Discovery of novel orally bioavailable GPR40 agonists

Article abstract of DOI:10.1016/j.bmcl.2013.03.060

The GPR40 (FFA1) has emerged as an attractive target for a novel insulin secretagogue with glucose dependency. A series of novel orally bioavailable GPR40 agonists was discovered. SAR study and structural optimization led to identification of compounds 28a and 30a as potent GPR40 agonists with superior physiochemical properties and robust in vivo efficacy in rhesus monkeys.

Full text of DOI:10.1016/j.bmcl.2013.03.060

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2920–2924
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel orally bioavailable GPR40 agonists
⇑
Hejun Lu , Hongbo Fei, Fanglong Yang, Suxin Zheng, Qiyue Hu, Lei Zhang, Jijun Yuan, Jun Feng,
Piaoyang Sun, Qing Dong
Shanghai Hengrui Pharmaceutical Co. Ltd, 279 Wenjing Rd., Shanghai 200245, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The GPR40 (FFA1) has emerged as an attractive target for a novel insulin secretagogue with glucose
dependency. A series of novel orally bioavailable GPR40 agonists was discovered. SAR study and struc-
tural optimization led to identification of compounds 28a and 30a as potent GPR40 agonists with supe-
rior physiochemical properties and robust in vivo efficacy in rhesus monkeys.
Received 29 December 2012
Revised 14 March 2013
Accepted 15 March 2013
Available online 26 March 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Diabetes
GPR40
Agonist
Orally bioavailable
Species specificity
GPR40 (also known as FFA1) is a free fatty acid-activated
G q-coupled class 1 GPCR that is found on the surfaces of pancreatic
orally bioavailable small molecular GPR40 agonists with improved
potency and physiochemical properties. Based on a published
a
b-cells, gastrointestinal enteroendocrine cells, immune cells and
also parts of the brain. Medium- (C6–C12) and long chain
(C14–C24) saturated and unsaturated fatty acids stimulate GPR40,
and evidence points to GPR40 being a mechanistic link to the
well-known effects of fatty acids to acutely stimulate insulin and
incretin secretion.1–3 The effect of fatty acid on insulin and incretin
(glucagon-like peptide-1 (GLP-1) and glucose-dependent insulino-
tropic peptide (GIP) secretion is blunted or eliminated in mice lack-
ing GPR40.4 GPR40 knockout mice also show impaired glucose and
arginine induced insulin secretion in vivo.5 Based on these studies,
GPR40 may serve as an attractive target to mediate insulin secre-
tion and agents that serve as GPR40 agonists may be useful for
the treatment of type-2 diabetes. Because activity of GPR40 ago-
nists on islet b-cells is glucose dependent, it is believed that
GPR40 may offer advantages to commonly used sulfonylurea drugs
which act independently of ambient glucose levels, resulting in
hypoglycemia in some patients.6
a
O
O
O
S
O
O
O
O
TAK-875
Introducing a fused dioxane ring
O
O
O
S
O
O
O
O
Potent small molecule GPR40 agonists based on scaffolds such
as aryalkanoic acids and thiazolidinediones have been reported
by several groups.7 More recently, research in Takeda Pharmaceu-
tical Co. Ltd led to discovery of TAK-875 (Fig. 1), a potent and orally
bioavailable small molecule GPR40 agonist, which is currently
undergoing phase III human clinical trials.8 This Letter describes
the design, synthesis and biological activity of a series of novel
O
8a
b
Figure 1. (a) The idea of introducing a fused dioxane ring to TAK-875. (b) The
conformation overlap between TAK-875 and 8a. The carbons of TAK-875 is shown
in yellow stick while the carbons of 8a in green.
⇑
Corresponding author. Tel.: +86 21 54759109; fax: +86 21 54759072.
E-mail address: lvhj@shhrp.com (H. Lu).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.060

H. Lu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2920–2924
2921
Table 1
Structures and GPR40 agonistic activities of compounds 8a–15
O
OH
i-iii
R2
H
OTBS
O
O
R3
R1
R4
S
O
S
O
O
O
O
O
O
O
O
S
O
R5
O
O
1
2
R6
OH
No.
