Discovery of thiophene-containing biaryl amide derivatives as novel glucagon receptor antagonists

Article abstract of DOI:10.1111/cbdd.13184

A novel series of thiophene-containing biaryl amide glucagon receptor (GCGR) antagonists were designed and synthesized. Two compounds of this series, 14f and 14h, exhibited good GCGR binding (IC₅₀?=?6.1 and 4.4?μm, respectively) and cAMP functional activities (IC₅₀?=?4.4 and 14.4?μm, respectively). The possible binding modes of compounds 14f and 14h with GCGR were explored by molecular simulation.

Full text of DOI:10.1111/cbdd.13184

PROFESSOR HONG LIU (Orcid ID : 0000-0003-3685-6268)
Article type : Research Article
Discovery of thiophene-containing biaryl amide derivatives as novel glucagon receptor
antagonists
a,b,c,d†
e,†
a,b,c,†
a,b,c
e
e
Jia Li
, Yang Feng , Huihui Li
, Shuangjie Shu , Antao Dai , Xiaoqing Cai ,
a,b,c
b,c,e
e
b,c,e*
a,b,c,d*
Jiang Wang , Dehua Yang , Dakota Ma , Mingwei Wang
and Hong Liu
a
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, People’s Republic of
China.
b
CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, People’s Republic of China
c
University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, China
d
School of Pharmacy, China Pharmaceutical University, Jiangsu, Nanjing 210009,
People’s Republic of China
e
The National Center for Drug Screening, 189 Guo Shou Jing Road, Shanghai 201203,
People’s Republic of China
*
Corresponding authors: Hong Liu (hliu@simm.ac.cn) and Mingwei Wang
(
mwwang@simm.ac.cn).
†
These authors contributed equally to this work.
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13184
This article is protected by copyright. All rights reserved.

Abstract: A novel series of thiophene-containing biaryl amide glucagon receptor (GCGR)
antagonists were designed and synthesized. Two compounds of this series, 14f and 14h,
exhibited good GCGR binding (IC50 = 6.1 μM and 4.4 μM, respectively) and cAMP
functional activities (IC50 = 4.4 μM and 14.4 μM, respectively). The possible binding modes
of compounds 14f and 14h with GCGR were explored by molecular simulation.
Key words: glucagon receptor antagonist, biary amide, molecular simulation
1
INTRODUCTION
Diabetes is a group of metabolic disorders characterized by hyperglycemia resulting
from defects in insulin action, insulin secretion, or both, and is affecting approximately 415
[
1]
million people around the globe in 2015. Type 2 diabetes mellitus (T2DM) is the most
[
2]
prevalent form and accounts for 90 to 95% of all diabetic patients. Despite the existence of
several classes of drugs for treating T2DM, such as dipeptidyl peptidase-4 (DPP-4) inhibitors
,
[
3]
[4]
sodium-glucose cotransporter-2 (SGLT-2) inhibitors, and glucagon-like peptide 1
[5]
(
GLP-1) receptor agonists, there remains a need to develop new therapies with improved
safety and efficacy.
Glucagon, secreted by the α-cells in the pancreas, is a 29-amino acids peptide hormone
[
6-7]
that counter-regulates the actions of insulin.
Glucagon binds to the glucagon receptor
(GCGR) and triggers a signal transduction cascade to stimulate the hepatic glucose
[8]
[9]
production via glycogenolysis and gluconeogenesis. In 2013, Siu et al. reported the
crystal structure of the seven transmembrane (7TM) helical domain of human GCGR, and
indicated that the glucagon-binding site is in the 7TM domain. It was shown that impairment
of insulin release and development of insulin resistance are often accompanied by absolute or
relative increases of glucagon in fasting and postprandial states of T2DM, and this situation
[
10]
leads to hyperglycemia.
Thus, it is postulated that GCGR antagonists may be useful in
[11]
mitigating glucagon-induced hyperglycemia, such as peptidic GCGR antagonists,
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[12]
[13]
[14]
antibodies,
antisense oligonucleotides,
and small-molecule antagonists
were
demonstrated to significantly decrease blood glucose levels. To data, a number of effective
small-molecule GCGR antagonists have been reported (Figure 1). The first active compound,
[
15]
(+)-Bay 27-9955,
was discovered in 2001 followed by several clinical compounds such as
[
16]
[17]
MK-0893
in phase 2 and LGD-6972 in phase 1. Recently, Jazayeril et al.
disclosed the
structure of human GCGR in complex with the antagonist MK-0893 (PDB ID: 5EE7).
MK-0893 was found to bind to an allosteric site outside the 7TM helical bundle.
In this paper, we chose compound 1 as the lead compound, which is one of the possible
structure of LGD-6972 according to the patent information filed by Ligand pharmaceuticals,
[
18-20]
and adopted bioisosteric strategy to design and synthesize a series of novel
thiophene-containing biaryl amide derivatives. We also described GCGR binding affinities
and cAMP functional activities of these compounds. Combined structure-activity relationship
(SAR) and molecular simulation studies were performed to validate their pharmacological
properties.
FIGURE 1. Structures of several glucagon receptor antagonists.
Thiophene is commonly used as a building block in drugs and has proven to be an
[
21]
[22]
attractive bioisostere to improve potency and selectivity (Fig. 2). Duloxetine
is an
anti-depressant, which functions in the central nervous system as a selective serotonin and
[
23]
norepinephrine reuptake inhibitor. Eprosartan,
a selective angiotensin II receptor blocker,
[
24]
is used for the treatment of essential hypertension. Rivaroxaban
is an oral anti-coagulant
indicated for prevention and treatment thrombo-embolic disorders.
FIGURE 2. Drugs that contain the thiophene ring.
Moreover, the structural characteristics of the thiophene-containing drugs exert an
important impact on the metabolic pathways. The main metabolites of thiophene are
[25]
thiophene S-oxides and thiophene epoxides, which may result in toxicity.
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There is no

thiophene reactive metabolite in some drugs following special modification. For example, the
main metabolic pathways of Rivaroxaban are cleavage of the amide bond and oxidative
[26]
degradation of the morpholine group (Figure 3),
while its structure is characteristic of
2
2
-CONH and 5-Cl at the thiophene ring. Therefore, we designed a novel series of
,5-disubstituted thiophene and investigated their antagonistic effects via different
1 2
substitutions at R and R (Figure 4).
FIGURE 3. Main metabolic pathways of Rivaroxaban.
FIGURE 4. Molecular design of novel glucagon receptor (GCGR) antagonists.
2
2
EXPERIMENTAL SECTION
.1 Material and methods
All commercially available compounds and solvents were used without further purification.
1
13
All products were characterized by their NMR and MS spectra. H spectra and C NMR
spectra were recorded in CDCl , CD OD, and DMSO-d with 400 MHz and 500 MHz
3
3
6
instruments. Chemical shifts were reported in parts per million (ppm, δ) downfield from
tetramethylsilane. Proton coupling patterns are described as singlet (s), doublet (d), triplet (t),
quartet (q), multiplet (m), and broad (br). Low- and high-resolution mass spectra (LRMS and
HRMS) were measured with a mass spectrometer. The purity of all compounds determined
by Agilent-1100 HPLC with binary pump, photodiode array detector (DAD) and C18 column
(
150×4.6 mm, 5 µm) was over 95%. Compounds 14a, 14c-14d, 14f, 14h-14j, 14l-14m, and
4o were analyzed using MeOH/H O = 70/30 (v/v) at 1 mL/min, and the peak areas were
calculated at 254 nm. Compounds 14b, 14e, 14g, 14k, and 14n were analyzed using
MeOH/H O = 80/20 (v/v) at 1 mL/min and the peak areas were calculated at 254 nm.
1
2
2
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2
2
.2 General procedure for synthesis of compounds 14a-14o
.2.1 Methyl 5-methylthiophene-2-carboxylate (3)
To a solution of 5-methylthiophene-2-carboxylic acid 2 (1.0 g, 7.0 mmol) in MeOH (50 mL)
under nitrogen, SOCl
2
(1.5 mL, 21.0 mmol) was added dropwise at 0 °C. the resulting
o
mixture was refluxed at 80 C for 3.5 h. After removal of the solvent, the crude product was
purified by flash chromatography eluting with petroleum ether/EtOAc (10:1) to afford
1
methyl 5-methylthiophene-2-carboxylate 3 as a yellow oil (1.0 g, 90.9%). H NMR (400
MHz, CDCl
3
) δ 7.60 (d, J = 3.7 Hz, 1H), 6.77–6.73 (m, 1H), 3.84 (s, 3H), 2.53–2.49 (m, 3H).
+
MS (ESI, m/z): 157.1 [M+H] .
