Design, synthesis and evaluation of 1-benzyl-1H-imidazole-5-carboxamide derivatives as potent TGR5 agonists

Article abstract of DOI:10.1016/j.bmc.2020.115972

TGR5 is emerging as an important and promising target for the treatment of diabetes, obesity and other metabolic syndromes. A series of novel 1-benzyl-1H-imidazole-5-carboxamide derivatives was designed, synthesized and evaluated in vitro and in vivo. The

Full text of DOI:10.1016/j.bmc.2020.115972

Bioorg. Med. Chem. 32 (2021) 115972
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and evaluation of 1-benzyl-1H-imidazole-5-carboxamide
derivatives as potent TGR5 agonists
Shizhen Zhaoa, Xinping Lia, Le Wanga, Wenjing Penga, Wenling Yea, Weiguo Lia,
,b,*
Yan-Dong Wangc, Wei-Dong Chena
a Key Laboratory of Receptors-Mediated Gene Regulation and Drug Discovery, People’s Hospital of Hebi, School of Medicine, Henan University, Henan, China
b Key Laboratory of Molecular Pathology, School of Basic Medical Science, Inner Mongolia Medical University, Hohhot, China
c State Key Laboratory of Chemical Resource Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
TGR5 is emerging as an important and promising target for the treatment of diabetes, obesity and other meta-
bolic syndromes. A series of novel 1-benzyl-1H-imidazole-5-carboxamide derivatives was designed, synthesized
and evaluated in vitro and in vivo. The most potent compounds 19d and 19e exhibited excellent agonistic ac-
tivities against hTGR5, which was superior to those of the reference drugs INT-777 and LCA. In addition,
compounds 19d and 19e exhibited good selectivity against FXR and presented significant glucose-lowering ef-
fects in vivo. Compound 19d could stimulate GLP-1 secretion by activating of TGR5.
TGR5
TGR5 agonists
Diabetes
Structure-activity relationship
1. Introduction
(2–5), which exhibited excellent TGR5 agonist activity and were orally
efficacious in lowering glucose levels in vivo.16–22 However, none of
Takeda G-protein-coupled receptor 5 (TGR5, GPBAR1, M-BAR, or
GPCR19) belongs to a G-protein-coupled receptor (GPCR) for bile acids.
The TGR5 receptor is expressed broadly in various tissues including
liver, intestine, pancreas, spleen and brown adipose tissue.1–5 Activation
of TGR5 results in the elevation of intracellular cyclic adenosine
monophosphate (cAMP) levels, which is recognized as an important
intracellular signaling cascades.6,7 TGR5 activation has also elevated
glucagon like peptide-1 (GLP-1) secretion, which activates intestinal cell
secretion and plays a key role in glucose metabolism and energy ho-
meostasis.8,9 In addition, TGR5 activation increases energy expenditure
and oxygen consumption in brown adipose tissue and muscle.10 There-
fore, TGR5 has become an attractive therapeutic target for the treatment
of metabolic disorders, such as non-alcoholic steatohepatitis, type 2
diabetes mellitus (T2DM) and obesity.11–13
these agonists have been successful in clinical trials.
In our effort to discover more potent TGR5 agonists for a treatment of
metabolic disease. A number of compounds in our library were evalu-
ated for their TGR5 agonist activity by computer-aided drug design
(CADD). Compound 6, which features 1- benzyl-1H-imidazole-5-car-
boxamide scaffolds, was displayed obvious TGR5 agonist activity, with
the EC50 values of 19.5 μM. The chemical scaffold of compound 6 is an
attractive starting point for generating novel TGR5 agonists. In order to
further explore the structure–activity relationships (SARs) and improve
TGR5 agonist activity, a series of 1-benzyl-1H-imidazole-5-carboxamide
derivatives have been synthesized and evaluated for their in vitro TGR5
agonist activity.
2. Results and discussion
Two categories of TGR5 agonists (steroidal and nonsteroidal) have
been discovered in the literature by pharmaceutical companies (Fig. 1).
One series is structurally based on bile acids (BAs), including cholic acid
(CA), lithocholic acid (LCA) and their semisynthetic derivatives such as
2.1. Chemistry
The key intermediates 12 was synthesized according to the proced-
6
α
-ethyl-23(S)-methylcholic acid (INT-777, 1).14,15 INT-777 is a selec-
ure which were previously reported in Scheme 1.23 2-fluoronitroben-
tive TGR5 agonist as an antidiabetic drug candidate. In addition, several
other nonsteroidal TGR5 agonists have been reported in the literature
zene 7 was used as the starting material, and treated with
cyclopropylamine to obtain intermediate 8. The intermediate 8 was
* Corresponding author at: Key Laboratory of Receptors-Mediated Gene Regulation and Drug Discovery, People’s Hospital of Hebi, School of Medicine, Henan
University, Henan, China.
E-mail address: wdchen666@163.com (W.-D. Chen).
https://doi.org/10.1016/j.bmc.2020.115972
Received 14 October 2020; Received in revised form 20 December 2020; Accepted 21 December 2020
Available online 27 December 2020
0968-0896/© 2020 Published by Elsevier Ltd.

S. Zhao et al.
Bioorganic & Medicinal Chemistry 32 (2021) 115972
treated with methyl oxalyl chloride in the presence of triethylamine by
acylation to produce the intermediate 9. Further transformation of 9 by
reduction reaction to afford 10, which on treatment with triphenyl-
phosphine provided the intermediate 11. Finally, intermediate 11 was
reduced with borane-tetrahydrofuran complex to obtain the key in-
termediates 12.
superior or comparable to those of the reference drug INT-777. Of these,
Compounds 19d and 19e with 2,4-di-Cl and 2,5-di-Cl substituents
showed the best hTGR5 agonist activity, with EC50 values of 6.8 and 9.5
nM on hTGR5, respectively.
Several potent hTGR5 agonists were further evaluated for their
ability to activate mTGR5 using a similar method described for hTGR5
(Table 2). Compared with hTGR5 agonism activity, most of the com-
pounds showed less agonist activity on mTGR5. The differences in po-
tencies may be mainly due to the weak sequence homology (83%)
between the two species. Among these, the potent compounds 19d and
19e, with EC50 values of 611 and 832 nM toward mTGR5 in our assay,
respectively. The CLogP values of these compounds are shown in
Table 2. Interestingly, there was a increase in TGR5 agonist activity
along with increasing lipophilicity within compounds 19a-f.
The target compounds 15a-p were synthesized as shown in Scheme
2. The intermediate 14 was synthesized from etomidate 13 via hydro-
lysis reaction with 2 N NaOH. Then, the intermediates 14 reacted with
the key intermediate 12, substituted aniline, substituted N-methylani-
line or substituted 1-phenylpiperazine in the presence of HATU, that
gave the target compounds 15a-p.
The target compounds 19a-f were formed according to the reaction
pathways illustrated in Scheme 3. Ethyl 1H-imidazole-5-carboxylate 16
was used as the starting material, and treated with substituted benzyl
bromide by nucleophilic substitution to obtain intermediate 17a-f.
Similar to the preparation of 15a-s, compounds 19a-f were synthesized
from intermediate 17a-f by hydrolysis and condensation reactions.
2.3. FXR activity assay
Because many bile acid derived TGR5 agonists can also activate the
nuclear bile acid receptor FXR (Farnesoid X Receptor).26,27 The most
potent compounds 19d and 19e were further evaluated by using ho-
mogeneous time resolved fluorescence (HTRF) assay. As shown in
Table 3, compounds 19d and 19e exhibited good selectivity against
FXR.
