Discovery of furan-2-carbohydrazides as orally active glucagon receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2014.07.025

Furan-2-carbohydrazides were found as orally active glucagon receptor antagonists. Starting from the hit compound 5, we successfully determined the structure activity relationships of a series of derivatives obtained by modifying the acidity of the phenol

Full text of DOI:10.1016/j.bmcl.2014.07.025

Bioorganic & Medicinal Chemistry Letters 24 (2014) 4266–4270
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of furan-2-carbohydrazides as orally active glucagon
receptor antagonists
⇑
Futoshi Hasegawa , Kazumi Niidome, Chiaki Migihashi, Makoto Murata, Toshiyuki Negoro,
Takafumi Matsumoto, Kaori Kato, Akihito Fujii
Dainippon Sumitomo Pharma. Co. Ltd, 3-1-98 Kasugade-naka, Konohana-ku, Osaka 554-0022, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Furan-2-carbohydrazides were found as orally active glucagon receptor antagonists. Starting from the hit
compound 5, we successfully determined the structure activity relationships of a series of derivatives
obtained by modifying the acidity of the phenol. We identified the ortho-nitrophenol as a good scaffold
for glucagon receptor inhibitory activity. Our efforts have led to the discovery of compound 7l as a potent
glucagon receptor antagonist with good bioavailability and satisfactory long half-life.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 19 May 2014
Revised 3 July 2014
Accepted 9 July 2014
Available online 24 July 2014
Keywords:
Glucagon
Type 2 diabetes
Furan-2-carbohydrazide
Glucagon receptor antagonist
Hyperglucagonemia
Type 2 diabetes is characterized not only by insulin resistance
and b-cell dysfunction but also by hyperglucagonemia in the fast-
ing state and lack of glucagon suppression following meal inges-
tion.1,2 It is therefore necessary for a complete treatment of type
2 diabetes to include agents that reverse hyperglucagonemia.
Glucagon, a peptide hormone consisting of 29 amino acid resi-
derivative (Fig. 1), as a hit compound with moderate binding
affinity for GCGR (50% inhibition at 10
M in rat hepatocyte).11
Our strategy for hit to lead generation focused on introducing the
l
acidic moiety (Fig. 2).
Initially we replaced the furyl group in 5 with various groups as
shown in Table 1. Since the phenyl compound 6a exhibited a GCGR
binding affinity similar to that of 5, we next introduced a hydroxy
group, as acidic moiety, at the phenyl group of 6a. The obtained
para-hydroxyphenyl compound 6b showed a slight improvement
in GCGR affinity, whereas the meta-hydroxyphenyl compound 6c
gave a loss in GCGR affinity. When the para-hydroxy group was
masked with a methyl group, the resulting compound 6d showed
a complete loss of GCGR affinity. Regarding the other acidic group,
benzoic acid 6e showed a slight loss in GCGR affinity compared to
the phenol 6b. Introduction of two hydroxy groups (6f) resulted in
no improvement in GCGR affinity. Remarkably, further improve-
ment was seen with the hydroxypyridine 6g, which showed a
10-fold IC50 value improvement compared to the hit compound
5. Based on these results, it became clear that the pKa values and
GCGR affinity of compounds 6b,c,f,g had similar variation. These
findings suggested that an acidic proton at the para-position is
needed for high GCGR affinity and that the acidity of the phenol
group relates to GCGR affinity. However, a too strong acid such
as benzoic acid 6e would not exceptionally be well tolerated.
To confirm the relationship between the pKa values and the
affinity for GCGR, we screened substituents at the meta-position
of the phenol shown as R2 in Table 2 and calculated the pKa values
dues and produced in the a-cells of the pancreas, acts in the liver
where it binds to the glucagon receptor (GCGR) to initiate gluco-
neogenesis and glycogenolysis.3 It has been reported that plasma
glucagon levels are abnormally high throughout the day in type
2 diabetic patients.2 This led to the idea that GCGR antagonists
may reduce hepatic glucose output and lower abnormal plasma
glucose levels.3b,4 In fact, Bayer reported that the GCGR antagonist,
Bay 27-9955 (1, Fig. 1), suppresses excess glucagon-induced high
plasma glucose levels in humans.5 These findings indicate that
GCGR antagonists may be useful in the treatment of type 2
diabetes.
