Design and optimization of 2,3-dihydrobenzo[b][1,4]dioxine propanoic acids as novel GPR40 agonists with improved pharmacokinetic and safety profiles

Article abstract of DOI:10.1016/j.bmc.2018.10.019

GPR40 has become a new potential therapeutic target for the treatment of diabetes due to its role in mediating the enhancement of glucose-stimulated insulin secretion in pancreatic β cells with a low risk of hypoglycemia. As an effort to extend the chemical space and identify structurally distinct GPR40 agonists with improved liver safety, a novel series of fused-ring phenyl propanoic acid analogues were designed. Comprehensive structure-activity relationship studies around novel scaffolds were conducted and led to several analogues exhibited potent GPR40 agonistic activities and high selectivity against other fatty acid receptors. Further evaluation of pharmacokinetic (PK) profiles and in vivo efficacy identified compound 40a with excellent PK properties and significant glucose-lowering efficacy during an oral glucose tolerance test. In addition, compound 40a displayed lower hepatobiliary transporter inhibition and favorable druggability. All results indicate that compound 40a is a promising candidate for further development.

Full text of DOI:10.1016/j.bmc.2018.10.019

Accepted Manuscript
Design and Optimization of 2,3-dihydrobenzo[b][1,4]dioxine Propanoic Acids
as Novel GPR40 Agonists with Improved Pharmacokinetic and Safety Profiles
Bin Guo, Shimeng Guo, Jing Huang, Jingya Li, Jia Li, Qian Chen, Xianli Zhou,
Xin Xie, Yushe Yang
PII:
S0968-0896(18)31457-3
https://doi.org/10.1016/j.bmc.2018.10.019
BMC 14578
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
14 August 2018
16 October 2018
20 October 2018
Please cite this article as: Guo, B., Guo, S., Huang, J., Li, J., Li, J., Chen, Q., Zhou, X., Xie, X., Yang, Y., Design
and Optimization of 2,3-dihydrobenzo[b][1,4]dioxine Propanoic Acids as Novel GPR40 Agonists with Improved
Pharmacokinetic and Safety Profiles, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.
2
018.10.019
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Design and Optimization of 2,3-dihydrobenzo[b][1,4]dioxine
Propanoic Acids as Novel GPR40 Agonists with Improved
Pharmacokinetic and Safety Profiles
a,||
b,||
c
b
b
a
Bin Guo , Shimeng Guo , Jing Huang , Jingya Li , Jia Li , Qian Chen , Xianli
c
b,
a,
Zhou , Xin Xie *, and Yushe Yang *
a
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, Shanghai 201203, China.
b
CAS Key Laboratory of Receptor Research, the National Center for Drug Screening,
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, shanghai
2
01203, China.
c
School of Life Science and Engineering, Southwest Jiao Tong University, Chengdu
6
10031, China.
Abstract

GPR40 has become a new potential therapeutic target for the treatment of diabetes
due to its role in mediating the enhancement of glucose-stimulated insulin secretion in
pancreatic β cells with a low risk of hypoglycemia. As an effort to extend the
chemical space and identify structurally distinct GPR40 agonists with improved liver
safety, a novel series of fused-ring phenyl propanoic acid analogues were designed.
Comprehensive structure-activity relationship studies around novel scaffolds were
conducted and led to several analogues exhibited potent GPR40 agonistic activities
and high selectivity against other fatty acid receptors. Further evaluation of
pharmacokinetic (PK) profiles and in vivo efficacy identified compound 40a with
excellent PK properties and significant glucose-lowering efficacy during an oral
glucose tolerance test. In addition, compound 40a displayed lower hepatobiliary
transporter inhibition and favorable druggability. All results indicate that compound
4
0a is a promising candidate for further development.
Keywords: GPR40 agonist; 2,3-dihydrobenzo[b][1,4]dioxine; Insulin secretion; Type
2
diabetes mellitus

*
*
Corresponding author.
for Xin Xie: Tel: 86-21-50801313-156; E-mail: xxie@simm.ac.cn
for Yushe Yang: Tel &Fax: 86-21-50806786; E-mail: ysyang@simm.ac.cn

1
. Introduction
Diabetes has become a serious worldwide health care problem in recent years.
The International Diabetes Federation estimates that in 2017 more than 425 million
1
people were living with diabetes. Type 2 diabetes mellitus (T2DM), representing 95%
of all diabetes cases, is characterized by insufficient insulin secretion and insulin
resistance which results in high blood glucose levels. Some of the available treatments
for T2DM have been associated with undesired adverse effects such as
gastrointestinal symptoms, weight gain, liver damage, and a high risk of
2
-3
hypoglycemia. Therefore, there is an urgent need for preferable hypoglycemic drugs
4
-5
with improved safety.
G protein-coupled receptor 40 (GPR40), also known as FFA1 (free fatty acid
receptor 1) is primarily expressed in the β-cells of the pancreas and enteroendocrine
cells. It is activated by medium- and long-chain free fatty acids, leading to the
enhancement of glucose-stimulated insulin secretion only in the presence of elevated
glucose levels. This characteristic makes this receptor an excellent antidiabetic drug
6
-7
target with little or no risk of hypoglycemia.
Considerable effort has been made to focus on the development of small
8
-10
molecule GPR40 agonists in the past few years.
TAK875 (compound 1), the most
advanced compound from Takeda, validates this target as an effective approach for
11-12
the treatment of T2DM in clinical trials.
Unfortunately, TAK875 was discontinued
13
during phase III clinical trials due to liver-associated adverse events in patients.
1
4
Because GPR40 is not expressed in the liver, the findings are unlikely to be a direct

result of GPR40 agonism. It is speculated that the hepatotoxicity might be
1
5-16
structure-related.
Therefore, it is an urgent mission for medicinal chemists to
extend the chemical space and identify structurally distinct GPR40 agonists with
improved liver safety.
Figure 1. Representative GPR40 agonists and endogenous ligands.
Several series of GPR40 agonists have already been published and we find that
there is a common structural moiety of the benzyloxy fragment in these
1
7-25
compounds.
However, this may result in poor oral pharmacokinetic profiles (PK)
and a potential safety concern due to the benzaldehyde fragment resulting from
26-27
metabolic oxidation at the benzyl position.
In addition, the biphenyl structure of
these compounds has been associated with cytotoxicity through the inhibition of
2
8-29
mitochondrial respiratory chain complex 1 in reported literatures.
Therefore, as an
effort to identify novel GPR40 agonists with improved PK properties and safety
profiles, we designed a structurally distinct series of GPR40 agonists with the
benzyloxy and biphenyl structure removed. In this novel skeleton, we introduced an
ethylene glycol ether fragment instead of the benzyloxy group, and further
constructed a fused-ring structure between the ortho-position of the left phenyl ring
and ethylene glycol ether (Figure 2). In this way, decreased molecular flexibility and