2.2. In vitro biological evaluation
The target compounds were evaluated for their ability to activate
hTGR5 by assessing for intracellular levels of cAMP, which used the
homogeneous time resolved fluorescence (HTRF) assay, similar to the
reported in the literature.24,25 As reference compound, the response of
2.4. In vitro human plasma stability assay
LCA at 10 μM was defined as 100% hTGR5 activation. Vehicle control
The stability of compounds in human plasma is an important
consideration in drug discovery. Based on their in vitro activities, the
most potent compounds 19d and 19e were incubated with human
Plasma.24 As shown in Table 4, compounds 19d and 19e exhibited
excellent metabolic profiles in human plasma at 120 min (remaining
104.0% and105.4%, respectively).
with 0.1% DMSO was set to 0% hTGR5 activation. As shown in Table 1,
the introduction of different the side chain amide had a notable influ-
ence on the hTGR5 activity.
Many of these compounds were found to have agonistic activities
against hTGR5 at the concentration of 10 μM and 40 μM. Interestingly,
compounds such as 4-F aniline (15a), N-methyl aniline (15g, 15h and
15i), substituted 1-Phenylpiperazine (15k and 15l) and tetrahydro-
quinoxaline (15n) exhibited agonistic activities with an Agonistic rate
more then 50% at 10 µM. Furtherly, the most potent compounds 15h,
15k, 15l and 15n were evaluated using a dose–response experiment and
displayed obvious TGR5 agonist activity, with the EC50 values of 1.05,
2.5. In vitro cytotoxicity assay
The most potent compounds 19d and 19e were further evaluated for
their in vitro cytotoxicity against A549 and Hela cells. Cytotoxicity was
measured using the CCK8 assay after incubation with the compounds for
24 h. As shown in Table 5, Compounds 19d and 19e showed no activities
1.09, 4.39 and 0.33 μM, respectively.
Based on the results above, we selected a scaffold of tetrahydro-
quinoxaline 15n as our starting point for further modification. Our
optimized efforts were directed toward replacing the phenylethyl group
with the substituted benzyl group to expand the SAR studies (Table 2).
Most of the compounds (19a-f) exhibited excellent hTGR5 agonist ac-
tivity with EC50 values in the range of 6.8 to 1751 nM, which are
toward tumor cell lines A549 and Hela with IC50 > 50 μM.
2.6. Oral glucose tolerance test in mice
An oral glucose tolerance test (OGTT) was carried out in C57 BL/6
Fig. 1. Structures of some known TGR5 agonists.
2

S. Zhao et al.
Bioorganic & Medicinal Chemistry 32 (2021) 115972
mice to examine the effect of compounds 19d and 19e on blood glucose
(Fig. 2). Mice were fasted overnight before glucose tolerance tests.
Compounds 19d and 19e at 30 mg/kg were orally administered 60 min
prior to the administration of 2 g/kg of glucose. Blood glucose was
measured at 0, 15, 30, 60 and 120 min after glucose administration. As
shown in Fig. 2, compounds 19d and 19e caused a significant 15.8% and
13.7% of reduction in blood glucose AUC0ꢀ 120 min compared with the
control group.
(Agilent, Palo Alto, CA, USA). High-resolution accurate mass de-
terminations (HRMS) were recorded on a Bruker Micromass Time of
Flight mass spectrometer equipped with electrospray ionisation (ESI).
Nuclear magnetic resonance (1H-NMR and 13C NMR) spectra were
recorded on a Bruker 400 MHz NMR spectrometer with TMS as an in-
ternal standard. The chemical shifts were reported in parts per million
(ppm), the coupling constants (J) were expressed in hertz (Hz). Peak
multiplicities were described as singlet (s), doublet (d), triplet (t),
quartet (q), multiplet (m) and broad (br).
2.7. GLP-1 secretion
4.2. Cyclopropyl-(2-nitro-phenyl)-amine (8)
To explore whether the glucose-lowering effect of compound 19d is
dependent on mediated GLP-1 secretion, a further GLP-1 levels of C57
BL/6 mice (WT) and TGR5ꢀ /ꢀ mice were detected by GLP-1(7–36)
ELISA assay. As shown in Fig. 3, compound 19d could increased the
secretion of GLP-1 compared to control in WT mice. In TGR5ꢀ /ꢀ mice,
there is no significant difference for GLP-1 concentration between con-
trol and compound 19d treated groups. All of these results indicated that
compound 19d could stimulate GLP-1 secretion by activating of TGR5.
Cyclopropylamine (27.31 g, 478.38 mmol) was added to a solution of
2-fluoronitrobenzene (30.00 g, 212.6 mmol), and the resulting mixture
was stirred for 12 h at ambient temperature. After confirming that the
reaction was complete by using TLC analysis, and the reaction was
extracted with ethyl acetate and washed with saturated sodium bicar-
bonate. The organic layer was dried over anhydrous Na2SO4 and
concentrated. The crude product was purified by silica gel column
chromatography to yield the target product 8 (34.20 g, yield: 90.3%).
3. Conclusion
4.3. N-Cyclopropyl-N-(2-nitro-phenyl)-oxalamic Acid methyl ester (9)
In summary, we have designed and synthesized a series of novel 1-
benzyl-1H-imidazole-5-carboxamide derivatives as potent TGR5 ago-
nists. Many of these compounds showed agonistic activities against
Methyl oxalyl chloride (23.37 g, 190.8 mmol) was slowly added to a
solution of cyclopropyl-(2-nitro-phenyl)-amine (34.00 g, 190.8 mmol)
and triethylamine (31.83 mL, 228.9 mmol) in dichloromethane cooled
TGR5 at the concentration of 10 μM and 40 μM. Among these, com-
◦
to <0 C using a salted ice bath. The reaction mixture was stirred at
pounds 19d and 19e displayed the most remarkable agonistic activities
against TGR5, which was superior to those of the reference drugs INT-
777 and LCA. In addition, compounds 19d and 19e exhibited good
selectivity against FXR and presented significant glucose-lowering ef-
fects in vivo studies. The GLP-1 ELISA assay indicated that compound
19d could stimulate GLP-1 secretion by activation of TGR5 in WT and
TGR5ꢀ /ꢀ mice. Further developments of compounds 19d and 19e are
ongoing in our laboratory.
ambient temperature for 12 h. The reaction mixture was extracted with
EtOAc and brine. The organic phase was dried over Na2SO4 overnight
and the solvent was removed under vacuum to give the target product 9
(42.60 g, yield: 84.5%).
4.4. 1-Cyclopropyl-4-hydroxy-1,4-dihydro-quinoxaline-2,3-dione (10)
To a solution of 9 (40.00 g, 151.4 mmol) in methanol (200 mL) was
added palladium on carbon (4.00 g, 10%w/w). The reaction flask was
evacuated under vacuum and back filled with hydrogen 3 times, before
being stirred under a hydrogen atmosphere at ambient temperature for
18 h. The reaction mixture was diluted with ethyl acetate (400 mL),
filtered and the solvent was removed in vacuo to give the target product
10 (28.42 g, yield: 86.0%).
4. Experimental section
4.1. General procedure for the synthesis of compounds
Unless otherwise noted, all reagents and solvents were obtained from
commercially available sources and were used without purification. TLC
analysis was performed on GF254 silica gel plates (Jiangyou, Yantai).
Column chromatography was carried out with silica gel (200–300 mesh)
from Qingdao Haiyang Chemicals (Qingdao, Shandong, China). Mass
spectrometry was performed using ESI mode on an Agilent 1200 LC-MS
4.5. 1-Cyclopropyl-1,4-dihydro-quinoxaline-2,3-dione (11)
A solution of 1-Cyclopropyl-4-hydroxy-1,4-dihydro-quinoxaline-2,3-
Scheme 1. Synthesis of intermediates 12. Reagents and conditions:(a) Cyclopropylamine, r.t.; (b) Methyl Oxalyl chloride, Triethylamine, CH2Cl2, r.t.; (c) Pd/C, H2,
MeOH, r.t.; (d) Triphenylphosphine, DMF, 135 ◦C; (e) BH3-THF, THF, r.t..
3

S. Zhao et al.
Bioorganic & Medicinal Chemistry 32 (2021) 115972
Scheme 2. General synthesis of the target compounds 15a-p. Reagents and conditions: (a) NaOH, MeOH/H2O, r.t.; (b) 12, substituted aniline, substituted N-
methylaniline or substituted 1-phenylpiperazine, HATU, DIEA, DMF, r.t..
