Synthesis of 5-chloro-N-aryl-1H-indole-2-carboxamide derivatives as inhibitors of human liver glycogen phosphorylase a

Article abstract of DOI:10.1016/j.bmc.2008.04.010

A series of 5-chloro-N-aryl-1H-indole-2-carboxamide derivatives were prepared and evaluated as inhibitors of human liver glycogen phosphorylase a (hLGPa). One compound, 5-chloro-N-[4-(1,2-dihydroxyethyl)phenyl]-1H-indole-2-carboxamide (2f), inhibited hLGPa with an IC₅₀ of 0.90 μM. The pyridine analogue of 2f showed inhibitory activity of glucagon-induced glucose output in cultured primary hepatocytes with an IC₅₀ of 0.62 μM and oral hypoglycemic activity in diabetic db/db mice. Crystallographic determination of the complex of 2f with hLGPa showed binding of the inhibitor in a solvent cavity at the dimer interface, with the two hydroxyl groups making favorable electrostatic interactions with hLGPa.

Full text of DOI:10.1016/j.bmc.2008.04.010

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5452–5464
Synthesis of 5-chloro-N-aryl-1H-indole-2-carboxamide
derivatives as inhibitors of human liver glycogen phosphorylase a
Kenichi Onda,* Takayuki Suzuki, Ryota Shiraki, Yasuhiro Yonetoku, Kenji Negoro,
Kazuhiro Momose, Naoko Katayama, Masaya Orita, Tomohiko Yamaguchi,
Mitsuaki Ohta and Shin-ichi Tsukamoto
Drug Discovery Research, Astellas Pharma Inc., 5-2-3 Toukoudai, Tsukuba, Ibaraki 300-2698, Japan
Received 13 February 2008; revised 7 April 2008; accepted 8 April 2008
Available online 11 April 2008
Abstract—A series of 5-chloro-N-aryl-1H-indole-2-carboxamide derivatives were prepared and evaluated as inhibitors of human
liver glycogen phosphorylase a (hLGPa). One compound, 5-chloro-N-[4-(1,2-dihydroxyethyl)phenyl]-1H-indole-2-carboxamide
(2f), inhibited hLGPa with an IC50 of 0.90 lM. The pyridine analogue of 2f showed inhibitory activity of glucagon-induced glucose
output in cultured primary hepatocytes with an IC50 of 0.62 lM and oral hypoglycemic activity in diabetic db/db mice. Crystallo-
graphic determination of the complex of 2f with hLGPa showed binding of the inhibitor in a solvent cavity at the dimer interface,
with the two hydroxyl groups making favorable electrostatic interactions with hLGPa.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
supplied to the tissues (Fig. 1).12–15 Therefore, inhibition
of liver GP (LGP) is an alternative target for develop-
ment of new drugs for type 2 diabetes.
Non-insulin-dependent diabetes mellitus (type 2 diabe-
tes) is a severe and prevalent metabolic disorder. Tight
control of blood glucose levels reduces the extent and
progression of diabetic complications such as nerve
and kidney damage, blindness, and heart disease,1,2
but it is difficult to achieve exact glycemic control with
oral hypoglycemic agents because of limited efficacy, tol-
erability, and hypoglycemic risk.3 In type 2 diabetic pa-
tients, high blood glucose levels are in part caused by
elevated hepatic glucose output (HGO) due to gluconeo-
genesis (de novo synthesis of glucose from 2- and 3-car-
bon precursors) and glycogenolysis (phosphorolysis of
a-(1,4)-linkages within glycogen molecules),4–7 and inhi-
bition of glycogenolysis has recently emerged as an
attractive therapeutic target for treatment of type 2 dia-
betes.8–11 Glycogen phosphorylase (GP) catalyzes the
first step of the breakdown of glycogen to yield glu-
cose-1-phosphate. In the liver, phosphoglucomutase
and glucose-6-phosphatase convert glucose-1-phosphate
to glucose, which is then released into the blood to be
GP is a homodimeric enzyme that exists in two confor-
mational states (the GPa and GPb forms) that are
interconverted by phosphorylation–dephosphorylation
reactions at Ser14.16 Only GPa has significant potency.16
The activity of GP is regulated by ligand binding at sev-
eral sites, including an allosteric site, a catalytic site and
a caffeine-binding site.8,17–22 Several studies have re-
vealed a further site (a new allosteric site or dimer inter-
face site) at which 5-chloroindole-2-carboxamide
derivatives are able to interact as GP inhibitors.23,24
Among this new class of inhibitors, CP-320626, which
has 5-chloroindole and phenylalanine moieties, shows
potent activity against human liver GPa (hLGPa) and
may act synergistically with glucose.10 As the blood glu-
cose concentration decreases, the potency of GP inhibi-
tors decreases, and so the risk of hypoglycemia seems to
be minimized. This hypothesis prompted us to modify
the structure of CP-320626 to identify novel and potent
GP inhibitors.
A crystal structure of the CP-320626 inhibitor–GP com-
plex shows that the planar 5-chloroindole moiety docks
into a hydrophobic pocket, and suggests that there is lit-
tle room for modification of this part of the structure.23
Keywords: Glycogen phosphorylase; Hepatic glucose output; Diabetes;
Crystallographic determination.
*
Corresponding author. Tel.: +81 29 847 8611; fax: +81 29 847
8313; e-mail: kenichi.onda@jp.astellas.com
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.04.010

K. Onda et al. / Bioorg. Med. Chem. 16 (2008) 5452–5464
5453
Glycogen
phosphorylase
Glucose-1-phosphate + (Glucosyl)
n-1
(Glucosyl)n + Pi
Phosphoglucomutase
Glucose-6-
phosphatase
Glucose-6-phosphate
Glucose
Figure 1. Hepatic pathways of glycogen metabolism.
However, the relatively flexible phenylalanine moiety is
located close to a solvent-filled gap between the two sub-
units of the GP dimer and could be modified to maintain
or improve the potency.23–25 The hydroxyl group and
the nitrogen atom of the 4-hydroxypiperidyl moiety
form a hydrogen bond to GP through a water mole-
cule.26 Therefore, to discover more potent inhibitors,
we focused on replacement of the phenylalanine of
CP-320626 with substituents having hydroxyl group(s)
that might form hydrophilic interactions with the en-
zyme (Fig. 2).
Coupling reactions of anilines 1a–n with 5-chloro-1H-in-
dole-2-carboxylic acid gave the corresponding products
in moderate to high yield. In the case of branched diol
derivative 2i, the starting material 1i was synthesized
from 4-nitrobenzaldehyde through three steps, as shown
in Scheme 2.27
Compound 1h as starting material for 2h was obtained
by hydrogenation of 2-methoxy-1-(4-nitrophenyl)etha-
nol28 under a hydrogen atmosphere. The substituted
4-(1,2-dihydroxyethyl) anilide derivatives 9a–j were syn-
thesized as shown in Scheme 3.
In this paper, we report the discovery of a novel class of
GP inhibitors and discuss their binding site based on a
crystal structure of one of the inhibitors with GP.
Coupling reactions of commercially available substi-
tuted 4-bromoanilines 6a– i with 5-chloro-1H-indole-
2-carboxylic acid or 5-chloro-1H-indole-2-carbonyl
chloride gave intermediate bromides 7a–i in moderate
to high yield. The desired products 9a–i were obtained
by Stille’s vinylation reaction29 followed by dihydroxy-
lation using osmium tetroxide and N-methylmorpho-
line-N-oxide. The 3-amino derivative 9j was obtained
from the corresponding nitro derivative 9i by reduction
using zinc dust.
2. Chemistry
The 5-chloro-N-phenyl-1H-indole-2-carboxamide ana-
logues 2a–i were prepared as shown in Scheme 1.
Cl
Cl
Compounds 13a–c with a pyridine or pyrazine ring in-
stead of the benzene ring in 2f were prepared from the
corresponding arylamines 10a–c by the same reactions
as those described for compounds 9a–i (Scheme 4).
O
H
N
H
N
N
H
N
N
H
OH
O
OH
Ar
O
CP-320626
F
Thiazole derivatives 15a,b were prepared from 1-(2-ami-
no-1,3-thiazol-4-yl) ethane-1,2-diol (14a) or its isomer
(14b) using the coupling reaction described above. Syn-
Figure 2. Design of N-aryl-5-chloroindolecarboxamides based on CP-
320626.
2a; X=4-CH2OH
b; X=4-(CH2)2OH
c; X=4-(CH2)3OH
Cl
X
H2N
a
H
N
X
d; X=2-(CH2)2OH
N
H
2
4
e; X=3-(CH2)2OH
3
O
4
2
f; X=4-CH(OH)CH2OH
3
1a-n
g; X=4-CH(CHOH)2
2
h; X=4-CH(OH)CH2OMe
i; X=4-CH(OH)CMe2OH
Scheme 1. Syntheses of compounds 2a–i. Reagents: (a) 5-chloroindole-2-carboxylic acid, WSCÆHCl, HOBt, DMF.
O2N
b
a
O2N
c
O2N
1i
OH
CHO
OH
3
4
5
Scheme 2. Syntheses of compound 1i. Reagents: (a) Ph3(i-Pr)PI, n-BuLi, THF; (b) OsO4, N-methylmorpholine-N-oxide, THF, H2O; (c) H2, 10% Pd–
C, MeOH.

