Synthesis and biological evaluation of novel potent FFA1 agonists containing 2,3-dihydrobenzo[b][1,4]dioxine

Article abstract of DOI:10.1016/j.bmcl.2019.01.014

FFA1 (free fatty acid receptor 1) has emerged as an attractive antidiabetic target due to its role in mediating the enhancement of glucose-stimulated insulin secretion in pancreatic β cells with a low risk of hypoglycemia. Many reported FFA1 agonists possessed somewhat pharmacokinetic and/or safety issues. Herein, we describe the identification of 2,3-dihydrobenzo[b][1,4]dioxine as a novel scaffold for FFA1 agonists. Comprehensive structure-activity relationship study based on this scaffold led to the discovery of (S)-3-(4-(((S)-7-(4-methoxyphenyl)-2,3-dihydrobenzo [b][1,4]dioxin-2-yl)methoxy) phenyl)hex-4-ynoic acid (26k), which displayed a potent FFA1 agonistic activity and good pharmacokinetic profiles. Subsequent in vivo studies demonstrated that compound 26k significantly improved the glucose tolerance in ICR mice. In summary, compound 26k is a promising drug candidate for further investigation.

Full text of DOI:10.1016/j.bmcl.2019.01.014

Accepted Manuscript
Synthesis and biological evaluation of novel potent FFA1 agonists containing
2,3-dihydrobenzo[b][1,4]dioxine
Deyu Kong, Shimeng Guo, Yushe Yang, Bin Guo, Xin Xie, Wenhao Hu
PII:
S0960-894X(19)30024-1
https://doi.org/10.1016/j.bmcl.2019.01.014
BMCL 26251
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
2 November 2018
7 December 2018
15 January 2019
Please cite this article as: Kong, D., Guo, S., Yang, Y., Guo, B., Xie, X., Hu, W., Synthesis and biological evaluation
of novel potent FFA1 agonists containing 2,3-dihydrobenzo[b][1,4]dioxine, Bioorganic & Medicinal Chemistry
Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.01.014
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Synthesis and biological evaluation of novel potent FFA1 agonists
containing 2,3-dihydrobenzo[b][1,4]dioxine
Deyu Konga,b, Shimeng Guoc, Yushe Yangb, Bin Guo b, *, Xin Xiec,*, Wenhao Hua,d*
a Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development,
School of Chemistry and Molecular Engineering, East China Normal University, Shanghai,
200062, PR China.
b
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, Shanghai 201203, PR China.
c CAS Key Laboratory of Receptor Research, the National Center for Drug Screening, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences, shanghai 201203, PR China.
d School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China.
∗Corresponding author:
Wenhao Hu. Tel: 86-20-39943117. E-mail: huwh9@mail.sysu.edu.cn.
Bin Guo. Tel: 86-21-5080 6600-3405. E-mail: guobin@simm.ac.cn
Abstract
FFA1 (free fatty acid receptor 1) has emerged as an attractive antidiabetic target due to its role in
mediating the enhancement of glucose-stimulated insulin secretion in pancreatic β cells with a low
risk of hypoglycemia. Many reported FFA1 agonists possessed somewhat pharmacokinetic and/or
safety issues. Herein, we describe the identification of 2,3-dihydrobenzo[b][1,4]dioxine as a novel
scaffold for FFA1 agonists. Comprehensive structure-activity relationship study based on this
scaffold led to the discovery of (S)-3-(4-(((S)-7-(4-methoxyphenyl)-2,3-dihydrobenzo
[b][1,4]dioxin-2-yl)methoxy) phenyl)hex-4-ynoic acid (26k), which displayed a potent FFA1
agonistic activity and good pharmacokinetic profiles. Subsequent in vivo studies demonstrated that
compound 26k significantly improved the glucose tolerance in ICR mice. In summary, compound
26k is a promising drug candidate for further investigation.
Keywords: FFA1 agonist; 2,3-dihydrobenzo[b][1,4]dioxine; Insulin secretion; Type 2 diabetes
mellitus
It is estimated that in 2017 more than 425 million people were living with diabetes all over