2
.2.2 Methyl 5-(bromomethyl)thiophene-2-carboxylate (4)
Methyl 5-methylthiophene-2-carboxylate 3 (5.0 g, 32.0 mmol), N-bromosuccinimide (6.3 g,
3
3
5.4 mmol), and benzoylperoxide (0.78 g, 0.32 mmol) were dissolved in CHCl . The mixture
o
was heated at 65 C for 7 h and then cooled to room temperature. The resulting crude product
was filtered and the filtrate was extracted with EtOAc, washed with saturated brine, dried
2 4
over anhydrous Na SO and concentrated under reduced pressure. The residue was purified
by flash chromatography eluting with petroleum ether/EtOAc (10:1)to yield 6.2 g (82.3%) of
1
methyl 5-(bromomethyl)thiophene-2-carboxylate 4 as a yellow oil. H NMR (400 MHz,
CDCl
3
) δ 7.57 (d, J = 3.8 Hz, 1H), 7.03 (dd, J = 3.8, 0.6 Hz, 1H), 4.62 (s, 2H), 3.83 (dd, J =
6
.2, 0.6 Hz, 3H).
2
.2.3 tert-Butyl 2-(4-bromophenyl)acetate (6)
To a solution of 2-(4-bromophenyl)acetic acid 5 (10.0 g, 46.5 mmol) in t-BuOH (50 mL)
under nitrogen, t-butyl pyrocarbonate (20.3 g, 93.1 mmol) and DMAP (1.7 g, 13.9 mmol)
were added. The mixture was stirred at room temperature for 24 h. After removal of the
solvent, the crude product was purified by flash chromatography eluting with petroleum
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ether/EtOAc (10:1) to afford tert-butyl 2-(4-bromophenyl)acetate 6 as a yellow oil (9.4 g,
1
7
3
4.5%). H NMR (400 MHz, CDCl
3
) δ 7.43 (d, J = 8.3 Hz, 2H), 7.14 (d, J = 8.3 Hz, 2H),
+
.47 (s, 2H), 1.43 (s, 9H). MS (ESI, m/z): 279.1 [M+H] .
2
.2.4 Methyl 5-[2-(4-bromophenyl)-3-(tert-butoxy)-3-oxopropyl]thiophene-2-carboxylate
(7)
A solution of tert-butyl 2-(4-bromophenyl)acetate 6 (9.4 g, 34.7 mmol) in anhydrous THF
o
was cooled to -78 C under nitrogen atmosphere. To this mixture, a solution of n-BuLi in
o
hexane (28.9 mL, 2.4 M) was added dropwise at -78 C, and the resulting mixture was stirred
for 1 h. A solution of methyl 5-(bromomethyl)thiophene-2-carboxylate 4 (8.2 g, 34.7 mmol)
in anhydrous THF was subsequently added dropwise and the mixture was stirred for 2.5 h.
The reaction was quenched with saturated ammonium chloride aqueous solution, followed by
extracted with EtOAc. The combined organic layer was washed with brine, dried over
2 4
anhydrous Na SO , and concentrated under reduced pressure. The crude residue was purified
by flash chromatography eluting with petroleum ether/EtOAc (10:1) to yield 6.2 g (42.0%) of
methyl 5-[2-(4-bromophenyl)-3-(tert-butoxy)-3-oxopropyl]thiophene-2-carboxylate 7 as a
1
yellow oil. H NMR (400 MHz, CDCl
3
) δ 7.54 (d, J = 3.7 Hz, 1H), 7.40 (d, J = 8.4 Hz, 2H),
7
1
.17–7.11 (m, 2H), 6.70 (dd, J = 7.1, 3.8 Hz, 1H), 3.80 (s, 3H), 3.72 (dd, J = 15.3, 7.0 Hz,
H), 3.52 (dd, J = 14.7, 8.4 Hz, 1H), 3.12 (dd, J = 14.8, 7.1 Hz, 1H), 1.33 (s, 9H). MS (ESI,
-
m/z): 423.0 [M-H] .
2
.2.5 Methyl 5-[2-(4-bromophenyl)-3-chloro-3-oxopropyl]thiophene-2-carboxylate (8)
A solution of the compound 7 (900 mg, 2.1 mmol) in CH
5 mL) at room temperature for 3 h. The mixture was concentrated under reduced pressure to
give a brown oil. The oil was dissolved in anhydrous CH Cl , and then to the solution was
2 2
Cl (10 mL) was treated with TFA
(
2
2
added oxalychloride (481.3 mg, 3.8 mmol) dropwise. The resulting mixture was stirred at
ambient tempreature for 14 h. After removal of the solvent under reduced pressure, the
resulting crude product was used in next reaction without further purification.
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2
.2.6 4'-Chloro-2'-methyl-biphenyl-4-amine (11a)
The protocol reported in ref. 19 was modified as follows: A mixture of 4-iodo-aniline 9 (1.0
g, 4.6 mmol), 2-methyl-4-chlorophenyl-boronic acid 10 (1.2 g, 5.9 mmol), PdCl (P(o-tolyl)
O (16/8/4 mL) was
2
3 2
)
2 3 2
(0.47 mg, 0.59 mmol), and Na CO (2.4 g, 22.6 mmol) in DME/EtOH/H
o
heated at 125 C for 2 h. The reaction mixture was cooled to room temperature, filtered and
washed with EtOAc. The solution was cancentrated under reduced pressure. The resulting
mixture was extracted with EtOAc. the combined organic layer was washed with brine,
2 4
dried over anhydrous Na SO and concentrated in vacuo. The resulting crude product was
purified by flash chromatography eluting with petroleum ether/EtOAc (10:1) to give 0.9 g
1
(
90.5%) of 4'-chloro-2'-methyl-biphenyl-4-amine 11a as a brown solid. H NMR (400 MHz,
DMSO-d
6
) δ 7.30 (d, J = 2.3 Hz, 1H), 7.22 (dd, J = 8.2, 2.3 Hz, 1H), 7.13 (d, J = 8.2 Hz, 1H),
7
.01 – 6.94 (m, 2H), 6.65 – 6.59 (m, 2H), 5.16 (s, 2H), 2.23 (s, 3H). MS (ESI, m/z): 218.1
+
[
M+H] .
2
.2.7 4'-Chloro-3'-methyl-biphenyl-4-amine (11b)
Compound 11b was prepared according to the procedure for the preparation of compound
1
1
1a. H NMR (400 MHz, DMSO-d
6
) δ 7.52 (m, 1H), 7.36 (d, J = 1.7 Hz, 3H), 7.34 (s, 1H),
+
6
.65 – 6.62 (m, 2H), 5.27 (s, 2H), 2.35 (s, 3H). MS (ESI, m/z): 218.0 [M+H] .
2
.2.8 2',4',6'-trimethyl-biphenyl-4-amine (11c)
Compound 11c was prepared according to the procedure for the preparation of compound
1
1
1a. H NMR (400 MHz, DMSO-d
6
) δ 6.86 (s, 2H), 6.76 – 6.69 (m, 2H), 6.63 – 6.58 (m,
+
2
H), 5.02 (s, 2H), 2.23 (s, 3H), 1.94 (s, 6H). MS (ESI, m/z): 212.1 [M+H] .
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2
5
.2.9 Methyl
-{2-(4-bromophenyl)-3-[(4'-chloro-2'-methyl-biphenyl-4-yl)amino]-3-oxopropyl}thioph
ene-2-carboxylate (12a)
2 2
A solution of the compound 8 (720 mg, 1.9 mmol) in CH Cl was treated with
4
'-chloro-2'-methyl-biphenyl-4-amine 11 (404 mg, 1.9 mmol) and N,N-diispropylethylamine
(2.9 mL, 17.8 mmol), ssuccessively. The resulting mixture was then stirred for 14 h at room
temperature. After removal of the solvent, the crude product was purified by flash
chromatography eluting with petroleum ether/EtOAc (4:1) to afford methyl
5
2
1
-{2-(4-bromophenyl)-3-[(4'-chloro-2'-methyl-biphenyl-4-yl)amino]-3-oxopropyl}thiophene-
1
-carboxylate 12a as a yellow solid (970 mg, 91.5%). H NMR (400 MHz, DMSO-d
6
) δ
0.33 (s, 1H), 7.61 (h, J = 4.2, 3.7 Hz, 3H), 7.59 – 7.50 (m, 2H), 7.44 – 7.39 (m, 2H), 7.36 (d,
J = 2.2 Hz, 1H), 7.29 – 7.22 (m, 3H), 7.16 (d, J = 8.2 Hz, 1H), 6.98 (t, J = 4.1 Hz, 1H), 4.06
(t, J = 7.6 Hz, 1H), 3.76 (s, 3H), 3.65 (dd, J = 14.6, 9.0 Hz, 1H), 3.29 (dd, J = 14.5, 6.1 Hz,
+
1
H), 2.19 (s, 3H). MS (ESI, m/z): 568.0 [M+H] .
2.2.10 Methyl
5
-{2-(4-bromophenyl)-3-[(4'-chloro-3'-methyl-biphenyl-4-yl)amino]-3-oxopropyl}thioph
ene-2-carboxylate (12b)
Compound 12b was prepared according to the procedure for the preparation of compound
1
1
2a. H NMR (400 MHz, DMSO-d
6
) δ 10.33 (s, 1H), 7.66 – 7.59 (m, 6H), 7.55 (d, J = 8.5
Hz, 2H), 7.46 – 7.38 (m, 4H), 6.98 (d, J = 3.8 Hz, 1H), 4.06 (dd, J = 8.7, 6.6 Hz, 1H), 3.75 (s,
3
H), 3.65 (dd, J = 14.7, 8.7 Hz, 1H), 3.29 (dd, J = 14.7, 6.5 Hz, 1H), 2.37 (s, 3H). MS (ESI,
+
m/z): 569.7 [M+H] .