5454
K. Onda et al. / Bioorg. Med. Chem. 16 (2008) 5452–5464
Cl
d
X
H2N
H
N
X
a, b or c
N
H
2
Br
O
2
3
Br
3
6a; X=2-Me
b; X=2-F
7a; X=2-Me
b; X=2-F
c; X=2-Cl
d; X=2-CF
c; X=2-Cl
3
d; X=2-CF
3
e; X=3-OMe
f; X=3-Ph
g; X=3-F
e; X=3-OMe
f; X=3-Ph
g; X=3-F
h; X=3-CF
3
h; X=3-CF
3
i; X=3-NO
2
i; X=3-NO
2
Cl
Cl
H
N
X
e
H
X
N
N
N
H
O
H
2
OH
O
2
3
OH
3
9a; X=2-Me
b; X=2-F
8a; X=2-Me
b; X=2-F
c; X=2-Cl
c; X=2-Cl
d; X=2-CF
d; X=2-CF
3
3
e; X=3-OMe
f; X=3-Ph
g; X=3-F
e; X=3-OMe
f; X=3-Ph
g; X=3-F
h; X=3-CF
3
h; X=3-CF
3
i; X=3-NO
2
i; X=3-NO
f
2
j; X=3-NH
2
Scheme 3. Synthesis of compounds 9a–j. Reagents and conditions: (a) 5-chloroindole-2-carboxylic acid, WSCÆHCl, HOBt, DMF (for 7a, e, f, and k);
(b) 5-chloroindole-2-carbonyl chloride, py. (for 7b, c, d, g, and h); (c) 5-chloroindole-2-carboxylic acid, POCl3, py., ꢀ25 ꢁC (for 7i, and j); (d) n-
Bu3SnCHCH2, Pd(PPh3)4, LiCl, DMF, THF, 80 ꢁC; (e) OsO4, N-methylmorpholine-N-oxide, THF, H2O; (f) Zn, AcOH.
Cl
Cl
Cl
b
c
a
H
N
X
Y
H2N
H
N
X
Y
H
N
X
Y
N
H
N
H
X
Y
N
H
Br
O
O
OH
Br
O
OH
10a; X=N, Y=CH
b; X=CH, Y=N
c; X=N, Y=N
11a; X=N, Y=CH
b; X=CH, Y=N
c; X=N, Y=N
12a; X=N, Y=CH
b; X=CH, Y=N
c; X=N, Y=N
13a; X=N, Y=CH
b; X=CH, Y=N
c; X=N, Y=N
Scheme 4. Syntheses of compounds 13a–c. Reagents and condition: (a) 5-chloroindole-2-carbonyl chloride, py.; (b) n-Bu3SnCHCH2, Pd(PPh3)4,
LiCl, DMF, THF, 80 ꢁC; (c) OsO4, N-methylmorpholine-N-oxide, THF, H2O.
Cl
theses of 14a and 14b were accomplished by a reduction
of the corresponding a-hydroxy acid ester (Scheme 5).30
a or b
N
H
N
4
H2N
N
4
N
H
R
S
R
5
S
O
5
3. Results and discussion
14a; R= 4-CH(OH)CH2OH
b; R= 5-CH(OH)CH2OH
15a; R= 4-CH(OH)CH2OH
b; R= 5-CH(OH)CH2OH
The structure of the synthesized compounds and their
hLGPa inhibitory activities in vitro are summarized in
Tables 1–4. The 4-hydroxymethyl (2a) and 4-(2-
hydroxyethyl) (2b) derivatives inhibited hLGPa with
Scheme 5. Synthesis of compounds 15a–b. Reagents: (a) 5-chloro-1H-
indole-2-carboxylic acid, WSCÆHCl, HOBt, DMF (for 15a); (b) 5-
chloro-1H-indole-2-carbonyl chloride, TEA, MeCN (for 15b).

K. Onda et al. / Bioorg. Med. Chem. 16 (2008) 5452–5464
Table 1. SAR of N-aryl-5-chloroindolecarboxamides Table 3. SAR of N-heteroaryl-5-chloroindolecarboxamides
5455
Cl
Cl
Cl
H
N
H
N
H
N
X
N
X
Y
4
N
H
N
H
N
H
R
S
O
O
O
5
4
OH
2
3
OH
Entry Compound Position
X
hLGPa
IC50 (lM)
13a; X=N, Y=CH
b ; X=CH, Y=N
c; X=N, Y=N
15a; R= 4-CH(OH)CH2OH
b; R= 5-CH(OH)CH2OH
1
2
2a
2b
4
4
4
2
3
4
4
4
4
CH2OH
(CH2)2OH
1.6
1.2
Entry
Compound
hLGPa IC50 (lM)
3
2c
2d
(CH2)3OH
(CH2)2OH
>10
>10
6.2
1
2
3
4
5
6
7
13a
13b
0.25
4.4
4
5
2e
(CH2)2OH
CH(OH)CH2OH
CH(CH2OH)2
13c
15a
0.44
>10
3.1
6
2f
2g
0.90
5.8
7
15b
2f
CP-320626
8
2h
2i
CP-320626
CH(OH)CH2OMe 3.6
CH(OH)CMe2OH >10
0.92
0.90
0.92
9
10
IC50 of 0.90 lM, which is similar to the activity of CP-
320626.
Table 2. SAR of N-aryl-5-chloroindolecarboxamides with
dihydroxyethyl moiety
a 1,2-
Cl
Lengthening the distance between the benzylic hydroxy
group and the benzene ring in 2f by addition of a one-
carbon unit and changing the primary hydroxy group
into a methoxy group resulted in a moderate decrease
in inhibitory activity (2f vs 2g, 2h). The presence of
branches in the 1,2-dihydroxyethyl chain (2i) was detri-
mental to inhibitory activity. These results suggested
that the hydroxy group(s) and their appropriate position
on the side chain played an important role in inhibitory
activity against hLGPa, and that the introduction of
substituents on the side chain did not seem to be well
tolerated, probably due to steric factors. We then exam-
ined the effects of substituents on the benzene ring of 2f
(Table 2).
H
N
X
N
H
O
2
OH
3
OH
9a; X=2-Me
b; X=2-CF3
c; X=2-F
d; X=2-Cl
e; X=3-OMe
f; X=3-Ph
g; X=3-CF
3
h; X=3-F
i; X=3-NO
2
Introduction of a methyl or trifluoromethyl group on
the 2-position of the benzene ring led to a slight decrease
of inhibitory activity (9a,b). In introduction of a halogen
atom to benzene ring, the chloro analogue (9d) was equi-
potent compared to parent compound 2f and the fluoro
analogue (9c) showed improved inhibitory activity. Sub-
stitution at the 3-position with a methoxy, phenyl or tri-
fluoromethyl group led to a slight decrease in inhibitory
activity (9e–g), but a fluoro group (9h) conferred potent
activity, as at the 2-position. Introduction of a nitro or
amino group resulted in a slight decrease of inhibitory
activity (9i,j). From these results, it appears that the ste-
ric properties of substituents are more important than
the electronic character.
j; X=3-NH
2
Entry
Compound
Position
X
hLGPa IC50 (lM)
1
2
9a
9b
2
2
2
2
3
3
3
3
3
3
—
Me
CF3
F
2.6
3.2
3
9c
9d
0.42
1.1
4
Cl
OMe
Ph
5
9e
2.5
7.4
6
9f
9g
7
CF3
F
3.3
8
9h
9i
0.34
2.1
9
NO2
NH2
H
10
11
12
9j
1.7
2f
CP-320626
0.90
0.92
Next, changing the benzene ring of 2f into other aro-
matic rings was attempted (Table 3). Among the com-
pounds with 6-membered aromatic rings containing
nitrogen, the 3-pyridine derivative (13b) had lower activ-
ity whereas the 2-pyridine derivative (13a) showed the
best in vitro activity (IC50 = 0.25 lM). The pyrazine
derivative (13c) was approximately two times more po-
tent than 2f. These results suggest that the nitrogen atom
in the pyridine ring of 13a might form an appropriate
electrostatic interaction with hLGPa. On the other
IC50 values of 1.6 and 1.2 lM, respectively, whereas the
4-(3-hydroxypropyl) derivative (2c) did not show strong
inhibitory activity. For 2-hydroxyethyl substitution, the
three positions showed different activities and the 4-po-
sition was the most effective (2d, 2e vs 2b). We then syn-
thesized the 4-(1,2-dihydroxyethyl) derivative (2f) with a
combination of the hydroxy groups of 2d and 2e at the
4-position. Interestingly, 2f inhibited hLGPa with an

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K. Onda et al. / Bioorg. Med. Chem. 16 (2008) 5452–5464
Table 4. Inhibition of glucagon-induced glucose output in cultured primary hepatocytes and oral hypoglycemic activity in diabetic db/db mice
Entry
Compound
hLGPa IC50 (lM)
HGO inh. IC50 (lM)
db/db Mice hypoglycemic activitya
Dose (mg/kg, po) % Glucose lowering
1
2
3
13a
0.25
0.92
0.62
1.7
50
30
50
26b
13
20b
CP-320626
a Percentage decrease in drug-treated blood glucose concentration at 2 h postdose in db/db mice, relative to vehicle-treated controls.
b P < 0.05 versus control.
hand, the corresponding nitrogen atom in 13b might not
be properly located to give potent activity because of
undesirable interactions with hydroxyl groups on 13b it-
self or on hLGPa. For 5-membered rings, the 2,5-substi-
tuted thiazole derivative (15b) slightly decreased
inhibitory activity whereas the corresponding 2,4-substi-
tuted analogue (15a) showed a complete loss of inhibi-
tory activity. We suspect that these effects were caused
by changing the distance and/or angle between the in-
dole ring and the diol moiety, both of which are thought
to play important roles in the inhibitory activity.
ety, we determined the crystal structure of the hLGPa–
˚
glucose–2f complex at 2.45 A resolution (Fig. 3).
As expected, the crystal structure showed that two mol-
ecules of 2f bind at the dimer interface of the enzyme
(Fig. 3A). The 5-chloroindole group is buried in a
hydrophobic pocket composed of lipophilic side chains
of amino acids including Leu63, Val64, and Trp67,
and the indole NH moiety acts as a hydrogen bond do-
nor to the backbone of Glu190. Furthermore, the amide
moiety establishes a hydrogen bond from NH to the
backbone of Thr38 and the carbonyl O makes two polar
contacts with the backbone oxygen of Glu190 and a
water molecule. This water molecule mediates two addi-
tional polar contacts between the carbonyl oxygen and
the backbone oxygens of Asn187 and Glu190. The
benzene ring appears to act as a junction between the
5-chloro-1H-indole-2-carboxamide and the 1,2-dihydr-
oxyethyl side chain, because no significant interaction
of the ring with the enzyme was observed. The two hy-
droxyl groups of 2f have direct electrostatic interactions
with the imidazole ring of His57 and the backbone of
Tyr185, respectively. This supports the observation that
both these two hydroxyl groups are both important for
inhibitory activity (Table 1). At the medium resolution
of the X-ray structure, the dominant configuration of
the asymmetric carbon of 2f was indistinguishable, but
Compound 13a, which was the most potent hLGPa
inhibitor in vitro, was examined for inhibition of gluca-
gon-induced glucose output in cultured primary hepato-
cytes and for oral hypoglycemic activity in diabetic db/
db mice (Table 4).
Compound 13a inhibited glucose output dose-depen-
dently with an IC50 value of 0.62 lM, showing approx-
imately three times greater potency than CP-320626. A
single oral administration of 13a at a dose of 50 mg/kg
significantly reduced the blood glucose level at 2 h
postdose.
To understand the enzyme binding of the 5-chloro-1H-
indole-2-carboxamides with a 1,2-dihydroxyethyl moi-
Figure 3. (A) Crystal structure of 2f bound to the hLGPa dimer interface. Glucose is situated at the catalytic site. (B) Schematic diagram of the
interactions made by 2f with hLGPa revealed by crystallographic analysis. Protein residues of the two hLGPa molecules in the dimer are
distinguished with labels (A) and (B), respectively. The figure was prepared using the ligand interactions application in MOE.31

