Synthesis and biological evaluation of glucagon-like peptide-1 receptor agonists

Article abstract of DOI:10.1007/s12272-013-0253-9

In this study, a series of fused-heterocyclic derivatives were systematically designed and synthesized using an efficient route, and evaluated in terms of GLP-1R agonist activity. We employed short synthetic steps and reactions that are tolerant of the presence of various functional groups and suitable for parallel operations to enable the rapid generation of libraries of diverse and structurally complex small molecules. Of the compounds synthesized, 3-(8-chloro-6-(trifluoromethyl)imidazo[1,2-a] pyridin-2-yl)phenyl methanesulfonate (8e) was the most potent agonist with an EC₅₀ of 7.89 μM, and thus is the compound with the greatest potential for application. These findings represent a valuable starting point for the design and discovery of small-molecule GLP-1R agonists that can be administered orally.

Full text of DOI:10.1007/s12272-013-0253-9

Arch. Pharm. Res.
DOI 10.1007/s12272-013-0253-9
RESEARCH ARTICLE
Synthesis and biological evaluation of glucagon-like peptide-1
receptor agonists
•
Yu-Juan Zhang Liu-Lan Shen Hyae-Gyeong Cheon
•
•
•
Yong-Nan Xu Jin-Hyun Jeong
Received: 9 April 2013 / Accepted: 23 September 2013
Ó The Author(s) 2013. This article is published with open access at Springerlink.com
Abstract In this study, a series of fused-heterocyclic
derivatives were systematically designed and synthesized
using an efficient route, and evaluated in terms of GLP-1R
agonist activity. We employed short synthetic steps and
reactions that are tolerant of the presence of various
functional groups and suitable for parallel operations to
enable the rapid generation of libraries of diverse and
structurally complex small molecules. Of the compounds
synthesized, 3-(8-chloro-6-(trifluoromethyl)imidazo[1,2-a]
pyridin-2-yl)phenyl methanesulfonate (8e) was the most
potent agonist with an EC50 of 7.89 lM, and thus is the
compound with the greatest potential for application. These
findings represent a valuable starting point for the design
and discovery of small-molecule GLP-1R agonists that can
be administered orally.
Introduction
Type 2 diabetes mellitus (DM2), a state of hormonal dis-
ruption and incretin deficiency, is increasingly becoming a
worldwide epidemic (Kwak and Ha 2013). Current drugs
utilized in the treatment of DM2 have well-established
shortcomings: (1) increasing body weight and (2) increas-
ing loss of b-cell function (Whitehouse 1997; Giugliano
et al. 2009). However, the recent emergence of incretin-
based therapies, which focus on glucagon-like peptide-1
(GLP-1), has attracted much interest.
GLP-1 is a peptide hormone of 30 amino acid residues.
As a peptide, it has a very short half-life (\2 min) (Deacon
et al. 1995). Such a short half-life has limited the utility of
native GLP-1 in the treatment of DM2. The effort to
identify GLP-1 analogues has resulted in the development
of the drugs exenatide (Sennik et al. 2011; Buse et al. 2004)
Keywords Small molecule agonists ꢀ GLP-1R ꢀ
Heterocycles ꢀ Structure–activity relationships ꢀ
Synthesis
¨
and liraglutide (Sjoholm 2010; Hribal and Sesti 2010).
However, the requirement for injection limits the clinical
utility of these peptide drugs. Therefore, orally active,
small-molecule agonists of the GLP-1 receptor (GLP-1R)
are highly sought after (Murphy and Bloom 2007).
Electronic supplementary material The online version of this
article (doi:10.1007/s12272-013-0253-9) contains supplementary
material, which is available to authorized users.
Figure 1 shows synthetic small molecule agonists
reported by several groups (Teng et al. 2000; Wang et al.
2009; Teng et al. 2007; Kopin 2004; Gong et al. 2010).
Compound 6b, characterized by a novel imidazopyridine hit
core, was identified from a library of 10,000 heterocyclic
small molecules (Gong et al. 2010). As a small and drug-like
active molecule, it represents an interesting starting point for
the development of novel drugs. Therefore, we selected this
compound as a model. In an effort to move away from the
labile ester group of the phenol, we planned a synthetic
pathway of new derivatives of imidazo[1,2-a]pyridine-
based molecules (Fig. 2). To evaluate the structure–activity
relationship, we designed and synthesized a series of
Y.-J. Zhang ꢀ Y.-N. Xu
School of Pharmaceutical Engineering, Shenyang
Pharmaceutical University, Shenyang 110016, Liaoning, China
Y.-J. Zhang ꢀ L.-L. Shen ꢀ J.-H. Jeong (&)
Yonsei Institute of Pharmaceutical Sciences and College
of Pharmacy, Yonsei University, 162-Songdo-dong, Yeonsu-gu,
Incheon 406-840, Republic of Korea
e-mail: organicjeong@yonsei.ac.kr
H.-G. Cheon
Department of Pharmacology and Pharmaceutical Sciences,
Gachon University of Medicine and Science, Incheon 406-799,
Republic of Korea
123

Y.-J. Zhang et al.
(b)
H
3C
R
O
N
N
N
X
O
N
S
N
19, 21a-21b,
27a-27d
N
S
(a)
X
N
Cl
Cl
S
R
F
3C
N
R
R
F
F
OAc
O
N
N
N
N
N
F
3C
F
37a, 37b, 37d
37c, 37e
OAc
O
N
6a-6b, 8a-8f,
12a-12c, 33a-33c
X
Cpd.1
6Cu
Cl
N
CH3
N
l
C
N
O2
N
N
N
Fig. 2 Structures of synthesized compounds. a Synthesized imi-
dazo[1,2-a]pyridine-based molecules. b Other synthesized heterocy-
cle-series compounds
N
CH3
N
F
3C
H
OH
Comounpd A
Comounpd 6b
(0.92 g, 4.7 mmol) in EtOH (50 mL) was added NaHCO3
(0.31 g, 4.7 mmol) at room temperature. The reaction mix-
ture was heated to reflux and monitored by TLC (hexane/
ethyl acetate: 2/1) until completion. After removing EtOH,
the residue was extracted with ethyl acetate and water. The
combined organic phases were washed with water, 1 N HCl,
and brine, dried, and filtered and concentrated in vacuo. The
residue was purified by silica gel column chromatography
(ethyl acetate/hexane = 10–20 %, Rf = 0.23).
Fig. 1 Known ago-allosteric modulators of GLP-1R
heterocyclic derivatives containing a ring-junction nitrogen
using a three-dimensional (3D) pharmacophore model
reported previously (Gong et al. 2010) (Fig. 2). For the first
stage, only combinations of five- and six-membered rings
are considered, including imidazo[1,5-a]pyridine, imi-
dazo[1,2-a]pyrimidine and imidazo[1,2-a]pyrazine. We
employed short synthetic steps and reactions that are tolerant
of the presence of various functional groups and suitable for
parallel operations to enable the rapid generation of libraries
of diverse, structurally complex, small molecules.
