Synthesis and anti-diabetic activity of novel biphenylsulfonamides as glucagon receptor antagonists

Article abstract of DOI:10.1111/cbdd.13928

Type 2 diabetes is characterized by chronic hyperglycemia. Insulin, a hormone secreted from pancreatic β-cells, decreases blood glucose levels, and glucagon, a hormone secreted from pancreatic α-cells, increases blood glucose levels by counterregulation of insulin through stimulation of hepatic glucose production. In diabetic patients, dysregulation of glucagon secretion contributes to hyperglycemia. Thus, inhibition of the glucagon receptor is one strategy for the treatment of hyperglycemia in type 2 diabetes. In this paper, we report a series of biphenylsulfonamide derivatives that were designed, synthesized, and then evaluated by cAMP and hepatic glucose production assays as glucagon receptor antagonists. Of these, compound 7aB-3 decreased glucagon-induced cAMP production and glucagon-induced glucose production in the in vitro assays. Glucagon challenge tests and glucose tolerance tests showed that compound 7aB-3 significantly inhibited glucagon-induced glucose increases and improved glucose tolerance. These results suggest that compound 7aB-3 has therapeutic potential for the treatment of type 2 diabetes.

Full text of DOI:10.1111/cbdd.13928

Received: 29 April 2021
DOI: 10.1111/cbdd.13928
Revised: 24 June 2021
Accepted: 10 July 2021
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R E S E A R C H A R T I C L E
Synthesis and anti-diabetic activity of novel biphenylsulfonamides
as glucagon receptor antagonists
Chang-Yong Lee1
Han-Joo Maeng1
Hojung Choi1,2
Eun-Young Park3
Hee-Sook Jun1,2,5
Thi-Thao-Linh Nguyen1
Dongyun Shin1
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Kyoung Mee Lee4
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1College of Pharmacy, Gachon Institute of
Pharmaceutical Science, Gachon University,
Incheon, Korea
Abstract
Type 2 diabetes is characterized by chronic hyperglycemia. Insulin, a hormone
secreted from pancreatic β-cells, decreases blood glucose levels, and glucagon, a
hormone secreted from pancreatic α-cells, increases blood glucose levels by counter-
regulation of insulin through stimulation of hepatic glucose production. In diabetic
patients, dysregulation of glucagon secretion contributes to hyperglycemia. Thus,
inhibition of the glucagon receptor is one strategy for the treatment of hyperglycemia
in type 2 diabetes. In this paper, we report a series of biphenylsulfonamide deriva-
tives that were designed, synthesized, and then evaluated by cAMP and hepatic glu-
cose production assays as glucagon receptor antagonists. Of these, compound 7aB-3
decreased glucagon-induced cAMP production and glucagon-induced glucose pro-
duction in the in vitro assays. Glucagon challenge tests and glucose tolerance tests
showed that compound 7aB-3 significantly inhibited glucagon-induced glucose in-
creases and improved glucose tolerance. These results suggest that compound 7aB-3
has therapeutic potential for the treatment of type 2 diabetes.
2Lee Gil Ya Cancer and Diabetes Institute,
Gachon University, Incheon, Korea
3College of Pharmacy, Mokpo National
University, Muan-gun, Jeollanam-do, Korea
4Kwangdong Pharmaceutical Co., Ltd,
Seoul, Korea
5Gachon Medical Research Institute, Gil
Hospital, Incheon, Korea
Correspondence
Dongyun Shin and Hee-Sook Jun,
College of Pharmacy, Gachon Institute of
Pharmaceutical Science, Gachon University,
Yeonsu-gu, Incheon, Korea.
Emails: dyshin@gachon.ac.kr;
hsjun@gachon.ac.kr
Funding information
Korea Health Industry Development
Institute
K E Y W O R D S
biphenyl, glucagon receptor antagonist, sulfonamide, Type 2 diabetes
1
INTRODUCTION
When glucagon binds to the glucagon receptor (GCGR),
Gsα activation leads to activation of adenylate cyclase, in-
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Diabetes mellitus (DM) is a group of metabolic diseases
caused by chronic hyperglycemia resulting from defects
in insulin action and/or insulin secretion (Kharroubi &
Darwish, 2015). Excess glucose production in the liver and
morbidly increased glucose reabsorption in the kidneys induce
hyperglycemia (DeFronzo, 2009; DeFronzo et al., 2014).
Glucagon, a 29 amino acid peptide hormone secreted
from pancreatic α cells (Authier & Desbuquois, 2008), in-
creases plasma glucose levels (Christensen et al. 2011).
creases in intracellular cAMP levels, and subsequent activa-
tion of protein kinase A (PKA) (Bagger et al., 2011; Jiang
& Zhang, 2003; Li & Zhuo, 2013; Quesada et al., 2008).
Activation of glucagon-signaling results in increased gly-
cogenolysis and gluconeogenesis, which are responsible for
increased hepatic glucose production (Qureshi et al., 2004).
Small molecules involved in GCGR antagonism are rec-
ognized as novel approaches for better glycemic control in
the diabetic state by inhibiting glucagon-induced hepatic
Chang-Yong Lee and Hojung Choi are contributed equally to this work.
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium,
provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
© 2021 The Authors. Chemical Biology & Drug Design published by John Wiley & Sons Ltd.
Chem Biol Drug Des. 2021;00:1–18.
wileyonlinelibrary.com/journal/cbdd
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glucose output (Sammons & Lee, 2015). Recently, many
pharmaceutical companies have designed a number of
GCGR antagonists and evaluated their biological activities.
Representative compounds are BAY 27–9955 (Petersen
& Sullivan, 2001), MK-0893 (Xiong et al., 2012), PF-
06291874 (Bergman et al., 2017), LY-2409021 (Guzman
et al., 2017; Kelly et al., 2015), and LGD-6972 (Vajda
et al., 2012, 2017). All of these compounds have phe-
nyl ether and β-alanine benzamide moieties in common.
Moreover, the crystal structure of GCGR complexed with
MK-0893 revealed the detailed binding mode of the β-
alanine benzamide moiety and other aryl groups (Figure 1;
Jazayeri et al., 2016).
Since Novo-Nordisk first discovered β-alanine ben-
zamide as an important pharmacophore of GCGR antago-
nism (Kodra et al., 2008; Lau et al., 2007; Ling et al. 2000;
Madsen et al., 2009), other research groups have adopted
this moiety or its sulfonic acid congener (e.g., LGD-6972)
as an essential structure and performed lead optimization
by modifying the hydrophobic region to generate novel
antagonists. By analyzing known potent antagonists, we
determined that their hydrophobic parts have a rotatable
carbon (Lee et al., 2014) and most of the reported com-
pounds contain a stereogenic carbon center at the benzylic
position, which could be disadvantageous during the drug
discovery process in terms of eutomer purity and cost of
synthesis. Considering the structural features of known
compounds and novelty of our designed antagonists, we
predicted that biphenylsulfonamides would be strong
candidates, as shown in Figure 2. In this planned struc-
ture, Novo-Nordisk's β-alanine benzamide was preserved
and a hydrophobic biphenyl group was introduced at the
para position of the benzamide via sulfonamide bond.
This compound is conformationally more rigid than the
carbon-linked compounds and has no stereogenic carbon
(Figure 2).
2
METHODS AND MATERIALS
Chemistry
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2.1
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The reaction solvents used for the synthesis were dried over
appropriate drying agents or purchased as anhydrous grade
or reagent grade. The reactions were performed under an N2
atmosphere or under designated conditions. Purification of
products was carried out using flash column chromatography
with silica gel (40–63 μm) purchased from ZEOprep or using
medium-performance liquid chromatography (Isolera™
by Biotage (Uppsala, Sweden)). Melting points were de-
termined on an OptiMelt-automated melting point system
(Stanford Research Systems) and were uncorrected. Proton
and carbon 13 magnetic resonance spectra (1H NMR and 13
C
NMR) were recorded on a Bruker 600 (1H NMR at 600 MHz
and 13C NMR at 150 MHz) spectrometer with solvent reso-
nance as the internal standard (1H NMR: CDCl3 at 7.26 ppm,
DMSO-d6 at 2.5 ppm; 13C NMR: CDCl3 at 77.0 ppm,
DMSO-d6 at 39.5 ppm). Mass spectra were obtained using a
Waters 3100 Mass Detector spectrometer using electrospray
ionization (Waters Technologies, Accurate–Mass QTOF LC-
MS, 1200 series LC with dual spray ESI source). Optical ro-
tation was determined on the Optical Activity Ltd. AA-10R
automatic polarimeter in CH2Cl2.
2.1.1
Ethyl 3-(4-nitrobenzamido)propanoate
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(2)
To a stirred solution of β-alanine ethyl ester hydrochloride
(4.1 g, 26.9 mmol) and TEA (8.3 ml, 59.3 mmol) in CHCl3
(135 ml) at 0℃, 4-nitrobenzoyl chloride (5 g, 26.9 mmol) was
successively added, and the mixture was stirred at room tem-
perature for 30 min. The reaction mixture was diluted with
EtOAc. The organic layer was washed with H2O and brine
FIGURE 1 GCGR antagonists

LEE Et aL.
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was stirred overnight under a hydrogen atmosphere at room
temperature. The reaction mixture was filtered and washed
with MeOH. After filtration, the solvent was removed under
reduced pressure and the residue was purified by SiO2 flash
column chromatography.
Ethyl 3-(4-(propylamino)benzamido)propanoate (4a)
1
Yield: 68%, a colorless oil. H NMR (600 MHz, CDCl3)
δ (ppm): 7.61 (d, J = 9 Hz, 2H), 6.71 (bs, 1H), 6.54 (d,
J = 9 Hz, 2H), 4.16 (q, J = 7.2 Hz, 2H), 4.11 (bs, 1H),
3.69 (q, J = 6 Hz, 2H), 3.10 (t, J = 7.2 Hz, 2H), 2.62 (t,
J = 6 Hz, 2H), 1.67–1.61 (m, 2H), 1.26 (t, J = 7.2 Hz, 3H),
0.99 (t, J = 7.2 Hz, 3H); 13C NMR (150 MHz, CDCl3)
δ (ppm): 173.0, 167.2, 151.1, 128.6, 122.1, 111.6, 60.7,
45.2, 35.1, 34.2, 22.5, 14.2, 11.6; HRMS (ESI-TOF) m/z
FIGURE 2 Design of biphenylsulfonamide as novel GCGR
antagonist
and then dried over MgSO4. After filtration, the solvent was
removed under reduced pressure and the residue was puri-
fied by SiO2 flash column chromatography. The synthesis
yielded 6.8 g (94%) of 3-(4-nitrobenzamido)propanoic acid
as a white solid. Mp: 86–87℃; 1H NMR (600 MHz, CDCl3)
δ (ppm): 8.26 (d, J = 8.4 Hz, 2H), 7.96 (d, J = 8.4 Hz, 2H),
7.37 (s, 1H), 4.17 (q, J = 7.2 Hz, 2H), 3.75 (q, J = 5.4 Hz,
2H), 2.68 (t, J = 6 Hz, 2H), 1.28 (t, J = 6.6 Hz, 3H); 13C
NMR (150 MHz, CDCl3) δ (ppm): 172.8, 165.4, 149.5,
139.9, 128.2, 123.7, 61.0, 35.7, 33.6, 14.1; HRMS (ESI-
[M + H]+ calculated for C15H23N2O3 279.1530, found
+
279.1541.
Ethyl 3-(4-(butylamino)benzamido)propanoate (4b)
1
Yield: 73%, a colorless oil. H NMR (600 MHz, CDCl3)
δ (ppm): 7.61 (d, J = 8.4 Hz, 2H), 6.66 (bs, 1H), 6.55 (d,
J = 9 Hz, 2H), 4.16 (q, J = 7.2 Hz, 2H), 4.00 (bs, 1H), 3.69 (q,
6.6 Hz, 2H), 3.14 (t, J = 7.2 Hz, 2H), 2.62 (t, J = 6 Hz, 2H),
1.63–1.58 (m, 2H), 1.46–1.40 (m, 2H), 1.27 (t, J = 6.6 Hz,
3H), 0.96 (t, J = 7.2 Hz, 3H); 13C NMR (150 MHz, CDCl3)
δ (ppm): 173.1, 167.2, 151.1, 128.6, 122.2, 111.6, 60.7,
43.2, 35.1, 34.2, 31.4, 20.2, 14.2, 13.9; HRMS (ESI-TOF)
TOF) m/z [M + H]+ calculated for C12H15N2O5 267.0903,
+
found 267.0913.
2.1.2
Ethyl 3-(4-aminobenzamido)
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+
propanoate (3)
m/z [M + H]+ calculated for C16H25N2O3 293.1787, found
293.1772.
To a stirred solution of compound 2 (6.15 g, 23.1 mmol)
in MeOH (75 ml), palladium (10% on carbon, 250 mg,
0.231 mmol) was added, and the mixture was stirred for 5 hr
under a hydrogen atmosphere at room temperature. The re-
action mixture was filtered and washed with MeOH. After
filtration, the solvent was removed under reduced pressure
and the residue was purified by SiO2 flash column chro-
matography. The synthesis yielded 5.3 g (97%) of ethyl
3-(4-aminobenzamido)propanoate as a colorless oil. 1H
NMR (600 MHz, CDCl3) δ (ppm): 7.59 (d, J = 8.4 Hz, 2H),
6.72 (s, 1H), 6.64 (d, J = 8.4 Hz, 2H), 4.15 (q, J = 7.2 Hz,
2H), 3.99 (bs, 2H), 3.68 (q, J = 6.6 Hz, 2H), 2.61 (t, J = 6 Hz,
2H), 1.26 (t, J = 7.2 Hz, 3H); 13C NMR (150 MHz, CDCl3)
δ (ppm): 173.1, 167.1, 149.6, 128.7, 123.9, 114.1, 60.7, 35.2,
34.1, 14.2; HRMS (ESI-TOF) m/z [M + H]+ calculated for
C12H17N2O3+ 237.1161, found 237.1153.
Ethyl 3-(4-((3,3,3-trifluoropropyl)amino)benzamido)
propanoate (4c)
1
Yield: 75%, a colorless oil. H NMR (600 MHz, CDCl3)
δ (ppm): 7.64 (d, J = 8.4 Hz, 2H), 6.69 (bs, 1H), 6.58 (d,
J = 8.4 Hz, 2H), 4.17 (q, J = 7.2 Hz, 2H), 3.70 (q, J = 6 Hz,
2H), 3.49 (q, J = 6.6 Hz, 2H), 2.63 (t, J = 6 Hz, 2H), 2.46–
2.39 (m, 2H), 1.27 (t, J = 7.2 Hz, 3H); 13C NMR (150 MHz,
CDCl3) δ (ppm): 173.1, 166.9, 149.6, 128.8, 123.5, 111.9,
60.7, 36.7, 35.1, 34.1, 33.6, 33.4, 33.2, 33.1, 14.2; HRMS
(ESI-TOF) m/z [M + H]+ calculated for C15H20F3N2O3
+
333.1348, found 333.1350.
2.1.4
General procedure for the
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synthesis of (5)
To a stirred solution of compound 4a (750 mg, 2.69 mmol)
in CH2Cl3 (15 ml) and TEA (0.6 ml, 4.04 mmol) in CH2Cl3
(15 ml) at 0℃, 4-iodobenzenesulfonyl chloride (978 mg,
3.23 mmol) was successively added, and the mixture was
stirred at room temperature overnight. The reaction mixture
was diluted with EtOAc. The organic layer was washed with
H2O and brine and then dried over MgSO4. After filtration,
2.1.3
General procedure for the
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synthesis of (4)
To a stirred solution of compound 3 (2 g, 8.46 mmol) in MeOH
(42 ml), palladium (10% on carbon, 450 mg, 0.846 mmol) and
propanal (0.909 ml, 12.7 mmol) were added, and the mixture