K. Onda et al. / Bioorg. Med. Chem. 16 (2008) 5452–5464
5457
an electron density map showed that both enantiomers
are able to bind to the enzyme (Fig. 3B).
5. Experimental
5.1. Chemistry
To study differences in the binding mode of 2f and CP-
320626, we overlaid the crystal structures of the two com-
pounds (Fig. 4). The 5-chloroindole group and the amide
moiety of 2f are situated in almost the same position as
those in the CP-320626 complex with muscle glycogen
phosphorylase b (MGPb) (PDB: 1H5U).26 Interestingly,
the 1,2-dihydroxyethyl moiety of 2f occupies a similar po-
sition to the 4-fluorophenyl moiety of CP-320626, with
van der Waals interactions with a hydrophobic pocket
containing Phe53, Pro188, and His57, indicating that this
pocket can accept both hydrophilic and hydrophobic
moieties. In contrast, the hydroxyl group of the 4-
hydroxypiperidyl moiety of CP-320626 makes an indirect
water-mediated hydrogen bond with rMGPb.9 Neither of
the hydroxyl groups in 2f mimics the role of the hydroxyl
group in CP-320626, but both play an important role in
the enzyme binding of 2f. Collectively, the results indicate
that hydrophobic and hydrophilic interactions observed
in the crystal structure stabilize the less active conforma-
tional state of the enzyme, which leads to inhibitory
activity.
1H NMR spectra were recorded on a JEOL JNM-
LA300 or a JEOL JNM-EX400 spectrometer and
the chemical shifts are expressed in d (ppm) values
with tetramethylsilane as an internal standard (in
NMR description, s, singlet; d, doublet; dd, double
doublet; t, triplet; q, quartet; m, multiplet; and br,
broad peak). Mass spectra were recorded on a JEOL
JMS-700T or micromass Q-Tof Ultima API spectrom-
eter. The elemental analyses were performed with a
Yanaco MT-5 microanalyzer (C, H, N) and were
within 0.4% of theoretical values. Drying of organic
solutions during work-up was done over anhydrous
MgSO4 or Na2SO4.
5.1.1. 5-Chloro-N-[4-(hydroxymethyl)phenyl]-1H-indole-
2-carboxamide (2a). To a solution of 4-hydroxymethy-
laniline (1a, 182 mg, 1.48 mmol) and 5-chloro-1H-in-
dole-2-carboxylic acid (290 mg, 1.48 mmol) in DMF
(10 mL) were added 1-ethyl-3-(30-dimethylaminopro-
pyl)carbodiimide hydrochloride (340 mg, 1.77 mmol)
and
1-hydroxybenzotriazole
hydrate
(272 mg,
1.77 mmol), and the mixture was stirred at room tem-
perature for 14 h. The resulting mixture was concen-
trated in vacuo and the residue was washed with
H2O (60 mL) to give 2a (385 mg, 96%) as colorless
solid: 1H NMR (400 MHz, DMSO-d6) d: 4.48 (2H,
d, J = 5.4 Hz), 5.14 (1H, t, J = 5.4 Hz), 7.22 (1H,
dd, J = 8.8, 2.0 Hz), 7.28 (2H, d, J = 8.3 Hz), 7.41
(1H, s), 7.47 (1H, d, J = 8.3 Hz), 7.74–7.81 (3H, m),
10.27 (1H, s), 11.94 (1H, s). FAB-MS m/z: 301
(M+1)+. Anal. Calcd for C16H13Cl N2O2Æ0.15H2O:
C, 63.33; H, 4.42; N, 9.23. Found: C, 63.20; H,
4.26; N, 9.57.
4. Conclusion
In conclusion, we synthesized a series of N-aryl-1H-in-
dole-2-carboxamide derivatives with a 1,2-dihydroxy-
ethyl moiety as a new class of hLGPa inhibitors. The
inhibitory activity of these compounds was explained
using the structure of the hLGPa– 2f complex. Com-
pound 13a, which was the most potent in vitro hLGPa
inhibitor in the study, inhibited glucose output from
hepatocytes and reduced blood glucose in a db/db
mouse model of diabetes.
Figure 4. A stereoview of a docking model of 2f with CP-320626.