To date, a number of non-peptidic GCGR antagonists with vari-
ous acidic moieties including, b-alanine (NNC 25-09266 and MK-
0893, 27), tetrazole (38), or ortho-cyanophenol (49) have been
reported (Fig. 1). Although some of these compounds proceeded
to clinical trials,7,10 none is clinically available. In our search for
new chemotypes of GCGR antagonists, we screened our chemical
library and found compound 5, 3,4-diphenylfuran-2-carbohydrazide
⇑
Corresponding author. Tel.: +81 6 6466 5947; fax: +81 6 6466 5287.
E-mail address: futoshi-hasegawa@ds-pharma.co.jp (F. Hasegawa).
http://dx.doi.org/10.1016/j.bmcl.2014.07.025
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

F. Hasegawa et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4266–4270
4267
Cl
Table 1
F
SARs following modification of the furyl ring in 5
Cl
O
O
OH
HO
N
H
N
N
O
O
H
N
R1
N
H
(+)
2
O
1
MK-0893
Bay 27-9955
GCGR binding IC50: 6.6 nM
Rat PK (3 mg/kg, iv)
half-life: 5.9 h
O
,b
c
Compound no.
R1
GCGR bindinga (%)
pKa
Cl
Cl
F: 43%
O
5
6a
6b
50
42
65
N
NH
N
N
O
O
N
N
N
HN
O
N
N
H
HO
CN
8.04
8.52
HO
HO
3
4
GCGR binding IC50: 24 nM
Rat PK (3 mg/kg, iv)
half-life: 8.3 h
GCGR binding IC50: 2.3 nM
Dog PK (0.5 mg/kg, iv)
half-life: 1.1 h
6c
21
F: 15%
F: 15%
O
O
H
N
O
6d
6e
No inhibition
61
MeO
N
H
O
5
3.14
7.96
HO2C
HO
Figure 1. Representative chemotypes of GCGR antagonists and hit compound 5.
6f
57
HO
HO
N
of the obtained compounds 6h–n. The use of a fluoride (6h)
resulted in a remarkable improvement in GCGR affinity with a
pKa value much lower than that of compound 6b. Similarly, a chlo-
ride (6i) or a bromide (6j) led to improved GCGR affinity. Com-
pounds possessing a strong electron withdrawing group, such as
a trifluoromethyl group (6k) or a nitro group (6l) showed dramat-
ically improved GCGR affinity, especially compound 6l exhibited
more than 100-fold improved affinity compared to the hit com-
pound 5. On the other hand, compounds with electron-donating
groups, a methoxy (6m) or a phenyl group (6n) showed no
improvement in GCGR affinity compared to 6b. These findings con-
firmed our hypothesis as good correlation was observed between
IC50 values and pKa values (correlation coefficient: r = 0.96, from
6b to 6n in Table 2).
Next, the effects of a phenyl group at the 3- and 4-positions of
the furan on GCGR affinity were investigated (Fig. 3). Surprisingly,
despite the lack of a 4-phenyl group at the furan, compound 7a
exhibited almost the same GCGR affinity as compound 6l. On the
other hand, when the phenyl group at the 3-position of the furan
was removed, GCGR affinity diminished (8, and 9). Therefore, only
the phenyl group at the 4-position of furan was removed from 6l to
decrease its molecular weight and lipophilicity.
6g
IC50 = 0.97
lM
7.83
a
b
c
Activities are shown as the percent inhibition at 10 lM in rat hepatocytes.
The assay was performed in duplicate (n = 2).
Predicted using ADMET Predictor (SimulationsPlus, Lancaster, CA, USA).