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2
.2.11
Methyl
-{2-(4-bromophenyl)-3-[(2',4',6'-trimethyl-biphenyl-4-yl)amino]-3-oxopropyl}thiophen
e-2-carboxylate (12c)
5
Compound 12c was prepared according to the procedure for the preparation of compound
1
1
2a. H NMR (400 MHz, DMSO-d
6
) δ 10.28 (s, 1H), 7.63 – 7.61 (m, 2H), 7.60 (d, J = 2.0
Hz, 1H), 7.57 – 7.54 (m, 2H), 7.45 – 7.41 (m, 2H), 7.02 – 6.98 (m, 3H), 6.88 (s, 2H), 4.07
(dd, J = 8.8, 6.5 Hz, 1H), 3.76 (s, 3H), 3.66 (dd, J = 14.7, 8.8 Hz, 1H), 3.29 (dd, J = 14.7, 6.4
-
Hz, 1H), 2.23 (s, 3H), 1.89 (s, 6H). MS (ESI, m/z): 561.8 [M-H] .
2
5
.2.12 Methyl
-{3-[(4'-chloro-2'-methyl-biphenyl-4-yl)amino]-2-(4'-methoxy-biphenyl-4-yl)-3-oxopro
pyl}thiophene-2-carboxylate (13c)
Compound 12a (200 mg, 0.35 mmol) was dissolved in DME, followed with
4
2 3 2
-methoxyphenyl-boronic acid (134 mg, 0.88 mmol), PdCl (P(o-tolyl) ) (82.9 mg, 0.11
o
mmol) and DIPEA. The mixture was heated at 85 C for 2 h. After cooling to room
temperature, the mixture was filtered through a pad of celite which was rinsed three times
with EtOAc and the combined organic layer was concentrated in vacuo. The resulting crude
product was purified by flash chromatography eluting with petroleum ether/EtOAc (4:1) to
yield 135 mg (64.4%) of
5
-{3-[(4'-chloro-2'-methyl-biphenyl-4-yl)amino]-2-(4'-methoxy-biphenyl-
1
4
-yl)-3-oxopropyl}thiophene-2-carboxylate 13c as a yellow solid. H NMR (400 MHz,
CDCl
3
) δ 7.65 (s, 1H), 7.57 (d, J = 3.7 Hz, 1H), 7.55–7.49 (m, 6H), 7.40 (d, J = 8.2 Hz, 2H),
7
.34 (d, J = 8.6 Hz, 2H), 7.16 (d, J = 7.7 Hz, 1H), 7.08 (d, J = 8.0 Hz, 1H), 6.97 (d, J = 8.8
Hz, 2H), 6.77 (d, J = 3.7 Hz, 1H), 3.89 (d, J = 3.7 Hz, 1H), 3.85 (s, 3H), 3.82 (s, 3H), 3.76 (d,
-
J = 11.7 Hz, 1H), 3.32 (td, J = 11.1, 7.3 Hz, 1H), 2.36 (s, 3H). MS (ESI, m/z): 594.1 [M-H] .
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2
5
.2.13 Methyl
-{3-[(4'-chloro-2'-methyl-biphenyl-4-yl)amino]-2-(4'-fluoro-biphenyl-4-yl)-3-oxopropyl
}
thiophene-2-carboxylate (13d)
Compound 13d was prepared according to the procedure for the preparation of compound
1
1
3c. H NMR (400 MHz, DMSO-d
6
) δ 10.34 (s, 1H), 7.72 – 7.66 (m, 2H), 7.66 – 7.60 (m,
5
H), 7.55 (d, J = 8.3 Hz, 2H), 7.35 (d, J = 2.3 Hz, 1H), 7.30 – 7.22 (m, 5H), 7.16 (d, J = 8.2
Hz, 1H), 7.03 (d, J = 3.8 Hz, 1H), 4.12 (dd, J = 9.3, 5.9 Hz, 1H), 3.76 (s, 3H), 3.74 – 3.65 (m,
-
1
H), 3.31 (d, J = 5.9 Hz, 1H), 2.19 (s, 3H).MS (ESI, m/z): 583.8 [M-H] .
2
5
.2.14 Methyl
-{3-[(4'-chloro-2'-methyl-biphenyl-4-yl)amino]-2-(4'-trifluoromethyl-biphenyl-4-yl)-3-o
xopropyl}thiophene-2-carboxylate (13e)
Compound 13e was prepared according to the procedure for the preparation of compound
1
1
3c. H NMR (400 MHz, DMSO-d
6
) δ 10.36 (s, 1H), 7.89 (d, J = 8.1 Hz, 2H), 7.80 (d, J =
8
.2 Hz, 2H), 7.74 (d, J = 8.0 Hz, 2H), 7.66 – 7.59 (m, 5H), 7.36 (d, J = 2.3 Hz, 1H), 7.26 (t, J
=
5.5 Hz, 3H), 7.16 (d, J = 8.2 Hz, 1H), 7.03 (d, J = 3.8 Hz, 1H), 4.17 – 4.12 (m, 1H), 3.76 (s,
-
3
H), 3.75 – 3.69 (m, 1H), 3.33 (s, 1H), 2.20 (s, 3H). MS (ESI, m/z): 633.8 [M-H] .
2
5
.2.15 Methyl
-{3-[(4'-chloro-2'-methyl-biphenyl-4-yl)amino]-2-(4'-trifluoromethoxy-biphenyl-4-yl)-3
-
oxopropyl}thiophene-2-carboxylate (13f)
Compound 13f was prepared according to the procedure for the preparation of compound
1
1
3c. H NMR (400 MHz, DMSO-d
6
) δ 10.35 (s, 1H), 7.88 (d, J = 8.0 Hz, 2H), 7.80 (d, J =
8
.2 Hz, 2H), 7.77 – 7.71 (m, 2H), 7.70 – 7.57 (m, 5H), 7.35 (d, J = 2.2 Hz, 1H), 7.32 – 7.21
(m, 3H), 7.16 (d, J = 8.2 Hz, 1H), 7.03 (d, J = 4.0 Hz, 1H), 4.19 – 4.12 (m, 1H), 4.02 (q, J =
7
.0 Hz, 1H), 3.76 (s, 3H), 3.71 (d, J = 9.1 Hz, 1H), 2.19 (s, 3H). MS (ESI, m/z): 649.8
-
[
M-H] .
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2
5
.2.16 Methyl
-{3-[(4'-chloro-2'-methyl-biphenyl-4-yl)amino]-2-(4'-methy-biphenyl-4-yl)-3-oxopropyl
}
thiophene-2-carboxylate (13g)
Compound 13g was prepared according to the procedure for the preparation of compound
1
1
3c. H NMR (400 MHz, DMSO-d
6
) δ 10.33 (s, 1H), 7.63 (m, 5H), 7.57 – 7.51 (m, 4H), 7.35
(d, J = 2.3 Hz, 1H), 7.25 (m, 5H), 7.16 (d, J = 8.3 Hz, 1H), 7.03 (d, J = 3.9 Hz, 1H), 4.11 (dd,
J = 9.1, 5.7 Hz, 1H), 3.76 (s, 3H), 3.74 – 3.68 (m, 1H), 3.30 (d, J = 5.7 Hz, 1H), 2.32 (s, 3H),
-
2
.19 (s, 3H). MS (ESI, m/z): 579.8 [M-H] .
2
5
.2.17 Methyl
-{3-[(4'-chloro-2'-methyl-biphenyl-4-yl)amino]-2-(4'-tert-butyl-biphenyl-4-yl)-3-oxopro
pyl}thiophene-2-carboxylate (13h)
Compound 13h was prepared according to the procedure for the preparation of compound
1
1
3c. H NMR (400 MHz, DMSO-d
6
) δ 10.34 (d, J = 3.0 Hz, 1H), 7.67 – 7.61 (m, 5H), 7.56
(
m, 4H), 7.48 – 7.44 (m, 2H), 7.36 (d, J = 2.5 Hz, 1H), 7.29 – 7.22 (m, 3H), 7.16 (dd, J = 8.3,
3
3
6
.2 Hz, 1H), 7.04 (d, J = 3.6 Hz, 1H), 4.13 – 4.09 (m, 1H), 3.76 (d, J = 3.0 Hz, 3H), 3.74 –
.66 (m, 1H), 3.32 – 3.26 (m, 1H), 2.20 (s, 3H), 1.30 (d, J = 3.1 Hz, 9H). MS (ESI, m/z):
-
21.7 [M-H] .