less rotatable bonds may improve the physiochemical properties while preserving
3
0
GPR40 agonistic potency.
Herein, we describe the design and optimization of fused-ring phenyl propanoic
acids as novel GPR40 agonists, leading to the discovery of compound 40a which
exhibits excellent pharmacokinetic properties, improved hepatobiliary transporter
inhibition, and significant glucose-lowering efficacy during an oral glucose tolerance
test (OGTT) in SD rats as well as good druggability.
Figure 2. Design of novel fused-ring scaffold GPR40 agonists
2
. Results and discussion
2
.1 Chemistry
The synthesis of compounds 20a-c is depicted in Scheme 1. Refluxing of
,4-dihydroxyacetophenone 10 with oxalic acid diethyl ester in absolute ethanol
2
employing sodium ethoxide as the base and subsequent acid cyclization afforded
compound 12. Hydrogenation of 12 under a pressure of 45 psi in acetic acid produced
compound 13. Then, 13 was protected by a benzyl group to obtain 14, which was
followed by LiAlH reduction to afford 15. The Mitsunobu reaction of compound 15
4
1
8
with ethyl 3-(2-fluoro-4-hydroxyphenyl) propanoate (compound 16) produced
intermediate 17. Deprotection of the benzyl group was performed under hydrogen in
the presence of palladium on charcoal, and the resulting phenol 18 was alkylated to

give ethyl esters 19a-c. Finally, ethyl esters 19a-c was hydrolyzed to carboxylic acids
0a-c.
2
Scheme 1. Synthesis of compounds 20a-c. Reagents and conditions. (a) NaOEt, EtOH,
reflux; (b) HCl, EtOH, reflux, 81%; (c) 10% Pd/C, H , AcOH, 45psi, 65%; (d) BnBr,
2
K CO , DMF, 80 °C, 83%; (e) LiAlH , THF, 0 °C, 91%; (f) ADDP, TBP, toluene,
2
3
4
rt-40 °C, 65%; (g) 10% Pd/C, H , MeOH, rt, 82%; (h) K CO , MeI for 19a or
2
2
3
CH OCH CH OMs for 19b or CH OCH CH Cl for 19c; (i) LiOH, THF/MeOH/H O,
3
2
2
3
2
2
2
rt; (j) HCl, rt.
The preparation of compounds 25a-d and 29 was outlined in Scheme 2.
Nucleophilic substitution of substituted salicylaldehyde 21a-d using epichlorohydrin
to afford compounds 22a-d. The oxidation of 22a-d with 3-chloroperbenzoic acid and
subsequent cyclization under basic condition provided substituted 1,4-benzodioxan
derivatives 23a-d. The Mitsunobu reaction of 23a-d with compound 16 gave esters
2
4a-d. On the other hand, compound 23b was further transformed into mesylate 26
3
1
and followed by substitution with ethyl 3-(4-amino-2-fluorophenyl) propanoate to

obtain ester 28. The resulting esters 24a-d and 28 were finally subjected to hydrolysis
to afford desired compounds 25a-d and 29, respectively.
Scheme 2. Synthesis of compounds 25a-d and 29. Reagents and conditions. (a)
epichlorohydrin, K CO , DMF, 80 °C; (b) mCPBA, DCM, reflux; (c) NaOH,
2
3
THF/H O, rt; (d) ADDP, TBP, toluene, rt-40 °C; (e) LiOH, THF/MeOH/H O, rt; (f)
2
2
HCl, rt; (g) MsCl, Et N, DCM, 0 °C; (h) K CO , DMF, 100 °C.
3
2
3
Compounds 32a-h and 35 were synthesized according to Scheme 3. Following
3
2
33
the similar route depicted in Scheme 2, the phenoxy derivatives 30a-h and 33 were
condensed with 23b using the Mitsunobu reaction and subsequent basic hydrolysis to
produce the desired carboxylic acids 32a-h and 35, respectively.

Scheme 3. Synthesis of compounds 32a-h and 35. Reagents and conditions. (a)
ADDP, TBP, toluene, rt-40 °C; (b) LiOH, THF/MeOH/H O, rt; (c) HCl, rt.
2
The synthetic routes utilized to prepare chiral isomers 40a-d of compound 32a
are shown in Scheme 4. Treatment of 5-chlorosalicylaldehyde 21b with chiral
oxiran-2-ylmethyl 4-methylbenzenesulfonates (R)-36 or (S)-36 in the presence of
potassium carbonate afforded 2-substituted salicylaldehydes (R)-37 and (S)-37,
respectively. Following a similar synthetic approach to the synthesis of compound
3
2a mentioned above, the four chiral isomers 40a-d can be successfully obtained
1
9
from (R)-38 and (S)-38 with (R)-30a and (S)-30a.
Scheme 4. Synthesis of compounds 40a-d. Reagents and conditions. (a) K CO , DMF,
2
3
8
0 °C; (b) mCPBA, DCM, reflux; (c) NaOH, THF/H O, rt; (d) ADDP, TBP, toluene,
2
rt-40 °C; (e) LiOH, THF/MeOH/H O, rt; (f) HCl, rt.
2
Different regio-isomers 45a-c of compounds 40a were synthesized according to a
similar route to the synthesis of compound 40a mentioned above, as outlined in
Scheme 5.

