Discovery of a potent and selective free fatty acid receptor 1 agonist with low lipophilicity and high oral bioavailability

Article abstract of DOI:10.1021/jm301470a

The free fatty acid receptor 1 (FFA1, also known as GPR40) mediates enhancement of glucose-stimulated insulin secretion and is emerging as a new target for the treatment of type 2 diabetes. Several FFA1 agonists are known, but the majority of these suffer from high lipophilicity. We have previously reported the FFA1 agonist 3 (TUG-424). We here describe the continued structure-activity exploration and optimization of this compound series, leading to the discovery of the more potent agonist 40, a compound with low lipophilicity, excellent in vitro metabolic stability and permeability, complete oral bioavailability, and appreciable efficacy on glucose tolerance in mice.

Full text of DOI:10.1021/jm301470a

Article
pubs.acs.org/jmc
Discovery of a Potent and Selective Free Fatty Acid Receptor 1
Agonist with Low Lipophilicity and High Oral Bioavailability
Elisabeth Christiansen,† Maria E. Due-Hansen,† Christian Urban,‡ Manuel Grundmann,§
Johannes Schmidt,§ Steffen V. F. Hansen,† Brian D. Hudson,⊥ Mohamed Zaibi,∥ Stine B. Markussen,†
Ellen Hagesaether,† Graeme Milligan,⊥ Michael A. Cawthorne,∥ Evi Kostenis,§ Matthias U. Kassack,‡
and Trond Ulven*,†
†Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark,
‡Institute of Pharmaceutical and Medicinal Chemistry, University of Dusseldorf, Universitatsstrasse 1, D-40225 Dusseldorf, Germany,
̈
̈
̈
§Institute for Pharmaceutical Biology, University of Bonn, Nussallee 6, D-53115 Bonn, Germany,
⊥Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow
G12 8QQ, Scotland, U.K.,
∥Clore Laboratory, University of Buckingham, Hunter Street, Buckingham MK18 1EG, U.K.
S
* Supporting Information
ABSTRACT: The free fatty acid receptor 1 (FFA1, also
known as GPR40) mediates enhancement of glucose-
stimulated insulin secretion and is emerging as a new target
for the treatment of type 2 diabetes. Several FFA1 agonists are
known, but the majority of these suffer from high lipophilicity.
We have previously reported the FFA1 agonist 3 (TUG-424).
We here describe the continued structure−activity exploration
and optimization of this compound series, leading to the discovery of the more potent agonist 40, a compound with low
lipophilicity, excellent in vitro metabolic stability and permeability, complete oral bioavailability, and appreciable efficacy on
glucose tolerance in mice.
secretion at high glucose levels through two independent
mechanisms.
INTRODUCTION
■
Type 2 diabetes (T2D) is characterized by insulin resistance
and insufficient insulin secretion by pancreatic β-cells, leading
to dysfunctional control of plasma glucose and numerous long-
term health consequences, such as increased risk of heart
disease and stroke, kidney failure, blindness, neuropathy, and
amputations. The global number of diabetics has now reached
350 million, of which 90% are type 2 diabetics.1,2 Besides a
healthy lifestyle, the most common treatments include insulin,
metformin, and sulfonylureas, all of which are associated with
problems such as weight gain, risk of hypoglycemia, and lack of
sustained efficacy, and there is an urgent need for improved
therapeutics.
A number of synthetic FFA1 agonists have been reported in
the literature (Chart 1),5,8−26 of which the compounds TAK-
875 and AMG-837 have reached clinical trials. A general
problem with the currently known FFA1 ligands is their
relatively high lipophilicity, which is associated with numerous
problems, such as poor pharmacokinetic properties, metabolic
instability, toxicity, and off-target effects, and correlates with
attrition in clinical trials.27−32 Studies have recommended that
ClogP values should not exceed 4−5,30,33 and ranking functions
such as ligand lipophilicity efficiency (LLE) have been
suggested to facilitate implementation of lipophilicity concerns
in the optimization process.27
The long-chain free fatty acid receptor 1 (FFA1, previously
known as GPR40) is highly expressed in pancreatic β-cells and
enhances glucose-stimulated insulin secretion (GSIS) but does
not affect insulin secretion at low glucose levels.3−6 This
mechanism provides the potential for boosting insulin levels of
type 2 diabetics without the risk of hypoglycemia associated
with sulfonylureas and insulin administration. FFA1 is also
expressed in enteroendocrine cells and has been implicated in
the secretion of the incretin hormones glucagon-like peptide-1
(GLP-1) and glucose-dependent insulinotropic polypeptide
(GIP).7 Thus, the receptor may potentially enhance insulin
We previously reported the discovery of the alkyne agonist
series with 3 (TUG-424, Chart 1) as the most potent
compound.13 The compound is, however, relatively lipophilic
and exhibited only moderate in vitro metabolic stability and is
thus not suitable as a drug candidate. We therefore aimed at
improving the lipophilicity and metabolic stability of the
compound by replacement of the terminal benzene ring by
nitrogen-containing heterocycles, which led to the identification
Received: October 10, 2012
Published: January 8, 2013
© 2013 American Chemical Society
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Chart 1. Representative FFA1 Agonists
of TUG-499 (Chart 1).19 Inspired by Takeda’s clinical
candidate TAK-875 (Chart 1), we recently explored the
attachment of a mesylpropoxy appendage on TUG-469
(Chart 1), the previously reported compound from another
series, and thereby identified TUG-905 (Chart 1), a compound
with significant reduction in lipophilicity.25 We have also
continued the further optimization and exploration of
structure−activity relationships (SAR) around 3 with focus
on lowering lipophilicity and improving metabolic stability by
the introduction of polar substituents. Herein, we report the
results from these studies, which led to the identification of 40,
a compound with improved potency, lower lipophilicity than
any previously reported FFA1 agonist, high selectivity, excellent
in vitro ADME properties, complete bioavailability, and
appreciable efficacy on glucose tolerance in mice.