2
5
.2.18 Methyl
-{3-[(4'-chloro-3'-methyl-biphenyl-4-yl)amino]-2-(4'-tert-butyl-biphenyl-4-yl)-3-oxopro
pyl}thiophene-2-carboxylate (13i)
Compound 13i was prepared according to the procedure for the preparation of compound
1
1
3c. H NMR (400 MHz, DMSO-d
6
) δ 10.35 (s, 1H), 7.67 (d, J = 8.5 Hz, 3H), 7.62 (d, J =
2
.9 Hz, 4H), 7.59 – 7.52 (m, 5H), 7.45 (d, J = 8.8 Hz, 4H), 7.02 (d, J = 3.8 Hz, 1H), 4.11 (dd,
J = 9.0, 6.1 Hz, 1H), 3.75 (s, 3H), 3.69 (d, J = 9.1 Hz, 1H), 3.30 (s, 1H), 2.37 (s, 3H), 1.29 (s,
-
9
H). MS (ESI, m/z): 621.8 [M-H] .
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2
5
.2.19
Methyl
-{3-[(4'-chloro-3'-methyl-biphenyl-4-yl)amino]-2-(4'-methoxy-biphenyl-4-yl)-3-oxopro
pyl}thiophene-2-carboxylate (13j)
Compound 13j was prepared according to the procedure for the preparation of compound
1
1
3c. H NMR (400 MHz, DMSO-d
6
) δ 10.32 (s, 1H), 7.66 (d, J = 8.7 Hz, 2H), 7.64 – 7.56
(m, 8H), 7.51 (m, 2H), 7.48 – 7.40 (m, 2H), 7.04 – 6.98 (m, 3H), 4.09 (dt, J = 8.9, 4.3 Hz,
1
H), 3.78 (s, 3H), 3.75 (s, 2H), 3.70 (dd, J = 14.8, 9.1 Hz, 1H), 3.30 (d, J = 6.1 Hz, 1H), 2.37
+
(
s, 3H). MS (ESI, m/z): 597.0[M+H] .
2
5
.2.20
Methyl
-{3-[(4'-chloro-3'-methyl-biphenyl-4-yl)amino]-2-(4'-chloro-2'-methyl-biphenyl-4-yl)-3-
oxopropyl}thiophene-2-carboxylate (13k)
Compound 13k was prepared according to the procedure for the preparation of compound
1
1
4
7
9
2
3c. H NMR (400 MHz, DMSO-d
6
) δ 10.36 (s, 1H), 7.71 – 7.66 (m, 2H), 7.65 – 7.58 (m,
H), 7.56 – 7.52 (m, 2H), 7.47 – 7.41 (m, 2H), 7.37 (d, J = 2.2 Hz, 1H), 7.36 – 7.31 (m, 2H),
.29 (dd, J = 8.3, 2.3 Hz, 1H), 7.19 (d, J = 8.3 Hz, 1H), 7.02 (d, J = 3.9 Hz, 1H), 4.11 (dd, J =
.4, 5.6 Hz, 1H), 3.76 (d, J = 1.4 Hz, 3H), 3.71 (d, J = 9.2 Hz, 1H), 3.30 (d, J = 5.6 Hz, 1H),
+
.37 (s, 3H), 2.21 (s, 3H). MS (ESI, m/z): 616.1[M+H] .
2
5
.2.21
Methyl
-{3-[(4'-chloro-3'-methyl-biphenyl-4-yl)amino]-2-(2',4',6'-trimethyl-biphenyl-4-yl)-3-ox
opropyl}thiophene-2-carboxylate (13l)
Compound 13l was prepared according to the procedure for the preparation of compound
1
1
3c. H NMR (400 MHz, DMSO-d
6
) δ 10.37 (s, 1H), 7.71 (d, J = 8.6 Hz, 2H), 7.65 – 7.58
(m, 4H), 7.50 (d, J = 7.9 Hz, 2H), 7.48 – 7.40 (m, 2H), 7.09 (d, J = 8.1 Hz, 2H), 7.00 (d, J =
3
3
.8 Hz, 1H), 6.90 (s, 2H), 4.07 (dd, J = 8.8, 6.3 Hz, 1H), 3.75 (s, 3H), 3.73 – 3.62 (m, 1H),
+
.34 – 3.27 (m, 1H), 2.37 (s, 3H), 2.24 (s, 3H), 1.90 (s, 6H). MS (ESI, m/z): 609.2[M+H] .
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2
5
.2.22
Methyl
-{3-[(2',4',6'-trimethyl-biphenyl-4-yl)amino]-2-(4'-tert-butyl-biphenyl-4-yl)-3-oxopropy
l}thiophene-2-carboxylate (13m)
Compound 13m was prepared according to the procedure for the preparation of compound
1
1
3c. H NMR (400 MHz, DMSO-d
6
) δ 10.37 (s, 1H), 7.71 (d, J = 8.6 Hz, 2H), 7.65 – 7.58
(m, 4H), 7.50 (d, J = 7.9 Hz, 2H), 7.48 – 7.40 (m, 2H), 7.09 (d, J = 8.1 Hz, 2H), 7.00 (d, J =
3
3
.8 Hz, 1H), 6.90 (s, 2H), 4.07 (dd, J = 8.8, 6.3 Hz, 1H), 3.75 (s, 3H), 3.73 – 3.62 (m, 1H),
+
.34 – 3.27 (m, 1H), 2.37 (s, 3H), 2.24 (s, 3H), 1.90 (s, 6H). MS (ESI, m/z): 616.0 [M+H] .
2
5
.2.23
Methyl
-{3-[(2',4',6'-trimethyl-biphenyl-4-yl)amino]-2-(4'-methy-biphenyl-4-yl)-3-oxopropyl}t
hiophene-2-carboxylate (13n)
Compound 13n was prepared according to the procedure for the preparation of compound
1
1
3c. H NMR (400 MHz, DMSO-d
6
) δ 10.28 (s, 1H), 7.65 – 7.63 (m, 2H), 7.63 – 7.59 (m,
3
H), 7.55 (d, J = 3.0 Hz, 2H), 7.53 (d, J = 2.7 Hz, 2H), 7.27 – 7.21 (m, 2H), 7.03 (d, J = 3.8
Hz, 1H), 7.02 – 6.97 (m, 2H), 6.88 (s, 2H), 4.11 (dd, J = 9.1, 6.0 Hz, 1H), 3.76 (s, 3H), 3.75 –
3
5
.67 (m, 1H), 3.32 (d, J = 8.8 Hz, 1H), 2.32 (s, 3H), 2.23 (s, 3H), 1.89 (s, 6H). MS (ESI, m/z):
+
74.0 [M+H] .
2
5
.2.24
Methyl
-{3-[(2',4',6'-trimethyl-biphenyl-4-yl)amino]-2-(4'-methoxy-biphenyl-4-yl)-3-oxopropyl
}
thiophene-2-carboxylate (13o)
Compound 13o was prepared according to the procedure for the preparation of compound
1
1
3
4
1
3c. H NMR (400 MHz, DMSO-d
6
) δ 10.28 (s, 1H), 7.65 – 7.61 (m, 4H), 7.60 – 7.58 (m,
H), 7.53 (d, J = 8.4 Hz, 2H), 7.04 – 7.01 (m, 3H), 7.00 (d, J = 1.1 Hz, 2H), 6.88 (s, 2H),
.10 (dd, J = 9.2, 6.1 Hz, 1H), 3.78 (s, 3H), 3.76 (s, 3H), 3.74 – 3.68 (m, 1H), 3.34 – 3.28 (m,
+
H), 2.23 (s, 3H), 1.89 (s, 6H). MS (ESI, m/z): 590.0 [M+H] .
This article is protected by copyright. All rights reserved.

2
2
.2.25
-(5-{3-[(4'-chloro-2'-methyl-biphenyl-4-yl)amino]-2-(4'-methoxy-bipheny-4-yl)-3-oxopr
opyl} thiophene-2-carboxamido)ethanesulfonic acid (14c)
A solution of compound 13c in 1,4-dioxane/MeOH (1:1) was treated with NaOH aq. (2 N).