the world according to the International Diabetes Federation.1 Type 2 diabetes mellitus (T2DM),
representing 95% of all diabetes cases, is characterized by impaired insulin secretion and
increased insulin resistance which results in high blood glucose levels. Due to limited efficacy of
current hypoglycemic drugs and associated some undesired adverse effects such as gastrointestinal
symptoms, weight gain, liver damage, and a high risk of hypoglycemia, there is an urgent need for
preferable hypoglycemic drugs with improved safety.2-5
FFA1 (free fatty acid receptor 1), also known as GPR40, is a new promising antidiabetic
target which 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. These characteristics
make this receptor an excellent antidiabetic drug target associated with little or no risk of
hypoglycemia.6-7
Various series of small molecule FFA1 agonists have been reported in the past few years, as
exampled in Fig.1.8-10 TAK-875 (3), the most advanced compound from Takeda, have provided
clinical proof-of-concept for effective glucose control in T2DM in phase II clinical trials.11-12
Unfortunately, TAK-875 was terminated during phase III clinical trials due to liver-associated
adverse events in patients.13 Because FFA1 is not expressed in the liver, the findings are unlikely
to be a direct result of FFA1 agonism.14 It is widely speculated that the hepatotoxicity might be
structure-related.15-16 Therefore, it is an urgent mission for medicinal chemists to identify
structurally distinct FFA1 agonists with improved liver safety.
O
OH
O
O
S
O
O
OH
OH
O
F
O
O
O
2
linoleic acid (1)
TAK-875 (3)
EC50: 80 nM
EC50: 30 nM
F
O
O
O
F3C
OH
O
OH
O
AMG-837 (4)
EC50: 60 nM
AMG-1638 (5)
EC50: 160 nM
Fig.1. Representative FFA1 agonists and endogenous ligands.
By analyzing reported FFA1 agonists, we find that there is a common structural moiety of the

benzyloxy fragment in these compounds.17-21 However, this moiety may result in poor oral
pharmacokinetic profiles (PK) and a potential safety concern due to the benzaldehyde fragment
resulting from metabolic oxidation at the benzyl position.22-23 Therefore, as an effort to identify
novel FFA1 agonists with improved PK and safety profiles, we designed a structurally distinct
series of FFA1 agonists with the benzyloxy moiety removed. In this novel skeleton, an ethylene
glycol ether fragment was introduced instead of the benzyloxy group, and further cyclized to
construct a 2,3-dihydrobenzo[b][1,4]dioxine structure (6, Fig.2). In this way, decreased molecular
flexibility may improve the physiochemical properties while preserving FFA1 agonistic potency.24
Removing the
benzyloxy moiety
O
O
O
O
R1
Introducing a
benzodioxane ring
O
F
O
F
R2
R3
O
SAR
R6
R5
O
metabolic oxidation
O
R4
O
2
7
6
OH
OH
OH
Fig.2. Design of novel benzodioxane scaffold FFA1 agonists
Herein, we describe the design and optimization of 2,3-dihydrobenzo[b][1,4]dioxine phenyl
propanoic acids as novel FFA1 agonists. These efforts ultimately led to the discovery of
compound 26k which exhibits satisfactory pharmacokinetic properties and significant glucose-
lowering efficacy in an oral glucose tolerance test (OGTT) in ICR mice.
The synthetic routes of compounds 16, 19 and 22 are depicted in Scheme 1. Treatment of 5-
bromo-2-hydroxybenzaldehyde 8 with chiral oxiran-2-ylmethyl 4-methylbenzenesulfonates (R)-9
or (S)-9 in the presence of potassium carbonate afforded 2-substituted salicylaldehydes (R)-10 and
(S)-10, respectively. The oxidation of (R)-10 and (S)-10 via Baeyer-Villiger oxidation in the
presence of 3-Chloroperbenzoic acid and subsequent cyclization under basic condition provided
substituted 1,4-benzodioxan derivatives (S)-11 and (R)-11, respectively. Suzuki coupling reaction
of (S)-11 and (R)-11 with (4-chlorophenyl)boronic acid produced compounds (S)-11 and (R)-11,
respectively. Then the Mitsunobu reaction of (S)-11 and (R)-11 with compound ethyl 3-(2-fluoro-
4-hydroxyphenyl)propanoate (14) gave esters (S)-15 and (R)-15, respectively. The resulting esters

(S)-15 and (R)-15 were finally subjected to hydrolysis to afford desired compounds (S)-16 and
(R)-16, respectively.
OTs