a
Scheme 1
a
Reagents and conditions: (a) method A, Pd(PPh3)2Cl2, CuI, aryl
halide, Et3N, DMF, 50 °C; method B, Na2PdCl4, PIntB, CuI, aryl
halide, TMEDA, water, 80 °C; method C, Na2PdCl4, PIntB, CuI, aryl
halide, TMEDA, water, 80 °C. (b) LiOH, THF, water, room
temperature.
RESULTS AND DISCUSSION
■
The majority of the alkyne ligands were synthesized directly by
Sonogashira cross-coupling between the alkyne intermediate 1
and aryl halides (Scheme 1).34 As the classical Sonogashira
protocol (method A) in most cases works unsatisfactorily with
1, we screened other methods and found that a method
reported by Beller and co-workers (method B) gave improved
results.35 This method however also often gave low yields,
especially with aryl iodides. We therefore optimized the method
further and found that addition of 10% water (method C)
resulted in suppression of Glaser−Hay dimerization of the
alkyne and very rapid cross-coupling in high yields with both
aryl bromides and iodides.34 Method C is more convenient and
has so far given significantly better results than other methods
in all cases where iodo- and bromobenzenes are used as
substrates.
(Scheme 2). Acrylic acid analogues 8 and 9 were synthesized
from 4-bromocinnamic acid via esterification and Sonogashira
cross-coupling reactions as for the propionic acids (Scheme 3).
a
Scheme 2
a
The phenylacetic acid (6) and 4-phenylbutanoic acid (7)
analogues were synthesized from the corresponding aryl
bromides by Sonogashira coupling with phenylacetylene
Reagents and conditions: (a) Na2PdCl4, PIntB, CuI, phenylacetylene,
TMEDA, water, 80 °C (78%). (b) LiOH, THF, water, room
temperature (95−99%).
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a
a
Scheme 3
Scheme 5
a
Reagents and conditions: (a) trimethylsilylacetylene, Na2PdCl4,
a
Reagents and conditions: (a) trimethylsilylacetylene, Na2PdCl4,
PIntB, CuI, TMEDA, water, 80 °C. (b) K2CO3, MeOH, room
temperature (97% over two steps). (c) Na2PdCl4, PIntB, CuI, aryl
halide, TMEDA (or TMEDA with 10% water for R = 2-CH2CN), 80
°C (36−49%). (d) LiOH, THF, water, room temperature (97−99%).
(e) Pd(PPh3)2Cl2, CuI, phenylacetylene, Et3N, DMF, 50 °C (53%).
PIntB, CuI, TMEDA, water, 80 °C (77%). (b) K2CO3, MeOH,
room temperature (95%). (c) Na2PdCl4, PIntB, CuI, 2-bromotoluene
or phenylacetylene, TMEDA, water, 80 °C (68−84%). (d) LiOH,
THF, water, room temperature (85−91%).
The β-methyl substituted analogue 10 was prepared by a
conjugated addition of methyl Gilman reagent to methyl 4-
bromocinnamate in the presence of chlorotrimethylsilane
(Scheme 4).36−38 All cyclopropyl analogues are racemic and
a
Scheme 6
a
Scheme 4
a
Reagents and conditions: (a) triethyl phosphonoacetate, 60% NaH,
toluene, 0 °C → reflux (45%). (b) Et3SiH, TFA, room temperature →
50 °C (58%). (c) Na2PdCl4, PIntB, CuI, phenylacetylene, TMEDA,
water, 80 °C (57%). (d) LiOH, THF, water, room temperature (91%).
a
Reagents and conditions: (a) MeMgCl, copper(I) thiophenolate,
TMSCl, THF, −78 °C → 0 °C (47%). (b) Na2PdCl4, PIntB, CuI,
phenylacetylene, TMEDA, water, 80 °C (57%). (c) LiOH, THF,
water, room temperature (64%).
a
Scheme 7
were prepared from methyl trans-2-(4-iodophenyl)-
cyclopropanoate (Scheme 5).19 The bicyclic alkyne 11 was
synthesized from 5-bromodihydroindanone by a Horner−
Wadsworth−Emmons reaction with triethyl phosphonoacetate,
reduction with triethylsilane and TFA, and Sonogashira
coupling with phenylacetylene followed by ester hydrolysis
(Scheme 6). Aryl halides used to prepare 27, 28, 42, and 43
were synthesized by alkylation of iodophenols. The methox-
ymethyl-substituted bromobenzene building blocks for 34 and
35 were synthesized following a FeSO4-promoted ether
synthesis of the corresponding bromobenzyl bromides.39 The
biphenylalkyne 21 was synthesized from the methyl ester 23a
by Suzuki cross-coupling (Scheme 7).
