Design, synthesis, and biological evaluations of phenylpropiolic acid derivatives as novel GPR40 agonists

Article abstract of DOI:10.1016/j.ejmech.2018.08.075

GPR40, also known as free fatty acid receptor 1 (FFAR1), is a member of G protein-coupled receptors (GPCR) family and has emerged as an attractive target for the treatment of type 2 diabetes mellitus. So far, most of the synthetic GPR40 agonists, including several drug candidates discontinued in clinical trials, were derived from the phenylpropionic acid scaffold. For discovering novel GPR40 agonists with diverse chemical structures, a series of phenylpropiolic acid derivatives were designed, synthesized, and evaluated under a battery of bioassays. Compound 9, the most potent compound in this series, exhibited submicromolar agonist activity and similar agonistic efficacy compared to that of TAK-875. In addition, compound 9 was able to dose-dependently amplify glucose-stimulated insulin secretion (GSIS) in pancreatic β-cell line MIN6, which could be reversed by a selective GPR40 antagonist GW1100. In addition, compound 9 was found to have potent glucose-lowering effects during an oral glucose tolerance test in normal C57BL/6 mice.

Full text of DOI:10.1016/j.ejmech.2018.08.075

Accepted Manuscript
Design, synthesis, and biological evaluations of phenylpropiolic acid derivatives as
novel GPR40 agonists
Xing-Wu Jiang, Bei-Er Jiang, Hao Liu, Zhi-Tao Liu, Long-Long Hu, Mingyao Liu,
Weiqiang Lu, Han-Kun Zhang
PII:
S0223-5234(18)30747-5
10.1016/j.ejmech.2018.08.075
EJMECH 10686
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 May 2018
Revised Date: 24 August 2018
Accepted Date: 27 August 2018
Please cite this article as: X.-W. Jiang, B.-E. Jiang, H. Liu, Z.-T. Liu, L.-L. Hu, M. Liu, W. Lu, H.-K.
Zhang, Design, synthesis, and biological evaluations of phenylpropiolic acid derivatives as novel GPR40
agonists, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.08.075.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, Synthesis, and Biological Evaluations of
Phenylpropiolic Acid Derivatives as Novel GPR40
Agonists
Xing-Wu Jiang†, Bei-Er Jiang†, Hao Liu, Zhi-Tao Liu, Long-Long Hu, Mingyao Liu, Weiqiang Lu*, and
Han-Kun Zhang*
Drug Discovery Unit, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences
and School of Life Sciences, East China Normal University, 500 Dongchuan Road, Shanghai 200241,
China
*To whom correspondence should be addressed. Phone: +86-21-24206642. Fax: +86-21-54344922. E-
mail: wqlu@bio.ecnu.edu.cn (W. Lu); hkzhang@bio.ecnu.edu.cn (H.-K. Zhang)
†These authors contributed equally to this work.
Keywords: Diabetes, GPR40 agonist, phenylpropionic acid, glucose, OGTT
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ABSTRACT
GPR40, also known as free fatty acid receptor 1 (FFAR1), is a member of G protein-coupled
receptors (GPCR) family and has emerged as an attractive target for the treatment of type 2 diabetes
mellitus. So far, most of the synthetic GPR40 agonists, including several drug candidates discontinued
in clinical trials, were derived from the phenylpropionic acid scaffold. For discovering novel GPR40
agonists with diverse chemical structures, a series of phenylpropiolic acid derivatives were designed,
synthesized, and evaluated under a battery of bioassays. Compound 9, the most potent compound in this
series, exhibited submicromolar agonist activity and similar agonistic efficacy compared to that of TAK-
875. In addition, compound 9 was able to dose-dependently amplify glucose-stimulated insulin secretion
(GSIS) in pancreatic
β-cell line MIN6, which could be reversed by a selective GPR40 antagonist
GW1100. In addition, compound 9 was found to have potent glucose-lowering effects during an oral
glucose tolerance test in normal C57BL/6 mice.
Introduction
G protein-coupled receptors (GPCRs), the largest and most versatile class of membrane receptors
in eukaryotes, represent the most common target of modern therapeutic drugs. They broadly participate
in physiological processes and pathophysiological conditions through coupling to specific G protein
families (Gαs, Gαi/o, Gαq/11 and others), which in turn trigger the production of second messengers such
as cAMP, inositol phosphates and Ca2+ to regulate different intracellular signaling pathways[1, 2]. G
protein-coupled receptor 40 (GPR40), also known as free fatty acid receptor 1 (FFAR1), belongs to
rhodopsin-like GPCR family and demonstrated to be highly expressed in pancreatic
β-cells and
gastrointestinal enteroendocrine cells. Activation of this receptor by either endogenous long-chain free
fatty acids or small-molecule agonists can promote glucose-stimulated insulin secretion (GSIS) by not
only directly acting on β-cells but also indirectly through regulation of incretin secretion[3]. Considering
that GPR40 only stimulate insulin release in the presence of elevated glucose levels, it has drawn
considerable attention from both academia and industry as a novel target for the treatment of type 2
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diabetes (T2D) with minimal risk of iatrogenic hypoglycemia[4, 5]. As shown in Fig. 1, a variety of
synthetic small-molecule GPR40 agonists have been reported based on the 3-phenylpropanoic acid
scaffold. Among them, the most successful example was TAK-875 (fasiglifam), which exhibited potent
agonist activity and high selectivity towards GPR40 and was advanced into clinical studies as an oral
active drug candidate. Treatment with TAK-875 led to improved insulin secretion in T2D patients with a
sharp reduction in glucose levels, including an average reduction in glycated haemoglobin (HbA1C) of
1.2%. At the 50 mg dose used in phase 3 clinical trials, TAK-875 primarily demonstrated improvement
on fasting plasma glucose levels with increased insulin secretion observed[6]. However, by the end of
2013, Takeda voluntarily decided to terminate the development activities for TAK-875 in phase 3
clinical studies as this compound demonstrated clear signs of liver toxicity in patients. Although it was
still unclear whether the hepatotoxicity of TAK-875 was a molecule-specific issue or a target-related
issue, a recent study indicated that TAK-875 may affect bile acid and bilirubin homeostasis through
inhibiting the efflux transporter and uptake transporters, and therefore produced off-target effects in the
liver[7]. Given that the failure of TAK-875 was most likely a molecule-specific issue and GPR40 played
a vital role in regulating glucose homeostasis, there was every expectation that some other GPR40
agonists might find their way into future clinical trials as anti-diabetic therapeutics.
Fig. 1. Selected examples of synthetic GPR40 agonists.
Through the analysis of various synthetic GPR40 agonists as reported in literature, we found that a
number of these compounds contained a phenylpropanoic acid motif (highlighted in red in Fig. 1) which
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was inspired from naturally occurring medium to long chain fatty acids. For instance, in the early period
of discovering TAK-875, it was found that lead compound 1 exhibited potent GPR40 agonist activity
and a significant insulinotropic effect in diabetic animal models. However, low sustainability of this
compound in rat, likely due to its susceptibility to β-oxidation at the 3-phenylpropanoic acid moiety
discouraged further advancement of this compound down the drug discovery pipeline[8]. Hence, for
improving the metabolic stability of GPR40 agonists bearing 3-phenylpropanoic acid moiety, successful
design strategies such as introducing small residues in the
β-position to the acid group or cyclizing the
β
-position to the benzene ring were applied to block the active site of metabolism. In the course of this
work, we initially employed a scaffold hopping strategy, replacing the propionic acid moiety in
compound 1 with different functional groups such as a propiolic acid fragment. We hypothesized that
this strategy would eliminate issues related to β-oxidaion of 3-phenylproponic acid moiety, leading to
investigation of novel GPR40 agonists with improved metabolic stability.
Chemistry
The syntheses of the designed ligands 4, 6a, 6b, 8a, 8b, and 9 are described in Scheme 1.
Intermediate 2 was synthesized according to a previously reported procedure[8] and followed by
reaction with tert-butyl-4-hydroxyphenylcarbamate to furnish compound 3. After removal of the Boc
group, the amino group was treated with ethyl chlorooxoacetate to give amide intermediate which was
then hydrolyzed to corresponding acid 4. Compound 2 underwent Br displacement with 4-iodophenol,
followed by an Ullmann coupling reaction with methyl L- or D-pyroglutamate to afford compound 6a
and 6b respectively after hydrolysis of ester group. Similarly, compound 2 was treated with methyl-4-
hydroxybenzoate or methyl-4-hydroxycinnamate under alkaline conditions to furnish ester 7a and 7b
respectively. After ester hydrolysis, compound 8a and 8b were prepared from acid form of 7a under
routine peptide coupling conditions. Dibromination of methyl cinnamate 7b using bromine in
dichloromethane at room temperature furnished dibromoester intermediate and the subsequent double
HBr elimination provided acid 9 in refluxing isopropanol as a solvent and KOH as a base[9].