o
Then the mixture was heated at 60 C for 2 h. After removal of the solvent, the residue was
acidified with 1 N HCl, and the resulting mixture was extracted with EtOAc, washed
withbrine, dried over anhydrous Na
2 4
SO , and concentrated in vacuo. The crude product
(
2-thiophenecarboxylic acid) was used as a yellow solid which was used in next reaction
without further purification. This crude 2-thiophenecarboxylic acid was dissolved in DMF,
followed with addition of taurine (28.4 mg, 0.23 mmol), EDCI (65.2 mg, 0.34 mmol), HOBt
(46.0 mg, 0.34 mmol) and DIPEA (0.2 mL). The reaction mixture was stirred at ambient
temperature for overnight. After the reaction was processed completely, EtOA cwas added,
and the organic layer was washed twice with1 N HCl and brine, successively. After removal
of the solvent, the residue was purified by flash chromatography eluting with DCM/MeOH
(
15:1) to afford
-(5-{3-[(4'-chloro-2'-methyl-biphenyl-4-yl)amino]-2-(4'-methoxy-bipheny-4-yl)-3-oxopropy
l}thiophene-2-carboxamido)ethanesulfonic acid 14c as a white solid (100 mg, 64.1%). mp
2
o
1
1
60-162 C. H NMR (400 MHz, DMSO-d
J = 8.6 Hz, 2H), 7.62–7.56 (m, 4H), 7.52 (d, J = 8.4 Hz, 2H), 7.41 (d, J = 3.7 Hz, 1H), 7.35
d, J = 2.0 Hz, 1H), 7.29–7.21 (m, 3H), 7.17 (d, J = 8.2 Hz, 1H), 7.01 (d, J = 8.8 Hz, 2H),
6
) δ 10.40 (s, 1H), 8.40 (t, J = 5.4 Hz, 1H), 7.66 (d,
(
6
1
.91 (d, J = 3.7 Hz, 1H), 4.13 (t, J = 5.3 Hz, 1H), 3.78 (s, 3H), 3.65 (dt, J = 20.7, 10.5 Hz,
1
3
H), 3.44 (dd, J = 14.1, 6.1 Hz, 2H), 3.27–3.20 (m, 1H), 2.67–2.60 (m, 2H), 2.20 (s, 3H). C
) δ 171.8, 161.9, 160.1, 148.1, 140.9, 139.9, 139.4, 139.1, 139.0,
38.5, 136.0, 133.3, 132.7, 132.4, 131.0, 130.5, 129.4, 128.9, 128.7, 127.7, 127.4, 127.0,
NMR (125 MHz, DMSO-d
6
1
-
1
20.0, 115.5, 56.3, 54.6, 51.5, 37.1, 34.2, 21.2. LRMS (ESI, m/z): 687.1 [M-H] . HRMS (ESI,
-
-
2 6 2
m/z): calcd for C36H32 ClN O S , 687.1396 [M-H] ; found 687.1406, purity: 98.8%.
This article is protected by copyright. All rights reserved.

2
.2.26 2-(5-{3-[(4'-chloro-2'-methyl-biphenyl-4-yl)amino]-3-oxo-2-phenylpropyl}
thiophene-2-carboxamido)ethanesulfonic acid (14a)
Compound 14a was prepared according to the procedure for the preparation of compound
o
1
1
7
3
1
6
4c. mp 151-153 C. H NMR (400 MHz, DMSO-d ) δ 10.37 (s, 1H), 8.39 (t, J = 5.2 Hz, 1H),
.64 (d, J = 8.4 Hz, 2H), 7.47 (d, J = 7.7 Hz, 2H), 7.40 (d, J = 3.5 Hz, 1H), 7.37–7.31 (m,
H), 7.29–7.21 (m, 4H), 7.16 (d, J = 8.2 Hz, 1H), 6.88 (d, J = 3.6 Hz, 1H), 4.10–4.01 (m,
H), 3.63 (dd, J = 14.6, 9.3 Hz, 1H), 3.44 (dd, J = 13.5, 6.4 Hz, 2H), 3.21 (dd, J = 14.9, 5.9
-
Hz, 1H), 2.70–2.58 (m, 2H), 2.20 (s, 3H). LRMS (ESI, m/z): 581.1 [M-H] . HRMS (ESI,
-
-
2 5 2
m/z): calcd for C29H26ClN O S , 581.0977 [M-H] ; found 581.0976, purity: 98.3%.
2
2
.2.27
-(5-{2-(4-bromophenyl)-3-[(4'-chloro-2'-methyl-biphenyl-4-yl)amino]-3-oxopropyl}thio
phene-2-carboxamido)ethanesulfonic acid (14b)
Compound 14b was prepared according to the procedure for the preparation of compound
o
1
1
4c. mp 135-137 C. H NMR (400 MHz, DMSO-d
6
) δ 10.39 (d, J = 20.2 Hz, 1H), 8.40 (s,
1
H), 7.63 (d, J = 7.5 Hz, 2H), 7.54 (d, J = 8.5 Hz, 2H), 7.44–7.38 (m, 3H), 7.36 (d, J = 1.8
Hz, 1H), 7.29–7.21 (m, 3H), 7.17 (d, J = 8.2 Hz, 1H), 6.87 (d, J = 3.5 Hz, 1H), 4.07 (t, J =
7
.5 Hz, 1H), 3.60 (dd, J = 14.6, 8.8 Hz, 1H), 3.45 (dd, J = 13.6, 6.4 Hz, 2H), 3.22 (dd, J =
-
1
4.7, 6.4 Hz, 1H), 2.65 (t, J = 7.3 Hz, 2H), 2.20 (s, 3H). LRMS (ESI, m/z): 659.0 [M-H] .
-
-
2 5 2
HRMS (ESI, m/z): calcd for C29H25BrClN O S , 659.0082 [M-H] ; found 659.0089, purity:
9
5.3%.
2
.2.28 2-(5-{3-[(4'-chloro-2'-methyl-biphenyl-4-yl)amino]-2-(4'-fluoro-bipheny-4-yl)-3-
oxopropyl} thiophene-2-carboxamido)ethanesulfonic acid (14d)
Compound 14d was prepared according to the procedure for the preparation of compound
o
1
1
4c. mp 151-153 C. H NMR (400 MHz, DMSO-d
6
) δ 10.48 (s, 1H), 8.41 (t, J = 5.4 Hz, 1H),
7
.73–7.60 (m, 6H), 7.56 (d, J = 8.3 Hz, 2H), 7.42 (d, J = 3.6 Hz, 1H), 7.35 (s, 1H), 7.31–7.21
This article is protected by copyright. All rights reserved.

(m, 5H), 7.17 (d, J = 8.2 Hz, 1H), 6.92 (d, J = 3.6 Hz, 1H), 4.14–4.09 (m, 1H), 3.66 (dd, J =
1
4.6, 9.2 Hz, 1H), 3.44 (dd, J = 13.7, 6.3 Hz, 2H), 3.26 (dd, J = 14.9, 6.0 Hz, 1H), 2.69–2.59
-
(
m, 2H), 2.20 (s, 3H). LRMS (ESI, m/z): 674.9 [M-H] . HRMS (ESI, m/z): calcd for
-
-
C
35 2 5 2
H29ClFN O S , 675.1196 [M-H] ; found 675.1199, purity: 99.5%.
2
2
.2.29
-(5-{3-[(4'-chloro-2'-methyl-biphenyl-4-yl)amino]-2-(4'-trifluoro-bipheny-4-yl)-3-oxopr
opyl} thiophene-2-carboxamido)ethanesulfonic acid (14e)
Compound 14e was prepared according to the procedure for the preparation of compound
o
1
1
7
8
6
1
6
4c. mp 148-150 C. H NMR (500 MHz, DMSO-d ) δ 10.36 (s, 1H), 8.38 (t, J = 5.5 Hz, 1H),
.89 (d, J = 8.2 Hz, 2H), 7.81 (d, J = 8.4 Hz, 2H), 7.74 (d, J = 8.3 Hz, 2H), 7.63 (dd, J = 22.3,
.5 Hz, 4H), 7.38 (dd, J = 19.7, 2.8 Hz, 2H), 7.31–7.23 (m, 3H), 7.18 (d, J = 8.2 Hz, 1H),
.91 (d, J = 3.7 Hz, 1H), 4.15–4.09 (m, 1H), 3.68 (dd, J = 14.6, 9.1 Hz, 1H), 3.44 (dd, J =
3.4, 6.5 Hz, 2H), 3.29 (dd, J = 14.7, 5.9 Hz, 1H), 2.65–2.58 (m, 2H), 2.20 (s, 3H). LRMS
-
-
-
(
ESI, m/z): 725.0 [M-H] . HRMS (ESI, m/z): calcd for C36
3 2 5 2
H29ClF N O S , 725.1164 [M-H] ;
found 725.1166, purity: 97.3%.
2
2
.2.30
-(5-{3-[(4'-chloro-2'-methyl-biphenyl-4-yl)amino]-2-(4'-trifluoromethoxy-bipheny-4-yl)
-
3-oxopropyl} thiophene-2-carboxamido)ethanesulfonic acid (14f)
Compound 14f was prepared according to the procedure for the preparation of compound
o
1
1
4c. mp 161-163 C. H NMR (400 MHz, DMSO-d
6
) δ 10.51 (s, 1H), 8.42 (t, J = 5.5 Hz, 1H),
7
.78 (d, J = 8.8 Hz, 2H), 7.67 (dd, J = 8.5, 1.2 Hz, 4H), 7.59 (d, J = 8.4 Hz, 2H), 7.47–7.41
(m, 3H), 7.35 (d, J = 1.9 Hz, 1H), 7.30–7.21 (m, 3H), 7.17 (d, J = 8.2 Hz, 1H), 6.92 (d, J =
3
1
.7 Hz, 1H), 4.15 (dd, J = 11.3, 6.0 Hz, 1H), 3.67 (dd, J = 14.8, 9.1 Hz, 1H), 3.44 (dd, J =
1
3
4.2, 6.1 Hz, 2H), 3.26 (dd, J = 14.7, 5.9 Hz, 1H), 2.68–2.60 (m, 2H), 2.20 (s, 3H). C NMR
) δ 171.6, 161.9, 149.0, 148.0, 140.9, 140.3, 140.3, 139.3, 139.2, 138.8,
(125 MHz, DMSO-d
6
This article is protected by copyright. All rights reserved.

