Synthesis and pharmacological evaluation of bis-3-(3,4-dichlorophenyl)acrylamide derivatives as glycogen phosphorylase inhibitors

Article abstract of DOI:10.1016/j.bmc.2008.08.010

During our research using a high-throughput screening system for discovery of a new class of human liver glycogen phosphorylase a (hLGPa) inhibitors, a series of 3-(3,4-dichlorophenyl)acrylamide derivatives were synthesized, and their inhibitory activities toward hLGPa were evaluated. Among the derivatives, (2E,2′E)-N,N′-pentane-1,5-diylbis[3-(3,4-dichlorophenyl)acrylamide] (6c) inhibited hLGPa with an IC₅₀ value of 0.023 μM. An X-ray crystallographic study of the enzyme-6c complex showed that the inhibitor is bound at the dimer interface site, where the 3,4-dichlorophenyl moiety interacts hydrophobically with the enzyme.

Full text of DOI:10.1016/j.bmc.2008.08.010

Bioorganic & Medicinal Chemistry 16 (2008) 8627–8634
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and pharmacological evaluation of bis-3-(3,4-dichlorophenyl)
acrylamide derivatives as glycogen phosphorylase inhibitors
*
Kenichi Onda , Ryota Shiraki, Yasuhiro Yonetoku, Kazuhiro Momose, Naoko Katayama,
Masaya Orita, Tomohiko Yamaguchi, Mitsuaki Ohta, Shin-ichi Tsukamoto
Drug Discovery Research, Astellas Pharma Inc., 5-2-3 Toukoudai, Tsukuba, Ibaraki 300-2698, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
During our research using a high-throughput screening system for discovery of a new class of human
liver glycogen phosphorylase a (hLGPa) inhibitors, a series of 3-(3,4-dichlorophenyl)acrylamide deriva-
tives were synthesized, and their inhibitory activities toward hLGPa were evaluated. Among the deriva-
tives, (2E,20E)-N,N0-pentane-1,5-diylbis[3-(3,4-dichlorophenyl)acrylamide] (6c) inhibited hLGPa with an
Received 11 June 2008
Revised 1 August 2008
Accepted 2 August 2008
Available online 7 August 2008
IC50 value of 0.023 lM. An X-ray crystallographic study of the enzyme–6c complex showed that the
inhibitor is bound at the dimer interface site, where the 3,4-dichlorophenyl moiety interacts hydropho-
bically with the enzyme.
Keywords:
Glycogen phosphorylase
X-ray crystallographic study
Bis-3-(3,4-dichlorophenyl)acrylamide
Type 2 diabetes
Ó 2008 Elsevier Ltd. All rights reserved.
High-throughput screening (HTS)
1. Introduction
ence of glucose, the enzyme activity of 1 increased to about 10
times (IC50 = 0.043 lM) that under glucose free conditions, which
Diabetes is a chronic and a severe metabolic disorder that
occurs when the body does not produce or properly use insulin.
There are several types of this disease, with Type 2 being the
most common form. Type 2 diabetes is defined by chronic
elevated blood glucose levels characterized by peripheral insulin
resistance and pancreatic defects associated with insulin
secretion.1,2
The hepatic glucose output (HGO) system plays an important
role in Type 2 diabetic therapy.3,4 The HGO rate in diabetic
patients is significantly higher than that in normal individuals,
and the current study indicates that the degradative phosphor-
ylation of glycogen (glycogenolysis) is an important contributor
to HGO.5–7 Human liver glycogen phosphorylase a (hLGPa, the
active form of human liver GP) is a homodimeric enzyme that
catalyzes glycogenolysis. This enzyme has been an attractive
molecular target through which HGO can be controlled for the
treatment of diabetes, and several kinds of compounds have
been discovered.8–18
indicates that the risk of hypoglycemia can be minimized. There-
fore, we considered 1 to be the lead compound in the search to find
much more potent compounds via structural modification, such as
a mono-amide analogue, bond analogues (at the trans double
bonds), and a Cn- or heteroatom-containing chain instead of the
C4 chain in 1 (Fig. 1).
It is noteworthy that GP inhibitors that may be structurally
related to 1 have also been reported. CP-526423 (2) has a
symmetrical bis-indole structure, and a single molecule of this
compound is situated at the dimer interface in a complex with
hLGPa.19 Compound 3, from GSK research group, has a 3-(3,4-
dichlorophenyl)acrylamide moiety and showed inhibitory
activity toward hLGPa in the presence of glucose; however, the
binding site of this compound in the enzyme was unrevealed.20
FR258900 (4), which was isolated from the culture broth of a
fungal strain and has a bis-3-phenylacrylester structure, showed
both enzymatic and in vivo activity.21,22 The X-ray crystallo-
graphic study of the rMGPa in complex with 4 showed that
the compound binds not at the dimer interface site, but at the
allosteric site on the enzyme.23 These reports piqued our
interest in the binding site on 1 (or its derivatives) considerably
(Fig. 2).
To find novel GP inhibitors, our compound library was sub-
jected to high-throughput screening (HTS). One compound (1),
which had a symmetric structure containing two 3-(3,4-dichloro-
phenyl)acrylamide moieties, was found to be a potent inhibitor
of hLGPa (IC50 = 0.42
l
M) and act as a glucose sensor. In the pres-
In this paper, we report the structure–activity relationships
(SARs) and binding site as well as binding mode for these
compounds determined via X-ray crystallographic analysis of the
enzyme–ligand complex.
* Corresponding author. Tel.: +81 29 847 8611; fax: +81 29 847 8313.
E-mail address: kenichi.onda@jp.astellas.com (K. Onda).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.08.010