The mesylalkoxy-substituted alkynes were prepared from 26a
by a Williamson ether synthesis to form the bromoalkylated
intermediate, which was subsequently mesylated by gentle
heating with sodium methanesulfinate in PEG-400 (Scheme 8).
Compounds were screened on the human FFA1 in a calcium
mobilization assay. Besides overall activity, ligand efficiency
a
Reagents and conditions: (a) PhB(OH)2, Pd(OAc)2, SPhos, K3PO4,
toluene, 100 °C (74%). (b) LiOH, THF, water, room temperature
(94%).
(LE)40 and calculated lipophilicity (ClogP), as well as LLE27
calculated as the difference between pEC50 and ClogP, were
taken advantage of in the evaluation of analogues. Since the
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a
Scheme 8
a
Reagents and conditions: (a) Br(CH2)n+1Br, K2CO3, acetone, reflux (79−93%). (b) MeSO2Na, PEG-400, 45 °C (38−51%). (c) LiOH, THF, water,
room temperature (58−80%).
Table 1. SAR Exploration of the Propionic Acid Chain
a
b
c
d
e
compd
R1
R2
FFA1, calcium pEC50 (% efficacy)
LE
ClogP
log D7.4
LLE
f
2
A
A
A
A
B
C
D
D
E
H
6.70 0.03 (106)
7.34 0.07 (103)
7.13 0.05 (97)
6.55 0.04 (109)
6.01 0.03 (93)
6.00 0.02 (99)
5.03 0.08 (83)
5.64 0.04 (104)
6.70 0.03 (103)
6.46 0.04 (99)
6.84 0.04 (107)
6.86 0.02 (99)
7.03 0.02 (102)
0.48
0.50
0.49
0.45
0.46
0.41
0.36
0.39
0.46
0.42
0.47
0.45
0.46
0.42
4.54
5.04
5.04
5.04
4.05
4.92
4.88
5.38
4.94
5.05
4.59
5.09
5.09
5.09
2.16
2.86
2.75
2.17
1.96
1.08
0.15
0.26
1.76
1.42
2.25
1.78
1.95
1.43
f
3
2-Me
3-Me
4-Me
H
2.44 0.01
2.13 0.02
f
4
f
5
6
7
H
g
8
H
9
2-Me
H
10
11
12
13
14
15
(Scheme 6)
F
F
F
F
H
2-Me
3-Me
4-Me
6.51 0.04 (107)
a
b
Efficacy is given as percent response relative to 10 μM TUG-20.18 Ligand efficiencies (LE) were calculated by LE = RT lnKD, presuming that EC50
≈ KD. Values are given in kcal mol−1 per non-hydrogen atom.40 Calculated by BioByte’s algorithm as implemented in ChemBioDraw Ultra 12.0 (the
c
“ClogP” option). Determined by the shake flask method.25 Ligand lipophilicity efficiencies (LLE) were calculated by the formula LLE = pEC50
−
d
e
ClogP.27 Previously reported.13 Tested with 0.05% bovine serum albumin (BSA).
f
g
ClogP calculation method was used extensively in the
optimization, a validation and comparison of alternative
methods using related compounds with experimental log D7.4
values was performed, and log D7.4 values from representative
new alkynes were subsequently added to strengthen the
validation. Of 11 methods, the ClogP method showed the
best correlation with experimental values (R2 = 0.96; see the
Supporting Information).
We have previously reported the discovery of the potent and
selective FFA1 agonist 3 (Chart 1) by screening of a focused
library of constrained fatty acid analogues and optimization of a
4-(phenylethynyl)phenoxyacetic acid hit.13 The optimization
study revealed the phenylpropanoic acid analogue 2 (Table 1)
to be clearly favored over the phenoxyacetic acid of the initial
hit and that introduction of methyl substituents at the terminal
phenyl showed the 2-position (3) was preferred over the 3-
position (4), while a 4-methyl (5) did not result in increased
potency relative to the unsubstituted 2.25 Shortening or
elongating the propionic acid chain by one methylene group
(6 and 7) resulted in significant decrease in potency. The
planar acrylic acid 8 showed a further order of magnitude
reduced potency, which to some degree was regained by
introduction of the 2-methyl on the terminal phenyl ring (9).
The introduction of a β-methyl group (10) did not affect
potency, but is disfavored relative to 2 as it results in lower LE
and higher lipophilicity and introduces a chiral center that
complicates synthesis. Constraining the propionic acid by
connecting the methyl group of 10 back to the benzene ring via
a methylene group to form a dihydroindane system (11,
Scheme 6), in analogy to the structures reported by Takeda,21
yielded reduced potency. In contrast, connecting the β-methyl
to the α-carbon to form a cyclopropyl constraint of the
propionic acid chain (12) results in a moderate gain of activity.
Unfortunately, the gain was not maintained with the
introduction of methyl substituents at the terminal benzene
ring (13−15).
