Identification of a potent and selective free fatty acid receptor 1 (FFA1/GPR40) agonist with favorable physicochemical and in vitro ADME properties

Article abstract of DOI:10.1021/jm2005699

The free fatty acid receptor 1 (FFA1, also known as GPR40) enhances glucose-stimulated insulin secretion from pancreatic β-cells and is recognized as an interesting new target for treatment of type 2 diabetes. Several series of selective FFA1 agonists are already known. Most of these are derived from free fatty acids (FFAs) or glitazones and are relatively lipophilic. Aiming for the development of potent, selective, and less lipophilic FFA1 agonists, the terminal phenyl of a known compound series was replaced by nitrogen containing heterocycles. This resulted in the identification of 37, a selective FFA1 agonist with potent activity on recombinant human FFA1 receptors and on the rat insulinoma cell line INS-1E, optimal lipophilicity, and excellent in vitro permeability and metabolic stability.

Full text of DOI:10.1021/jm2005699

ARTICLE
pubs.acs.org/jmc
Identification of a Potent and Selective Free Fatty Acid Receptor 1
(FFA1/GPR40) Agonist with Favorable Physicochemical and in Vitro
ADME Properties
Elisabeth Christiansen,† Christian Urban,‡ Manuel Grundmann,§ Maria E. Due-Hansen,† Ellen Hagesaether,†
Johannes Schmidt,§ Leonardo Pardo,|| Susanne Ullrich,^ Evi Kostenis,§ Matthias Kassack,‡ and
Trond Ulven*,†
†Department of Physics and Chemistry, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark
‡Institute of Pharmaceutical and Medicinal Chemistry, University of D€usseldorf, Universit€atsstrasse 1, D-40225 D€usseldorf, Germany
§Institute for Pharmaceutical Biology, University of Bonn, Nussallee 6, D-53115 Bonn, Germany
Laboratori de Medicina Computacional, Unitat de Bioestadística, Facultat de Medicina, Universitat Autꢀonoma de Barcelona,
E-08193 Bellaterra, Barcelona, Spain
^Department of Internal Medicine, Division of Endocrinology, Diabetology and Clinical Chemistry, University of T€ubingen,
Otfried-M€uller-Strasse 10, D-72076 T€ubingen, Germany
S Supporting Information
b
ABSTRACT: The free fatty acid receptor 1 (FFA1, also known as
GPR40) enhances glucose-stimulated insulin secretion from pan-
creatic β-cells and is recognized as an interesting new target for
treatment of type 2 diabetes. Several series of selective FFA1 agonists
are already known. Most of these are derived from free fatty acids
(FFAs) or glitazones and are relatively lipophilic. Aiming for the
development of potent, selective, and less lipophilic FFA1 agonists,
the terminal phenyl of a known compound series was replaced by
nitrogen containing heterocycles. This resulted in the identification
of 37, a selective FFA1 agonist with potent activity on recombinant
human FFA1 receptors and on the rat insulinoma cell line INS-1E,
optimal lipophilicity, and excellent in vitro permeability and meta-
bolic stability.
’ INTRODUCTION
FFA-mediated incretin secretion and therefore may potentially
enhance GSIS through a second mechanism.13 FFA1 has
attracted much interest as an antidiabetic target with the poten-
tial of increasing insulin secretion only when needed, and several
series of selective agonists and antagonists have already been
published (Chart 1).11,14ꢀ22 TAK-875 and AMG-837 are both
in clinical trials for treatment of type 2 diabetes.21,23
Lipophilicity and molecular size are factors that typically
increase during the ligand optimization process and that are
found to negatively influence pharmacokinetic properties and to
correlate with attrition.24ꢀ26 The concept of ligand efficiency
(LE), calculated as the estimated binding energy per non-
hydrogen atom, has recently become a popular measure to avoid
undue increase in molecular size in the optimization process.27
Lipophilicity, nominated as the most important druglike physical
property, is found to correlate with poor oral absorption, high
The free fatty acid receptor 1 (FFA1/GPR40)1 is highly
expressed in pancreatic β-cells and is activated by long-chain
free fatty acids (FFAs), resulting in enhancement of glucose-
stimulated insulin secretion (GSIS).2ꢀ4 It is well established that
FFAs enhance GSIS but also that prolonged exposure to FFAs
results in toxic effects on the β-cells. While an early study with
FFA1 knockout mice suggested that FFA1 also mediates the
lipotoxic effect of FFAs,5 several other studies involving knockout
mice or specific agonists have concluded that the enhancing effect
on GSIS but not the lipotoxic effect is mediated by FFA1.6ꢀ12
For example, Tan and co-workers observed that a selective FFA1
agonist improved glucose tolerance in diet-induced obese mice
and that this effect was maintained after 10 days of chronic
treatment.11 Moreover, the observations that the receptor is
coexpressed with various peptide hormones including the
incretins GLP-1 and GIP in endocrine cells of the gastrointestinal
tract and that FFA1 deficient mice exhibit reduced GLP-1
and GIP secretion suggest that FFA1 may also be involved in
Received: May 5, 2011
Published: August 22, 2011
r
2011 American Chemical Society
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Chart 1. Structures, Activities, and ClogP of Leading FFA1 Modulatorsa
aClogP values are calculated with ChemBioDraw 12.0 using the “ClogP” option.
Scheme 1a
promiscuity, high metabolic rate, toxicity, and attrition in clinical
phases.28 On the other hand, a too high hydrophilicity can result
in poor permeability and rapid renal clearance. A meta-analysis of
several studies on the correlation between lipophilicity and
ADME properties, toxicity, promiscuity, and attrition concludes
that the optimal lipophilicity when all factors are taken into
account lies within the narrow log D7.4 range between 1 and 3.29
The nature of the FFAs indicates that the orthosteric binding site
of FFA1 would be lipophilic, and compounds published hitherto,
often derived from FFAs or glitazones, are indeed on the
lipophilic side (Chart 1). We therefore aimed to develop potent
FFA1 agonists with lower lipophilicity. Replacing a phenyl by
pyridyl or a similar heterocycle generally results in significant
reduction of lipophilicity. Thus, rather than extending the
structure with polar substituents, we decided to investigate the
effect of replacing the terminal phenyl of our recently published
4-alkynyldihydrocinnamic acid series of FFA1 agonists by more
hydrophilic nitrogen-containing heterocycles.17
a Reagents and conditions: (a) pyridyl halide, method A (Pd(PPh3)2Cl2,
CuI, Et3N, DMF, 50 ꢀC), method B (Na2PdCl4, PIntB, CuI, TMEDA,
80 ꢀC), or method C (Na2PdCl4, PIntB, CuI, TMEDA, H2O, 80 ꢀC);
(b) LiOH, THF, H2O, room temp; (c) 2,4-dichloropyrimidine, Pd-
(PPh3)2Cl2, PPh3, CuI, THF, Et3N, reflux; (d) NaOMe, MeOH,
room temp.
’ RESULTS AND DISCUSSION
however failed in the coupling with pyrimidyl chlorides. In these
cases, a protocol by Deng and co-workers worked satisfactorily,
although yields were low.32
The compounds were synthesized from the previously de-
scribed central alkyne intermediate 130 by Sonogashira coupling
with pyridyl halides (Scheme 1). We initially used a classical
Sonogashira protocol (method A), but 1 proved to be a challen-
ging substrate and we observed varying results and frequently
failed reactions. A protocol reported by Beller and co-workers
(method B)31 provided the desired product in many cases where
the original protocol failed but still had limitations, in particular
with aryl iodides. We developed this method further, using water
together with TMEDA as reaction medium, which solved the
problem for the iodo substrates and turned out as a highly
efficient and reliable protocol for Sonogashira coupling with pyridyl
bromides and iodides (Scheme 1, method C).30 The protocol
As only a few of the desired halopyridines were commercially
available, additional required halopyridines (27, 30, 32, 34, 38)
were synthesized using LDA promoted halogen migration, also
referred to as the “halogen dance”, followed by quenching with
water or electrophiles (Scheme 2).33,34 The 2-fluoropyridyl inter-
mediate 21a was converted to the corresponding methoxy and
phenoxy analogues by nucleophilic aromatic fluoride substitution
(Scheme 3), and the 2-chloropyrimidine alkyne 12 was likewise
converted to the corresponding 2-methoxy analogue 13 (Scheme 1).34
The 2-chloropyridine methyl ester 22a was converted further to
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Scheme 2a
Scheme 4a
a Reagents and conditions: (a) KIO3, I2, H2SO4, H2O, AcOH, reflux;
(b) MeOH, HCl (cat.), room temp; (c) trimethylsilylacetylene,
Na2PdCl4, PIntB, CuI, TMEDA, H2O, 80 ꢀC, then K2CO3, MeOH,
room temp; (e) 2,6-dichloro-4-iodopyridine, Na2PdCl4, PIntB, CuI,
TMEDA, 80 ꢀC; (e) LiOH, H2O, THF, room temp.
a Reagents and conditions: (a) LDA, electrophile (H2O/MeI/allyl
bromide/BnBr), THF, ꢀ78 ꢀC.
presence of the ortho-nitrogen also found to be unfavorable in 3.
The direction of the nitrogen electron lone-pairs on the terminal
ring appears to have significant influence on activity. The
5-thiazolyl analogue (16), with a lone-pair vector intermediate
of 4 and 5, exhibited an activity similar to that of 4-pyridyl 5. To
investigate the effect of the sulfur atom of 16, the corresponding
2-thienyl analogue 17 and the 3-methyl-2-thienyl 18, correspond-
ing to the previously reported TUG-424 (19),17 were synthe-
sized. The compounds exhibited activities similar to those of 2
and 19, respectively, but did not result in improved activity. As
sterically unhindered pyridines are known to frequently interact
with CYP enzymes, we directed the focus toward compounds
with 2-substituted pyridines. Methyl (20), fluoro (21), chloro
(22), and methoxy (23) substituted analogues of 5 were
explored, of which 22 stood out as the more potent.