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Accordingly, compounds 10 – 27 were synthesized from appropriate benzyl bromide analogs by the
same sequence of steps as described for compound 9.
Scheme 1.a
Me
I
Me
R1
O
O
f, d
N
N
O
O
or
Me
Me
R1
=
O
O
HO
HO
6a, 6b
5
a
b
e
O
H
Me
Me
NHBoc
Me
N
OH
O
b, c, d
a
Br
O
O
O
Me
Me
Me
2
3
4
O
Me
N
N
R2
g
R2
=
or
O
d, h, d
O
HO
HO
Me
a
b
Me
X
O
8a, 8b
O
O
O
OH
Me
Me
7a X = 0
7b X = -CH=CH-
i, j
O
Me
9
aReagents and conditions: (a) tert-butyl-4-hydroxyphenylcarbamate, K2CO3, DMF, 50 °C; (b) HCl, 1,4-
dioxane, rt; (c) ethyl chlorooxoacetate, Et3N, THF, 0 °C; (d) LiOH, MeOH/H2O, rt; (e) 4-iodophenol,
K2CO3, DMF, 50 °C; (f) CuI, N,N′-dimethylethylenediamine, Cs2CO3, 1,4-dioxane, methyl L- or D-
pyroglutamate, rt; (g) methyl-4-hydroxybenzoate or methyl-4-hydroxycinnamate, K2CO3, DMF, 50 °C;
(h) EDCI, DMAP, CH2Cl2, L- or D-proline methyl ester hydrochloride, rt; (i) Br2, CH2Cl2, 0 °C; (j) i.
KOH, 2-propanol, 90 °C; ii. HCl, H2O.
Results and Discussion
GPR40 receptor is generally considered to be a Gq-coupled receptor[10-12]. After Gq-coupled
GPCR activation, the membrane lipid phosphatidylinositol 4, 5-bisphosphate to generate inositol
triphosphate (IP3) and diacylglycerol (DAG), IP3 can activate IP3 receptor (IP3R) and release Ca2+ from
the endoplasmic reticulum (ER)[13]. The agonistic activity of these synthetic compounds was
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investigated by a calcium flux assay using HEK293-GPR40 cells. Two known GPR40 agonists, TAK-
875 and linolenic acid, were served as positive controls[14]. Maximal efficacy (Emax) was determined as
percent response relative to that of 30 µM linolenic acid. All of the tested compounds were initially
tested at a concentration of 10 µM and those demonstrated more than 50% agonist response shown by
linolenic acid were further evaluated for their EC50 values. We firstly evaluated the agonistic activity of
compounds 4, 6a, 6b, 8a, 8b, and 9, which were designed to avoid potential β-oxidation. None of these
compounds displayed more than 50% Emax as compared to linolenic acid, with the exception of
compound 9 which demonstrated 90% Emax and an EC50 value of 0.4 µM. As shown in Fig. 1,
introducing one more methyl group or removing both methyl groups in the terminal phenyl ring of
compound 9 led to a decrease in potency (10, EC50 = 2.87 µM; 12, EC50 = 3.77 µM), while compound
11 having only one methyl group retained the agonist activity (11, EC50: 0.61 µM). Replacement of the
methyl group in compound 11 with a trifluoromethoxy or isopropyl group yielded 13 and 15 which were
less potent than parent compound (13, EC50: 2.85 µM; 15, EC50: 3.98 µM), however compound 14 with
a chlorine substitution maintained the activity indicating that a substituent having a similar size to a
methyl group was preferred. With the exception of compound 18 showing moderate agonist activity
(EC50 = 1.80 µM), compounds bearing substitution at the meta-position or para-position in the terminal
phenyl ring generally lost their activities. Similarly, 2,4- or 2,5-substitution analogs 23 and 24 were
found to exhibit poor agonist activity. In addition, two diphenyl ether analogs prepared only displayed
modest to weak activity (25, EC50: 1.10 µM; 26, EC50: 4.02 µM), and introducing a methylene group to
the terminal phenyl ring of compound 25 completely lost its activity (27, EC50 >10 µM). Collectively,
we have identified a series of novel GPR40 agonists bearing phenylpropiolic acid scaffold and the best
three compounds in this series 9, 11, and 14 exhibited submicromolar agonistic activity and similar
agonist response (Emax) to that of positive control TAK-875 (Fig. 2A). Moreover, when incubated with
rat liver microsomes, at least 80% of compounds 9, 11, and 14 remained unchanged after 1 h incubation
at 10 µM (data not shown), suggesting that these compounds were likely devoid of
β
-oxidation occurred
in compound 1.
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Table 1. Structures and in Vitro Agonistic Activities of Synthetic Compounds.
EC50(µM)a
EC50(µM)
Mean SEM
(Emax, %)
Compd
R
Compd
20
R
Mean SEM
(Emax, %)b
0.38 0.09 (90)
2.87 0.73 (100)
>10/ND
>10/ND
9
10
21
0.61 0.14 (90)
3.77 0.36 (95)
2.85 0.68 (100)
0.69 0.05 (120)
>10/ND
8.08 1.21 (70)
>10/ND
11
12
13
14
22
23
24
25
F
F
1.10 0.11 (65)
3.98 0.29 (95)
4.02 0.56 (80)
15
26
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>10/NDa
>10/ND
16
17
27
Linoleni
>10/ND
--
--
11.09 1.6(100)
0.039 0.006 (90)
c
acid
TAK-
875
1.80 0.32 (60)
>10/ND
18
19
b
aEC50 values were determined by calcium flux assay and calculated with GraphPad Prism 5 software. Maximal
efficacy (Emax) was determined as percent response relative to that of 30 µM linolenic acid. cND: not determined.
To further investigate if GPR40 was the functional target of these phenylpropiolic acid analogs,
GW-1100, a selective antagonist of GPR40-mediated Ca2+ elevations in HEK293 cells with an IC50
value equal to 1µM, was used to inhibit agonist-induced intracellular calcium flux [15]. HEK293-
GPR40 cells were pretreated with GW-1100 (10 µM) for 15 min, then stimulated with TAK-875,
compounds 9, 11, and 14 respectively. As presented in Fig. 2B, GW-1100 was capable of blocking the
increase of Ca2+ level induced by TAK-875. Similarly, GW-1100 also reversed the agonist response
elicited by compounds 9, 11, and 14. It has been well established that the activation of GPCR could
often stimulate mitogen activated protein kinase (MAPK) signaling pathway. Specifically, the
phosphorylation of ERK has been proven to be a frequent consequence of the activation of GPCR[16].
Indeed, the activation of GPR40 by TAK875 resulted in the phosphorylation of ERK1/2 (Fig. 1C). In the
same way, compounds 9, 11, and 14 can also induce the phosphorylation of ERK1/2 in HEK293-GPR40
cells which could be completely reversed by GPR40 antagonist GW-1100. In contrast, only epidermal
growth factor (EGF) can activated ERK1/2 in HEK293 cells transfected with empty plasmid (Fig. 1D).
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Taken together, these results confirmed that the agonist activity of compounds 9, 11, and 14 was
mediated directly through GPR40 receptor.
Fig. 2. Compounds 9, 11, and 14 activated GPR40-mediated intracellular signaling pathway. (A) Dose-response
curve of 9, 11, 14, and TAK-875 in calcium flux assay. GPR40 agonists TAK-875 and linolenic acid were used as
positive controls. All data represented mean ± SD and were from three independent replicates. (B) GPR40 antagonist
GW-1100 inhibit calcium flux induced by 9, 11, 14, and TAK-875 in HEK293-GPR40 cells. GW-1100 (10 µM) were
added into the cells after loading with calcium 5 assay kit, and incubation for 15 min, then stimulated with TAK-875,
9, 11, and 14. TAK-875 was used as a positive control. All data represent mean ± SEM, data were assessed by
unpaired two-tailed Student’s t test, ***, p<0.001. (C-D) Representative western blot analysis of phosphorylated
ERK1/2 in HEK-293-GPR40 (C) or HEK293 (D) with empty vectors. HEK293-GPR40 or HEK293 cells with empty
vectors were starved for 2 hours, and then treated with indicated compounds at 10 µM for 20 min. (EGF, 50 ng/mL)
was used as a positive control.
It is well recognized that repeated administration of an agonist would result in the desensitization
of a GPCR response and subsequent the loss of GPCR function[17]. To determine whether compounds
9, 11, and 14 could induce GPR40 receptor desensitization, HEK293-GPR40 cells were firstly
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stimulated with tested compounds (10 µM) or DMSO solution (0.2%). As shown in Fig. 3, TAK875 and
three synthetic compounds 9, 11, and 14 all produced a significant increase of intracellular Ca2+ level.