4
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the solvent was removed under reduced pressure and the resi-
due was purified by SiO2 flash column chromatography.
(ESI-TOF) m/z [M + H]+calculated for C23H29BrN2O5S+
524.0981, found 524.0966.
Ethyl 3-(4-((4-iodo-N-propylphenyl)sulfonamido)
Ethyl 3-(4-((4-bromo-3,5-dimethyl-N-(3,3,3-
trifluoropropyl)phenyl)sulfonamido)benzamido)
propanoate (5cB)
benzamido)propanoate (5aA)
1
Yield: 60%, a colorless oil. H NMR (600 MHz, CDCl3) δ
1
(ppm): 7.81 (d, J = 8.4 Hz, 2H), 7.74 (d, J = 9 Hz, 2H),
7.26 (d, J = 8.4 Hz, 2H), 7.13 (d, J = 9 Hz, 2H), 7.00 (t,
J = 6 Hz, 1H), 4.18 (q, J = 7.2 Hz, 2H), 3.72 (q, J = 6 Hz,
2H), 3.51 (t, J = 6.6 Hz, 2H), 2.65 (t, J = 6 Hz, 2H), 1.45–
1.69 (m, 2H), 1.28 (t, J = 7.2 Hz, 3H), 0.89 (t, J = 7.2 Hz,
3H); 13C NMR (150 MHz, CDCl3) δ (ppm): 172.8, 166.4,
141.6, 138.2, 137.6, 133.8, 128.9, 128.5, 127.6, 100.3, 60.8,
60.4, 52.0, 35.4, 33.8, 21.4, 21.1, 14.2, 10.9; HRMS (ESI-
TOF) m/z [M + H]+ calculated for C21H26IN2O5S+ 545.0529,
found 545.0512.
Yield: 64%, a colorless oil. H NMR (600MHz, CDCl3)
δ(ppm): 7.84 (d, J = 8.9 Hz, 1H), 7.76 (d, J = 8.6 Hz, 1H),
7.48 (d, J = 8.9 Hz, 1H), 7.33 (s, 1H), 7.25 (s, 1H), 7.17 (d,
J = 8.7 Hz, 1H), 6.92–6.84 (m, 1H), 4.19 (qd, J = 7.1, 1.0 Hz,
2H), 3.75 (dd, J = 15.9, 9.4 Hz, 2H), 2.66 (dd, J = 11.6,
5.6 Hz, 2H), 2.52–2.37 (m, 6H), 1.33–1.24 (m, 3H); 13C NMR
(150MHz, CDCl3) δ(ppm): 128.5, 128.1, 126.6, 121.2, 115.7,
60.9, 33.8, 31.8, 24.0, 14.2; HRMS (ESI-TOF) m/z [M + H]+
calculated for C23H26BrF3N2O5S+ 578.0698, found 578.0686.
Ethyl 3-(4-((4-iodo-N-(3,3,3-trifluoropropyl)phenyl)
2.1.5
General procedure for the
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sulfonamido)benzamido)propanoate (5cA)
synthesis of (6)
1
Yield: 63%, a colorless oil. H NMR (600 MHz, CDCl3) δ
(ppm): 7.84 (d, J = 8.4 Hz, 2H), 7.75 (d, J = 9 Hz, 2H), 7.27–
7.25 (m, 3H), 7.14 (d, J = 8.4 Hz, 2H), 6.89 (t, J = 5.4 Hz,
1H), 4.19 (q, J = 7.2 Hz, 2H), 3.79 (t, J = 7.2 Hz, 2H), 3.73
(q, J = 6 Hz, 2H) 2.65 (t, J = 6 Hz, 2H), 2.40–2.33 (m, 2H),
1.29 (t, J = 7.2 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ
(ppm): 173.0, 166.1, 141.2, 138.4, 136.8, 134.5, 128.9, 128.5,
128.2, 101.0, 61.0, 44.4, 35.4, 33.8, 33.7, 33.5, 14.2; HRMS
(ESI-TOF) m/z [M + H]+ calculated for C21H23F3IN2O5S+
598.0246, found 598.0250.
Compound 5a (50 mg, 0.092 mmol), boronic acid (17 mg,
0.138 mmol), and 20% K2CO3 (2.2 ml) were dissolved in an-
hydrous THF (2.2 ml). The mixture was purged with nitrogen
for 5 min. Pd(PPh3)4 (5 mg, 4.6 µmol) was added, and the
mixture was stirred at 80℃ overnight. The mixture was parti-
tioned between EtOAc and water, and the aqueous layer was
extracted with EtOAc. The pooled organic layer was washed
with H2O and brine and then dried over MgSO4. After filtra-
tion, the solvent was removed under reduced pressure and the
residue was purified by SiO2 flash column chromatography.
Ethyl 3-(4-((4-bromo-3,5-dimethyl-N-propylphenyl)
sulfonamido)benzamido)propanoate (5aB)
Ethyl 3-(4-(N-propyl-[1,1′-biphenyl]-4-sulfonamido)
benzamido)propanoate (6aA-1)
1
Yield: 58%, a colorless oil. H NMR (600 MHz, CDCl3)
1
δ (ppm): 7.73 (d, J = 8.4 Hz, 2H), 7.25 (s, 2H), 7.16 (d,
J = 9 Hz, 2H), 6.88 (t, J = 6Hz, 1H), 4.18 (q, J = 6.6 Hz,
2H), 3.73 (q, J = 5.4 Hz, 2H), 3.51 (t, J = 7.2 Hz, 2H), 2.65
(t, J = 5.4 Hz, 2H), 2.42 (s, 6H), 1.46–1.40 (m, 2H), 1.29 (t,
J = 7.2 Hz, 3H), 0.89 (t, J = 7.2 Hz, 3H); 13C NMR (150 MHz,
CDCl3) δ (ppm): 173.0, 166.4, 141.9, 139.6, 136.2, 133.7,
132.9, 128.6, 127.7, 126.6, 60.9, 52.0, 35.4, 33.8, 24.0, 21.4,
14.2, 10.9; HRMS (ESI-TOF) m/z [M + H]+ calculated for
C23H30BrN2O5S+ 525.0981, found 525.0968.
Yield: 73%, a colorless oil. H NMR (600 MHz, CDCl3) δ
(ppm): 7.73 (d, J = 8.4, 2H), 7.67 (d, J = 8.4 Hz, 2H), 7.63–
7.61 (m, 4H), 7.48 (t, J = 7.4 Hz, 2H), 7.42 (t, J = 7.4 1H),
7.18 (d, J = 8.4 Hz, 2H), 6.87 (t, J = 5.4 Hz, 1H), 4.18 (q,
J = 7.2 Hz, 2H), 3.73 (q, J = 6 Hz, 2H), 3.56 (t, J = 7.2 Hz,
2H), 2.65 (t, J = 6 Hz, 2H), 1.48–1.42 (m, 2H), 1.28 (t,
J = 7.2 Hz, 3H), 0.91 (t, J = 7.2 Hz, 3H); 13C NMR (150 MHz,
CDCl3) δ (ppm): 173.0, 166.4, 141.9, 139.6, 136.2, 133.7,
132.9, 128.6, 127.7, 126.6, 60.9, 52.0, 35.4, 33.8, 24.0, 21.4,
14.2, 10.9; HRMS (ESI-TOF) m/z [M + H]+ calculated for
C27H31N2O5S+ 495.1875, found 495.1887.
Ethyl 3-(4-((4-bromo-N-butyl-3,5-dimethylphenyl)
sulfonamido)benzamido)propanoate (5bB)
Yield: 67%, a colorless oil. 1H NMR (600MHz, CDCl3)
δ(ppm): 7.73 (d, J = 8.6 Hz, 2H), 7.25 (s, 2H), 7.16 (d,
J = 8.6 Hz, 2H), 6.88 (t, J = 5.7 Hz, 1H), 4.18 (q, J = 7.1 Hz,
2H), 3.73 (dd, J = 11.8, 6.0 Hz, 2H), 3.53–3.48 (m, 2H),
2.67–2.63 (m, 2H), 2.42 (s, 6H), 1.46–1.39 (m, 2H), 1.29 (t,
J = 7.2 Hz, 3H), 0.89 (t, J = 7.4 Hz, 3H); 13C NMR (150MHz,
CDCl3) δ(ppm): 173.0, 142.0, 139.6, 133.7, 128.6, 127.7,
126.6, 60.9, 52.0, 35.4, 33.8, 24.1, 21.4, 14.2, 11.0; HRMS
Ethyl 3-(4-((4′-chloro-N-propyl-[1,1′-biphenyl])-4-
sulfonamido)benzamido)propanoate (6aA-2)
1
Yield: 65%, a colorless oil. H NMR (600 MHz, CDCl3) δ
(ppm): 7.72 (d, J = 8.4 Hz, 2H), 7.64–7.61 (m, 4H), 7.54 (d,
J = 8.4 Hz, 2H), 7.45 (d, J = 8.4 Hz, 2H), 7.17 (d, J = 8.4 Hz,
2H), 6.87 (t, J = 5.4 Hz, 1H), 4.18 (q, J = 7.2 Hz, 2H), 3.73
(q, J = 6 Hz, 2H), 3.56 (t, J = 7.2 Hz, 2H), 2.65 (t, J = 6 Hz,
2H), 1.48–1.42 (m, 2H), 1.28 (t, J = 7.2 Hz, 3H), 0.91 (t,

