3-Substituted 3-(4-aryloxyaryl)-propanoic acids as GPR40 agonists

Article abstract of DOI:10.1016/j.bmcl.2011.03.114

The design, synthesis, and structure-activity relationship (SAR) for a series of β-substituted 3-(4-aryloxyaryl)propanoic acid GPR40 agonists is described. Systematic replacement of the pendant aryloxy group led to identification of potent GPR40 agonists. In order to identify candidates suitable for in vivo validation of the target, serum shifted potency and pharmacokinetic properties were determined for several compounds. Finally, further profiling of compound 7 is presented, including demonstration of enhanced glucose tolerance in an in vivo mouse model.

Full text of DOI:10.1016/j.bmcl.2011.03.114

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3390–3394
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
3-Substituted 3-(4-aryloxyaryl)-propanoic acids as GPR40 agonists
⇑
Shawn P. Walsh , Alexandra Severino, Changyou Zhou, Jiafang He, Gui-Bai Liang, Carina P. Tan, Jin Cao,
George J. Eiermann, Ling Xu, Gino Salituro, Andrew D. Howard, Sander G. Mills, Lihu Yang
Merck Research Laboratories, Merck & Co., Inc., 126 E. Lincoln Ave., PO Box 2000, Rahway, NJ 07065-0900, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The design, synthesis, and structure–activity relationship (SAR) for a series of b-substituted 3-(4-aryloxy-
aryl)propanoic acid GPR40 agonists is described. Systematic replacement of the pendant aryloxy group
led to identification of potent GPR40 agonists. In order to identify candidates suitable for in vivo valida-
tion of the target, serum shifted potency and pharmacokinetic properties were determined for several
compounds. Finally, further profiling of compound 7 is presented, including demonstration of enhanced
glucose tolerance in an in vivo mouse model.
Received 1 March 2011
Revised 29 March 2011
Accepted 31 March 2011
Available online 8 April 2011
Keywords:
GPR40
Ó 2011 Elsevier Ltd. All rights reserved.
Agonist
3-(4-Aryloxy)-propanoic acids
Diabetic
GDIS
Type 2 diabetes is a growing public health epidemic. Given the
worldwide spread of this debilitating condition, there is a contin-
uing need for novel therapies. Recently, renewed focus has been
placed on pancreatic islet-based insulin secretion as a novel mech-
anism for the treatment of type 2 diabetes. This approach has the
potential for stabilization and restoration of b-cell function.1 In this
regard several orphan G-protein coupled receptors (GPCR’s) have
been identified that are preferentially expressed in the b-cells
and are implicated in glucose stimulated insulin secretion (GSIS).2
GPR40, which is a cell surface GPCR, is highly expressed in human
(and rodent) islets as well as in insulin-secreting cell lines. Several
naturally occurring medium to long-chain fatty acids (FA’s)3 as
well as synthetic compounds, including members of the thiazolid-
Figure 1. Small molecule GPR40 agonists (human GPR40 activity from FLIPR based
inone class of PPARc
agonists4 and other small molecule fatty acid
functional assay).9
mimetics,5 have recently been identified as ligands for GPR40. The
activation of the GPR40 receptor leads to inositol triphosphate
(IP3) production, release of intracellular Ca+2 from the endoplasmic
reticulum, and Ca+2 influx through membrane channels, collec-
tively resulting in enhanced insulin secretion. Therefore, under
hyperglycemic conditions, GPR40 agonists are capable of augment-
ing the release of insulin from islet cells. The specificity of this re-
sponse is suggested by data showing that the inhibition of GPR40
by siRNA attenuates FA-induced amplification of GSIS.6
risk of hypoglycemia. Second, the limited tissue distribution of
GPR40 (mainly in islets)2 suggests that there may be less chance
for side effects associated with GPR40 inhibition in other tissues.
Third, GPR40 agonists that are active in islets may have the poten-
tial to restore or preserve islet function.1b–d This would be highly
advantageous because long term diabetes therapy often leads to
the gradual diminution of islet activity, so that after extended peri-
ods of treatment, it is often necessary for patients to receive daily
insulin injections. By restoring or preserving islet cell function,
GPR40 agonists may delay or prevent the loss of islet cell function
in type 2 diabetic patients. Identification of a small molecule with
sufficient pharmacokinetic and pharmacodynamic properties to
effect islet GPR40 stimulation in vivo would allow for validation
of this target in animal models, and potentially in the clinic. This
There are several potential advantages of GPR40 as a target for
the treatment of type 2 diabetes. First, since GPR40 induced insulin
secretion acts in a glucose dependent manner, there is little or no
⇑
Corresponding author. Tel.: +1 732 594 7152; fax: +1 732 594 3007.