Scheme 5. Synthesis of compounds 45a-c. Reagents and conditions. (a) K CO , DMF,
2
3
8
0 °C; (b) mCPBA, DCM, reflux; (c) NaOH, THF/H O, rt; (d) ADDP, TBP, toluene,
2
rt-40 °C; (e) LiOH, THF/MeOH/H O, rt; (f) HCl, rt.
2
Starting from commercially available salicylaldehyde derivatives, more target
compounds 50a-g, 55a-b and 59a-d were synthesized according to the synthetic route
mentioned above by applying minor changes and optimizations as shown in Scheme 6.
For compounds 50a-g, the synthetic routes were the same as that for the synthesis of
compound 40a. For compounds 55a-b, intermediate 48c was first protected by TBS to
give compound 51, and then coupling of 51 with K [Fe(CN) ]·3H O or NaSO Me
4
6
2
2
followed by deprotection provided intermediates 53a-b. For compounds 59a-d,
compound 51 was converted to formyl and acetyl compounds 56a-b by employing
n
BuLi as the base in the presence of DMF and N, N-dimethylethanamide, respectively.
5
6a was further oxidized into acid 56c, which was condensed with dimethylamine
hydrochloride or morpholine, using HATU in the presence of sodium bicarbonate as
the base to produce amide 56d-e. The TBS-deprotection of compounds 56a-b and
5
6d-e was achieved under TBAF conditions to give compounds 57a-d. Finally,
compounds 53a-b and 57a-d were subjected to the Mitsunobu reaction with (S)-30a
and subsequent basic hydrolysis to provide 55a-b and 59a-d, respectively.

Scheme 6. Synthesis of compounds 50a-g, 55a-b and 59a-d. Reagents and conditions.
a) K CO , DMF, 80 °C; (b) mCPBA, DCM, reflux; (c) NaOH, THF/H O, rt; (d)
(
2
3
2
ADDP, TBP, toluene, rt-40 °C; (e) LiOH, THF/MeOH/H O, rt; (f) HCl, rt; (g)
2
TBDMSCl, Imidazole, DMF; (h) for 52a: K [Fe(CN) ]·3H O, DBU, Pd(PPh ) ,
4
6
2
3 4
8
5 °C; for compound 52b: NaSO Me, N,N'-Dimethyl-1,2-ethanediamine,
2
n
(
CuOTf) PhH, DMSO, 110 °C; (i) TBAF, THF; (j) BuLi, -78 °C, DMF for 56a or
2
t
DMA for 56b; (k) NaClO , 2-methyl-2-butene, NaH PO , BuOH/H O; (l) HATU,
2
2
4
2
NaHCO , dimethylamine hydrochloride for 56d or morpholine for 56e; (m) NaBH ,
3
4
MeOH, rt.
The di-substituted analogues 65a-c were synthesized by similar routes as shown
in Scheme 7.
Scheme 7. Synthesis of compounds 65a-c
α

α
Reagents and conditions. (a) TFA, hexamethylenetetramine, 105 °C; (b) K CO ,
2
3
DMF, 80 °C; (c) mCPBA, DCM, reflux; (d) NaOH, THF/H O, rt; (e) ADDP, TBP,
2
toluene, rt-40 °C; (f) LiOH, THF/MeOH/H O, rt; (g) HCl, rt.
2
2
.2 GPR40 agonistic activity and SAR study
A comprehensive structure-activity relationship (SAR) study was conducted
around the distinct fused-ring scaffold as illustrated in Figure 2.
The first round of structural modification focused on the fused-ring A and
substituents on phenyl ring B. Initially, a fused tetrahydropyran ring was introduced,
and various substituents were screened on the phenyl ring B. As shown in Table 1,
analogues with a fused tetrahydropyran ring only displayed weak GRP40 agonistic
activities (20a-c) regardless of the substituents. Then, the fused tetrahydropyran was
replaced by a fused dioxane ring. To our delight, the dioxane analogue showed
slightly improved activity (25a vs 20a). Agonistic activity further increased if
methoxy was converted to chlorine (25b vs 25a). However, bromo-substitution
resulted in decreased GPR40 agonistic activity (25c vs 25b). The substitution position
had some effect on the activity and the 6-substitution analogue seemed to exhibit
higher activity than the 7-substitution analogue (25c vs 25d). Then, we examined the

effect of the linker atom Z between the benzodioxanemethyl and the phenyl propanoic
acid moiety. Replacement of the oxygen atom with a nitrogen atom markedly reduced
the agonistic activity (29 vs 25b).
Table 1. In vitro GPR40 agonistic activity of compounds 20a-c, 25a-d and 29
b
compound
R2
R3
X
Z
hGPR40
Emax (%)
α
EC (μM)
5
0
2
0a
0b
0c
5a
5b
5c
5d
H
H
H
H
H
H
Br
H
/
OMe
CH
2
2
2
O
O
O
O
O
O
O
2.11 ± 0.54
36.27 ± 7.55
2.37 ± 0.56
1.47 ± 0.64
0.83 ± 0.29
2.24 ± 1.10
0.98 ± 0.08
58 ± 5
38 ± 6
36 ± 2
80 ± 10
50 ± 5
81± 3
67 ± 3
69 ± 1
92 ± 5
100
2
O(CH
2
)
3
SO
2
Me CH
2
O(CH
2
)
2
OMe
CH
O
O
O
O
O
/
2
OMe
Cl
Br
H
2
2
2
2
9
Cl
/
NH 13.57 ± 1.03
1
/
0.08 ± 0.02
4.80 ± 0.48
linoleic acid
α
EC50 values for hGPR40 activities measured by calcium mobilization assay.
b
Maximal efficacy (Emax) was determined compared to linoleic acid. Data are
presented as the mean±SD (n=3).

After fixing the benzodioxane ring, we screened various substituents at R on this
5
more potent scaffold with the knowledge that the introduction of substituents could
21
reduce the potential of β-oxidation. As shown in Table 2, propynyl (32a) was
preferred to the ethoxy (32b) and epoxy butyl group (35) at R . Then, we replaced the
5
propynyl by more polar nitrogen-containing heterocycle with the aim to reduce the
lipophilicity of the molecules. However, the resulting analogues (32c-h) displayed a
dramatic decrease in activity.
Table 2. In vitro GPR40 agonistic activity of compounds 32a-h and 35
α
b
compound
2a
2b
2c
R5
hGPR40 EC (μM)
Emax (%)
104 ± 7
46 ± 11
63 ± 7
50
3
0.10 ± 0.03
0.64 ± 0.34
1.03 ± 0.35
3
OEt
3
3
2d
5.88 ± 0.64
3.87 ± 1.47
3.06 ± 0.38
68 ± 2
70 ± 5
62 ± 4
3
2e
3
2f
3
2g
4.31 ± 5.80
1.17 ± 0.03
83 ± 10
62 ± 4
3
2h