Scheme 3a
Assuming that the central alkyne of this compound series
corresponds to the methyleneamino linkers found in GW950814,15
and TUG-469 (Chart 1),22 the m-phenoxypyridyl 24, the biphenyl
25, and the 20-methylbiphenyl 26 were synthesized and tested.
The increased size was, however, not accompanied by increased
activity, resulting in pronounced lower LE values. The lower
activity of 26 compared with 25 demonstrates that the structureꢀ
activity relationships of the methyleneamino compounds cannot
be transferred directly to the present series.
a Reagents and conditions: (a) NaOMe, MeOH, reflux or PhOH,
K2CO3, DMSO, 100 ꢀC; (b) LiOH, THF, H2O, room temp;
(c) arylboronic acid, Pd(OAc)2, SPhos, K3PO4, PhMe, 100 ꢀC.
biphenyl analogues by Suzuki cross-couplings (Scheme 3). The
racemic cyclopropyl carboxylic acid 43 was synthesized as out-
lined in Scheme 4.
Compounds were tested in a functional calcium mobilization
assay on 1321N1 cells stably transfected with the human FFA1
receptor. Replacing the terminal phenyl of parent compound 2
by 2-pyridyl (3) resulted in a significant drop of activity (Table 1). A
terminal 3-pyridyl (4) gave a less pronounced drop in activity,
whereas the 4-pyridyl (5) was best tolerated. Some activity could
be regained for the 2-pyridyl analogue by introducing a meta-
methyl substituent (relative to the alkyne attachment) on the
pyridine ring (6). Similarly, chloro (8 and 9) or methyl and
fluoro (10) substituents on the 3-pyridyl brought the activity
back to around pEC50 = 6.2. Ligand efficiencies were closely
monitored in the optimization process, and calculated log P
values (ClogP) were used as a convenient guide of lipophilicity,
despite the fact that such values often are inaccurate.35 As
expected, an estimated decrease in lipophilicity on the order of
1 log P unit was observed upon substitution of the terminal
phenyl by pyridyl (Table 1).
The 2-fluoro (21) and 2-chloro (22) analogues appeared as the
most promising for further optimization. The o-methyl analogues 28,
29, and 31 showed increased potency, paralleling the SARs of 2 and
19. The larger 3-allyl 33 and 3-benzyl 35 resulted in reduced activity
relative to 31, whereas chloroquinoline 36 exhibited increased activity.
The 2,6-dichloropyridine 37 provided a significant leap in
activity up to pEC50 = 7.39, making the compound equipotent
with 19. Introducing an o-methyl substituent (39) did not result
in further improvement. Conformational constraint of the pro-
pionate chain by a cyclopropyl has been reported to improve the
activity of the GW9508 series;15 however, a similar constraint in
43 did not improve the activity relative to 37.
The preferred compound 37 exhibited >100-fold selectivity
over the related receptors FFA2 and FFA3, which was expected
despite their high homology with FFA1 because the receptors are
activated by short-chain FFAs and other small carboxylic acids
but not by FFAs with more than six carbon atoms or larger
carboxylic acids.36 The compound furthermore exhibited >100-
fold selectivity over the nuclear receptor PPARγ and 55 other
diverse receptors, ion channels, and transporters. Compounds 37
A terminal pyrimidine ring further decreased the lipophilicity
of the compounds. However, none of the analogues explored
(12ꢀ15) exhibited the desired potency, probably because of the
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Table 1. Agonist Activities of Alkynedihydrocinnamic Acids with Heterocyclic Western Parts on FFA1
a Efficacy is given as % maximal response relative to 10 μM 3-(4-benzyloxyphenyl)propionic acid (TUG-20).22 All values were obtained in the same
laboratory under identical conditions. b Ligand efficiencies (LE) were calculated by ꢀΔG = RT ln KD, presuming that EC50 ≈ KD. Values are given in kcal
molꢀ1 per non-hydrogen atom.27 c Calculated by ChemBioDraw Ultra 12.0 with the “ClogP” option. d Previously reported (pEC50 = 7.49 ( 0.05,
obtained in a different laboratory).17 e Experimentally determined log D (n-octanol/water, pH 7.4).
and the previously published 19 were also evaluated in a panel of
in vitro ADME assays. Whereas 19 only exhibited moderate
stability toward human liver microsomes, 37 displayed excellent
properties in all assays (Table 2). Notably, 37 has good aqueous
solubility and moderate lipophilicity with a log D7.4 in the middle
of the ideal range. The compound also demonstrated high
chemical stability, no inhibition of selected CYP enzymes or
P-glycoprotein, and excellent Caco-2 permeability.
The permeability of 37 was, moreover, evaluated using the
mucus-secreting cell-line HT29-MTX,37 more closely mimicking
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the intestinal epithelium. The compound was found to have equal or
higher permeablility than ketoprofen, which is known to be rapidly
and completely absorbed (37, Papp = (1.64 ( 0.08) ꢁ 10ꢀ5 cm/s,
63% recovered; ketoprofen, Papp = (1.51 ( 0.05) ꢁ 10ꢀ5 cm/s,
80% recovered).
The activity of 37 on FFA1 transfected HEK-293 cells was
investigated in a dynamic mass redistribution (DMR) assay,
enabling label-free real-time monitoring of cell activity.38 The
compound produced robust and potent activity in FFA1 trans-
fected HEK cells, and the resulting concentration effect curve was
superimposable with that obtained with the inositol monopho-
sphate (IP1) accumulation assay (Figure 1). The DMR assay was
furthermore used to evaluate the activity of 37 on insulin
secreting INS-1E cells. The compound again produced a potent
and robust response in the cells. The activity could be blocked with
the FFA1-selective antagonist 44 (see Supporting Information),20
demonstrating that it is mediated through FFA1. The response
translates to enhanced insulin secretion by 60% at high glucose level
but does not, as expected, significantly affect insulin secretion at low
glucose level (Figure 2).
The complex of 37 with human FFA1 was modeled using the
crystal structure of the β2-adrenergic receptor (β2AR, PDB code
2RH1)39 as template (see Experimental Section). However,
hFFA1 contains the Ser-x-Pro60II:18/2.58 motif in TM2, homo-
logous to the Thr-x-Pro92II:18/2.58 motif of the crystal structure
of chemokine CXCR4 (PDB code 3OE0)40 that induces a tight
Table 2. Physicochemical and in Vitro ADME Properties of
19 and 37
assay
19
37
aqueous solubility (PBS, pH 7.4)
log D (n-octanol/PBS, pH 7.4)
chemical stability (PBS, 37 ꢀC, 12 days)
plasma protein binding (human)
metabolic stability (human liver S9)
CYP inhibition (10 μM)
CYP1A2
174 μM
2.34
199 μM
1.97
99.9%
97.7%
26%
99.9%
96.9%
97%
ꢀ8%
ꢀ25%
ꢀ1%
0%
ꢀ7%
ꢀ4%
ꢀ7%
5%
CYP2C9
CYP1C19
CYP2D6
CYP3A4
ꢀ4%
6.6%
87 ꢁ
5%
P-gp inhibition (100 μM)
Caco-2 (A to B, TC7, pH 6.5/7.4)
ꢀ1.9%
112 ꢁ
10ꢀ6 cm/s
10ꢀ6 cm/s
Figure 1. Activity of 37 on FFA1 transfected HEK-293 cells and on the rat β-cell line INS-1E that endogenously express FFA1. (A) Representative
traces from the dynamic mass redistribution (DMR) assay of 37 on HEK-FFA1 cells. (B) Dose response curves of 37 on FFA1-HEK cells from the DMR
assay (pEC50 = 7.06 ( 0.08, n = 3) and an inositol phosphate assay (pEC50 = 7.05 ( 0.06, n = 5). (C) Traces from the DMR assay of 37 on INS-1E cells.
(D) Dose response curve of 37 on INS-1E cells (pEC50 = 6.63 ( 0.07, n = 4) and inhibition of 37 (0.3 μM) by the FFA1 antagonist trans-1-oxo-3-(4-
phenoxyphenyl)-2-propyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (44, pIC50 = 5.10 ( 0.07, n = 4).
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helical turn of TM2. Thus, TM2 of hFFA1 was modeled in the
conformation observed in CXCR4, resulting in ∼120ꢀ rotation
of its extracellular end compared with other 7TM receptor
structures. This rotation positions Lys62II:20/2.60 toward
the TM bundle. The model depicted in Figure 3 shows the
carboxylic acid of 37 anchored between Arg183V:05/5.39 and
Arg258VII:02/7.35, and the rigid body of the ligand was extended
toward TM2 in close contact with all helices except TM1 and
TM4. The central benzene ring forms aromaticꢀaromatic inter-
actions with Phe87III:09/3.33, and a twist of almost 90ꢀ between
the aromatic rings places the pyridyl favorably for interaction with
His86III:08/3.32. This mode of binding of 37 explains the observed
structureꢀactivity relationships. The o-methyl analogues (19,
28, 29, 31) at the terminal aromatic ring are favored because
of the presence of the hydrophobic Leu262VII:06/7.39. Accord-
ingly, the presence of the polar o-nitrogen (2-pyridyl) in 3 is
unfavorable. In contrast, the presence of a polar binding pocket
within TMs 2, 3, and 7, formed by Lys62II:20/2.60, His86III:02/3.32
,
and Lys259VII:03/7.36, permits replacement of the terminal phenyl
ring by 4-pyridyl and addition of chloro or fluoro substituents.