Scheme 3. General synthesis of the target compounds 19a-f. Reagents and conditions: (a) 10% NaOH, THF, 0 ◦C; (b) 2 N NaOH, MeOH/H2O; (c) 12, HATU, DIEA,
DMF, r.t..
dione (28.00 g, 128.3 mmol) and triphenylphosphine (50.48 g, 192.47
mmol) in DMF was heated to 135 ◦C for 6 h. After confirming that the
reaction was complete by using TLC analysis, the solution was cooled to
room temperature and dichloromethane (400 mL) was added. The sus-
pension was stirred for 30 min, filtered and washed with dichloro-
methane to give the desired compound 11 (21.10 g, yield: 81.3%). 1H
NMR (300 MHz, DMSO‑d6) δ 11.92 (s, 1H), 7.63 (dd, J = 6.5, 1.8 Hz,
1H), 7.30–7.10 (m, 3H), 2.99–2.86 (m, 1H), 1.30–1.14 (m, 2H),
0.81–0.66 (m, 2H).
(R)-1-(1-phenylethyl)-1H-imidazole-5-carboxylic acid 14 (1 equiv.) and
substituted aniline (1 equiv.) in anhydrous DMF. The solution was
heated to 70 ◦C for 6 h and then cooled to room temperature. The re-
action mixture was poured into ice water, and the resulting solid was
filtered. The crude product was purified by silica gel column chroma-
tography to give the target product 15a-p.
4.8.1. (R)-N-(4-fluorophenyl)-1-(1-phenylethyl)-1H-imidazole-5-
carboxamide (15a)
Light white solid; yield: 72.8%; 1H NMR (400 MHz, DMSO‑d6) δ
10.09 (s, 1H), 8.22 (s, 1H), 7.77 (s, 1H), 7.69–7.60 (m, 2H), 7.32 (t, J =
7.4 Hz, 2H), 7.25 (d, J = 7.3 Hz, 1H), 7.23–7.19 (m, 2H), 7.15 (t, J = 8.9
Hz, 2H), 6.40 (q, J = 7.2 Hz, 1H), 1.85 (d, J = 7.3 Hz, 3H). 13C NMR
(100 MHz, DMSO‑d6) δ 158.6, 157.1, 142.6, 139.8, 135.0, 133.6, 128.6
(2C), 127.6, 126.0 (2C), 125.0, 122.1, 122.0, 115.4 , 115.1, 54.3, 21.7.
HRMS calcd for C18H17FN3O, [M + H]+, 310.1356; found 310.1349
4.6. 1-Cyclopropyl-1,2,3,4-tetrahydro-quinoxaline (12)
1 M solution of boranete-trahydrofuran complex (207.8 mL, 207.8
mmmol) was slowly added dropwise to a solution of 1-cyclopropyl-1,4-
dihydro-quinoxaline-2,3-dione (21.00 g, 103.9 mmol) in THF (500 mL),
and the resulting mixture was stirred for 18 h at ambient temperature.
The solvent was removed under reduced pressure, and the residue was
extracted with ethyl acetate and washed with brine. The organic layer
was dried over anhydrous Na2SO4 and concentrated. The crude product
was purified by silica gel column chromatography to yield the target
product 12 as a white solid (8.60 g, yield: 47.5%). 1H NMR (300 MHz,
DMSO‑d6) δ 6.90 (d, J = 7.6 Hz, 1H), 6.57–6.32 (m, 3H), 5.47 (s, 1H),
3.29–3.27 (m, 2H), 3.14–3.12 (m, 2H), 2.14–2.12 (m, 1H), 0.76–0.74
(m, 2H), 0.47–0.45 (m, 2H).
4.8.2. (R)-N-(3-fluorophenyl)-1-(1-phenylethyl)-1H-imidazole-5-
carboxamide (15b)
Light white solid; yield: 75.1%; 1H NMR (400 MHz, DMSO) δ 10.20
(s, 1H), 8.25 (s, 1H), 7.80 (s, 1H), 7.60 (d, J = 11.8 Hz, 1H), 7.43 (d, J =
8.4 Hz, 1H), 7.39–7.29 (m, 3H), 7.23 (dd, J = 17.5, 7.2 Hz, 3H), 6.90 (td,
J = 8.4, 2.2 Hz, 1H), 6.39 (q, J = 7.2 Hz, 1H), 1.86 (d, J = 7.2 Hz, 3H).
13C NMR (101 MHz, DMSO) δ 163.7, 159.2, 143.0, 141.0, 140.5, 134.5,
130.8, 129.1(2C), 128.0, 126.5 (2C), 125.6, 116.2, 110.4, 107.4, 54.8,
22.1.HRMS calcd for C18H17FN3O, [M + H]+, 310.1356; found
310.1351.
4.7. (R)-1-(1-phenylethyl)-1H-imidazole-5-carboxylic acid (14)
To a solution of etomidate 13 (1.0 g, 4.09 mmol) in methanol was
added 2 N sodium hydroxide (5 mL) at ambient temperature. The re-
action mixture was stirred for 6 h and the methanol was removed by
rotary evaporation. The resultant mixture was adjusted to pH = 5–6 with
1 N HCl. The precipitated white solid was collected by filtration and
dried to give the carboxylic acid intermediate 14 (0.81 g, yield: 91.5%).
4.8.3. (R)-N-(2-fluorophenyl)-1-(1-phenylethyl)-1H-imidazole-5-
carboxamide (15c)
Light white solid; yield: 73.2%; 1H NMR (400 MHz, DMSO‑d6) δ 9.90
(s, 1H), 8.24 (s, 1H), 7.82 (s, 1H), 7.47 (t, J = 7.7 Hz, 1H), 7.32 (t, J =
7.3 Hz, 2H), 7.28–7.14 (m, 6H), 6.41 (q, J = 7.2 Hz, 1H), 1.84 (d, J = 7.3
Hz, 3H). 13C NMR (101 MHz, DMSO) δ 159.1, 157.4, 142.9, 140.3,
134.45, 129.0 (2C), 128.0, 127.6, 127.4, 126.5 (2C), 125.4, 125.2,
124.7, 116.2, 54.7, 22.1. HRMS calcd for C18H17FN3O, [M + H]+,
310.1356; found 310.1343.
4.8. General procedure for the synthesis of compounds 15a-p
HATU (1.1 equiv.) and DIEA (2 equiv.) were added to a solution of
4

S. Zhao et al.
Bioorganic & Medicinal Chemistry 32 (2021) 115972
Table 1
In vitro biological evaluation.a
Compd.
15a
R1
Agonistic rate at 10
51.29
μM (%)
Agonistic rate at 40
85.21
μM (%)
hTGR5
EC50
(
μ
M)b
–
15b
15c
20.14
16.52
56.18
48.40
–
–
15d
15e
15f
37.71
20.16
25.34
63.13
84.13
63.33
66.51
71.88
–
–
–
–
15g
15h
15i
70.88
62.12
85.36
86.67
1.05±0.21
–
15j
41.72
79.34
81.64
95.34
–
15k
1.09±0.11
15l
79.08
17.39
89.65
49.18
4.39±0.16
15m
–
15n
89.79
97.36
0.33±0.08
(continued on next page)
5

S. Zhao et al.
Bioorganic & Medicinal Chemistry 32 (2021) 115972
Table 1 (continued)
Compd.
R1
Agonistic rate at 10
μM (%)
Agonistic rate at 40
μM (%)
hTGR5
EC50
(μ
M)b
15o
48.32
54.12
77.14
74.53
–
–
15p
a
The response to 10 µM LCA was set to 100% and 0.1% DMSO was set to 0% hTGR5 activation. Data represent mean of three independent experiments. EC50 values
given are expressed as mean ± SEM of three independent experiments.
b
–, not tested.
Table 2
Table 5
In vitro cytotoxicity of compounds on A549 and Hela cells.
Compd.