After 10 min incubation, cells were washed with HBSS buffer and re-treated with 10 µM of TAK-875.
Cells pretreated with 0.2% DMSO responded sufficiently to TAK-875 stimulation, while cells pretreated
with TAK-875, compounds 9, 11, and 14 led to receptor desensitization and displayed a reduced
calcium response at the second stimulation of TAK-875 (Fig. 3).
Fig. 3. Compounds 9, 11, and 14 induced desensitization of GPR40 receptor. HEK293-GPR40 cells were loaded
with calcium 5 assay kit and activated (first arrow) with 10 µM TAK-875, 9, 11, and 14. After 10 min, cells were
washed once with HBSS buffer and stimulated again (second arrow) with the same concentration of TAK-875.
Intracellular calcium flux was measured by three independent replicate. All data represent mean ± SEM (n=3), data
were assessed by unpaired two-tailed Student’s t test. ***, p<0.001, 1st: first treatment of each group vs vehicle
(DMSO treatment, first green bar); ***, p<0.001, 2nd: Second treatment of each group vs vehicle (second green bar).
Previous studies have reported that GPR40 is predominantly expressed in pancreatic β-cells, and
activation of GPR40 by endogenous long-chain fatty acids or synthetic small-molecule compounds is
capable of producing a glucose-dependent increase of insulin secretion in β-cells [15, 18, 19]. Thus, we
evaluated the effects of compounds 9, 11, and 14 on glucose-stimulated insulin secretion using the
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MIN6 mouse insulinoma cell line. As shown in Fig. 4A, high concentration of glucose (25 mM)
promoted insulin release in MIN6 cells. Similar to TAK-875, our compounds 9, 11, and 14 remarkably
induced insulin secretion under high-glucose conditions, while they were less effective or had no effect
under low-glucose conditions (2.5 mM). In addition, GPR40 antagonist GW-1100 (10 µM) could
effectively block the effects of tested compounds on stimulating insulin release under high-glucose
conditions indicating that these compounds can induce insulin secretion through activation of GPR40.
Of note, compound 9 produced a dose-dependent (0.1 − 10 µM) increase in the glucose-stimulated
insulin secretion at high glucose levels (25 mM), and the capability of compound 9 to stimulate insulin
secretion was comparable to that found for positive control TAK-875 (Fig. 4B).
Further in vivo efficacy of compound 9 was evaluated by an oral glucose tolerance test (OGTT) in
C57BL/6 mice. As shown in Fig. 5A, oral administration of compound 9 at 3 or 10 mg/kg can both
significantly reduce plasma glucose concentrations at 15, 30, and 60 min respectively. Notably, the
capacity of compound 9 to decrease glucose levels was similar to that of TAK-875 (Fig. 5B): 21% at 3
mg/kg vs vehicle (9, p<0.001), 34% at 10 mg/kg vs vehicle (9, p<0.001), 24% at 3mg/kg vs vehicle
(TAK-875, p<0.001), respectively. Taken together, these results indicated that compound 9 was a
potential therapeutic agent for type 2 diabetes by inducing insulin secretion through the activation of
GRP40.
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Figure 4. Stimulation of insulin secretion in MIN6 cells by compounds 9, 11, and 14. MIN6 cells were treated in medium
supplemented with 2.5 or 25 mM glucose and the indicated compounds for 2 hours and then insulin level in the medium
was detected. (A) TAK-875, 9, 11, and 14 produced a 2-fold increase in glucose-stimulated insulin secretion in MIN6 cells
in the presence of 25 mM glucose. No effect of these compounds was observed in the presence of 2.5 mM glucose. GW1100
(10 µM) significantly reduced GPR40 activation induced insulin secretion in the presence of 25 mM glucose. All data
represent mean ±SEM (n=3). (B) Compound 9 and TAK-875 produced a dose-dependent increase in glucose-stimulated
insulin secretion in MIN6 cells in the presence of 25 mM glucose. All data represent mean ± SEM, data were analyzed by
one-way ANOVA and multiple comparison test.
glucose).
P<0.05 vs control (25 mM glucose); ***, P<0.001 vs control (25 mM
**,
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Fig. 5. Effect of compound 9 on oral glucose tolerance test in normal C57BL/6 mice. (A) Compound 9 (3 mg/kg and
10 mg/kg), or TAK-875 (3 mg/kg) were orally administered to mice 60 min prior to glucose challenge (2 g/kg). Left
panels show plasma glucose concentration curves, and right panels (B) show areas under the curves (AUC). Means ±
SD (n = 8) are shown, data were analyzed by one-way ANOVA and multiple comparison test. (*, p < 0.05; **, p <
0.01, *** p < 0.001).
Conclusion
In summary, a series of novel GPR40 agonists bearing phenylpropiolic acid motif with favorable
metabolic stability were prepared and evaluated for their activities as GPR40 agonists. Among them,
compound 9 was identified as a structurally distinct GPR40 agonist possessing potent activity in vitro,
and was effective in cellular insulin secretion in MIN6 cells under high-glucose conditions which could
be reversed by a selective GPR40 antagonist GW1100. In vivo, compound 9 significantly lowered
plasma glucose levels in normal mice during an oral glucose tolerance test suggesting that this
compound served as a valuable research tool to explore the therapeutic utility of GPR40 agonists.
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Experimental Section
General. All chemicals were purchased from Adamas-beta, J&K Scientific, Sigma-Aldrich, or Aladdin-
Reagents, and solvents were used as obtained from Tansoole or Sigma-Aldrich without further
purification. All non-aqueous reactions were run under an inert atmosphere (Ar or N2) with exclusion of
moisture from reagents, and all reaction vessels were oven-dried. The progress of reactions was
monitored by TLC on SiO2. Spots were visualized by their quenching of the fluorescence of an indicator
admixed to the SiO2 layer, or by dipping into I2/SiO2 mixture. Products were purified by column
chromatography on 200−300 mesh SiO2. Proton and carbon NMR spectra were generated on Bruker 400
or 500 MHz instruments. NMR chemical shifts were reported in δ (ppm) using the δ 7.26 signal of
CDCl3 (1H NMR), δ 2.50 signal of DMSO-d6 (1H NMR), and the δ 77.23 signal of CDCl3 (13C NMR), δ
39.50 signal of DMSO-d6 (13C NMR) as internal standards. High-resolution mass spectra (HRMS) were
gathered on a Bruker Micro TOF-Q II LC-MS instrument operating in electrospray ionization (ESI).
Purities of final compounds were established by analytical HPLC, which was carried out on an Agilent
1200 HPLC system with an Eclipse XDB C18 column (5 µm, 4.6 mm × 150 mm), with detection at 254
or 280 nm on a variable wavelength detector G1314B; flow rate = 1.5 mL/min; solvent A, water; solvent
B, methanol; gradient of 40 − 90% B (0 − 10 min), 90% B (10 − 15 min), 90 − 40% B (15 − 20 min).
3'-(bromomethyl)-2, 6-dimethyl-1,1'-biphenyl (2) was prepared according to the procedure reported in
reference 8.
Tert-butyl (4-((2',6'-dimethyl-[1,1'-biphenyl]-3-yl)methoxy)phenyl)carbamate (3).
To a mixture of compound 2 (90 mg, 0.33 mmol), tert-butyl (4-hydroxyphenyl) carbamate (140 mg,
0.66 mmol), and DMF (5.0 mL) was added K2CO3 (456 mg, 3.3 mmol). The mixture was stirred at 50
℃ for 5 h. After cooling, the resulting mixture was extracted with EtOAc and water. The organic layer
was washed with brine, dried over anhydrous sodium sulfate and concentrated. The residue was purified
by silica gel column chromatography (hexane/EtOAc = 10/1) to afford compound 3 (94 mg, 70.7%) as a
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1
white solid. H NMR (500 MHz, CDCl3) δ 7.49 – 7.40 (m, 2H), 7.32 – 7.08 (m, 7H), 7.01 – 6.86 (m,
2H), 6.37 (s, 1H), 5.11 (s, 2H), 2.05 (s, 6H), 1.54 (s, 9H).
2-((4-((2', 6'-dimethyl-[1, 1'-biphenyl]-3-yl)methoxy)phenyl)amino)-2-oxoacetic acid (4).
Step 1: To a mixture of compound 2 and 1, 4-dioxane (5.0 mL) was added a solution of HCl in 1, 4-
dioxane (0.5 mL, 2.65 mol/L). After the mixture was stirred at room temperature for 6 h, saturated
NaHCO3 aqueous solution was added to adjust the pH to 7.4, and the resulting mixture was extracted
with EtOAc and water. The organic layer was washed with brine, dried over anhydrous sodium sulfate
and concentrated to afford 4-((2', 6'-dimethyl-[1, 1'-biphenyl] -3-yl)methoxy)aniline (56 mg, 82.2%) as a
yellow oil which was used for the next step without purification.