8628
K. Onda et al. / Bioorg. Med. Chem. 16 (2008) 8627–8634
Cl
Cl
Cl
Cl
O
Cl
Cl
O
O
H
bond
X
bond
Cl
Cl
N
N
N
H
N
H
H
O
1
Figure 1. Design of amide derivatives based on 1.
O
Cl
O
H
Cl
Cl
N
H
N
N
H
H
O
N
Cl
Cl
N
N
N
O
H
N
H
O
O
Cl
3
2: CP-526423
HO2C
O
OH
O
O
O
HO
CO2H
4: FR258900
Figure 2. Examples of GP inhibitors.
some cases followed by the conversion of the substituent parts,
as shown in Scheme 1.
2. Chemistry
Carboxylic acids were prepared as the starting material for the
cis double and triple bond analogues of 6c shown in Scheme 2.
Compounds1, 5, 6a–f, 7a–c, and 8a–f were synthesized via cou-
pling reactions between carboxylic acids and various amines, in
O
Cl
Cl
a
Cl
Cl
CO2H
N
H
1: n=4
5
6a: n=2
b: n=3
c: n=5
d: n=6
e: n=7
f: n=8
O
O
Cl
Cl
(CH2)n
Cl
a
N
N
H
H
Cl
O
O
a
Cl
Cl
Cl
Cl
Cl
Cl
RCO H
R =
2
R
N
N
R
H
H
7a
7b
7c
O
O
Cl
Cl
A
a
Cl
Cl
Cl
Cl
CO2H
N
N
H
H
8a: A=(CH2)2NBoc(CH2)2
b
.
b: A=(CH2)2NH(CH)
2HCl
2
c: A=(CH2)2O(CH )2
2
d: A=(CH2)2S(CH2)2
e: A=(CH2)2S(O)(CH2)2
f: A=(CH2)2S(O)2(CH2)2
c
d
Scheme 1. Synthesis of compounds 1, 5, 6a–f, 7a–c, and 8a–f. Reagents and conditions: (a) amine, WSCꢀHCl, HOBt, DMF; (b) 4 M HCl/AcOEt, EtOH; (c) MCPBA (1.0 equiv),
CH2Cl2, 0 °C; (d) MCPBA (4.0 equiv), CH2Cl2, rt.