3
5
/
8.06 ± 2.63
4.80 ± 0.48
66 ± 7
100
linoleic acid
α
EC50 values for hGPR40 activities measured by calcium mobilization assay.
b
Maximal efficacy (Emax) was determined compared to linoleic acid. Data are
presented as the mean±SD (n=3).
After identification of the propynyl moiety as the favored substituent at R , we
5
turned to optimization of the chirality of the two chiral centers A and B in the
molecules. Four isomers 40a-d of compound 32a were synthesized through an
asymmetric route (Scheme 4). As illustrated in Table 3, the chirality had a significant
effect on the GPR40 agonistic activity, especially at the chiral center B. In general,
(
S)-isomers displayed more potent than (R)-isomers (40a vs 40d or 40b vs 40c) for
chiral center A. In terms of chiral center B, (S)-isomers were also superior to
R)-isomers (40a vs 40b or 40c vs 40d). Then, we turned our attention to the effect of
(
the chloride atom position in the left benzene ring on the agonistic activity of GPR40.
Relocating the chloride atom from 7-position to 8-position afforded a 10-fold loss in
potency (40a vs 45c). Meanwhile, a shift of the chloride atom from 7-positon to
5
-position or 6-position also resulted in a more or less decrease in activity (40a vs 45a;
0a vs 45b). The general trend of activity is 7-Cl > 6-Cl > 5-Cl > 8-Cl, which was a
4
little different from the above SAR.
Table 3. In vitro GPR40 agonistic activity of compounds 32a, 40a-d and 45a-c

b
compound
R
Chiral A
Chiral B
hGPR40
Emax (%)
α
EC (μM)
5
0
3
4
2a
0a
7-Cl
7-Cl
7-Cl
7-Cl
7-Cl
5-Cl
6-Cl
8-Cl
racemic
racemic
0.10 ± 0.03
0.05 ± 0.01
3.40 ± 1.46
5.04 ± 1.49
0.26 ± 0.05
0.17 ± 0.05
0.11 ± 0.01
0.52 ± 0.26
4.80 ± 0.48
100
S
S
R
R
S
S
S
S
R
R
S
S
S
S
86 ± 3
47 ± 4
41 ± 8
65 ± 6
81 ± 8
80 ± 2
95 ± 9
100
4
0b
0c
0d
5a
5b
5c
linoleic acid
4
4
4
4
4
α
EC50 values for hGPR40 activities measured by calcium mobilization assay.
b
Maximal efficacy (Emax) was determined compared to linoleic acid. Data are
presented as the mean±SD (n=3).
Next, we shifted our focus on an examination of various substituents at the left
benzene ring. As shown in Table 4, a simple hydrogen substitution at R maintained
3
satisfactory activity. (50a vs 40a). Compared with the chloride atom, other halogen
atoms (F, Br) displayed lower activity (50b-c vs 40a). The small alkyl group such as

methyl (50d) led to a slight decrease in activity. Additionally, we found that
replacement of the methyl group with a different alkyloxy (50e-f) or
Trifluoromethoxy (50g) group resulted in further loss in activity. When
electron-withdrawing or more polar groups were introduced at R , the agonistic
3
activity substantially decreased or even disappeared (55a-b, 59a-d). Furthermore, we
introduced another halogen atom at the R site to suppress potential metabolic
2
oxidation of the left benzene, but this led to a decrease in the agonistic activity
(65a-c).
Table 4. In vitro GPR40 agonistic activity of compounds 50a-g, 55a-b and 59a-d
α
b
compound
0a
0b
0c
0d
0e
0f
R2
H
H
H
H
H
H
H
H
R3
H
hGPR40 EC (μM)
Emax (%)
70 ± 4
86 ± 5
69 ± 7
78 ± 3
91 ± 4
90 ± 3
73 ± 5
60 ± 8
50
5
0.09 ± 0.01
0.21 ± 0.02
0.21 ± 0.07
0.12 ± 0.05
0.13 ± 0.04
0.28 ± 0.04
0.70 ± 0.14
2.10 ± 0.86
5
F
5
Br
5
Me
OMe
5
i
5
O Pr
5
5
0g
5a
OCF
3
CN

5
5b
9a
9b
9c
H
H
H
H
SO
2
Me
OH
27.2 ± 16.2
> 30
55 ± 6
42 ± 4
51 ± 3
32 ± 6
5
CH
2
5
COCH
3
> 30
5
CON(Me)
2
> 30
5
9d
H
> 50
33 ± 1
6
5a
5b
5c
linoleic acid
F
Cl
Cl
Cl
0.21 ± 0.12
0.15 ± 0.03
0.20 ± 0.07
4.80 ± 0.48
46 ± 10
33 ± 5
82 ± 6
100
6
Cl
Br
6
α
EC50 values for hGPR40 activities measured by calcium mobilization assay.
b
Maximal efficacy (Emax) was determined compared to linoleic acid. Data are
presented as the mean±SD (n=3).
2
.3 Glucose-lowering effect of compounds in SD rats
Due to the good in vitro GPR40 agonistic activity, compounds 40a, 50a and 50d
were selected to further investigate their in vivo efficacy by an oral glucose tolerance
test (OGTT) in Sprague-Dawly (SD) rats. These compounds were orally administered
6
0 min prior to a glucose challenge (3.0 g/kg). As shown in Figure 3, all three
compounds significantly reduced blood glucose excursion (Figure 3A and C) and the
area under the curve of blood glucose (AUC0-60min or AUC0-120min) (Figure 3B and D)
compared with the control. They exhibited similar in vivo blood glucose-lowering
efficacies to TAK875 at the same dose.