5458
K. Onda et al. / Bioorg. Med. Chem. 16 (2008) 5452–5464
5.1.2. 5-Chloro-N-[4-(2-hydroxyethyl)phenyl]-1H-indole-
2-carboxamide (2b). The title compound was prepared in
the same manner as described for 2a using 1b instead of
1a, in 96% yield. 1H NMR (400 MHz, DMSO-d6) d: 2.71
(2H, t, J = 7.1 Hz), 3.55–3.68 (2H, m), 4.63 (1H, t,
J = 5.2 Hz), 7.15–7.28 (3H, m), 7.40 (1H, d,
J = 1.8 Hz), 7.47 (1H, d, J = 8.8 Hz), 7.69 (2H, d,
J = 8.3 Hz), 7.77 (1H, d, J = 1.8 Hz), 10.23 (1H, s),
11.93 (1H, s). FAB-MS m/z: 315 (M+1)+. Anal. Calcd
for C17H15ClN2O2: C, 64.87; H, 4.80; N, 8.90. Found:
C, 64.61; H, 4.75; N, 8.92.
pound was prepared in the same manner as described
for 2a using 1g instead of 1a, in 59% yield. H NMR
1
(400 MHz, DMSO-d6) d: 2.75–2.82 (1H, m), 3.56–3.61
(2H, m), 3.66–3.72 (2H, m), 4.52 (2H, t, J = 4.6 Hz),
7.21–7.23 (3H, m), 7.40 (1H, d, J = 1.7 Hz), 7.47 (1H,
d, J = 8.8 Hz), 7.67–7.69 (2H, m), 7.76 (1H, d,
J = 1.7 Hz), 10.21 (1H, s), 11.93 (1H, br s). FAB-MS
m/z: 345 (M+1)+. Anal. Calcd for C18H17ClN2O3
Æ0.04CHCl 3: C, 61.98; H, 4.91; N, 8.01. Found: C,
61.76; H, 4.80; N, 7.94.
5.1.8. 5-Chloro-N-[4-(1-hydroxy-2-methoxyethyl)phenyl]-
1H-indole-2-carboxamide (2h). The title compound was
prepared in the same manner as described for 2a using
5.1.3. 5-Chloro-N-[4-(3-hydroxypropyl)phenyl]-1H-indole-
2-carboxamide (2c). The title compound was prepared in
the same manner as described for 2a using 1c instead of
1
1h instead of 1a, in 56% yield. H NMR (400 MHz,
1
1a, in 40% yield. H NMR (400 MHz, DMSO-d6) d:
DMSO-d6) d: 3.27 (3H, s), 3.35–3.44 (2H, m), 4.66–
4.70 (1H, m), 5.31 (1H, d, J = 3.4 Hz), 7.22 (1H, dd,
J = 8.8, 2.0 Hz), 7.34 (2H, d, J = 8.3 Hz), 7.41 (1H, s),
7.47 (1H, d, J = 8.8 Hz), 7.73 (2H, d, J = 8.3 Hz), 7.77
(1H, d, J = 2.0 Hz), 10.26 (1H, br s), 11.94 (1H, br s).
FAB-MS m/z: 345 (M+1)+. Anal. Calcd for
C18H17ClN2O3: C, 62.70; H, 4.97; N, 8.12. Found: C,
62.32; H, 4.83; N, 8.02.
1.65–1.75 (2H, m), 2.59 (2H, t, J = 7.8 Hz), 3.43 (2H,
q, J = 5.8 Hz), 4.46 (1H, t, J = 5.4 Hz), 7.15–7.25 (3H,
m), 7.39 (1H, s), 7.46 (1H, d, J = 8.8 Hz), 7.68 (2H, d,
J = 8.3 Hz), 7.76 (1H, s), 10.21 (1H, s), 11.92 (1H, s).
FAB-MS m/z: 329 (M+1)+. Anal. Calcd for
C18H17ClN2O2: C, 65.75; H, 5.21; N, 8.52. Found: C,
65.82; H, 5.36; N, 8.54.
5.1.4. 5-Chloro-N-[2-(2-hydroxyethyl)phenyl]-1H-indole-
2-carboxamide (2d). The title compound was prepared in
the same manner as described for 2a using 1d instead of
1a, in 94% yield. 1H NMR (400 MHz, DMSO-d6) d: 2.83
(2H, t, J = 6.1 Hz), 3.64–3.78 (2H, m), 5.44 (1H, t,
J = 4.2 Hz), 7.10–7.36 (5H, m), 7.47 (1H, d,
J = 8.8 Hz), 7.64 (1H, d, J = 7.8 Hz), 7.73 (1H, s),
10.39 (1H, s), 11.99 (1H, s). FAB-MS m/z: 315
(M+1)+. Anal. Calcd for C17H15ClN2O2: C, 64.87; H,
4.80; N, 8.90. Found: C, 64.53; H, 4.73; N, 9.02.
5.1.9. 5-Chloro-N-[4-(1,2-dihydroxy-2-methylpropyl)-
phenyl]-1H-indole-2-carboxamide (2i). The title com-
pound was prepared in the same manner as described
for 2a using 1i instead of 1a, in 74% yield. H NMR
1
(400 MHz, DMSO-d6) d: 0.98 (3H, s), 1.07 (3H, s),
4.21 (1H, s), 4.31 (1H, d, J = 4.4 Hz), 5.13 (1H, d,
J = 4.4 Hz), 7.22 (1H, dd, J = 8.8, 2.4 Hz), 7.33 (2H, d,
J = 8.8 Hz),
7.38–7.50 (2H, m), 7.69 (2H, d,
J = 8.8 Hz), 7.77 (1H, d, J = 2.0 Hz), 10.23 (1H, s),
11.93 (1H, s). FAB-MS m/z: 359 (M+1)+. Anal. Calcd
for C19H19ClN2O3: C, 63.60; H, 5.34; N, 7.81. Found:
C, 63.33; H, 5.28; N, 7.73.
5.1.5. 5-Chloro-N-[3-(2-hydroxyethyl)phenyl]-1H-indole-
2-carboxamide (2e). The title compound was prepared in
the same manner as described for 2a using 1e instead of
1a, in 97% yield. 1H NMR (400 MHz, DMSO-d6) d: 2.74
(2H, t, J = 7.1 Hz), 3.57–3.69 (2H, m), 4.68 (1H, t,
J = 5.2 Hz), 6.98 (1H, d, J = 7.9 Hz), 7.23 (1H, dd,
J = 8.7, 2.3 Hz), 7.28 (1H, d, J = 7.9 Hz), 7.42 (1H, d,
J = 1.7 Hz), 7.48 (1H, d, J = 8.7 Hz), 7.62 (1H, s),
7.65–7.73 (1H, m), 7.78 (1H, d, J = 1.7 Hz), 10.22 (1H,
s), 11.92 (1H, s). FAB-MS m/z: 315 (M+1)+. Anal. Calcd
for C17H15ClN2O2Æ0.25H 2O: C, 63.95; H, 4.89; N, 8.77.
Found: C, 63.91; H, 4.89; N, 8.87.
5.1.10. 2-Methyl-1-(4-nitrophenyl)propane-1,2-diol (5).
To a mixture of isopropyltriphenylphosphonium iodide
(9.43 g, 21.8 mmol) and THF (60 mL) was added n-BuLi
(1.55 M in n-hexane, 14.0 mL, 21.7 mmol) at ꢀ20 ꢁC,
then the mixture was stirred at same temperature for
45 min. To this reaction mixture was added to a solution
of 4-nitrobenzaldehyde (2.64 g, 17.5 mmol) in THF
(10.0 mL), then the mixture was stirred at room temper-
ature for 1 h, and then under reflux for 1.5 h. The result-
ing mixture was concentrated in vacuo and the residue
was partitioned between AcOEt (2· 100 mL) and satd
NH4Cl (100 mL), and the AcOEt layer was washed with
satd NaCl, then dried and concentrated in vacuo. The
residue was purified by column chromatography on sil-
ica gel (n-hexane/AcOEt = 5:1) to give a reddish oil
5.1.6. 5-Chloro-N-[4-(1,2-dihydroxyethyl)phenyl]-1H-indole-
2-carboxamide (2f). The title compound was prepared in
the same manner as described for 2a using 1f instead of
1
1a, in 76% yield. H NMR (400 MHz, DMSO-d6) d:
3.40–3.47 (2H, m), 4.53 (1H, dd, J = 5.8, 4.4 Hz), 4.69
(1H, t, J = 5.8 Hz), 5.18 (1H, d, J = 4.4 Hz), 7.22 (1H,
dd, J = 8.8, 2.0 Hz), 7.32 (2H, d, J = 8.8 Hz), 7.40 (1H,
d, J = 1.5 Hz), 7.46 (1H, d, J = 8.8 Hz), 7.73 (2H, d,
J = 8.8 Hz), 7.77 (1H, d, J = 2.0 Hz), 10.25 (1H, s),
11.93 (1H, s). FAB-MS m/z: 331 (M+1)+. Anal. Calcd
for C17H15ClN2O3: C, 61.73; H, 4.57; N, 8.47. Found:
C, 61.63; H, 4.41; N, 8.54.
(900 mg). To a solution of this reddish oil in
THF/H2O (6:1, 7.0 mL) were added osmium tetroxide
(0.08 Mt-t-BuOH solution; 5.0 mL, 0.4 mmol) and N-
methylmorpholine-N-oxide (860 mg, 7.34 mmol), and
the mixture was stirred at room temperature for 72 h.
The resulting mixture was concentrated in vacuo and
the residue was partitioned between AcOEt (60 mL)
and 10% NaHSO3 (30 mL), and the AcOEt layer was
washed with satd NaCl, then dried and concentrated
in vacuo. The residue was purified by column chroma-
tography on silica gel (CHCl3/MeOH = 50:1) to give 5
5.1.7. 5-Chloro-N-{4-[2-hydroxy-1-(hydroxymethyl)ethyl]-
phenyl}-1H-indole-2-carboxamide (2g). The title com-

K. Onda et al. / Bioorg. Med. Chem. 16 (2008) 5452–5464
5459
1
(325 mg, 9% for 2 steps) as a colorless solid. H NMR
(300 MHz, CDCl3) d: 1.09 (3H, s), 1.28 (3H, s), 2.00 (1H,
s), 2.83 (1H, s), 4.64 (1H, s), 7.57 (2H, d, J = 9.0 Hz), and
8.19 (2H, d, J = 10.1 Hz). FAB-MS m/z: 211 (M)+.
6b, in 96% yield. 1H NMR (400 MHz, DMSO-d6) d: 3.87
(3H, s), 7.24 (1H, dd, J = 8.3, 2.0 Hz), 7.40–7.50 (3H,
m), 7.55 (1H, d, J = 8.3 Hz), 7.63 (1H, d, J = 2.0 Hz),
7.79 (1H, d, J = 2.0 Hz), 10.38 (1H, s), and 11.97 (1H,
s). FAB-MS m/z: 380 (M+1)+.
5.1.11. 1-(4-Aminophenyl)-2-methylpropane-1,2-diol (1i).
To a solution of 5 (316 mg, 1.50 mmol) in EtOH/THF
(5:1, 12.0 mL) was added Pd/C (10 w/w %, 60 mg),
and the mixture was stirred under hydrogen atmosphere
at room temperature for 2.5 h. The catalyst was filtrated
on Celite and the filtrate was concentrated in vacuo to
give 1i (259 mg, 95%) as a colorless solid. 1H NMR
(400 MHz, DMSO-d6) d: 0.93 (3H, s), 0.98 (3H, s),
4.01 (1H, s), 4.13 (1H, d, J = 3.4 Hz), 4.78 (1H, d,
J = 3.9 Hz), 4.84 (2H, s), 6.46 (2H, d, J = 8.3 Hz), and
6.97 (2H, d, J = 8.3 Hz). EI-MS m/z: 181 (M)+.
5.1.17. N-(6-Bromobiphenyl-3-yl)-5-chloro-1H-indole-2-
carboxamide (7f). The title compound was prepared in
the same manner as described for 2a using 6f instead
1
of 6b, in 84% yield. H NMR (400 MHz, DMSO-d6) d:
7.22 (1H, dd, J = 8.8, 2.0 Hz), 7.40–7.53 (7H, m),
7.74–7.85 (4H, m), 10.43 (1H, s), and 11.95 (1H, s).
FAB-MS m/z: 426 (M+1)+.
5.1.18. N-(4-Bromo-3-fluorophenyl)-5-chloro-1H-indole-
2-carboxamide (7g). The title compound was prepared
in the same manner as described for 7b using 6g instead
1
5.1.12. N-(4-Bromo-2-methylphenyl)-5-chloro-1H-indole-
2-carboxamide (7a). The title compound was prepared in
the same manner as described for 2a using 6a instead of
of 6b, in 66% yield. H NMR (400 MHz, DMSO-d6) d:
7.25 (1H, dd, J = 8.8, 2.0 Hz), 7.43 (1H, s), 7.49 (1H,
d, J = 8.8 Hz), 7.58 (1H, dd, J = 8.8, 2.0 Hz), 7.70 (1H,
t, J = 8.3 Hz), 7.80 (1H, d, J = 1.5 Hz), 7.95 (1H, dd,
J = 11.2, 2.0 Hz), 10.56 (1H, s), 12.00 (1H, s). FAB-
MS m/z: 368 (M+1)+.
1
1a, in 72% yield. H NMR (400 MHz, DMSO-d6) d:
2.27 (3H, s), 7.23 (1H, dd, J = 8.8, 1.5 Hz), 7.32–7.40
(2H, m), 7.44 (1H, dd, J = 8.3, 1.5 Hz), 7.47 (1H, d,
J = 8.7 Hz), 7.53 (1H, d, J = 1.9 Hz), 7.76 (1H, d,
J = 1.4 Hz), 9.99 (1H, s), and 11.96 (1H, s). FAB-MS m/
z: 364 (M+1)+.
5.1.19. N-[4-Bromo-3-(trifluoromethyl)phenyl]-5-chloro-
1H-indole-2-carboxamide (7h). The title compound was
prepared in the same manner as described for 7b using
1
5.1.13. N-(4-Bromo-2-fluorophenyl)-5-chloro-1H-indole-
2-carboxamide (7b). To a mixture of 5-chloro-1H-in-
dole-2-carbonyl chloride (428 mg, 2.00 mmol) and
4-bromo-2-fluoroaniline (6b, 380 mg, 2.00 mmol) was
added pyridine (20 mL) and the mixture was stirred at
room temperature for 21 h. The resulting mixture was
concentrated in vacuo and the residue was washed with
H2O, 0.5 M HCl, and 0.5 M NaOH, then ether to give
7b (453 mg, 62%) as an orange solid. 1H NMR
(400 MHz, DMSO-d6) d: 7.24 (1H, dd, J = 8.8,
2.0 Hz), 7.39 (1H, d, J = 1.5 Hz), 7.47 (2H, d,
6h instead of 6b, in 57% yield. H NMR (400 MHz,
DMSO-d6) d: 7.25 (1H, dd, J = 8.8, 2.0 Hz), 7.44 (1H,
d, J = 1.6 Hz), 7.48 (1H, d, J = 8.8 Hz), 7.81 (1H, d,
J = 2.0 Hz), 7.89 (1H, d, J = 8.8 Hz), 8.08 (1H, dd,
J = 8.8, 2.4 Hz), 8.35 (1H, d, J = 2.4 Hz), 10.66 (1H,
s), and 12.03 (1H, s). FAB-MS m/z: 418 (M+1)+.
5.1.20. N-(4-Bromo-3-nitrophenyl)-5-chloro-1H-indole-
2-carboxamide (7i). To a solution of 5-chloro-1H-
indole-2-carboxylic acid (780 mg, 3.99 mmol) and
4-bromo-3-nitroaniline (830 mg, 4.00 mmol) in pyridine
(15 mL) was added phosphoric trichloride (0.41 mL,
4.41 mmol) at ꢀ25 ꢁC, then the mixture was stirred at
room temperature for 30 min. To this reaction mixture
was added ice-water (150 mL) and extracted with
AcOEt (200 mL). The organic layer was washed with
0.5 M HCl (100 mL) and satd NaCl then dried and con-
centrated in vacuo. The obtained solid was washed with
ether and MeCN, then dried to give 7i (390 mg, 25%) as
J = 8.8 Hz), 7.61–7.69 (2H, m), 7.77 (1H, d,
J = 1.5 Hz), 10.25 (1H, s), and 11.99 (1H, s). FAB-MS
m/z: 368 (M+1)+.
5.1.14. N-(4-Bromo-2-chlorophenyl)-5-chloro-1H-indole-
2-carboxamide (7c). The title compound was prepared
in the same manner as described for 7b using 6c instead
1
of 6b, in 76% yield. H NMR (400 MHz, DMSO-d6) d:
1
7.23 (1H, dd, J = 8.8, 1.0 Hz), 7.38 (1H, d, J = 2.0 Hz),
7.47 (1H, d, J = 8.8 Hz), 7.56–7.65 (2H, m), 7.77 (1H,
d, J = 2.0 Hz), 7.86 (1H, d, J = 2.0 Hz), 10.18 (1H, s),
and 12.00 (1H, s). FAB-MS m/z: 384 (M+1)+.
a reddish powder: H NMR (400 MHz, DMSO-d6) d:
7.26 (1H, dd, J = 8.8, 2.0 Hz), 7.45–7.51 (2H, m),
7.80–8.07 (3H, m), 8.57 (1H, d, J = 2.4 Hz), 10.86 (1H,
s), 12.08 (1H, s). FAB-MS m/z: 395 (M+1)+.
5.1.15. N-[4-Bromo-2-(trifluoromethyl)phenyl]-5-chloro-
1H-indole-2-carboxamide (7d). The title compound was
prepared in the same manner as described for 7b using
6d instead of 6b, in 61% yield. 1H NMR (400 MHz,
DMSO-d6) d: 7.20–7.22 (1H, m), 7.34 (1H, s), 7.45
(1H, d, J = 8.4 Hz), 7.55 (1H, d, J = 8.4 Hz), 7.73–8.20
(3H, m), 10.24 (1H, s), and 11.98 (1H, s). FAB-MS m/
z: 418 (M+1)+.
5.1.21. 5-Chloro-N-(2-methyl-4-vinylphenyl)-1H-indole-
2-carboxamide (8a). To a mixture of N-(4-bromo-2-
methylphenyl)-5-chloro-1H-indole-2-carboxamide
7a
(436 mg, 1.20 mmol) and tributyl(vinyl)stannane (1.14 g,
3.60 mmol) in DMF-THF (1:1, 8 mL) were added
Pd(PPh3)4 (70 mg, 0.061 mmol) and LiCl (153 mg,
3.60 mmol), then the mixture was refluxed for 8 h under
Ar atmosphere. The resulting mixture was concentrated
in vacuo and the residue was dissolved in satd NaCl,
then extracted with AcOEt (80 mL). The extract was
dried and concentrated in vacuo. The obtained solid
was washed with ether to give 8a (168 mg, 45%) as a col-
5.1.16. N-(4-Bromo-3-methoxyphenyl)-5-chloro-1H-indole-
2-carboxamide (7e). The title compound was prepared in
the same manner as described for 2a using 6e instead of