Table 2
SARs following meta-substitution at the phenyl ring
O
O
H
N
R2
HO
N
H
O
a
b
Compound no.
R2
GCGR binding IC50
(l
M)
pKa
6b
6h
6i
6j
6k
6l
H
F
Cl
Br
CF3
NO2
MeO
Ph
9.5
1.4
8.04
6.41
6.71
6.78
6.11
6.21
8.02
8.08
0.43
0.27
0.16
0.087
9.5
Finally, we optimized the substituents at the ortho-, meta- or
para-position of the phenyl ring (shown as R3, R4, R5 in Table 3).
The para-methyl compound 7d exhibited high affinity compared
to the ortho-isomer 7b or the meta-isomer 7c. As the para-substitu-
ent was critical for good affinity, we fixed a mono substituent at
the para-position of the phenyl ring. The para-methoxy compound
7e had weak GCGR affinity compared to the methyl compound 7d.
6m
6n
>10
a
The assay was performed in duplicate using rat hepatocytes (n = 2).
Predicted using ADMET Predictor (SimulationsPlus, Lancaster, CA, USA).
b
HO
HO2C
O
O
O
O
H
N
H
N
O
N
H
N
H
Hybrid
O
O
R
5
R: OH or CO2H
Figure 2. Strategy for hit to lead generation.

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F. Hasegawa et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4266–4270
a Predicted using ADMETP redictor (SimulationsPlus, Lancaster, CA,USA)
Removal
O
4
3
O
H
N
O
O
H
N
O2N
HO
O2N
HO
N
H
N
H
O
O
6l
7a
Retention
GCGR binding IC50: 0.087 µM
GCGR binding IC50: 0.089 µM
MW: 443.42
MW: 367.32
cLogD7.4: 2.3a
cLogD7.4: 0.87a
4
O
O
O
O
H
N
H
N
3
N
H
N
H
O
O
O
O
8
Removal
9
Decrease
GCGR binding: 73% inhibition at 10 µM
GCGR binding: 6% inhibition at 10 µM
Figure 3. Effects of the furan phenyl group on GCGR affinity.
Table 3
Table 4
Profiles of compound 7l
SARs following substitution at the phenyl ring
O
O
Pharmacological profiles
GCGR binding IC50 (nM)
cAMP IC50 rat/dog/human (nM)
GLP-1R IC50 (nM)
R3
H
N
9.6
5.3/183/292
>1000
O2N
HO
N
H
R4
O
R5
Safety profiles
hERG IC50 (nM)
Sodium channel, site 2 IC50 (nM)
>1000
>1000
a
Compound no.
R3
R4
R5
GCGR binding IC50 (nM)
Pharmacokinetic profiles in rats
1 mg/kg, iv
Clearance (min/mL/kg)
T1/2 (h)
7a
7b
7c
7d
7e
7f
7g
7h
7i
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
Me
MeO
F
Cl
Et
n-Pr
i-Pr
n-Bu
n-Pen
89
180
100
27
93
69
270
44
2.9
24
5.5
9.6
0.28
5.9
0.59
Vdss (L/kg)
1 mg/kg, po
Cmax (ng/mL)
T1/2 (h)
411
6.1
37
BA (%)
7j
7k
7l
a
The assay was performed in duplicate using rat hepatocytes (n = 2).
Similarly, the fluoride compound 7f and the chloride compound 7g
showed lower affinity than the methyl compound 7d. Since an
alkyl group seemed to be preferred to a halogen group, we
extended the alkyl chain at the methyl group. As a result, the linear
alkyl-chain derivatives 7i,k,l showed more potent GCGR binding
affinity than 7d.