1
38.5, 136.1, 132.7, 132.4, 131.0, 130.5, 129.7, 129.6, 128.7, 128.2, 127.8, 127.0, 122.6,
-
1
20.1, 54.6, 51.5, 37.1, 34.2, 21.2. LRMS (ESI, m/z): 741.0 [M-H] . HRMS (ESI, m/z): calcd
-
-
3 2 6 2
for C36H29ClF N O S , 741.1113 [M-H] ; found 741.1117, purity: 99.8%.
2
2
.2.31
-(5-{3-[(4'-chloro-2'-methyl-biphenyl-4-yl)amino]-2-(4'-methyl-bipheny-4-yl)-3-oxopro
pyl} thiophene-2-carboxamido)ethanesulfonic acid (14g)
Compound 14g was prepared according to the procedure for the preparation of compound
o
1
1
4c. mp 150-152 C. H NMR (400 MHz, DMSO-d
6
) δ 10.33 (s, 1H), 8.39 (t, J = 5.3 Hz, 1H),
7
.63 (dd, J = 10.5, 8.6 Hz, 4H), 7.54 (dd, J = 7.8, 3.6 Hz, 4H), 7.41 (d, J = 3.6 Hz, 1H), 7.35
(s, 1H), 7.31–7.20 (m, 5H), 7.17 (d, J = 8.2 Hz, 1H), 6.91 (d, J = 3.5 Hz, 1H), 4.19–3.99 (m,
1
H), 3.67 (dd, J = 14.5, 9.1 Hz, 1H), 3.46 (dd, J = 13.6, 6.3 Hz, 2H), 3.25 (dd, J = 14.6, 5.6
-
Hz, 1H), 2.67 (t, J = 7.3 Hz, 2H), 2.33 (s, 3H), 2.20 (s, 3H). LRMS (ESI, m/z): 671.0 [M-H] .
-
-
2 5 2
HRMS (ESI, m/z): calcd for C36H32ClN O S , 671.1147 [M-H] ; found 671.1458, purity:
9
8.2%.
2
2
.2.32
-(5-{3-[(4'-chloro-2'-methyl-biphenyl-4-yl)amino]-2-(4'-t-butyl-bipheny-4-yl)-3-oxopro
pyl} thiophene-2-carboxamido)ethanesulfonic acid (14h)
Compound 14h was prepared according to the procedure for the preparation of compound
o
1
1
1
3
1
7
1
1
6
4c. mp 160-162 C. H NMR (400 MHz, DMSO-d ) δ 10.41 (s, 1H), 8.39 (d, J = 5.2 Hz,
H), 7.60 (ddd, J = 19.3, 12.6, 8.4 Hz, 8H), 7.50–7.30 (m, 4H), 7.26 (dd, J = 12.8, 9.6 Hz,
H), 7.17 (d, J = 8.3 Hz, 1H), 6.91 (d, J = 3.5 Hz, 1H), 4.11 (d, J = 6.2 Hz, 1H), 3.67 (dd, J =
4.3, 9.2 Hz, 1H), 3.44 (dd, J = 13.7, 6.5 Hz, 3H), 3.25 (dd, J = 14.8, 5.6 Hz, 1H), 2.64 (t, J =
1
3
6
.3 Hz, 2H), 2.20 (s, 3H), 1.30 (s, 9H). C NMR (125 MHz, DMSO-d ) δ 171.7, 161.8,
51.0, 148.1, 140.2, 139.9, 139.5, 139.4, 139.2, 138.1, 137.6, 134.8, 132.3, 132.1, 131.2,
30.8, 129.4, 129.3, 128.6, 127.8, 127.8, 127.5, 126.9, 119.9, 54.8, 51.5, 37.2, 35.4, 32.3,
This article is protected by copyright. All rights reserved.

-
-
2
7
1.4. LRMS (ESI, m/z): 713.2 [M-H] . HRMS (ESI, m/z) calcd for C39
H38ClN
2
O
5
S
2
,
-
13.1916 [M-H] ; found 713.1915, purity: 99.6%.
2
2
.2.33
-(5-{2-(4'-t-butyl-bipheny-4-yl)-3-[(4'-chloro-3'-methyl-biphenyl-4-yl)amino]-3-oxopro
pyl} thiophene-2-carboxamido)ethanesulfonic acid (14i)
Compound 14i was prepared according to the procedure for the preparation of compound
o
1
1
7
4
6
4c. mp 139-141 C. H NMR (400 MHz, DMSO-d ) δ 10.41 (s, 1H), 8.39 (t, J = 5.3 Hz, 1H),
.68 (d, J = 8.7 Hz, 2H), 7.64–7.51 (m, 8H), 7.49–7.37 (m, 5H), 6.90 (d, J = 3.6 Hz, 1H),
.14–4.06 (m, 1H), 3.66 (dd, J = 14.7, 9.0 Hz, 1H), 3.43 (dd, J = 13.7, 6.1 Hz, 2H), 3.26 (dd,
J = 14.6, 5.9 Hz, 1H), 2.62 (t, J = 7.3 Hz, 2H), 2.35 (d, J = 13.8 Hz, 3H), 1.30 (s, 9H). LRMS
-
-
-
(
ESI, m/z): 713.2 [M-H] . HRMS (ESI, m/z): calcd for C39
2 5 2
H38ClN O S , 713.1916 [M-H] ;
found 713.1928, purity: 96.9%.
2
2
.2.34
-(5-{3-[(4'-chloro-3'-methyl-biphenyl-4-yl)amino]-2-(4'-methoxy-bipheny-4-yl)-3-oxopr
opyl} thiophene-2-carboxamido)ethanesulfonic acid (14j)
Compound 14j was prepared according to the procedure for the preparation of compound
o
1
1
7
6
3
3
6
6
4c. mp 158-160 C. H NMR (400 MHz, DMSO-d ) δ 10.38 (s, 1H), 8.37 (t, J = 5.4 Hz, 1H),
.67 (d, J = 8.8 Hz, 2H), 7.64–7.55 (m, 7H), 7.54–7.37 (m, 5H), 7.00 (d, J = 8.9 Hz, 2H),
.89 (d, J = 3.7 Hz, 1H), 4.12–4.08 (m, 1H), 3.78 (s, 3H), 3.65 (dd, J = 14.7, 8.9 Hz, 1H),
.43 (dd, J = 13.7, 6.4 Hz, 2H), 3.25 (dd, J = 14.6, 6.0 Hz, 1H), 2.65–2.58 (m, 2H), 2.37 (s,
-
-
H). LRMS (ESI, m/z): 687.1 [M-H] . HRMS (ESI, m/z): calcd for C36
H32ClN
2
O
6
S
2
,
-
87.1396 [M-H] ; found 687.1401, purity: 97.6%.
This article is protected by copyright. All rights reserved.

2
2
3
.2.35
-(5-{3-[(4'-chloro-3'-methyl-biphenyl-4-yl)amino]-2-(4'-chloro-2'-methyl-bipheny-4-yl)-
-oxopropyl} thiophene-2-carboxamido)ethanesulfonic acid (14k)
Compound 14k was prepared according to the procedure for the preparation of compound
o
1
1
4c. mp 166-169 C. H NMR (400 MHz, DMSO-d
6
) δ 10.44 (s, 1H), 8.37 (t, J = 5.2 Hz, 1H),
7
.69 (d, J = 8.7 Hz, 2H), 7.62 (dd, J = 15.0, 10.0 Hz, 4H), 7.54 (d, J = 8.2 Hz, 2H), 7.49–7.43
(
m, 2H), 7.43–7.39 (m, 1H), 7.37 (d, J = 1.7 Hz, 1H), 7.35–7.29 (m, 3H), 7.28 (d, J = 1.9 Hz,
H), 7.20 (d, J = 8.2 Hz, 1H), 6.90 (d, J = 3.6 Hz, 1H), 4.12 (dd, J = 9.1, 3.4 Hz, 1H), 3.68
dd, J = 14.5, 9.4 Hz, 1H), 3.45 (dd, J = 13.5, 6.3 Hz, 2H), 3.25 (dd, J = 14.6, 5.6 Hz, 1H),
1
(
1
3
2
1
1
3
C
3
.65 (t, J = 7.3 Hz, 2H), 2.37 (s, 3H), 2.21 (s, 3H). C NMR (125 MHz, CD OD) δ 171.1,
62.5, 147.4, 139.5, 139.5, 138.7, 137.6, 137.4, 136.8, 136.0, 135.5, 135.2, 132.3, 132.1,
30.3, 129.1, 128.6, 128.4, 128.3, 128.0, 127.0, 126.3, 126.1, 125.0, 124.7, 119.7, 54.2, 49.5,
-
5.0, 33.3, 18.7, 18.3. LRMS (ESI, m/z): 705.1 [M-H] . HRMS (ESI, m/z): calcd for
-
-
36 2 2 5 2
H31Cl N O S , 705.1057 [M-H] ; found 705.1065, purity: 95.8%.