TAK-875
R1
R2
R3
R4
R5
R6
Chirality
EC50 (nM)
Emax (%)
a
HO
HO
O
41.8
55.9
282
100
124
111
87
83
83
77
89
71
53
v
iv
8a
8b
9a
H
H
F
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
F
Me
Me
Me
Me
Me
Me
Cl
Cl
Me
Cl
Me
Me
Me
Me
Me
Me
H
H
H
H
H
H
H
H
H
F
R
S
R
S
R
S
R
S
R/S
R/S
R/S
R/S
I
O
O
2
46.5
67.5
82.4
120.7
94.8
145.2
122.6
56.2
205.1
125
O
O
9b
F
10a
10b
11a
11b
12
H
H
H
H
H
F
3
4
F
H
H
H
H
H
H
O
O
vii
13
102
69
66
O
S
O
14
15
H
H
Cl
Cl
O
R
O
I
O
H
H
a
Values are means of three experiments.
O
5, R = TBS
6, R = H
vi
2 under typical Mitsnobu conditions, followed by TBAF deprotec-
tion, giving intermediate 6. Palladium catalyzed intra-molecular
cyclization of 6, followed by sodium hydroxide hydrolysis giving
the final product 8a. Compound 8b is a diastereomer of 8a and is
synthesized via similar procedures except using AD-mix bas chiral
reagent in step ii.
Compounds 8a,b were tested for GPR40 activity in a functional
assay monitoring calcium flux in CHO cells transiently transfected
with human GPR40 gene. As shown in Table 1, compound 8a was
found to be a nanomolar GPR40 agonist, while compound 8b was
much less potent than 8a. To explore the structure activity rela-
tionship of this new scaffold, a series of compounds with different
substitution patterns on the left and middle phenyl ring were syn-
thesized and tested as GPR40 agonists. The results were summa-
rized in Table 1.
2,6-Dimethyl substitution on left phenyl ring was found to be
optimal for GPR40 agonistic activity (8a, 9a), while introduction
of fluorine at R1 position improved activity (9a vs 8a, 13 vs
11a,b). Introduction of fluorine substitutions at R2, R3, and R6 posi-
tions gave mixed results, while in all cases, the R diastereomers
were more potent than its S counterparts.
Compound 9a and 9b were selected for further profiling in vivo
(Fig. 2). Oral administration of 9b (50 mg/kg) and TAK-875 (20 mg/
kg) in high fat feasted ICR mice 15 min prior to dextrose challenge
in an oral glucose tolerance test (OGTT) significantly reduced blood
glucose excursion. Compounds 9a failed to exhibit any efficacy in
this test, probably due to high clearance of the compound in mice
(data not shown).
Although 9b exhibited some oral efficacy in mice OGTT test, it
appeared to be less potent than TAK-875. We then went further
to modify these compounds to improve their in vitro and in vivo
GPR40 activity. Elimination of the 3-(methylsulfonyl)propan-1-ol
tails in compounds 8a–15 gave another series of GPR40 agonists.
These compounds were synthesized similarly as 8a and tested as
GPR40 agonists (Table 2).
To our surprise, compounds without the 3-(methylsulfonyl)pro-
pan-1-ol tails generally exhibited improved GPR40 agonistic
activity. Also noteworthy in this series of compounds is the activity
difference between the S and R diastereomers were generally smal-
ler, opposite to the SAR in compounds with the sulfone tails in
Table 1. We hypothesized that the lack of the sulfone tail could
O
O
O
O
S
O
O
O
O
R'
7, R' = Me
8a, R' = H
viii
Scheme 1. Synthesis of 8a. Reagents and conditions: (i) CH2 = PPh3, dioxane/H2O,
100 °C, 12 h, 80%; (ii) AD-mix , tBuOH/H2O (1/1), 0 °C–rt, 4 h, 100%; (iii) TBSCl,
a
imidazole, DMAP (cat.), DMF, rt, 1 h, 70%; (iv) ICl, Et2O, 0 °C–rt, 0.5 h, 66.5%; (v) 1,10-
(1,2-diazenediyl)bis[1-(1-piperidinyl)-methanone, P(n-Bu)3, toluene, 50 °C, 1 h,
87%; (vi) TBAF, THF, rt, 1 h, 97%; (vii) Pd(OAc)2, 1,10-[(1S)-[1,10-binaphthalene]-
2,20-diyl]bis[1,1-di-tert-butyl-phosphine (cat.), Cs2CO3, toluene, 50 °C, 12 h, 46%;
(viii) NaOH, THF/MeOH (1/5), 60 °C, 1 h, 64%.