Three potent compounds that is, 7i,k,l were selected and evalu-
ated for their inhibition of cAMP production using rat, dog and
human primary hepatocytes.12 As cAMP is known as an intracellu-
lar second messenger for downstream signals of GCGR stimulation
by glucagon,2a,13 GCGR antagonists are believed to inhibit cAMP
production in hepatocytes. Among the three selected compounds,
compound 7l14 showed the most potent inhibitory activity of cAMP
Figure 4. Compound 7l suppression of glucagon-induced glucose elevation in
normal rats. Compound 7l was orally administered 30 min before intravenous
administration of glucagon (3 lg/kg). Blood samples were collected 6 h after
accumulation (Table 4). In addition, compound 7l (1 lM) showed
no noteworthy binding affinity for over 100 off-targets, including
enzymes, receptors, ion channels and GLP-1R (Table 4),15 indicat-
ing that compound 7l has more than 100-fold selectivity for GCGR.
To evaluate the in vivo efficacy of compound 7l, a rat pharmacoki-
netic study of this compound was performed. As shown in Table 4,
compound 7l showed good bioavailability with satisfactory long
half-life.
Figure 4 shows the results of glucagon challenge test of
compound 7l in normal rats (n = 6). Compound 7l was orally
administered 30 min before intravenous administration of
glucagon administration. Data are given as the mean values SEM (n = 6/group).
⁄⁄P <0.01 (Dunnett multiple comparison test).
glucagon (3 lg/kg). Compound 7l at 30 mg/kg significantly
inhibited glucagon-induced glucose elevation. Compound 7l
suppressed glucose elevation for up to 6 h after administration
(data not shown).
We further investigated the glucose lowering effect of com-
pound 7l using Goto-Kakizaki (GK) rats, which are non-obese type

F. Hasegawa et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4266–4270
4269
O
O
R3
R5
R3
R5
H
N
O
O
a
c
b
H2N
O
Br
R4
R4
O
O
O
11
12
13
3
3
3
3
:
:
=
=
=
=
=
=
=
:
:
R
=
=
=
=
=
=
7a
7b
R
R
R4
R5
H,
H
=
=
R
R
R4
R4
R5 Cl
O
O
H,
H,
=
7g
7h
H,
H,
H,
H,
R3
H
N
R4
=
R5
R5
H
H
R5
5 = n
Et
-Pr
O2N
HO
Me,
N
H
R4
R4
R4
R4
R4
R
3
3
3
:
:
:
=
=
=
=
=
7c
7d
7i
R
R
H,
H,
H,
H,
Me,
H,
H,
=
H,
H,
R4
O
R4
R4
R4
R5
Me
R
R
R5 i-Pr
5=n
3
=
=
=
=
:
=
H,
H,
H,
7j
R
R5 MeO
R
R
-Butyl
3
3
R5
:
:
=
:
7e
7f
7k
H,
H,
7a-l
R5
F
3
3
=
=
:
=
=
5=n
R
-Pentyl
7l
R
H,
H,
Scheme 2. Reagents and conditions: (a) phenylboronic acid, Pd(PPh3)4, Cs2CO3,
THF/H2O, reflux, 84–99%; (b) N2H4ÁH2O, EtOH, reflux, 47–99%; (c) (i) 3-nitro-4-
acetoxybenzoic acid, WSCÁHCl, DMF, rt; (ii) 10% NaOH aq, MeOH, rt, 51–77% (2
steps).
Figure 5. Glucose lowering effect of compound 7l in GK rats. Compound 7l was
orally administered at 30 mg/kg. Blood samples were collected at 2, 4 and 6 h after
compound 7l administration. Data are given as the mean values SEM (n = 8–9/
group). ⁄⁄⁄P <0.001 (Student’s t-test)
O
a
O
O
O
NH2
H
N
N
H
HO
N
H
O
O
9
O
O
14
15
Scheme 3. Reagents and conditions: (a) BOP, Et3N, DMF, rt, 32%.
O
O
O
O
O
a
b
H
N
H
N
H
N
R2
HO
R1
N
H
H2N
N
H
O
O
O
Acknowledgments
6a,c-e,g,8
10
6b,f,h-n
6a: R1 = Ph
6b: R2 = H
6c: R1 = 3-hydroxyphenyl
6f: R2 = OH
We are thankful to Dr. Yoshiaki Isobe, Dr. Masanori Tobe and Dr.