Although methyl substituents in both 2- and 3-position
resulted in increased potency, the initial studies showed that the
2,3-dimethyl as well as the 3,5-dimethyl pattern gave a
compound with lower potency than the corresponding
monomethyl analogues.13 To complete this study, we
synthesized the 2,5-dimethyl (16), 2,6-dimethyl (17), and 4-
chloro-2-methyl (18) analogues, all of which exhibited reduced
activity relative to 3 (Table 2).
We found that 3 had only moderate stability toward human
liver microsomes (HLM, see below) and suspected that the 2-
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Table 2. Exploration of Substitution on the Terminal Phenyl Ring
a
b
c
d
e
compd
R
FFA1, calcium pEC50 (efficacy, %)
LE
ClogP
log D7.4
LLE
16
17
18
19
20
21
22
23
24
25
26
27
28
29
2-Me, 5-Me
2-Me, 6-Me
2-Me, 4-Cl
4-F
6.91 0.06 (102)
6.38 0.04 (103)
6.86 0.02 (103)
6.28 0.05 (96)
7.05 0.03 (101)
6.00 0.02 (91)
6.82 0.04 (107)
7.08 0.06 (99)
6.47 0.02 (98)
4.79 0.07 (83)
6.35 0.04 (103)
6.66 0.03 (99)
7.15 0.03 (101)
6.65 0.03 (106)
7.02 0.04 (103)
6.73 0.04 (101)
6.24 0.02 (111)
5.69 0.02 (106)
7.39 0.03 (108)
7.42 0.04 (99)
6.09 0.03 (100)
6.37 0.03 (106)
6.33 0.02 (103)
7.40 0.03 (105)
7.70 0.04 (103)
6.72 0.04 (98)
7.33 0.03 (105)
6.58 0.03 (105)
0.45
0.42
0.45
0.43
0.46
0.33
0.47
0.49
0.39
0.33
0.43
0.44
0.47
0.38
0.40
0.42
0.41
0.35
0.46
0.46
0.31
0.31
0.39
0.45
0.48
0.42
0.44
0.39
0.45
5.54
5.54
5.75
4.68
5.57
6.43
5.25
5.40
5.42
3.87
3.87
4.46
4.46
5.57
5.57
3.98
3.50
3.73
4.34
4.34
3.55
3.33
4.47
4.47
3.96
3.96
3.45
3.45
4.01
1.37
0.84
1.11
1.60
1.48
−0.43
1.57
1.68
1.05
0.92
2.48
2.20
2.69
1.08
1.45
2.75
2.74
1.96
3.05
3.08
2.55
3.04
1.86
2.93
3.76
2.76
3.89
3.14
3.44
2-Et
2-Ph
2-Cl
2-Br
2-CF3
2-OH
3-OH
2-OMe
3-OMe
2-OCF3
2.66 0.01
2.83 0.01
f
30
3-OCF3
31
32
33
34
35
36
37
38
39
40
41
42
43
44
2-COMe
2-CH2OH
2-C2H4OH
2-CH2OMe
3-CH2OMe
3-O(CH2)3Ms
3-O(CH2)4Ms
2-CN, 5-Me
2-Me, 5-CN
2-CH2CN
3-CH2CN
2-OCH2CN
3-OCH2CN
(Scheme 5)
1.29 0.00
1.43 0.01
0.88 0.01
1.75 0.01
1.28 0.01
0.98 0.01
7.45 0.03 (99)
a
b
Efficacy is given as percent response relative to 10 μM TUG-20.18 Ligand efficiencies (LE) were calculated by LE = RT lnKD, presuming that EC50
≈ KD. Values are given in kcal mol−1 per non-hydrogen atom.40 Calculated by ChemBioDraw Ultra 12.0 with the “ClogP” option. Determined by
c
d
the shake flask method.25 Calculated by the formula LLE = pEC50 − ClogP.27 Tested with 0.05% BSA.
e
f
methyl group might be implicated. A 4-fluoro substituent on
the terminal phenyl ring (19) to stabilize the terminal phenyl
ring toward oxidation came with a penalty in terms of lower
potency. Increasing the steric bulk of the 2-methyl group of 3
by replacement with ethyl (20) or phenyl (21) resulted in
reduced potency. These analogues also had increased lip-
ophilicity and were unlikely to improve the HLM stability.
Replacement of the 2-methyl by metabolically stable chloro
(22), bromo (23), or trifluoromethyl (24) substituents did not
preserve potency, while lipophilicity was increased in all cases.
It is well-known that lipophilicity generally correlates with
poor metabolic stability, a consequence of the increased
tendency of more lipophilic molecules to seek out of the
aqueous phase and into the active sites of the metabolic
enzymes. We have previously described our efforts to optimize
3 by replacing the terminal phenyl group with hydrophilic
heterocycles,19 and we recently described the effects of
introducing a polar substituent on an FFA1 agonist, resulting
in reduced lipophilicity and higher HLM stability together with
preserved potency.25 Although initial attempts to introduce
polar substituents on the terminal phenyl ring of 3 had resulted
in compounds with low or no activity,13 we decided to continue
the exploration of this strategy. Introduction of a hydroxyl
group in the 2-position (25) resulted in a 2 orders of
magnitude erosion of activity. A 3-hydroxy substituent (26)
gave a moderate reduction in potency relative to 2, but a higher
LLE. It is thus clear that the positioning of a polar group is
critical. A rationale for this was provided by the modeling study
below. Methoxy groups in the 2- and 3-positions (27, 28)
improved the situation significantly compared to the hydroxyls.