O
OH
OH
Cl
B
(R)-9
(S)-9
O
O
O
OH
O
O
(R)-10
(S)-10
O
O
12

b, c


OH
F
H
H
OH
a
d
Br
Br
Br
O
Cl
(S)-13
(R)-13
8
(S)-11
(R)-11
O
HO
e
O
14
O
O
O
O


O
F
O
F
f, g
Cl
Cl
(S)-16
(R)-16
(S)-15
(R)-15
O
OH
O
O
O
O
O
O
O
O
O
f, g
HO
O
Cl
Cl
18
19
17
O
O
e
e
O
OH
(S)-13
O
O
O
O
O
O
O
O
f, g
O
O
HO
O
Cl
Cl
21
22
20
O
O
O
OH
Scheme 1. Synthesis of compounds 16, 19 and 22. Reagents and conditions. (a) K2CO3, DMF,
80 °C; (b) m-CPBA, DCM, reflux; (c) NaOH, THF/H2O, rt; (d) Pd(PPh3)4, Cs2CO3, dioxane/H2O,
80°C; (e) ADDP, TBP, toluene, rt-40 °C; (f) LiOH, THF/MeOH/H2O, rt; (g) HCl, rt.
The preparations of compounds 26a-n and 30 were outlined in Scheme 2. Following the
similar route depicted in Scheme 1, Suzuki coupling reaction of compound (S)-11 with various
substituted phenylboronic acid 23a-n and pyridin-3-ylboronic acid (27) afforded compounds 24a-
n and 28, respectively. Then these compounds were further treated with 20 through the Mitsunobu
reaction and subsequent basic hydrolysis to produce the desired carboxylic acids 26a-n and 30,
respectively.

R2
R1
R4
OH
OH
R3
B
O
HO
O
O
O
R1
R1
O
20
23a-n
R2
R3
O
R2
R3
OH
OH
O
O
b
Br
O
a
R4
R4 24a-n
25a-n
(S)-11
O
c, d
OH
O
B
a
N
OH
O
27
R1
R2
R3
O
O
O
O
R4
20
26a-n
OH
O
O
O
O
OH
R1 R2 R3 R4
b
29
N
28
N
O
c, d
23-26
R1 R2 R3 R4
23-26
O
a
b
c
d
e
Cl
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
h
i
H
H
H
Cl
H
CH3O
H
H
H
H
H
H
O
H
H
CF3O
CF3
Cl
H
j
O
O
F
k
l
H
CH3
Cl
Cl
N
30
O
OH
f
H
H
H
H
CN
H
H
m
n
F
H
H
CH3O
H
H
g
CH3O
CH3
CH3
Scheme 2. Synthesis of compounds 26a-n and 30. Reagents and conditions. (a) Pd(PPh3)4,
Cs2CO3, dioxane/H2O, 80°C; (b) ADDP, TBP, toluene, rt-40 °C; (c) LiOH, THF/MeOH/H2O, rt;
(d) HCl, rt.
The agonistic activities of target compounds were investigated by monitoring Ca2+ signal
using a calcium mobilization assay. Aiming to pursue novel potent FFA1 agonists without the
benzyloxy moiety, we designed a novel 2,3-dihydrobenzo[b][1,4]dioxine scaffold and a
comprehensive structure-activity relationship (SAR) study was conducted around this structure as
illustrated in Fig.2.
Compound 2 was chosen as lead structure, because it has robust efficacy and simple scaffold
to avoid excessive increased molecular size in the optimization process. To suppress the oxidation
of the benzyloxy group in compound 2, we firstly replaced the -CH2O- structure with -
OCH2CH2O- and further constructed a 2,3-dihydrobenzo[b][1,4]dioxine ring (compound 6). A
chlorine atom was introduced at the para-position of terminal biphenyl ring instead of the
dimethyl group due to the potential oxidative metabolism at the methyl group (compound 16). As
illustrated in Table 1, the absolute configuration of carbon A significantly affected the FFA1
agonistic activity. (S)-isomer displayed 6-fold more potent than (R)-isomer ((S)-16 vs (R)-16).
Besides the benzyloxy group, β-position of phenylpropanoic acid moiety was also easily oxidized
[16], which could be avoided by blocking with a propynyl (19) or epoxy butyl group (22). As
expected, propynyl group (19) could improve the FFA1 agonistic activity by 4-fold compared with
compound (S)-16. However, epoxy butyl group (22) led to a significant decrease for FFA1

agonistic activity.
Table 1. FFA1 agonistic activity of compounds (R)/(S)-16, 19 and 22
O
R5
A