Tomoya Shiro for their informative advice and continuous support.
: R1 = 4-methoxyphenyl
: R1 = 4-carboxyphenyl
: R1 = 4-hydroxy-3-pyridyl
: R2 = F
: R2 =Cl
: R2 = Br
6d
6e
6g
6h
6i
6j
8: R1 = 3-furyl
6k: R2 = CF3
6l: R2 = NO2
6m: R2 = MeO
6n: R2 = Ph
References and notes
1. Baron, A. D.; Schaeffer, L.; Shragg, P.; Kolterman, O. G. Diabetes 1987, 36, 274.
2. Reaven, G. M.; Chen, Y.-D. I.; Golay, A.; Swislocki, A. L. M.; Jaspan, J. B. J. Clin.
Endocrinol. Metab. 1987, 64, 106.
3. (a) Jiang, G.; Zhang, B. B. Am. J. Physiol. Endocrinol. Metab. 2003, 284, E671; (b)
Sloop, K. W.; Michael, M. D.; Moyers, J. S. Expert Opin. Ther. Targets 2005, 9, 593.
4. Ling, A. Drugs Future 2002, 27, 987.
Scheme 1. Reagents and conditions: (a) carboxylic acid, BOP, Et3N, DMF, rt, 17–
95%; (b) (i) benzoic acid, BOP, Et3N, DMF, rt, 18–100%; (ii) BBr3, CH2Cl2, rt, 13–96%.
5. Petersen, K. F.; Sullivan, J. T. Diabetologia 2001, 44, 2018.
2 diabetic rats (Fig. 5).16 Compound 7l (30 mg/kg) significantly
decreased glucose levels in GK rats. This decreasing effect was sus-
tained for up to 6 h after administration. These results indicate that
compound 7l acts as a long-term GCGR antagonist not only in nor-
mal rats but also in GK diabetic rats.
The 3,4-diphenyl furan derivatives were prepared as illustrated
in Scheme 1. The acylhydrazide 1017 was condensed with various
carboxylic acids to afford compounds 6a,c–e,g, and 8. Similarly,
compound 10 was condensed with various benzoic acids followed
by deprotection of the methyl group by BBr3, when necessary, to
afford compounds 6b,f,h–n.
Compounds 7a–l were synthesized as shown in Scheme 2.
Suzuki–Miyaura reaction of the ethyl 3-bromofuran-2-carboxylate
1118 with various phenylboronic acids gave compound 12 in high
yields. The ester group of compound 12 was converted to a hydra-
zide by hydrazine monohydrate under reflux conditions. Conden-
sation of 13 with 3-nitro-4-acetoxybenzoic acid19 followed by
deprotection of the acetyl group afforded the desired compounds
7a–l.
6. (a) Ling, A.; Plewe, M. B.; Truesdale, L. K.; Lau, J.; Madsen, P.; Sams, C.; Behrens,
C.; Vagner, J.; Christensen, I. T.; Lundt, B. F.; Sidelmann, U. G.; Thøgersen, H. WO
Patent 069810, 2000.; (b) Lau, J.; Behrens, C.; Sidelmann, U. G.; Knudsen, L. B.;
Lundt, B.; Sams, C.; Ynddal, L.; Brand, C. L.; Pridal, L.; Ling, A.; Kiel, D.; Plewe,
M.; Shi, S.; Madsen, P. J. Med. Chem. 2007, 50, 113; (c) 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. J. Med. Chem. 2008, 51, 5387.
7. Xiong, Y.; Guo, J.; Candelore, M. R.; Liang, R.; Miller, C.; Dallas-Yang, Q.; Jiang,
G.; McCann, P. E.; Qureshi, S. A.; Tong, X.; Xu, S. S.; Shang, J.; Vincent, S. H.; Tota,
L. M.; Wright, M. J.; Yang, X.; Zhang, B. B.; Tata, J. R.; Parmee, E. R. J. Med. Chem.
2012, 55, 6137.