The progressively reduced activity of 33 and 35 compared to 31
is explained by increased steric interactions of the allyl and benzyl
substituents with Leu262VII:06/7.39 and TM7, leading to displace-
ment of the ligands toward TM3. Docking of cyclopropyl
analogue 43 resulted in a conformation similar to that of the
unconstrained 37 but with a slightly bent ligand, explaining that its
activity is preserved but not increased. Moreover, the >100-fold
selectivity of compound 37 over the highly homologous FFA2
and FFA3 receptors is attributed to the fact that these receptors
replace the polar His86III:08/3.32 by Phe and the positively charged
Lys259VII:03/7.36 by Ser or Ile, respectively (see Figure S2 in the
Supporting Information).
’ CONCLUSION
With the aim of developing potent FFA1 agonists with
reduced lipophilicity and with our previously described alkyne
ligands as starting point,17 we have identified a new series of
pyridyl FFA1 agonists. The preferred compound 37 exhibits
potent full agonist activity on FFA1 and produces a potent effect
in insulin secreting INS-1E cells. The compound is highly
selective, is stable toward human liver microsomes, and does
not inhibit any of the most important CYP enzymes. The
lipophilicity of the compound is in the ideal range, and in vitro
assays indicate excellent penetration. The compound therefore
appears as a useful tool for in vivo exploration of FFA1 and as a
lead for development of new therapeutics for treatment of type 2
diabetes.
Figure 2. Effect of 37 on glucose-stimulated insulin secretion. Mean (
SE (n = 8) values are shown (///, p < 0.005, to 2.8 mM glucose; ##, p <
0.05, to 12 mM glucose).
Figure 3. Model of 37 in complex with the human FFA1. Residues in direct contact with the ligand are labeled with sequence number and with
SchwartzꢀBaldwin41 and BallesterosꢀWeinstein42 notations given as superscript.
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’ EXPERIMENTAL SECTION
3-(4-(Pyridin-2-ylethynyl)phenyl)propanoic Acid (3). 3 was
prepared from 3a (54 mg, 0.20 mmol) by general procedure II to give
39 mg (77%) of a yellow solid: Rf = 0.16 ([EtOAc with 1.25% AcOH]/
petroleum ether, 1:1); 1H NMR (CDCl3) δ 8.64ꢀ8.63 (m, 1H),
7.74ꢀ7.68 (m, 1H), 7.63ꢀ7.49 (m, 3H), 7.29ꢀ7.21 (m, 3H), 3.00
(t, J = 7.8 Hz, 2H), 2.71 (t, J = 7.8 Hz, 2H); 13C NMR (CDCl3) δ 177.0,
149.4, 142.9, 141.9, 136.8, 132.2, 128.4, 127.2, 122.9, 119.9, 90.3, 87.6,
35.2, 30.6; MALDIꢀHRMS calcd for C16H13NO2 (M+) 252.1019,
found 252.1008.
All commercially available starting materials and solvents were used
without further purification, unless otherwise stated. THF was freshly
distilled from sodium/benzophenone. DMF, Et3N, and TMEDA were
dried over 4 Å sieves. Purification by flash chromatography was carried
out using silica gel 60 (0.040ꢀ0.063 mm, Merck). TLC analysis was
performed on silica gel 60 F254 plates. 1H and 13C NMR spectra were
calibrated relative to TMS internal standard or residual solvent peak.
High-resolution mass spectra (HRMS) were obtained on Thermo
Finnigan TSQ 700 using electrospray ionization (ESI), Bruker micrO-
TOF-Q II (ESI) or IonSpec 4.7 T Ultima FTMS using DHB matrix
(MALDI). Electron ionization mass spectra were obtained on a Thermo
Finnigan SSQ 710 (EI). Purity was determined by HPLC and confirmed
by inspection of NMR spectra. All test compounds were of g95% purity
unless otherwise stated. HPLC analysis was performed using a Dionex
120 C18 column (5 μm, 4.6 mm ꢁ 150 mm) with 10% acetonitrile in
water (0ꢀ1 min), 10ꢀ100% acetonitrile in water (1ꢀ10 min), 100%
acetonitrile (11ꢀ15 min), both solvents containing 0.05% TFA as
modifier, with a flow of 1 mL/min and UV detection at 230 and 254 nm.
General Procedure I-A: Sonogashira Coupling. A flame-
dried Schlenk flask charged with alkyne (1 equiv), pyridyl halide
(1.1 equiv), CuI (2 mol %), Et3N (2.4 equiv), and DMF (2 mL/mmol)
was evacuated and backfilled with inert gas three times before addition of
Pd(PPh3)2Cl2 (1 mol %) under nitrogen flow. The mixture was heated
to 50 ꢀC and stirred until consumption of the alkyne as indicated by
TLC, typically overnight, then cooled to room temperature. Water was
added, and the aqueous layer was extracted with EtOAc. The combined
organic phases were washed with brine, dried (MgSO4), and concentrated.
General Procedure I-B: Sonogashira Coupling31. A flame-
dried Schlenk flask filled with argon was charged with Na2PdCl4 (1 mol %),
2-(di-tert-butylphosphino)-N-phenylindole (PIntB, 2 mol %), CuI
(2 mol %), alkyne (1 equiv), pyridyl halide (1.1ꢀ1.5 equiv), and
TMEDA (2 mL/mmol). The reaction mixture was stirred at 80 ꢀC until
consumption of the alkyne, typically overnight, then cooled to room
temperature. Water was added, and the aqueous layer was extracted with
EtOAc. The combined organic phases were washed with brine, dried
(MgSO4), and concentrated.
Methyl 3-(4-(Pyridin-3-ylethynyl)phenyl)propanoate (4a).
4a was prepared from 130 (102 mg, 0.54 mmol) and 3-iodopyridine
(122 mg, 0.60 mmol) by general procedure I-A to give 27 mg (20%
isolated yield, 43% based on recovered starting material) of an orange
brown solid after purification by flash chromatography (SiO2, EtOAc/
1
petroleum ether, 1:3): Rf = 0.09 (EtOAc/petroleum ether, 1:5); H
NMR (CDCl3) δ 8.76ꢀ7.75 (m, 1H), 7.54ꢀ7.52 (m, 1H), 7.81ꢀ7.77
(m, 1H), 7.48ꢀ7.45 (m, 2H), 7.29ꢀ7.25 (m, 1H), 7.22ꢀ7.19 (m, 2H),
3.67 (s, 3H), 2.98 (t, J = 7.8 Hz, 2H), 2.65 (t, J = 7.8 Hz, 2H); 13C NMR
(CDCl3) δ 173.0, 152.2, 148.4, 141.5, 138.3, 131.8, 128.4, 123.0, 120.5,
120.4, 92.5, 85.7, 51.6, 35.3, 30.8; EI-MS m/z 265 (M+)
3-(4-(Pyridin-3-ylethynyl)phenyl)propanoic Acid (4). 4 was
prepared from 4a (24 mg, 0.09 mmol) by general procedure II to give
18 mg (79%) of an orange solid: Rf = 0.16 ([EtOAc with 1.25% AcOH]/
petroleum ether, 1:1); 1H NMR (acetone-d6) δ 12.60ꢀ10.70 (br s, 1H),
8.73ꢀ8.72 (m, 1H), 8.57ꢀ8.55 (m, 1H), 7.93ꢀ7.89 (m, 1H), 7.52ꢀ
7.49 (m, 2H), 7.44ꢀ7.40 (m, 1H), 7.36ꢀ7.33 (m, 2H), 2.96 (t, J =
7.8 Hz, 2H), 2.65 (t, J = 7.8 Hz, 2H); 13C NMR (acetone-d6) δ 173.8,
152.6, 149.6, 143.4, 139.1, 132.5, 129.6, 124.3, 121.1, 121.0, 93.2, 86.3,
35.5, 31.4; MALDI-HRMS calcd for C16H13NO2 (M+) 252.1019, found
252.1015.
Methyl 3-(4-(Pyridin-4-ylethynyl)phenyl)propanoate (5a).
5a was prepared from 130 (94 mg, 0.50 mmol) and 4-bromopyridine
hydrochloride (108 mg, 0.55 mmol) by general procedure I-B to give
72 mg (54%) of a white solid after purification by flash chromatography
(SiO2, EtOAc/petroleum ether, 1:5): Rf = 0.26 (EtOAc/petroleum
ether, 1:1); 1H NMR (CDCl3) δ 8.52 (dd, J = 4.5, 1.5 Hz, 2H),
7.45ꢀ7.39 (m, 2H), 7.29 (dd, J = 4.5, 1.6 Hz, 2H), 7.14 (d, J = 8.3 Hz,
2H), 3.60 (s, 3H), 2.90 (t, J = 7.7 Hz, 2H), 2.57 (t, J = 7.7 Hz, 2H); 13C
NMR (CDCl3) δ 172.0, 148.8, 141.0, 131.0, 130.5, 127.5, 124.5, 119.0,
92.9, 85.5, 50.7, 34.3, 29.8; ESI-MS m/z 288.1 (M + Na+).
3-(4-(Pyridin-4-ylethynyl)phenyl)propanoic Acid (5). 5 was
prepared from 5a (63 mg, 0.24 mmol) by general procedure II to give
18 mg (30%) of a white solid: Rf = 0.04 (EtOAc/petroleum ether, 1:1);
1H NMR (DMSO-d6) δ 12.15 (s, 1H), 8.62 (dd, J = 4.6, 1.3 Hz, 2H),
7.58ꢀ7.46 (m, 4H), 7.33 (d, J = 8.1 Hz, 2H), 2.87 (t, J = 7.5 Hz, 2H),
2.57 (t, J = 7.6 Hz, 2H); 13C NMR (DMSO-d6) δ 173.6 149.9, 142.9,
131.7, 130.3, 128.8, 125.3, 118.8, 93.7, 86.3, 34.7, 30.2; ESI-HRMS calcd
for C16H14NO2 (M + H+) 252.1019, found 252.1016.