O
R6
O
O
Cl
16, 19 and 22
(R)/(S)-
OH
compd
(R)-16
(S)-16
19
A (R/S)
R5
F
R6
H
hFFA1 EC50(μM)α
1.78 ± 0.09
Emax (%)b
26 ± 2
R
S
S
F
H
0.46 ± 0.44
39 ± 5
H
0.08 ± 0.04
86 ± 6
O
22
S
H
1.02 ± 0.28
4.80 ± 0.48
77 ± 14
100
linoleic acid
b
αEC50 values for hFFA1 activities measured by calcium mobilization assay. 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 R6, we turned to
optimization of various substituents at the left benzene ring. As shown in Table 2, movement of
the chlorine atom from the para-position to the ortho-position resulted in similar potency (19 vs
26a). Meanwhile, a shift of the chloride atom from para-position to meta-position resulted in a 5-
fold loss in activity (19 vs 26b). Therefore, the general trend of activity is para-Cl ≈ ortho-Cl >
meta-Cl. When one more chlorine atom was introduced to compound 19, the resulting 2,4-
dichloride derivative 26k displayed a higher activity, whereas the 3,4-dichloride analogue 26l
showed a significantly decreased potency. Moreover, activity would decrease by 3-fold when the
chloride atom was removed (26c vs 19). Compared to the chlorine atom, other halogen atom (F)
could result in lower activity (26d vs 19). Additionally, we found that the electronic effect of the
substituent in para-position of the biphenyl group might have no direct influence on potency for
FFA1. For example, analogue (26g) containing a more electron-donating methoxy group exhibited
more potent than methyl analogue (26e), while analogues (26f and 26j) bearing a more electron-
withdrawing CN or CF3 group also displayed more potency than methyl analogue. When the
methoxy group was transferred to meta-position, agonistic activity also significantly decreased as

seen above (26h vs 26g). Furthermore, trifluoromethoxy group was inferior to methoxy group (26i
vs 26g). Introducing another ortho-fluorine substituent into the most potent compound 26g led to a
decrease of activity by 4-fold (26m vs 26g). Finally, changing the left benzene ring to a 3-
pyridine ring resulted in a slightly loss in activity (30 vs 26c).
Table 2. FFA1 agonistic activity of compounds 26a-n and 30
O
R1
O
O
R2
R3
O
OH
R4
26a-n and 30
X
compd
R1
R2
R3
R4
X
hFFA1
Emax (%)b
EC50(μM)α
0.08 ± 0.03
0.40 ± 0.06
0.16 ± 0.03
0.12 ± 0.02
0.24 ± 0.01
0.15 ± 0.01
0.04 ± 0.02
6.50 ± 1.21
0.29 ± 0.13
0.22 ± 0.13
0.07 ± 0.04
0.30 ± 0.15
0.20 ± 0.05
0.12 ± 0.05
0.22 ± 0.08
0.08 ± 0.02
4.80 ± 0.48
26a
26b
26c
26d
26e
26f
Cl
H
H
Cl
H
H
H
H
H
H
H
H
H
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
112 ± 2
116 ± 6
131 ± 1
114 ± 2
84 ± 2
112 ± 1
94 ± 2
149 ± 1
79 ± 9
102 ± 9
119 ± 5
105 ± 6
46 ± 3
89 ± 9
95 ± 9
92 ± 5
100
H
H
H
H
F
H
H
CH3
CN
CH3O
H
H
H
H
26g
26h
26i
H
H
H
CH3O
H
H
H
CF3O
CF3
Cl
H
26j
H
H
H
26k
26l
Cl
H
H
H
Cl
H
Cl
H
26m
26n
30
F
CH3O
H
H
CH3
H
H
CH3
H
H
H
3
linoleic
acid
b
αEC50 values for hFFA1 activities measured by calcium mobilization assay. Maximal efficacy