Step 2: 4-((2', 6'-dimethyl-[1, 1'-biphenyl] -3-yl)methoxy)aniline (30 mg, 0.1 mmol) and TEA (12
mg,0.12 mmol) were dissolved in THF (5.0 mL). Ethyl chlorooxoacetate (0.012 mL, 0.1 mmol) was
added dropwise to the solution at 0 °C and then stirred at the same temperature for 2 h. Water was added
to the mixture and extracted with EtOAc. The organic layer was washed with brine, dried over
anhydrous sodium sulfate, concentrated and purified by silica gel column chromatography
(hexane/EtOAc
=
10/1) to
afford
ethyl
2-((4-((2',6'-dimethyl- [1,1'- biphenyl]-3-
yl)methoxy)phenyl)amino)-2-oxoacetate (32 mg, 79.4%) as a white solid.
Step 3: To a mixture of ethyl 2-((4-((2',6'-dimethyl-[1,1'-biphenyl]-3-yl)methoxy) phenyl)amino)-2-
oxoacetate (32 mg, 0.74 mmol) in MeOH (2.5 mL) and water (0.5 mL) was added lithium hydroxide
monohydrate (31 mg, 0.74 mmol). The mixture was stirred at 0 °C for 3 h. Then 3M aqueous solution of
hydrochloric acid was added to the mixture until the pH = 3‒4. The mixture was extracted with EtOAc
and water. The organic layer was washed with brine, dried over anhydrous sodium sulfate, and
evaporated to dryness. Then the residue was purified by column chromatography (CHCl3/MeOH = 20/1)
to afford 2-((4-((2',6'-dimethyl-[1,1'-biphenyl] -3-yl)methoxy) phenyl)amino)-2-oxoacetic acid (4) (15
mg,53.7%) as a white solid. 1H NMR (500 MHz, DMSO-d6) δ 14.06 (s, 1H), 10.59 (s, 1H), 7.68 – 7.64
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(m, 2H), 7.50 – 7.45 (m, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.19 (s, 1H), 7.17 – 7.12 (m, 1H), 7.12 – 7.07 (m,
3H), 7.02 – 6.98 (m, 2H), 5.15 (s, 2H), 1.94 (s, 6H). 13C NMR (126 MHz, DMSO-d6) δ 164.9 , 163.2 ,
155.1 , 141.6 , 140.9, 137.9 , 135.6 , 132.1 , 129.1 , 128.7 , 128.4 , 127.8 , 127.5 , 126.5 , 121.7 , 115.3 ,
69.7 , 21.0 .Purity: 96.7%. HRMS (ESI): calcd for C23H21NO4 [M + H]+, 375.1471; found, 375.1497.
3'-((4-iodophenoxy) methyl)-2, 6-dimethyl-1, 1'-biphenyl (5).
Compound 2 (1100 mg, 4.0 mmoL), 4-iodophenol (880 mg, 4.0 mmol), and K2CO3 (5.52 g, 40.0 mmol)
were dissolved in DMF (15.0 mL) and the mixture was stirred for 6 h at 50 °C. The resulting mixture
was extracted with EtOAc and water. The organic layer was washed with brine, dried over anhydrous
sodium sulfate and concentrated. The residue was purified by silica gel column chromatography
(hexane/EtOAc = 15/1) to afford 3'-((4-iodophenoxy)methyl)-2,6-dimethyl-1,1'-biphenyl (5) (868 mg,
1
52.4%) as a white solid. H NMR (500 MHz, CDCl3) δ 7.54 (d, J = 8.9 Hz, 2H), 7.47 – 7.42 (m, 1H),
7.38 (d, J = 7.7 Hz, 1H), 7.20 – 7.14 (m, 2H), 7.14 – 7.08 (m, 3H), 6.78 – 6.71 (m, 2H), 5.08 (s, 2H),
2.01 (s, 6H).
(S)-1-(4-((2', 6'-dimethyl-[1, 1'-biphenyl]-3-yl)methoxy)phenyl)-5-oxopyrrolidine-2-carboxylic acid
(6a).
Step 1: A mixture of compound 5 (621 mg, 1.5 mmol), methyl L-pyroglutamate (143 mg, 1.0 mmol),
CuI (95 mg, 0.5 mmol), Cs2CO3 (815 mg, 2.5 mmol), and N, N′-dimethylethylenediamine (0.1 mL, 1.0
mmol) in 1,4- dioxane (2.0 mL) was stirred under nitrogen atmosphere at room temperature for 12 h.
Then the mixture was extracted with EtOAc and water. The organic layer was washed with brine, dried
over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column
chromatography (hexane/EtOAc = 10/1) to afford (S)-methyl 1-(4-((2',6'-dimethyl-[1,1'-biphenyl]-3-
yl)methoxy)phenyl)-5-oxopyrrolidine-2-carboxylate (322 mg, 75.0%) as a white solid.
16

ACCEPTED MANUSCRIPT
Step 2: To a mixture of (S)-methyl 1-(4-((2',6'-dimethyl-[1,1'-biphenyl]-3-yl)methoxy) phenyl)-5-
oxopyrrolidine-2-carboxylate (216 mg, 0.5 mmol) in MeOH (4.0 mL), water (2.0 mL) and THF (4.0
mL) was added lithium hydroxide monohydrate (105 mg, 2.5 mmol) at 0 °C, and the mixture was stirred
at the same temperature for 3 h. Then 3M HCl aqueous solution was added to the mixture until the pH =
3‒4. The mixture was extracted with EtOAc and water. The organic layer was washed with brine, dried
over anhydrous sodium sulfate, and evaporated to dryness. Then the residue was purified by silica gel
column chromatography (CHCl3/MeOH = 20/1) to afford compound 6a (120 mg, 57.8%) as a white
solid. 1H NMR (500 MHz, DMSO-d6) δ 13.04 (s, 1H), 7.49-7.46 (m, 1H), 7.43 (d, J = 7.5 Hz, 1H), 7.38
(d, J = 8.9 Hz, 2H), 7.19 (s, 1H), 7.16-7.08 (m, 4H), 7.01 (d, J = 8.9 Hz, 2H), 5.15 (s, 2H), 4.75 (d, J =
7.4 Hz, 1H), 2.43 (t, J = 10.2 Hz, 2H), 2.08-1.99 (m, 2H), 1.94 (s, 6H). 13C NMR (126 MHz, CDCl3) δ
175.3 , 175.0, 156.9 , 141.5 , 141.4, 137.0 , 136.0 , 130.9 , 128.8 , 128.7 , 128.0 , 127.3, 127.1, 125.6 ,
124.5 , 115.4 , 70.2 , 62.3 , 30.5 , 23.4, 20.9 . Purity: 97.0%. HRMS (ESI): calcd for C26H25NO4 [M +
H]+, 415.1784; found, 415.1766.
(R)-1-(4-((2', 6'-dimethyl-[1,1'-biphenyl]-3-yl)methoxy)phenyl)-5-oxopyrrolidine-2-carboxylic acid
(6b).
The title compound was prepared by the same sequence as described for 6a using methyl D-
pyroglutamate as a starting material (192 mg, 89.2%). 1H NMR (500 MHz, DMSO-d6) δ 7.47 (t, J = 7.5
Hz, 1H), 7.42 (d, J = 7.7 Hz, 1H), 7.38 (d, J = 9.0 Hz, 2H), 7.19 (s, 1H), 7.15 (dd, J = 8.5, 6.3 Hz, 1H),
7.12 – 7.07 (m, 3H), 7.01 (d, J = 9.0 Hz, 2H), 5.15 (s, 2H), 4.82 – 4.65 (m, 1H), 2.46 – 2.36 (m, 2H),
2.12 – 1.98 (m, 2H), 1.94 (s, 6H). 13C NMR (126 MHz, DMSO-d6) δ 174.1 , 173.9 , 155.8 , 141.6 ,
140.9 , 137.8 , 135.6 , 132.4 , 129.2, 128.7 , 128.5 , 127.8 , 127.5 , 126.5 , 123.3 , 115.4, 69.7, 61.6 ,
30.9 , 23.0 , 21.0 . Purity: 98.9%. HRMS (ESI): calcd for C26H25NO4 [M + H]+, 415.1784; found,
415.1767.
Methyl 3-(4-((2',6'-dimethyl-[1,1'-biphenyl]-3-yl)methoxy)phenyl) benzoate (7a).