Methyl 3-(4-((2-Chloropyridin-3-yl)ethynyl)phenyl)pro-
panoate (8a). 8a was prepared from 130 (95 mg, 0.50 mmol) and
3-bromo-2-chloropyridine (106 mg, 0.55 mmol) by general procedure
I-C to give 113 mg (75%) of a pale yellow oily product after purification
by flash chromatography (SiO2, EtOAc/petroleum ether, 1:8): Rf = 0.24
(EtOAc/petroleum ether, 1:2); 1H NMR (CDCl3) δ 8.33 (dd, J = 4.8,
1.9 Hz, 1H), 7.84 (dd, J = 7.7, 1.9 Hz, 1H), 7.54ꢀ7.47 (m, 2H),
7.26ꢀ7.19 (m, 3H), 3.68 (s, 3H), 2.98 (t, J = 7.7 Hz, 2H), 2.65 (t, J =
7.7 Hz, 2H); 13C NMR (CDCl3) δ 173.0, 152.3, 148.1, 142.0, 141.1,
132.0, 128.5, 121.9, 120.7, 120.2, 96.8, 84.0, 51.7, 35.3, 30.9; ESI-MS m/
z 322.1 (M + Na+).
3-(4-((2-Chloropyridin-3-yl)ethynyl)phenyl)propanoic Acid
(8). 8 was prepared from 8a (89 mg, 0.30 mmol) by general procedure II
to give 79 mg (92%) of a white solid: tR = 11.21 min (HPLC); 1H NMR
(DMSO-d6) δ 12.17 (s, 1H), 8.43 (dd, J = 4.8, 1.9 Hz, 1H), 8.12 (dd,
J = 7.7, 1.9 Hz, 1H), 7.56ꢀ7.46 (m, 3H), 7.34 (d, J = 8.3 Hz, 2H), 2.87
General Procedure I-C: Sonogashira Coupling30. A Schlenk
flask filled with argon was charged with Na2PdCl4 (1 mol %), PIntB
(2 mol %), CuI (2 mol %), alkyne (1 equiv), pyridyl halide (1.1ꢀ1.5 equiv),
TMEDA (1.8 mL/mmol), and water (0.2 mL/mmol). The reaction
mixture was stirred at 80 ꢀC until consumption of the alkyne, typically
5ꢀ30 min, then cooled to room temperature. Water was added, and the
aqueous layer was extracted with EtOAc. The combined organic phases
were washed with brine, dried (MgSO4), and concentrated.
General Procedure II: Ester Hydrolysis. The ester (1 equiv)
dissolved in THF (∼5 mL/mmol) was added to LiOH H2O (2ꢀ3 equiv)
3
in H2O(∼2 mL/mmol). The mixture was stirred at room temperature until
complete consumption of the starting material, as indicated by TLC,
typically after 1ꢀ12 h. To the mixture was added water. The mixture was
acidified with 3% HCl until pH < 1, and the aqueous layer was extracted
with EtOAc. The combined extracts were washed with brine, dried
(MgSO4), and concentrated under vacuum.
Methyl 3-(4-(Pyridin-2-ylethynyl)phenyl)propanoate (3a).
3a was prepared from methyl 3-(4-ethynylphenyl)propanoate30 (1, 99 mg,
0.52 mmol) and 2-iodopyridine (0.07 mL, 0.66 mmol) by general pro-
cedure I-A to give 69 mg (50%) of a pale yellow solid after purification by
flash chromatography (SiO2, EtOAc/petroleum ether, 1:3): Rf = 0.09
(EtOAc/petroleum ether, 1:5); 1H NMR (CDCl3) δ 8.62ꢀ8.60 (m, 1H),
7.70ꢀ7.64 (m, 1H), 7.54ꢀ7.49 (m, 3H), 7.25ꢀ7.18 (m, 3H), 3.67 (s, 3H),
2.97 (t, J = 7.8 Hz, 2H), 2.64 (t, J = 7.8 Hz, 2H); 13C NMR (CDCl3) δ
173.0, 150.0, 143.5, 141.6, 136.1, 132.2, 128.4, 127.1, 122.6, 120.2, 89.2,
88.4, 51.6, 35.2, 30.8; EIꢀMS m/z 265 (M+).
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(t, J = 7.5 Hz, 2H), 2.57 (t, J = 7.6 Hz, 2H); 13C NMR (DMSO-d6) δ
173.5, 150.7, 148.9, 142.8, 141.9, 131.4, 128.8, 123.0, 119.3, 118.8, 96.4,
83.8, 34.6, 30.1; ESI-HRMS calcd for C16H12ClNO2Na (M + Na+)
308.0449, found 308.0464.
Methyl 3-(4-((2-Methylpyridin-4-yl)ethynyl)phenyl)pro-
panoate (20a). 20a was prepared from 130 (95 mg, 0.50 mmol) and
4-iodo-2-methylpyridine (0.07 mL, 0.59 mmol) by general procedure
I-B to give 62 mg (44%) of a pale yellow solid after purification by flash
chromatography (SiO2, EtOAc/petroleum ether, 1:5): Rf = 0.50 (EtOAc/
petroleum ether, 1:2); 1H NMR (CDCl3) δ 8.48ꢀ8.46 (m, 1H),
7.48ꢀ7.45 (m, 2H), 7.25ꢀ7.20 (m, 4H), 3.67 (s, 3H), 2.98 (t, J =
7.8 Hz, 2H), 2.64 (t, J = 7.8 Hz, 2H), 2.56 (s, 3H); 13C NMR (CDCl3) δ
173.1, 158.6, 149.3, 142.0, 132.1, 131.8, 128.6, 125.1, 122.7, 120.3, 93.4,
86.9, 51.8, 35.4, 31.0, 24.5; ESI-MS m/z 302.1 (M + Na+).
Methyl 3-(4-((6-Chloropyridin-3-yl)ethynyl)phenyl)pro-
panoate (9a). 9a was prepared from 130 (94 mg, 0.50 mmol) and
2-chloro-5-iodopyridine (133 mg, 0.55 mmol) by general procedure I-C
to give 107 mg (72%) of a white solid product after purification by flash
chromatography (SiO2, EtOAc/petroleum ether, 1:8): Rf = 0.35
1
(EtOAc/petroleum ether, 1:2); H NMR (CDCl3) δ 8.52 (dd, J =
2.3, 0.6 Hz, 1H), 7.74 (dd, J = 8.3, 2.3 Hz, 1H), 7.50ꢀ7.43 (m, 2H), 7.31
(dd, J = 8.3, 0.7 Hz, 1H), 7.24ꢀ7.18 (m, 2H), 3.67 (s, 3H), 2.98 (t, J =
7.7 Hz, 2H), 2.64 (t, J = 7.7 Hz, 2H); 13C NMR (CDCl3) δ 173.0, 152.0,
150.3, 141.8, 140.8, 131.9, 128.5, 123.9, 120.1, 119.5, 93.7, 84.5, 51.7,
35.3, 30.8; ESI-MS m/z 322.1 (M + Na+).
3-(4-((6-Chloropyridin-3-yl)ethynyl)phenyl)propanoic Acid
(9). 9 was prepared from 9a (80 mg, 0.27 mmol) by general procedure II
to give 74 mg (100%) of a white solid: tR = 11.73 min (HPLC); 1H NMR
(DMSO-d6) δ 12.17 (s, 1H), 8.60 (dd, J = 2.4, 0.6 Hz, 1H), 8.03 (dd,
J = 8.3, 2.4 Hz, 1H), 7.59 (dd, J = 8.3, 0.6 Hz, 1H), 7.55ꢀ7.47 (m, 2H),
7.36ꢀ7.29 (m, 2H), 2.87 (t, J = 7.5 Hz, 2H), 2.57 (t, J = 7.6 Hz, 2H); 13C
NMR (DMSO-d6) δ 173.5, 151.8, 149.5, 142.6, 141.7, 131.4, 128.7,
124.3, 119.0, 118.8, 93.5, 84.4, 34.7, 30.1; ESI-HRMS calcd for
C16H12ClNO2Na (M + Na+) 308.0449, found 308.0463.
3-(4-((2-Methylpyridin-4-yl)ethynyl)phenyl)propanoic Acid
(20). 20 was prepared from 20a (55 mg, 0.20 mmol) by general
procedure II to give 58 mg (70%) of a white solid: Rf = 0.25 ([EtOAc
with 1.25% AcOH]/petroleum ether, 1:1); 1H NMR (DMSO-d6) δ 8.74
(d, J = 7.6 Hz, 1H), 8.01 (s, 1H), 7.90ꢀ7.88 (m, 1H), 7.61ꢀ7.59 (m,
2H), 7.39ꢀ7.37 (m, 2H), 2.89 (t, J = 7.4 Hz, 2H), 2.73 (s, 3H), 2.58 (t,
J = 7.6 Hz, 2H); 13C NMR (DMSO-d6) δ 173.4, 154.0, 144.3, 141.5,
138.3, 132.2, 129.1, 128.7, 125.5, 117.7, 100.2, 85.5, 34.6, 30.3, 19.3; ESI-
HRMS calcd for C17H15NO2Na (M + Na+) 288.0996, found 288.0989.
Methyl 3-(4-((2-Fluoropyridin-4-yl)ethynyl)phenyl)pro-
panoate (21a). 21a was prepared from 130 (352 mg, 1.87 mmol) and
4-bromo-2-fluoropyridine (210 μL, 2.04 mmol) by general procedure
I-C to give 388 mg (73%) of a pale yellow solid after purification by flash
chromatography (SiO2, EtOAc/petroleum ether, 1:4): Rf = 0.32 (EtOAc/
petroleum ether, 1:2); 1H NMR (CDCl3) δ 8.19 (d, J = 5.2 Hz, 1H),
7.51ꢀ7.45 (m, 2H), 7.26ꢀ7.20 (m, 3H), 7.04ꢀ6.99 (m, 1H), 3.68 (s,
3H), 2.99 (t, J = 7.7 Hz, 2H), 2.65 (t, J = 7.7 Hz, 2H); 13C NMR
(CDCl3) δ 173.0, 163.8 (d, J = 239.4 Hz), 147.7 (d, J = 16.2 Hz), 142.4,
136.5 (d, J = 9.1 Hz), 132.2, 128.6, 123.4 (d, J = 4.0 Hz), 119.6, 111.4 (d,
J = 39.4 Hz), 95.2, 85.5 (d, J = 5.1 Hz), 51.7, 35.2, 30.9; ESI-MS m/z
306.1 (M + Na+).