LEE Et aL.
5
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J = 7.2 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ (ppm):
173.0, 129.3, 128.6, 128.2, 127.8, 127.3, 60.9, 52.0, 35.4,
33.8, 21.4, 14.2, 10.9; HRMS (ESI-TOF) m/z [M + H]+ cal-
culated for C27H30ClN2O5S+ 529.1486, found 529.1466.
J = 9 Hz, 1H), 7.19 (d, J = 8.4 Hz, 2H), 6.88 (t, J = 6 Hz,
1H), 4.18 (q, J = 7.2 Hz, 2H), 3.73 (q, J = 6 Hz, 2H), 3.57 (t,
J = 6.6 Hz, 2H), 2.65 (t, J = 8.4 Hz, 2H), 1.49–1.43 (m, 2H),
1.28 (t, J = 7.2 Hz, 3H), 0.91 (t, J = 7.8 Hz, 3H); 13C NMR
(150 MHz, CDCl3) δ (ppm): 173.0, 166.4, 141.9, 138.7,
137.9, 133.8, 129.5, 128.6, 128.1, 127.1, 127.8, 117.2, 117.1,
60.9, 52.0, 35.4, 33.8, 21.5, 14.2, 11.0; HRMS (ESI-TOF)
m/z [M + H]+ calculated for C28H29F4N2O5S+ 581.1655,
found 581.1650.
Ethyl 3-(4-((N-propyl-4′-(trifluoromethyl)-[1,1′-
biphenyl])-4-sulfonamido)benzamido)propanoate
(6aA-3)
1
Yield: 80%, a colorless oil. H NMR (600 MHz, CDCl3)
δ (ppm): 7.73 (m, 6H), 7.67 (q, J = 7.8 Hz, 4H), 7.18 (d,
J = 8.5 Hz, 2H), 6.91 (t, J = 5.8 Hz, 1H), 4.18 (q, J = 7.1 Hz,
2H), 3.73 (q, J = 5.9 Hz, 2H), 3.57 (t, J = 7.1 Hz, 2H), 2.65 (t,
J = 5.9 Hz, 2H), 1.46 (q, J = 7.2 Hz, 2H), 1.28 (t, J = 7.2 Hz,
3H), 0.91 (t, J = 7.4 Hz, 3H); 13C NMR (150 MHz, CDCl3)
δ (ppm): 173.0, 166.4, 144.0, 142.7, 141.9, 137.6, 133.8,
128.6, 128.3, 127.8, 127.7, 126.0, 60.9, 52.0, 35.4, 33.8,
21.5, 14.2, 11.0; HRMS (ESI-TOF) m/z [M + H]+ calculated
for C28H30F3N2O5S+ 563.1749, found 563.1741.
Ethyl 3-(4-((4′-chloro-N-(3,3,3-trifluoropropyl)-[1,1′-
biphenyl])-4-sulfonamido)benzamido)propanoate
(6cA-1)
1
Yield: 66%, a colorless oil. H NMR (600 MHz, CDCl3) δ
(ppm): 7.76 (d, J = 8.6 Hz, 2H), 7.63 (q, J = 9.5 Hz, 4H),
7.54 (d, J = 8.6 Hz, 2H), 7.46 (d, J = 8.6 Hz, 2H), 7.18 (d,
J = 8.5 Hz, 2H), 6.89 (t, J = 5.8 Hz, 1H), 4.18 (q, J = 7.1 Hz,
2H), 3.83 (t, J = 7.6 Hz, 2H), 3.73 (q, J = 5.9 Hz, 2H), 2.65
(t, J = 5.8 Hz, 2H), 2.43–2.35 (m, 2H), 1.29 (t, J = 7.2 Hz,
3H); 13C NMR (150 MHz, CDCl3) δ (ppm): 173.0, 166.2,
144.9, 141.5, 137.4, 136.1, 135.0, 134.4, 129.3, 128.6, 128.5,
128.3, 128.1, 127.5, 60.9, 44.3, 35.4, 33.8, 30.9, 14.2; HRMS
(ESI-TOF) m/z [M + H]+ calculated for C27H27ClF3N2O5S+
583.1203, found 583.1199.
Ethyl 3-(4-((4′-(tert-butyl)-N-propyl-[1,1′-biphenyl])-4-
sulfonamido)benzamido)propanoate (6aA-4)
1
Yield: 60%, a colorless oil. H NMR (600 MHz, CDCl3) δ
(ppm): 7.72 (d, J = 8.4 Hz, 2H), 7.66 (d, J = 9 Hz, 2H),
7.60 (d, J = 9 Hz, 2H), 7.56 (d, J = 8.4 Hz, 2H), 7.50 (d,
J = 8.4 Hz, 2H), 7.18 (d, J = 8.4 Hz, 2H), 6.86 (t, J = 5.4 Hz,
1H), 4.18 (q, J = 7.2 Hz, 2H), 3.73 (q, J = 6 Hz, 2H), 3.56 (t,
J = 7.2 Hz, 2H), 2.65 (t, J = 6 Hz, 2H), 1.47–1.41 (m, 2H),
1.37 (s, 8H), 1.28 (t, J = 6.6 Hz, 3H), 0.90 (t, J = 7.2 Hz, 3H);
13C NMR (150 MHz, CDCl3) δ (ppm): 173.0, 166.5, 145.4,
142.1, 136.2, 133.6, 128.6, 128.1, 127.7, 127.2, 126.9, 126.0,
60.9, 51.9, 35.4, 34.7, 33.9, 31.3, 21.4, 14.2, 11.0; HRMS
(ESI-TOF) m/z [M + H]+ calculated for C31H39N2O5S+
551.2501, found 551.2600.
Ethyl 3-(4-((4′-(trifluoromethyl)-N-(3,3,3-
trifluoropropyl)-[1,1′-biphenyl])-4-sulfonamido)
benzamido)propanoate (6cA-2)
1
Yield: 72%, a colorless oil. H NMR (600 MHz, CDCl3) δ
(ppm): 7.77–7.65 (m, 10H), 7.19 (d, J = 8.6 Hz, 2H), 6.91 (t,
J = 5.8 Hz, 1H), 4.18 (q, J = 7.1 Hz, 2H), 3.85 (t, J = 7.6 Hz,
2H), 3.73 (q, J = 5.9 Hz, 2H), 2.65 (t, J = 5.8 Hz, 2H), 2.44–
2.36 (m, 2H), 1.29 (t, J = 7.2 Hz, 3H); 13C NMR (150 MHz,
CDCl3) δ (ppm): 166.1, 144.6, 142.5, 141.4, 136.8, 134.4,
128.5, 128.4, 128.2, 127.9, 127.7, 126.1, 124.9, 60.9,
60.4, 44.4, 35.4, 33.8, 33.7, 33.5, 21.1, 14.2, 14.2; HRMS
(ESI-TOF) m/z [M + H]+ calculated for C28H27F6N2O5S+
617.1467, found 617.1417.
Ethyl 3-(4-((3′,5′-dichloro-N-propyl-[1,1′-biphenyl])-4-
sulfonamido)benzamido)propanoate (6aA-5)
1
Yield: 71%, a colorless oil. H NMR (600 MHz, CDCl3) δ
(ppm): 7.73 (d, J = 8.4 Hz, 2H), 7.63 (q, J = 9 Hz, 4H),
7.47 (d, J = 1.8 Hz, 2H), 7.42 (t, J = 1.8 Hz, 1H), 7.16 (d,
J = 8.4 Hz, 2H), 6.88 (t, J = 5.4 Hz, 1H), 4.18 (q, J = 7.2 Hz,
2H), 3.73 (q, J = 6 Hz, 2H), 3.56 (t, J = 7.2 Hz, 2H), 2.65
(t, J = 6 Hz, 2H), 1.48–1.42 (m, 2H), 1.29 (t, J = 7.2 Hz,
3H), 0.91 (t, J = 7.2 Hz, 3H); 13C NMR (150 MHz, CDCl3)
δ (ppm): 173.0, 166.4, 142.8, 142.1, 141.8, 137.9, 135.7,
133.8, 128.6, 128.4, 128.3, 127.8, 127.5, 125.9, 60.9, 52.0,
35.4, 33.8, 21.5, 14.2, 11.0; HRMS (ESI-TOF) m/z [M + H]+
calculated for C27H29Cl2N2O5S+ 563.1095, found 563.1088.
Ethyl 3-(4-((4′-(tert-butyl)-N-(3,3,3-trifluoropropyl)-
[1,1′-biphenyl])-4-sulfonamido)benzamido)propanoate
(6cA-3)
1
Yield: 66%, a colorless oil. H NMR (600 MHz, CDCl3)
δ (ppm): 7.75 (d, J = 8.6 Hz, 2H), 7.68 (d, J = 8.6 Hz,
2H), 7.60 (d, J = 8.6 Hz, 2H), 7.56 (d, J = 8.6 Hz, 2H),
7.51 (d, J = 8.6 Hz, 2H), 7.18 (d, J = 8.6 Hz, 2H), 6.88
(t, J = 5.8 Hz, 1H), 4.18 (q, J = 7.1 Hz, 2H), 3.83 (t,
J = 7.6 Hz, 2H), 3.73 (q, J = 5.9 Hz, 2H), 2.65 (t, J = 5.9 Hz,
2H), 2.43–2.35 (m, 2H), 1.28 (t, J = 7.1 Hz, 3H); 13C
NMR (150 MHz, CDCl3) δ (ppm): 128.6, 128.2, 128.1,
127.4, 127.0, 126.1, 60.9, 35.4, 33.8, 31.3, 14.2; HRMS
(ESI-TOF) m/z [M + H]+ calculated for C31H36F3N2O5S+
605.2219, found 605.2111.
Ethyl 3-(4-((2′-fluoro-N-propyl-5′-(trifluoromethyl)-
[1,1′-biphenyl])-4-sulfonamido)benzamido)propanoate
(6aA-6)
1
Yield: 76%, a colorless oil. H NMR (600 MHz, CDCl3)
δ (ppm): 7.75–7.73 (m, 3H), 7.69–7.64 (m, 5H), 7.32 (t,

6
LEE Et aL.
|
Ethyl 3-(4-((N-butyl-4′-chloro-2,6-dimethyl-[1,1′-
biphenyl])-4-sulfonamido)benzamido)propanoate
(6bB-1)
2H), 6.91 (t, J = 5.7 Hz, 1H), 4.18 (q, J = 7.2 Hz, 2H), 3.84 (t,
J = 7.6 Hz, 2H), 3.73 (q, J = 5.9 Hz, 2H), 2.65 (t, J = 5.9 Hz,
2H), 2.42–2.36 (m, 2H), 2.03 (s, 6H), 1.28 (t, J = 7.1 Hz,
3H); 13C NMR (150 MHz, CDCl3) δ (ppm): 173.0, 166.1,
145.8, 137.7, 137.6, 134.2, 133.6, 129.7, 129.1, 128.5, 128.1,
126.3, 60.9, 35.4, 33.8, 21.0, 14.2; HRMS (ESI-TOF) m/z
[M + H]+ calculated for C29H31ClF3N2O5S+ 611.1516, found
611.1415.
1
Yield: 75%, a colorless oil. H NMR (600 MHz, CDCl3) δ
(ppm): 7.75 (d, J = 8.5 Hz, 2H), 7.44 (d, J = 8.3 Hz, 2H),
7.30 (s, 2H), 7.22 (d, J = 8.5 Hz, 2H), 7.06 (d, J = 8.3 Hz,
2H), 6.91 (d, J = 5.5 Hz, 1H), 4.18 (q, J = 7.1 Hz, 2H),
3.73 (q, J = 5.9 Hz, 2H), 3.60 (t, J = 6.9 Hz, 2H), 2.65 (t,
J = 5.8 Hz, 2H), 2.01 (s, 6H), 1.43–1.37 (m, 2H), 1.36–1.31
(m, 2H), 1.28 (t, J = 7.1 Hz, 3H), 0.87 (t, J = 7.3 Hz, 3H);
13C NMR (150 MHz, CDCl3) δ (ppm): 207.0, 173.0, 166.5,
145.2, 142.2, 137.8, 137.3, 136.7, 133.5, 129.8, 129.1, 128.6,
127.7, 126.3, 60.9, 50.9, 50.0, 35.4, 33.8, 30.9, 30.2, 24.0,
20.9, 19.6, 14.2, 13.6, 13.5; HRMS (ESI-TOF) m/z [M + H]+
calculated for C30H36ClN2O5S+ 571.1955, found 571.1910.
Ethyl 3-(4-((2,6-dimethyl-4′-(trifluoromethyl)-N-
(3,3,3-trifluoropropyl)-[1,1′-biphenyl])-4-sulfonamido)
benzamido)propanoate (6cB-2)
1
Yield: 63%, a colorless oil. H NMR (600 MHz, CDCl3) δ
(ppm): 7.80–7.74 (m, 3H), 7.33 (s, 2H), 7.27–7.24 (m, 4H),
7.17 (d, J = 8.5 Hz, 1H), 6.99 (t, J = 6 Hz, 1H), 4.20–4.16 (m,
2H), 3.86 (t, J = 7.6 Hz, 2H), 3.79 (t, J = 7.5 Hz, 1H), 3.73
(q, J = 5.9 Hz, 2H), 2.65 (t, J = 5.8 Hz, 2H), 2.41–2.34 (m,
2H), 2.02 (s, 5H), 1.30–1.27 (m, 3H); 13C NMR (150 MHz,
CDCl3) δ (ppm): 173.0, 166.2, 145.5, 143.0, 141.7, 141.4,
140.0, 137.5, 136.4, 135.4, 134.3, 134.2, 133.5, 130.0, 129.8,
128.8, 128.5, 128.1, 126.6, 126.4, 125.9, 125.0, 123.2, 60.9,
53.4, 50.8, 44.3, 35.4, 33.9, 33.8, 33.7, 33.5, 33.4, 29.7, 24.1,
22.7, 20.9, 14.2, 14.1; HRMS (ESI-TOF) m/z [M + H]+ cal-
culated for C30H31F6N2O5S+ 645.1780, found 645.1654.
Ethyl 3-(4-((N-butyl-2,6-dimethyl-4′-(trifluoromethyl)-
[1,1′-biphenyl])-4-sulfonamido)benzamido)propanoate
(6bB-2)
1
Yield: 69%, a colorless oil. H NMR (600 MHz, CDCl3) δ
(ppm): 7.77–7.23 (m, 4H), 7.32 (s, 2H), 7.26–7.23 (m, 4H),
6.91 (t, J = 5.9 Hz, 1H), 4.18 (q, J = 7.2 Hz, 2H), 3.73 (q,
J = 5.9 Hz, 2H), 3.62 (t, J = 6.9 Hz, 2H), 2.65 (t, J = 5.9 Hz,
2H), 2.01 (s, 6H), 1.44–1.38 (m, 2H), 1.37–1.32 (m, 3H),
1.28 (t, J = 7.1 Hz, 3H), 0.87 (t, J = 7.3 Hz, 3H); 13C NMR
(150 MHz, CDCl3) δ (ppm): 173.0, 166.5, 144.9, 143.2,
142.2, 137.1, 133.5, 128.9, 128.6, 127.7, 126.6, 126.4, 125.8,
60.9, 53.4, 50.9, 50.0, 35.4, 33.8, 30.2, 30.1, 24.0, 20.9, 19.6,
14.2, 13.6, 13.5; HRMS (ESI-TOF) m/z [M + H]+ calculated
for C31H36F3N2O5S+ 605.2219, found 605.2185.
Ethyl 3-(4-((4′-(tert-butyl)-2,6-dimethyl-N-(3,3,3-
trifluoropropyl)-[1,1′-biphenyl])-4-sulfonamido)
benzamido)propanoate (6cB-3)
1
Yield: 73%, a colorless oil. H NMR (600 MHz, CDCl3) δ
(ppm): 7.79 (d, J = 8 Hz, 2H), 7.45 (d, J = 7.8 Hz, 2H), 7.29
(s, 2H), 7.25 (d, J = 8.2 Hz, 2H), 7.02 (d, J = 7.7 Hz, 2H),
6.90 (t, J = 5.8 Hz, 1H), 4.18 (q, J = 7.1 Hz, 2H), 3.85 (t,
J = 7.6 Hz, 2H), 3.74 (q, J = 5.8 Hz, 2H), 2.65 (t, J = 5.7 Hz,
2H), 2.44–2.36 (m, 2H), 2.04 (s, 5H), 1.37 (s, 7H), 1.28 (t,
J = 7.1 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ (ppm): 173.0,
147.3, 141.9, 138.0, 136.1, 134.1, 128.6, 128.0, 127.9, 126.1,
125.6, 60.9, 35.4, 34.6, 33.8, 31.4, 30.9, 21.1, 14.2; HRMS
(ESI-TOF) m/z [M + H]+ calculated for C33H40F3N2O5S+
633.2532, found 633.2510.
Ethyl 3-(4-((4′-(tert-butyl)-N-butyl-2,6-dimethyl-[1,1′-
biphenyl])-4-sulfonamido)benzamido)propanoate
(6bB-3)
1
Yield: 74%, a colorless oil. H NMR (600 MHz, CDCl3) δ
(ppm): 7.75 (d, J = 8.6 Hz, 2H), 7.45 (d, J = 8.5 Hz, 2H), 7.29
(s, 2H), 7.24 (d, J = 8.6 Hz, 2H), 7.02 (d, J = 8.5 Hz, 2H),
6.87 (t, J = 5.9 Hz, 1H), 4.18 (q, J = 7.2 Hz, 2H), 3.73 (q,
J = 5.9 Hz, 2H), 3.60 (t, J = 6.9 Hz, 2H), 2.65 (t, J = 5.9 Hz,
2H), 2.03 (s, 6H), 1.42–1.33 (m, 12H), 1.28 (t, J = 7.1 Hz,
3H), 0.87 (t, J = 7.2 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ
(ppm): 173.0, 166.5, 150.2, 146.7, 142.4, 137.6, 136.3, 136.1,
133.4, 128.6, 128.0, 127.6, 126.1, 125.5, 60.9, 49.9, 35.3,
34.6, 33.9, 31.4, 30.2, 21.1, 19.6, 14.2, 13.6; HRMS (ESI-
TOF) m/z [M + H]+ calculated for C34H45N2O5S+ 593.2971,
found 593.2966.
2.1.6
General procedure for the
|
synthesis of (7)
LiOH·H2O (6.0 mg, 0.135 mmol) was added to a solu-
tion of compound 6a (33 mg, 0.068 mmol) in THF/H2O
(0.3 ml/0.2 ml). The reaction mixture was stirred at room tem-
perature overnight. After completion, the reaction mixture was
diluted with H2O and acidified to pH 2 with 1 N HCl. The mix-
ture was diluted with EtOAc, and the organic layer was washed
with H2O and brine and then dried over MgSO4. After filtra-
tion, the solvent was removed under reduced pressure and the
residue was purified by SiO2 flash column chromatography.
Ethyl 3-(4-((4′-chloro-2,6-dimethyl-N-(3,3,3-
trifluoropropyl)-[1,1′-biphenyl])-4-sulfonamido)
benzamido)propanoate (6cB-1)
1
Yield: 65%, a colorless oil. H NMR (600 MHz, CDCl3) δ
(ppm): 7.78 (d, J = 8.6 Hz, 2H), 7.45 (d, J = 8.5 Hz, 2H),
7.30 (s, 2H), 7.23 (d, J = 8.6 Hz, 2H), 7.06 (d, J = 8.5 Hz,