IC50
(μ
M)a
In vitro biological evaluation.a
A549
Hela
19d
19e
>50
>50
>50
>50
a
The mean values of three independent experiments ± SE are reported.
Compd.
R2
hTGR5
mTGR5
CLogPb
EC50 (nM)
EC50 (nM)
11.8, 8.6, 6.7 Hz, 5H), 7.25–7.21 (m, 2H), 6.49 (q, J = 7.1 Hz, 1H), 1.88
(d, J = 7.1 Hz, 3H). 13C NMR (100 MHz, DMSO‑d6) δ 158.6, 142.6,
139.9, 137.7, 133.9, 128.6 (2C), 128.6 (2C), 127.5, 127.2, 126.0 (2C),
125.2, 121.7 (2C), 54.4, 21.7. HRMS calcd for C18H17ClN3O, [M + H]+,
326.1060; found 326.1062.
19a
19b
19c
19d
19e
19f
H
1751 ± 52
568 ± 68
118 ± 11
6.8 ± 0.43
9.5 ± 0.61
338 ± 21
820 ± 43
2.8 ± 0.19
3500 ± 35
>10,000
2.99
3.13
3.70
4.42
4.42
3.28
4-F
2-Cl
3690 ± 86
611 ± 33
832 ± 47
>10,000
2,4-di-Cl
2,5-di-Cl
2,4-di-F
INT-777
132 ± 24
3.2 ± 0.09
5
4.8.5. (R)-1-(1-phenylethyl)-N-(p-tolyl)-1H-imidazole-5-carboxamide
(15e)
a
EC50 values given are expressed as mean ± SEM of three independent
Light white solid; yield: 72.5%; 1H NMR (400 MHz, DMSO‑d6) δ 9.94
(s, 1H), 8.20 (s, 1H), 7.75 (s, 1H), 7.51 (d, J = 8.4 Hz, 2H), 7.32 (t, J =
7.3 Hz, 2H), 7.25 (d, J = 7.1 Hz, 1H), 7.21 (d, J = 7.2 Hz, 2H), 7.11 (d, J
= 8.3 Hz, 2H), 6.42 (q, J = 7.2 Hz, 1H), 2.25 (s, 3H), 1.85 (d, J = 7.3 Hz,
3H). 13C NMR (100 MHz, DMSO‑d6) δ 158.5, 142.6, 139.6, 136.1, 133.4,
132.6, 129.0 (2C), 128.6 (2C), 127.5, 126.0 (2C), 125.5, 120.2 (2C),
54.2, 21.7, 20.5. HRMS calcd for C19H20N3O, [M + H]+, 306.1606;
found 306.1600.
experiments.
b
Calculated from ChemBioDraw Ultra 12.0 by CambridgeSoft.
Table 3
FXR Agonistic activities of the compounds 19d and 19e by HTRF assay.a
Compd.
FXR (% effect at
10 M)
FXR (% effect at
20 M)
FXR (% effect at
50 M)
μ
μ
μ
19d
1.1
2.3
–
1.6
ꢀ 1.1
0.31
19e
ꢀ 0.8
4.8.6. (R)-N-(4-methoxyphenyl)-1-(1-phenylethyl)-1H-imidazole-5-
carboxamide (15f)
CDCA
–
100%
Light white solid; yield: 66.5%; 1H NMR (400 MHz, DMSO‑d6) δ 9.93
(s, 1H), 8.19 (s, 1H), 7.75 (s, 1H), 7.58–7.50 (m, 2H), 7.32 (t, J = 7.3 Hz,
2H), 7.27–7.18 (m, 3H), 6.89 (d, J = 9.0 Hz, 2H), 6.44 (q, J = 7.2 Hz,
1H), 3.73 (s, 3H), 1.85 (d, J = 7.3 Hz, 3H). 13C NMR (101 MHz,
DMSO‑d6) δ 158.4, 155.5, 142.7, 139.5, 133.2, 131.7, 128.6 (2C), 127.5,
126.0 (2C), 125.5, 121.9 (2C), 113.8 (2C), 55.2, 54.2, 21.7.HRMS calcd
for C19H20N3O2, [M + H]+, 322.1556; found 322.1554.
a
The response to 50 µM CDCA was set to 100% and 0.1% DMSO was set to 0%
FXR activation. – , not tested.
Table 4
In vitro human Plasma Stability of compounds 19d and 19e.
Compd.
Stablity in Human Blood Plasma
% Remaining at 60min
% Remaining at 120min
4.8.7. (R)-N-(4-chlorophenyl)-N-methyl-1-(1-phenylethyl)-1H-imidazole-
5-carboxamide (15g)
19d
19e
107.2
111.6
104.0
105.4
Light white solid; yield: 60.5%; 1H NMR (400 MHz, DMSO‑d6) δ 8.00
(s, 1H), 7.41 (t, J = 7.5 Hz, 2H), 7.33 (t, J = 7.2 Hz, 1H), 7.26 (d, J = 8.5
Hz, 2H), 7.17 (d, J = 7.6 Hz, 2H), 6.67 (d, J = 8.4 Hz, 2H), 6.37 (s, 1H),
6.01 (q, J = 7.2 Hz, 1H), 3.19 (s, 3H), 1.83 (d, J = 7.2 Hz, 3H). 13C NMR
(100 MHz, DMSO‑d6) δ 160.9, 143.2, 142.5, 137.5, 133.7, 131.3, 129.1,
128.7, 128.6, 127.9, 126.3 (2C), 125.4, 54.7, 37.2, 21.2. HRMS calcd for
4.8.4. (R)-N-(4-chlorophenyl)-1-(1-phenylethyl)-1H-imidazole-5-
carboxamide (15d)
Light white solid; yield: 75.9%; 1H NMR (400 MHz, CDCl3) δ 7.99 (s,
1H), 7.71 (d, J = 11.9 Hz, 2H), 7.50 (d, J = 8.9 Hz, 2H), 7.32 (ddd, J =
6

S. Zhao et al.
Bioorganic & Medicinal Chemistry 32 (2021) 115972
Fig. 2. Oral glucose tolerance test (OGTT) of compounds 19d and 19e in male C57BL/6 mice. (A) Blood glucose concentration; (B) blood glucose AUC0–120 min
.
Compounds 19d and 19e (30 mg/kg in 0.25%CMC) or 0.25% CMC (Control) was orally administered at ꢀ 60 min of OGTT followed by oral glucose challenge at 2.0
g/kg at 0 min. n = 7–8 animals/group. *P < 0.05 vs. control. Error bar indicates SEM (standard error of the mean).
(100 MHz, DMSO‑d6) δ 161.0, 142.6, 141.8, 137.2, 136.5, 133.5, 129.7
(2C), 128.7 (2C), 127.8, 126.6 (2C), 126.4 (2C), 125.6, 54.6, 37.4, 21.2,
20.5. HRMS calcd for C20H22N3O, [M + H]+, 320.1763; found 320.1759.
4.8.10. (R)-(1-(1-phenylethyl)-1H-imidazol-5-yl)(4-phenylpiperazin-1-yl)
methanone (15j)
Light white solid; yield: 74.8%; 1H NMR (400 MHz, DMSO‑d6) δ 8.12
(s, 1H), 7.28 (t, J = 7.6 Hz, 2H), 7.23–7.16 (m, 4H), 7.10 (d, J = 7.2 Hz,
2H), 6.85 (d, J = 8.0 Hz, 2H), 6.80 (t, J = 7.3 Hz, 1H), 5.77 (q, J = 7.1
Hz, 1H), 3.39–3.34 (m, 4H), 2.94–2.89 (m, 4H), 1.83 (d, J = 7.2 Hz, 3H).
13C NMR (100 MHz, DMSO‑d6) δ 160.4, 150.7, 142.6, 137.7, 130.7,
129.0 (2C), 128.7 (2C), 127.6, 126.1 (2C), 124.9, 119.5, 116.0 (2C),
56.0, 54.8 (2C), 48.3 (2C), 21.2. HRMS calcd for C20H22N3O3SNa, [M +
Na]+, 383.1848; found 383.1842.