In the case of the 3-MeO analogue 28, the reduction in
lipophilicity compensated for the lower potency and gave the
compound an LLE of 2.69 based on ClogP, compared to 2.86
for 3. As methoxy substituents imply a risk of metabolic
instability, the more stable trifluoromethoxy group was
explored in the same positions (29, 30). The activities of 29
and 30 paralleled those of methoxy analogues 27 and 28, but
the increased lipophilicity and decreased LE and LLE made the
compounds less attractive. Continuing the screen for more
polar substituents, the 2-acetyl (31) turned out equipotent with
the unsubstituted 2, but significantly less potent than 3. Fishing
for hydrogen-bond interactions further away from the scaffold,
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Figure 1. Complex of 3 (left) and 40 (right) with a homology model of the human FFA1. Residues situated close to the ligands are labeled with
sequence number, and Schwartz−Baldwin41 and Ballesteros−Weinstein42 notations are given as superscripts.
Figure 2. Activity of 40 on FFA1-transfected HEK-293 cells and in the rat β-cell line INS-1E that endogenously expresses FFA1. (A) Representative
traces from the dynamic mass redistribution (DMR) assay of 40 on HEK-FFA1 cells. (B) Concentration−response curves of 40 in FFA1-HEK cells
from the DMR assay (pEC50 = 7.4 0.05, n = 3). (C) Traces from the DMR assay of 40 in INS-1E cells. (D) Concentration−response curve of 40
in INS-1E cells (pEC50 = 5.6 0.16, n = 5) and inhibition of 40 (3 μM) by the FFA1 antagonist PPTQ (pIC50 = 5.3 0.13, n = 3).
the 2-hydroxymethyl (32) and 2-hydroxyethyl (33) analogues
were investigated, but these did not display sufficient potency.
The methoxymethyl substituent in the 2- or 3-position (34,
35), however, brought the activity up to the same level as 3
while lower lipophilicity was maintained, thus increasing LLE.
Mesylalkoxy substituents, successfully applied on another
compound series,25 were investigated with various chain lengths
(36, 37). The 3-mesylbutoxy substituent (37) resulted in a
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small decrease in activity relative to 2 but more than an order of
magnitude reduced lipophilicity, and thereby a significantly
increased LLE relative to both 2 and 3. The concept of
introducing mesylalkoxy substituents to lower lipophilicity
while potency was maintained thus seems to work well,
although the potencies of these analogues were too low to place
them among the preferred compounds.
(4-phenoxyphenyl)-2-propyl-1,2,3,4-tetrahydroisoquinoline-4-
carboxylic acid (PPTQ).44
Counterscreens demonstrated >100-fold selectivity for FFA1
over FFA2 (GPR43, pEC50 < 5), FFA3 (GPR41, pEC50 < 5),
GPR120 (pEC50 = 5.32 0.03), PPARγ (pEC50 < 5), and 55
other receptors, enzymes, and transporters (see the Supporting
Information). Physicochemical and in vitro ADME properties
of 40 were obtained for a full comparison with the lead
compound 3 (Table 3). Both 3 and 40 have good solubility and
The introduction of a polar cyano substituent on the
terminal ring together with a methyl group (38, 39) resulted in
39 being equipotent with 3 with somewhat reduced lip-
ophilicity. Gratifyingly, combining the cyano and the methyl
into a 2-cyanomethyl substituent (40) produced a significant
increase in potency and a pronounced reduction in lipophilicity
relative to 3. The 3-cyanomethyl analogue (41) turned out an
order of magnitude less potent. Thus, the substituent position
is important, as for the hydroxyl analogues 25 and 26, but the
favored position is reversed. Extending the substituent to
cyanomethoxy (42, 43) led to reduced potency to the same
degree in both the 2- and 3-postion together with further
reduced lipophilicity. The preference for the 2-position is thus
in agreement with the methyl (3, 4) and cyanomethyl (40, 41)
analogues, but reversed in comparison to the other analogues
where oxygen is attached directly to the phenyl ring (25−29).
A combination of 2-cyanomethyl with the cyclopropyl
constraint of the propionic acid chain (44) resulted in a drop
in potency, as for the methyl substituents. Altogether, 40
remained the preferred compound, despite the slightly higher
LLE of 42.
A molecular modeling study was performed to explore the
basis for the interesting effects observed with the different
substituent positions. The complex of 3 and 40 with a
homology model of hFFA1 was generated as described
previously.19 The carboxylic acid of the compounds interacts
with the two arginine residues Arg183 and Arg258 and the
diphenylacetylene part extends toward TM2 (Figure 1). The
benzene rings are twisted with almost 90° between them, as
also observed in the crystal structure of 3,13 which favors
interaction between the central ring and Phe87 and places the
methyl group in a small hydrophobic cavity created by Leu262.