LEE Et aL.
7
|
3-(4-(N-propyl-[1,1′-biphenyl]-4-sulfonamido)
3-(4-((3′,5′-dichloro-N-propyl-[1,1′-biphenyl])-4-
benzamido)propanoic acid (7aA-1)
sulfonamido)benzamido)propanoic acid (7aA-5)
1
Yield: 93% (29 mg) of 3-(4-(N-propyl-[1,1′-biphenyl]-4-
sulfonamido)benzamido)propanoic acid as a colorless oil.
1H NMR (600 MHz, CDCl3) δ (ppm): 7.72 (d, J = 8.6 Hz,
2H), 7.67 (d, J = 8.6 Hz, 2H), 7.62–7.60 (m, 3H), 7.48 (t,
J = 7.5 Hz, 2H), 7.43–7.41 (m, 1H), 7.19 (d, J = 8.6 Hz, 2H),
6.77 (t, J = 6 Hz, 1H), 3.75 (q, J = 5.9 Hz, 2H), 3.56 (t,
J = 7.1 Hz, 2H), 2.74 (t, J = 5.8 Hz, 2H), 1.48–1.42 (m,
2H), 0.90 (t, J = 7.4 Hz, 3H); 13C NMR (150 MHz, CDCl3)
δ (ppm): 129.1, 128.6, 128.1, 127.8, 127.5, 127.3, 51.9, 35.2,
33.3, 21.4, 11.0; HRMS (ESI-TOF) m/z [M + H]+ calculated
for C33H40F3N2O5S+ 467.1562, found 467.1506.
Yield: 85%, a colorless oil. H NMR (600 MHz, CDCl3) δ
(ppm): 7.73 (d, J = 8.5 Hz, 2H), 7.62 (q, J = 7.8 Hz, 4H),
7.47 (d, J = 1.9 Hz, 2H), 7.41 (t, J = 1.8 Hz, 1H), 7.17 (d,
J = 8.6 Hz, 2H), 6.78 (bs, 1H), 3.75 (q, J = 5.9 Hz, 2H),
3.56 (t, J = 7.1 Hz, 2H), 2.74 (t, J = 5.8 Hz, 2H), 1.48–1.42
(m,2H), 0.91 (t, J = 7.4 Hz, 3H); 13C NMR (150 MHz, CDCl3)
δ (ppm): 128.6, 128.3, 127.8, 127.6, 125.9, 11.0; HRMS
(ESI-TOF) m/z [M + H]+ calculated for C25H25Cl2N2O5S+
535.0783, found 535.0775.
3-(4-((2′-fluoro-N-propyl-5′-(trifluoromethyl)-[1,1′-
biphenyl])-4-sulfonamido)benzamido)propanoic acid
(7aA-6)
3-(4-((4′-chloro-N-propyl-[1,1′-biphenyl])-4-
1
sulfonamido)benzamido)propanoic acid (7aA-2)
Yield: 81%, a colorless oil. H NMR (600 MHz, CDCl3)
Yield: 87%, a colorless oil. 1H NMR (600 MHz, DMSO-d6)
δ (ppm): 8.58 (t, J = 5.3 Hz, 1H), 7.89 (d, J = 8.4 Hz, 2H),
7.79 (q, J = 7.9 Hz, 4H), 7.61 (d, J = 8.4 Hz, 2H), 7.57 (d,
J = 8.5 Hz, 2H), 7.21 (d, J = 8.5 Hz, 2H), 3.58 (t, J = 6.9 Hz,
2H), 3.45 (q, J = 6.5 Hz, 2H), 1.32 (q, J = 7.1 Hz, 2H),
1.22 (s, 1H), 0.83 (t, J = 7.3 Hz, 3H); 13C NMR (150 MHz,
DMSO-d6) δ (ppm): 173.4, 165.9, 143.5, 141.5, 137.5, 137.0,
134.1, 133.9, 129.6, 129.3, 128.4, 128.4, 127.8, 51.6, 36.1,
34.2, 21.4, 11.3; HRMS (ESI-TOF) m/z [M + H]+ calculated
for C25H26ClN2O5S+ 501.1173, found 501.1123.
δ (ppm): 7.75–7.72 (m, 3H), 7.67–7.64 (m, 5H), 7.32 (t,
J = 9.2 Hz, 1H), 7.19 (d, J = 8.6 Hz, 2H), 6.82 (t, J = 6 Hz,
1H), 3.74 (q, J = 5.9 Hz, 2H), 3.57 (t, J = 7.1 Hz, 2H), 2.74 (t,
J = 5.8 Hz, 2H), 1.48–1.42 (m, 2H), 0.90 (t, J = 7.4 Hz, 3H);
13C NMR (150 MHz, CDCl3) δ (ppm): 166.8, 142.0, 138.8,
137.9, 133.5, 129.5, 129.5, 128.6, 128.1, 127.9, 127.9, 117.3,
117.1, 52.0, 35.3, 33.5, 21.4, 11.0; HRMS (ESI-TOF) m/z
[M + H]+ calculated for C26H25F4N2O5S+ 553.1342, found
553.1355.
3-(4-((4′-chloro-N-(3,3,3-trifluoropropyl)-[1,1′-
biphenyl])-4-sulfonamido)benzamido)propanoic acid
(7cA-1)
3-(4-((N-propyl-4′-(trifluoromethyl)-[1,1′-biphenyl])-4-
sulfonamido)benzamido)propanoic acid (7aA-3)
1
1
Yield: 78%, a colorless oil. H NMR (600 MHz, CDCl3) δ
Yield: 89%, a colorless oil. H NMR (600 MHz, CDCl3) δ
(ppm): 7.75–7.70 (m, 5H), 7.66 (q, J = 7.9 Hz, 4H), 7.19 (d,
J = 8.5 Hz, 2H), 6.79 (t, J = 5.8 Hz, 1H), 3.75 (q, J = 5.9 Hz,
2H), 3.56 (t, J = 7.1 Hz, 2H), 2.74 (t, J = 5.8 Hz, 2H), 1.48–
1.42 (m, 2H), 0.91 (t, J = 7.4 Hz, 3H); 13C NMR (150 MHz,
CDCl3) δ (ppm): 166.7, 144.1, 142.1, 137.5, 128.6, 128.3,
127.8, 127.7, 126.0, 52.0, 35.2, 33.4, 21.4, 11.0; HRMS
(ESI-TOF) m/z [M + H]+ calculated for C26H26F3N2O5S+
535.1436, found 535.1331.
(ppm): 7.76 (d, J = 8.5 Hz, 2H), 7.63 (q, J = 9.8 Hz, 4H),
7.54 (d, J = 8.5 Hz, 2H), 7.46 (d, J = 8.5 Hz, 2H), 7.19 (d,
J = 8.5 Hz, 2H), 6.80 (t, J = 5.8 Hz, 1H), 3.83 (t, J = 7.5 Hz,
2H), 3.75 (q, J = 5.9 Hz, 2H), 2.74 (t, J = 5.8 Hz, 2H),
2.43–2.35 (m, 2H); 13C NMR (150 MHz, CDCl3) δ (ppm):
129.3, 128.6, 128.3, 128.2, 127.5, 30.9; HRMS (ESI-TOF)
m/z [M + H]+ calculated for C25H23ClF3N2O5S+ 555.0890,
found 555.0873.
3-(4-((4′-(tert-butyl)-N-propyl-[1,1′-biphenyl])-4-
sulfonamido)benzamido)propanoic acid (7aA-4)
3-(4-((4′-(trifluoromethyl)-N-(3,3,3-trifluoropropyl)-
[1,1′-biphenyl])-4-sulfonamido)benzamido)propanoic
acid (7cA-2)
1
Yield: 89%, a colorless oil. H NMR (600 MHz, CDCl3) δ
(ppm): 7.72 (d, J = 8.6 Hz, 2H), 7.66 (d, J = 8.6 Hz, 2H), 7.59
(d, J = 8.6 Hz, 2H), 7.55 (d, J = 8.6 Hz, 2H), 7.50 (d, J = 8.6 Hz,
2H), 7.18 (d, J = 8.6 Hz, 2H), 6.78 (t, J = 5.4 Hz, 1H), 3.74 (q,
J = 5.9 Hz, 2H), 3.55 (t, J = 7.1 Hz, 2H), 2.74 (t, J = 5.8 Hz,
2H), 1.47–1.41 (m, 2H), 1.37 (s, 9H), 0.90 (t, J = 7.4 Hz, 3H);
13C NMR (150 MHz, CDCl3) δ (ppm): 151.8, 142.3, 136.1,
128.6, 128.1, 127.7, 127.2, 126.9, 126.1, 51.9, 35.2, 34.7, 33.4,
31.3, 21.4, 11.0; HRMS (ESI-TOF) m/z [M + H]+ calculated
for C29H35N2O5S+ 523.2188, found 523.2149.
Yield: 91%, a colorless oil. 1H NMR (600 MHz, DMSO-d6)
δ (ppm): 8.59 (t, J = 5.4 Hz, 1H), 7.96 (q, J = 4.2 Hz, 4H),
7.86 (d, J = 8.3 Hz, 2H), 7.82 (d, J = 8.5 Hz, 2H), 7.66 (d,
J = 8.4 Hz, 2H), 7.24 (d, J = 8.5 Hz, 2H), 3.92 (t, J = 6.6 Hz,
2H), 3.44 (q, J = 6.5 Hz, 2H), 3.32 (s, 2H), 2.43 (m, 2H); 13
C
NMR (150 MHz, DMSO-d6) δ (ppm): 173.3, 165.8, 143.6,
142.6, 140.9, 137.0, 134.3, 128.6, 128.5, 128.5, 126.5, 126.5,
36.1, 34.2; HRMS (ESI-TOF) m/z [M + H]+ calculated for
C26H23F6N2O5S+ 589.1154, found 589.1132.