3-(6-(Trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)phenyl
acetate (6a)
Pale yellow solid; Yield: 64 %;1H NMR (500 MHz,
CDCl3): d 2.33 (s, 3H), 7.10 (d, J = 10.1 Hz, 1H), 7.32 (d,
J = 11.9 Hz, 1H), 7.45 (t, J = 10.0 Hz, 1H), 7.70–7.73
(m, 2H), 7.80 (d, J = 9.7 Hz, 1H), 7.94(s, 1H), 8.49 (s,
1H);13C NMR (125 MHz, CDCl3); d 21.1, 109.8, 117.0,
117.3, 118.0, 119.5, 121.1, 121.8, 123.6, 124.8, 129.9,
134.2, 145.2, 146.2, 151.2, 169.6; EI-HRMS calculated for
(C16H11F3N2O2?H)? 321.0851, found 321.0860.
Materials and methods
Chemistry
All the chemicals used in synthesis were supplied by Al-
drichand TCI, and were used without further purification.
All solvents were purified and stored in a dry condition.
Reaction progress was determined by thin-layer chroma-
tography (TLC) on Merck TLC Silica gel 60 F245 plates.
Column chromatography was carried out using a silica gel
60 (63–200 mesh, Merck). NMR spectra were recorded on
3-(8-Chloro-6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-
yl)phenyl acetate (6b)
Pale yellow solid; Yield: 29 %;1H NMR (500 MHz,
CDCl3): d 2.34 (s, 3H), 7.10 (dd, J = 10.1, 2.1 Hz, 1H),
7.41–7.47 (m, 2H), 7.76 (t, J = 2.2 Hz, 1H), 7.81 (d,
J = 10.2 Hz, 1H), 7.98 (s, 1H), 8.43 (s, 1H);13C NMR
(125 MHz, CDCl3): d 21.2,111.2, 119.62, 119.65, 119.68,
122.0, 123.3, 123.4, 123.7, 124.3, 129.8, 134.0, 142.7,
147.0, 151.1, 169.6; EI-HRMS calculated for (C16H10
ClF3N2O2?H)? 355.0461, found 355.0470.
1
Agilent 400 instruments operating at 400 MHz for H and
100 MHz for 13C, and Agilent 500 instruments operating at
500 MHz for 1H and 125 MHz for 13C. Chemical shifts are
expressed as parts per million (ppm) with tetramethylsilane
as the internal standard. MS spectra were recorded on an
Agilent G6530A Q-TOF.
General synthetic procedure for (6a–b)
General synthetic procedure for (8a and 8d)
To a stirred solution of bromomethylketone 3 (1.21 g,
4.7 mmol) and 2-amino-5-trifuoromethylpyridine 4 (0.61 g,
4.7 mmol) or 2-amino-3-chloro-5-trifluoromethylpyridine 5
To a mixture of 7a (99 mg, 0.36 mmol) or 7b (113 mg,
0.36 mmol) and K2CO3 (250 mg, 1.81 mmol) in acetone
123

Synthesis of GLP-1R agonists
(10 mL) was added 1-chloroacetone (1 mL, 34.83 mmol)
at room temperature. The reaction mixture was heated to
reflux for 6 h. After removing acetone and 1-chloroace-
tone, the residue was extracted with ethyl acetate and
water. The combined organic phases were washed with
water and brine, dried, and filtered and concentrated in
vacuo. The residue was purified by silica gel column
chromatography (ethyl acetate/hexane = 10–20 %, Rf =
0.25).
J = 9.9 Hz, 1H), 7.72 (d, J = 11.7 Hz, 1H), 7.89–7.92 (m,
2H), 7.98 (s, 1H), 8.51 (s, 1H);13C NMR (125 MHz,
CDCl3): d 37.6, 109.9, 118.3, 119.7, 121.09, 121.11,
122.0, 124.77, 124.81, 125.0, 130.5, 135.3, 145.3, 145.9,
149.8; EI-HRMS calculated for (C15H11F3N2O3S ? Na)?
379.0340, found 379.0360.
3-(6-(Trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)phenyl
4-methylbenzenesulfonate (8c)
General synthetic procedure for (8b and 8e)
Pale yellow solid; Yield: 64 %;1H NMR (500 MHz,
CDCl3): d 2.43 (s, 3H), 6.91 (d, J = 8.2 Hz, 1H),
7.30–7.35 (m, 4H), 7.62 (s, 1H), 7.67 (m, 1H), 7.75 (d,
J = 7.4 Hz, 2H), 7.84–7.89 (m, 2H), 8.48(s, 1H);13C NMR
(125 MHz, CDCl3): d 21.7, 109.8, 118.2, 120.2, 120.9,
122.0, 124.8, 128.5, 129.8, 130.0, 132.2, 134.9, 145.3,
145.5, 146.1, 150.1; EI-HRMS calculated for (C21H15
F3N2O3S ? Na)? 455.0653, found 455.0656.
To a solution of 7a (98 mg, 0.35 mmol) or 7b (121 mg,
0.45 mmol) in pyridine (5 mL) was added methanesulfonyl
chloride (66 mg, 0.60 mmol) dropwise with stirring over-
night in an ice bath. The reaction mixture was quenched
with water in an ice bath and extracted with ethyl acetate
(3 9 30 mL). The combined organic phases were washed
with water, 1 N HCl, and brine, dried, and filtered and
concentrated in vacuo. The residue was purified by silica
gel column chromatography (hexane/ethyl acetate = 4/1,
Rf = 0.25).
1-(3-(8-Chloro-6-(trifluoromethyl)imidazo[1,2-a]pyridin-
2-yl)phenoxy)propan-2-one (8d)
Pale yellow solid; Yield: 50 %;1H NMR (500 MHz,
CDCl3): d 2.32 (s, 3H), 4.64 (s, 2H), 6.89 (dd, J = 8.2,
2.7 Hz, 1H), 7.35 (t, J = 7.9 Hz, 1H), 7.40 (s, 1H),
7.54–7.57 (m, 2H), 7.97 (s, 1H), 8.43 (s, 1H);13C NMR
(125 MHz, CDCl3): d 26.8, 73.0, 111.3, 112.6, 115.0,
116.6, 116.9, 119.6, 119.7, 123.3, 124.3, 130.1, 134.1,
142.7, 147.6, 158.1, 205.5; EI-HRMS calculated for
(C17H12ClF3N2O2?H)? 369.0618, found 369.0669.
General synthetic procedure for (8c and 8f)
To a solution of 7a (90 mg, 0.32 mmol) or 7b (100 mg,
0.32 mmol) in pyridine (10 mL) was added toluenesulfo-
nyl chloride (80 mg, 0.42 mmol) dropwise in an ice bath.