5460
K. Onda et al. / Bioorg. Med. Chem. 16 (2008) 5452–5464
1
orless powder. H NMR (400 MHz, DMSO-d6) d: 2.27
(3H, s), 5.25 (1H, d, J = 11.2 Hz), 5.82 (1H, d,
J = 17.6 Hz), 6.72 (1H, dd, J = 17.6, 11.2 Hz), 7.22
(1H, dd, J = 8.8, 2.0 Hz), 7.30–7.68 (5H, m), 7.45 (1H,
d, J = 1.9 Hz), 9.94 (1H, s), and 11.93 (1H, s). FAB-
MS m/z: 311 (M+1)+.
1.9 Hz), 7.43 (1H, s), 7.48 (1H, d, J = 8.8 Hz), 7.58
(1H, dd, J = 8.8, 2.0 Hz), 7.66 (1H, t, J = 8.8 Hz),
7.79–7.83 (2H, m), 10.50 (1H, s), and 12.00 (1H, s).
FAB-MS m/z: 313 (Mꢀ1)ꢀ.
5.1.28. 5-Chloro-N-[3-(trifluoromethyl)-4-vinylphenyl]-1H-
indole-2-carboxamide (8h). The title compound was pre-
pared in the same manner as described for 8a using 7h
instead of 7a. This compound was an inseparable
mixture with 7h, therefore further purification was not
attempted.
5.1.22. 5-Chloro-N-(2-fluoro-4-vinylphenyl)-1H-indole-2-
carboxamide (8b). The title compound was prepared in
the same manner as described for 8a using 7b instead
1
of 7a, in 91% yield. H NMR (400 MHz, DMSO-d6) d:
5.33 (1H, d, J = 10.8 Hz), 5.91 (1H, d, J = 17.6 Hz),
6.74 (1H, dd, J = 17.6, 10.8 Hz), 7.23 (1H, dd, J = 8.8,
2.0 Hz), 7.32–7.77 (6H, m), 10.19 (1H, s), and 11.98
(1H, s). FAB-MS m/z: 315 (M+1)+.
5.1.29. 5-Chloro-N-(3-nitro-4-vinylphenyl)-1H-indole-2-
carboxamide (8i). The title compound was prepared in
the same manner as described for 8a using 7i instead of
7a, in 46% yield. 1H NMR (400 MHz, DMSO-d6) d:
5.47 (1H, d, J = 11.7 Hz), 5.87 (1H, d, J = 17.4 Hz), 7.00
(1H, dd, J = 17.2, 11.7 Hz), 7.25 (1H, dd, J = 17.4,
2.2 Hz), 7.45–7.52 (2H, m), 7.81–7.88 (2H, m), 8.12 (1H,
dd, J = 8.8, 2.4 Hz), 8.52 (1H, d, J = 2.2 Hz), 10.73 (1H,
s), and 12.03 (1H, s). FAB-MS m/z: 340 (Mꢀ1)ꢀ.
5.1.23. 5-Chloro-N-(2-chloro-4-vinylphenyl)-1H-indole-2-
carboxamide (8c). The title compound was prepared in
the same manner as described for 8a using 7c instead
1
of 7a, in 68% yield. H NMR (400 MHz, DMSO-d6) d:
5.35 (1H, d, J = 11.2 Hz), 5.94 (1H, d, J = 17.6 Hz),
6.76 (1H, dd, J = 17.6, 11.2 Hz), 7.23 (1H, dd, J = 8.8,
2.0 Hz), 7.39 (1H, d, J = 2.0 Hz), 7.47 (1H, d,
J = 8.8 Hz), 7.52 (1H, dd, J = 8.3, 2.0 Hz), 7.62 (1H, d,
J = 8.3 Hz), 7.70 (1H, d, J = 2.0 Hz), 7.77 (1H, d,
J = 1.5 Hz), 10.11 (1H, s), and 11.99 (1H, s). FAB-MS
m/z: 331 (M+1)+.
5.1.30. 5-Chloro-N-[4-(1,2-dihydroxyethyl)-2-methyl-
phenyl]-1H-indole-2-carboxamide (9a). To a solution of
8a (160 mg, 0.52 mmol) in THF/H2O (4:1, 7.5 mL) were
added osmium tetroxide (0.08 Mt-t-BuOH solution;
0.65 mL, 0.052 mmol) and N-methylmorpholine-N-oxide
(90 mg, 0.77 mmol), and the mixture was stirred at room
temperature for 22 h. The resulting mixture was concen-
trated in vacuo and the residue was partitioned between
AcOEt (30 mL) and 10% NaHSO3 (10 mL), and the
AcOEt layer was washed with satd NaCl, then dried
and concentrated in vacuo. The residue was purified
by column chromatography on silica gel (CHCl3/
MeOH = 95:5) to give 9a (45 mg, 25%) as a colorless so-
lid. 1H NMR (400 MHz, DMSO-d6) d: 2.25 (3H, s), 3.45
(2H, t, J = 5.8 Hz), 4.50–4.56 (1H, m), 4.72 (1H, t,
J = 5.9 Hz), 5.20 (1H, d, J = 4.4 Hz), 7.17–7.36 (5H,
m), 7.46 (1H, d, J = 8.8 Hz), 7.75 (1H, d, J = 1.5 Hz),
9.92 (1H, s), and 11.92 (1H, s). FAB-MS m/z: 345
(M+1)+. Anal. Calcd for C18H17ClN2O3: C, 62.70; H,
4.97; N, 8.12. Found: C, 62.39; H, 5.02; N, 8.10.
5.1.24. 5-Chloro-N-[2-(trifluoromethyl)-4-vinylphenyl]-1H-
indole-2-carboxamide (8d). The title compound was pre-
pared in the same manner as described for 8a using 7d in-
1
stead of 7a, in 54% yield. H NMR (400 MHz, DMSO-
d6) d: 5.43 (1H, d, J = 10.8 Hz), 6.02 (1H, d,
J = 18.0 Hz), 6.83 (1H, dd, J = 18.0, 10.8 Hz), 7.23 (1H,
dd, J = 8.8, 1.9 Hz), 7.33 (1H, s), 7.47 (1H, d,
J = 8.8 Hz), 7.57 (1H, d, J = 8.3 Hz), 7.77 (1H, d,
J = 1.9 Hz), 7.87–7.89 (2H, m), 10.18 (1H, s), and 11.97
(1H, s). FAB-MS m/z: 365 (M+1)+.
5.1.25. 5-Chloro-N-(3-methoxy-4-vinylphenyl)-1H-indole-
2-carboxamide (8e). The title compound was prepared in
the same manner as described for 8a using 7e instead of
7a, in 33% yield. 1H NMR (400 MHz, DMSO-d6) d: 3.83
(3H, s), 5.19 (1H, d, J = 10.8 Hz), 5.74 (1H, d,
J = 18.0 Hz), 6.91 (1H, dd, J = 18.0, 10.8 Hz), 7.23
(1H, dd, J = 8.8, 2.0 Hz), 7.41–7.56 (5H, m), 7.78 (1H,
d, J = 1.6 Hz), 10.32 (1H, s), and 11.95 (1H, s). FAB-
MS m/z: 327 (M+1)+.
5.1.31. 5-Chloro-N-[4-(1,2-dihydroxyethyl)-2-fluorophenyl]-
1H-indole-2-carboxamide (9b). The title compound was
prepared in the same manner as described for 9a using
1
8b instead of 8a, in 34% yield. H NMR (400 MHz,
DMSO-d6) d: 3.45–3.49 (2H, m), 4.57 (1H, dt, J = 5.8,
5.4 Hz), 4.77 (1H, dd, J = 5.8, 5.4 Hz), 5.38 (1H, d,
5.1.26. 5-Chloro-N-(6-vinylbiphenyl-3-yl)-1H-indole-2-car-
boxamide (8f). The title compound was prepared in the
same manner as described for 8a using 7f instead of 7a,
J = 4.9 Hz),
7.18–7.27 (3H, m), 7.38 (1H, d,
J = 0.9 Hz), 7.46 (1H, d, J = 8.8 Hz), 7.51–7.56 (1H,
m), 7.76 (1H, d, J = 1.9 Hz), 10.15 (1H, s), and 11.96
(1H, s). FAB-MS m/z: 349 (M+1)+. Anal. Calcd for
C17H14ClFN2O3Æ0.2H2OÆ0.02CHCl3: C, 57.62; H, 4.10;
N, 7.90. Found: C, 57.65; H, 3.86; N, 7.89.
1
in 57% yield. H NMR (400 MHz, DMSO-d6) d: 5.19
(1H, d, J = 11.2 Hz), 5.75 (1H, d, J = 17.5 Hz), 6.59
(1H, dd, J = 17.6, 10.7 Hz), 7.20–7.98 (12H, m), 10.39
(1H, s), and 11.94 (1H, s). FAB-MS m/z: 371 (Mꢀ1)ꢀ.
5.1.32. 5-Chloro-N-[2-chloro-4-(1,2-dihydroxyethyl)-
phenyl]-1H-indole-2-carboxamide (9c). The title com-
pound was prepared in the same manner as described
5.1.27. 5-Chloro-N-(3-fluoro-4-vinylphenyl)-1H-indole-2-
carboxamide (8g). The title compound was prepared in
the same manner as described for 8a using 7g instead
1
for 9a using 8c instead of 8a, in 63% yield. H NMR
1
of 7a, in 65% yield. H NMR (400 MHz, DMSO-d6) d:
5.37 (1H, d, J = 11.5 Hz), 5.86 (1H, d, J = 17.9 Hz),
6.80 (1H, dd, J = 17.9, 11,5 Hz), 7.24 (1H, dd, J = 8.8,
(400 MHz, DMSO-d6) d: 3.45–3.52 (2H, m), 4.57 (1H,
dt, J = 5.9, 5.3 Hz), 4.79 (1H, dd, J = 5.9, 5.4 Hz), 5.41
(1H, d, J = 4.4 Hz), 7.23 (1H, dd, J = 8.8, 2.0 Hz), 7.35