8
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3-(4-((4′-(tert-butyl)-N-(3,3,3-trifluoropropyl)-[1,1′-
biphenyl])-4-sulfonamido)benzamido)propanoic acid
(7cA-3)
3-(4-((N-butyl-4′-chloro-2,6-dimethyl-[1,1′-biphenyl])-4-
sulfonamido)benzamido)propanoic acid (7bB-1)
Yield: 85%, a colorless oil. 1H NMR (600 MHz, CDCl3) δ
(ppm): 7.75 (d, J = 8.6 Hz, 2H), 7.44 (d, J = 8.5 Hz, 2H),
7.30 (s, 2H), 7.23 (d, J = 8.6 Hz, 2H), 7.05 (d, J = 8.5 Hz,
2H), 6.82 (t, J = 6 Hz, 1H), 3.75 (q, J = 5.9 Hz, 2H), 3.60
(t, J = 6.9 Hz, 2H), 2.74 (t, J = 5.8 Hz, 2H), 2.01 (s, 6H),
1.43–1.38 (m, 2H), 1.37–1.31 (m, 2H), 0.86 (t, J = 7.3 Hz,
3H); 13C NMR (150 MHz, CDCl3) δ (ppm): 137.7, 137.4,
136.7, 133.5, 133.2, 129.8, 129.1, 128.6, 127.7, 126.3,
49.9, 35.2, 30.2, 21.0, 19.6, 13.6; HRMS (ESI-TOF) m/z
[M + H]+ calculated for C28H32ClN2O5S+ 543.1642, found
543.1622.
1
Yield: 84%, a colorless oil. H NMR (600 MHz, CDCl3) δ
(ppm): 7.76 (m, 2H), 7.72–7.65 (m, 3H), 7.59 (d, J = 8.6 Hz,
1H), 7.55 (d, J = 8.6 Hz, 1H), 7.51 (d, J = 8.6 Hz, 1H),
7.19 (q, J = 4.7 Hz, 2H), 6.81 (t, J = 6.2 Hz, 1H), 3.83 (q,
J = 7.9 Hz, 2H), 3.75 (q, J = 5.7 Hz, 2H), 2.74 (t, J = 5.5 Hz,
2H), 2.43–2.35 (m, 2H), 1.37 (s, 6H); 13C NMR (150 MHz,
CDCl3) δ (ppm): 128.6, 128.3, 128.2, 128.1, 127.7, 127.4,
127.0, 126.1, 34.7, 31.3; HRMS (ESI-TOF) m/z [M + H]+
calculated for C31H36F3N2O5S+ 605.2219, found 605.2200.
3-(4-((4′-chloro-2,6-dimethyl-N-propyl-[1,1′-biphenyl])-
4-sulfonamido)benzamido)propanoic acid (7aB-1)
3-(4-((2,6-dimethyl-N-propyl-4′-(trifluoromethyl)-[1,1′-
biphenyl])-4-sulfonamido)benzamido)propanoic acid
(7bB-2)
1
Yield: 87%, a colorless oil. H NMR (600 MHz, CDCl3) δ
(ppm): 8.14 (d, J = 8.3 Hz, 2H), 7.75 (d, J = 8.6 Hz, 1H),
7.49 (d, J = 8.3 Hz, 1H), 7.44 (d, J = 8.5 Hz, 1H), 7.30 (s,
1H), 7.24 (d, J = 8.6 Hz, 2H), 7.05 (d, J = 8.5 Hz, 2H),
6.80 (t, J = 6 Hz, 1H), 3.75 (q, J = 5.9 Hz, 2H), 3.57 (t,
J = 7.1 Hz, 2H), 2.74 (t, J = 5.8 Hz, 2H), 2.01 (s, 6H), 1.49–
1.43 (m, 2H), 0.91 (t, J = 7.4 Hz, 3H); 13C NMR (150 MHz,
CDCl3) δ (ppm): 137.4, 137.0, 135.0, 129.8, 129.1, 128.6,
128.5, 127.7, 126.3, 21.0; HRMS (ESI-TOF) m/z [M + H]+
calculated for C27H30ClN2O5S+ 529.1486, found 529.1770.
1
Yield: 90%, a colorless oil. H NMR (600 MHz, CDCl3) δ
(ppm): 7.77–7.73 (m, 4H), 7.32 (s, 2H), 7.24 (d, J = 8.5 Hz,
4H), 6.82 (t, J = 5.9 Hz, 1H), 3.75 (q, J = 5.9 Hz, 2H), 3.61
(t, J = 7 Hz, 2H), 2.74 (t, J = 5.7 Hz, 2H), 2.01 (s, 6H),
1.43–1.39 (m, 2H), 1.36–1.31 (m, 2H), 0.87 (t, J = 7.2 Hz,
3H); 13C NMR (150 MHz, CDCl3) δ (ppm): 137.1, 133.3,
128.9, 128.6, 127.7, 126.4, 125.8, 49.9, 35.2, 30.2, 24.0,
20.9, 19.6, 13.6; HRMS (ESI-TOF) m/z [M + H]+ calculated
for C29H32F3N2O5S+ 577.1906, found 577.1886.
3-(4-((2,6-dimethyl-N-propyl-4′-(trifluoromethyl)-[1,1′-
biphenyl])-4-sulfonamido)benzamido)propanoic acid
(7aB-2)
3-(4-((4′-(tert-butyl)-N-butyl-2,6-dimethyl-[1,1′-
biphenyl])-4-sulfonamido)benzamido)propanoic acid
(7bB-3)
1
Yield: 83%, a colorless oil. H NMR (600 MHz, CDCl3)
1
δ (ppm): 7.76–7.73 (m, 3H), 7.68–7.65 (m, 2H), 7.56 (t,
J = 7.4 Hz, 1H), 7.49–7.46 (m, 2H), 7.32 (s, 2H), 7.24 (d,
J = 8.6 Hz, 1H), 6.88 (t, J = 5.8 Hz, 1H), 3.74 (q, J = 5.9 Hz,
2H), 3.58 (t, J = 7.1 Hz, 2H), 2.72 (t, J = 5.7 Hz, 2H), 2.01 (s,
6H), 1.49–1.43 (m, 2H), 0.91 (t, J = 7.4 Hz, 3H); 13C NMR
(150 MHz, CDCl3) δ (ppm): 166.7, 137.1, 132.2, 132.1,
132.1, 128.9, 128.6, 127.7, 126.4, 125.8, 52.0, 35.3, 33.4,
21.5, 20.9, 11.0; HRMS (ESI-TOF) m/z [M + H]+ calculated
for C28H30F3N2O5S+ 563.1749, found 563.1724.
Yield: 88%, a colorless oil. H NMR (600 MHz, CDCl3) δ
(ppm): 7.75 (d, J = 8.6 Hz, 2H), 7.44 (d, J = 8.3 Hz, 2H), 7.29
(s, 2H), 7.25 (d, J = 8.6 Hz, 2H), 7.01 (d, J = 8.3 Hz, 2H), 6.81
(t, J = 5.9 Hz, 1H), 3.75 (q, J = 5.9 Hz, 2H), 3.60 (t, J = 7 Hz,
2H), 2.74 (t, J = 5.8 Hz, 2H), 2.02 (s, 6H), 1.43–1.31 (m, 12H),
0.86 (t, J = 7.2 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ (ppm):
137.6, 136.0, 128.6, 127.9, 127.7, 126.1, 125.5, 49.9, 35.2, 34.6,
31.4, 30.2, 21.1, 19.6, 13.6; HRMS (ESI-TOF) m/z [M + H]+
calculated for C32H41N2O5S+ 565.2658, found 565.2666.
3-(4-((4′-(tert-butyl)-2,6-dimethyl-N-propyl-[1,1′-
biphenyl])-4-sulfonamido)benzamido)propanoic acid
(7aB-3)
3-(4-((4′-chloro-2,6-dimethyl-N-(3,3,3-trifluoropropyl)-
[1,1′-biphenyl])-4-sulfonamido)benzamido)propanoic
acid (7cB-1)
1
1
Yield: 86%, a colorless oil. H NMR (600 MHz, CDCl3) δ
Yield: 85%, a colorless oil. H NMR (600 MHz, CDCl3) δ
(ppm): 7.75 (d, J = 8.6 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H), 7.29
(s, 2H), 7.25 (d, J = 8.8 Hz, 2H), 7.01 (d, J = 8.5 Hz, 2H), 6.82
(t, J = 6.1 Hz, 1H), 3.74 (q, J = 5.9 Hz, 2H), 3.57 (t, J = 7.1 Hz,
2H), 2.73 (t, J = 5.8 Hz, 2H), 2.02 (s, 6H), 1.48–1.44 (m, 2H),
1.37 (s, 9H), 0.91 (t, J = 7.4 Hz, 3H); 13C NMR (150 MHz,
CDCl3) δ (ppm): 166.8, 146.7, 142.5, 137.6, 136.2, 136.1, 135.6,
128.6, 127.9, 127.7, 126.1, 125.5, 125.0, 51.9, 35.2, 34.6, 33.5,
31.4, 31.2, 21.5, 21.1, 11.0; HRMS (ESI-TOF) m/z [M + H]+
calculated for C31H39N2O5S+ 551.2501, found 551.2500.
(ppm): 7.78 (d, J = 8.5 Hz, 2H), 7.45 (d, J = 8.3 Hz, 2H),
7.30 (s, 2H), 7.24 (d, J = 8.5 Hz, 2H), 7.05 (d, J = 8.3 Hz,
2H), 6.84 (t, J = 5.8 Hz, 1H), 3.84 (t, J = 7.5 Hz, 2H), 3.75
(q, J = 5.7 Hz, 2H), 2.74 (t, J = 5.7 Hz, 2H), 2.40–2.36 (m,
2H), 2.02 (s, 6H); 13C NMR (150 MHz, CDCl3) δ (ppm):
141.9, 137.7, 137.5, 136.0, 129.7, 129.2, 128.6, 128.1,
126.3, 44.3, 35.3, 33.4, 29.7, 21.0; HRMS (ESI-TOF) m/z
[M + H]+ calculated for C27H27ClF3N2O5S+ 583.1203,
found 583.1213.

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9
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3-(4-((2,6-dimethyl-4′-(trifluoromethyl)-N-(3,3,3-
trifluoropropyl)-[1,1′-biphenyl])-4-sulfonamido)
benzamido)propanoic acid (7cB-2)
H2O (14 ml) at −5℃. After stirring for 30 min at −5℃, the
solution prepared in (A) was added dropwise at the same
temperature. The reaction temperature was increased slowly,
and the reaction mixture was stirred at room temperature
overnight. The reaction mixture was diluted with EtOAc, and
the organic layer was washed with H2O and brine, and then
dried over MgSO4. After filtration, the solvent was removed
under reduced pressure and the residue was purified by SiO2
flash column chromatography. The synthesis yielded 1.5 g
(52%) of 4-bromo-3,5-dimethylbenzenesulfonyl chloride. 1H
NMR (600 MHz, CDCl3) δ (ppm): 7.73 (s, 2H), 2.54 (s, 6H);
13C NMR (150 MHz, CDCl3) δ (ppm): 144.3, 140.8, 135.8,
125.7, 24.2; HRMS (ESI-TOF) m/z [M + H]+ calculated for
C8H9BrClO2S+ 282.9117, found 282.9079.
1
Yield: 89%, a colorless oil. H NMR (600 MHz, CDCl3) δ
(ppm): 7.79 (d, J = 8.5 Hz, 2H), 7.74 (d, J = 8 Hz, 2H),
7.32 (s, 2H), 7.26–7.24 (m, 4H), 6.84 (bs, 1H), 3.85 (q,
J = 5 Hz, 2H), 3.75 (d, J = 5.6 Hz, 2H), 2.74 (s, 2H), 2.40–
2.36 (m, 2H), 2.02 (s, 6H); 13C NMR (150 MHz, CDCl3) δ
(ppm): 137.5, 128.8, 128.6, 128.1, 126.4, 125.9, 20.9; HRMS
(ESI-TOF) m/z [M + H]+ calculated for C28H27F6N2O5S+
617.1467, found 616.1450.
3-(4-((4′-(tert-butyl)-2,6-dimethyl-N-(3,3,3-
trifluoropropyl)-[1,1′-biphenyl])-4-sulfonamido)
benzamido)propanoic acid (7cB-3)
1
Yield: 81%, a colorless oil. H NMR (600 MHz, CDCl3) δ
(ppm): 7.78 (d, J = 8.5 Hz, 2H), 7.45 (d, J = 8.3 Hz, 2H),
7.29 (s, 2H), 7.25 (s, 2H), 7.01 (d, J = 8.3 Hz, 2H), 6.82 (t,
J = 5.6 Hz, 1H), 3.84 (t, J = 5 Hz, 2H), 3.75 (q, J = 5.9 Hz,
2H), 2.74 (t, J = 5.7 Hz, 2H), 2.44–2.36 (m, 2H), 2.03 (s,
5H), 1.37 (s, 8H); 13C NMR (150 MHz, CDCl3) δ (ppm):
128.6, 128.0, 127.9, 126.1, 125.6, 34.6, 31.4, 21.1; HRMS
(ESI-TOF) m/z [M + H]+ calculated for C31H36F3N2O5S+
605.2219, found 605.2201.
2.2
Animals
|
Five-week-old male C57BL/6 mice were purchased from
Orient Bio Inc. (Seongnam, Kyounggido, Korea). The mice
were maintained under specific pathogen-free conditions in
a temperature-controlled room under a 12 hr light/dark cycle
with ad libitum access to food and water at the Animal Care
Center, Lee Gil Ya Cancer and Diabetes Institute, Gachon
University, South Korea. The mice were fed HFD (60%
fat composed primarily of lard, Research Diet, Inc., New
Brunswick, NJ, USA, #D12492). After 8 weeks (at 14 weeks
of age), the mice were randomly divided into three groups:
9% Kolliphor® HS 15 with 10% DMSO-treated group
(Vehicle), 7aB-3-treated group (7aB-3), and LGD-6972-
treated group (LGD-6972). Fifty milligrams per kilogram
of 7aB-3 or LGD-6972 (dissolved in 9% Kolliphor® HS 15
with 10% DMSO in sterilized water) was administered orally
by gavage once daily for 6 weeks. LGD-6972 was synthe-
sized in the laboratory. Body weight and food intake were
measured daily. Fasting blood glucose level was measured
every two weeks, and non-fasting blood glucose level was
measured at weeks 3 and 4 after administration. All animal
experiments were approved by the Institutional Animal Care
and Use Committee of the Lee Gil Ya Cancer and Diabetes
Institute (LCDI-2015-0006).
2.1.7
Synthesis of 4-bromo-3,5-
|
dimethylbenzenesulfonyl chloride
4-bromo-3,5-dimethylaniline (9)
3,5-Dimethylaniline (5 ml, 40 mmol) was dissolved in ace-
tonitrile (60 ml). NBS (7 g, 40 mmol) was added, and the reac-
tion mixture was stirred overnight at room temperature. The
reaction mixture was then quenched with H2O and diluted
with EtOAc. The organic phase was separated, washed with
H2O and brine, and then dried over MgSO4. After filtration,
the solvent was removed under reduced pressure and the resi-
due was purified by SiO2 flash column chromatography. The
reaction yielded 6 g (75%) of 4-bromo-3,5-dimethylaniline.
1H NMR (600 MHz, CDCl3) δ (ppm): 6.44 (s, 2H), 3.53
(bs, 2H), 2.31 (s, 6H); 13C NMR (150 MHz, CDCl3) δ
(ppm): 144.9, 138.8, 115.9, 115.1, 23.8; HRMS (ESI-TOF)
m/z [M + H]+ calculated for C8H11BrN+ 199.9997, found
199.9999.
2.3
Cell culture and isolation of
|
hepatocytes
4-bromo-3,5-dimethylbenzenesulfonyl chloride (10)
(A) Thionyl chloride (5 ml, 70 mmol) was added dropwise
to copper chloride (l) (99 mg, 1 mmol) dissolved in H2O
(20 ml) on ice. The reaction temperature was raised slowly
to room temperature, and the mixture was stirred overnight.
(B) Sodium nitrite (759 mg, 11 mmol) in H2O (16 ml) was
added dropwise to a mixture of 4-bromo-3,5-dimethylaniline
(2 g, 10 mmol), concentrated HCl (10 ml, 330 mmol), and
The cAMP Hunter™ CHO-K1 GCGR Gs cell line (CHO-K1
GCGR Gs cells) expressing human GCGR were purchased
from DiscoverX (Fremont, USA). The cells were maintained
in CHO-K1 medium (DiscoverX). Isolation of hepatocytes
was performed as previously described (Yoon et al., 2017).
Briefly, ten-week-old male C57BL/6N mice were anes-
thetized with ketamine. The liver was perfused through the