After stirring for 2 h at room temperature, the reaction
mixture was quenched with water in an ice bath and then
extracted with ethyl acetate (3 9 30 mL). The combined
organic phases were washed with water, 1 N HCl, and
brine, dried, and filtered and concentrated. The residue was
purified by silica gel column chromatography (hexane/
ethyl acetate = 8/1, Rf = 0.23).
3-(8-Chloro-6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-
yl)phenyl methanesulfonate (8e)
Pale yellow solid;Yield: 45 %;1H NMR (500 MHz,
CDCl3): d 3.20 (s, 3H), 7.31 (d, J = 10.2 Hz, 1H),
7.43 (s, 1H), 7.50 (t, J = 10.0 Hz, 1H), 7.91 (m, 1H),
7.94 (d, J = 9.8 Hz, 1H), 8.03 (s, 1H), 8.45(s,
1H);13C NMR (125 MHz, CDCl3): d 37.7, 111.5, 116.9,
117.2, 120.0, 122.2, 123.38, 123.43, 124.5, 125.3, 130.4,
134.8, 142.8, 146.4, 149.7; EI-HRMS calculated for
(C15H10ClF3N2O3S?H)? 391.0131, found 391.0135.
1-(3-(6-(Trifluoromethyl)imidazo[1,2-a]pyridin-2-
yl)phenoxy)propan-2-one (8a)
Pale yellow solid; Yield: 63 %;1H NMR (500 MHz,
CDCl3): d 2.32 (s, 3H), 4.64 (s, 2H), 6.91 (d, J = 10.1 Hz,
1H), 7.32–7.40 (m, 2H), 7.55–7.57 (m, 2H), 7.73 (d,
J = 11.8 Hz, 1H), 7.94 (s, 1H), 8.50 (s, 1H);13C NMR
(125 MHz, CDCl3): d 26.7, 73.0, 109.6, 112.2, 114.8,
118.1, 119.6, 120.7, 122.4, 124.6, 126.7, 130.1, 134.6,
145.2, 147.1, 158.2, 205.5; EI-HRMS calculated for
(C17H13F3N2O2?H)? 335.1007, found 335.1019.
3-(8-Chloro-6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-
yl)phenyl 4-methylbenzene sulfonate (8f)
Pale yellow solid; Yield: 38 %;1H NMR (500 MHz,
CDCl3): d 2.43 (s, 3H), 6.91 (d, J = 8.2 Hz, 1H),
7.27–7.33 (m, 3H), 7.39 (m, 1H), 7.61 (s, 1H), 7.74 (d,
J = 8.2 Hz, 2H), 7.87 (d, J = 7.8 Hz, 1H), 7.94 (s, 1H),
8.48(s, 1H);13C NMR (125 MHz, CDCl3): d 21.7, 111.5,
119.8, 120.3, 122.2, 123.5, 124.3, 125.1, 126.9, 128.5,
130.0, 132.3, 134.4, 138.6, 145.6, 145.8, 146.5, 149.8,
3-(6-(Trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)phenyl
methanesulfonate (8b)
Pale yellow solid; Yield: 44 %;1H NMR (500 MHz,
CDCl3): d 3.20 (s, 3H), 7.29–7.36 (m, 2H), 7.50 (t,
123

Y.-J. Zhang et al.
4-Methyl-N-(3-(6-(trifluoromethyl)imidazo[1,2-a]pyridin-
150.0. EI-HRMS calculated for (C21H14ClF3N2O3S ?
Na)? 489.0263, found 489.0274.
2-yl)phenyl)benzene sulfonamide (12c)
Pale yellow solid;Yield: 21 %;1H NMR (500 MHz,
CDCl3): d 2.30 (s, 3H), 7.14–7.19 (m, 3H), 7.26–7.29 (m,
2H), 7.63 (dd, J = 7.7, 0.9 Hz, 1H), 7.66–7.70 (m, 4H),
7.85 (brs, NH), 7.89 (s, 1H), 8.45(s, 1H);13C NMR
(125 MHz, CDCl3): d 21.5, 109.9, 118.0, 118.8, 120.7,
121.0, 122.7, 124.7, 127.3, 129.7, 129.9, 134.0, 136.0,
137.5, 143.9, 145.2, 146.5; EI-HRMS calculated for
(C21H16ClF3N3O2S?H)? 432.0994, found 432.1020.
General synthetic procedure for (12a–c)
Pyridium bromide perbromide (1.79 g, 5.60 mmol) was
added to a solution of 10a–c (0.9 g, 5.08 mmol) in AcOH
(100 mL) with stirring for 3 h at room temperature. The
reaction mixture was poured into ice-cold water and then
extracted with ethyl acetate (3 9 50 mL). The combined
organic phases were washed with saturated aqueous
NaHCO3, water, and brine, dried, and filtered and con-
centrated in vacuo to give crude 11a–c as a yellow oil
(1.29 g, 98 %). The resulting crude 11a–c could be used
without further purification. To a stirred solution of bro-
momethylketone 11a–c (1.30 g, 5.1 mmol) and amino-
pyridine 4 (0.82 g, 5.1 mmol) in EtOH (80 mL) was added
NaHCO3 (0.43 g, 5.1 mmol) at room temperature. The
reaction mixture was heated to reflux for 8 h. After
removing EtOH, the residue was extracted with ethyl
acetate and water. The combined organic phases were
washed with water, 1 N HCl, and brine, dried, and filtered
and concentrated in vacuo. The residue was purified by
silica gel column chromatography (12a–b, hexane/ethyl
acetate = 1/1, Rf = 0.24; 12c, hexane/ethyl acetate = 4/1,
Rf = 0.22).
3-(8-Chloro-6-(trifluoromethyl)imidazo[1,5-a]pyridin-3-
yl)phenyl acetate (19)
To a solution of 18 (300 mg, 0.80 mmol) in benzene
(10 mL) was added POCl3 (1.2 mL, 13.04 mmol) dropwise
at room temperature. The reaction mixture was heated to
reflux for 6 h. After cooling to room temperature, the
mixture was poured into iced-water and then extracted with
ethyl acetate (3 9 50 mL). The combined organic phases
were washed with water and brine, dried, and filtered and
concentrated in vacuo. The residue was purified by silica
gel column chromatography (hexane/ethyl acetate = 12/1,
Rf = 0.25). White solid; Yield: 90 %;1H NMR (400 MHz,
CDCl3): d 2.35 (s, 3H), 6.92 (s, 1H), 7.25 (d, J = 8.6 Hz,
1H), 7.54–7.61 (m, 3H), 7.77 (s, 1H), 8.51(s, 1H); 13C
NMR (100 MHz, CDCl3): d 21.1, 113.9, 117.6, 117.9,
119.6, 121.5, 121.8, 122.5, 122.9, 124.9, 125.3, 126.5,
126.8, 129.7, 130.1, 130.5, 151.3, 169.2; EI-HRMS cal-
culated for (C16H10ClF3N2O2?H)? 355.0461, found
355.0474.