4.8.11. (R)-(4-(2-fluorophenyl)piperazin-1-yl)(1-(1-phenylethyl)-1H-
imidazol-5-yl)methanone (15k)
Light white solid; yield: 68.2%; 1H NMR (400 MHz, CDCl3) δ 7.79 (s,
1H), 7.22 (dd, J = 13.2, 5.5 Hz, 2H), 7.15 (t, J = 7.3 Hz, 1H), 7.10 (s,
1H), 7.04 (d, J = 7.2 Hz, 2H), 7.01–6.96 (m, 1H), 6.97–6.85 (m, 2H),
Fig. 3. In vivo GLP-1 secretion study of compound 19d at 30 mg/kg in C57 BL/
6.77–6.64 (m, 1H), 5.86 (q, J = 7.1 Hz, 1H), 3.85–3.31 (m, 4H),
2.72–2.70 (m, 4H), 1.81 (d, J = 7.1 Hz, 3H). 13C NMR (100 MHz,
DMSO‑d6) δ 160.4, 142.6, 139.4, 137.7, 130.6, 128.7 (2C), 127.7, 126.1
(2C), 124.9, 124.8, 123.0, 119.5, 116.1, 115.9, 54.9, 49.9 (2C), 49.9
(2C), 21.2. HRMS calcd for C22H24FN4O, [M + H]+, 379.1934; found
379.1947.
6 mice (WT) and TGR5ꢀ /ꢀ mice (TGR5 KO). n = 7–8 animals/group. **P < 0.01
vs. control. Error bar indicates SEM.
C19H19ClN3O, [M + H]+, 340.1217; found 340.1218.
4.8.8. (R)-N-(4-fluorophenyl)-N-methyl-1-(1-phenylethyl)-1H-imidazole-
5-carboxamide (15h)
4.8.12. (R)-(4-(4-methoxyphenyl)piperazin-1-yl)(1-(1-phenylethyl)-1H-
imidazol-5-yl)methanone (15l)
Light white solid; yield: 69.5%; 1H NMR (400 MHz, DMSO‑d6) δ 7.98
(s, 1H), 7.40 (t, J = 7.3 Hz, 2H), 7.33 (dd, J = 8.4, 6.0 Hz, 1H), 7.16 (d, J
= 7.1 Hz, 2H), 7.05 (t, J = 8.8 Hz, 2H), 6.71 (dd, J = 8.4, 5.0 Hz, 2H),
6.33 (s, 1H), 6.02 (q, J = 7.1 Hz, 1H), 3.18 (s, 3H), 1.83 (d, J = 7.2 Hz,
3H). 13C NMR (100 MHz, DMSO‑d6) δ 161.1, 142.6, 140.6, 140.6, 137.4,
133.5, 129.0, 128.9, 128.7 (2C), 127.9, 126.4 (2C), 125.4, 116.1, 115.8,
54.7, 37.5, 21.2. HRMS calcd for C19H19FN3O, [M + H]+, 324.1512;
found 324.1513.
Light white solid; yield: 74.4%; 1H NMR (400 MHz, DMSO‑d6) δ 8.55
(s, 1H), 7.44 (s, 1H), 7.31 (t, J = 7.5 Hz, 2H), 7.21 (t, J = 7.3 Hz, 1H),
7.14 (d, J = 7.2 Hz, 2H), 6.85–6.77 (m, 4H), 5.80 (q, J = 7.0 Hz, 1H),
3.68 (s, 3H), 3.44–3.31 (m, 4H), 2.75–2.71 (m, 4H), 1.85 (d, J = 7.1 Hz,
3H). 13C NMR (100 MHz, DMSO‑d6) δ 159.8, 153.9, 145.3, 142.2, 137.6,
129.2 (2C), 128.4, 128.2, 126.7 (2C), 125.6, 118.7 (2C), 114.7 (2C),
56.1 (2C), 55.6 (2C), 50.1 (2C), 21.6. HRMS calcd for C23H27N4O2, [M +
H]+, 391.2134; found 391.2127.
4.8.9. (R)-N-methyl-1-(1-phenylethyl)-N-(p-tolyl)-1H-imidazole-5-
carboxamide (15i)
4.8.13. (R)-(4-(2-methoxyphenyl)piperazin-1-yl)(1-(1-phenylethyl)-1H-
imidazol-5-yl)methanone (15m)
Light white solid; yield: 71.7%; 1H NMR (300 MHz, DMSO‑d6) δ 7.96
(s, 1H), 7.36 (dq, J = 14.3, 7.1 Hz, 3H), 7.16 (d, J = 6.9 Hz, 2H), 7.00 (d,
J = 8.1 Hz, 2H), 6.54 (d, J = 8.0 Hz, 2H), 6.28 (s, 1H), 6.06 (q, J = 7.3
Hz, 1H), 3.17 (s, 3H), 2.23 (s, 3H), 1.82 (d, J = 7.2 Hz, 3H). 13C NMR
Light white solid; yield: 70.1%; 1H NMR (400 MHz, DMSO‑d6) δ 8.12
(s, 1H), 7.31 (t, J = 7.0 Hz, 2H), 7.22 (t, J = 7.0 Hz, 1H), 7.18 (s, 1H),
7

S. Zhao et al.
Bioorganic & Medicinal Chemistry 32 (2021) 115972
7.11 (d, J = 7.2 Hz, 2H), 7.01–6.81 (m, 3H), 6.73 (d, J = 7.5 Hz, 1H),
5.79 (q, J = 6.5 Hz, 1H), 3.75 (s, 3H), 3.45–3.31 (m, 4H), 2.76–2.65 (m,
4H), 1.83 (d, J = 6.8 Hz, 3H). 13C NMR (100 MHz, DMSO‑d6) δ 160.8,
152.4, 143.1, 141.0, 138.1, 131.1, 129.1 (2C), 128.1, 126.6 (2C), 125.4,
123.4, 121.2, 118.7, 112.3, 55.8 (2C), 55.6 (2C), 50.4 (2C), 21.7. HRMS
calcd for C23H27N4O2, [M + H]+, 391.2134; found 391.2134.
3H).
4.10. General procedure for the synthesis of compounds 18 a-f
Sodium hydroxide (2 N) was added to a solution of intermediate 17a-
f (1 equiv.) in methanol at ambient temperature. The reaction mixture
was stirred for 6 h and the methanol was removed by rotary evaporation.
The resultant mixture was adjusted to pH = 5–6 with 1 N HCl solution.
The precipitated white solid was collected by filtration and dried to give
the 18a-f.
4.8.14. (R)-(4-cyclopropyl-3,4-dihydroquinoxalin-1(2H)-yl)(1-(1-
phenylethyl)-1H-imidazol-5-yl)methanone (15n)
Light white solid; yield: 65.2%; 1H NMR (300 MHz, DMSO‑d6) δ 8.11
(s, 1H), 7.38–7.25 (m, 3H), 7.12 (d, J = 7.0 Hz, 3H), 6.98 (dd, J = 12.0,
4.9 Hz, 1H), 6.90 (s, 1H), 6.59 (d, J = 7.5 Hz, 1H), 6.43 (t, J = 7.6 Hz,
1H), 5.76 (q, J = 7.1 Hz, 1H), 4.06–3.90 (m, 1H), 3.43–3.39 (m, 1H),
3.28–3.23 (m, 1H), 2.93–2.89 (m, 1H), 2.44–2.33 (m, 1H), 1.76 (d, J =
7.1 Hz, 3H), 0.84–0.78 (m, 2H), 0.57–0.47 (m, 2H). 13C NMR (100 MHz,
DMSO‑d6) δ 159.9, 142.4, 139.6, 137.9, 132.9, 128.7 (2C), 127.8, 126.0
(2C), 125.7, 125.6, 124.9, 123.7, 115.9, 112.9, 54.9, 54.8, 48.1, 31.1,
21.18, 7.9, 7.5. HRMS calcd for C23H25N4O, [M + H]+, 373.2028; found
373.2021.