Figure 3. In vivo efficacy of compounds 40a, 50a and 50d during an OGTT in SD
rats. (A) Time-dependent changes of glucose levels for 40a. (B) Area under the curve
(AUC0-60min) of plasma glucose for 40a. (C) Time-dependent changes of glucose
levels for 50a and 50d. (D) Area under the curve (AUC0-120min) of plasma glucose for
5
0a and 50d. Statistical significance compared with the vehicle group is *P < 0.05,
*P < 0.01. Data are expressed as mean values with standard deviation from eight
*
independent experiments (n = 8)
2
.4 Pharmacokinetic evaluation of compounds 40a, 50a and 50d
Because compounds 40a, 50a and 50d possessed significant in vivo blood
glucose-lowering efficacies, we further evaluated their PK properties in SD rats. As
outlined in Table 5 and 6, compound 40a exhibited excellent metabolic stability with

a half-life of 16.1 h, and a good bioavailability of 65.5% at an oral dose of 3 mg/kg.
Both maximal plasma concentration (Cmax) and plasma exposure (AUC ) of
0-t
compound 40a improved by 3-5 fold compared with TAK875 (compound 1) at a dose
of 10 mg/kg. In addition, the AUC and Cmax values of compound 40a increased as the
administration dose. When administered via the intravenous route, 40a displayed low
clearance with CL of 0.17 mL/min/kg. However, compared with 40a, compounds 50a
and 50d displayed inferior PK profiles, which was partly due to the insufficient
3
4
stability at the methyl group of the benzene ring such as in compound 50d.
Table 5. PK parameters of selected compounds in SD Rats after oral
α
administration
Cmax ± SD
ng/mL)
AUC ± SD
Tmax
(h)
t1/2
F
0-t
Compound
dose
(
(h*ng/mL)
(h)
(%)
po 3 mg/kg
po 10 mg/kg
po 10 mg/kg
po 10 mg/kg
po 10 mg/kg
9308 ± 3279
47821 ± 9309
36736 ± 8010
23684 ± 3697
19427± 5008
140301 ± 43179
656406 ± 43179
274813 ± 37477
65249 ± 8184
3.00
4.00
1.17
0.417
3.50
16.1
25.7
3.14
1.79
3.94
65.5
4
0a
5
0a
0d
1
5
137326 ± 19193
α
Abbreviations: Cmax: peak plasma concentration of a drug after administration;
AUC: area under the concentration–time curve; Tmax: time to reach Cmax; t1/2:
elimination half-life; F: bioavailability; p.o.: per oral. Data are expressed as the
mean values with standard deviation from three independent experiments (n = 3)；
Table 6. PK parameters of compound 40a in SD Rats after intravenous injection
α

CL± SD
AUC0-t ± SD
MRT
(h)
t
1/2
Vdss
Compound
dose
(
mL/min/kg) (h*ng/mL)
(h)
(L/kg)
40a
iv 1 mg/kg
0.17±0.008
71430 ± 6708
17.9
13.2 0.182 ± 0.0339
α
Abbreviations: CL: plasma clearance; MRT: Mean Residence Time; Vdss: volume
of distribution at steady state; iv: intravenous. Data are expressed as the mean
values with standard deviation from three independent experiments (n = 3)；
As a result of its high potency for GPR40, the selectivity of compound 40a was
examined against other related free fatty acid receptors. Differing from its excellent
GPR40 agonistic potency, compound 40a performed no agonistic activity over
GPR41, GPR120 and GPR119, which indicated a low off-target adverse effect. The
result was shown in Table 7.
Table 7. Selectivity of compound 40a
EC (μM)
50
compound
GPR40
0.046
GPR41
GPR120
GPR119
> 100
α
α
4
0a
NR
NR
α
NR, no response at concentrations up to 100 μM.
Although the definite mechanism about the hepatotoxicity caused by TAK875
was unclear, recent studies have revealed some clues to explain the possible
molecular basis. For example, Chen et al. speculated that the hepatobiliary transporter
1
5
inhibition of TAK875 might contribute to cholestatic hepatotoxicity. Hence, the
influence of compound 40a on bile acid transport was evaluated in sandwich-cultured

Rat Hepatocytes. Moreover, the biliary excretion index (BEI) was calculated to
demonstrate the fraction of the accumulated bile acid that is excreted into bile. As
shown in Figure 4, compound 40a displayed higher BEI than TAK875 at the same
concentration, indicating that compound 40a had lower hepatobiliary transport
inhibition. Furthermore, the inhibitory activities of compound 40a over multiple
human hepatobiliary transporters such as MRP2, OATP1BA and OATP1B3 were
tested. The result showed that compound 40a exhibited very weak inhibitory activity,
with IC > 100 μM against MRP2 and OATP1BA, and IC values of 39.49 μM
5
0
50
against OATP1B3, while TAK875 inhibited MRP2, OATP1B1 and OATP1B3
transporters with IC values of 2.41 μM, 2.28 μM and 11.2 μM, respectively.
5
0
Figure 4. Inhibitory effects of compound 40a on the concentration and BEI of
d8-TCA. (A) The concentration of d8-TCA in cells incubated in standard buffer
2
+
2+
(
containing Ca ) or Ca -free buffer. (B) Effect on BEI of d8-TCA. Data are
expressed as mean values from three independent experiments (n = 3).
In addition, the parameters of liability profiling were tested to evaluate the
+
druggability of compound 40a. 40a showed no obvious inhibition of the hERG K
channel (IC > 40 μM, patch clamp assay) and CYP450 isozymes (summarized in
5
0

Table 8). In addition, 40a displayed no genetic toxicity in the Ames mutagenicity test
and micronucleus formation assay. Besides, 40a was administered orally once a day
for two weeks at doses of 50 mg/kg and 100 mg/kg to evaluate its subacute toxicity in
SD rats. We found that 40a was well tolerated at both doses with no obvious toxic
reaction in terms of survival rate, behavior, food intake and organ coefficients.
Table 8. Inhibition of CYP450 Isozymes by 40a
CYP450: IC (μM)
5
0
compound
3
A4
3A4
1A2
2C19
> 100
2D6
2B6
84.7
2C9
60.5
4
0a
> 100
> 100
> 100
> 100
3
. Conclusion
In summary, considering possible causes about the hepatotoxicity caused by
TAK875 reported in literatures, a series of novel benzo-fused ring propanoic acid
derivatives were designed in order to extend the chemical space and identify
structurally distinct GPR40 agonists with improved liver safety. Extensive structural
modification and SAR study around these scaffolds led to several novel analogues
with high in vitro GPR40 agonistic activities, exampled by 40a, 50a and 50d. Further
pharmacokinetic profiles evaluation in SD rats demonstrated that compound 40a
displayed a favorable PK profiles with the longest half-life and the highest plasm
exposure. Compound 40a exhibited an obvious plasma glucose-lowering effect at an
oral dose of 10 mg/kg at an OGTT in SD rats. Furthermore, compound 40a showed
high selectivity over the relevant receptors GPR41, GPR120 and GPR119, which