8. DeMong, D.; Dai, X.; Hwa, J.; Miller, M.; Lin, S.-I.; Kang, L.; Stamford, A.;
Greenlee, W.; Yu, W.; Wong, M.; Lavey, B.; Kozlowski, J.; Zhou, G.; Yang, D.-Y.;
Patel, B.; Soriano, A.; Zhai, Y.; Sondey, C.; Zhang, H.; Lachowicz, J.; Grotz, D.;
Cox, K.; Morrison, R.; Andreani, T.; Cao, Y.; Liang, M.; Meng, T.; McNamara, P.;
Wong, J.; Bradley, P.; Feng, K.-I.; Belani, J.; Chen, P.; Dai, P.; Gauuan, J.; Lin, P.;
Zhao, H. J. Med. Chem. 2014, 57, 2601.
9. Madsen, P.; Ling, A.; Plewe, M.; Sams, C. K.; Knudsen, L. B.; Sidelmann, U. G.;
Ynddal, L.; Brand, C. L.; Andersen, B.; Murphy, D.; Teng, M.; Truesdale, L.; Kiel,
D.; May, J.; Kuki, A.; Shi, S.; Johnson, M. D.; Teston, K. A.; Feng, J.; Lakis, J.;
Anderes, K.; Gregor, V.; Lau, J. J. Med. Chem. 2002, 45, 5755.
10. Kelly, R. P.; Garhyan, P.; Abu-Raddad, E. J.; Fu, H.; Lim, C.; Prince, M. J.; Pinaire, J.
A.; Loh, M.; Deeg, M. A. Diabetes 2011, 60, A84.
11. The protocol for GCGR binding affinity test is as follows: A liver membrane
specimen was prepared from rat liver according to the method described in
Bioorg. Med. Chem. Lett. 1992, 12, 915, and compounds binding affinity was
measured using [125I] glucagon. The liver was extirpated and added to the 20-
fold volume of 50 mM Tris–HCl buffer (pH 7.2) relative to liver wet weight. The
Preparation of compound 9 is shown in Scheme 3. Two com-
mercially available fragments 14 and 15 were condensed by BOP
reagent to give compound 9.
In summary, we have found furan-2-carbohydrazides as novel
scaffold of GCGR antagonists by determining SARs of a series of
derivatives obtained by modifying the acidity of the phenol moiety.
We have also found that the ortho-nitrophenol is a good scaffold
for GCGR antagonistic activity. The most promising compound 7l
in our series demonstrated comparatively long-term suppression
of glucose level in vivo. Further investigations are currently
ongoing.
mixture was then homogenized using
a glass-Tefron homogenizer. The
homogenate was centrifuged at 30,000g for 15 min, and 20-fold volume of
50 mM Tris–HCl buffer (pH 7.2) relative to liver wet weight was added to the
pellet. The mixture was further centrifuged at 30,000g for 15 min and the
obtained pellet was used as liver membrane specimen. The specimen was
suspended in the 100-fold volume of 1 mg/mL BSA relative to liver wet weight
and 50 mM Tris–HCl buffer (pH7.2) containing 0.1 mg/mL bacitracin. [125I]
glucagon (final concd: 50 pM) was added to the specimen mixture. The
mixture was then incubated at 25 °C for 30 min. [125I] glucagon bound to the
specimen was recovered by suction filtration using a GF/C filter pretreated

4270
F. Hasegawa et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4266–4270
with 0.1% polyethylenimine, and the residues were washed with 50 mM
Tris–HCl buffer (pH 7.4) for three times. Radioactivity was measured using an
ARC-360 -counter. Specific binding volume was calculated by subtracting the
non-specific binding volume in the presence of 1 mM glucagon from the total
binding volume. Inhibition rates were calculated at each concentration of test
compounds with binding volume in the absence of test compound considered
as 100%. IC50 values were calculated using the pseudo-Hill plot.