K. Onda et al. / Bioorg. Med. Chem. 16 (2008) 5452–5464
5461
(1H, dd, J = 8.3, 1.5 Hz), 7.37 (1H, s), 7.46 (1H, d,
J = 8.8 Hz), 7.51 (1H, d, J = 1.5 Hz), 7.53 (1H, d,
J = 8.3 Hz), 7.76 (1H, d, J = 1.9 Hz), 10.09 (1H, s),
and 11.97 (1H, s). FAB-MS m/z: 365 (M+1)+. Anal.
Calcd for C17H14Cl2N2O3: C, 54.56; H, 4.04; N, 7.49.
Found: C, 54.35; H, 3.92; N, 7.44.
J = 4.3 Hz), 7.24 (1H, dd, J = 8.6, 2.2 Hz), 7.44 (1H, d,
J = 1.6 Hz), 7.48 (1H, d, J = 8.6 Hz), 7.76 (1H, d,
J = 8.6 Hz), 7.80 (1H, d, J = 2.2 Hz), 8.09 (1H, dd,
J = 8.6, 1.6 Hz), 8.17 (1H, d, J = 2.2 Hz), 10.54 (1H,
s), and 11.99 (1H, s). FAB-MS m/z: 399 (M+1)+. Anal.
Calcd for C18H14ClF3N2O3Æ0.4H2O: C, 53.25; H, 3.67;
N, 6.90. Found: C, 53.25; H, 3.36; N, 6.98.
5.1.33. 5-Chloro-N-[4-(1,2-dihydroxyethyl)-2-(trifluoro-
methyl)phenyl]-1H-indole-2-carboxamide (9d). The title
compound was prepared in the same manner as de-
5.1.38. 5-Chloro-N-[4-(1,2-dihydroxyethyl)-3-nitrophenyl]-
1H-indole-2-carboxamide (9i). The title compound was
prepared in the same manner as described for 9a using
8i instead of 8a, in 62% yield. 1H NMR (400 MHz,
DMSO-d6) d: 3.42–3.50 (2H, m), 4.88 (1H, t, J = 5.9
Hz), 5.09 (1H, q, J = 5.4 Hz), 5.59 (1H, d, J = 4.4 Hz),
7.25 (1H, dd, J = 8.8, 2.0 Hz), 7.44–7.50 (2H, m), 7.77–
7.81 (2H, m), 8.08 (1H, dd, J = 8.8, 2.0 Hz), 8.41 (1H, d,
J = 1.9 Hz), 10.65 (1H, s), and 12.01 (1H, s). ESI-MS m/
z: 376 (M+1)+. Anal. Calcd for C17H14ClN3O5ÆH2O: C,
51.85;H,4.10;N,10.67.Found:C, 51.87;H,3.85;N,10.58.
1
scribed for 9a using 8d instead of 8a, in 87% yield. H
NMR (400 MHz, DMSO-d6) d: 3.46–3.58 (2H, m),
4.67 (1H, q, J = 5.4 Hz), 4.84 (1H, dd, J = 5.8, 5.4 Hz),
5.52 (1H, d, J = 4.9 Hz), 7.23 (1H, dd, J = 8.8, 2.0 Hz),
7.34 (1H, d, J = 1.4 Hz), 7.46 (1H, d, J = 8.8 Hz), 7.50
(1H, d, J = 8.3 Hz), 7.69 (1H, d, J = 8.3 Hz), 7.76–
7.78 (2H, m), 10.14 (1H, s), and 11.96 (1H, s). FAB-
MS m/z: 399 (M+1)+. Anal. Calcd for C18H14ClF3
N2O3Æ0.1H 2OÆ0.02CHCl3: C, 53.71; H, 3.56; N, 6.95.
Found: C, 53.36; H, 3.41; N, 6.99.
5.1.39. N-[3-Amino-4-(1,2-dihydroxyethyl)phenyl]-5-chloro-
1H-indole-2-carboxamide (9j). To a solution of 5-chloro-
N-[4-(1,2-dihydroxyethyl)-3-nitrophenyl]-1H-indole-2-
carboxamide 9i (137 mg, 0.36 mmol) in AcOH (5 mL)
were added Zn powder (240 mg, 3.67 mmol), and the
mixture was stirred at room temperature for 1.5 h. The
excess Zn was removed by filtration and the filtrate
was concentrated in vacuo. The residue was purified
by column chromatography on silica gel (CHCl3/
MeOH/NH4OH = 100:10:1) to give 9j (37 mg, 30%) as
5.1.34. 5-Chloro-N-[4-(1,2-dihydroxyethyl)-3-methoxy-
phenyl]-1H-indole-2-carboxamide (9e). The title com-
pound was prepared in the same manner as described
1
for 9a using 8e instead of 8a, in 24% yield. H NMR
(400 MHz, DMSO-d6) d: 3.20–3.50 (2H, m), 3.80 (3H,
s), 4.63 (1H, t, J = 5.9 Hz), 4.83–4.89 (1H, m), 5.00
(1H, d, J = 4.9 Hz), 7.23 (1H, dd, J = 8.8, 2.0 Hz),
7.34–7.49 (5H, m), 7.77 (1H, d, J = 1.5 Hz), 10.23 (1H,
s), and 11.93 (1H, s). FAB-MS m/z: 361 (M+1)+. Anal.
Calcd for C18H17ClN2O4: C, 59.92; H, 4.75; N, 7.76.
Found: C, 60.01; H, 4.82; N, 7.86.
1
slightly yellow solid. H NMR (400 MHz, DMSO-d6)
d: 3.43–3.50 (2H, m), 4.55–4.65 (2H, m), 5.03 (2H, s),
5.16 (1H, d, J = 3.9 Hz), 6.92 (1H, dd, J = 8.0, 2.0 Hz),
7.04 (1H, d, J = 8.3 Hz), 7.10 (1H, d, J = 2.0 Hz), 7.20
(1H, dd, J = 8.8, 1.9 Hz), 7.37 (1H, d, J = 1.5 Hz), 7.47
(1H, d, J = 8.8 Hz), 7.74 (1H, d, J = 2.0 Hz), 9.99 (1H,
s), and 11.85 (1H, s). FAB-MS m/z: 346 (M+1)+. Anal.
Calcd for C17H16ClN3O3: C, 59.05; H, 4.66; N, 12.15.
Found: C, 58.85; H, 4.67; N, 12.14.
5.1.35. 5-Chloro-N-[6-(1,2-dihydroxyethyl)biphenyl-3-yl]-
1H -indole-2-carboxamide (9f). The title compound was
prepared in the same manner as described for 9a using
8f instead of 8a, in 4% yield. 1H NMR (400 MHz,
DMSO-d6) d: 3.34–3.43 (2H, m), 4.58–4.63 (2H, m),
5.06 (1H, d, J = 3.9 Hz), 7.21 (1H, dd, J = 8.8, 1.9 Hz),
7.37–7.50 (7H, m), 7.54 (1H, d, J = 8.8 Hz), 7.61 (1H, d,
J = 2.4 Hz), 7.77 (1H, d, J = 2.0 Hz), 7.84 (1H, dd,
J = 8.8, 2.0 Hz), 10.24 (1H, s), and 11.91 (1H, s). FAB-
MS m/z: 407 (M+1)+. Anal. Calcd for C23H19ClN2O3:
C, 67.90; H, 4.71; N, 6.89. Found: C, 67.80; H, 4.75; N, 6.92.
5.1.40. N-(5-Bromopyridin-2-yl)-5-chloro-1H-indole-2-
carboxamide (11a). The title compound was prepared in
the same manner as described for 7b using 10a instead of
6b, in 70% yield. 1H NMR (400 MHz, DMSO-d6) d: 7.24
(1H, dd, J = 8.8, 1.9 Hz), 7.48 (1H, d, J = 8.8 Hz), 7.62
(1H, d, J = 1.5 Hz), 7.74 (1H, d, J = 2.0 Hz), 8.09 (1H,
dd, J = 8.8, 2.5 Hz), 8.22 (1H, d, J = 8.8 Hz), 8.54 (1H,
d, J = 2.0 Hz), 11.09 (1H, s), and 12.01 (1H, s). FAB-
MS m/z: 351 (M+1)+.
5.1.36. 5-Chloro-N-[4-(1,2-dihydroxyethyl)-3-fluorophenyl]-
1H-indole-2-carboxamide (9g). The title compound was
prepared in the same manner as described for 9a using
8g instead of 8a, in 68% yield. 1H NMR (400 MHz,
DMSO-d6) d: 3.40–3.51 (2H, m), 4.77–4.83 (2H, m), 5.33
(1H, d, J = 4.4 Hz), 7.23 (1H, dd, J = 8.8, 2.0 Hz), 7.41–
7.48 (3H, m), 7.54 (1H, dd, J = 8.8, 2.0 Hz), 7.71 (1H,
dd, J = 13.0, 1.9 Hz), 7.79 (1H, d, J = 2.0 Hz), 10.41
(1H, s), and 11.98 (1H, s). FAB-MS m/z: 349 (M+1)+.
Anal. Calcd for C17H14ClFN2O3Æ0.2H 2OÆ0.05CHCl 3: C,
57.15; H, 4.06; N, 7.82. Found: C, 57.45; H, 4.27; N, 7.73.
5.1.41. N-(6-bromopyridin-3-yl)-5-chloro-1H-indole-2-
carboxamide (11b). The title compound was prepared
in the same manner as described for 7b using 10b instead
1
of 6b, in 74% yield. H NMR (400 MHz, DMSO-d6) d:
7.25 (1H, dd, J = 8.7, 1.4 Hz), 7.43 (1H, s), 7.48 (1H,
d, J = 8.7 Hz), 7.66 (1H, d, J = 8.3 Hz), 7.81 (1H, d,
J = 1.0 Hz), 8.18 (1H, dd, J = 8.3, 2.4 Hz), 8.81 (1H, d,
J = 2.4 Hz), 10.62 (1H, s), and 12.04 (1H, s). FAB-MS
m/z: 351 (M+1)+.
5.1.37. 5-Chloro-N-[4-(1,2-dihydroxyethyl)-3-(trifluoro-
methyl)phenyl]-1H -indole-2-carboxamide (9h). The title
compound was prepared in the same manner as de-
scribed for 9a using 8h instead of 8a, in 29% yield for
two steps. 1H NMR (400 MHz, DMSO-d6) d: 3.37–
3.46 (2H, m), 4.84–4.90 (2H, m), 5.50 (1H, d,
5.1.42. N-(5-Bromopyrazin-2-yl)-5-chloro-1H-indole-2-car-
boxamide (11c). The title compound was prepared in the
same manner as described for 7b using 10c instead of 6b,