K. Onda et al. / Bioorg. Med. Chem. 16 (2008) 8627–8634
8629
TMS
CO2H
Cl
Cl
Cl
Cl
Cl
Cl
a
c
Cl
Cl
I
b
Cl
Cl
d
CO2H
9
10
11
12
13
Scheme 2. Synthesis of carboxylic acids 12 and 13. Reagents and conditions: (a) TMSCCH, PdCl2(PPh3)2, CuI, NHEt2; (b) KOH, MeOH, H2O; (c) n-BuLi, CO2, THF; (d) H2/Pd–
BaSO4, quinoline, MeOH.
Table 2
The coupling reaction between 1,2-dichloro-4-iodobenzene (9)
with trimethylsilylacetylene, followed by deprotection under basic
conditions, yielded 1,2-dichloro-4-ethynylbenzene (11).24 The car-
boxylation of 11, followed by partial hydrogenation, afforded 12
and 13, respectively.
SAR of compounds 7a–ca
Cl
O
Cl
Cl
O
bond
bond
N
N
Cl
H
H
Entry
1
Compound
Bond
CH2-CH2
hLGPa (ꢁGlu) IC50 (lM)
45
3. Results and discussion
7a
H
C
H
C
Compounds were evaluated for their inhibitory activity toward
hLGPa. The structures and activities are listed in Tables 1–3.
The bis-amide structure in 1 seemed to be critical to inhibitory
activity, because the mono-amide compound (5) showed no activ-
ity. Shortening the butylene chain into an ethylene (6a) or propyl-
ene chain (6b) caused a decline in inhibitory activity, while the
presence of a pentylene (6c) chain caused it to increase twofold
in both the absence and presence of glucose (ꢁGlu and +Glu).
The hexylene (6d), heptylene (6e), and octylene (6f) analogues
were not tolerated. Therefore, the length of the chain between
the two amide parts was strictly limited, with the five-carbon
chain being preferable (Table 1). In addition, the trans double
bonds in the acrylamide part were clearly essential moieties since
the activity with the single bond analogue (7a), cis double bond
analogue (7b), and triple bond analogue (7c) were all dramatically
less (Table 2). When heteroatoms were introduced into the pentyl-
ene chain in 6c, a nitrogen atom in the center of the chain (8a,b) led
to a loss of inhibitory activity, but both an oxygen atom (8c) and a
sulfur atom (8d–f) were tolerated (Table 3).
2
7b
7c
36%
59
3
C
C
a
Inhibitory activities are expressed as the IC50 or as % inhibition at a concen-
tration of 10 lM.
Table 3
SAR of compounds 8a–fa
O
O
Cl
X
Cl
N
N
H
H
Cl
Cl
Entry Compound
X
hLGPa (ꢁGlu) IC50
48%
(l
M) hLGPa (+Glu) IC50 (lM)
1
2
3
4
5
6
8a
8b
8c
8d
8e
8f
NBoc
NT
NT
0.026
0.012
0.026
NT
NHꢀHCl 51%
O
S
SO
SO2
0.43
0.45
0.31
1.5
To determine the binding site and binding mode of the com-
pounds mentioned above, the crystal structure of the hLGPa–6c
complex was elucidated at a resolution of 2.45 Å (Fig. 3A, B). The
structure showed that 6c existed not at the allosteric site where
a
Inhibitory activities are expressed as the IC50 or as % inhibition at a concen-
tration of 10 M.
l
4 binds, but at the dimer interface site. In addition, one molecule
of 6c acted as a bridge between the two dimers, which would sta-
bilize the less active conformational state of the enzyme and lead
to inhibitory activity.
Table 1
SAR of compounds 1, 5, and 6a–fa
O
The structures also indicate that the 3,4-dichlorophenyl moie-
ties interact with a hydrophobic pocket formed by amino acid res-
idues such as Arg60, Val64, Trp67, Asp227, Pro229, Pro188, Trp189,
Glu190, Lys191, Phe370, Thr380, and Val400, where the prime (0)
indicates the second subunit. No significant hydrophilic interac-
tions between the enzyme and either of the two amide moieties
were observed.
The role of the trans double bond in the inhibitory activity of the
3-(3,4-dichlorophenyl) acrylamide moieties could not be clearly
determined from this structure. However, it probably has the abil-
ity to form a favorable conformation because the other bond ana-
logues (7a–c) do not show potent activity.
Cl
N
H
Cl
5
O
O
Cl
Cl
(CH2)n
Cl
Cl
N
N
H
H
1, 6a-f
Entry
Compound
n
hLGPa (ꢁGlu) IC50
(l
M)
hLGPa (+Glu) IC50 (lM)
The reason for the inactivity of the amine analogues (8a,b) could
not be definitively determined from this structure either; however,
it was deduced that the Boc moiety in 8a contains steric hindrance,
and the nitrogen atom in 8b might have undesirable electrostatic
interactions with the hydrophilic regions in the dimer interface,
which would lead to an inappropriate bridge form.
Since the position of 6c in hLGPa was very similar to that of in-
dole-type inhibitors, the superposition of the crystal structure of 6c
and 5-chloro-N-[4-(1,2-dihydroxyethyl)phenyl]-1H-indole-2-car-
1
2
3
4
5
6
7
8
5
0%
20
55
0.42
0.26
18
11
33
NT
NT
NT
0.043
0.023
NT
NT
NT
6a
6b
1
6c
6d
6e
6f
2
3
4
5
6
7
8
a
Inhibitory activities are expressed as the IC50 or as % inhibition at a concen-
M.
tration of 10
l