This hydrophobic cavity provides a rationale for the preference
of the 2-methyl substituent (3) over the 3-methyl (4) and the
unsubstituted compound (2), and for the highly disfavored
interaction with the 2-hydroxy-substituted 25 compared to the
3-hydroxy analogue 26. Likewise, the lower potency of the 2-
cyano-5-methyl-substituted 38 compared to the 5-cyano-2-
methyl-substituted 39 can be rationalized by the disfavored
polar ortho-substituent. The 2-cyanomethyl moiety of 40 fit
well into the hydrophobic cavity and, in addition, provides a
hydrogen bond acceptor that is perfectly situated to form a
hydrogen bond interaction with Lys259 (distance ∼ 2.0 Å) with
Lys62 as an alternative interaction point, thus providing a
rationale for the high potency of this compound.
Table 3. Physicochemical and in Vitro ADME Properties of
3 and 40
assay
3
40
188 μM
a
aqueous solubility (PBS, pH 7.4)
174 μM
chemical stab. (PBS, 37 °C, 12 days) 99.9%
99.8%
b
log D (n-octanol/PBS, pH 7.4)
2.44 (2.34)
97.7%
1.28 (1.32)
>99.9%
81%
c
plasma protein binding (human)
a
metabolic stability (HLM)
26%
a
CYP inhibition (10 μM)
CYP1A2
CYP2C9
CYP2C19
CYP2D6
CYP3A4
−8%
−25%
−1%
−3%
11%
−2%
5%
0%
−4%
6.6/23.3
8%
a
a
P-gp inhibition (% @ 30/100 μM)
−4.0/−1.8
Caco-2 (A to B, TC7, pH 6.5/7.4)
87 × 10−6 cm/s 91 × 10−6 cm/s
a
b
Determined at Cerep Inc. Determined by the shake-flask method.25
The values given in parentheses were determined at Cerep Inc.
c
Determined by equilibrium dialysis at Cerep Inc.
demonstrated excellent chemical stability. The experimental
lipophilicity of 40 as measured by log D7.4 was reduced by an
order of magnitude relative to 3. Stability toward HLM was
drastically improved, supporting that the 2-methyl group was
involved in the insufficient stability observed with 3 and
demonstrating that reduction of lipophilicity is an efficient
means for mending unsatisfactory metabolic stability. No
significant cell toxicity or inhibition of the most important
CYP enzymes or P-glycoprotein (P-gp) was found. Both
compounds exhibited high permeability in Caco-2 cells. The
permeability of 40 was also examined on the mucus-secreting
cell-line HT29-MTX,45 an improved model system for the
intestinal epithelium, and was found to be significantly higher
[Papp = (2.54 0.07) × 10−5 cm/s, 78% recovered] than the
readily absorbed drug ketoprofen [Papp = (1.51 0.05) × 10−5
cm/s, 80% recovered].
Pharmacokinetic investigations of 3 and 40 in mice revealed
rapid and complete absorption of both compounds after oral
dosing (Table 4). The half-life in mice is as expected rather
short, but 40 showed a somewhat longer half-life than 3. The
exposure of 40 after oral dosing was 3.5-fold higher than for 3.
The relatively low volume of distribution for 40 can be
rationalized by the high plasma protein binding and contributes
to 40 having a half-life only moderately longer than 3 despite
higher metabolic stability. A low clearance was confirmed with
40 compared to a quite high clearance for 3, corresponding to
approximately 15% and 60% of the hepatic blood flow in mice,
respectively. Both compounds were well-tolerated by the mice
in acute exposures up to 250 mg/kg po. A satisfactory overall
pharmacokinetic profile was found for 40.
The 2-cyanomethyl analogue 40 was the most potent agonist
by good margin and also had lower lipophilicity and was thus
chosen for further examination. The compound was evaluated
using a dynamic mass redistribution (DMR) assay, which
monitors real-time protein activity in the living cell without the
need of labeling.43 Potent activity in HEK293 cells transfected
with hFFA1 was confirmed [Figures 2 and S1 (Supporting
Information)]. Further testing of 40 with the insulin-secreting
rat β-cell line INS-1E endogenously expressing FFA1 showed a
concentration-dependent effect that was confirmed to be FFA1-
mediated by treatment with the FFA1 antagonist trans-1-oxo-3-
The effects of 3 and 40 on glucose tolerance in normal mice
after oral administration 30 min prior to glucose challenge were
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Journal of Medicinal Chemistry
Article
a
thus be ascribed mainly to the improved pharmacokinetic
properties of 40.
Table 4. Pharmacokinetic Profiles of 3 and 40 in Mice
3
40
Intravenous
CONCLUSION
■
Cmax (ng/mL)
tmax (min)
2284
5
5071
5
In the continued SAR exploration of the alkyne series of FFA1
agonists represented by 3, we focused especially on lowering
the lipophilicity of the compounds. Introduction of polar
substituents on the terminal phenyl ring led to the discovery of
40, a compound with significantly improved potency and
reduced lipophilicity relative to 3 (EC50 = 20 vs 46 nM, log D7.4
1.3 vs 2.4). Compound 40 exhibited high selectivity over a
panel of enzymes, receptors, and transporters, and in vitro
ADME-tox studies indicated high absorption, good metabolic
stability, and no inhibition of enzymes implicated in drug−drug
interactions. Pharmacokinetic evaluation in mice indicated
complete bioavailability and >3-fold higher exposure of 40
compared to 3. The effect of 40 in a glucose tolerance test in
mice indicated an effect comparable to sitagliptin at 10 mg/kg
after oral administration and maximal effect reached at 50 mg/
kg. Altogether, 40 appears as a promising candidate for more
advanced animal studies and further development of improved
therapeutics for T2D.