(Emax) was determined compared to linoleic acid. Data are presented as the mean ± SD (n=3).
Based on the in vitro data, compounds 26g and 26k were selected for further pharmacokinetic
profiling in Sprague Dawley (SD) rats and the results are summarized in Table 3. The PK
properties of 26k in rats were outstanding, with relatively lower clearance (CL: 1.2±0.2
mL/min/kg), higher plasma exposure (AUC0−t: 14032 ± 2558 ng·h/mL) after intravenous
administrationd at the same dose, compared to those of 26g. When administered via the oral route,
compound 26k also displayed high plasma exposure (AUC0−t: 71697 ± 10997 ng·h/mL), and
moderate oral bioavailability (F: 53.7%) at a dose of 10 mg/kg. While, Compound 26g exhibited
acceptable plasma exposure (AUC0−t: 8377 ± 1049 ng·h/mL) at an oral dose of 3 mg/kg.
Table 3. Pharmacokinetic Properties of Compounds 26g and 26k in SD Ratsα
Cmax ± SD
(ng/mL)
1977 ± 207
/
CL ± SD
AUC0-t ± SD
(ng·h/mL)
Tmax
(h)
1.00
/
T1/2
(h)
F
compd
26g
dose
(mL/min/kg)
(%)
po 3 mg/kg
iv 1 mg/kg
8377 ± 1049
4882 ± 342
4.00 57.1
3.88
3.39±0.24
1.2±0.2
po 10 mg/kg 10516 ± 2719
iv 1 mg/kg
71697 ± 10997 1.67
14032 ± 2558
3.09 53.7
4.06
26k
αAbbreviations: 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; CL: plasma clearance; iv: intravenous. Data are expressed as the
mean values with standard deviation from three independent experiments (n = 3)；
To assess the pharmacological effects of compounds 26g and 26k, we performed an oral
glucose tolerance test (OGTT) in ICR (Institute of Cancer Research) mice. Compounds (10
mg/kg) were orally administered 30 min prior to an oral glucose challenge (2.5 g/kg). TAK-875
was chosen as positive control. As shown in Fig.3, compounds 26g and 26k significantly reduced
blood glucose excursion (Fig.3A) and the area under the curve of blood glucose (AUC0-120min
)
(Fig.3B) compared with the vehicle. Moreover, compound 26k exhibited slightly superior in vivo
blood glucose-lowering efficacy to TAK-875 at the same dose of 10 mg/kg, while compound 26g
displayed comparable efficacy with TAK-875.

Fig.3. In vivo efficacy of compound 26g and 26k during an OGTT in ICR mice at an oral dose of
10 mg/kg. (A) Time-dependent changes of glucose levels for 26g and 26k. (B) Area under the
curve (AUC0-120min) of plasma glucose for 26g and 26k. Statistical significance compared with the
vehicle group is *P < 0.05, **P < 0.01. Data are expressed as mean values with standard deviation
from eleven independent experiments (n = 11).
In conclusion, we have developed two novel FFA1 agonists with improved potency and PK
profiles. By removing the benzyloxy moiety of lead compound that may cause poor oral
pharmacokinetic profiles and a potential safety concern, we constructed a unique 2,3-
dihydrobenzo[b][1,4]dioxine scaffold. Further structural modification and SAR study around this
scaffold afforded several potent FFA1 agonists. Particularly, two most potent compounds 26g and
26k showed an EC50 of 40 nM and 70nM, respectively. Furthermore, both compounds displayed
good oral absorption and metabolic stability in SD rats. Further in vivo studies demonstrated that
compound 26g and 26k both exhibited significant plasma glucose-lowering effects at an oral dose
of 10 mg/kg during an OGTT in ICR mice, in which 26k presented better potency. These qualities
made compound 26k a promising lead compound for the identification of FFA1 agonist candidate
and worth for further investigation.
Declaration of interest
The authors declare no competing financial interest.
Acknowledgements
The work was supported by the National Natural Science Foundation of China (21402222,
21332003), the “Personalized Medicines – Molecular Signature-Based Drug Discovery and

Development”, Strategic Priority Research Program of Chinese Academy of Sciences
(XDA12040307), the Youth Innovation Promotion Association of Chinese Academy of Sciences
(2016262), and the Program for Guangdong Introducing Innovative and Entrepreneurial Teams
(No. 2016ZT06Y337).
Supplementary data
Experimental details and spectroscopic characterization of compounds involved in the synthesis
are available online.
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Graphical Abstract
Synthesis and biological evaluation of novel potent FFA1 agonists
containing 2,3-dihydrobenzo[b][1,4]dioxine
Deyu Konga,b, Shimeng Guoc, Yushe Yangb, Bin Guo b, *, Xin Xiec,*, Wenhao Hua,d*
a Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development,
School of Chemistry and Molecular Engineering, East China Normal University, Shanghai,
200062, PR China.
b
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, Shanghai 201203, PR China.
c CAS Key Laboratory of Receptor Research, the National Center for Drug Screening, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences, shanghai 201203, PR China.
d School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China.
O
Cl
SAR
F
O
O
Cl
26k
O
O
EC50: 70nM
E
max: 119%
OH
OH
In order to develop novel FFA1 agonists with improved metabolic and safety profiles,
a series of novel 2,3-dihydrobenzo[b][1,4]dioxine phenylpropanoic acid derivatives
were designed and synthesized. Structural modification and SAR study led to the
identification of compound 26k as a potent FFA1 agonist with good pharmacokinetic
profiles and significant glucose-lowering efficacy.

 Corresponding author.
E-mail address: huwh9@mail.sysu.edu.cn. (Wenhao Hu); guobin@simm.ac.cn
(Bin Guo).
25.