E-mail address: shawn_walsh@merck.com (S.P. Walsh).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.114

S. P. Walsh et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3390–3394
3391
article describes the results of efforts to identify novel GPR40
agonists.
position (Scheme 2). A range of biaryl ethers (6–15, 19; Table 1)
were formed through nucleophilic aromatic substitution of the
corresponding aryl halides. Alternatively, Mitsunobu coupling with
the corresponding alkyl alcohols provided 16–18. In both cases,
hydrolysis of the esters completed the desired b-(2-propynyl)dihy-
drocinnamic acids.
Cis- and trans- cyclopropyl hexynoic acids 20 and 21 were pre-
pared as shown in Scheme 3. Addition of propynyl magnesium bro-
mide to the biaryl ether ketone prepared as described in Scheme 1,
followed by elimination with catalytic p-toluene sulfonic acid fur-
nished the eneyne. This unstable intermediate was immediately
subjected to cyclopropanation using ethyl diazoacetate under cop-
per catalysis.10 Chromatographic separation of the cis and trans
diastereomers, followed by hydrolysis of the esters provided the
desired targets.11
The synthesis of compounds 22–25, in which the carboxylic
acid has been replaced by an oxazolidinedione, is depicted in
Scheme 4. Condensation of 4-hydroxybenzaldehyde with 2,2-di-
methyl-1,3-dioxane-4,6-dione was followed by conjugate addition
of propynyl magnesium bromide. Thermal hydrolysis of the
dimethylacetal with concomitant decarboxylation was followed
by methyl ester formation and bezyl protection of the phenol to
complete the oxazolidinedione precursor. This transformation
Following directed screening of the Merck compound collection,
thiazolidinedione 1 was identified as a modestly potent GPR40
agonist (Fig. 1). Extensive modifications were carried out in the
thiazolidinedione series which led to potent compounds,7 although
off target ion channel activity plagued some members of this class.
A full account of this research will be presented elsewhere.
Replacement of the thiazolidinedione functionality led to dihydro-
cinnamic acid derivatives typified by 2 and 3, as well as the malon-
ic acid 4. These carboxylic acids possess modest GPR40 agonist
activity, however, carboxylic acids of this type failed to provide
activity in our in vivo models. We speculated that these types of
fatty acid mimetics may undergo extensive protein binding in vivo,
leading to low free fractions and subsequent lack of efficacy. Addi-
tion of a methyl substituent at the b-carbon (5) led to about a two-
fold loss in potency, however, further exploration of this position
led to significant potency enhancement.8 This letter describes the
synthesis and biological activity of a series of b-substituted propi-
onic acids with improved potency.
A general synthesis of the carboxylic acids 2–5 is shown in
Scheme 1. Initially, the p-substituted phenols were coupled to
the appropriate aryl fluorides. For the synthesis of dihydrocinnam-
ic acids 2 and 3, and the b-methyl dihydrocinnamic acid 5, Wittig
homologation was followed by catalytic reduction of the resulting
olefins and finally ester hydrolysis. For the synthesis of malonic
acid 4, the 4-aryloxyaryl aldehyde intermediate was reduced to
the alcohol and subsequently converted to the primary bromide.
The resulting bromide was used in the alkylation of diethyl malon-
ate, followed again by ester hydrolysis to provide 4.
was accomplished through a-oxygenation utilizing the Davis oxaz-
iridine, conversion of the ester to the primary amide, and finally
cyclization. Hydrolysis of the benzyl ether revealed the phenol,
which was coupled to appropriate aryl fluoride to provide 23 as a
2:1 mixture of trans:cis diastereomers. Careful chromatographic
separation provided 24 and 25 as pure diastereomers.
The GPR40 activities of selected 4-aryloxyaryl and 4-alkyloxya-
ryl (3-phenyl)hexynoic acids are shown in Table 1. In general com-
pounds displayed similar levels of agonist activity across species
when mouse and human GPR40 was compared, and observed
IC50’s in the human GPR40 binding assay in most cases paralleled
the agonist EC50’s. As previously mentioned, b-methylation of 2
to provide 5, resulted in a slight loss in GPR40 activity, however
incorporation of the 2-propynyl substituent (6) resulted in a signif-
icant potency increase. Alternative aryl substitutions led to com-
pounds with similar potency levels, including the larger 4-
cyanonaphthyloxy group (10). Replacement of the cyano substitu-
ent in 10 with the trifluoromethyl group (11) resulted in a surpris-
ing loss in potency. In an effort to increase the polarity of aryl
substituent, a series of heteroaryl subunits were examined (12–
15). Most of these heterocycles resulted in a loss of GPR40 agonist
activity with the notable exception of the pyridyl substituent in 12.