t1/2 (min)
10
17
AUC0‑∞ (μg/mL·min)
Vd (L/kg)
47
174
0.80
0.35
CLtotal (mL/min/kg)
Oral
53
14
Cmax (ng/mL)
tmax (min)
2591
30
7757
30
t1/2 (min)
48
50
AUC0‑∞ (μg/mL·min)
F (%)
205
109
732
105
a
Data are mean concentrations in mouse plasma (n = 3) following a
single 2.5 mg/kg intravenous dose or 10 mg/kg oral dose.
studied. 3 showed a significant improvement in glucose
tolerance at a 50 mg/kg dose (Figure 3), similar to the effect
of the oral antihyperglycemic DPP-4 inhibitor sitagliptin (10
mg/kg), a compound on the market for treatment of T2D. 40
exhibited a dose-dependent response with a significant effect
similar to that of sitagliptin at 10 mg/kg and a maximal effect
reached at 50 mg/kg, sustained at 250 mg/kg (Figure 3).
Compound 40 is confirmed to be a full agonist also of the
murine FFA1, but with an order of magnitude lower potency
(pEC50 = 6.40 on mFFA1) than on the human orthologue. The
considerable difference in efficacy and to some degree potency
between the two compounds in the glucose tolerance test can
EXPERIMENTAL SECTION
■
All commercial starting materials and solvents were used without
further purification, unless otherwise stated. THF was freshly distilled
from sodium/benzophenone. Purification by flash chromatography
was carried out using silica gel 60 (0.040−0.063 mm, Merck). TLC
analysis was performed on silica gel 60 F254 plates. H and 13C NMR
1
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) or
Figure 3. Effect of 3 and 40 on glucose tolerance in normal mice. Male C57Bl/6 mice were dosed po with test compound, vehicle, or positive
control (sitagliptin, 10 mg/kg) 30 min prior to a 2 g/kg glucose challenge. Top panels show plasma glucose concentration curves as a function of
time, and bottom panels show areas under the curves. Means standard errors (n = 6) are shown (*, p < 0.05; **, p < 0.01).
989
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Journal of Medicinal Chemistry
Article
Bruker micrOTOF-Q II (ESI). Purity was determined by HPLC and
confirmed by inspection of NMR spectra. HPLC analysis was
performed using a Dionex 120 C18 column (5 μm, 4.6 × 150 mm)
with 10% acetonitrile in water (0−1 min), 10−100% acetonitrile in
water (1−10 min), 100% acetonitrile (11−15 min), with both solvents
containing 0.05% TFA as modifier; a flow of 1 mL/min; and UV
detection at 230 and 254 nm. All test compounds were of ≥95% purity
unless otherwise stated.
general procedure I to give 312 mg (77%) of a white solid after
purification by flash chromatography (SiO2, EtOAc:petroleum ether,
1
1:4): Rf = 0.08 (EtOAc:petroleum ether, 1:4); H NMR (CDCl3) δ
7.55−7.46 (m, 4H), 7.37−7.34 (m, 2H), 7.21 (d, J = 8.4 Hz, 2H), 3.96
(s, 2H), 3.67 (s, 3H), 2.97 (t, J = 7.7 Hz, 2H), 2.64 (t, J = 7.5 Hz, 2H);
13C NMR (CDCl3) δ 173.0, 141.6, 132.3, 131.7, 131.6, 128.9, 128.5,
128.1, 122.9, 120.4, 117.4, 95.6, 85.7, 51.7, 35.3, 30.8, 22.7; ESI-MS m/
z 326.1 (M + Na+).
General Procedure I: Sonogashira Coupling. A Schlenk flask
charged with Na2PdCl4 (1 mol %), 2-(di-tert-butylphosphino)-N-
phenylindole (PIntB, 2 mol %), CuI (2 mol %), alkyne (1 equiv), aryl
halide (1.1−1.5 equiv), H2O (0.2 mL/mmol), and TMEDA (1.8 mL/
mmol) was evacuated and back-filled with argon three times and then
heated to 80 °C. After consumption of the alkyne, the reaction was
cooled to room temperature, water was added, and the mixture was
extracted with EtOAc (×3). The organic phases were combined,
washed with brine, dried over MgSO4, and concentrated under
vacuum. The residue was purified by flash chromatography and dried
under vacuum to give the desired product.
Step 2. 40 was prepared from 40a (292 mg, 0.96 mmol) according
to the general procedure II to give 216 mg (77%) of a white solid (tR =
11.44, purity: 99.9% by HPLC) after purification by flash
chromatography [SiO2, EtOAc (with 1% AcOH):petroleum ether,
1
1:2]; H NMR (DMSO-d6) δ 11.87 (br s, OH), 7.31−7.21 (m, 4H),
7.16−7.12 (m, 2H), 7.03−7.00 (m, 2H), 3.87 (s, 2H), 2.57 (t, J = 7.5
Hz, 2H), 2.27 (t, J = 7.5 Hz, 2H); 13C NMR (DMSO-d6) δ 173.6,
142.3, 132.8, 132.0, 131.4, 129.3, 128.9, 128.7, 128.3, 122.2, 119.6,
118.4, 95.3, 85.9, 34.8, 30.3, 22.1; ESI-HRMS calcd for C19H15NO2Na
(M + Na+) 312.0996, found 312.1002.