N-(3-(6-(Trifluoromethyl)imidazo[1,2-a]pyridin-2-
yl)phenyl)acetamide(12a)
Pale yellow solid;Yield: 39 %;1H NMR (500 MHz,
CDCl3): d 2.19 (s, 3H), 7.31 (d, J = 11.6 Hz, 1H), 7.39
(t, J = 9.8 Hz, 1H), 7.51 (s, NH), 7.60 (d, J = 9.8 Hz,
1H), 7.68 (d, J = 11.1 Hz, 2H), 7.93 (s, 1H), 8.07 (s,
1H), 8.47(s, 1H);13C NMR (125 MHz, CDCl3): d 24.7,
109.6, 117.4, 118.1, 119.9, 120.7, 122.0, 124.6, 124.7,
129.6, 133.7, 138.5, 145.2, 147.1, 168.5; EI-HRMS
calculated for (C16H12F3N3O ? Na)? 342.0830, found
342.0835.
3-(8-Chloro-6-(trifluoromethyl)imidazo[1,5-a]pyridin-3-
yl)phenyl cyclohexane carboxylate (21a)
To a solution of 19 (257 mg, 0.72 mmol) in THF (20 mL)
was added a solution of NaOH (50 mg, 1.25 mmol) in
water (10 mL) with stirring for 3 h at room temperature.
After removing THF, the resulting mixture was extracted
with ethyl acetate. The combined organic phases were
washed with water and brine, dried, and filtered and con-
centrated in vacuo. The resulting crude 20 could be used
without further purification. Cyclohexanecarboxylic chlo-
ride (28 mg, 0.19 mmol) was added to a solution of 20
(50 mg, 0.16 mmol), TEA (19 mg, 0.19 mmol), and
DMAP (4 mg, 0.03 mmol) in anhydrous CH2Cl2 (20 mL)
slowly in an ice bath. After stirring for 3 h at room tem-
perature, the reaction mixture was poured into ice water
and then extracted with CH2Cl2 (3 9 20 mL). The com-
bined organic phases were washed with 1 N HCl, water,
and brine, dried, and filtered and concentrated in vacuo.
N-(3-(6-(Trifluoromethyl)imidazo[1,2-a]pyridin-2-
yl)phenyl)methanesulfonamide (12b)
Pale yellow solid;Yield: 20 %;1H NMR (500 MHz,
CDCl3): d 3.05 (s, 3H), 7.30 (dt, J = 7.0, 1.3 Hz, 1H),
7.34 (dd, J = 9.5, 1.8 Hz, 1H), 7.40 (t, J = 7.9 Hz, 1H),
7.69–7.74 (m, 3H), 7.82 (t, J = 1.9 Hz, 1H), 7.99 (s,
1H), 8.51(s, 1H);13C NMR (125 MHz, CDCl3): d 39.4,
110.0, 118.0, 118.5, 120.7, 121.3, 123.0, 124.8, 124.9,
130.2, 130.3, 134.2, 137.6, 145.2, 146.2; EI-HRMS
calculated for (C15H12F3N3O2S?H)? 356.0681, found
356.0705.
123

Synthesis of GLP-1R agonists
N-(3-(8-Chloro-6-(trifluoromethyl)imidazo[1,5-a]pyridin-
The residue was purified by silica gel column chromatog-
raphy (hexane/ethyl acetate = 20/1, Rf = 0.23) to afford
21a as a pale yellow solid (64 mg, 94 %). 1H NMR
(400 MHz, CDCl3): d 1.25–1.39 (m, 4H), 1.57–1.65 (m,
2H), 1.81–1.84 (m, 2H), 2.06–2.09 (m, 2H), 2.58 (t,
J = 10.1 Hz, 1H), 6.92 (s, 1H), 7.24 (m, 1H), 7.52–7.59
(m, 3H), 7.78 (s, 1H), 8.52(s, 1H);13C NMR (100 MHz,
CDCl3): d 25.3, 25.6, 28.8, 43.1, 113.9, 117.7, 119.6,
121.4; EI-HRMS calculated for (C21H18ClF3N2O2 ? Na)?
445.0907, found 445.0907.
3-yl)phenyl)cyclohexane carboxamide (27c)
Pale yellow solid; Yield: 32 %;1H NMR (400 MHz,
CDCl3): d 1.28–1.35 (m, 2H), 1.50–1.59 (m, 2H), 1.71 (m,
2H), 1.83–1.85 (m, 2H), 1.94–1.97 (m, 2H), 2.26 (t,
J = 11.6 Hz, 1H), 6.90 (s, 1H), 7.44–7.52 (m, 2H), 7.55 (s,
1H), 7.71 (d, J = 7.6 Hz, 1H), 7.75 (s, 1H), 7.95 (s, 1H),
8.54 (s, 1H);13C NMR (100 MHz, CDCl3): d 25.6, 29.6,
46.5, 113.8, 117.4, 117.8, 119.7, 119.8, 121.0, 122.2,
123.3, 126.3, 129.4, 129.6, 130.0, 139.1, 141.5, 174.6; EI-
HRMS calculated for (C21H19ClF3N3O ? Na)? 444.1066,
found 444.1075.
1-(3-(8-Chloro-6-(trifluoromethyl)imidazo[1,5-a]pyridin-
3-yl)phenoxy)propan-2-one (21b)
1-(3-(8-Chloro-6-(trifluoromethyl)imidazo[1,5-a]pyridin-
Using the same method as for the preparation of 8a,
starting with 20 (73 mg, 0.23 mmol), 1-chloroacetone
(0.5 mL, 17.41 mmol) and K2CO3 (161 mg, 1.17 mmol),
21b was generated as a pale yellow solid (30 mg, 35 %).
1H NMR (400 MHz, CDCl3): d 2.30 (s, 3H), 4.66 (s, 2H),
6.91 (s, 1H), 7.06 (d, J = 7.9 Hz, 1H), 7.28 (s, 1H), 7.35
(d, J = 7.5 Hz, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.76 (s,
1H), 8.48 (s, 1H);13C NMR (100 MHz, CDCl3): d 26.6,
73.0, 113.9, 114.6, 116.5, 117.5, 117.9, 119.6, 120.9,
121.4, 122.1, 124.1, 126.5, 129.6, 130.2, 130.6, 141.3,
158.5, 204.6; EI-HRMS calculated for (C17H12ClF3N3
O2?H)? 369.0618, found 369.0670.