4.11. General procedure for the synthesis of compounds 19a-f
HATU (1.1 equiv.) and DIEA (2 equiv.) were added to a solution of
the intermediate acid compound 18a-f (1 equiv.) and 1-Cyclopropyl-
1,2,3,4-tetrahydro-quinoxaline 12 (1 equiv.) in anhydrous DMF. The so-
lution was heated to 70 ◦C for 6 h and then cooled to room temperature.
The reaction mixture was poured into ice water, and the resulting solid
was filtered. The crude product was purified by silica gel column
chromatography to give the target product 19a-f.
4.8.15. (R)-(3,4-dihydroquinolin-1(2H)-yl)(1-(1-phenylethyl)-1H-
imidazol-5-yl)methanone (15o)
4.11.1. (1-benzyl-1H-imidazol-5-yl)(4-cyclopropyl-3,4-
Light white solid; yield: 64.8%; 1H NMR (400 MHz, DMSO‑d6) δ 8.11
(s, 1H), 7.40–7.32 (m, 2H), 7.32–7.26 (m, 1H), 7.18 (d, J = 7.2 Hz, 2H),
7.14 (d, J = 7.5 Hz, 1H), 6.99 (t, J = 7.1 Hz, 1H), 6.86 (t, J = 7.3 Hz, 1H),
6.79 (s, 1H), 6.47 (d, J = 8.0 Hz, 1H), 5.89 (q, J = 7.1 Hz, 1H), 3.84 (dt,
J = 12.4, 6.1 Hz, 1H), 3.41 (ddd, J = 12.8, 7.4, 5.5 Hz, 1H), 2.77–2.58
(m, 2H), 1.83 (d, J = 7.2 Hz, 3H), 1.77–1.58 (m, 2H). 13C NMR (100
MHz, DMSO‑d6) δ 161.1, 142.4, 138.6, 137.9, 132.9, 131.2, 128.7 (2C),
128.6, 127.8, 126.2, 126.2 (2C), 125.3, 124.5, 124.3, 54.8, 44.7, 25.9,
23.4, 21.2. HRMS calcd for C21H22N3O, [M + H]+, 332.1763; found
332.1760.
dihydroquinoxalin-1(2H)-yl)methanone (19a)
Light white solid; yield: 73.1%; 1H (400 MHz, DMSO‑d6) δ 7.98 (s,
1H), 7.39–7.24 (m, 3H), 7.13 (d, J = 7.4 Hz, 3H), 6.99 (t, J = 7.7 Hz,
1H), 6.88 (s, 1H), 6.66 (d, J = 7.6 Hz, 1H), 6.45 (t, J = 7.5 Hz, 1H), 5.35
(s, 2H), 3.74 (t, J = 5.2 Hz, 2H), 3.12 (t, J = 5.3 Hz, 2H), 2.40 (ddd, J =
10.1, 6.7, 3.7 Hz, 1H), 0.81 (q, J = 6.5 Hz, 2H), 0.59–0.46 (m, 2H). 13
C
NMR (100 MHz, DMSO‑d6) δ 159.8, 140.9, 139.5, 137.5, 133.5, 128.7
(2C), 127.8, 127.3 (2C), 125.6, 125.0, 124.9, 123.7, 115.9, 113.0, 48.6,
48.1, 43.0, 31.2, 7.7 (2C). HRMS calcd for C22H23N4O, [M + H]+,
359.1872; found 359.1872.
4.8.16. (R)-(2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)(1-(1-
phenylethyl)-1H-imidazol-5-yl)methanone (15p)
4.11.2. (4-cyclopropyl-3,4-dihydroquinoxalin-1(2H)-yl)(1-(4-
fluorobenzyl)-1H-imidazol-5-yl)methanone (19b)
Light white solid; yield: 69.1%; 1H NMR (400 MHz, DMSO‑d6) δ 8.20
(s, 1H), 7.33 (t, J = 7.3 Hz, 2H), 7.28 (d, J = 7.1 Hz, 1H), 7.24 (s, 1H),
7.19 (d, J = 8.0 Hz, 1H), 7.14 (d, J = 7.2 Hz, 2H), 7.02–6.94 (m, 1H),
6.87–6.81 (m, 1H), 6.73 (t, J = 7.7 Hz, 1H), 5.86 (q, J = 7.1 Hz, 1H),
4.21–4.12 (m, 1H), 3.92–3.83 (m, 1H), 3.68–3.47 (m, 2H), 1.85 (d, J =
7.1 Hz, 3H). 13C NMR (100 MHz, DMSO‑d6) δ 160.3, 145.9, 142.6,
138.8, 133.6, 128.7 (2C), 127.8, 126.0 (2C), 125.4, 125.2, 125.1, 123.7,
119.6, 117.0, 65.4, 54.9, 44.1, 21.3. HRMS calcd for C20H19N3O2Na, [M
+ Na]+, 356.1375; found 356.1381.
Light white solid; yield: 71.3%; 1H NMR (400 MHz, DMSO‑d6) δ 7.98
(s, 1H), 7.27–7.10 (m, 5H), 7.00 (t, J = 7.8 Hz, 1H), 6.88 (s, 1H),
6.71–6.63 (m, 1H), 6.46 (t, J = 7.5 Hz, 1H), 5.34 (s, 2H), 3.75 (t, J = 5.4
Hz, 2H), 3.14 (t, J = 5.3 Hz, 2H), 2.43–2.36 (m, 1H), 0.82 (q, J = 6.4 Hz,
2H), 0.60–0.49 (m, 2H). 13C NMR (100 MHz, DMSO‑d6) δ 159.8, 140.9,
139.6, 133.8, 133.7, 133.6, 129.7, 129.6, 125.7, 124.9, 124.9, 123.8,
115.9, 115.6, 115.4, 113.0, 48.1, 47.9, 43.0, 31.2, 7.7 (2C). HRMS calcd
for C22H22FN4O, [M + H]+, 377.1778; found 377.1792.
4.11.3. (1-(2-chlorobenzyl)-1H-imidazol-5-yl)(4-cyclopropyl-3,4-
dihydroquinoxalin-1(2H)-yl)methanone (19c)
4.9. General procedure for the synthesis of compounds 17a-f
Light white solid; yield: 66.7%; 1H NMR (400 MHz, DMSO‑d6) δ 7.98
(s, 1H), 7.25–7.10 (m, 5H), 7.04–6.96 (m, 1H), 6.88 (s, 1H), 6.67 (d, J =
7.3 Hz, 1H), 6.46 (t, J = 7.3 Hz, 1H), 5.34 (s, 2H), 3.75 (t, J = 5.4 Hz,
2H), 3.14 (t, J = 5.4 Hz, 2H), 2.44–2.36 (m, 1H), 0.85–0.78 (m, 2H),
0.58–0.52 (m, 2H). 13C NMR (100 MHz, DMSO‑d6) δ 159.5, 141.6,
139.6, 135.1, 133.6, 131.7, 129.5, 128.4, 127.6, 125.9, 125.7, 125.1,
124.8, 123.8, 116.0, 113.0, 48.1, 46. 7, 43.2, 31.2, 7.8 (2C). HRMS calcd
for C22H22ClN4O, [M + H]+, 393.1482; found 393.1476.
To a solution of ethyl 1H-imidazole-5-carboxylate (1 equiv) 16 and
the substituted benzyl bromide (1 equiv) in Tetrahydrofuran was added
10%NaOH and tetra-n-butylammonium bromide (0.2 equiv) at 0 ◦C. The
reaction was stirred for 6–8 h and was then concentrated under reduced
pressure. The residue was extracted with EtOAc and then brine. The
organic phase was dried over Na2SO4 overnight and and the solvent was
removed in vaccuo. The crude product was purified by silica gel column
chromatography to yield the target product 17a-f.