2
2
.2.36
-(5-{3-[(4'-chloro-3'-methyl-biphenyl-4-yl)amino]-2-(2',4',6'-trimethyl-bipheny-4-yl)-3-
oxopropyl} thiophene-2-carboxamido)ethanesulfonic acid (14l)
Compound 14l was prepared according to the procedure for the preparation of compound
o
1
1
4c. mp 159-161 C. H NMR (400 MHz, DMSO-d
6
) δ 10.36 (s, 1H), 8.34 (t, J = 5.4 Hz, 1H),
7
.70 (d, J = 8.8 Hz, 2H), 7.63 (dd, J = 13.0, 9.0 Hz, 3H), 7.50 (d, J = 8.2 Hz, 2H), 7.47–7.40
(
m, 2H), 7.37 (d, J = 3.7 Hz, 1H), 7.09 (d, J = 8.2 Hz, 2H), 6.90 (s, 2H), 6.86 (d, J = 3.7 Hz,
H), 4.06 (dd, J = 9.1, 6.3 Hz, 1H), 3.67 (dd, J = 14.5, 9.1 Hz, 1H), 3.44 (dd, J = 13.7, 6.2
Hz, 2H), 3.25 (dd, J = 14.5, 5.7 Hz, 1H), 2.66–2.58 (m, 2H), 2.38 (s, 3H), 2.25 (s, 3H), 1.90
1
-
-
(
s, 6H). LRMS (ESI, m/z): 699.2 [M-H] . HRMS (ESI, m/z): calcd for C38
H
36ClN
2
O
5
S
2
,
-
6
99.1760 [M-H] ; found 699.1765, purity: 98.9%.
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2
2
.2.37
-(5-{2-(4'-t-butyl-bipheny-4-yl)-3-oxo-3-[(2',4',6'-trimethyl-bipheny-4-yl)amino]-propyl
}
thiophene-2-carboxamido)ethanesulfonic acid (14m)
Compound 14m was prepared according to the procedure for the preparation of compound
o
1
1
4c. mp 165-168 C. H NMR (400 MHz, DMSO-d
6
) δ 10.32 (s, 1H), 8.39 (t, J = 5.5 Hz, 1H),
7
.63 (dd, J = 8.4, 4.5 Hz, 4H), 7.56 (dd, J = 11.7, 8.4 Hz, 4H), 7.46 (d, J = 8.4 Hz, 2H), 7.41
(d, J = 3.7 Hz, 1H), 7.00 (d, J = 8.5 Hz, 2H), 6.92–6.83 (m, 3H), 4.10 (dd, J = 11.8, 6.6 Hz,
1
H), 3.72–3.58 (m, 1H), 3.44 (dd, J = 14.0, 6.3 Hz, 2H), 3.25 (dd, J = 14.6, 5.9 Hz, 1H),
.66–2.59 (m, 2H), 2.24 (s, 3H), 1.90 (s, 6H), 1.31 (s, 9H). LRMS (ESI, m/z): 707.2 [M-H] .
-
2
-
-
43 2 5 2
HRMS (ESI, m/z): calcd for C41H N O S , 707.2619 [M-H] ; found 707.2627, purity:
9
5.2%.
2
2
.2.38
-(5-{2-(4'-methyl-bipheny-4-yl)-3-oxo-3-[(2',4',6'-trimethyl-bipheny-4-yl)amino]propyl
}
thiophene-2-carboxamido)ethanesulfonic acid (14n)
Compound 14n was prepared according to the procedure for the preparation of compound
o
1
1
4c. mp 180-182 C. H NMR (400 MHz, DMSO-d
6
) δ 10.29 (s, 1H), 8.38 (t, J = 5.4 Hz, 1H),
7
.65–7.59 (m, 4H), 7.54 (dd, J = 8.2, 2.6 Hz, 4H), 7.40 (d, J = 3.7 Hz, 1H), 7.25 (d, J = 8.0
Hz, 2H), 7.00 (d, J = 8.5 Hz, 2H), 6.94–6.86 (m, 3H), 4.08 (d, J = 6.2 Hz, 1H), 3.67 (dd, J =
1
4.9, 9.1 Hz, 1H), 3.44 (dd, J = 13.9, 6.2 Hz, 2H), 3.27–3.22 (m, 1H), 2.68–2.59 (m, 2H),
-
2
.33 (s, 3H), 2.23 (s, 3H), 1.90 (s, 6H). LRMS (ESI, m/z): 665.2 [M-H] . HRMS (ESI, m/z):
-
-
37 2 5 2
calcd for C38H N O S , 665.2149 [M-H] ; found 665.2155, purity: 96.3%.
This article is protected by copyright. All rights reserved.

2
2
.2.39
-(5-{2-(4'-methoxy-bipheny-4-yl)-3-oxo-3-[(2',4',6'-trimethyl-bipheny-4-yl)amino]prop
yl}thiophene-2-carboxamido)ethanesulfonic acid (14o)
Compound 14o was prepared according to the procedure for the preparation of compound
o
1
1
4c. mp 179-180 C. H NMR (400 MHz, DMSO-d
15.6, 8.2 Hz, 6H), 7.52 (d, J = 8.2 Hz, 2H), 7.40 (d, J = 3.7 Hz, 1H), 7.00 (dd, J = 8.6, 2.9
Hz, 4H), 6.92–6.84 (m, 3H), 4.07 (d, J = 6.9 Hz, 1H), 3.79 (s, 3H), 3.66 (dd, J = 14.4, 9.2 Hz,
6
) δ 10.27 (s, 1H), 8.36 (s, 1H), 7.61 (dd, J
=
1
H), 3.44 (dd, J = 13.3, 6.4 Hz, 2H), 3.25 (dd, J = 14.7, 6.0 Hz, 1H), 2.63 (t, J = 7.2 Hz, 2H),
-
2
.24 (s, 3H), 1.90 (s, 6H). LRMS (ESI, m/z): 681.3 [M-H] . HRMS (ESI, m/z): calcd for
-
-
C
38 37 2 6 2
H N O S , 681.2099 [M-H] ; found 681.2105, purity: 95.8%.
2
2
.3 Biological evaluation method
.3.1 Construction of GCGR vector
The wild-type human GCGR was cloned into the EcoRI and HindIII sites of the
pcDNA3.1/V5-His-TOPO vector (Invitrogen, Carlsbad, CA, USA). Sequences of receptor
clones were confirmed by sequencing with ABI 3730xl DNA Analyzer (Applied Biosystems,
Foster City, CA, USA).
2
.3.2 GCGR binding assay
According to the previously reported method, cells were harvested 24 h after
[
9]
transfections with GCGR and incubated with blocking buffer (F12 supplemented with 33 mM
HEPES and 0.1% bovine serum albumin (BSA), pH 7.4) for 2 h. Then the binding assay were
performed at room temperature for 3 h by incubating the cells with constant concentration of
1
25
[
I]-glucagon (40 pM, PerkinElmer, Boston, MA, USA) and different concentrations of
unlabeled peptides (glucagon, 3.57 pM-1 μM) or compounds (1.28 nM - 100 μM). After cells
were washed by ice-cold PBS for three times, they were lysed and counted for radioactivity
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(counts per minute, CPM) in a scintillation counter (MicroBeta Plate Counter, PerkinElmer)
using a scintillation cocktail (OptiPhase SuperMix, PerkinElmer).
2
.3.3 cAMP assays
[
27]
Based on the manufacturer’s instructions and the previously published method,
cAMP accumulation was measured using HTRF-cAMP dynamic kit (Cisbio International,
Gif sur Yvette Cedex, France). Firstly, HEK293T cells were transfected with GCGR. Then,
cells were transferred to 384-well plates at a density of 3,000 cells per well and incubated for
o
a further 24 h at 37 C. After cells were incubated for 30 min in assay buffer (DMEM, 1 mM
o
3
-isobutyl-1-methylxanthine) with different concentration of compounds at 37 C, lysis buffer
containing HTRF reagents was added to stop the reactions. Then, plates were incubated for
0 min at room temperature, and time-resolved FRET signals were measured after excitation
at 620 nm and 650 nm by EnVision (PerkinElmer).
6
2
.4 Molecular docking studies
Docking modes was generated by the Glide program from Schrӧdinger Suite. The
[28]
receptor protein was obtained from the Protein Data Bank (PDB ID: 5EE7), which was
reported to bind with MK-0893, and processed by removing solvents and unwanted
[
29]
molecules.
The receptor grid was sized to 15 Å in each direction and MK-0893 was
selected as the reference. The low energy 3-dementional structures of all compounds were
generated by OPLS2001 force field under its default parameters and were refined by LigPrep.
[
30]
Docking studies were carried out using Glide in standard precision mode, with up to 40
conformers saved per molecule. According to assessment of the Glide score, the top scoring
conformer for each compound was exported to a Maestro-formatted output file.