3-(4-Ethynylphenyl)propanoic Acid (11). 11 was prepared
from 130 (188 mg, 1.00 mmol) by general procedure II to give 165 mg
1
(95%) of a pale yellow solid: tR = 12.53 min (HPLC); H NMR
(CDCl3) δ 11.44 (s, 1H), 7.47ꢀ7.39 (m, 2H), 7.21ꢀ7.12 (m, 2H),
3.05 (s, 1H), 2.96 (t, J = 7.7 Hz, 2H), 2.68 (t, J = 7.7 Hz, 2H); 13C
NMR (CDCl3) δ 178.6, 141.0, 132.4, 128.3, 120.2, 83.5, 77.0, 35.2,
30.4; ESI-HRMS calcd for C11H10O2Na (M + Na+) 197.0574, found
197.0570.
3-(4-((2-Fluoropyridin-4-yl)ethynyl)phenyl)propanoic Acid
(21). The title compound was prepared from 21a (70 mg, 0.25 mmol)
3-(4-((2-Chloropyrimidin-4-yl)ethynyl)phenyl)propanoic
Acid (12). A flame-dried Schlenk flask was charged with Pd(PPh3)2Cl2
(3 mg, 0.004 mmol, 0.5 mol %), PPh3 (2 mg, 0.008 mmol, 1 mol %),
THF (1 mL), Et3N (1.5 mL), and 2,4-dichloropyrimidine (146 mg,
0.98 mmol) under a continuous N2 flow. The solution was bubbled with
N2 for 15 min, and to it were added CuI (2 mg, 0.01 mmol, 1 mol %) and
11 (152 mg, 0.87 mmol). The reaction mixture was heated to reflux until
consumption of the starting material, typically overnight, then cooled
to room temperature. Water and aqueous HCl (1 M) were added, and
the aqueous layer was extracted with EtOAc. The combined organic
phases were washed with brine, dried (MgSO4), and concentrated.
The residue was recrystallized from AcOH/acetone (1:1) to give
71 mg (28%) of an orange solid: tR = 10.73 min (HPLC); 1H NMR
(DMSO-d6) δ 12.19 (s, 1H), 8.83 (d, J = 5.1 Hz, 1H), 7.76 (d, J =
5.1 Hz, 1H), 7.67ꢀ7.58 (m, 2H), 7.43ꢀ7.33 (m, 2H), 2.89 (t, J = 7.5
Hz, 2H), 2.58 (t, J = 7.5 Hz, 2H); 13C NMR (DMSO-d6) δ 173.5,
160.9, 160.1, 152.2, 144.2, 132.3, 129.0, 122.5, 117.3, 95.3, 85.5, 34.5,
30.2; ESI-HRMS calcd for C15H11ClN2O2Na (M + Na+) 309.0402,
found 309.0400.
3-(4-((2-Methoxypyrimidin-4-yl)ethynyl)phenyl)propanoic
Acid (13). To a dry round bottomed flask with a magnetic stirring bar
and argon atmosphere were added 12 (86 mg, 0.30 mmol) and NaOMe
in MeOH (0.67 mmol, 2 mL, 0.336 M). The mixture was stirred
overnight. Water and aqueous HCl (1 M) were added, and the aqeuous
layer was extracted with EtOAc. The organic phases were combined,
washed with brine, dried (MgSO4), and concentrated to give 77 mg
(23%) of a white solid: tR = 10.18 min (HPLC); 1H NMR (DMSO-d6) δ
12.19 (s, 1H), 8.66 (d, J = 4.9 Hz, 1H), 7.63ꢀ7.54 (m, 2H), 7.40ꢀ7.30
(m, 3H), 3.94 (s, 3H), 2.88 (t, J = 7.5 Hz, 2H), 2.58 (t, J = 7.5 Hz, 2H);
13C NMR (DMSO-d6) δ 173.5, 165.0, 160.3, 151.4, 143.7, 132.1, 128.9,
117.7, 117.5, 92.9, 86.5, 54.6, 34.5, 30.2; ESI-HRMS calcd for
C16H14N2O3Na (M + Na+) 305.0897, found 305.0911.
by general procedure II to give 61 mg (92%) of a white solid: tR
=
11.18 min (HPLC); 1H NMR (acetone-d6) δ 10.66 (s, 1H), 8.27 (d, J =
5.1 Hz, 1H), 7.58ꢀ7.51 (m, 2H), 7.45ꢀ7.35 (m, 3H), 7.20 (s, 1H), 2.98
(t, J = 7.6 Hz, 2H), 2.66 (t, J = 7.6 Hz, 2H); 13C NMR (acetone-d6) δ
173.7, 164.8 (d, J = 236.3 Hz), 149.0 (d, J = 16.2 Hz), 144.3, 137.2 (d, J =
10.1 Hz), 132.9, 129.8, 124.4 (d, J = 4.0 Hz), 120.1, 111.9 (d, J = 40.4
Hz), 95.8, 86.0 (d, J = 6.1 Hz), 35.4, 31.4; ESI-HRMS calcd for
C16H12FNO2Na (M + Na+) 292.0745, found 292.0745.
Methyl 3-(4-((2-Chloropyridin-4-yl)ethynyl)phenyl)pro-
panoate (22a). 22a was prepared from 130 (104 mg, 0.55 mmol)
and 2-chloro-4-iodopyridine (141 mg, 0.59 mmol) by general procedure
I-A to give 96 mg (58%) of a pale yellow solid after purification by flash
chromatography (SiO2, EtOAc/petroleum ether, 1:5): Rf = 0.22
1
(EtOAc/petroleum ether, 1:5); H NMR (CDCl3) δ 8.36ꢀ8.35 (m,
1H), 7.48ꢀ7.45 (m, 2H), 7.42ꢀ7.41 (m, 1H), 7.29ꢀ7.26 (m, 1H),
7.24ꢀ7.21 (m, 2H), 3.67 (s, 3H), 2.99 (t, J = 7.8 Hz, 2H), 2.65 (t, J =
7.8 Hz, 2H); 13C NMR (CDCl3) δ 172.9, 151.7, 149.5, 142.4, 134.4, 132.1,
128.6, 125.9, 124.1, 95.4, 85.3, 51.8, 35.2, 30.8; EI-MS m/z 299 (M+).
3-(4-((2-Chloropyridin-4-yl)ethynyl)phenyl)propanoic Acid
(22). The title compound was prepared from 22a (73 mg, 0.24 mmol)
by general procedure II to give 62 mg (90%) of a pale yellow solid: Rf =
1
0.09 (EtOAc/petroleum ether, 1:1); H NMR (DMSO-d6) δ 12.17
(s, 1H), 8.46ꢀ8.45 (m, 1H), 7.69ꢀ7.68 (m, 1H), 7.55ꢀ7.53 (m, 3H),
7.36ꢀ7.33 (m, 2H), 2.88 (t, J = 7.8 Hz, 2H), 2.58 (t, J = 7.8 Hz, 2H); 13C
NMR (DMSO-d6) δ 173.5, 150.6, 150.2, 143.3, 133.6, 131.8, 128.8,
125.4, 124.6, 95.3, 85.1, 34.6, 30.2; MALDI-HRMS calcd for C16H12ClNO2
(M+) 286.0629, found 286.0626.
4-Bromo-2-fluoro-5-methylpyridine (27). To a flame-dried
round-bottom flask equipped with a magnetic stirring bar filled with
argon was added dry THF (5 mL). The mixture was cooled to ꢀ78 ꢀC.
LDA (1.8 M in THF/heptanes/ethylbenzene, 0.8 mL) was added, and
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the mixture was stirred for 10 min before dropwise addition of the
3-bromo-2-fluoro-5-methylpyridine (274 mg, 1.44 mmol) in dry THF
(1.4 mL). The mixture was stirred for 1 h before addition of water
(0.5 mL, 31.3 mmol). Stirring was continued overnight. The reaction
was quenched with water/THF (1:1, 1 mL), and the mixture was
allowed to reach 0 ꢀC before addition of water (2 mL) and extraction
with EtOAc. The combined organic phases were washed with brine,
dried (MgSO4), and concentrated. Purification by flash chromatography
(SiO2, EtOAc/petroleum ether, 1:8) provided 257 mg (94%) of 27: Rf =
0.37 (EtOAc/petroleum ether, 1:4); 1H NMR (CDCl3) δ 8.04 (s, 1H),
7.16 (d, J = 2.9 Hz, 1H), 2.35 (s, 3H); 13C NMR (CDCl3) δ 161.9 (d, J =
241.4 Hz), 147.4 (d, J = 15.2 Hz), 138.0 (d, J = 9.1 Hz), 131.6 (d, J =
5.1 Hz), 113.0 (d, J = 40.4 Hz), 18.6.
(CDCl3) δ 8.22ꢀ8.16 (m, 1H), 7.52ꢀ7.45 (m, 2H), 7.28 (d, J =
5.0 Hz, 1H), 7.25ꢀ7.20 (m, 2H), 3.68 (s, 3H), 2.99 (t, J = 7.7 Hz, 2H),
2.65 (t, J = 7.7 Hz, 2H), 2.57 (s, 3H); 13C NMR (CDCl3) δ 173.0, 152.1,
146.3, 142.3, 133.9, 133.5, 132.0, 128.6, 124.7, 119.9, 98.4, 85.2, 51.7,
35.3, 30.9, 17.9; ESI-MS m/z 336.1 (M + Na+).