3-yl)phenyl)pyrrolidine-2,5-dione (27d)
Pale yellow solid; Yield: 28 %;1H NMR (400 MHz,
CDCl3): d 2.95 (s, 4H), 6.93 (s, 1H), 7.52 (d, J = 7.9 Hz,
1H), 7.69 (t, J = 7.8 Hz, 1H), 7.76–7.82 (m, 3H), 8.63 (s,
1H);13C NMR (100 MHz, CDCl3): d 28.4, 114.0, 117.7,
118.0, 119.7, 121.5, 122.6, 124.2, 125.7, 126.4, 127.1,
128.2, 129.8, 130.3, 132.7, 140.7, 175.8; EI-HRMS cal-
culated for (C18H11ClF3N3O2?H)? 394.0570, found
394.0608.
tert-Butyl (3-(8-chloro-6-(trifluoromethyl)imidazo[1,5-
a]pyridin-3-yl)phenyl) carbamate (27a)
General synthetic procedure for (27a–d)
Pale yellow solid;Yield: 13 %;1H NMR (400 MHz,
CDCl3): d 1.53 (s, 9H), 6.71 (s, 1H), 6.90 (s, 1H),
7.41–7.48 (m, 3H), 7.76 (s, 1H), 7.88 (s, 1H), 8.58 (s,
1H);13C NMR (100 MHz, CDCl3): d 28.2, 113.7, 117.3,
117.6, 118.3, 119.7, 119.9, 122.3, 122.5, 124.2, 126.3,
129.5, 130.0, 139.4, 141.6, 152.6; EI-HRMS calculated for
(C19H17ClF3N3O2 ? Na)? 434.0859, found 434.0865.
POCl3 (0.3 mL, 3.40 mmol) was added to a mixture of 24
or 26a–c (0.17 mmol) and pyridine (0.93 mL,
11.60 mmol) in anhydrous dichloroethane (14 mL) at room
temperature. The reaction mixture was heated to reflux for
7 h. After cooling to room temperature, the reaction mix-
ture was concentrated, filtered, and extracted with ethyl
acetate. The combined organic phases were washed with
1 N HCl, water, and brine, dried, and filtered and con-
centrated in vacuo. The residue was purified by silica gel
column chromatography (hexane/ethyl acetate = 2/1,
Rf = 0.24).
N-(3-(8-Chloro-6-(trifluoromethyl)imidazo[1,2-a]pyridin-
2-yl)phenyl)acetamide (33a)
Acetic anhydride (20 mg, 0.20 mmol) was added to a
mixture of 32 (50 mg, 0.16 mmol) and DMAP (3 mg,
0.02 mmol) in anhydrous CH2Cl2 (10 mL) with stirring for
1 h at room temperature. After removing the solvent, the
residue was extracted with ethyl acetate and water. The
combined organic phases were washed with 1 N HCl,
saturated Na2CO3, water, and brine, dried, and filtered and
concentrated in vacuo. The residue was purified by silica
gel column chromatography (ethyl acetate/hexane =
33–50 %, Rf = 0.23). Pale yellow solid; Yield: 50 %;1H
NMR (400 MHz, CDCl3): d 2.19 (s, 3H), 7.35–7.39 (m,
2H), 7.62–7.66 (m, 2H), 7.72 (s, 1H), 7.94 (s, 1H), 8.06 (s,
N-(3-(8-Chloro-6-(trifluoromethyl)imidazo[1,5-a]pyridin-
3-yl)phenyl)acetamide (27b)
Pale yellow solid; Yield: 96 %;1H NMR (400 MHz,
CDCl3): d 2.17 (s, 3H), 6.90 (s, 1H), 7.44–7.48 (m, 2H),
7.63 (d, J = 6.7 Hz, 1H), 7.74 (s, 1H), 7.96 (s, 1H), 8.16 (s,
1H), 8.53(s, 1H);13C NMR (100 MHz, CDCl3): d 24.5,
113.9, 117.5, 117.9, 119.7, 119.8, 121.1, 121.5, 122.1,
123.6, 124.2, 126.4, 129.2, 129.6, 130.0, 139.1, 141.5,
168.9; EI-HRMS calculated for (C16H11ClF3N3O ? Na)?
376.0440, found 376.0447.
123

Y.-J. Zhang et al.
1H), 8.39 (s, 1H);13C NMR (100 MHz, CDCl3): d 24.6,
111.3, 116.6, 117.6, 119.6, 120.3, 121.5, 122.2, 123.4,
124.2, 129.5, 133.1, 138.5, 142.6, 147.5, 168.8; EI-HRMS
calculated for (C16H11ClF3N3O ? Na)? 376.0440, found
376.0453.
room temperature, ethyl acetate was added, washed with
water and brine, dried over calcium oxide, and filtered and
concentrated in vacuo. The residue was purified by silica
gel column chromatography (gradient eluent: ethyl acetate/
hexane = 20–50 %, Rf = 0.21).
General synthetic procedure for (33b–c)
3-(6-Bromoimidazo[1,2-a]pyrimidin-2-yl)phenyl acetate
(37a)
Cyclohexanecarboxylic chloride (28 mg, 0.19 mmol) or
toluenesulfonyl chloride (39 mg, 0.21 mmol) was added to
a solution of 32 (50 mg, 0.16 mmol), TEA (19 mg,
0.19 mmol), and DMAP (4 mg, 0.03 mmol) in anhydrous
CH2Cl2 (10 mL) slowly in an ice bath. After stirring for
3 h at room temperature, the reaction mixture was poured
into ice water and then extracted with CH2Cl2
(3 9 20 mL). The combined organic phases were washed
with 1 N HCl, water, and brine, dried, and filtered and
concentrated in vacuo. The residue was purified by silica
gel column chromatography (hexane/ethyl acetate = 9/1,
Rf = 0.22).
1
White solid;Yield: 11 %; H NMR (400 MHz, CDCl3): d
2.33 (s, 3H), 7.10 (d, J = 7.9 Hz, 1H), 7.45 (t, J = 7.9 Hz,
1H), 7.75 (s, 1H), 7.77 (s, 1H), 7.84 (d, J = 7.6 Hz, 1H),
8.51 (s, 1H), 8.55 (s, 1H);13C NMR (100 MHz, CDCl3): d
21.2, 104.8, 106.6, 119.6, 122.1, 123.7, 129.8, 132.5,
134.2, 150.7, 151.2, 158.6, 169.5; EI-HRMS calculated for
(C14H10BrN3O2?H)? 332.00346, found 332.00372.