docking study8,9 of TAK-875 in a GPR40 homology model, the mid-
dle phenyl ring is orthogonal to the right dihydrobenzofuran ring
as well as the left 2,6-dimethylphenyl ring, this conformation al-
lowed this molecule to fit into the active pocket of GPR40 with
multiple hydrophilic as well as hydrophobic interactions.
To design a novel series of GPR40 agonist, we tried to introduce
conformational restrictions into the TAK-875 skeleton, which may
result in decreased molecular flexibility and less rotatable bonds,
and in this way improve the physiochemical properties while pre-
serving GPR40 agonistic potency. Based on the predicted binding
conformation of TAK-875,8,9 introduction of a fused dioxane ring
into TAK-875 provided
a pair of two diastereoisomers 8a,b
(Fig. 1a). Comparison of the energy minimized conformation of
8a and TAK-875 resulted in high percentage of overlap, indicating
good chance of 8a being a potent GPR40 agonist (Fig. 1b). We then
set up to synthesis compounds 8a,b and tested them as GPR40 ago-
nists. Synthesis of 8a was outlined in Scheme 1.
Compounds 1 and 3 were synthesized via published proce-
dures.8 Wittig reaction of 1, followed by Sharpless asymmetric
di-hydroxylation with AD-mix
a and selective TBS protection of
the primary alcohol led to intermediate 2. Iodination of compound
3 with equimolar ICl gave intermediate 4, which was coupled with

2922
H. Lu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2920–2924
Table 3
Structures and GPR40 agonistic activities of compounds 28–35
R3
O
R2
O
O
R1
O
OH
R1
R2
R3
Chirality
EC50 (nM)
Emax (%)
a
No.
28
Br
Br
Br
H
Cl
Cl
H
H
H
Br
H
H
Cl
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
R/S
R
S
R/S
R
S
R
S
R
S
54.7
13.5
89
120
105
38
87
72
129
61
99.5
55.6
66
28a
28b
29
30a
30b
31a
31b
32a
32b
33
111.1
>10,000
34.2
219.8
16.0
190.5
11.3
30.4
Cl
Cl
Figure 2. ICR mice OGTT results of compound 9b and TAK-875.
CF3
CF3
Cyclopropyl
Br
CF3
R/S
R/S
R/S
36.4
105.1
63.7
Table 2
34
35
38
79
Structures and GPR40 agonistic activities of compounds 16a–27
F
a
Values are means of three experiments.
R1
R2
R3
O
O
O
R5
ated (29). R diastereomers were generally more potent than S dia-
stereomers. 3,5-Disubstitution resulted in diminished GPR40
agonistic activity (34, 35).
Compounds 28a, 30a, 31a and 32a were selected for pharmaco-
kinetic profiling in rats, dogs and monkeys (Table 4). These com-
pounds are characterized by low clearance and volume of
distribution, consistent with high plasma protein binding, as well
as long half-life and good oral bioavailability.
Compounds 28a, 30a, 31a and 32a were then pushed forward to
in vivo profiling. To our surprise, none of them exhibited significant
in vivo efficacy in our ICR mice OGTT test after a 50 mg/kg single
dosage (28a: 4.9% inhibition of AUCGlu, p <0.005; 30a: 10.98% inhi-
bition of AUCGlu, p <0.005; 31a: 4.9% inhibition of AUCGlu, p <0.005;
32a: 13.3% inhibition of AUCGlu, p <0.005). This discrepancy be-
tween in vitro and in vivo results promoted us to conjecture that
their GPR40 activity maybe different among species tested.