3-(4-((2-Chloro-3-methylpyridin-4-yl)ethynyl)phenyl)pro-
panoic Acid (31). 31 was prepared from 31a (71 mg, 0.23 mmol) by
general procedure II to give 58 mg (85%) of a white solid: tR = 12.08 min
(HPLC); 1H NMR (DMSO-d6) δ 12.18 (s, 1H), 8.30ꢀ8.24 (m, 1H),
7.59ꢀ7.53 (m, 2H), 7.53ꢀ7.49 (m, 1H), 7.38ꢀ7.31 (m, 2H), 2.88 (t, J =
7.5 Hz, 2H), 2.57 (t, J = 7.6 Hz, 2H), 2.52 (s, 3H); 13C NMR (DMSO-
d6) δ 173.5, 151.0, 146.8, 143.2, 133.1, 132.6, 131.7, 128.8, 125.0, 118.5,
98.3, 84.7, 34.6, 30.2, 17.5; ESI-HRMS calcd for C17H14ClNO2Na (M +
Na+) 322.0606, found 322.0599.
Methyl 3-(4-((7-Chloroquinolin-4-yl)ethynyl)phenyl)pro-
panoate (36a). 36a was prepared from 130 (54 mg, 0.29 mmol) and
7-chloro-4-iodoquinoline (93 mg, 0.32 mmol) by general procedure I-A
to give 54 mg (54%) of a beige solid after purification by flash chro-
matography (SiO2, EtOAc/petroleum ether, 1:4): Rf = 0.06 (EtOAc/
petroleum ether, 1:4); 1H NMR (CDCl3) δ 8.88 (d, J = 4.8 Hz, 1H),
8.27 (d, J = 9.0 Hz, 1H), 8.11 (m, 1H), 7.59ꢀ7.52 (m, 4H), 7.28ꢀ7.25
(m, 2H), 3.69 (s, 3H), 3.01 (t, J = 7.8 Hz, 2H), 2.67 (t, J = 7.8 Hz, 2H);
13C NMR (CDCl3) δ 172.9, 150.8, 148.5, 142.4, 135.8, 132.1, 129.9,
128.8, 128.6, 128.1, 127.4, 126.1, 123.5, 119.9, 99.2, 84.4, 51.7, 35.2,
30.9; ESI-MS m/z 372.1 (M + Na+).
Methyl 3-(4-((2-Fluoro-5-methylpyridin-4-yl)ethynyl)phenyl)-
propanoate (28a). 28a was prepared from 130 (95 mg, 0.51 mmol)
and 27 (153 mg, 0.81 mmol) by general procedure I-C to give 83 mg
(55%) of a pale yellow solid after purification by flash chromatography
(SiO2, EtOAc/petroleum ether, 1:4): Rf = 0.29 (EtOAc/petroleum
1
ether, 1:2); H NMR (CDCl3) δ 8.06 (s, 1H), 7.52ꢀ7.45 (m, 2H),
7.26ꢀ7.21 (m, 2H), 6.98 (d, J = 2.0 Hz, 1H), 3.68 (s, 3H), 2.99 (t, J =
7.7 Hz, 2H), 2.65 (t, J = 7.7 Hz, 2H), 2.43 (s, 3H); 13C NMR (CDCl3)
δ 173.0, 162.1 (d, J = 237.4 Hz), 147.4 (d, J = 15.2 Hz), 142.4, 136.1 (d,
J = 9.1 Hz), 132.1, 131.9 (d, J = 4.0 Hz), 128.6, 119.8, 110.7 (d, J =
39.4 Hz), 98.7, 84.7 (d, J = 5.1 Hz), 51.7, 35.3, 30.9, 16.8; ESI-MS m/z
320.1 (M + Na+).
3-(4-((2-Fluoro-5-methylpyridin-4-yl)ethynyl)phenyl)pro-
panoic Acid (28). 28 was prepared from 28a (67 mg, 0.23 mmol) by
general procedure II to give 63 mg (98%) of a white solid: tR = 11.66 min
(HPLC); 1H NMR (DMSO-d6) δ 12.19 (s, 1H), 8.20 (s, 1H),
7.59ꢀ7.52 (m, 2H), 7.38ꢀ7.33 (m, 2H), 7.31 (d, J = 2.1 Hz, 1H),
2.87 (d, J = 7.5 Hz, 2H), 2.57 (t, J = 7.6 Hz, 2H), 2.41 (s, 3H); 13C NMR
(DMSO-d6) δ 173.5, 161.5 (d, J = 234.3 Hz), 147.5 (d, J = 16.2 Hz),
143.2, 135.3 (d, J = 10.1 Hz), 132.1 (d, J = 5.1 Hz), 131.7, 128.8, 118.5,
110.3 (d, J = 39.4 Hz), 98.7, 84.3, 34.6, 30.2, 16.2; ESI-HRMS calcd for
C17H14FNO2Na (M + Na+) 306.0902, found 306.0906.
Methyl 3-(4-((2-Fluoro-3-methylpyridin-4-yl)ethynyl)phenyl)-
propanoate (29a). 29a was prepared from 130 (95 mg, 0.50 mmol)
and 2-fluoro-4-iodo-3-methylpyridine34 (134 mg, 0.56 mmol) by gen-
eral procedure I-C to give 106 mg (71%) of a pale yellow solid after
purification by flash chromatography (SiO2, EtOAc/petroleum ether,
1:5): Rf = 0.37 (EtOAc/petroleum ether, 1:2); 1H NMR (CDCl3) δ 8.00
(d, J = 5.1 Hz, 1H), 7.52ꢀ7.46 (m, 2H), 7.25ꢀ7.19 (m, 3H), 3.68 (s,
3H), 2.99 (t, J = 7.7 Hz, 2H), 2.65 (t, J = 7.7 Hz, 2H), 2.43 (s, 3H); 13C
NMR (CDCl3) δ 173.0, 162.4 (d, J = 238.4 Hz), 144.1 (d, J = 16.2 Hz),
142.3, 135.9 (d, J = 7.1 Hz), 132.0, 128.6, 123.6 (d, J = 4.0 Hz), 121.1 (d,
J = 33.3 Hz), 119.9, 98.5, 84.6 (d, J = 6.1 Hz), 51.7, 35.3, 30.9, 12.6; ESI-
MS m/z 320.1 (M + Na+).
3-(4-((2-Fluoro-3-methylpyridin-4-yl)ethynyl)phenyl)pro-
panoic Acid (29). 29 was prepared from 29a (81 mg, 0.27 mmol) by
general procedure II to give 72 mg (93%) of a pale yellow solid: tR =
11.63 min (HPLC); 1H NMR (DMSO-d6) δ 12.19 (s, 1H), 8.09 (d, J =
5.1 Hz, 1H), 7.59ꢀ7.53 (m, 2H), 7.43 (d, J = 5.0 Hz, 1H), 7.37ꢀ7.32
(m, 2H), 2.88 (t, J = 7.5 Hz, 2H), 2.57 (t, J = 7.6 Hz, 2H), 2.39 (s, 3H);
13C NMR (DMSO-d6) δ 173.5, 161.6 (d, J = 234.3 Hz), 144.5 (d, J =
16.2 Hz), 143.2, 134.9 (d, J = 7.1 Hz), 131.7, 128.8, 123.7 (d, J = 4.0 Hz),
120.3 (d, J = 34.3 Hz), 118.5, 98.5, 84.2 (d, J = 6.1 Hz), 34.6, 30.2, 12.3;
ESI-HRMS calcd for C17H14FNO2Na (M + Na+) 306.0902, found
306.0893.
Methyl 3-(4-((2-Chloro-3-methylpyridin-4-yl)ethynyl)phenyl)-
propanoate (31a). 31a was prepared from 130 (94 mg, 0.50 mmol)
and 4-bromo-2-chloro-3-methylpyridine43 (30, 114 mg, 0.55 mmol) by
general procedure I-C to give 105 mg (67%) of a pale yellow oily product
after purification by flash chromatography (SiO2, EtOAc/petroleum
ether, 1:10): Rf = 0.28 (EtOAc/petroleum ether, 1:2); 1H NMR
3-(4-((7-Chloroquinolin-4-yl)ethynyl)phenyl)propanoic Acid
(36). The title compound was prepared from 36a (50 mg, 0.14 mmol)
by general procedure II to give 43 mg (90%) of a beige solid: Rf = 0.12
([EtOAc with 1.25% AcOH]/petroleum ether, 1:2); 1H NMR (DMSO-
d6) δ 12.20 (s, 1H), 8.96 (d, J = 4.5 Hz, 1H), 8.37 (d, J = 8.7 Hz, 1H),
8.15ꢀ8.14 (m, 1H), 7.78ꢀ7.74 (m, 2H), 7.68 (d, J = 8.1 Hz, 2H), 7.39
(d, J = 8.1 Hz, 2H), 2.91 (t, J = 7.5 Hz, 2H), 2.60 (t, J = 7.7 Hz, 2H); 13C
NMR (DMSO-d6) δ 173.6, 151.5, 147.9, 143.4, 134.8, 132.0, 128.9,
128.6, 128.3, 128.2, 127.8, 125.4, 124.0, 118.6, 99.3, 84.0, 34.7, 30.3; ESI-
HRMS calcd for C20H14ClNO2Na (M + Na+) 336.0786, found
336.0785.