3-(Imidazo[1,2-a]pyrimidin-2-yl)phenyl acetate (37b)
Pale yellow solid; Yield: 21 %;1H NMR (400 MHz,
CDCl3): d 2.32 (s, 3H), 6.82 (t, J = 5.6 Hz, 1H), 7.07 (d,
J = 7.7 Hz, 1H), 7.42 (t, J = 7.8 Hz, 1H), 7.76 (m, 2H),
7.83 (d, J = 7.7 Hz, 1H), 8.39 (d, J = 6.3 Hz, 1H), 8.49 (s,
1H);13C NMR (100 MHz, CDCl3): d 21.2, 106.5, 108.9,
119.5, 121.7, 123.6, 129.7, 133.1, 134.7, 146.2,
148.6, 150.1, 151.2, 169.5; EI-HRMS calculated for
(C14H11 N3O2?H)? 254.09295, found 254.09322.
N-(3-(8-Chloro-6-(trifluoromethyl)imidazo[1,2-a]pyridin-
2-yl)phenyl)cyclohexane carboxamide (33b)
Pale yellow solid; Yield: 54 %; 1H NMR (400 MHz,
CDCl3): d 1.28–1.33 (m, 2H), 1.51–1.60 (m, 2H), 1.72 (m,
2H), 1.84–1.86 (m, 2H), 1.96–1.99 (m, 2H), 2.25 (m, 1H),
7.35–7.39 (m, 2H), 7.48 (s, 1H), 7.66 (d, J = 7.0 Hz,
2H),7.98 (s, 1H), 8.13 (s, 1H), 8.40 (s, 1H);13C NMR
(100 MHz, CDCl3): d 25.6, 25.7, 29.7, 46.6, 111.3, 116.6,
116.9, 117.5, 119.6, 120.2, 122.0, 123.3, 124.2, 129.5,
133.0, 138.7, 142.7, 147.6, 174.7; EI-HRMS calculated for
(C21H19ClF3N3O?H)? 422.12470, found 422.12446.
3-(Imidazo[1,2-a]pyrazin-2-yl)phenyl acetate (37c)
Pale yellow solid;Yield: 11 %;1H NMR (400 MHz,
CDCl3): d 2.34 (s, 3H), 7.12 (d, J = 7.6 Hz, 1H), 7.47 (t,
J = 7.5 Hz, 1H), 7.74 (s, 1H), 7.83 (d, J = 7.3 Hz, 1H),
7.89 (s, 1H), 7.95 (s, 1H), 8.07 (s, 1H), 9.10 (s, 1H);13C
NMR (100 MHz, CDCl3): d 21.1, 109.4, 118.6, 119.6,
122.0, 123.7, 129.8, 129.9, 134.4, 140.9, 143.8, 146.8,
151.2, 169.4; EI-HRMS calculated for (C14H11 N3O2?H)?
254.09295, found 254.09288.
N-(3-(8-Chloro-6-(trifluoromethyl)imidazo[1,2-a]pyridin-
2-y l)phenyl)-4-toluenesulfonamide (33c)
Pale yellow solid;Yield: 68 %;1H NMR (400 MHz,
CDCl3): d 2.46 (s, 3H), 7.00 (d, J = 7.5 Hz, 1H),
7.33–7.35 (m, 3H), 7.40 (s, 1H), 7.58 (s, 1H), 7.83–7.87
(m, 4H), 8.10 (d, J = 7.4 Hz, 1H), 8.40 (s, 1H);13C NMR
(100 MHz, CDCl3): d 21.7, 111.3, 116.6, 117.0, 119.7,
123.3, 124.4, 128.2, 128.5, 128.6, 129.4, 129.6, 129.7,
129.8, 131.5, 133.9, 134.9, 136.5, 142.7, 145.1, 146.6; EI-
HRMS calculated for (C21H15ClF3N3O2S?H)? 466.06038,
found 466.07034.
2-(3-Nitrophenyl)imidazo[1,2-a]pyrimidine (37d)
A mixture of 2-aminopyrazine 34 (195 mg, 2.05 mmol)
and bromoacetone 29 (660 mg, 2.70 mmol) in ethanol
(20 mL) was stirred until reflux for 3 h, After cooling to
room temperature, the mixture was concentrated. The res-
idue was dissolved in ethyl acetate, washed with 1 N HCl,
water, and brine, dried over calcium oxide, and filtered and
concentrated in vacuo. The residue was purified by silica
gel column chromatography (methanol/methylene chlo-
ride = 0–1 %, Rf = 0.21) to afford 37d as a yellow solid
(24 mg, 5 %). 1H NMR (400 MHz, CDCl3): d 6.95 (t,
J = 1.7 Hz, 1H), 7.65 (t, J = 8.0 Hz, 1H), 7.97 (s, 1H),
8.21 (d, J = 7.9 Hz, 1H), 8.45 (d, J = 7.6 Hz, 1H), 8.50
General synthetic procedure for (37a–c)
A mixture of 5-bromopyrimidin-2-amine 35–36 (222 mg,
1.27 mmol) and bromoacetone 3 (257 mg, 1.0 mmol) in
dioxane (10 mL) with or without NaHCO3 (84 mg,
1.00 mmol) was stirred until reflux for 7 h. After cooling to
123

Synthesis of GLP-1R agonists
(d, J = 5.9 Hz, 1H), 8.61 (d, J = 1.2 Hz, 1H), 8.80 (s,
1H);13C NMR (100 MHz, CDCl3): d 107.1, 108.8, 109.4,
113.6, 113.9, 120.9, 123.1, 129.9, 132.2, 133.3, 134.9,
150.8; EI-HRMS calculated for (C12H8N4O2?H)?
241.07255, found 241.07288.
2-(3-Nitrophenyl)imidazo[1,2-a]pyrazine (37e)
A mixture of 2-aminopyrazine 36 (95 mg, 1.0 mmol) and
bromoacetone 29 (488 mg, 2.0 mmol) in ethanol (10 mL)
was stirred until reflux for 3 h. After cooling to room tem-
perature, the mixture was concentrated. Then the residue was
dissolved in ethyl acetate and washed with water. The
combined aqueous phase was extracted with ethyl acetate.
The combined organic phase was washed with brine, dried
over calcium oxide, and filtered and concentrated in vacuo.
The residue was purified by silica gel column chromatog-
raphy (methylene chloride/methanol = 100/1, Rf = 0.21)
1
to afford 37e as a yellow solid (40 mg, 17 %). H NMR
(400 MHz, CDCl3): d 7.68 (t, J = 7.6 Hz, 1H), 7.97 (s, 1H),
8.11 (s, 1H), 8.13 (s, 1H), 8.24 (d, J = 7.3 Hz, 1H), 8.37 (d,
J = 6.7 Hz, 1H), 8.80 (s, 1H), 9.18 (s, 1H);13C NMR
(100 MHz,CDCl3): d 102.6, 110.0, 114.3, 117.1, 118.9,
121.2, 123.4, 129.7, 130.1, 132.2, 134.6, 144.1; EI-HRMS
calculated for (C12H8N4O2?H)? 241.07255, found
241.07260.