17

ACCEPTED MANUSCRIPT
To a mixture of compound 2 (225 mg, 1.0 mmol) and methyl 4-hydroxybenzoate (183 mg, 1.2 mmol) in
MeCN (5.0 mL) was added Cs2CO3 (815 mg, 2.5 mmol), and the resulting mixture was stirred at 60 °C
for 3 h. After cooling, the resulting mixture was extracted with EtOAc and water. The organic layer was
washed with brine, dried over anhydrous sodium sulfate and concentrated. The residue was purified by
silica gel column chromatography (hexane/EtOAc = 10/1) to furnish compound 7a (268 mg, 77.5%) as a
1
colorless oil. H NMR (500 MHz, CDCl3) δ 8.06 – 7.99 (m, 2H), 7.52 – 7.45 (m, 1H), 7.43 (d, J = 7.8
Hz, 1H), 7.24 (s, 1H), 7.22 – 7.09 (m, 4H), 7.05 – 6.99 (m, 2H), 5.20 (s, 2H), 3.91 (s, 3H), 2.04 (s, 6H).
Methyl 3-(4-((2', 6'-dimethyl-[1, 1'-biphenyl]-3-yl)methoxy)phenyl)acrylate (7b).
To a mixture of compound 2 (550 mg, 2.0 mmol), methyl-4-hydroxycinnamate (434 mg, 2.3 mmol) in
DMF (10.0 mL) was added K2CO3 (1.38 g, 10.0 mmol) and the mixture was stirred for 6 h at 50 °C.
After cooling, the resulting mixture was extracted with EtOAc and water. The organic layer was washed
with brine, dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel
column chromatography (hexane/EtOAc = 15/1) to afford compound 7b (457 mg, 63.8%) as a white
1
solid. H NMR (500 MHz, CDCl3) δ 7.68 (d, J = 16.0 Hz, 1H), 7.52 – 7.41 (m, 4H), 7.25 – 7.11 (m,
5H), 7.05 – 6.97 (m, 2H), 6.34 (d, J = 16.0 Hz, 1H), 5.18 (s, 2H), 3.82 (s, 3H), 2.04 (s, 6H).
(4-((2',6'-dimethyl-[1,1'-biphenyl]-3-yl)methoxy)benzoyl)-D-proline (8a)
Step 1: To a mixture of compound 7a (346 mg, 1.0 mmol) in MeOH (2.5 mL) and water (0.5 mL) was
added lithium hydroxide monohydrate (120 mg, 5.0 mmol). The mixture was stirred at 0 °C for 3 h. 3N
aqueous HCl was added to the mixture to arrive at a pH of 3‒4. The mixture was extracted with EtOAc
and water and the organic layer was washed with brine, dried over anhydrous sodium sulfate, and
concentrated to dryness. The residue was purified by column chromatography (CHCl3/MeOH = 20/1) to
afford methyl 3-(4-((2',6'-dimethyl-[1,1'-biphenyl]-3-yl)methoxy)phenyl)benzoic acid (318 mg, 95.8%)
as a colorless oil.
18

ACCEPTED MANUSCRIPT
Step 2: To a mixture of methyl 3-(4-((2',6'-dimethyl-[1,1'-biphenyl]-3-yl)methoxy) phenyl)benzoic acid
(318 mg, 1.0 mmol) and (D)-methyl prolinate (142 mg, 1.1 mmol) in DMF (5.0 ml) was added HATU
(456 mg, 1.2 mmol), followed by DIPEA (0.4 ml, 2.5 mmol) and the mixture was stirred at room
temperature for 5h. The mixture was extracted with EtOAc and water, then the organic layer was
washed with brine, dried over anhydrous sodium sulfate, and concentrated to dryness. Then the residue
was purified by column chromatography (hexane/EtOAc = 5/1) to afford methyl (4-((2',6'-dimethyl-
[1,1'-biphenyl]-3-yl)methoxy)benzoyl)-D-prolinate (242 mg, 54.6%) as a colorless oil.
Step 3: To a mixture of methyl (4-((2',6'-dimethyl-[1,1'-biphenyl]-3-yl)methoxy) benzoyl)-D-prolinate
(242 mg, 0.5 mmol) in MeOH (2.5 mL) and water (0.5 mL) was added lithium hydroxide monohydrate
(60 mg, 2.5 mmol), and the resulting mixture was stirred at 0 for 3 h. 3N aqueous HCl was added to
the mixture to arrive at a pH of 3‒4. The mixture was extracted with EtOAc and water. The organic
layer was washed with brine, dried over anhydrous sodium sulfate, concentrated to dryness. The residue
was purified by column chromatography (CHCl3/MeOH = 20/1) to afford compound 8a (220 mg,
1
93.1%) as a white solid. H NMR (500 MHz, DMSO-d6) δ 12.45 (s, 1H), 7.55 – 7.42 (m, 4H), 7.21 (s,
1H), 7.18 – 7.02 (m, 6H), 5.23 (s, 2H), 4.41 – 4.36 (m, 1H), 3.57 – 3.51 (m, 2H), 1.94 (s, 6H), 1.89 –
1.80 (m, 2H). 13C NMR (126 MHz, DMSO-d6) δ 173.9 , 168.2, 160.0, 141.5, 141.0, 137.5, 135.6, 129.7,
129.2, 129.0, 128.9, 128.8, 128.5, 127.8, 127.5, 126.6, 114.9, 69.6, 59.6, 50.1, 29.4, 25.6, 21.0. Purity:
98.3%. HRMS (ESI): calcd for C27H27NO4 [M + H]+, 429.1940; found, 429.1949.
(4-((2', 6'-dimethyl-[1, 1'-biphenyl]-3-yl) methoxy) benzoyl)-L-proline (8b)
The title compound was prepared by the same procedure described for 8a ]using (L)-methyl prolinate as
1
a starting material (134 mg, 89.5%). H NMR (500 MHz, DMSO-d6) δ 12.45 (s, 1H), 7.55 – 7.42 (m,
4H), 7.21 (s, 1H), 7.18 – 7.02 (m, 6H), 5.23 (s, 2H), 4.41 – 4.36 (m, 1H), 3.57 – 3.51 (m, 2H), 1.94 (s,
13
6H), 1.89 – 1.80 (m, 2H). C NMR (126 MHz, DMSO-d6) δ 173.9, 168.2, 160.0, 141.5, 141.0, 137.5,
135.6, 129.7, 129.2, 129.0, 128.9, 128.8, 128.5, 127.8, 127.6, 126.6, 114.9, 69.6, 59.6, 50.1, 29.4, 25.6,
21.0 . Purity: 95.3%. HRMS (ESI): calcd for C27H27NO4 [M + H]+, 429.1940; found, 429.1934.
19

ACCEPTED MANUSCRIPT
3-(4-((2',6'-dimethyl-[1,1'-biphenyl]-3-yl)methoxy)phenyl)propiolic acid (9).
Step 1: To a solution of compound 7b (183 mg, 0.51 mmol) in DCM (5.0 mL) was dropwise added Br2
(0.2 mL, 3.9 mmol) at 0 and the mixture was stirred at the same temperature for 10 min. Na2S2O3
(1.90 g, 12.0 mmol) and water (10.0 mL) were added to the solution respectively and the mixture was
stirred for another 5 min. The reaction mixture was extracted with EtOAc and water, and the organic
layer was washed with brine, dried over anhydrous sodium sulfate, and dried to furnish dibromoester as
a pale yellow solid (291 mg) which was used for the next step without purification.
Step 2: KOH (224 mg, 4.0 mmol) was added to the solution of the dibromoester (291 mg) in 2-propanol
(10.0 mL), and the mixture was stirred at 90 °C for 6 h. After cooling, 3N aqueous HCl was added to the
mixture to arrive at a pH of 3‒4. The mixture was extracted with EtOAc and water, and the organic layer
was washed with brine, dried over anhydrous sodium sulfate, and concentrated to dryness. The residue
was purified by silica gel column chromatography (CHCl3/MeOH = 10/1) to afford compound 9 (86 mg,
47.2%, two steps) as a white solid. 1H NMR (500 MHz, DMSO-d6): δ 13.61 (s, 1H), 7.57 (d, J = 8.1 Hz,
2H), 7.52 – 7.47 (m, 1H), 7.45 (d, J = 7.4 Hz, 1H), 7.20 (s, 1H), 7.18 – 7.14 (m, 1H), 7.13 – 7.03 (m,
5H), 5.25 (s, 2H), 1.94 (s, 6H); 13C NMR (126 MHz, DMSO-d6) δ 160.7 , 155.0 , 141.5 , 141.0 , 137.2 ,
135.6 , 135.0 , 129.3 , 128.9 , 128.6 , 127.8 , 127.6 , 126.6 , 116.1 , 111.3 , 85.6 , 81. 7 , 69.8 , 21.0
.Purity: 95.3%. HRMS (ESI): calcd for C24H21O3 [M + H]+, 357.1485; found, 357.1506.
3-(4-((2',4',6'-trimethyl-[1,1'-biphenyl]-3-yl)methoxy)phenyl)propiolic acid (10).