4.11.4. (4-cyclopropyl-3,4-dihydroquinoxalin-1(2H)-yl)(1-(2,4-
dichlorobenzyl)-1H-imidazol-5-yl)methanone (19d)
4.9.1. Ethyl 1-(2,4-dichlorobenzyl)-1H-imidazole-5-carboxylate (17d)
1H NMR (400 MHz, DMSO‑d6) δ 8.08 (s, 1H), 7.77 (d, J = 0.8 Hz,
1H), 7.70 (d, J = 2.1 Hz, 1H), 7.38 (dd, J = 8.4, 2.1 Hz, 1H), 6.52 (d, J =
8.4 Hz, 1H), 5.59 (s, 2H), 4.15 (q, J = 7.1 Hz, 2H), 1.16 (t, J = 7.1 Hz,
3H).
Light white solid; yield: 69.4%; 1H NMR (400 MHz, DMSO‑d6) δ 7.93
(s, 1H), 7.67 (d, J = 2.1 Hz, 1H), 7.42 (dd, J = 8.4, 2.1 Hz, 1H), 7.16 (d, J
= 8.2 Hz, 1H), 7.01 (dd, J = 11.3, 4.2 Hz, 1H), 6.96 (s, 1H), 6.88 (d, J =
7.9 Hz, 1H), 6.82 (d, J = 8.4 Hz, 1H), 6.52 (t, J = 7.6 Hz, 1H), 5.47 (s,
2H), 3.81 (t, J = 5.3 Hz, 2H), 3.20 (t, J = 5.3 Hz, 2H), 2.46–2.36 (m, 1H),
0.88–0.78 (m, 2H), 0.61–0.50 (m, 2H). 13C NMR (100 MHz, DMSO‑d6) δ
159.4, 141.6, 139.6, 134.3, 133.7, 133.1, 132.7, 129.8, 128.9, 127.7,
125.8, 125.0, 124.8, 123.8, 116.0, 113.0, 48.1, 46.3, 43.1, 31.2, 7.7
(2C).HRMS calcd for C22H21Cl2N4O, [M + H]+, 427.1092; found
4.9.2. Ethyl 1-(2,5-dichlorobenzyl)-1H-imidazole-5-carboxylate (17e)
1H NMR (400 MHz, DMSO‑d6) δ 8.09 (s, 1H), 7.78 (d, J = 0.8 Hz,
1H), 7.57 (d, J = 8.5 Hz, 1H), 7.43 (dd, J = 8.5, 2.5 Hz, 1H), 6.49 (d, J =
2.5 Hz, 1H), 5.61 (s, 2H), 4.16 (q, J = 7.1 Hz, 2H), 1.16 (t, J = 7.1 Hz,
8

S. Zhao et al.
Bioorganic & Medicinal Chemistry 32 (2021) 115972
427.1075.
4.15. GLP-1 secretion
4.11.5. (4-cyclopropyl-3,4-dihydroquinoxalin-1(2H)-yl)(1-(2,5-
dichlorobenzyl)-1H-imidazol-5-yl)methanone (19e)
To examine the in vivo effect of compound 19d on GLP-1 secretion,
vehicle (0.25% CMC) or test compound 19d at 30 mg/kg was admin-
istered orally to overnight-fasted eight-week-old wild-typ (WT) (C57BL/
6J) or TGR5ꢀ /ꢀ mice (TGR5 KO). 1 h later, all of the mice were received
an oral glucose (2.0 g/kg) challenge. Blood samples were collected at 5
min after the glucose challenge and placed into eppendorf tubes con-
taining the dipeptidyl peptidase IV (DPP IV) inhibitor with a final con-
centration of 1% blood samples and 25 mg/mL EDTA to measure serum
active GLP-1[7–36 amide] levels.
Light white solid; yield: 72.1%; 1H NMR (400 MHz, DMSO‑d6) δ 7.95
(s, 1H), 7.55 (d, J = 8.5 Hz, 1H), 7.44 (dd, J = 8.5, 2.5 Hz, 1H), 7.16 (d, J
= 7.9 Hz, 1H), 7.03 (t, J = 7.7 Hz, 1H), 6.95 (s, 1H), 6.87 (d, J = 7.8 Hz,
1H), 6.76 (d, J = 2.4 Hz, 1H), 6.52 (t, J = 7.4 Hz, 1H), 5.49 (s, 2H), 3.82
(t, J = 5.4 Hz, 2H), 3.25 (t, J = 5.4 Hz, 2H), 2.46–2.36 (m, 1H), 0.83 (q, J
= 6.5 Hz, 2H), 0.62–0.51 (m, 2H). 13C NMR (100 MHz, DMSO‑d6) δ
159.4, 141.7, 139.7, 137.4, 133.7, 132.1, 131.2, 130.4, 129.3, 127.9,
125.9, 124.9, 124.8, 123.8, 115.9, 113.1, 48.2, 46.5, 43.0, 31.2, 7.7
(2C).HRMS calcd for C22H21Cl2N4O, [M + H]+, 427.1092; found
427.1082.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
4.11.6. (4-cyclopropyl-3,4-dihydroquinoxalin-1(2H)-yl)(1-(2,4-
difluorobenzyl)-1H-imidazol-5-yl)methanone (19f)
Light white solid; yield: 61.6%; 1H NMR (400 MHz, DMSO‑d6) δ 7.92
(s, 1H), 7.33–7.24 (m, 1H), 7.19–6.98 (m, 4H), 6.89 (s, 1H), 6.76 (d, J =
7.3 Hz, 1H), 6.49 (t, J = 7.6 Hz, 1H), 5.41 (s, 2H), 3.78 (t, J = 5.4 Hz,
2H), 3.19 (t, J = 5.3 Hz, 2H), 2.45–2.36 (m, 1H), 0.82 (q, J = 6.5 Hz,
2H), 0.56 (dd, J = 6.4, 3.6 Hz, 2H). 13C NMR (100 MHz, DMSO‑d6) δ
159.5, 141.2, 139.6, 133.3, 131.1, 131.0, 125.7, 125.0, 124.8, 123.7,
120.9, 120.8, 115.9, 113.0, 111.8, 104.2, 56.0, 48.1, 42.5, 31.18, 7.7
(2C). HRMS calcd for C22H21F2N4O, [M + H]+, 395.1683; found
395.1672.
Acknowledgements
This research was supported by the National Natural Science Foun-
dation of China (Grant No. 81970726 and No. 81472232), Henan Pro-
vincial Natural Science Foundation (Grant No.182300410323), First
Class Discipline Cultivation Project of Henan University (Grant No.
2019YLZDYJ19) and Plan for Scientific Innovation Talent of Henan
Province (Grant No. 154100510004), Key Program for Science &
Technology from Henan Education Department (Grant No. 21A310001)
to W.-D.C., the National Natural Science Foundation of China (Grant
No.81903444), China Postdoctoral Science Foundation (Grant No.
2018M640674), Key Science and Technology Program of Henan Prov-
ince (Grant No. 192102310145) and Henan Postdoctoral Foundation
(Grant No. 201902027) to S.Z., Major Scientific and Technological
Innovation Project of Hebi to W.L., the National Natural Science Foun-
dation of China (Grant No. 81970551 and No. 81672433) and the
Fundamental Research Funds for the Central Universities and Research
projects on biomedical transformation of China-Japan Friendship Hos-
pital (Grant No. PYBZ1803) to Y.-D.W.
4.12. Cell-Based TGR5 agonism assay
A lentivirus expressing human TGR5 (NM_001077191.1) and mouse
(NM_174985) were obtained from Hanbio Biotechnology Co. Ltd.
HEK293T cells were transfected with lentivirus and a stable cell line was
isolated using drug selection following standard techniques. LCA and
INT-777 was used as reference compounds. The test compounds and
reference compounds were prepared in DMSO at stock solutions of 10
mM. TGR5-mediated cAMP generation was assayed using a HTRF (Ho-
mogeneous Time-Resolved Fluorescence) detection method (HTRF
cAMP dynamic 2 Assay Kit; Cisbio cat # 62AM4PEB) according to the
manufacturer’s protocol.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmc.2020.115972.