Scheme 1 Reagents and conditions: (a) acetic anhydride, DMAP,
anhydrous CH2Cl2, RT; (b) Br2, AlCl3, Et2O, ice bath; (c) NaHCO3,
EtOH, reflux; (d) NaOH, THF/H2O, RT; (e) R1Cl, K2CO3, acetone,
reflux or R2Cl, pyridine, 0 °C or RT
Biology
acetophenone 1 was converted into 3-acetoxy acetophe-
none 2 by acetylation. Treatment of substituted acetophe-
none 2 with bromine in the presence of AlCl3 in Et2O
In vitro GLP-1R activation assay (Chen et al. 2007)
´
(Bunders et al. 2010) afforded a-bromomethylketone 3.
CHO-K1 cells (4 9 106/100 mm dish) were transiently
transfected with the pCMV6-GLP-1R (Origene #SC124060)
and pCRE-Luc (Promega #631911) plasmids using Lipo-
fectamine 2000 (Invitrogen, Carlsbad, CA, USA). After 24 h
incubation at 37 °C, cells were seeded into 96-well culture
plates (2 9 104/well), and further incubated at 37 °C over-
night. At the time of assay, GLP-1 (7–37) (Sigma, St. Louis,
MO) or test compounds in DMSO were added to the plate.
After 8 h incubation, cells were lysed and luciferase activity
quantified using the Steady-Glo luciferase assay system
(Promega #E2550).
Subsequently, 3 and the substituted 2-aminopyridines 4, 5
were allowed to react in the presence of sodium bicar-
bonate in refluxing ethanol (Fookes et al. 2008), resulting
in the generation of compounds 6a and 6b. The deacety-
lation of compounds 6a and 6b with sodium hydroxide
afforded compounds 7a and 7b, respectively. The akylation
or acylation of 7a and 7b furnished the target compounds
8a–8f.
The derivatives 12a–12c were readily prepared in three
steps, as illustrated in Scheme 2. 3-Aminoacetophenone 9
was first acylated with acyl chloride or sulfonyl chloride, as
appropriate, to produce compounds 10a–10c. Subsequent
bromination of 10a–10c with PBB (pyridinium bromide
perbromide) in acetic acid (Yu et al. 2008) afforded com-
pounds 11a–11c, which were then cyclized with 5-tri-
fluoro-2-aminopyridine in refluxing ethanol to give the
desired derivatives 12a–12c.
Data were analyzed in Excel and EC50 values were
determined graphically from dose–response curves in
OriginPro.
Results and discussion
Next we synthesized the imidazo[1,5-a]pyridine deriv-
atives 19, 21a–21b, and 27a–27d, as detailed in Scheme 3.
The intermediate 2-aminomethylpyridine 15, prepared
from 2,3-dichloro-5-triflouropyridine 14 in two steps using
a method reported elsewhere (Stolting 2004), was treated
Chemistry
The general synthetic pathway yielding the novel deriva-
tives 6a, 6b, and 8a–8f is outlined in Scheme 1. 3-Hydroxy
123

Y.-J. Zhang et al.
resulting compound 20 with appropriate acyl chloride or
1-chloroacetone resulted in the generation of the desired
compounds 21a–21b. For the synthesis of derivatives 27a–
27d, the first step is the amidation of intermediate 15 with
benzoic acid 23 in the presence of DCC and DMAP in
dichloromethane, resulting in the generation of compound
24. Then, deprotection of 24 with trifluoroacetic acid (Mu
2001) followed by acylation of the resulting compound 25
afforded the compounds 26a–26c. Finally, the amides 24
and 26a–26c were cyclized in the presence of POCl3 and
pyridine in refluxing dichloroethane (Cookson et al. 1986),
resulting in the generation of the target derivatives 27a–
27d, respectively.
Scheme 4 describes the synthesis of derivatives 33a–
´
33c. The formation of a-diazoketone intermediate 30 was
Scheme 2 Reagents and conditions: (a) acetic anhydride, DMAP,
anhydrous CH2Cl2, RT or R3Cl, pyridine, 0 °C or RT; (b) pyridium
bromide perbromide, AcOH, RT; (c) NaHCO3, EtOH, reflux
´
achieved from a-bromomethylketone 28 by treatment with
N,N0-ditosylhydrazine and DBU (Toma et al. 2007). Sub-
´
sequent coupling of a-diazoketone with 3-chloro-5-tri-
with 3-acetoxybenzoinc acid 17, followed by cyclization in
the presence of POCl3 in refluxing benzene (Bower and
Ramage 1955) to give intermediate 19. Then, deacylation
of 19 and subsequent acylation or alkylation of the
fluoro-2-aminopyridine in the presence of 10 mol %
Cu(OTf)2 in dichloroethane (DCE) (Yadav et al. 2007)
afforded substituted 2-arylimidazo[1,2-a]pyridine 31.
Scheme 3 Reagents and
conditions: (a) CH3NO2, KOH,
DMSO, RT; (b) SnCl2, conc.
HCl, EtOH, reflux; (c) acetic
anhydride, reflux; (d) DCC,
DMAP, anhydrous CH2Cl2, RT;
(e) POCl3, benzene, reflux;
(f) NaOH, THF/H2O, RT;
(g) R4Cl, TEA, DMAP,
anhydrous CH2Cl2, RT, or R5Cl,
K2CO3, acetone, reflux. (h) di-
tert-butyl dicarbonate, TEA,
dioxane/H2O; (i) DCC, DMAP,
anhydrous CH2Cl2, RT;
(j) CF3COOH, anhydrous
CH2Cl2, RT; (k) acetic
anhydride, DMAP, anhydrous
CH2Cl2, RT or R6Cl, TEA,
DMAP, anhydrous CH2Cl2, RT
or succimyl chloride, K2CO3,
CH3CN, reflux; (l) POCl3,
pyridine, dichloroethane, reflux
123

Synthesis of GLP-1R agonists
respectively. The cyclization of 2-anmonoprimidine 34 and
2-aminopyrazine 36 with intermediate 29 yielded the target
derivatives 37d–37e, respectively.
Biology
The compounds prepared in this study were evaluated in
terms of GLP-1R agonist activity using an in vitro acti-
vation efficacy assay in CHO-K1 cells (Chen et al. 2007),
and the magnitude of the responses have been compared at
two concentrations of compounds used. GLP-1 (7–37) was
used as the positive control and DMSO (0.1 %) was used
as the negative control. Induction values represent lucif-
erase activities driven by CRE (cAMP response element).
Compounds were grouped into three series according to
fused-heterocyclic ring type.