13. Jelinik, L. J.; Lok, S.; Rosenberg, G. B.; Smith, R. A.; Grant, F. J.; Biggs, S.; Bensch,
P. A.; Kuijper, J. L.; Sheppard, P. O.; Sprecher, C. A.; O’Hara, P. J.; Foster, D.;
Walker, K. M.; Chen, L. H. J.; McKernan, P. A.; Kindsvogel, W. Science 1993, 259,
1614.
c
14. Compound 7l: Mp 159–160 °C; 1H NMR (DMSO-d6, 400 MHz) d 11.75 (1H, br s),
10.51 (1H, s), 10.35 (1H, s), 8.47 (1H, d, J = 2.4 Hz), 8.06 (1H, dd, J = 8.7, 2.4 Hz),
7.94 (1H, d, J = 1.8 Hz), 7.66 (2H, d, J = 8.7 Hz), 7.23 (1H, d, J = 8.7 Hz), 7.19 (2H,
d, J = 8.7 Hz), 6.94 (1H, d, J = 1.8 Hz), 2.57 (2H, t, J = 7.8 Hz), 1.60–1.53 (2H, m),
1.34–1.22 (4H, m), 0.85 (3H, t, J = 7.3 Hz); 13C NMR (DMSO-d6, 100 MHz) d
163.9, 158.3, 155.0, 144.6, 142.5, 140.2, 136.7, 134.1, 131.3, 129.2, 128.8, 128.1,
125.1, 123.3, 119.4, 114.1, 35.0, 31.1, 30.8, 22.1, 14.1; HRMS (ESI) calcd for
12. The protocol for inhibitory activity of cAMP accumulation is as follows: Thawed
hepatocytes were added to HCM™ Bulletkit™ (Takara Bio Inc.) and the mixture
was centrifuged at 700 rpm, 4 °C for 3 min. After the supernatant was removed,
the hepatocytes were suspended in HCM™ Bulletkit™ and the mixture was
centrifuged at 700 rpm, 4 °C for 3 min. After the supernatant was removed, the
hepatocytes were suspended in HCM™ Bulletkit™ and cell number was
counted. The suspension was diluted to an appropriate living cell
concentration, and the cells were seeded on a 24 well collagen-coat plate
and incubated for 5 h. The test compound in cAMP assay buffer (Dulbecco’s
Modified Eagle Medium with 20 mM HEPES, 0.1% BSA and 0.5 mM IBMX) was
added to the hepatocytes and the mixture was incubated at room temperature
for 15 min. Glucagon or cAMP assay buffer was added, and the mixture was
incubated at room temperature for 5 min. After the solvent was removed,
0.5 M-HCl was added, and the mixture was kept at room temperature for
10 min. 1 M Tris–HCl (pH 8.0) was then added for neutralization, and the
solution was collected. The amount of cAMP in the supernatant was measured
by a cAMP dynamic2 kit (Cisbio Bioassays).
C23H24N3O6 [M+H] 438.1660, found 438.1665.
15. The assay was performed by Eurofin Panlabs.
16. (a) Goto, Y.; Kakizaki, M.; Masaki, N. Tohoku. J. Exp. Med. 1976, 119, 85; (b)
Galli, J.; Fakhrai-Rad, H.; Kamel, A.; Marcus, C.; Norgren, S.; Luthman, H.
Diabetes 1999, 48, 2463.
17. Yoshina, S.; Yamamoto, K. Yakugaku Zasshi 1974, 94, 1312.
18. (a) Chadwick, D. J.; Chambers, J.; Macrae, R.; Meakins, G. D.; Snowden, R. L. J.
Chem. Soc., Perkin Trans. 2 1976, 597; (b) Chadwick, D. J.; Chambers, J.; Macrae,
R.; Meakins, G. D.; Snowden, R. L. J. Chem. Soc., Perkin Trans. 1 1973, 1766.
19. Fujii, A.; Negoro, T.; Migihashi, C.; Murata, M.; Nakamura, K.; Nukuda, T.;
Matsumoto, T.; Konno, K. WO Patent 064404, 2003.