Carbocycles were also tolerated, resulting in potent GPR40 agonists
16–18. SAR in related series revealed the central phenyl ring was
less tolerant of substitution, however, the fluoro substituted 19
proved to be a potent GPR40 agonist. Finally, disubstituted cyclo-
propyl carboxylic acids have previously been reported as potent
GPR40 agonists. Unfortunately, no synergistic effect was observed
The synthesis of a series of b-(2-propynyl)dihydrocinnamic
acids 6–19 is shown in Scheme 2. Condensation of the appropriate
anisaldehyde with 2,2-dimethyl-1,3-dioxane-4,6-dione was fol-
lowed by conjugate addition of propynyl magnesium bromide.
Heating of the resulting product in the appropriate alcohol solvent
led to decarboxylation and concomitant transesterification. Hydro-
lysis of the aryl methoxy group provided the phenols, which were
coupled using one of two methods to provide diversity at the R3
Scheme 1. Synthesis of dihydrocinnamic acids: R1 = H or Me, Ar = 2,4-di-ClPh or 2-
Cl,4-CF3Ph. Reagents and conditions: (a) Ar-F, CsCO3, 80 °C, DMF (61–79%); (b)
(MeO)2P(O)CH2CO2Me, NaH, THF, 0 °C; (c) Pd/C, H2, EtOAc; (57–72%, two steps) (d)
LiOH, MeOH/H2O (83–97%); (e) LiBH4, THF, 0 °C; (f) CBr4, PPh3, imidazole, CH2Cl2
(48% two steps); (g) (EtO2C)2CH2, NaH, DMF (51%).
Scheme 2. Synthesis of hexynoic acids: R1 = H or F, R2 = Me or Et, for R3 see Table 1 below, X = F, Cl, or Br. Reagents and conditions: (a) 2,2-dimethyl-1,3-dioxane-4,6-dione,
H2O (78–99%); (b) CH3CHCMgBr, THF, 0 °C; (c) R2OH, reflux; (d) BBr3, CH2Cl2, ꢀ78 °C ? rt (11–62%, three steps); (e) Ar-X, CsCO3, 80 °C, DMF (25–78%); (f) LiOH, THF, water,
0 °C (19–83%); (g) DIAD, PPh3, THF (17–75%).

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S. P. Walsh et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3390–3394
Table 1
GPR40 agonist activities for compounds 4–20a
Compd #
R1
—
R2
—
hGPR40b EC50 (nM)
2484
mGPR40c EC50 (nM)
Na
hSpad IC50 (nM)
Na
5
6
H
94
127
40
7
H
H
H
H
71
42
85
53
77
48
88
93
12
23
36
13
8
9
10
11
12
H
H
1401
108
1248
95
211
30
13
14
H
H
1568
1578
1484
985
433
2487
15
H
H
394
31
149
47
342
42
16e
17f
H
51
100
23
18
19
H
F
114
113
124
104
312
49
20
21
—
—
—
—
2820
24,490
871
>40,000
3435
>10,000
a
b
c
d
e
f
All compounds racemic unless otherwise noted.
Human GPR40 activity from FLIPR based functional assay.9
Mouse GPR40 activity from FLIPR based functional assay.9
Human GPR40 activity from SPA based binding assay.12
R stereochemistry at bezyl alcohol, mixture of diastereomers.
Racemic, mixture of diastereomers.13
when this functionality was combined with our pharmacophore to
provide the trisubstituted cyclopropyl acids.
In order to examine the role of the carboxylic acid in poten-
tial plasma protein binding interactions, we also prepared a set
of oxazolidinediones (OZD’s, 22–25; Table 2). This heterocycle
has previously been shown to act as a carboxylic acid surrogate
due the similar pKa of the NH. The benzyloxy OZD (22) was
Scheme 3. Synthesis of cyclopropyl hexynoic acids: Ar = 2-Cl,4-CF3Ph. Reagents
and conditions: (a) CH3CHCMgBr, THF, 0 °C; (b) MePhSO3H, CH2Cl2, rt (51%, two
steps) (c) Cu(OTf)2, N2CCO2Et, (+)-2,20-isopropylidenebis[(4R)-4-phenyl-2-oxazo-
line], CH2Cl2, 0 °C; (d) LiOH, THF, water, 0 °C (23–25%, two steps).