4.13. FXR activity assay
Biotinylated SRC1 peptide (5′-biotin-CPSSHSSLTERHKILHRLL-
QEGSPS-CONH2) was synthesized by GL Biochem (Shanghai) Ltd.
References
(China). Human FXR ligand binding domain (GST-FXRαLBD) was
1 Tiwari A, Maiti P. TGR5: an emerging bile acid G-protein-coupled receptor target for
the potential treatment of metabolic disorders. Drug Discovery Today. 2009;14:
523–530.
expressed and purified by Shanghai Shengong Biotechonology
(Shanghai, China). The assay mixture contained 10 nM GST- FXR LBD,
α
100 nM biotin-SRC1, 0.83 nM Eu-labeled anti-GST, and 41.75 nM
Streptavidin-XL665 (Cisbio, USA) in HTRF buffer. The HTRF buffer was
composed of 50 mM Hepes pH 7.0, 125 mM KF, 0.125% CHAPS, and
0.05% dry milk. The white 384-well microplates were incubated at room
temperature and then read with a CLARIOstar Microplate Reader (BMG
LABTECH) and calculated using the equation (665 nm/620 nm) *
10,000.
2 Zhong Min. TGR5 as a therapeutic target for treating obesity. Current Top Med Chem.
2010;10:386–396.
3 Pols TWH, Noriega LG, Nomura M, et al. The bile acid membrane receptor TGR5 as
an emerging target in metabolism and inflammation. J Hepatol. 2011;54:1263–1272.
4 Guo C, Qi H, Yu YJ, et al. The G-protein-coupled bile acid receptor Gpbar1 (TGR5)
inhibits gastric inflammation through antagonizing NF-kappa B signaling pathway.
Front Pharmacol. 2015;6:287.
5 Fryer RM, Ng KJ, Mazurek SGN, et al. G protein-coupled bile acid receptor 1
stimulation mediates arterial vasodilation through a KCa1.1 (BKCa)-dependent
mechanism (vol 348, pg 421, 2014). J Pharmacol Exp Therapeut. 2014;349, 174–174.
6 Klindt C, Reich M, Hellwig B, et al. The G protein-coupled bile acid receptor TGR5
(Gpbar1) modulates endothelin-1 signaling in liver. Cells. 2019;8:21.
7 Keitel V, Stindt J, Haussinger D. Bile acid-activated receptors: GPBAR1 (TGR5) and
other G protein-coupled receptors. Handb Exp Pharmacol. 2019;256:19–49.
8 Katsuma S, Hirasawa A, Tsujimoto G. Bile acids promote glucagon-like peptide-1
secretion through TGR5 in a murine enteroendocrine cell line STC-1. Biochem Biophys
Res Commun. 2005;329:386–390.
4.14. Oral Glucose Tolerance Test (OGTT)
Male C57L/6J mice (6–8 weeks old) were obtained from Beijing Vital
River Laboratory Animal Technologies Co. Ltd and randomly assigned to
3 groups (n = 8 in each group). Overnight-fasted C57L/6J mice were
orally administered the vehicle (0.25% CMC) or test compounds 19d
and 19e at 30 mg/kg, followed by an oral glucose load (4 g/kg) at 60
min post compound dose. Blood glucose levels were measured via blood
drops obtained by clipping the tail of the mice before compound dosing
and 0, 15, 30, 60, and 120 min post glucose loading. Blood glucose levels
and Glucose AUC were carried out in GraphPad Prism.
9 Ullmer C, Sanchez RA, Sprecher U, et al. Systemic bile acid sensing by G protein-
coupled bile acid receptor 1 (GPBAR1) promotes PYY and GLP-1 release. Br J
Pharmacol. 2013;169:671–684.
10 Watanabe M, Houten SM, Mataki C, et al. Bile acids induce energy expenditure by
promoting intracellular thyroid hormone activation. Nature. 2006;439:484–489.
9

S. Zhao et al.
Bioorganic & Medicinal Chemistry 32 (2021) 115972
11 Zhang XQ, Wall M, Sui ZH, et al. Discovery of orally efficacious
tetrahydrobenzimidazoles as TGR5 agonists for type 2 diabetes. ACS Med Chem Lett.
2017;8:560–565.
20 Agarwal S, Patil A, Aware U, et al. Discovery of a potent and orally efficacious TGR5
receptor agonist. ACS Med Chem Lett. 2016;7:51–55.
21 Agarwal S, Sasane S, Kumar J, et al. Evaluation of novel TGR5 agonist in
combination with Sitagliptin for possible treatment of type 2 diabetes. Bioorg Med
Chem Lett. 2018;28:1849–1852.
12 Duboc H, Tache Y, Hofmann AF. The bile acid TGR5 membrane receptor: From basic
research to clinical application. Dig Liver Dis. 2014;46:302–312.
13 Hodge RJ, Nunez DJ. Therapeutic potential of Takeda-G-protein-receptor-5 (TGR5)
agonists. Hope or hype? Diabetes Obes Metab. 2016;18:439–443.
22 Duan HL, Ning MM, Chen XY, et al. Design, synthesis, and antidiabetic activity of 4-
phenoxynicotinamide and 4-phenoxypyrimidine-5-carboxamide derivatives as
potent and orally efficacious TGR5 agonists. J Med Chem. 2012;55:10475–10489.
23 Martin RE, Bissantz C, Gavelle O, et al. 2-Phenoxy-nicotinamides are potent agonists
at the bile acid receptor GPBAR1 (TGR5). ChemMedChem. 2013;8:569–576.
24 Chen T, Reich NW, Bell N, et al. Design of gut-restricted thiazolidine agonists of G
protein-coupled bile acid receptor 1 (GPBAR1, TGR5). J Med Chem. 2018;61:
7589–7613.
14 Pellicciari R, Gioiello A, Macchiarulo A, et al. Discovery of 6 alpha-Ethyl-23(S)-
methylcholic Acid (S-EMCA, INT-777) as a potent and selective agonist for the TGR5
receptor, a novel target for diabesity. J Med Chem. 2009;52:7958–7961.
15 Thomas C, Gioiello A, Noriega L, et al. TGR5-mediated bile acid sensing controls
glucose homeostasis. Cell Metab. 2009;10:167–177.
16 Evans KA, Budzik BW, Ross SA, et al. Discovery of 3-aryl-4-isoxazolecarboxamides as
TGR5 receptor agonists. J Med Chem. 2009;52:7962–7965.
25 Nakhi A, McDermott CM, Stoltz KL, et al. 7-Methylation of chenodeoxycholic acid
derivatives yields a substantial increase in TGR5 receptor potency. J Med Chem.
2019;62:6824–6830.
17 Herbert MR, Siegel DL, Staszewski L, et al. Synthesis and SAR of 2-aryl-3-amino-
methylquinolines as agonists of the bile acid receptor TGR5. Bioorg Med Chem Lett.
2010;20:5718–5721.
26 Sepe V, Renga B, Festa C, et al. Modification on ursodeoxycholic acid (UDCA)
scaffold. Discovery of bile acid derivatives as selective agonists of cell-surface G-
protein coupled bile acid receptor 1 (GP-BAR1). J Med Chem. 2014;57:7687–7701.
27 Piotrowski DW, Futatsugi K, Warmus JS, et al. Identification of tetrahydropyrido 4,3-
d pyrimidine amides as a new class of orally bioavailable TGR5 agonists. ACS Med
Chem Lett. 2013;4:63–68.
18 Budzik BW, Evans KA, Wisnoski DD, et al. Synthesis and structure-activity
relationships of a series of 3-aryl-4-isoxazolecarboxamides as a new class of TGR5
agonists. Bioorg Med Chem Lett. 2010;20:1363–1367.
19 Zou QA, Duan HL, Ning MM, et al. 4-Benzofuranyloxynicotinamide derivatives are
novel potent and orally available TGR5 agonists. Eur J Med Chem. 2014;82:1–15.
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