The title compound was prepared by the same procedure described for 9 using 3'-(bromomethyl)-2,4,6-
trimethyl-1,1'-biphenyl as a starting material. 1H NMR (500 MHz, DMSO-d6) δ 13.62 (s, 1H), 7.55 (d, J
= 8.8 Hz, 2H), 7.49 – 7.45 (m, 1H), 7.42 (d, J = 7.7 Hz, 1H), 7.17 (s, 1H), 7.09 (d, J = 8.8 Hz, 3H), 6.92
(s, 2H), 5.23 (s, 2H), 2.26 (s, 3H), 1.89 (s, 6H). 13C NMR (101 MHz, DMSO-d6): δ 172.4, 170.7, 160.6,
20

ACCEPTED MANUSCRIPT
154.9, 141.0, 137.3, 136.8, 135.1, 130.1, 129.3, 128.6, 126.9, 125.2, 116.1, 111.3, 85.5, 81.6, 69.7, 26.8,
21.2. Purity: 96.5%. HRMS (ESI): calcd for C25H23O3 [M + H]+, 371.1642; found, 371.1631.
3-(4-((2'-methyl-[1,1'-biphenyl]-3-yl)methoxy)phenyl)propiolic acid (11).
The title compound was prepared by the same procedure described for 9 using 3'-(bromomethyl)-2-
methyl-1,1'-biphenyl as a starting material. 1H NMR (500 MHz, DMSO-d6): δ 13.59 (s, 1H), 7.58 (d, J =
8.8 Hz, 2H), 7.46 (m, 2H), 7.41 (s, 1H), 7.33-7.23 (m, 4H), 7.22-7.17 (m, 1H), 7.12 (d, J = 8.8 Hz, 2H),
5.24 (s, 2H), 2.20 (s, 3H); 13C NMR (101 MHz, DMSO-d6): δ 160.7 , 154.9 , 141.9, 141.4 , 136.9 ,
135.1 , 130.8 , 129.9, 129.0 , 128.9 , 128.8 , 127.9 , 126.8 , 126.4 , 116.0, 115.3 , 111.2 , 85.7 , 81.5 ,
69.8 , 20.6 . Purity: 96.3%. HRMS (ESI): calcd for C23H19O3 [M + H]+, 343.1329; found, 343.1331.
3-(4-([1,1'-biphenyl]-3-ylmethoxy)phenyl)propiolic acid (12).
The title compound was prepared by the same procedure described for 9 using 3-(bromomethyl)-1,1'-
biphenyl as a starting material. 1H NMR (500 MHz, DMSO-d6): δ 13.61 (s, 1H), 7.75 (s, 1H), 7.68-7.67
(m, 2H), 7.64 (d, J = 7.6 Hz, 1H), 7.59 (d, J = 8.8 Hz, 2H), 7.51-7.44 (m, 4H), 7.38 (dd, J = 7.4, 7.4 Hz,
13
1H), 7.13 (d, J = 8.9 Hz, 2H), 5.25 (s, 2H); C NMR (101 MHz, DMSO-d6): δ 160.7 , 155.1 , 140.9 ,
140.3 , 137.7 , 135.1 , 129.6 , 129.5 , 128.1 , 127.3 , 127.2, 126.9 , 126.7 , 116.0 , 111.5 , 85.2 , 82.0,
69.9 . Purity: 96.3%. HRMS (ESI): calcd for C22H17O3 [M + H]+, 329.1172; found, 329.1194.
3-(4-((2'-(trifluoromethoxy)-[1,1'-biphenyl]-3-yl)methoxy)phenyl)propiolic acid (13).
The title compound was prepared by the same procedure described for 9 using 3'-(bromomethyl)-2-
(trifluoromethoxy)-1,1'-biphenyl as a starting material. 1H NMR (500 MHz, DMSO-d6): δ 13.63 (s, 1H),
7.57 (d, J = 8.7 Hz, 2H), 7.54 (d, J = 5.2 Hz, 1H), 7.44-7.40 (m, 3H), 7.37-7.33 (m, 1H), 7.29-7.27 (m,
1H), 7.11 (d, J = 8.7 Hz, 3H), 7.03 (dd, J = 7.5, 7.4 Hz, 1H), 5.22 (s, 2H); δ 160.2 , 154.5 , 143.8 , 138.8
21

ACCEPTED MANUSCRIPT
, 137.4 , 134.6 , 129.3 , 128.1 , 127.8 , 127.5 , 126.7 , 126.5 , 125.8 , 125.7 , 123.0 , 115.5 , 110.9 , 85.0 ,
81.2 , 69.3 .Purity: 95.3%. HRMS (ESI): calcd for C23H16F3O4 [M + H]+, 413.0995; found, 413.1003.
3-(4-((2'-chloro-[1,1'-biphenyl]-3-yl)methoxy)phenyl)propiolic acid (14).
The title compound was prepared by the same procedure described for 9 using 3'-(bromomethyl)-2-
1
chloro-1,1'-biphenyl as a starting material. H NMR (500 MHz, DMSO-d6): δ 13.65 (s, 1H), 7.57-7.55
(m, 3H), 7.53-7.47 (m, 3H), 7.45-7.38 (m, 4H), 7.11 (d, J = 8.4 Hz, 2H), 5.24 (s, 2H); 13C NMR (101
MHz, DMSO-d6): δ 160.7 , 155.0 , 140.0 , 139.3 , 137.0 , 135.1 , 132.0 , 131.7 , 130.3, 129.8, 129.4 ,
129.0 , 128.9 , 128.0 , 127.7 , 116.1 , 111.4 , 85.5 , 81.7 , 69.7. Purity: 95.4%. HRMS (ESI): calcd for
C22H16ClO3 [M + H]+, 363.0782; found, 363.0791.
3-(4-((2'-isopropyl-[1,1'-biphenyl]-3-yl)methoxy)phenyl)propiolic acid (15).
The title compound was prepared by the same procedure described for 9 using 3'-(bromomethyl)-2-
isopropyl-1,1'-biphenyl as a starting material. 1H NMR (500 MHz, DMSO-d6): δ 13.55 (s, 1H), 7.56 (d,
J = 7.7 Hz, 2H), 7.46 (d, J = 7.3 Hz, 2H), 7.41 (d, J = 7.8 Hz, 1H), 7.38 – 7.31 (m, 2H), 7.28 – 7.19 (m,
2H), 7.17 – 7.05 (m, 3H), 5.25 (s, 2H), 3.09 – 2.78 (m, 1H), 1.08 (d, J = 6.5 Hz, 6H); 13C NMR (126
MHz, DMSO-d6): δ 160.5 , 155.2 , 146.2 , 142.0 , 140.6 , 136.9 , 135.0 , 130.0 , 129.0 , 128.9 , 128.7,
128.3 , 126.7 , 126.0 , 125.9 , 116.0 , 111.7, 84.6 , 82.4 , 69.7 , 29.4 , 24.5 . Purity: 96.6%. HRMS (ESI):
calcd for C25H23O3 [M + H]+, 371.1642; found, 371.1650.
3-(4-((3'-methyl-[1,1'-biphenyl]-3-yl)methoxy)phenyl)propiolic acid (16).
The title compound was prepared by the same procedure described for 9 using 3-(bromomethyl)-3'-
1
methyl-1,1'-biphenyl as a starting material. H NMR (500 MHz, DMSO-d6): δ 13.62 (s, 1H), 7.74 (s,
1H), 7.63 (d, J = 7.4 Hz, 1H), 7.59 (d, J = 8.7Hz, 2H), 7.50-7.44 (m, 4H), 7.36 (dd, J = 7.2, 7.6 Hz, 1H),
7.20 (d, J = 7.5 Hz, 1H), 7.13 (d, J = 8.8 Hz, 2H), 5.25 (s, 2H), 2.39 (s, 3H); 13C NMR (126 MHz,
22

ACCEPTED MANUSCRIPT
DMSO-d6): δ160.3 , 154.5 , 141.4 , 140.9 , 136.4 , 134.6 , 130.3 , 129.4 , 128. 6 , 128.4 , 128.3 , 127.4 ,
126.3 , 126.0 , 115.6 , 114.8 , 110.8 , 85.2 , 81.1 , 69.3 , 20.1 . Purity: 95.4%. HRMS (ESI): calcd for
C23H19O3 [M + H]+, 343.1329; found, 343.1351.
3-(4-((3'-(trifluoromethyl)-[1,1'-biphenyl]-3-yl)methoxy)phenyl)propiolic acid (17).