Species specificity of small molecular GPR40 agonists have been
reported by Takeda scientist.9 In the binding pocket of TM5, a
Leu186 in human GPR40/FFA1 is replaced with Phe in rat, resulting
in dramatic inter-species GPR40 activity discrepancy in certain
scaffolds of small molecular GPR40 agonists.
O
R4
R3
OH
a
No.
R1
R2
R4
R5
Chirality
EC50 (nM)
Emax (%)
16a
16b
17a
17b
18a
18b
19
20
21
22
23
Me
Me
Me
Me
Cl
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
F
Me
Me
H
H
F
H
H
H
H
H
H
H
H
H
H
Me
Me
H
Me
Me
H
H
H
H
H
H
H
Me
H
H
H
H
R
S
R
S
R
S
R/S
R/S
R/S
R/S
R/S
R/S
R/S
R/S
R/S
52.9
30.1
39.3
19.1
66.7
56.7
65.4
35.9
38.7
61.6
88
85
72
76
91
99
97
105
77
90
103
89
Cl
H
Me
Me
Me
Me
H
Cl
F
CF3
24
25
26
27
132
68
74.7
73.2
74.5
103
115
104
H
H
H
H
H
a
Values are means of three experiments.
render the molecule with more flexibility to fit the binding pocket,
therefore minimized the potency differences between the two dia-
stereomers. Compounds with one or two methyl substitution at 2,6
position of the left phenyl ring were found to be most potent in
GPR40 assay (16b, 17b, 20). Chlorine, Fluorine or trifluoromethyl
substitution at 2,6 positions resulted in less potent compounds
(18a, b, 25, 26, 27). Compounds without 2,6 substitutions at the left
ring were less potent in GPR40 assay (19, 24).
Compound 17b was tested in high fat feasted ICR mice OGTT
test and was found only moderately active in this in vivo model
(9.7% inhibition of AUCGlu, p <0.005).
During profiling of the above compounds, a compound without
a left phenyl ring (28, Table 3) was noticed, exhibiting good GRP40
agonistic activity, indicating the left phenyl ring may not be neces-
sary for GPR40 activity. This observation was further explored by
introducing different substitutions onto the middle phenyl ring
and the results were summarized in Table 3.
To further understand the species difference, we also built an in
house homology model of GPR40 as shown in Figure 3, using the
similar procedure.9 There is a loop spanning across the possible
binding site of TAK-875 and was highlighted in blue. For conve-
nience, we will call it the Blue Loop in the rest of the article.
The residue similarity for the Blue Loop (between a.a. 121 and
180) among human, rat, mouse and monkey was compared.
(Fig. 3b) Although whole sequence similarity among these species
are all more than 90%, the blue Loop among human, rat and mouse
are quite different. At the same time, the blue loops between
human and monkey are almost identical with only one exception
at residue 143.
In our GPR40 homology model, the residue 143 (shown in blue
stick) was found quite far away from the putative ligand binding
site (Fig. 4), In summary, both sequence and structural evidences
suggested that monkey being a better animal model for the trans-
lation between in vitro and in vivo activity.
Compounds with a meta bromo-, chloro-, or trifluoromethyl
substitution or meta, para di-chloro substitution at the middle
phenyl ring were found to be highly potent in GPR40 assay (28a,
30a, 31a, 32a), however, para bromo-substitution was not toler-
The conjecture of species specificity of compounds 28a, 30a,
31a and 32a were confirmed by a rat GSIS INS-1 assay. The results
were summarized in Table 5. TAK-875 was used as a reference
compound in this assay. Although 28a exhibited comparable EC50

H. Lu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2920–2924
2923
Table 4
Pharmacokinetic parameters of selected GPR40 agonistsa
No.