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3
3
Results and Discussion
.1 Chemistry
The synthesis route to thiophene-containing GCGR antagonists (14a-14o) is shown in
Scheme 1. It mainly consisted of three fragments. The first fragment methyl 5-(bromomethyl)
thiophene-2-carboxylate 4 was prepared by esterification and bromination of commercially
available compound 2, and esterification of 4-bromophenyl acetic acid 5 afforded the second
fragment 6. Then, benzeneacetyl chloride 8 was generated by alkylation of compound 6 with
compound 4 followed by hydrolysis and chloroformylation. Compound 9 experienced a
classical Suzuki coupling reaction with various boric acids 10 to provide the third fragment
1
1. Suzuki coupling reaction of amide 12 with R
1 2
-B(OH) , hydrolysis, and condensation gave
the target compounds 14a-14o.
3
.2 Biological evaluation
Compounds 14a-14o were evaluated by GCGR binding and cAMP functional assays in
vitro, using compound 1 as the positive control. As shown in Table 1, the preliminary
structure and activity relationship (SAR) at the hydrophobic pocket was firstly investigated.
The effects of regiochemical substitution and electronics on the phenyl ring (R
1
) were
explored. Compounds 14a (R = H) and 14b (R = Br) exhibited similar GCGR binding
1
1
activities (IC50 = 38.4 μM and 39.9 μM, respectively), while 4-Br substituted derivate 14b
demonstrated more potent than 14a in cAMP functional assay. Then, various substituents on
the phenyl group were investigated. Compound 14c containing 4-methoxyphenyl improved
GCGR binding activity (IC50 = 6.9 μM), which was similar to the positive control compound
1
(IC50 = 6.5 μM). In addition, introducing electron-withdrawing group, compounds 14d-14f
displayed potent GCGR binding affinity and cAMP accumulation effects, with IC50 values of
2.9 μM, 16.3 μM, and 6.1 μM for GCGR binding, respectively. Among these derivatives,
compound 14f showed better GCGR binding activity and cAMP activity (IC50 = 6.1 μM and
.4 μM, respectively) than compound 1. Introducing tert-butylphenyl substitute on the phenyl
2
4
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group, the compound 14h displayed good GCGR binding affinity (IC50 = 4.4 μM) with good
cAMP activity (IC50 = 13.4 μM). It was shown that the introduction of bulky groups might
improve receptor binding affinity with GCGR. To investigate the importance of the R
2
group
of these compounds, compounds 14i-14o were designed and synthesized. Replacement of
2
2
-methyl-4-chlorophenyl group (R
2
) in compound 14h to 3-methyl-4-chlorophenyl and
,4,6-trimethylphenyl group, compounds 14i-14o led to sharply reduced GCGR binding
affinity and cAMP functional activity.
3
.3 Molecular docking of compounds 1, 14f, and 14 h with GCGR
Figure 5 displayed the putative binding modes of compounds 1, 14f, and 14h with
GCGR. The binding mode of positive control compound 1 indicates that the
tert-butylbiphenyl moiety formed hydrophobic interactions with residue Phe345, and the
4
-chloro-2-methylbiphenyl moiety formed hydrophobic interactions with residues Pro356,
Leu357, and Val360. In addition, 2-aminoethanesulfurous acid moiety occupied in the polar
TM6-TM7 cleft, which was consisted of residues Arg346, Lys349, Ser350, and Lys405
(Figure 5A).
Overlay view of compounds 1, 14f, and 14h suggests that these three compounds
occupied the allosteric site and with similar orientation (Figure 5B). Moreover, the polar and
nonpolar moieties of compounds 14f and 14h extended into the polar cleft and hydrophobic
pocket, respectively.
Compared with the tert-butylbiphenyl moiety of compound 1, the
4
-trifluoromethoxybiphenyl moiety of compound 14f (Figure 5C) and the t-butylbiphenyl
moiety of compound 14h (Figure 5D) formed more hydrophobic interactions with residues
Leu329 and Leu352, and the thiophene ring formed extra hydrophobic interaction with
residue Leu399. Furthermore, within the polar TM6-TM7 cleft, amide group of compounds
1
4f and 14h could form a hydrogen bond with residues Ser350 and Lys349, respectively; the
sulfonic acid group formed two hydrogen bonds with residues Arg346 and Lys349, which
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were not observed in compound 1. Clearly, the above distinctive interactions imply that
introduction of thiophene ring could be the main reason for the improvement of GCGR
binding affinity.
FIGURE 5. (A) Binding mode of compound 1. (B) Superimposition of compounds 14f (yellow), 14h
(
green) and 1 (purple) with glucagon receptor (GCGR). (C) Binding mode of compound 14f. (D)
Binding mode of compound 14h. All figures were prepared using PyMol (http://www.pymol.org).
4
CONCLUSIONS
In summary, we discovered a novel series of thiophene-containing derivatives as GCGR
antagonists. SAR studies of this series of compounds focused on the two parts of phenyl
rings, among them, compounds 14f and 14h showed better binding activities than lead
compound 1 with IC50 values of 6.1 μM and 4.4 μM, respectively. Moreover, the binding
modes of these three compounds were built to describe their interaction with GCGR.
Acknowledgments
We gratefully acknowledge the financial support from the National Natural Science
Foundation of China (21632008, 81620108027, 21672231, and 21472209), the Major Project
of Chinese National Programs for Fundamental Research and Development
(2015CB910304), SA-SIBS scholarship program, Shanghai Science and Technology
Development Fund (15DZ2291600 and 15QA1404400) and the Thousand Talents Program in
China, and the Strategic Priority Research Program of the Chinese Academy of Sciences
(XDA12040201 and XDA12040202).
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Figure legends
FIGURE 1. Structures of several glucagon receptor antagonists.
FIGURE 2. Drugs that contain the thiophene ring.
FIGURE 3. Main metabolic pathways of Rivaroxaban.
FIGURE 4. Molecular design of novel glucagon receptor (GCGR) antagonists.
FIGURE 1S. NMR and HPLC spectrums of compound 14a
FIGURE 2S. NMR and HPLC spectrums of compound 14b
FIGURE 3S. NMR and HPLC spectrums of compound 14c
FIGURE 4S. NMR and HPLC spectrums of compound 14d
FIGURE 5S. NMR and HPLC spectrums of compound 14e
FIGURE 6S. NMR and HPLC spectrums of compound 14f
FIGURE 7S. NMR and HPLC spectrums of compound 14g
FIGURE 8S. NMR and HPLC spectrums of compound 14h
FIGURE 9S. NMR and HPLC spectrums of compound 14i
FIGURE 10S. NMR and HPLC spectrums of compound 14j
FIGURE 11S. NMR and HPLC spectrums of compound 14k
FIGURE 12S. NMR and HPLC spectrums of compound 14l
FIGURE 13S. NMR and HPLC spectrums of compound 14m
FIGURE 14S. NMR and HPLC spectrums of compound 14n
FIGURE 15S. NMR and HPLC spectrums of compound 14o
1
FIGURE 16S. H NMR spectra for intermediates.
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Conflict of Interest statement
The authors declare that they have no conflict of interest
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8. Glide version 5.7; Schrödinger, LLC, New York, NY, USA, available at:
http://www.schrodinger.com.
9. Schrödinger Suite 2011 Schrödinger Suite; Epik version 2.2, Schrödinger, LLC, New York, NY,
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3
0. LigPrep, version 2.5, Schrödinger, LLC, New York, NY, 2011.
Scheme 1. Reagents and conditions: a) SOCl
Boc
2
, MeOH, reflux; b) NBS, BPO, CHCl
3
, reflux; c)
, DCM, r.t.; g)
-B(OH)2,
2
O, r.t.; k) Taurine, HOBt,
o
2
O, DMAP, t-BuOH, r.t.; d) n-BuLi, THF, -78 C; e) TFA, DCM, r.t.; f) CO
2
Cl
2
o
PdCl
PdCl
2
2
(P(o-tolyl)
3
3
)
2
2
, Na
2
CO
3
2 1
, DME/EtOH/H O, 125 C; h) DIPEA, DCM, r.t.; i) R
o
(P(o-tolyl)
)
, DIPEA, DME, 85 C; j) NaOH, 1,4-dioxane/MeOH/H
EDCI, DIPEA, r.t..
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Table 1. In vitro glucagon receptor (GCGR) binding and cAMP functional activities of
compounds 14a-14o.
a
a
GCGR Binding
GCGR cAMP
Compd.
R
1
R
2
IC50 (μM)
IC50 (μM)
b
1
4a
4b
4c
4d
4e
4f
H
38.4 ± 13.2
39.9 ± 8.3
6.9 ± 5.6
NA
1
1
1
1
1
1
Br
25.8 ± 2.1
24.6 ± 3.1
28.0 ± 8.4
7.1±1.0
22.9 ± 10.9
16.3 ± 6.1
6.1 ± 1.8
4.4 ± 0.6
13.5 ± 2.2
4g
115.7 ± 69.1
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