indicated a low off-target adverse activity. TAK875 was an inhibitor of multiple
hepatobiliary transporters, which may result in cholestatic hepatotoxicity. Compared
with TAK875, compound 40a demonstrated lower hepatobiliary transporter inhibition.
In addition, compound 40a had promising druggability, such as low CYP450
inhibition, no genetic toxicity, and no hERG inhibition. In a two-week subacute
toxicity study in rats, 40a was well tolerated. These attributes render compound 40a a
suitable candidate for further evaluation.
4
4
. Experimental Section
.1 Chemsity. All reactions were carried out in oven-dried glassware with magnetic
stirring, unless otherwise specified. All regents were weighed and handled in air at
room temperature. Column chromatography was performed on silica gel (200 – 300
1
13
mesh). All new compounds were characterized by H NMR, C NMR, and low/high
resolution mass spectroscopy. NMR spectra were recorded on Bruker AVANCE III
3
00, 400, 500 or 600 NMR spectrometer. Chemical shifts (δ) are reported in parts per
million (ppm) and coupling constants (J) are reported in Hertz (Hz). EI-MS spectra
were obtained on a Thermo DFS spectrometer and ESI-MS spectra were obtained on
a LTQ linear ion trap mass spectrometer. All final compounds were purified to > 95%
purity as determined by an Agilent 1100 series LC system (PLATISIL ODS 5μm 250
×
4.6 mm) with two solvent systems (acetonitrile/water or acetonitrile/buffer (0.1%
CF COOH in water)).
3
ethyl 7-hydroxy-4-oxo-4H-chromene-2-carboxylate (12). Diethyl oxalate (24.16

mL, 178.6 mmol) and 2,4-dihydroxyacetophenone (8.0 g, 52.6 mmol) were dissolved
in absolute ethanol (80 mL) and added to the sodium ethanolate (19.2 g, 282.1 mmol)
in absolute ethanol (80 mL). The solution was stirred for 2 hour under reflux. After
cooling, the mixture was treated with 6N-HCl (80 mL) for 10 min and was extracted
with CH Cl (3 x 100 mL). The organic layer was dried over anhydrous Na SO and
2
2
2
4
concentrated in vacuo. The residue was dissolved in EtOH (100 mL) and treated with
c-HCl (3 mL). The mixture was stirred at reflux for 24 h. The mixture was
concentrated in vacuo and purified by column chromatography (Ethyl acetate:
1
Petroleum ether =1:2) to give compound 12 (10 g, 81%) as a light yellow solid. H
NMR (CDCl , 300 MHz) δ 7.86 (d, J = 8.76 Hz, 1 H), 6.86 (s, 1 H), 6.82 (d, J = 8.4
3
Hz, 1 H), 6.80 (s, J = 8.4 Hz, 1 H), 4.33 (q, J = 7.1 Hz, 2H), 1.30 (t, J = 7.1 Hz, 3 H).
+
MS (EI) m/z: 234 [M] .
ethyl 7-hydroxychromane-2-carboxylate (13). A solution of compound 12 (1.46 g,
6
.24 mmol) in acetic acid and THF is hydrogenated over 10% Pd/C (450 mg) at 45 psi
H for 16 hr at ambient temperatures. The catalyst is removed by filtration and the
2
filtrate is concentrated in vacua to give a residue. The residue is dissolved in EtOAc,
washed with saturated NaHCO , dried over Na SO and purified by column
3
2
4
1
chromatography to give compound 13 (900 mg, 65%) as a white solid. H NMR (400
MHz, DMSO-d ) δ 9.19 (s, 1 H), 6.81 (d, J = 8.2 Hz, 1 H), 6.28 (dd, J = 8.2, 2.4 Hz, 1
6
H), 6.20 (d, J = 2.4 Hz, 1 H), 4.80 (dd, J = 6.6, 3.8 Hz, 1 H), 4.21 – 4.08 (m, 2 H),
2
1
.71 – 2.60 (m, 1 H), 2.53 – 2.44 (m, 1 H), 2.15 – 2.06 (m, 1 H), 2.05 – 1.94 (m, 1 H),
+
.20 (t, J = 7.1 Hz, 3 H). MS (EI) m/z: 222 [M] .

ethyl 7-(benzyloxy)chromane-2-carboxylate (14). To an acetone (50 mL) solution
of ethyl 7-hydroxy-chromane-2-carboxylate 13 (5.0 g, 22.5 mmol) were added
potassium carbonate powder (4.67 g, 33.8 mmol) and benzyl bromide (4.01 mL, 33.8
mmol). The resulting suspension was heated to reflux for 16 h. The reaction mixture
was filtered through a pad of Celite. The filtrate was concentrated to give solid
material, which was redissolved in AcOEt, washed with water to remove residual salt,
dried over anhydrous Na SO . The mixture was concentrated in vacuo and purified by
2
4
column chromatography (ethyl acetate: petroleum ether = 1:10) to give
1
compound 14 (5.83 g, 83%) as an off-white solid. H NMR (500 MHz,
CDCl ) δ 7.33-7.46 (m, 5 H), 6.96 (d, 1 H), 6.621 (d, 1 H), 6.58 (dd, 1 H), 5.05 (s, 2
3
H), 4.72 (q, 1 H), 4.29 (q, 2 H), 2.69- 2.84 (m, 2 H), 2.29 (m, 1 H), 2.21 (m, 1 H),
1
.33 (t, 3 H).
7-(benzyloxy)chroman-2-yl)methanol (15). To a cooled solution of ethyl
-(benzyloxy)chromane-2-carboxylate 14 (1.9 g, 6.1 mmol) in dry THF (25 mL) was
added LiAlH (1.0 M in THF, 7.3 mL, 7.3 mmol) dropwise. The mixture was stirred at
(
7
4
0
°C under nitrogen for 2 hr, then quenched with methanol, treated with water, and
extracted with ethyl acetate. The extracts are combined, dried over anhydrous Na SO4.
2
The mixture was concentrated in vacuo and purified by column chromatography
(ethyl acetate: petroleum ether =1:5) to give compound 15 (1.5 g, 91%) as a colorless
1
oil. H NMR (400 MHz, CDCl ) δ 7.44 – 7.29 (m, 5 H), 6.94 (d, J = 8.4 Hz, 1 H),
3
6
1
.53 (dd, J = 8.3, 2.6 Hz, 1 H), 6.48 (d, J = 2.5 Hz, 1 H), 5.01 (s, 2 H), 4.14 – 4.06 (m,
H), 3.87 – 3.79 (m, 1 H), 3.79 – 3.70 (m, 1 H), 2.86 – 2.76 (m, 1 H), 2.75 – 2.66 (m,