5462
K. Onda et al. / Bioorg. Med. Chem. 16 (2008) 5452–5464
in 47% yield. 1H NMR (400 MHz, DMSO-d6) d: 7.26 (1H,
dd, J = 8.8, 2.0 Hz), 7.48 (1H, J = 8.8 Hz), 7.65 (1H, d,
J = 1.6 Hz), 7.77 (1H, d, J = 1.6 Hz), 8.71 (1H, d,
J = 1.2 Hz), 9.29 (1H, d, J = 1.2 Hz), 11.43 (1H, s), and
12.08 (1H, s). FAB-MS m/z: 352 (M+1)+.
C16H14ClN3O3: C, 57.93; H, 4.25; N, 12.67. Found: C,
57.55; H, 4.46; N, 12.46.
5.1.48. 5-Chloro-N-[5-(1,2-dihydroxyethyl)pyrazin-2-yl]-
1H-indole-2-carboxamide (13c). The title compound
was prepared in the same manner as described for 9a
using 12c instead of 8a, in 35% yield. 1H NMR
(400 MHz, DMSO-d6) d: 3.57–3.63 (1H, m), 3.70–3.74
(1H, m), 4.68 (1H, q, J = 4.9 Hz), 4.75 (1H, t,
J = 5.9 Hz), 5.57 (1H, d, J = 5.3 Hz), 7.25 (1H, dd,
J = 8.8, 1.9 Hz), 7.48 (1H, d, J = 8.3 Hz), 7.64 (1H, d,
J = 1.2 Hz), 7.76 (1H, d, J = 2.0 Hz), 8.52 (1H, d,
J = 1.5 Hz), 9.36 (1H, d, J = 1.4 Hz), 11.23 (1H, s),
and 12.06 (1H, s). FAB-MS m/z: 333 (M+1)+. Anal.
5.1.43. 5-Chloro-N-(5-vinylpyridin-2-yl)-1H-indole-2-car-
boxamide (12a). The title compound was prepared in the
same manner as described for 8a using 11a instead of 7a,
1
in 69% yield. H NMR (400 MHz, DMSO-d6) d: 5.34
(1H, d, J = 10.8 Hz), 5.92 (1H, d, J = 17.6 Hz), 6.77
(1H, dd, J = 17.6, 10.8 Hz), 7.24 (1H, dd, J = 8.8,
2.4 Hz), 7.48 (1H, d, J = 8.3 Hz), 7.62 (1H, d,
J = 1.4 Hz), 7.73 (1H, d, J = 1.9 Hz), 8.03 (1H, dd,
J = 8.8, 2.4 Hz), 8.22 (1H, d, J = 8.3 Hz), 8.48 (1H, d,
J = 2.4 Hz), 10.99 (1H, s), and 11.99 (1H, s). FAB-MS
m/z: 298 (M+1)+.
Calcd for C15H13ClN4O3 Æ0.1H OÆ0.12CHCl 3: C,
2
52.05; H, 3.85; N, 16.06. Found: C, 52.10; H, 3.75; N,
16.00.
5.1.44. 5-Chloro-N-(6-vinylpyridin-3-yl)-1H-indole-2-car-
boxamide (12b). The title compound was prepared in the
same manner as described for 8a using 11b instead of 7a,
5.1.49. 5-Chloro-N-[4-(1,2-dihydroxyethyl)-1,3-thiazol-2-
yl]-1H-indole-2-carboxamide (15a). The title compound
was prepared in the same manner as described for 2a
using 14a instead of aniline, in 27% yield. 1H NMR
(400 MHz, DMSO-d6) d: 3.47–3.55 (1H, m), 3.60–3.75
(1H, m), 4.58–4.64 (1H, m), 4.64–4.72 (1H, m), 5.25–
5.32 (1H, m), 7.01 (1H, s), 7.25 (1H, dd, J = 8.8,
1.9 Hz), 7.47 (1H, d, J = 8.8 Hz), 7.62 (1H, s), 7.76
(1H, d, J = 1.9 Hz), 12.07 (1H, s), and 12.76 (1H, s).
FAB-MS m/z: 338 (M+1)+. Anal. Calcd for
C14H12ClN3O3S: C, 49.78; H, 3.58; N, 12.44. Found:
C, 49.81; H, 3.83; N, 12.43.
1
in 72% yield. H NMR (400 MHz, DMSO-d6) d: 5.41
(1H, dd, J = 10.8, 1.6 Hz), 6.15 (1H, dd, J = 17.6,
1.2 Hz), 6.79 (1H, dd, J = 17.6, 10.8 Hz), 7.24 (1H, dd,
J = 8.8, 2.0 Hz), 7.44 (1H, s), 7.48 (1H, d, J = 8.4 Hz),
7.54 (1H, d, J = 8.4 Hz), 7.80 (1H, d, J = 2.0 Hz), 8.21
(1H, dd, J = 8.4, 2.4 Hz), 8.93(1H, d, J = 2.4 Hz),
10.54 (1H, s), and 12.02 (1H, s). FAB-MS m/z: 298
(M+1)+.
5.1.45. 5-Chloro-N-(5-vinylpyrazin-2-yl)-1H-indole-2-car-
boxamide (12c). The title compound was prepared in
the same manner as described for 8a using 11c instead
5.1.50. 5-Chloro-N-[5-(1,2-dihydroxyethyl)-1,3-thiazol-2-
yl]-1H-indole-2-carboxamide (15b). The title compound
was prepared in the same manner as described for 7b
using 14b instead of 6b, in 51% yield. 1H NMR
(400 MHz, DMSO-d6) d: 3.48–3.64 (2H, m), 4.70–4.80
(1H, m), 4.85–4.97 (1H, m), 5.63 (1H, d, J = 3.9 Hz),
7.25 (1H, dd, J = 8.8, 2.0 Hz), 7.38 (1H, s), 7.47 (1H,
d, J = 8.3 Hz), 7.60 (1H, s), 7.75 (1H, d, J = 2.0 Hz),
12.08 (1H, s), and 12.59 (1H, s). FAB-MS m/z: 338
(M+1)+. Anal. Calcd for C14H12ClN3O3S: C, 49.78; H,
3.58; N, 12.44. Found: C, 49.85; H, 3.92; N, 12.47.
1
of 7a, in 70% yield. H NMR (400 MHz, DMSO-d6) d:
5.54 (1H, d, J = 10.8 Hz), 6.28 (1H, d, J = 17.6 Hz),
6.88 (1H, dd, J = 17.8, 11.0 Hz), 7.25 (1H, dd, J = 8.8,
2.0 Hz), 7.49 (1H, d, J = 9.0 Hz), 7.64–7.80 (2H, m),
8.59 (1H, s), 9.44 (1H, s), 11.30 (1H, s), and 12.07 (1H,
s). FAB-MS m/z: 299 (M+1)+.
5.1.46. 5-Chloro-N-[5-(1,2-dihydroxyethyl)pyridin-2-yl]-
1H-indole-2-carboxamide (13a). The title compound
was prepared in the same manner as described for 9a using
1
12a instead of 8a, in 78% yield. H NMR (400 MHz,
5.2. Pharmacology
DMSO-d6) d: 3.43–3.49 (1H, m), 3.50–3.56 (1H, m),
4.58 (1H, t, J = 5.7 Hz), 4.79 (1H, s), 5.38 (1H, s), 7.23
(1H, d, J = 7.4 Hz), 7.47 (1H, d, J = 8.3 Hz), 7.59 (1H,
s), 7.72 (1H, s), 7.79 (1H, dd, J = 8.8, 2.4 Hz), 8.16 (1H,
d, J = 8.3 Hz), 8.35 (1H, d, J = 1.9 Hz), 10.88 (1H, s),
and 11.98 (1H, s). ESI-MS m/z: 332 (M+1)+. Anal. Calcd
for C16H14ClN3O3Æ0.1H2O: C, 57.61; H, 4.29; N, 12.60.
Found: C, 57.63; H, 4.41; N, 12.26.
5.2.1. Glycogen phosphorylase activity. The activity of re-
combinant hLGPa in the forward direction was mea-
sured by determination of NADPH according to the
methods of Pesce et al. modified.32 The enzyme activity
was assayed at pH 6.8 in a phosphate buffer containing
KH2PO4 (45 mM), b-NADP (22.5 lM), a-glucose 1,6-
bisphosphate (4 · 10ꢀ4%), glucose-6-phosphate dehy-
drogenase (385 U/L), phosphoglucomutase (77 U/L),
glycogen (0.24%), and glucose (10.85 mM). The test
compounds were added as 10 lL of solution in DMSO
prior to the addition of the enzyme. The basal rate of
hLGPa enzyme activity in the absence of inhibitors
(Control) is determined by adding 10 lL of DMSO
and a fully inhibited rate of hLGPa enzyme activity is
obtained by adding 10 lL of 200 mM of the positive
control test substance, caffeine. Following, the phos-
phate buffer was added as 216.5 lL, and the reaction
was started by addition 23.5 lL hLGPa solution
5.1.47. 5-Chloro-N-[6-(1,2-dihydroxyethyl)pyridin-3-yl]-
1H-indole-2-carboxamide (13b). The title compoundwas
prepared in the same manner as described for 9a using
1
12b instead of 8a, in 20% yield. H NMR (400 MHz,
DMSO-d6) d: 3.46–3.53 (1H, m), 3.64–3.72 (1H, m),
4.56–4.61 (1H, m), 4.67 (1H, s), 5.35 (1H, s), 7.24 (1H,
dd, J = 8.3, 2.0), 7.42 (1H, s), 7.46–7.52 (2H, m), 7.79
(1H, d, J = 1.9 Hz), 8.16 (1H, dd, J = 8.3, 2.4 Hz), 8.87
(1H, d, J = 2.4 Hz), 11.72 (1H, s), and 12.00 (1H, s).
FAB-MS m/z: 332 (M+1)+. Anal. Calcd for