10
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|
portal vein using perfusion buffer I (142 mm NaCl, 6.7 mm
KCl, 0.01 M HEPES, and 2.5 mm EGTA, pH 7.4) for 10 min
and then perfusion buffer II (66.7 mm NaCl, 6.7 mm KCl,
10 mm HEPES, and 4.8 mm CaCl2, pH 7.6) with 0.5 mg/ml of
Collagenase IV for 5 min. The liver was excised and dissected
in cold high-glucose DMEM to separate the hepatic cells. The
cells were centrifuged at 50 g for 5 min at 4℃, washed using
cold high-glucose DMEM, and then centrifuged again at 50 g
for 5 min at 4℃. Hepatocytes were isolated by Percoll gra-
dient centrifugation (250 g for 5 min without brake at 4℃).
The protocol was approved by the Institutional Animal Care
and Use Committee of the Lee Gil Ya Cancer and Diabetes
Institute (LCDI-2015–0006). The isolated primary mouse
hepatocytes were grown in HepatoZYME-SFM (Gibco-BRL,
Grand Island, NY, USA) with 10% fetal bovine serum (FBS;
Gibco-BRL) and 1% antibiotics (100 unit/ml penicillin and
100 μg/ml streptomycin; Gibco-BRL). All cells were main-
tained under subconfluent conditions in a humidified incuba-
tor at 37℃ with 95% ambient air and 5% CO2.
Santa Cruz Biotechnology, sc-203972). Five independent ex-
periments were performed in triplicate.
2.6
Glucagon-induced glucose
|
excursion and intraperitoneal glucose tolerance
test (ipGTT)
For the glucagon-induced glucose excursion test, after
5 weeks of treatment with 7aB-3 or LGD-6972, the mice
were fasted for 5 hr, and then, 50 mg/kg of 7aB-3 or LGD-
6972 was orally administered by gavage. After 1 hr, glucagon
was injected intraperitoneally at a dose of 15 μg/kg. Blood
glucose levels were measured at 15, 30, 45, and 60 min after
glucagon injection. For the ipGTT, after 6 weeks of treatment
with 7aB-3 or LGD-6972, the mice were fasted for 14 hr, and
then, 50 mg/kg of 7aB-3 or LGD-6972 was orally adminis-
tered by gavage. After 1 hr, glucose (2 g/kg) was injected
intraperitoneally, and blood glucose levels were measured at
30, 60, 90, and 120 min afterward.
2.4
Measurement of cAMP production
|
2.7
In vivo pharmacokinetic studies and
|
Briefly, 1 × 104 CHO-K1 GCGR Gs cells/well were seeded
in 96-well plates in CHO-K1 medium (DiscoverX). The next
day, the cells were treated with GCGR antagonist candidates
in phosphate-buffered saline for 15 min at 37℃ in a 5% CO2
incubator. After incubation, 0.1 nm glucagon and 10 μm for-
skolin were added, and the cells were incubated for 30 min at
37℃ in a 5% CO2 incubator. As a positive control, the CHO-
K1 GCGR Gs cells were treated with 10 μm of GA (GCGR
antagonist I, Santa Cruz Biotechnology, sc-203972). cAMP
was measured using the HitHunter® cAMP Assay for Small
Molecules Kit (DiscoverX) according to the manufacturer's
instructions. Luminescence was measured using a Victor3
multilabel plate reader (PerkinElmer, Waltham, MA, USA).
bioanalytical assessment by LC-MS/MS
Rats were fasted overnight prior to the pharmacokinetic studies
but were allowed free access to water. Briefly, after cannulation
with a PE50 tube of the femoral vein for IV administration and
femoral artery for blood collection under anesthesia, similar
to our previous studies (Jeong et al., 2019; Song et al., 2020),
the systemic and oral pharmacokinetics of the test compounds
were investigated at doses of 2 and 5 mg/kg, respectively.
After recovering from anesthesia, the fasted rats were IV or
PO administered compound 7aB-3. At predetermined time in-
tervals (i.e., 1 (IV only), 5, 15, 30, 60, 120, 240, and 480 min),
~100 μl of blood was collected from the femoral artery can-
nula and replaced with an equal volume of normal saline to
compensate for blood loss. Plasma samples were prepared by
centrifugation of the blood at 12,000 g and 4℃ for 10 min,
and then, the supernatant was stored at −20℃ until the LC-
MS/MS assay. A simple deproteinization method was used by
adding a 2-fold volume of methanolic internal standard solu-
tion. The internal standard was used with the other test com-
pounds. After vigorous vortexing, followed by centrifugation
at 12,000 g and 4℃ for 15 min, the supernatant was injected
into the LC-MS/MS system. The LC-MS/MS consisted of an
Agilent HPLC system (1290 Infinity, Agilent Technologies,
Santa Clara, CA, USA) and Agilent 6490 QQQ mass spec-
trometer with a positive electrospray ionization (ESI+) Agilent
Jet Stream ion source (Agilent Technologies). To separate the
test compounds from endogenous substances in plasma, a
Synergi™ 4 μm polar-RP 80A column (150 mm × 2.0 mm,
4 μm, Phenomenex, Torrance, CA, USA) was utilized. The
2.5
Measurement of hepatic
|
glucose production
Mouse primary hepatocytes were seeded at 2.5 × 105 cells/
well in 12-well plates. After 24 hr, the cells were washed
twice in pre-warmed glucose-free DMEM and incubated for
3 hr in glucose-free DMEM. The cells were treated with vari-
ous concentrations of GCGR antagonist candidates, gluco-
neogenic substrates (20 mm sodium lactate and 2 mm sodium
pyruvate), and 10 nm glucagon. After 30 min, glucose in the
medium was quantified using a glucose assay kit (GAGO-20,
Sigma-Aldrich, St. Louis, MO, USA) and normalized to total
cellular protein concentration (Choi et al., 2018; J. Y. Yoon
et al., 2017). As a positive control, mouse primary hepato-
cytes were treated with 20 μm of GA (GCGR antagonist I,

LEE Et aL.
11
|
mobile phase was a mixture of 0.1% formic acid and acetoni-
trile (65:35, v/v) at a flow rate of 200 μl/s. Multiple reaction
monitoring (MRM) mode was chosen as follows: 7aB-3, m/z
551.2 → 462.0; internal standard (IS), m/z 403.4 → 138.2.
The calibration curves were linear with a range from 5 to
10,000 ng/ml (coefficient of determination R2 = 0.9986) with
weighing of 1/x. For the calculation of pharmacokinetic pa-
rameters, non-compartmental analysis was performed using
WinNonlin 5.0.1 (Pharsight, Cary, NC, USA). The absolute
oral bioavailability (BA) was calculated by dividing the dose-
normalized total area under the plasma concentration-time
curve from time zero to time infinity (AUC) after PO admin-
istration using the dose-normalized AUC after IV administra-
tion (Han et al., 2021; Maeng et al., 2019; Yoon et al., 2020).
All data are expressed as the mean standard deviation (SD).
3
RESULTS AND DISCUSSION
Synthesis
|
3.1
|
The synthesis of the biphenylsulfonamide derivatives is
shown in Scheme 1. 4-Nitrobenzoyl chloride was treated with
β-alanine ethyl ester to give amide 2, of which the nitro group
was reduced to form amine 3 with H2 and Pd/C in MeOH.
Several alkyl groups, such as n-Pr, i-Bu, n-Bu, n-Hexyl,
and 3,3,3-trifluoropropyl, were conjugated to the amine by
reductive amination to produce N-alkylanilines 4, which
were converted to 4-halosulfonamides 5A and 5B using
4-iodosulfonyl chloride or 4-bromo-3,5-dimethylsulfonyl
chloride, respectively. The halides were exchanged with vari-
ous substituted phenyl groups through the Suzuki reaction
to afford biphenyl compounds 6 in good yields. Finally, the
ethyl ester was hydrolyzed to obtain the corresponding car-
boxylic acids 7. Eighteen analogs were synthesized and are
presented in Table 1.
2.8
Statistical analysis
|
Multiple group comparisons were performed by analysis
of variance (ANOVA) followed by Fisher's protected least
significant difference test (* p < .05, ** p < .01, and ***
p < .001) using Graphpad Prism software.
The first step in the synthesis of 4-bromo-3,5-dimethylben
zenesulfonyl chloride (shown in Scheme 2) was carried out by
bromination of commercially available 3,5-dimethylaniline
in the para position with NBS (Zysman-Colman et al., 2009)
SCHEME 1 Synthesis of biphenylsulfonamides. Reagents and conditions: (a) β-alanine ethyl ester hydrochloride, TEA, CHCl3, rt,
94%; (b) Pd/C, H2, MeOH, rt, 98%; (c) alkyl aldehyde, Pd/C, H2, MeOH, rt, 68 ~ 75%; (d) 4-iodobenzenesulfonyl chloride or 4-bromo-3,5-
dimethylbenzenesulfonyl chloride, TEA, CH2Cl2, rt, 58 ~ 67%; (e) substituted phenylboronic acid, 20% K2CO3, Pd(PPh3)4, THF, 80℃, 60%–80%;
(f) LiOH·H2O, THF:H2O (1.5:1), rt, 78 ~ 93%

12
LEE Et aL.
|
to yield compound 9, followed by diazotization (Hogan &
Cox, 2009) and treatment with thionyl chloride to afford sul-
fonyl chloride 10 (Rennison et al., 2013).
the compounds. The cells were treated with 20 μm of each
compound for 24 hr, and cell viability was examined. As
shown in Figure 3, we selected 10 compounds that produced
over 95% cell viability in the CHO-K1 GCGR Gs cells.
3.2
Biological activity
|
3.2.2
Screening of biphenylsulfonamide
|
3.2.1
Screening of biphenylsulfonamide
compounds by in vitro efficacy
|
compounds for cytotoxicity
When GCGR is activated by glucagon, intracellular cAMP
levels are increased by stimulation of adenylate cyclase, and
the cAMP-dependent protein kinase (PKA) pathway is subse-
quently activated (Han et al., 2015; Jiang et al., 2001; Kimball
& Murlin, 1923; Li et al., 2006). We then tested the 10 se-
lected synthetic compounds for inhibition of glucagon-induced
cAMP production in the CHO-K1 GCGR Gs cells. Three
compounds showed over 70% inhibition of glucagon-induced
cAMP production compared to that of the vehicle-treated
group (Figure 4a). Substitution of R1 of the sulfonyl amide
with n-propyl (-CH2CH2CH3; 7aB-3) and 3,3,3-trifluoropropyl
(-CH2CH2CF3; 7cA-3 and 7cB-3) moieties was found to en-
hance potency in the cAMP assay. The introduction of a t-butyl
(4-t-Bu) group at the para position of R3 or methyl (CH3) substi-
tution at R2 resulted in excellent inhibitory activity (Figure 4a).
To examine the dose-dependent effects on glucagon-induced
cAMP production, we treated the CHO-K1 GCGR Gs cells with
various concentration of each compound (5, 10, or 20 μm of
7cA-3, 7aB-3, or 7cB-3) in the presence of glucagon (0.1 nm).
Except for compound 7cA-3, the other compounds showed
concentration-dependent inhibition of glucagon-induced cAMP
production. Compound 7aB-3 showed the lowest cAMP pro-
duction among the three compounds at 20 μm (Figure 4b).
We synthesized 18 compounds as GCGR antagonist candi-
dates based on the biphenylsulfonamide structure. The cyto-
toxic effects of the synthesized compounds were evaluated
in CHO-K1 GCGR Gs cells. These cells overexpress human
GCGR and were used for screening the biological activity of
TABLE 1 Biphenylsulfonamide derivatives
R2
R3
O
HO
2C
N
H
O
R2
S
O
N
R1
Entry
Compound R1
R2
R3
1
2
3
4
5
6
7
8
7aA-1
7aA-2
7aA-3
7aA-4
7aA-5
7aA-6
7cA-1
7cA-2
7cA-3
7aB-1
7aB-2
7aB-3
7bB-1
7bB-2
7bB-3
7cB-1
7cB-2
7cB-3
CH2CH2CH3
CH2CH2CH3
CH2CH2CH3
CH2CH2CH3
CH2CH2CH3
CH2CH2CH3
CH2CH2CF3
CH2CH2CF3
CH2CH2CF3
CH2CH2CH3
CH2CH2CH3
CH2CH2CH3
CH2CH2CH2CH3
CH2CH2CH2CH3
CH2CH2CH2CH3
CH2CH2CF3
CH2CH2CF3
CH2CH2CF3
H
H
H
H
H
H
H
H
H
4-Cl
4-CF3
4-t-Bu
3,5-Di-Cl
2-F, 5-CF3
4-Cl
4-CF3
4-t-Bu
4-Cl
4-CF3
4-t-Bu
4-Cl
4-CF3
4-t-Bu
4-Cl
9
H
3.2.3
Effect of the three selected
10
11
12
13
14
15
16
17
18
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
|
biphenylsulfonamide compounds on glucagon-
induced glucose production in primary
hepatocytes
Activation of GCGR by glucagon promotes glucose produc-
tion by inducing both glycogenolysis and gluconeogenesis in
the liver (Brubaker & Drucker, 2002; Christensen et al. 2011).
We then tested the selected three compounds for glucagon-
induced glucose production in mouse primary hepatocytes.
4-CF3
4-t-Bu
SCHEME 2 Synthesis of 4-bromo-3,5-
dimethylbenzenesulfonyl chloride. Reagents
and conditions: (a) NBS, acetonitrile, 0℃
to rt, 72%; (b) CuCl, SOCl2, HCl, sodium
nitrite, H2O, −5℃ to rt, 52%