General Procedure II: Ester Hydrolysis. A solution of
LiOH·H2O (2−3 equiv) in H2O (∼2 mL/mmol ester) was added
to the ester dissolved in THF (∼5 mL/mmol ester). The reaction was
stirred at room temperature until complete consumption of the
starting material as indicated by TLC, typically after 1−12 h. The
reaction had water added, was acidified with 3% HCl until pH <1, and
was extracted with EtOAc (×3). The combined extracts were washed
with brine, dried over MgSO4, and concentrated under vacuum.
3-(4-((3-(Methoxymethyl)phenyl)ethynyl)phenyl)propanoic Acid
(35). Step 1. 35a was prepared from 134 (93 mg, 0.49 mmol) and
1-bromo-3-(methoxymethyl)benzene (99 mg, 0.49 mmol) according
to the general procedure I to give 96 mg (64%) of a clear oily product
after purification by flash chromatography (SiO2, EtOAc:petroleum
ether, 1:10): Rf = 0.22 (EtOAc:petroleum ether, 1:4); 1H NMR
(CDCl3) δ 7.51 (s, 1H), 7.47−7.42 (m, 3H), 7.36−7.27 (m, 2H),
7.21−7.16 (m, 2H), 4.45 (s, 2H), 3.67 (s, 3H), 3.40 (s, 3H), 2.96 (t, J
= 7.7 Hz, 2H), 2.64 (t, J = 7.8 Hz, 2H); 13C NMR (CDCl3) δ 173.1,
140.9, 138.5, 131.8, 130.8, 128.44, 128.36, 127.5, 123.5, 121.2, 89.4,
89.0, 74.2, 58.2, 51.7, 35.4, 30.8.
ASSOCIATED CONTENT
* Supporting Information
Synthetic procedures and compound characterization, evalua-
tion of log P calculation methods, and biological assays. This
material is available free of charge via the Internet at http://
pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Tel: +45 6550 2568. Fax: +45 6615 8780. E-mail: ulven@sdu.
■
dk.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Lone Overgaard Storm for excellent technical
assistance and the Danish Council for Independent Research|
Technology and Production (grant 09-070364) and the Danish
Council for Strategic Research (grant 11-116196) for financial
support.
Step 2. 35 was prepared from 35a (76 mg, 0.25 mmol) according to
the general procedure II to give 61 mg (84%) of a white solid (tR =
1
11.96, purity 98.7% by HPLC); H NMR (acetone-d6) δ 7.55−7.43
(m, 4H), 7.42−7.29 (m, 4H), 4.46 (s, 2H), 3.36 (s, 3H), 2.95 (t, J =
7.6 Hz, 2H), 2.65 (t, J = 7.6 Hz, 2H); 13C NMR (acetone-d6) δ 173.8,
142.9, 140.3, 132.4, 131.19, 131.15, 129.6, 129.4, 128.3, 124.1, 121.7,
90.0, 89.6, 74.3, 58.2, 35.5, 31.4; ESI-HRMS calcd for C19H18O3Na (M
+ Na+) 317.1148, found 317.1159.
ABBREVIATIONS USED
■
DMR, dynamic mass redistribution; DPP-4, dipeptidyl
peptidase 4; FFA1, free fatty acid receptor 1 (GPR40); FFA2,
free fatty acid receptor 2 (GPR43); FFA3, free fatty acid
receptor 3 (GPR41); GSIS, glucose-stimulated insulin
secretion; HLM, human liver microsomes; LLE, ligand
lipophilicity efficiency; PIntB, 2-(di-tert-butylphosphino)-1-
phenylindole; SPhos, 2-dicyclohexylphosphino-2′,6′-dimethox-
ybiphenyl; T2D, type 2 diabetes.
3-(4-((5-Cyano-2-methylphenyl)ethynyl)phenyl)propanoic Acid
(39). Step 1. 39a was prepared from 134 (98 mg, 0.52 mmol) and
3-iodo-4-methylbenzonitrile (136 mg, 0.56 mmol) according to the
general procedure I to give 104 mg (68%) of a clear oily product after
purification by flash chromatography (SiO2, EtOAc:petroleum ether,
1
1:10): Rf = 0.20 (EtOAc:petroleum ether, 1:5); H NMR (CDCl3) δ
7.75 (d, J = 1.7 Hz, 1H), 7.49 (d, J = 1.7 Hz, 1H), 7.48−7.44 (m, 2H),
7.33 (d, J = 8.0 Hz, 1H), 7.24−7.18 (m, 2H), 3.68 (s, 3H), 2.98 (t, J =
7.7 Hz, 2H), 2.65 (t, J = 7.7 Hz, 2H), 2.56 (s, 3H); 13C NMR (CDCl3)
δ 173.0, 145.5, 141.7, 135.1, 131.8, 131.2, 130.3, 128.5, 124.8, 120.5,
118.4, 110.0, 95.6, 85.7, 51.7, 35.3, 30.9, 21.2; ESI-MS m/z 326.1 (M +
Na+).
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