1
H), 1.97 – 1.89 (m, 1 H), 1.88 – 1.76 (m, 1 H).
ethyl 3-(4-((7-(benzyloxy)chroman-2-yl)methoxy)-2-fluorophenyl)propanoate
17). To a solution of (7-(benzyloxy)chroman-2-yl)methanol 15 (2.65 g, 9.80 mmol)
and ethyl 3-(2-fluoro-4-hydroxyphenyl)propanoate (1.6 g, 7.54 mmol) in dry toluene
50 mL) was added tributyl phosphine (3.77 mL, 15.08 mmol) and azodicarboxylic
(
(
acid dipiperidide (3.8 g, 15.08 mmol). The mixture was stirred at 40°C for 5 hr, then
filtrated. The residue was concentrated in vacuo and purified by column
chromatography (ethyl acetate: petroleum ether =1:10) to give compound 17 (2.27 g,
1
6
5%) as a colorless oil. H NMR (400 MHz, CDCl ) δ 7.43 – 7.28 (m, 5 H), 7.10 (dd,
3
J = 11.0, 6.4 Hz, 1 H), 6.95 (d, J = 8.3 Hz, 1 H), 6.68 – 6.61 (m, 2 H), 6.53 (dd, J =
8
4
2
.3, 2.6 Hz, 1 H), 6.49 (d, J = 2.5 Hz, 1 H), 5.00 (s, 2 H), 4.39 – 4.31 (m, 1 H), 4.19 –
.01 (m, 4 H), 2.90 (t, J = 7.6 Hz, 2 H), 2.86 – 2.78 (m, 1 H), 2.78 – 2.69 (m, 1 H),
.58 (t, J = 7.7 Hz, 2 H), 2.18 – 2.08 (m, 1 H), 1.96 – 1.84 (m, 1 H), 1.23 (t, J = 7.1
+
Hz, 3 H). MS(EI) m/z: 464(M ).
ethyl 3-(2-fluoro-4-((7-hydroxychroman-2-yl)methoxy)phenyl)propanoate (18). A
solution of compound 17 (1.2 g, 2.6 mmol) in methanol (20 mL) is hydrogenated over
1
0% Pd/C (0.12 g) at H for 6 hr at ambient temperatures. The catalyst is removed by
2
filtration and the filtrate is concentrated in vacua and purified by column
chromatography (ethyl acetate: petroleum ether =1:5) to give compound 18 (800 mg,
1
8
6
2%) as a white solid. H NMR (400 MHz, CDCl ) δ 7.09 (dd, J = 11.1, 6.5 Hz, 1 H),
3
.89 (d, J = 8.2 Hz, 1 H), 6.67 – 6.60 (m, 2 H), 6.37 (dd, J = 8.2, 2.5 Hz, 1 H), 6.32 (d,
J = 2.5 Hz, 1 H), 5.39 (s, 1 H), 4.39 – 4.31 (m, 1 H), 4.18 – 4.01 (m, 4 H), 2.90 (t, J =

7
1
.6 Hz, 2 H), 2.86 – 2.66 (m, 2 H), 2.58 (t, J = 7.6 Hz, 2 H), 2.15 – 2.07 (m, 1 H),
+
.95 – 1.83 (m, 1 H), 1.23 (t, J = 7.1 Hz, 3 H). MS(EI) m/z: 374(M ).
ethyl 3-(2-fluoro-4-((7-methoxychroman-2-yl)methoxy)phenyl)propanoate (19a).
To a DMF (5 mL) solution of compound 18 (100 mg, 0.27 mmol) were added
potassium carbonate powder (75 mg, 0.54 mmol) and MeI (0.025 mL, 0.41 mmol).
The resulting suspension was heated to 60°C for 3 h. The reaction mixture was cooled
to ambient temperature and added AcOEt and water. The extracts are combined, dried
over anhydrous Na SO . The mixture was concentrated in vacuo and purified by
2
4
column chromatography (ethyl acetate: petroleum ether =1:10) to give
1
compound 19a (89 mg, 85%) as a colorless oil. H NMR (400 MHz, CDCl ) δ 7.11
3
(dd, J = 11.1, 6.5 Hz, 1 H), 6.95 (d, J = 8.3 Hz, 1 H), 6.69 – 6.62 (m, 2 H), 6.46 (dd, J
=
8.3, 2.6 Hz, 1 H), 6.42 (d, J = 2.5 Hz, 1 H), 4.41 – 4.31 (m, 1 H), 4.20 – 4.03 (m, 4
H), 3.75 (s, 3 H), 2.91 (t, J = 7.7 Hz, 2 H), 2.88 – 2.69 (m, 2 H), 2.58 (t, J = 7.7 Hz, 2
H), 2.17 – 2.09 (m, 1 H), 1.97 – 1.84 (m, 1 H), 1.24 (t, J = 7.1 Hz, 3 H). MS(EI) m/z:
+
3
88(M ).
ethyl
3-(2-fluoro-4-((7-(3-(methylsulfonyl)propoxy)chroman-2-yl)methoxy)
phenyl)propanoate (19b). Following the similar to the synthesis of compound 19a,
compound 19b (121 mg, 91%) was prepared from compound 18 (100 mg, 0.27mmol)
and 3-(methylsulfonyl)propyl methanesulfonate (88 mg, 0.41 mmol) as a a colorless
1
oil. H NMR (400 MHz, CDCl ) δ 7.11 (dd, J = 10.8, 6.6 Hz, 1 H), 6.95 (d, J = 8.3 Hz,
3
1
4
H), 6.69 – 6.62 (m, 2 H), 6.43 (dd, J = 8.3, 2.6 Hz, 1 H), 6.39 (d, J = 2.5 Hz, 1 H),
.40 – 4.33 (m, 1 H), 4.19 – 4.09 (m, 3 H), 4.05 (dd, J = 10.6, 5.3 Hz, 3 H), 3.26 –