LEE Et aL.
13
|
140%
120%
100%
80%
60%
40%
20%
0%
e
1
2
3
4
5
6
1
2
3
1
2
3
1
2
3
-
A
-
-
-
-
-
-
-
-
-
-1
-2
-3
-
-
-
-
-
G
A
A
A
A
A
A
A
A
A
B
B
B
B
B
B
B
B
B
a
a
a
a
a
a
c
c
c
a
a
a
c
c
c
b
b
b
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
Vehicl
FIGURE 3 Screening of biphenylsulfonamide compounds for cytotoxicity. CHO-K1 GCGR Gs cells were treated with 20 μm of indicated
compounds for 24 hr, and cell viability was determined by CCK-8 assay. As a positive control, CHO-K1 GCGR Gs cells were treated with 20 μm of
GCGR antagonist I (GA). Black bars indicate the compounds selected for further analysis. N = 3 independent experiments. The data are presented
as the mean standard error (SE)
FIGURE 4 Screening of biphenylsulfonamide compounds by glucagon-induced cAMP production assay. (a). cAMP production was measured
after treatment of CHO-K1 GCGR Gs cells with 10 μm of the indicated compound in the presence of 0.1 nm glucagon. (b) cAMP production was
measured after treatment of CHO-K1 GCGR Gs cells with 5, 10, or 20 μm of the indicated compound in the presence of 0.1 nm glucagon. As a
positive control, CHO-K1 GCGR Gs cells were treated with 10 μm of GCGR antagonist I (GA). N = 3 independent experiments. The data are
presented as the mean standard error (SE)
We tested the cells with the compounds at 20 μm in the pres-
ence of glucagon. The three compounds (7cA-3, 7aB-3, and
7cB-3) produced 72%, 74%, and 71% inhibition of glucagon-
induced glucose production, respectively (Figure 5a). Next,
we examined the concentration-dependent effects of the
compounds on glucagon-induced glucose production in
mouse primary hepatocytes. We treated the cells with 1, 5,
10, or 20 μm of each compound in the presence of glucagon
(10 nm). Except for compound 7cB-3, the other compounds
inhibited glucose production in a concentration-dependent
manner (Figure 5b). Based on the results of the cytotoxicity,
cAMP production, and glucagon-induced glucose production
assays, we selected compound 7aB-3 for in vivo further ex-
periments. We then evaluated the concentration-dependent
inhibitory effect of compound 7aB-3 on glucagon-induced
cAMP production in more detail (Figure 6). The obtained
IC50 value for 7aB-3 was 8.4 μm.
3.2.4
In vivo glucose-lowering effect of
|
compound 7aB-3
We selected compound 7aB-3 that inhibited glucagon-
induced glucose production to the greatest extent in vitro.
To evaluate the blood glucose-lowering effect of compound
7aB-3, high-fat diet (HFD)-induced obese mice were orally
administered 50 mg/kg of 7aB-3 or LGD-6972 as a con-
trol once daily for 6 weeks. To investigate whether the
effect of 7aB-3 on the control of blood glucose levels is
mediated by inhibition of GCGR, we performed glucagon

14
LEE Et aL.
|
FIGURE 5 Screening of three selected biphenylsulfonamide compounds by glucagon-induced glucose production in primary hepatocytes. (a)
Mouse primary hepatocytes were pretreated with 20 μm of the indicated compound in glucose-free Dulbecco's modified Eagle medium (DMEM).
After 3 hr, cells were treated with 20 μm of the indicated compound in the presence of 10 nm glucagon and gluconeogenic substrates (2 mm sodium
pyruvate and 20 mm sodium lactate). Glucose production was measured 30 min after glucagon treatment. (b) Mouse primary hepatocytes were
treated with 1, 5, 10, or 20 μm of the indicated compound in the presence of 10 nm glucagon and gluconeogenic substrates. Twenty μm of GCGR
antagonist I (GA) was used as a positive control. N = 5. The data are presented as the mean standard error (SE). ***p < .001 versus Vehicle,
##p < .01 versus Vehicle
150%
from the beginning (week 0) to the end (week 6) of the ex-
periment (Figure 7c). We also monitored daily food intake
over the 6 weeks, which did not change among all groups
(Figure 7d). During the experiment, we monitored non-
100%
fasting blood glucose levels at weeks 3 and 4 during the
experiment. In the group treated with 50 mg/kg 7aB-3, the
non-fasting blood glucose level decreased on weeks 3 and
50%
4 of treatment (Figure 7e). On week 6 of treatment with
7aB-3 or LGD-6972, we performed a glucose tolerance test
to confirm the ameliorative effect of 7aB-3 on hyperglyce-
0%
mia. Treatment with 7aB-3 or LGD-6972 decreased blood
glucose levels in the HFD-induced obese mice administered
2 g/kg of glucose (Figure 7f). From both the in vivo and in
vitro data, we conclude that 7aB-3 is more effective than the
other derivatives. Compound 7aB-3 was then recommended
for further investigation.
C o n c e n tra tio n (μ M )
FIGURE 6 Concentration-dependent inhibition of the selected
7aB-3 compound on glucagon-induced cAMP production. cAMP
production was measured after treatment of CHO-K1 GCGR Gs cells
with the indicated concentration of 7aB-3 in the presence of 0.1 nm
glucagon. N = 3. The data are presented as the mean standard error
(SE)
3.2.5
In vivo pharmacokinetic profiles and
|
characteristics of 7aB-3
To characterize the pharmacokinetic properties of 7aB-3
in vivo, we further investigated the plasma concentration-
time profiles of 7aB-3 in rats following intravenous (IV)
and oral (PO) administration, as shown in Figure 8 (n = 3–4
for each administration). The plasma concentration was
measured up to 480 min for both administrations. The cal-
culated pharmacokinetic parameters are listed in Table 2.
After IV administration, 7aB-3 showed relatively moderate
total clearance (CL) (i.e., 23.6 ml min−1 kg−1) with a small
challenge tests. On the first day of the experiment, treat-
ment with 7aB-3 significantly decreased glucagon-induced
blood glucose levels at all time points measured in the HFD-
induced obese mice, and the effect was similar to that of
LGD-6972 (Figure 7a). The area under the curve (AUC)
was also significantly decreased in the 7aB-3- and LGD-
6972-treated mice (Figure 7b). Body weight was measured
daily, and there was no significant change in body weight

LEE Et aL.
15
|
FIGURE 7 Blood glucose-lowering effect of 7aB-3 in HFD-induced obese mice. HFD-induced obese mice were orally administered 50 mg/
kg of 7aB-3 or LGD-6972 once daily for 6 weeks. A. On the first day, the mice were fasted for 5 hr and then 50 mg/kg of 7aB-3 or LGD-6972
was orally administered. After 1 hr, 15 μg/kg of glucagon was intraperitoneally injected. Blood glucose levels were measured at the indicated
time points after glucagon injection (n = 7/group). B. AUC of glucagon-induced glucose excursion test. C. Body weight comparison between
experimental beginning (week 0) and end (week 6) (n = 4–6/group). D. Food intake was measured daily (n = 4–6/group). E. Non-fasting blood
glucose levels were measured on weeks 0, 3, and 4 (n = 4–6/group). F. Mice were fasted for 14 hr on week 6 of the experiment and 2 g/kg of
glucose was intraperitoneally injected. Blood glucose levels were measured at the indicated time points after glucose injection (n = 4–6/group).
Data are presented as the mean standard error (SE). * p < .05, ** p < .01, or *** p < .001 versus. Vehicle group
volume of distribution at steady state (Vss = 371.4 ml/kg).
The elimination half-life was 90 min. However, the oral phar-
macokinetics of 7aB-3 showed poor bioavailability (BA) of
approximately 3.7% in vivo. Collectively, compound 7aB-3
possesses poor oral pharmacokinetic properties, although the
mechanism of its low oral absorption remains unknown.
Thus, pharmacokinetic optimization to enhance oral absorp-
tion may be required.

16
LEE Et aL.
|
FIGURE 8 Mean plasma
concentration–time profiles of 7aB-3 after
IV and PO administration at doses 2 mg/kg
and 5 mg/kg, respectively, in rats (n = 3–4).
Vertical bars represent standard deviations
Intravenous
Oral
10000
1000
100
10
1
0
60
120
180
240
300
360
420
480
Time (min)
TABLE 2 Pharmacokinetic parameters of 7aB-3 after IV and PO
administration in rats (n = 3–4)
ACKNOWLEDGMENTS
This study was supported by a grant from the Kwangdong
Pharmaceutical Co., Ltd. and a grant from the Korea Health
Technology R&D Project through the Korea Health Industry
Development Institute (KHIDI), funded by the Ministry for
Health and Welfare of Korea (HR14C0001).
7aB-3
Pharmacokinetic
parameters
IV (2 mg/kg)
PO (5 mg/kg)
AUC0→∞ (µg min−1 ml−1
Terminal t1/2 (min)
MRT (min)
Cmax (μg/ml)
Tmax (min)
)
86.3 13.7
90.0 15.6
15.8 2.0
–
8.0 0.5
–
183.2 19.1
0.0353 0.0067
80.0 34.6
CONFLICTS OF INTEREST
There are no competing financial, professional, or personal
interests that might have influenced the presentation of the
work described in this manuscript.
–
Vss (ml/kg)
371.4 67.1
23.6 3.3
CL (ml min−1 kg−1
BA (%)
)
AUTHOR CONTRIBUTIONS
3.7
Dongyun Shin and Hee-Sook Jun conceived and designed
the experiments. Chang-Yong Lee, Hojung Choi, Eun-
Young Park, Thi-Thao-Linh Nguyen, and Han-Joo Maeng
performed the experiments and analyzed the data. In par-
ticular, Chang-Young Lee prepared all compounds. Hojung
Choi and Eun-Young Park performed the biological assays
and in vivo experiments. Han-Joo Maeng performed the
pharmacokinetic analysis. Kyoung Mee Lee, Dongyun Shin,
and Hee-Sook Jun conducted the data analyses. Chang-Yong
Lee, Hojung Choi, Dongyun Shin, and Hee-Sook Jun sum-
marized the work and wrote the manuscript.
4
CONCLUSION
|
In summary, we synthesized 18 novel biphenylsulfonamide de-
rivatives. Among them, several compounds significantly inhib-
ited GCGR-dependent, glucagon-induced cAMP, and glucose
production. Based on the inhibitory efficacy of glucagon-
induced cAMP production in CHO-K1 GCGR Gs cells and
glucagon-induced glucose production in mouse primary hepat-
ocytes, we selected compound 7aB-3 for further study. 7aB-3
decreased glucagon-induced cAMP production and glucose
production in in vivo assays. In addition, the GCGR antago-
nist 7aB-3 showed efficacy equivalent to that of LGD-6972
in glucagon-signaling attenuation and blood glucose-lowering
in a diabetic animal model. However, the pharmacokinetics of
the GCGR antagonist, 7aB-3, showed low oral bioavailability,
requiring further pharmacokinetic optimization for oral drug
development. Our results suggest that compound 7aB-3 is a
potential glucose-lowering agent for treating type 2 diabetes.
ETHICAL APPROVAL
All animal experiments were approved by the Institutional
Animal Care and Use Committee of the Lee Gil Ya Cancer
and Diabetes Institute (LCDI-2015-0006).
DATA AVAILABILITY STATEMENT
The data used to support the findings of this study are avail-
able from the corresponding author upon request.