S. P. Walsh et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3390–3394
3393
Scheme 4. Synthesis of oxazolidinediones: Ar = 2-OCF2H,4-CF3Ph. Reagents and conditions: (a) 2,2-dimethyl-1,3-dioxane-4,6-dione, H2O; (b) CH3CHCMgBr, THF, 0 °C (84%,
two steps); (c) 3-pentanone, H2O, reflux; (d) SO2Cl, MeOH, 0 °C; (e) BnBr, NaH, DMF, 0 °C ? rt (56%, three steps); (f) (i) KHMDS, THF, ꢀ78 °C; (ii) PhSO2(N–O–CH)Ph,
ꢀ78 °C ? rt; (g) NH3, MeOH, rt; (h) CDI, CH2Cl2, rt (15%, three steps); (i) BBr3, CH2Cl2, ꢀ78 °C ? 0 °C; (j) Ar-F, CsCO3, 80 °C, DMF (43%, two steps).
Table 2
GPR40 agonist activities for compounds 22–25a
Compd #
Stereochem.
hGPR40b EC50 (nM)
mGPR40c EC50 (nM)
h Spad IC50 (nM)
22
23
24
25
cis/trans mix
cis/trans mix
Trans
954
124
7581
130
3707
266
7058
136
4399
19
2414
151
Cis
a
b
c
All compounds racemic.
Human GPR40 activity from FLIPR based functional assay.9
Mouse GPR40 activity from FLIPR based functional assay.9
Human GPR40 activity from SPA based binding assay.12
d
Table 3
GPR40 activity for selected compounds in the presence of serum
Compd
#
hGPR40a EC50
(nM)
hIP3 + 10% HS EC50
(nM)
hIP3 + 0.4% MSA EC50
(nM)
7
8
10
12
19
23
71
42
53
108
113
124
436
519
172
409
306
224
455
597
247
NA
349
249
a
Human GPR40 activity from FLIPR based functional assay.9
shown to be a modest GPR40 agonist with respect to both hu-
man and mouse receptors. Conversion of the benzyl group to
one of the preferred biaryl ethers from our carboxylic acid SAR
(23) led to a compound with comparable potency to the corre-
sponding carboxylic acid. Separation of the diastereomers re-
vealed that the majority of the GPR40 activity resulted from
the cis diastereomer.
Figure 2. Lean mouse ipGTT results.
In order to assess the potential effect of plasma protein in vivo,
an assay was developed which measured IP3 production from hu-
man cells in the presence of either human serum (10%), or rat ser-
um albumin (0.4%).14 Table 3 summarizes the serum shifted
potencies of selected GPR40 agonists in this assay. In this assay,
the relative polarity of the aryl group had little effect on the serum
shifted potency, however, fluorination of the central ring slightly
reduced the serum shift (7 vs 19). In agreement with our earlier
prediction, conversion of the carboxylic acid to the OZD does ap-
pear to reduce the serum shift (7 vs 23).
From the above set, two compounds were selected for pharma-
cokinetic profiling in mouse (Table 4). These compounds are char-
acterized by low clearance and volume of distribution, consistent
with high plasma protein binding, as well as long half-life and good
oral bioavailability.
Table 4
Pharmacokinetic properties of selected GPR40 agonistsa
Compd #
Clp (mL/min/kg)
Vd (L/kg)
t1/2 (h)
PO AUCN (lMꢁhꢁkg/mg)
F (%)
Cmax
(l
M)
Tmax (h)
7
25
0.49
0.85
0.31
0.47
7.8
7.0
83.8
42.6
112
93
12
5.5
4.0
4.7
a
Dose.

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S. P. Walsh et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3390–3394
Table 5
Off targets activity of selected GPR40 agonists
Compd #
CYP2C9 IC50
(
lM)
CYP2D6 IC50
(
lM)
CYP3A4 IC50
(l
M)
hERG IC50
(l
M)
CAV 1.2 IC50 (lM)
7
19
25
>50
28.0
2.4
>50
>50
>50
>50
>50
>50
>60
25.0
3.4
>30
25.6
5.1
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Compound 7 was selected for further profiling in vivo (Fig. 2).
Oral administration of 7 in normal lean mice one hour prior to dex-
trose challenge in an intraperitoneal glucose tolerance test
(IPGTT)15 significantly reduced blood glucose excursion (66% inhi-
bition of AUCGlu, p <0.004) at a dose of 10 mpk.