3
.20 (m, 2 H), 2.94 (s, 3 H), 2.91 (t, J = 7.6 Hz, 2 H), 2.87 – 2.78 (m, 1 H), 2.76 (dd, J
=
5.4, 3.4 Hz, 1 H), 2.58 (t, J = 7.7 Hz, 2 H), 2.35 – 2.27 (m, 2 H), 2.17 – 2.09 (m, 1
+
H), 1.96 – 1.84 (m, 1 H), 1.23 (t, J = 7.1 Hz, 3 H). MS(EI) m/z: 388(M ).
ethyl
3-(2-fluoro-4-((7-(2-methoxyethoxy)chroman-2-yl)methoxy)phenyl)
propanoate (19c). Following the similar route to the synthesis of compound 19a,
compound 19c (70 mg, 60%) was prepared from compound 18 (100 mg, 0.27mmol)
1
and 1-chloro-2-methoxyethane (0.04 mL, 0.43 mmol) as a a colorless oil. H NMR
(
400 MHz, CDCl ) δ 7.11 (dd, J = 10.9, 6.5 Hz, 1 H), 6.94 (d, J = 8.4 Hz, 1 H), 6.69 –
3
6
.62 (m, 2 H), 6.50 (dd, J = 8.4, 2.6 Hz, 1 H), 6.42 (d, J = 2.5 Hz, 1 H), 4.40 – 4.32
m, 1 H), 4.19 – 4.08 (m, 3 H), 4.08 – 4.02 (m, 3 H), 3.75 – 3.70 (m, 2 H), 3.44 (s, 3
H), 2.90 (t, J = 7.7 Hz, 2 H), 2.88 – 2.78 (m, 1 H), 2.74 (dd, J = 12.6, 9.1 Hz, 1 H),
(
2
.58 (t, J = 7.7 Hz, 2 H), 2.16 – 2.09 (m, 1 H), 1.96 – 1.84 (m, 1 H), 1.23 (t, J = 7.1
+
Hz, 3 H). MS(EI) m/z: 432(M ).
3
-(2-fluoro-4-((7-methoxychroman-2-yl)methoxy)phenyl)propanoic acid (20a). To
a solution of compound 19a (89 mg, 0.23 mmol) in methanol and THF was added
LiOH (1.15 mL, 1.0 M aqueous solution). The mixture was stirred at room
temperature for 5 hr, and then Acidified by 1N HCl Aqueous solution. The solution
was extracted with ethyl acetate and the extracts are combined, dried over anhydrous
Na SO . The mixture was concentrated in vacuo and purified by column
2
4
chromatography (dichloromethane: methanol =30:1) to give compound 20a (70 mg,
1
8
8
5%) as a white solid. H NMR (500 MHz, DMSO-d ) δ 12.15 (s, 1 H), 7.22 (t, J =
6
.8 Hz, 1 H), 6.97 (d, J = 8.3 Hz, 1 H), 6.85 (d, J = 12.2 Hz, 1 H), 6.78 (dd, J = 8.5,

2
.3 Hz, 1 H), 6.44 (dd, J = 8.4, 2.6 Hz, 1 H), 6.37 – 6.34 (m, 1 H), 4.37 – 4.31 (m, 1
H), 4.16 (d, J = 5.1 Hz, 2 H), 3.68 (s, 3 H), 2.83 – 2.73 (m, 3 H), 2.74 – 2.59 (m, 1 H),
1
3
2
.49 (t, J = 7.5 Hz, 2 H), 2.09 – 2.01 (m, 1 H), 1.84 – 1.74 (m, 1 H). C NMR (126
MHz, DMSO-d ) δ 173.47, 161.76, 159.82, 158.55, 158.06, 154.68, 130.90, 129.93,
6
119.27, 113.76, 110.60, 106.98, 102.07, 101.19, 73.79, 70.23, 54.97, 34.04, 23.56,
-
2
3
3
3.11. MS (ESI) m/z: 359.1(M-H) . HRMS (ESI): Anal. Calcd for C H O F,
20 20 5
59.1300; found, 359.1301.
-(2-fluoro-4-((7-(3-(methylsulfonyl)propoxy)chroman-2-yl)methoxy)phenyl)pro
panoic acid (20b). Following the similar route to the synthesis of compound 20a,
compound 20b (77 mg, 72%) was prepared from compound 19b (114 mg, 0.23mmol)
1
as a white solid. H NMR (500 MHz, DMSO-d ) δ 12.16 (s, 1 H), 7.22 (t, J = 8.8 Hz,
6
1
H), 6.98 (d, J = 8.4 Hz, 1 H), 6.85 (dd, J = 12.0, 2.5 Hz, 1 H), 6.78 (dd, J = 8.5, 2.5
Hz, 1 H), 6.46 (dd, J = 8.4, 2.5 Hz, 1 H), 6.37 (dd, J = 2.6, 1.4 Hz, 1 H), 4.38 – 4.32
m, 1 H), 4.20 – 4.14 (m, 2 H), 4.02 (t, J = 6.3 Hz, 2 H), 3.27 – 3.21 (m, 2 H), 3.01 (s,
H), 2.83 – 2.74 (m, 3 H), 2.72 – 2.65 (m, 1 H), 2.49 (t, J = 7.5 Hz, 3 H), 2.15 – 2.02
(
3
1
3
(
m, 3 H), 1.85 – 1.74 (m, 1 H). C NMR (126 MHz, DMSO-d ) δ 173.48, 161.76,
6
1
1
59.82, 158.15, 157.48, 154.68, 130.91, 130.85, 130.00, 119.15, 114.13, 110.62,
07.59, 102.08, 101.96, 73.80, 70.21, 65.57, 50.54, 34.04, 23.53, 23.11, 22.98, 21.93.
-
MS (ESI) m/z: 465.2(M-H) . HRMS (ESI): Anal. Calcd for C H FO S, 347.0692;
2
3
26
7
found, 347.0696.
-(2-fluoro-4-((7-(2-methoxyethoxy)chroman-2-yl)methoxy)phenyl)propanoic
acid (20c). Following the similar route to the synthesis of compound 20a, compound
3