Dosage (mg/kg)
Clp (L/h/kg)
Vd (L/kg)
T1/2 (h)
PO AUC (ng h/ml)
F (%)
Cmax (ng/ml)
Rat
28a
30a
31a
32a
5.0
5.0
5.0
5.0
0.007
0.005
0.01
0.25
0.21
0.25
0.23
26.8
27.5
13.3
16.1
529,516
665,884
337,172
427,069
17,775
21,950
18,925
19,375
0.01
100
Beagle dog
28a
30a
1.0
1.0
1.0
0.03
0.02
—
0.93
0.71
—
26.4
21.1
19.1
30,554
35,634
47,414
2273
2673
2582
32a
Cynomolgus monkey
28a
30a
32a
6.0
6.0
6.0
0.04
0.05
0.10
1.02
0.85
2.38
18.0
12.9
16.4
136,301
126,223
54,660
5357
19,712
7705
a
Data are average of three animals.
Figure 4. Docking of TAK-875 inside of a homology GPR40 model.
Table 5
Rat GSIS INS-1 assay of selected GPR40 agonists
R3
O
R2
R1
O
O
Figure 3. (a) Full homology model of human GPR40. (b) The blue loop sequence
O
OH
alignment among human, Cynomolgus monkey (MACFA), mouse and rat GPR40.
R1
R2
R3
EC50 (nM)
Emax (%)
a
No.
TAK-875
28a
30a
31a
32a
93
77
—
—
—
100
40
36.8
46.8
43.6
Br
Cl
Cl
H
H
Cl
H
H
H
H
H
value in this assay (77 nM vs 93 nM), its maximum efficacy was
only 40% of TAK-875, indicating partial agonistic activity of 28a
to rat GPR40. Similarly, none of compounds 30a, 31a and 32a
exhibited over 50% of maximum efficacy in this assay.
CF3
a
Values are means of three experiments.
Considering the high probability of species specificity and the
potential of monkey as a reasonable in vivo model, compounds
28a and 30a were then tested in an obese type 2 diabetes rhesus
monkey IVGTT model (Oral administration of 28a (6 mg/kg), 30a
(6 and 20 mg/kg) and TAK-875 (20 mg/kg) in high fat feasted male
monkeys 1 or 2 h prior to dextrose challenge in an intravenous glu-
cose tolerance test). Both 28a and 30a intensively reduced the
blood glucose excursion during the test, confirming our conjecture
of GPR40 species specificity. The detailed results were summarized
in Table 6.
GPR43 are activated by short-chain FFAs, GPR40 and GPR120 are
activated by medium- to long-chain FFAs and some eicosanoids.
Compounds 28a and 30a were tested against GPR41, GPR43 and
GPR120 and none of them were active up to 10 lM concentration,
indicating high GPR40 selectivity of this new scaffold.
Both compounds 28a and 30a have a molecular weight of less
than 400, relatively low logD (<3) and only 3 rotatable bonds,
which is quite ideal in the sense of drug ability. Compounds 28a
and 30a exhibited no hERG inhibition in a patch-clamp assay
GPR40 belongs to a family of FFAs binding GPCRs, which in-
cludes GPR40, GPR41, GPR43, and GPR120.10,11 While GPR41 and

2924
H. Lu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2920–2924
Table 6
Cao Guoqing for his stimulating discussions and feedback on this
manuscript.
IVGTT of selected GPR40 agonists in an obese type 2 diabetes rhesus monkey modela
No.
Dosage (mg/kg)
Inhibition of AUCGlu
0–60 min (%) 0–120 min (%)
References and notes
TAK-875
28a
30a
20.0
6.0
6.0
14.73
32.09
7.71
15.02
24.45
10.71
13.16
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30a
20.0
16.43
3. Latour, M. G.; Alquier, T.; Oseid, E.; Tremblay, C.; Jetton, T. L. Diabetes 2007, 56,
1087.
a
Values are means of three experiments.
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l
mal liabilities in potential drug combination use.
In conclusion, we discovered a series of novel conformationally
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in vitro and in vivo activities. Species specificity was observed dur-
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IVGTT model. These compounds may offer additional choices for
future treatment of diabetes.
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Acknowledgments
The authors gratefully acknowledge all our colleagues in the
GPR40 program for their technical support. We also thank Dr.