Finally, the carboxylic acid 7 as well as the related fluoro substi-
tuted carboxylic acid 19 and OZD 25 were profiled in a series of off
target assays (Table 5). For all three compounds, P450 inhibition
was found to be minimal, with 25 demonstrating <5 lM inhibition
of the CYP2C9 isoform. Consistent with previous results, the two
carboxylic acids showed minimal ion channel activity against the
hERG and CAV 1.2 channels, although the OZD 25 displayed mod-
est activity.
6. Itoh, Y.; Kawamata, Y.; Harada, M.; Kobayashi, M.; Fujii; Fukusumi, S.; Ogi, K.;
Hosoya, M.; Tanaka, Y.; Uejima, H.; Tanaka, H.; Maruyama, M.; Satoh, R.;
Okubo, S.; Kizawa, H.; Komatsu, H.; Matsumura, F.; Noguchi, Y.; Shinohara, T.;
Hinuma, S.; Fujisawa, Y.; Fujino, M. Nature 2003, 422, 173.
7. (a) Zhou, C.; Tang, C.; Chang, E.; Ge, M.; Lin, S.; Cline, E.; Tan, C. P.; Feng, Y.;
Zhou, Y.-P.; Eiermann, G. J.; Petrov, A.; Salitoro, G.; Meinke, P.; Mosley, R.;
Akiyama, T. E.; Einstein, M.; Kumar, S.; Berger, J.; Howard, A. D.; Thornberry, N.;
Mills, S. G.; Yang, L. Bioorg. Med. Chem. Lett. 2010, 20, 1298; (b) Tan, C. P.; Feng,
Y.; Zhou, Y.-P.; Eiermann, G. J.; Petrov, A.; Zhou, C.; Lin, S.; Salituro, G.; Meinke,
P.; Mosley, R.; Akiyama, T. E.; Einstein, M.; Kumar, S.; Berger, J.; Mills, S. G.;
Thornberry, N. A.; Yang, L.; Howard, A. D. Diabetes 2008, 57, 2211.
8. For other examples of b-substituted propionic acid GPR40 agonists see:; (a)
Ackerman, M.; Houze, J.; Lin, D. C. H.; Liu, J.; Liu, J.; Luo, J.; Ma, Z.; Medina, J. C.;
Qiu, W.; Reagan, J. D.; Sharma, R.; Schmitt, M. J.; Shuttleworth, S. J.; Sun, Y.;
Wang, Y.; Zhang, J.; Zhu, L. US20070142384A1, 2007.; (b) Ackerman, M.;
Brown, S.; Houze, J.; Liu, J.; Liu, J.; Ma, Z.; Medina, J. C.; Qiu, W.; Schmitt, M. J.;
Sharma, R.; Wang, Y.; Zhu, L. US20070066647A1, 2007.; (c) Ackerman, M.;
Houze, J.; Lin, D. C. H.; Liu, J.; Luo, J.; Medina, J. C.; Qiu, W.; Reagan, J. D.;
Sharma, R.; Schmitt, M. J.; Shuttleworth, S. J.; Sun, Y.; Wang, Y.; Zhang, J.; Zhu, L.
US20060004012A1, 2006.
In conclusion, a series of b-substituted 3-(4-aryloxyaryl)propa-
noic acids were prepared and their activities as GPR40 agonists
were evaluated. The OZD group was found to be a suitable acid sur-
rogate, which reduced the serum shifted EC50 in vitro, but also led
to modest ion channel activity. Following SAR optimization, com-
pound 7 was selected for in vivo evaluation, where it demonstrated
the potential of this class of small molecule GPR40 agonists as glu-
cose lowering agents.
Acknowledgment
The authors thank Eric Cline for the preparation of compound 3.
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11. Cis and trans diastereomers were assigned based on 1H NMR spectra and are in
agreement with previously biological activity of previously reported GPR40
agonists (Ref. 5b).
12. SPA binding assay was carried out using receptor isolated from CHO cells
overexpressing human GPR40. All reported values are means of P3
experiments, with standard deviations <75% of the average.
13. The coupling partner alcohol was prepared as follows:
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Baumann, C.; Lenhard, J.; Murray, W. V.; Demarest, K. T.; Kuo, G.-H. J. Med.
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(a) NaNO2, H3PO4, HNO3, CuBr, H2O, 0 °C ? rt (77%); (b) 4-CH3C6H4B(OH)2,
Pd(PPh3)4, Na2CO3, DME, EtOH, 140 °C, MW (68%); (c) LiBH4, THF, 0 °C (72%).
14. IP3 assay protocol is described in Ref. 7b.
15. For a full account of the experimental protocol used in the IPGTT assay see Ref.
7b.