K. Onda et al. / Bioorg. Med. Chem. 16 (2008) 5452–5464
5463
(40 mM b-glycerophosphate, 80 mM cystein, pH 6.8).
The reaction was followed at room temperature by mea-
suring the conversion of oxidized b-NADP to reduced b-
NADPH at 340 nm for 2 h. The hLGPa inhibition IC50
values were calculated using the logistic regression meth-
od with SAS software.
ly administered to mice at the rate of 10 mL/kg. After
2 h, blood glucose levels (2 h) were measured. Hypogly-
cemic activity of the test compounds was determined by
statistical analysis (unpaired t-test) of the mean blood
glucose levels between the test compound group and
vehicle group.
5.2.2. Isolation and culturing of hepatocytes. Male Spra-
gue–Dawley rats (150–200 g, purchased from Japan
CLEA, Inc., Tokyo, Japan) were anesthtized with a
freshly prepared pentobarbital intraperitoneally (65 mg/
kg). Hepatocytes were isolated from rats fed ad libtum
as described in the literature.33 Cell viability, assessed by
Trypan blue exclusion, was consistently greater than
85%. Cells were plated on to collagen-coated 24-well
plates in basal medium (William’s medium E containing
100 U/mL penicillin/streptomycin, 1 nM insulin, 1 nM
dexamethazone, and 2.2 g/L NaHCO3) supplemented
with 10 % FBS at a cell density of 2.5 · 105 cells/well.
5.3. Crystallization and data collection
Crystal of hLGP in complex with 2f was obtained at
4 ꢁC by the hanging drop vapor diffusion method. The
hanging drop consisted of equal volume of the concen-
trated protein solution (20 mg/mL) and reservoir solu-
tion containing 100 mM NaMES (pH 6), 22.5% MPD,
60 mM D-glucose, and 0.9 mM 2f. The crystal belongs
to the trigonal space group P31, with cell dimensions
˚
˚
of a = b = 123.8 A and c=123.5 A. The diffraction data
˚
set is 99% complete to 2.4 A. The structure was solved
with molecular replacement method using published
Code
hLGP/CP-403700 complex structure (PDB
1EXV) as the starting model,18 and refined using the
5.2.3. Glucagon-induced glycogenolysis in primary hepa-
tocytes. After attachment, the cells were washed two times
with pre-warmed, oxygen-saturated, glucose-free/cal-
cium-free Hanks buffer (137 mM NaCl, 5.4 mM KCl,
0.5 mM NaH2PO4, 0.42 mM Na2HPO4,10 mM Hepes,
0.017 mM phenol red, and 4.17 mM NaHCO3) and sub-
sequently incubated for 30 min at 37 ꢁC in a final volume
of 500 lL in glucose-free/calcium-free Hanks buffer,0.1%
DMSO, and respective inhibitor at a concentration within
the range 0.1–10 lM in the absence (basal condition) or
presence of 10 nM glucagon (stimulated condition). Med-
ium (100 lL) was removed, and glucagon-induced glu-
cose release into the medium was determined using
glucose assay reagent (0.1 M potassium phosphate solu-
tion containing 2.8 U/mL glucoseoxydase, 1 U/mL per-
oxydase, 0.25 mM 4-aminoantipyrine, and 1.5 lL/mL
dimethylaniline, pH 5.6). The IC50 values of glucagon-in-
duced glucose output were calculated using the logistic
regression method with SAS software.
program CNX34 to 2.45 A resolution and a final R-fac-
˚
tor of 0.261(Rfree 0.307). The coordinate for the complex
described in the paper has been deposited in the PDB
(Accession Code 2ZB2) for immediate release on
publication.
Acknowledgments
The authors wish to thank Ms. Akiko Matsuyama-Yok-
ono for helpful support in the preparation of this man-
uscript and the staff of the Division of Analytical
Science Laboratories for the elemental analysis and
spectral measurements. The authors also wish to thank
Dr. Hitoshi Sakashita, Dr. Tetsuo Matsui, Mr. Tatsuya
Maruyama and Dr. Minoru Okada for their support of
this work.
5.2.4. Hypoglycemic activity of glycogen phosphorylase
inhibitor. The acute hypoglycemic activities of glycogen
phosphorylase inhibitor compounds were determined
using 6 week old male C57BL/KsJ Jcl-dbm +db/+db
mice (purchased from Japan CLEA, Inc., Tokyo, Japan)
housed under conventional conditions with controlled
temperature, humidity, and lighting, and provided with
a commercial diet (CE-2; Oriental Yeast, Tokyo, Japan)
and water ad libitum. All procedures were conducted
according to the Yamanouchi Animal care Committee’s
Guideline. After a one-week acclimation period, the ani-
mals were weighted and 10 lL of blood was collected
from the tail veins of mice. The blood was mixed with
0.33 M perchloric acid solution (100 lL) in tubes and
stirred. The mixtures were centrifuged at 3000 rpm for
10 min, and the glucose levels in the supernatants were
measured using glucose CII-Test reagent (Wako Pure
Chemical Industries, Ltd, Osaka, Japan). Animals were
then grouped to make the blood glucose levels and body
weight uniform for all groups (n = 6). Next day, blood
glucose levels (pre) were measured in non-fasted mice.
Either the vehicle or test compounds, dissolved in 5%
propylenglycol–5% Tween 80–water solution, were oral-
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