The title compound was prepared by the same procedure described for 9 using 3-(bromomethyl)-3'-
1
(trifluoromethyl)-1,1'-biphenyl as a starting material. H NMR (500 MHz, DMSO-d6): δ 13.63 (s, 1H),
8.02-7.99 (m, 2H), 7.86 (s, 1H), 7.76-7.71 (m, 3H), 7.59-7.51 (m, 4H), 7.13 (d, J = 8.8 Hz, 2H), 5.26 (s,
13
2H); C NMR (126 MHz, DMSO-d6): δ 160.3 , 153.0 , 143.9 , 141.4 , 139.2 , 138.0 , 134.9 , 134.8 ,
133.7 , 131.4 , 130.6 , 129.8 , 128.2 , 127.1 , 127.0 , 124.7 (d, J = 3.7 Hz), 123.6 (d, J = 4.1 Hz), 115.9 ,
97.2 , 94.1 , 69.8 .Purity: 97.3%. HRMS (ESI): calcd for C23H16F3O3 [M + H]+, 397.1046; found,
397.1033.
3-(4-((3'-fluoro-[1,1'-biphenyl]-3-yl)methoxy)phenyl)propiolic acid (18).
The title compound was prepared by the same procedure described for 9 using 3-(bromomethyl)-3'-
1
fluoro-1,1'-biphenyl as a starting material. H NMR (500 MHz, DMSO-d6): δ 13.64 (s, 1H), 7.80 (s,
1H), 7.69 (d, J = 7.2 Hz, 1H), 7.57 (d, J = 8.8 Hz, 2H), 7.55-7.49 (m, 5H), 7.23-7.20 (m, 1H), 7.12 (d, J
= 8.8 Hz, 2H), 5.24 (s, 2H); 13C NMR (126 MHz, DMSO-d6): δ 164.1 , 162.2 , 160.4 , 142.8 (d, J = 7.8
Hz), 139.4 , 137.8 , 134.9 , 131.4 (d, J = 8.5 Hz), 129.7 , 128.0 , 127.0, 126.8 , 123.3 , 116.0 , 114.8 (d, J
= 21.0 Hz), 113.9 (d, J = 22.1 Hz), 112.1 , 84.4 , 83.1 , 69.8 . Purity: 95.5%. HRMS (ESI): calcd for
C22H16FO3 [M + H]+, 347.1078; found, 347.1091.
3-(4-((4'-fluoro-[1,1'-biphenyl]-3-yl)methoxy)phenyl)propiolic acid (19).
The title compound was prepared by the same procedure described for 9 using 3-(bromomethyl)-4'-
1
fluoro-1,1'-biphenyl as a starting material. H NMR (500 MHz, DMSO-d6) δ 7.78 – 7.65 (m, 3H), 7.61
(d, J = 7.2 Hz, 1H), 7.51 – 7.41 (m, 2H), 7.36 (d, J = 8.1 Hz, 2H), 7.33 – 7.26 (m, 2H), 7.03 (d, J = 8.4
23

ACCEPTED MANUSCRIPT
Hz, 2H), 5.19 (s, 2H); 13C NMR (126 MHz, DMSO-d6): δ 163.4 , 161.4 , 159.0 (d, J = 42.6 Hz), 139.8 ,
138.0 (d, J = 9.8 Hz), 136.8 , 133.7 (d, J = 11.6 Hz), 129.6 , 129.2 (d, J = 8.1 Hz), 127.3 , 126.7 , 126.5 ,
116.2 (d, J = 21.3 Hz), 115.6(d, J = 5.9 Hz), 114.4 , 83.9 , 79.8 , 69.7 .Purity: 96.2%. HRMS (ESI):
calcd for C22H16FO3 [M + H]+, 347.1078; found, 347.1072.
3-(4-((4'-(tert-butyl)-[1,1'-biphenyl]-3-yl)methoxy)phenyl)propiolic acid (20).
The title compound was prepared by the same procedure described for 9 using 3-(bromomethyl)-4'-(tert-
butyl)-1,1'-biphenyl as a starting material. 1H NMR (500 MHz, DMSO-d6): δ 13.63 (s, 1H), 7.72 (s, 1H),
7.62-7.57 (m, 5H), 7.50-7.46 (m, J = 8.0 Hz, 3H), 7.42 (d, J = 7.5 Hz, 1H), 7.12 (d, J = 8.8 Hz, 2H),
13
5.24 (s, 2H), 1.32 (s, 9H); C NMR (126 MHz, DMSO-d6) δ 159.7, 150.5, 140.7, 137.7, 137.5, 134.4,
129.6, 127.4, 127.0, 126.9, 126.6, 126.4 , 126.2, 115.8, 113.4, 82.3, 79.1, 69.9, 34.7, 31.6. Purity: 97.1%.
HRMS (ESI): calcd for C26H25O3 [M + H]+, 385.1798; found, 385.1787.
3-(4-((4'-(methylsulfonyl)-[1,1'-biphenyl]-3-yl)methoxy)phenyl)propiolic acid (21)
The title compound was prepared by the same procedure described for 9 using 3-(bromomethyl)-4'-
(methylsulfonyl)-1,1'-biphenyl as a starting material. 1H NMR (500 MHz, DMSO-d6): δ 8.01 (d, J = 8.4
Hz, 2H), 7.96 (d, J = 8.4 Hz, 2H), 7.84 (s, 1H), 7.73 (d, J = 6.3 Hz, 1H), 7.58-7.52 (m, 2H), 7.37 (d, J =
8.1 Hz, 2H), 7.04 (d, J = 8.5 Hz, 2H), 5.22 (s, 2H), 3.26 (s, 3H); 13C NMR (101 Hz, DMSO-d6): δ 160.4,
155.4, 145.2, 140.1, 139.0, 138.0, 134.9, 129.9, 128.5, 128.1, 128.1, 127.3, 127.1, 115.9, 111.9, 88.0,
82.8, 69.7, 44.0. Purity: 95.7%. HRMS (ESI): calcd for C23H19O5S [M + H]+, 407.0948; found,
407.0931.
3-(4-((4'-(trifluoromethoxy)-[1,1'-biphenyl]-3-yl)methoxy)phenyl)propiolic acid (22)
The title compound was prepared by the same procedure described for 9 using 3-(bromomethyl)-4'-
1
(trifluoromethoxy)-1,1'-biphenyl as a starting material. H NMR (500 MHz, CDCl3) δ 7.62 – 7.52 (m,
6H), 7.50 – 7.46 (m, 1H), 7.42 (d, J = 7.4 Hz, 1H), 7.29 (d, J = 8.3 Hz, 2H), 6.99 (d, J = 8.3 Hz, 2H),
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13
5.16 (s, 2H); C NMR (101 Hz, DMSO-d6): δ 160.5, 148.4, 148.4, 139.6, 139.4, 137.8, 134.9, 133.7,
129.7, 129.1, 127.7, 126.9, 126.7, 121.9, 115.9, 115.6, 84.4, 82.3, 69.8. Purity: 95.9%. HRMS (ESI):
calcd for C23H16F3O4 [M + H]+, 413.0995; found, 413.0981.
3-(4-((2'-chloro-4'-methyl-[1,1'-biphenyl]-3-yl)methoxy)phenyl)propiolic acid (23)
The title compound was prepared by the same procedure described for 9 using 3'-(bromomethyl)-2-
1
chloro-4-methyl-1,1'-biphenyl as a starting material. H NMR (500 MHz, DMSO-d6): δ 13.60 (s, 1H),
7.74 (s, 1H), 7.62 (d, J = 7.6 Hz, 1H), 7.59 (d, J = 8.8 Hz, 2H), 7.49-7.45 (m, 3H), 7.36 (dd, J = 7.6, 7.7
13
Hz, 1H), 7.19 (d, J = 7.4 Hz, 1H), 7.13 (d, J = 8.8 Hz, 2H), 5.25 (s, 2H), 2.38 (s, 3H); C NMR (101
Hz, DMSO-d6): δ 159.1, 139.6, 139.2, 137.1, 137.0, 133.7, 131.6, 131.3, 130.5, 129.3, 128.9, 128.8,
128.7, 127.3, 115.8, 115.6, 114.4, 83.9, 79.7, 69.6, 20.7. Purity: 95.4%. HRMS (ESI): calcd for
C23H18ClO3 [M + H]+, 377.0939; found, 377.0951.
3-(4-((2',5'-difluoro-[1,1'-biphenyl]-3-yl)methoxy)phenyl)propiolic acid (24)
The title compound was prepared by the same procedure described for 9 using 3'-(bromomethyl)-2,5-
1
difluoro-1,1'-biphenyl as a starting material. H NMR (500 MHz, DMSO-d6): δ 13.55 (s, 1H), 7.67 (s,
1H), 7.58-7.52 (m, 5H), 7.44-7.39 (m, 2H), 7.29-7.25 (m, 1H), 7.12 (d, J = 8.8 Hz, 2H), 5.25 (s, 2H);
13C NMR (126 Hz, DMSO-d6): δ 160.6 , 159.8 , 157.3 (d, J = 152.2 Hz), 155.0 (d, J = 69.0 Hz), 137.5,
135.0 , 134.7 , 133.7 , 129.4, 129.0 , 128.7 (d, J = 2.4 Hz), 128.2 , 118.2 (dd, J = 25.8, 9.1 Hz), 117.4
(dd, J = 25.0, 2.9 Hz), 116.4 (dd, J = 24.5, 8.7 Hz), 116.0 , 111.7 , 69.7.Purity: 97.0 %. HRMS (ESI):
calcd for C22H15F2O3 [M + H]+, 365.0984; found, 365.0991.