LEE Et aL.
17
|
interactions of procainamide and N-acetylprocainamide with cimeti-
dine, an inhibitor of rOCT2 and rMATE1, in rats. Pharmaceutics,
11(3), 108. https://doi.org/10.3390/pharmaceutics11030108
Jiang, G., & Zhang, B. B. (2003). Glucagon and regulation of glucose
metabolism. American Journal of Physiology - Endocrinology and
Metabolism, 284(4), E671–E678. https://doi.org/10.1152/ajpen
do.00492.2002
Jiang, Y., Cypess, A. M., Muse, E. D., Wu, C. R., Unson, C. G.,
Merrifield, R. B., & Sakmar, T. P. (2001). Glucagon receptor ac-
tivates extracellular signal-regulated protein kinase 1/2 via cAMP-
dependent protein kinase. Proceedings of the National Academy of
Sciences, 98(18), 10102–10107. https://doi.org/10.1073/pnas.13120
0398
Kelly, R. P., Garhyan, P., Raddad, E., Fu, H., Lim, C. N., Prince, M.
J., Pinaire, J. A., Loh, M. T., & Deeg, M. A. (2015). Short-term
administration of the glucagon receptor antagonist LY2409021 low-
ers blood glucose in healthy people and in those with type 2 diabe-
tes. Diabetes, Obesity and Metabolism, 17(4), 414–422. https://doi.
org/10.1111/dom.12446
Kharroubi, A. T., & Darwish, H. M. (2015). Diabetes mellitus: The ep-
idemic of the century. World Journal of Diabetes, 6(6), 850–867.
https://doi.org/10.4239/wjd.v6.i6.850
Kimball, C. P., & Murlin, J. R. (1923). Aqueous extracts of pancreas: III.
Someprecipitationreactionsofinsulin.JournalofBiologicalChemistry,
58(1), 337–346. https://doi.org/10.1016/S0021-9258(18)85474-6
Kodra, J. T., Jørgensen, A. S., Andersen, B., Behrens, C., Brand, C. L.,
Christensen, I. T., Guldbrandt, M., Jeppesen, C. B., Knudsen, L. B.,
Madsen, P., Nishimura, E., Sams, C., Sidelmann, U. G., Pedersen,
R. A., Lynn, F. C., & Lau, J. (2008). Novel glucagon receptor an-
tagonists with improved selectivity over the glucose-dependent in-
sulinotropic polypeptide receptor. Journal of Medicinal Chemistry,
51(17), 5387–5396. https://doi.org/10.1021/jm7015599
REFERENCES
Authier, F., & Desbuquois, B. (2008). Glucagon receptors. Cellular
and Molecular Life Sciences, 65(12), 1880–1899. https://doi.
org/10.1007/s00018-008-7479-6
Bagger, J. I., Knop, F. K., Holst, J. J., & Vilsbøll, T. (2011). Glucagon
antagonism as a potential therapeutic target in type 2 diabetes.
Diabetes, Obesity and Metabolism, 13(11), 965–971. https://doi.
org/10.1111/j.1463-1326.2011.01427.x
Bergman, A., Tan, B., Somayaji, V. R., Calle, R. A., & Kazierad, D. J.
(2017). A 4-week study assessing the pharmacokinetics, pharma-
codynamics, safety, and tolerability of the glucagon receptor antag-
onist PF-06291874 administered as monotherapy in subjects with
type 2 diabetes mellitus. Diabetes Research and Clinical Practice,
126, 95–104. https://doi.org/10.1016/j.diabres.2017.01.019
Brubaker, P. L., & Drucker, D. J. (2002). Structure-function of the glu-
cagon receptor family of G protein-coupled receptors: The gluca-
gon, GIP, GLP-1, and GLP-2 receptors. Receptors and Channels,
8(3–4), 179–188. https://doi.org/10.3109/10606820213687
Choi, H., Lee, C. Y., Park, E. Y., Lee, K. M., Shin, D., & Jun, H. S.
(2018). Design, synthesis, and effects of novel phenylpyrimidines as
glucagon receptor antagonists. Bioorganic & Medicinal Chemistry,
26(21), 5701–5710. https://doi.org/10.1016/j.bmc.2018.10.013
Christensen, M., Bagger, J. I., Vilsboll, T., & Knop, F. K. (2011). The
alpha-cell as target for Type 2 diabetes therapy. Review of Diabetic
Studies, 8(3), 369–381. https://doi.org/10.1900/RDS.2011.8.369
DeFronzo, R. A. (2009). From the triumvirate to the ominous octet: A
new paradigm for the treatment of type 2 diabetes mellitus. Diabetes,
58(4), 773–795. https://doi.org/10.2337/db09-9028
DeFronzo, R. A., Triplitt, C. L., Abdul-Ghani, M., & Cersosimo,
E. (2014). Novel agents for the treatment of type 2 diabetes.
Diabetes Spectrum, 27(2), 100–112. https://doi.org/10.2337/diasp
ect.27.2.100
Lau, J., Behrens, C., Sidelmann, U. G., Knudsen, L. B., Lundt, B.,
Sams, C., & Madsen, P. (2007). New beta-alanine derivatives are
orally available glucagon receptor antagonists. Journal of Medicinal
Chemistry, 50(1), 113–128. https://doi.org/10.1021/jm058026u
Lee, E. C. Y., Tu, M., Stevens, B. D., Bian, J., Aspnes, G., Perreault,
C., Sammons, M. F., Wright, S. W., Litchfield, J., Kalgutkar, A.
S., Sharma, R., Didiuk, M. T., Ebner, D. C., Filipski, K. J., Brown,
J., Atkinson, K., Pfefferkorn, J. A., & Guzman-Perez, A. (2014).
Identification of a novel conformationally constrained glucagon
receptor antagonist. Bioorganic & Medicinal Chemistry Letters,
24(3), 839–844. https://doi.org/10.1016/j.bmcl.2013.12.090
Li, X. C., Carretero, O. A., Shao, Y., & Zhuo, J. L. (2006). Glucagon
receptor-mediated extracellular signal-regulated kinase 1/2 phos-
phorylation in rat mesangial cells: Role of protein kinase A and
phospholipase C. Hypertension, 47(3), 580–585. https://doi.
org/10.1161/01.HYP.0000197946.81754.0a
Li, X. C., & Zhuo, J. L. (2013). Current insights and new perspectives
on the roles of hyperglucagonemia in non insulin-dependent type
2 diabetes. Current Hypertension Reports, 15(5), 522–530. https://
doi.org/10.1007/s11906-11013-10383-y
Ling, A., Plewe, M. B., Truesdale, L. K., Lau, J., Madsen, P., Sams, C.
J., Thogersen, H. (2000). Glucagon antagonist/inverse agonist. WO
2000/069810.
Guzman, C. B., Zhang, X. M., Liu, R., Regev, A., Shankar, S., Garhyan,
P., Pillai, S. G., Kazda, C., Chalasani, N., & Hardy, T. A. (2017).
Treatment with LY2409021, a glucagon receptor antagonist, in-
creases liver fat in patients with type 2 diabetes. Diabetes, Obesity
and Metabolism, 19(11), 1521–1528. https://doi.org/10.1111/
dom.12958
Han, D.-G., Cha, E., Joo, J., Hwang, J. S., Kim, S., Park, T., Jeong, Y.-S.,
Maeng, H.-J., Kim, S.-B., & Yoon, I.-S. (2021). Investigation of the
factors responsible for the poor oral bioavailability of acacetin in rats:
Physicochemical and biopharmaceutical aspects. Pharmaceutics,
13(2), https://doi.org/10.3390/pharmaceutics13020175
Han, J., Zhang, M., Froese, S., Dai, F. F., Robitaille, M., Bhattacharjee,
A., Huang, X., Jia, W., Angers, S., Wheeler, M. B., & Wei, L. I.
(2015). The identification of novel protein-protein interactions
in liver that affect glucagon receptor activity. PLoS One, 10(6),
e0129226. https://doi.org/10.1371/journal.pone.0129226
Hogan, P. J., & Cox, B. G. (2009). Aqueous process chemistry: The
preparation of aryl sulfonyl chlorides. Organic Process Research &
Development, 13(5), 875–879. https://doi.org/10.1021/op9000862
Jazayeri, A., Doré, A. S., Lamb, D., Krishnamurthy, H., Southall, S.
M., Baig, A. H., Bortolato, A., Koglin, M., Robertson, N. J., Errey,
J. C., Andrews, S. P., Teobald, I., Brown, A. J. H., Cooke, R. M.,
Weir, M., & Marshall, F. H. (2016). Extra-helical binding site of a
glucagon receptor antagonist. Nature, 533(7602), 274–277. https://
doi.org/10.1038/nature17414
Madsen, P., Kodra, J. T., Behrens, C., Nishimura, E., Jeppesen, C. B.,
Pridal, L., Andersen, B., Knudsen, L. B., Valcarce-Aspegren, C.,
Guldbrandt, M., Christensen, I. T., Jørgensen, A. S., Ynddal, L.,
Brand, C. L., Bagger, M. A., & Lau, J. (2009). Human glucagon
Jeong, Y. S., Balla, A., Chun, K. H., Chung, S. J., & Maeng, H. J. (2019).
Physiologically-based pharmacokinetic modeling for drug-drug

18
LEE Et aL.
|
receptor antagonists with thiazole cores. A novel series with supe-
rior pharmacokinetic properties. Journal of Medicinal Chemistry,
52(9), 2989–3000. https://doi.org/10.1021/jm8016249
LGD-6972 in healthy subjects and subjects with type 2 diabetes
mellitus. Diabetes, Obesity and Metabolism, 19(1), 24–32. https://
doi.org/10.1111/dom.12752
Maeng, H. J., Doan, T. N. K., & Yoon, I. S. (2019). Differential regula-
tion of intestinal and hepatic CYP3A by 1alpha,25-dihydroxyvitamin
D3: Effects on in vivo oral absorption and disposition of buspirone
in rats. Drug Development Research, 80(3), 333–342. https://doi.
org/10.1002/ddr.21505
Petersen, K. F., & Sullivan, J. T. (2001). Effects of a novel glucagon
receptor antagonist (Bay 27–9955) on glucagon-stimulated glucose
production in humans. Diabetologia, 44(11), 2018–2024. https://
doi.org/10.1007/s001250100006
Quesada, I., Tudurí, E., Ripoll, C., & Nadal, Á. (2008). Physiology of
the pancreatic α-cell and glucagon secretion: Role in glucose ho-
meostasis and diabetes. Journal of Endocrinology, 199(1), 5–19.
https://doi.org/10.1677/joe-08-0290
Qureshi, S. A., Rios Candelore, M., Xie, D., Yang, X., Tota, L. M.,
Ding, V.- D.-H., Li, Z., Bansal, A., Miller, C., Cohen, S. M., Jiang,
G., Brady, E., Saperstein, R., Duffy, J. L., Tata, J. R., Chapman,
K. T., Moller, D. E., & Zhang, B. B. (2004). A Novel Glucagon
Receptor Antagonist Inhibits Glucagon-Mediated Biological
Effects. Diabetes, 53(12), 3267–3273. https://doi.org/10.2337/diabe
tes.53.12.3267
Rennison, D., Conole, D., Tingle, M. D., Yang, J., Eason, C. T., &
Brimble, M. A. (2013). Synthesis and methemoglobinemia-inducing
properties of analogues of para-aminopropiophenone designed as
humane rodenticides. Bioorganic & Medicinal Chemistry Letters,
23(24), 6629–6635. https://doi.org/10.1016/j.bmcl.2013.10.046
Sammons, M. F., & Lee, E. C. Y. (2015). Recent progress in the develop-
ment of small-molecule glucagon receptor antagonists. Bioorganic
& Medicinal Chemistry Letters, 25(19), 4057–4064. https://doi.
org/10.1016/j.bmcl.2015.07.092
Vajda, E. G., Potter, S. C., Fujitaki, J. M., Reddy, R. K., Van Poelje,
P. D., Lee, Y.-H., & Marschke, K. B. (2012). LGD-6972, a potent,
orally-bioavailable, small molecule glucagon receptor antagonist for
the treatment of type 2 diabetes. Paper presented at the Diabetes.
Xiong, Y., Guo, J., Candelore, M. R., Liang, R., Miller, C., Dallas-
Yang, Q., & Parmee, E. R. (2012). Discovery of a novel glucagon
receptor antagonist N-[(4-{(1S)-1-[3-(3, 5-dichlorophenyl)-5-(6-m
ethoxynaphthalen-2-yl)-1H-pyrazol-1-yl]ethyl}phenyl)carbonyl]-β-
alanine (MK-0893) for the treatment of type II diabetes. Journal of
Medicinal Chemistry, 55(13), 6137–6148. https://doi.org/10.1021/
jm300579z
Yoon, J. Y., Choi, H., & Jun, H. S. (2017). The effect of phloroglucinol,
a component of ecklonia cava extract, on hepatic glucose produc-
tion. Mar Drugs, 15(4), 106. https://doi.org/10.3390/md15040106
Yoon, J. H., Nguyen, T. T., Duong, V. A., Chun, K. H., & Maeng, H. J.
(2020). Determination of KD025 (SLx-2119), a selective ROCK2
Inhibitor, in rat plasma by high-performance liquid chromatography-
tandem mass spectrometry and its pharmacokinetic application.
Molecules, 25(6), 1369. https://doi.org/10.3390/molecules25061369
Zysman-Colman, E., Arias, K., & Siegel, J. S. (2009). Synthesis of aryl-
bromides from arenes and N-bromosuccinimide (NBS) in acetonitrile
— A convenient method for aromatic bromination. Canadian Journal
of Chemistry, 87(2), 440–447. https://doi.org/10.1139/V08-176
SUPPORTING INFORMATION
Additional supporting information may be found online in
the Supporting Information section.
Song, Y.-K., Yoon, J.-H., Woo, J. K., Kang, J.-H., Lee, K.-R., Oh, S. H.,
Chung, S.-J., & Maeng, H.-J. (2020). Quercetin is a flavonoid breast
cancer resistance protein inhibitor with an impact on the oral phar-
macokinetics of sulfasalazine in rats. Pharmaceutics, 12(5), 397.
https://doi.org/10.3390/pharmaceutics12050397
Vajda, E. G., Logan, D., Lasseter, K., Armas, D., Plotkin, D. J., Pipkin,
J. D., Li, Y.-X., Zhou, R., Klein, D., Wei, X., Dilzer, S., Zhi, L., &
Marschke, K. B. (2017). Pharmacokinetics and pharmacodynamics
of single and multiple doses of the glucagon receptor antagonist
How to cite this article: Lee, C.-Y., Choi, H., Park,
E.-Y., Nguyen, T.-T., Maeng, H.-J., Mee Lee, K., Jun,
H.-S., & Shin, D. (2021). Synthesis and anti-diabetic
activity of novel biphenylsulfonamides as glucagon
receptor antagonists. Chem Biol Drug Des, 00, 1–18.
https://doi.org/10.1111/cbdd.13928