Scheme 4 Reagents and conditions: (a) pyridium bromide perbromide,
AcOH, reflux; (b) TsNHNHTs, DBU, THF, RT; (c) 10 mol %Cu(OTf)2,
dichloroethane, 80 °C; (d) SnCl2, conc. HCl, EtOH, reflux; (e) acetic
anhydride, DMAP, anhydrous CH2Cl2, RT or R8Cl, TEA, DMAP,
anhydrous CH2Cl2, RT
In general, the first series of compounds, 6a–6b, 8a–8f,
12a–12c, and 33a–33c (Fig. 3), based on the imidazo[1,2-
a]pyridine structure and containing various substituted
groups, generated higher responses than those of the sec-
ond series. Compound 6b is the model compound, in which
replacement of the acetyl group with propanyl-2-one 8d,
mesyl 8e, or tosyl 8f resulted in a significant increase in
magnitude of the response, suggesting that the hydrogen-
bond donor is preferred to be this region and that the length
of linker affects binding to the ago-allosteric binding site of
GLP-1R. To determine whether the chlorine in imi-
dazo[1,2-a]pyridine is essential for its activity, compounds
6a and 8a–8c were synthesized. Compounds 8a and 8b
showed good responses similar to that of compounds 8d
and 8e at 10 lM. However, a loss of response was
observed for compounds 6a and 8c. The bioisosteric
replacement of the ester group (6a, 8b–8c) with an amide
group (12a–12c) resulted in comparable activities; in par-
ticular, compound 12a exhibited a two fold enhanced
response. On the contrary, compounds 33a and 33c
exhibited decreased responses. However, compound 33b,
which contains acyclohexanecarboxamide group, exhibited
a moderate response. Further increases in concentration
resulted in a significant drop in the responses of com-
pounds 8a, 6a–6b, 8f, and 12a–12b to an about \1-fold
increase, likely due to cytotoxicity at a high concentration
(100 lM). Surprisingly, compound 8e, which generated the
highest response at 10 lM, also exhibited the greatest
response at a high concentration (100 lM).
Scheme 5 Reagents and conditions: (a) NBS, CH3CN, reflux;
(b) dioxane, reflux or NaHCO3, dioxane, reflux; (c) EtOH, reflux
Reduction of 31 with stannous chloride in a refluxing
mixture of ethanol and concentrated hydrochloride (Denora
et al. 2008) resulted in the generation of compound 32.
Finally, the acylation of 32 with the corresponding acyl
chloride afforded derivatives 33a–33c.
In the second series of compounds, 19, 21a–21b, and
27a–27d (Fig. 4), the imidazo[1,2-a]pyridine structure was
changed to an imidazo[1,5-a]pyridine structure. Unfortu-
nately, all derivatives exhibited low responses at a low
concentration (10 lM). Compounds 21a–21b and 27b–27d
showed moderate responses at 100 lM. Generally, both
first- and second-series compounds with substituted ester
The syntheses of compounds 37a–37e are detailed in
Scheme 5. The preparation of intermediate 35 was
achieved by bromination of 2-aminopyrimidine 34 with
NBS in refluxing acetonitrile. The subsequent cyclization
of 35, 2-anmonoprimidine 34, and 2-aminopyrazine 36
with intermediate 3 yielded the target derivatives 37a–37c,
123

Y.-J. Zhang et al.
Fig. 3 In vitro responses of compounds 6a–6b, 8a–8f, 12a–12c and
33a–33c on CHO-K1 cells at 10 and 100 lM. The cells were
transfected with the pCMV6-GLP-1R and pCRE-Luc plasmids. The
transfected and cultured cells were incubated with different com-
pounds for 8 h, and luciferase activity quantified using the Steady-Glo
luciferase assay system. Vertical and horizontal axes show the fold
increases compared to the control and the synthesized compounds,
respectively. Values shown are mean SD of three independent
experiments. Significant difference from 0.1 % DMSO treated group:
*p B 0.05, **p B 0.01 and ***p \ 0.001
Fig. 4 In vitro responses of compounds 19, 21a–21b, 27a–27d, and
37a–37e on CHO-K1 cells at 10 and 100 lM. Experimental details
are described in Fig. 3. Vertical and horizontal axes show the fold
increases compared to the control and the synthesized compounds,
respectively. Values shown are mean SD of three independent
experiments. Significant difference from 0.1 % DMSO treated group:
*p B 0.05, **p B 0.01, and ***p \ 0.001
groups showed higher responses than those with a substi-
tuted amide group.
mesyl group of benzene ring, was found to be a potent
GLP-1R agonist exhibiting an EC50 of 7.89 lM. Com-
pounds 8a and 8b, without chlorine substitution of imi-
dazo[1,2-a]pyridine ring, were about threefold less potent,
with EC50 values of 20 lM and 17 lM, respectively
(Table 1). Concentration–response curves of selected
compounds are shown in Fig. 5. The concentration are in a
range from 1 lM to 100 lM. Compounds 8b and 8e
showed above 50 % response (8b, 52 %; 8e, 58 %) at their
EC50 values, while compound 8a showed lower response
43 % at EC50 value (Fig. 5). Thus, compound 8e may serve
as a GLP-1R agonist with potential for application.
Finally, a nitrogen atom was introduced into the six-
membered fused-heterocyclic ring to evaluate the effect of
electron density on activity (Fig. 4). The majority of the
compounds 37a–37e thus generated showed loss of
responses compared to the first series. We speculated that
the loss of responses might be attributable to a decreased
interaction between the p-electron and the receptor.
Over the half of the synthesized compounds, the effects
did not appear concentration-dependent. However the
effects of the compounds on coupling the GLP-1R to the
signaling way may well be concentration-dependent, but
the responses measured did not appear concentration-
dependent due to cytotoxicity.
In conclusion, these new compounds, synthetic meth-
odology developed and preliminary biological evaluation
results could be helpful in further design and discovery of
more potent GLP-1R agonists for the treatment of DM2.
In addition, selected compounds 8a, 8b and 8e which
showed[2.5-fold increases at 10 lM were assayed further
to determine concentration–response curves (Fig. 5) and
calculate EC50 values (Table 1). Compound 8e, bearing
chlorine substitution imidazo[1,2-a]pyridine ring and
Acknowledgments This work was supported by the National
Research Foundation of Korea (NRF) grant funded by the Korea
government (MEST) (No. 2011-000-7061).
123

Synthesis of GLP-1R agonists
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Fig. 5 Concentration-response curves of agonists 8a, 8b and 8e of
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Vertical axes show the response percentage of GLP-1 response.
Values shown are mean SD of three independent experiments. For
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Table 1 Potency of agonists 8a, 8b and 8e at GLP-1R
X
F3C
N
Y
N
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Compounds
X
Y
EC50 (lM)a
O
8a
H
19.75 0.64
O
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O
8b
H
16.96 0.16
7.89 2.26
O
S
S
O
O
8e
Cl
O
O
a
Values are reported as mean SD
Open Access This article is distributed under the terms of the
Creative Commons Attribution License which permits any use, dis-
tribution, and reproduction in any medium, provided the original
author(s) and the source are credited.
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