3-(4-((3-phenoxybenzyl)oxy)phenyl)propiolic acid (25)
The title compound was prepared by the same procedure described for 9 using 1-(bromomethyl)-3-
phenoxybenzene as a starting material. 1H NMR (500 MHz, DMSO-d6): δ 13.56 (s, 1H), 7.57 (d, J = 8.5
Hz, 2H), 7.42-7.38 (m, 3H), 7.22 (d, J = 7.5 Hz, 1H), 7.15 (dd, J = 7.2, 7.6 Hz, 1H), 7.09-7.06 (m, 3H),
7.01 (d, J = 7.9 Hz, 2H), 6.96 (d, J = 7.7 Hz, 1H), 5.17 (s, 2H); 13C NMR (126 Hz, DMSO-d6): δ 159.8,
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157.3, 156.9, 156.2, 139.3, 134.5, 130.6, 130.5, 124.1, 123.0, 119.2, 118.4, 118.0, 115.8, 113.1, 84.9,
81.3, 69.3. Purity: 96.5%. HRMS (ESI): calcd for C22H17O4 [M + H]+, 345.1121; found, 345.1109.
3-(4-((4-phenoxybenzyl)oxy)phenyl)propiolic acid (26)
The title compound was prepared by the same procedure described for 9 using 1-(bromomethyl)-4-
phenoxybenzene as a starting material. 1H NMR (500 MHz, DMSO-d6): δ 13.61 (s, 1H), 7.58 (d, J = 8.1
Hz, 2H), 7.47 (d, J = 7.1 Hz, 2H), 7.41-7.39 (m, 2H), 7.16-7.14 (m, 1H), 7.09 (d, J = 8.4 Hz, 2H), 7.02
(d, J = 6.6 Hz, 4H), 5.13 (s, 2H); 13C NMR (101 Hz, DMSO-d6): δ 160.7, 157.0, 156.9, 155.0, 135.1,
131.7, 130.5, 130.4, 124.0, 119.2, 118.9, 115.9, 111.2, 85.6, 81.4, 69.5. Purity: 95.6%. HRMS (ESI):
calcd for C22H17O4 [M + H]+, 345.1121; found, 345.1138.
3-(4-((3-(benzyloxy)benzyl)oxy)phenyl)propiolic acid (27)
The title compound was prepared by the same procedure described for 9 using 1-(benzyloxy)-3-
(bromomethyl)benzene as a starting material. 1H NMR (500 MHz, DMSO-d6) δ 13.67 (s, 1H), 7.58-7.56
(m, 4H), 7.43 (d, J = 7.6 Hz, 2H), 7.40 – 7.36 (m, 2H), 7.33 (d, J = 6.9 Hz, 1H), 7.26 (d, J = 2.4 Hz, 1H),
7.10 (d, J = 8.3 Hz, 2H), 7.02 – 6.97 (m, 1H), 5.12 (d, J = 4.0 Hz, 4H). 13C NMR (101 Hz, DMSO-d6):
δ 172.4, 170.7, 160.7, 158.9, 154.9, 138.5, 137.4, 135.1, 130.1, 128.8, 128.1, 120.5, 116.0, 114.6, 111.2,
85.6, 81.5, 69.7, 69.6. Purity: 95.9%. HRMS (ESI): calcd for C23H19O4 [M + H]+, 359.1278; found,
359.1293.
Calcium flux assay. HEK293 stably expressing human GPR40 (HEK293-GPR40) was a gift from Prof.
Naiming Zhou (Zhejiang University, China). HEK293-GPR40 was maintained in DMEM medium
containing 10% fetal bovine serum (FBS) at 37 in a humidified incubator with 5% CO2. 25,000 cells
in 100 µL cell cluture medium were seeded into 96-well black/clear tissue culture treated plates (Costar,
USA) and incubated overnight in 5% CO2 at 37 . Cells were loaded with 100 µL of Calcium-5 assay
kit (Molecular Devices, USA) in HBSS (Gibco, USA) containing 20 mM HEPES, and incubated at 37
for 45 min. After incubation at room temperature for 15 min, 50 µL of compounds were injected using
the Flexstation III instrument (Molecular Devices, CA) and the intracellular calcium flux was
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continuously measured at an excitation wavelength of 485 nm and an emission wavelength of 525 nm
for 120 s. The agonistic activities on HEK293-GPR40 were expressed as: Response% = [(RFU -
Rmin)/(Rmax - Rmin)] ×100%, ( (RFU) in compounds-treated cells, (Rmin) in vehicle-treated cells, and
(Rmax) in 30 µM linolenic acid treated cells). The EC50 values were calculated with GraphPad Prism 5
software (GraphPad, USA). Data are representative of three independent experiments.
Western blotting. HEK293 cells and HEK293-GPR40 cells were serum starved for two hours and then
treated with designed compounds at 37
for 20 min. Cells were lysed with modified RIPA buffer
which contain 20 mM Tris, 2.5 mM EDTA, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 40 mM
NaF, 10 mM Na4V2O7, and 1 mM PMSF supplemented with proteinase inhibitor cocktail and
phosphorylated cocktail (Calbiochem, CA). Protein concentration was determined using BCA assay
(bicinchoninic acid, Thermo, USA). 30 µg aliquots of protein samples were electrophoresed on 10%
SDS-PAGE gels and transferred to Nitrocellulose membranes. The membranes were blocking at room
temperature for 1 hour and then incubated with the primary anti-ERK1/2 antibody (1:1000; Cell
Signaling Technology, USA), anti-phospho-ERK1/2 antibody (Thr202/Tyr204) (1:1000; Cell Signaling
Technology, USA) and anti-GAPDH (1:10000; Sigma Aldrich, USA) antibody overnight at 4 . The
membranes were washed 4 times for 5 min each in PBST buffer (Phosphate Buffered Saline and 0.05%
Tween 20) before the secondary antibody incubated for another 1 hour. The immune reactive bands were
visualized with the Odyssey Imager (Li-COR Biosciences, NE).
Insulin secretion assay. Mouse β-cell line MIN6 cells were cultured in DMEM (high glucose) medium
supplemented with 15% FBS and 72 µM β-mercaptoethanol. Cells were seeded in 48-well cell culture
plate at 50,000 cells/well and cultured for 48 hours. Then wash the cells with PBS buffer twice, and
glucose free DMEM containing 15% FBS were added to all wells and incubated at 37 for 30 min.
Plates were washed twice with PBS buffer, then 100 µL glucose free DMEM containing 2.5mM or 25
mM glucose and tested compounds were added. After incubation for two hours, insulin released into the
supernatant was determined by Mouse Insulin ELISA Kit (Mercodia, Sweden).
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Oral Glucose Tolerance Test Assay (OGTT). 8-week-old male C57BL/6 mice were obtained from
National Rodent Laboratory Animal Resources (Shanghai, China) and feed in a laminar airflow cabinet
under specific-pathogen-free conditions. All experiments were performed according to the guidelines of
Institution Animal Care and Use Committee and all the protocols were approved by East China Normal
University (Shanghai, China). Mice were allowed to acclimate to the surroundings for a week and fasted
overnight before the study. Next day, baseline plasma glucose values and body weights were collected at
-60 min. Mice were randomized based on fasting plasma glucose and body weight. C57BL/6 mice were
orally administered vehicle, TAK-875 at 3mg/kg, compound 9 at 3mg/kg or 10mg/kg prior to an oral
gavage of glucose (2 g/kg, 8 mL/kg). Blood samples are collected from the end of tail at the 15, 30, 60,
and 120ꢀmin after oral gavage and the plasma glucose was determined via glucometer (Accu-check,
Aviva, Roche).
Statistical data analysis. Data were analyzed with GraphPad Prism software. Nonlinear regression
analysis was performed to generate dose-response curves and to calculate the concentration for 50% of
the maximal effect (EC50). Calcium flux and insulin secretion data was shown as the mean ± SEM.
OGTT data was shown as mean ± SD. Statistical comparisons between two groups were assessed by
two-tailed Student’s t test. More than two groups were analyzed by one-way ANOVA and Scheffé
multiple comparison test. P value of < 0.05 was considered statistically significant.
Acknowledgments
This research was supported by the National Key R&D Program of China [2018YFA0507000], the
Natural Science Foundation of Shanghai (14ZR1411200), and the Innovation Program of Shanghai
Municipal Education Commission (2017-01-07-00-05-E00011).
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