rotating around the alkyne and the
three rotatable bonds of the propionic acid may be as likely to
represent the receptor bound conformation as the one of the
crystal structure.
Figure 1. Sensograms of 20 from DMR assay. A pEC50 of 7.04 (
0.07 was derived from the optical recordings. ntc denotes nontransfected
cells.
In conclusion, we have identified a new series of 4-phen-
ethynyldihydrocinnamic acid agonists of FFA1 with no activity
on related receptors. The compound series exhibited high
tolerance to substituents around the terminal phenyl ring.
Introduction of a 2-methyl substituent on this ring produced
the full agonist 20, which exhibited an EC50 of 32 nM. The
compound enhanced glucose-stimulated insulin secretion in a
rat ꢀ-cell line already at 100 nM and from isolated mouse islets
through FFA1 and thus compares favorably to FFA1 agonists
described hitherto. This compound is expected to be useful in
the further exploration of FFA1 and may also be valuable as a
lead structure for new potential antidiabetic therapeutics.
Acknowledgment. The authors thank Ulrike Rick, Elisabeth
Metzinger, Sieglinde Haug, and Brian Gregersen for expert
technical assistance. We gratefully acknowledge Euroscreen for
kindly providing 1321N1 cells stably expressing FFA1, Prof.
C. B. Wollheim (University of Geneva, Switzerland) for
providing INS-1E cells, and Dr. Klaus Seuwen (Novartis Pharma
AG, Basel, Switzerland) for providing FFA1 knockout mice.
The study was supported by the Danish Natural Science
Research Council, the Danish Medical Research Council, and
DFG Grants UL140/7-1 and GRK677.
Figure 2. Activity of 20 in glucose-stimulated insulin secretion from
rat INS-1E cells (top) and islets isolated from wild-type or FFA1(-/
-) mice (bottom). Shown are mean values ( SEM of at least three
independent experiments: (/, //) significant effect to 2.8 mM glucose;
(§, §§) significant effect to 12 mM glucose; (/, §) p < 0.05; (//, §§)
p < 0.005.
Supporting Information Available: Synthetic procedures and
compound characterization data, purity data, procedures for calcium
assay, sensogram measurements, islet isolation, and insulin secretion
assays. This material is available free of charge via the Internet at
http://pubs.acs.org. Crystallographic data for 20 have been deposited
at the Cambridge Crystallographic Data Centre (CCDC 698298).
BSA) in the presence of the same glucose concentration (data
not shown). Interestingly, basal insulin secretion at 2.8 mM
glucose was slightly but significantly reduced by 20. Compound
20 was furthermore evaluated on islets isolated from wild-type
mice and exhibited significant enhancement of GSIS here also,
whereas lack of effect in islets isolated from their FFA1-deleted
litter mates demonstrates that the effect is mediated by FFA1
(Figure 2, bottom).
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7064 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22
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JM8010178