3-(4-((2,6-Dichloropyridin-4-yl)ethynyl)phenyl)propanoic
Acid (37). The title compound was prepared from methyl 3-(4-((2,6-
dichloropyridin-4-yl)ethynyl)phenyl)propanoate30 (296 mg, 0.90 mmol)
by general procedure II to give 280 mg (99%) of a white solid: Rf = 0.54
([EtOAc with 1.25% AcOH]/petroleum ether, 1:1); 1H NMR (CDCl3)
δ 7.50ꢀ7.45 (m, 2H), 7.34 (s, 2H), 7.27ꢀ7.22 (m, 2H), 3.00 (t, J = 7.6
Hz, 2H), 2.71 (t, J = 7.6 Hz, 2H); 13C NMR (CDCl3) δ 177.7, 150.7,
142.5, 136.5, 132.3, 128.7, 124.4, 119.3, 96.8, 84.5, 34.9, 30.5; ESI-
HRMS calcd for C16H11Cl2NO2Na (M + Na+) 342.0060, found
342.0067.
trans-2-(4-Iodophenyl)cyclopropanecarboxylate (41). To
a solution of concentrated H2SO4 (0.1 mL) in water (1 mL) and AcOH
(4 mL) were added 2-phenylcyclopropanecarboxylic acid (40, 200 mg,
1.23mmol), iodine (173mg, 0.68 mmol), and KIO3 (61 mg, 0.28 mmol).
The reaction mixture was heated to reflux, and to it was added AcOH
(6 mL) in portions of 2 mL to rinse iodine from the condenser. After
4 h, when no further color changes appeared, the mixture was cooled to
room temperature. The reaction was quenched with 1 M Na2S2O4,
and water was added. The mixture was extracted with EtOAc, and
the combined extracts were washed with brine, dried (MgSO4), and
concentrated. The crude product was recrystallized from petroleum
ether to provide 157 mg (44%) of 41 as a white solid: tR = 11.33 min
(HPLC); 1H NMR (CDCl3) δ 7.62ꢀ7.58 (m, 2H), 6.87ꢀ6.84 (m, 2H),
2.56ꢀ2.51 (m, 1H), 1.89ꢀ1.84 (m, 1H), 1.69ꢀ1.64 (m, 1H),
1.39ꢀ1.34 (m, 1H); 13C NMR (CDCl3) δ 179.3, 139.2, 137.6, 128.3,
91.7, 26.5, 23.9, 17.4; ESI-MS m/z 311.0 (M + Na+).
Methyl 2-(4-Ethynylphenyl)cyclopropanecarboxylate (42).
Methanol (16 mL) under argon at 0 ꢀC was added to AcCl (1.3 mL,
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Journal of Medicinal Chemistry
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18.3 mmol). The mixture was stirred for 10 min before slow addition of 41
(1.65 g, 5.74 mmol). The mixture was stirred for an additional 3 h at room
temperature before the mixture was concentrated under vacuum, redis-
solved in MeOH, and concentrated to give 1.68 g (97%) of the methyl ester
as a pure white solid: tR = 13.24 min (HPLC).
A Schlenk flask charged with the methyl ester (2.56 g, 8.47 mmol),
Na2PdCl4 (25 mg, 0.08 mmol), 2-(di-tert-butylphosphino)-1-phenylin-
dole (PIntB, 57 mg, 0.17 mmol), CuI (32 mg, 0.17 mmol), water
(1.7 mL), and TMEDA (15.3 mL) was evacuated and backfilled with
argon three times and heated to 70 ꢀC. Trimethylsilylacetylene (2.1 mL,
16.4 mmol) was added, and the temperature was elevated to 80 ꢀC. After
30 min, consumption of the starting material was confirmed by HPLC,
and the reaction mixture was cooled to room temperature. Water was
added, and the aqueous layer was extracted with EtOAc. The combined
extracts were washed with brine, dried (MgSO4), concentrated, and used
directly in the next step: tR = 14.56 min (HPLC).
high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemen-
ted with 10% FCS, 100 U/mL penicillin, 100 μg/mLstreptomycin, 100 μg/
mL hygromycin B, and 15 μg/mL blasticidin (InvivoGen, Toulouse,
France). Expression of FFA1 from the Flp-In locus was induced by
treatment with 1 μg/mL doxycycline (Sigma-Aldrich, Taufkirchen,
Germany) for 16 h. All cells were cultivated with 5% CO2 at 37 ꢀC in
a humidified atmosphere.
Calcium Mobilization Assays. Calcium measurements were
performed using a NOVOstar microplate reader with a built-in pipetor
(BMG LabTech, Offenburg, Germany). Cells were seeded in 96-well
tissue-culture plates at a density of 30000ꢀ40000 cells per well. After
24 h, cells were washed twice in Krebs-HEPES buffer (KHB, 118.6 mM
NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 4.2 mM NaHCO3, 11.7 mM
D-glucose, 10 mM HEPES (free acid), 1.3 mM CaCl2, and 1.2 mM
MgSO4, pH 7.4) and loaded with 1.5 μM Oregon Green 488 BAPTA-1/
AM (Molecular Probes, Eugene, OR) and 0.03% Pluronic F-127
(Invitrogen, Karlsruhe, Germany) for 1 h (37 ꢀC, 5% CO2). After
addition of KHB, microplates were directly transferred to Novostar and
kept at 37 ꢀC under exclusion of light for 15 min until the measurement
was started. For testing of agonists, 20 μL of a 10-fold concentrated test
compound solution was injected sequentially into separate wells and
fluorescence wasmeasured at 520 nm (bandwidth 25 nm) for50 intervals
of 0.4 s each. The excitation wavelength was 485 nm (bandwidth 25 nm).
Label-Free Dynamic Mass Redistribution (DMR) Assay. A
beta version of the label-free Corning Epic biosensor was used consisting
of an optical detection unit, a temperature-control unit, and an inte-
grated robotic liquid handling device. The method has been validated
recently in Schr€oder et al.38 Refractive waveguide grating optical bio-
sensors, integrated in Epic 384-well microplates (Corning, NY, U.S.),
allow extremely sensitive measurements of the index of refraction in a
detecting zone 150 nm above the surface of the sensor. Mass movements
of cellular constituents (dynamic mass redistribution, DMR) induced
upon GPCR activation can be detected by illuminating the underside of
the microplate with broadband light. The Epic instrument measures
changes in wavelength of the outgoing light that is a sensitive function of
the index of refraction. The magnitude of this wavelength shift (in
picometers) is directly proportional to the amount of DMR. At 24 h
before the assay INS-1E and FFA1-HEK cells were seeded onto
fibronectin-coated 384-well Epic sensor microplates at a density of
30 000 (INS-1E) and 15 000 (FFA1-HEK) cells/well and cultured for
20ꢀ24 h (37 ꢀC, 5% CO2) to obtain confluent monolayers. After
removal of medium, cells were washed twice with Hank’s buffered salt
solution (HBSS) containing 20 mM HEPES and kept for 1 h in the Epic
reader at a constant temperature of 28 ꢀC. Hereafter, the sensor plate
was scanned and a baseline optical signature was recorded. Compound
solutions were then transferred into the sensor plate, and DMR was
monitored for at least 5000 s.
To methyl 3-(4-((trimethylsilyl)ethynyl)phenyl)cyclopropanecar-
boxylate (2.24 g, 8.47 mmol) and potassium carbonate (2.34 g, 16.95 mmol)
was added MeOH (85 mL), and the mixture was stirred vigorously for
1 h at room temperature. To the mixture was added water, and the
aqueous layer was extracted with EtOAc. The organic phases were
combined, washed with brine, dried (MgSO4), and concentrated. The
residue was purified by flash chromatography (SiO2, EtOAc/petroleum
ether, 1:5) to give 1.59 g (97%) of 42 as a pale yellow solid: Rf = 0.60
(EtOAc/petroleum ether, 1:1); 1H NMR (CDCl3) δ 7.43ꢀ7.37
(m, 2H), 7.07ꢀ7.01 (m, 2H), 3.72 (s, 3H), 3.06 (s, 1H), 2.51 (ddd, J =
9.2, 6.5, 4.2 Hz, 1H), 1.91 (ddd, J = 8.5, 5.4, 4.2 Hz, 1H), 1.63 (ddd, J = 9.2,
5.3, 4.7 Hz, 1H), 1.32 (ddd, J = 8.4, 6.5, 4.7 Hz, 1H); 13C NMR (CDCl3) δ
173.5, 141.0, 132.3, 126.1, 120.2, 83.4, 77.2, 52.0, 26.1, 24.2, 17.2.
Methyl 2-(4-((2,6-Dichloropyridin-4-yl)ethynyl)phenyl)-
cyclopropanecarboxylate (43a). 43a was prepared from 42 (80 mg,
0.40 mmol) and 2,6-dichloro-4-iodopyridine (121 mg, 0.44 mmol) by
general procedure I-B to give 19 mg (23%) of a white solid after purification
by flash chromatography (SiO2, EtOAc/petroleum ether, 1:10): Rf = 0.36
(EtOAc/petroleum ether, 1:4); 1H NMR (CDCl3) δ 7.45 (d, J = 8.2 Hz,
2H), 7.34 (s, 2H), 7.11 (d, J = 8.2 Hz, 2H), 3.73 (s, 3H), 2.61ꢀ2.48 (m,
1H), 2.02ꢀ1.90 (m, 1H), 1.67 (dt, J = 10.0, 5.1 Hz, 1H), 1.35 (ddd, J = 8.4,
6.4, 4.8 Hz, 1H); 13C NMR (CDCl3) δ 173.4, 150.7, 142.5, 136.5, 132.2,
126.4, 124.4, 119.2, 96.7, 84.7, 52.1, 26.1, 24.4, 17.3; ESI-MS m/z368.1 (M +
Na+).
2-(4-((2,6-Dichloropyridin-4-yl)ethynyl)phenyl)cyclopro-
panecarboxylic Acid (43). 43 was prepared from 43a (16 mg,
0.05 mmol) by general procedure II to give 14 mg (88%) of a white solid:
Rf = 0.37 ([EtOAc with 1.25% AcOH]/petroleum ether, 1:1); 1H NMR
(acetone-d6) δ 7.47ꢀ7.40 (m, 4H), 7.17 (d, J = 8.3 Hz, 2H), 2.43ꢀ2.33
(m, 1H), 1.84 (ddd, J = 9.3, 5.2, 4.2 Hz, 1H), 1.44 (dt, J = 9.6, 4.9 Hz,
1H), 1.35ꢀ1.29 (m, 1H); 13C NMR (acetone-d6) δ 173.9, 151.2, 144.2,
137.7, 133.1, 127.4, 125.4, 119.9, 97.5, 85.3, 26.5, 25.0, 17.7; ESI-HRMS
calcd for C18H13Cl2NO2Na (M + Na+) 354.0060, found 354.0055.
Materials, Cell Culture, and Cell Lines. Tissue culture media
reagents were purchased from Invitrogen, Karlsruhe, Germany, unless
otherwise explicitly specified. 1321N1 cells stably transfected with
human FFA1 were grown in Dulbecco’s modified Eagle’s medium
supplemented with 10% (v/v) heat-inactivated fetal calf serum, 1%
sodium pyruvate, 100 U/mL penicillin, 100 μg/mL streptomycin, and
400 μg/mL G418. Rat INS-1E cells for DMR studies were cultivated in
RPMI 1640+ GlutaMAXꢀI medium supplemented with 10% (v/v) fetal
calf serum (FCS), 100 U/mL penicillin, 100 μg/mL streptomycin,
and 50 μM 2-mercaptoethanol, and INS-1E cells for insulin secretion
studies were cultivated in RPMI 1640 medium supplemented with
10% (v/v) fetal calf serum (FCS), 10 mM HEPES, 2 mM glutamine,
1 mM Na pyruvate, 50 μM 2-mercaptoethanol. Flp-In T-Rex
HEK293 cells (Invitrogen) stably expressing human FLAG-tagged FFA1
(FFA1-HEK) in a doxycycline-dependent manner22 were maintained in
IP1 Accumulation Assays. The HTRF-IP One kit (CIS Bio
International, Gif-sur-Yvette Cedex, France) was used for measuring
IP1 production in cells expressing FFA1. In a 384-well format, the cell
suspension was dispensed at (100 000 cells/7 μL)/well. Following
20 min of incubation at 37 ꢀC, an amount of 7 μL of stimulation buffer
(HBSS, 10 mM HEPES and 50 mM LiCl) containing various concen-
trations of ligands was added. After 30 min, an amount of 3 μL of IP1-d2
conjugate followed by 3 μL of Europium cryptate-labeled anti-IP1
antibody was added to each well. After incubation at room temperature
for 60 min, time-resolved fluorescence at 620 and 665 nm was measured
with the Mithras LB 940 multimode reader (Berthold Technologies, Bad
Wildbad, Germany). Results were calculated from the 665 nm/620 nm
ratio and expressed as ΔF (ΔF (%) = [(standard or sample ratio ꢀ
rationeg)/rationeg] ꢁ 100). Data were normalized to the maximum IP1
response obtained with 10 μM 37.
Insulin Secretion. INS-1E cells were seeded at a density of 100 000
cells/mL in 24-well plates for 2 days and incubated as described
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Journal of Medicinal Chemistry
ARTICLE
previously.44 In brief, cells were incubated for 1 h at 37 ꢀC in a solution
containing 136 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM
MgSO4, 1 mM CaCl2, 5 mM NaHCO3, 10 mM HEPES, 0.5 g/L bovine
serum albumin (fatty acid free, Sigma, Deisenhofen, Germany), pH 7.4,
and the test substance as indicated. Insulin released into the supernatant
and insulin content after acid ethanol extraction were determined by
radioimmunoassay (Linco, U.S.).
’ ASSOCIATED CONTENT
S
Supporting Information. Synthetic procedures and com-
b
pound characterization, activity of 19 compared to 37 in IP1 and
DMR assays, and receptor sequence alignments. This material is
available free of charge via the Internet at http://pubs.acs.org.
Permeability in Mucus-Expressing Cells37. The HT29-MTX
cell line for permeability studies was kindly provided by Dr. Thꢀecla
Lesuffleur (INSERM UMR S 938, Paris, France). Details regarding cell
medium, growth, and seeding, as well as the permeability experiments
including monolayer integrity and calculations of Papp (cm/s), are
described elsewhere.45 Cells of passage number 32 were grown for
’ AUTHOR INFORMATION
Corresponding Author
*Phone: +45 6550 2568. Fax: +45 6615 8780. E-mail: ulven@
ifk.sdu.dk.
23 days (TEER > 750 Ω cm2) before permeability studies were
3
’ ACKNOWLEDGMENT
conducted. The permeation of 37 and ketoprofen (the drugs were
dissolved at 10 mM in dimethylsulfoxide followed by diluting 1:100 in
buffer, giving a concentration of 100 μM) was tested at 15, 30, 45, and
60 min at 37 ꢀC and 50 rpm. The drugs were quantified using HPLC
analysis. Nonpermeated drug was also measured to estimate the
recovery. At the end of the experiments, the integrity of the cell
monolayers were monitored by testing the permeation of the para-
cellular marker carboxyfluorescein (20 μM) after 1 h and measuring
TEER. The results for the permeation experiments are presented as the
mean and standard deviation of three parallels.
We thank Sieglinde Haug, Elisabeth Metzinger, and Lone
Overgaard Storm for excellent technical help with insulin mea-
surements (S.H. and E.M.) and synthesis (L.O.S.), Corning for
support on the Epic biosensor, and the Danish Council for Indepen-
dent Research|Technology and Production (Grant No. 09-070364)
for financial support. S.U. was supported by a grant from the German
Federal Ministry of Education and Research (BMBF) to the German
Center for Diabetes Research (DZD e.V).
Counterscreens. Counterscreening on a panel of 56 receptors, ion
channels, and transporters (PPARγ, A1, A2A, A3, α1, α2, β1, β2, AT1,
BZD (central), B2, CB1, CCK1, D1, D2S, ETA, GABA, GAL2, CXCR2,
CCR1, H1, H2, MC4, MT1, M1, M2, M3, NK2, NK3, Y1, Y2, NTS1, δ2
(DOP), k (KOP), μ (MOP), NOP (ORL1), TP (TXA2/PGH2), 5-HT1A,
5-HT1B, 5-HT2A, 5-HT2B, 5-HT3, 5-HT5a, 5-HT6, 5-HT7, sst, VPAC1
(VIP1), V1a, Ca2+ channel (L, verapamil site, phenylalkylamine), KV channel,
SKCa channel, Na+ channel (site 2), Clꢀ channel (GABA-gated), nore-
pinephrine transporter, dopamine transporter, and 5-HT transporter)
was performed at Cerep Inc. Counterscreens on FFA2 and FFA3 were
performed on HEK293 cells stably transfected to express hFFA2 and
hFFA3, respectively, using the label-free DMR assay essentially as
described above for FFA1-HEK cells. The test compound was dissolved
in DMSO to a 10 mM stock solution, which was diluted with water or
HBSS to a final test concentration of 10 μM.
’ ABREVIATIONS USED
ADME, absorption, distribution, metabolism, and excretion;
AR, adrenergic receptor; CYP, cytochrome P450; DHB, 2,5-
dihydroxybenzoic acid; DMR, dynamic mass redistribution;
ECL, extracellular loop; FFA, free fatty acid; FFA1, free fatty
acid receptor 1 (GPR40); FFA2, free fatty acid receptor 2
(GPR43); FFA3, free fatty acid receptor 3 (GPR41); HBSS,
Hank’s balanced salt solution;HEK, human embryonic kidney; GIP,
gastric inhibitory polypeptide; GLP-1, glucagon-like peptide 1;
GSIS, glucose-stimulated insulin secretion; LE, ligand efficiency;
PBS, phosphate buffered saline; P-gp, P-glycoprotein; PIntB,
2-(di-tert-butylphosphino)-1-phenylindole; PPARγ, peroxisome
proliferator-activated receptor γ; SAR, structureꢀactivity rela-
tionship; SPhos, 2-dicyclohexylphosphino-20,60-dimethoxybiphenyl;
TM, transmembrane; TMEDA, N,N,N0,N0-tetramethylethy-
lenediamine
Molecular Modeling. Modeller 9v846 was used to build a homo-
logy model of hFFA1 using both the crystal structure of chemokine
CXCR440 (PDB code 3OE0) for TM2 and the crystal structure of the
β2-adrenergic receptor39 (PDB code 2RH1) for the remaining receptor.
The rationale for this approach is that the longer TMs 6 and 7 of CXCR4
are incompatible with hFFA1. TM1 of CXCR4 is bent toward the center
of the bundle because of a disulfide bridge between the N terminus and
Cys7.25 that is absent in hFFA1, and hFFA1 contains the Ser-x-
Pro60II:18/2.58 motif in TM2, homologous to the Thr-x-Pro92II:18/2.58
motif of CXCR4, that induces a tight helical turn of TM2 (see Figure S2
in the Supporting Information for sequence alignments). The second
extracellular loop of hFFA1 was arbitrarily modeled as in the β2AR,
formed by a helical segment, instead of as in CXCR4, formed by two
β-strands. The Glu145-Arg183V:05/5.39 and Glu172-Arg258VII:02/7.35
ionic locks previously reported by Sum et al.,47 together with the
disulfide bridge between Cys79III:01/3.25 and Cys170, were included in
the model. The ligand was minimized using Gaussian 0348 and manually
docked so that the carboxylic group interacted with Arg183V:05/5.39 and
Arg258VII:02/7.35, as described by mutational studies49 using PyMOL50
as graphical interface. Afterward, the ligandꢀreceptor complex was
energy-optimized using the Sander module of AMBER 9.51 The Duan
et al. force field52 was used for the receptor, and the general AMBER
force field (GAFF) and HF/6-31G*-derived RESP atomic charges were
used for the ligands.
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