8630
K. Onda et al. / Bioorg. Med. Chem. 16 (2008) 8627–8634
Figure 3. (A) Crystal structure of 6c bound to the hLGPa dimer interface. Glucose and the essential cofactor pyridoxal 50-phosphate (PLP) are situated at the catalytic site. (B)
Schematic diagram of the interaction of 6c with hLGPa as revealed by crystallographic analysis. The protein residues of the two hLGPa molecules in the dimer are labeled as A
and B, respectively. The figure was prepared using the ligand interactions application in MOE.25
boxamide (14) was examined in order to compare their binding
styles (Fig. 4).
It has been suggested that this is due to the lack of interaction be-
tween the amide moieties and the enzyme. The alkylene chain on
6f is too flexible to link with the dimer in an inactive conformation;
therefore, the shorter chain seems to be necessary. Binding with
the C5 derivative (6c) resulted in tight control of the dimer and po-
tent activity.
Unfortunately, this series of compounds did not inhibit gluca-
gon-induced glucose output in cultured primary hepatocytes,
probably because of their poor solubility and/or membrane perme-
ability. Efforts to modify the structures of these compounds to
achieve cell-based activity will continue.
As shown in Figure 4, the planar 3,4-dichlorophenyl moiety is
well-situated in proximity to the center of the indole ring in 14,
and the 3,4-dichlorophenyl ring itself seems to interact
hydrophobically with hLGPa, as would a 5-chloroindole ring. It
should be noted that, in the case of 14, the hydrophobic interac-
tions between the 5-chloroindole moiety and the enzyme, as
well as the hydrophilic interactions between the amide moiety
and the two hydroxyl groups with the enzyme, were both
important to inhibitory activity.26 This indicates that 6c and 14
utilize the same binding site on GP, but their means of inhibition
are different.
The activity of 2 and the derivative mentioned above were then
compared. According to the literature, 2 has very potent inhibitory
activity against hLGPa (IC50 = 6 nM),19 while the activity of the C8
derivative (6f), which has the same chain length as 2, is very weak.
4. Conclusion
To summarize, a series of bis-3-(3,4-dichlorophenyl)acrylamide
derivatives were synthesized as a new class of hLGPa inhibitors.

K. Onda et al. / Bioorg. Med. Chem. 16 (2008) 8627–8634
8631
Cl
H
N
N
H
OH
OH
O
14
Figure 4. (A) Chemical structure of 14. (B) An overview of the docking model for 6c with 14.
The bis-amide structures and the presence of a trans double bond
in the acrylamides were found to be essential. The length of the
carbon chain connecting the two amide moieties was also found
to be important to activity. An X-ray crystallographic study of
the enzyme–6c complex suggested that this compound is the first
example of interaction with the dimer interface site on an enzyme,
except for that seen with indole-type compounds or related deriv-
atives. The hydrophobic interaction between the 3,4-dichloro-
phenyl moiety and hLGPa was also important to inhibitory activity.
(M+1)+. Anal. Calcd for C12H13Cl2NO: C, 55.83; H, 5.08; N, 5.43.
Found: C, 55.83; H, 4.91; N, 5.43.
5.1.3. (2E,20E)-N,N0-Ethane-1,2-diylbis[3-(3,4-dichlorophenyl)-
acrylamide] (6a)
This compound was prepared in the same manner as 1 using 1,2-
ethylenediamine instead of 1,4-butylenediamine to obtain a quanti-
tative yield. 1H NMR (400 MHz, DMSO-d6) d: 3.27–3.33 (4H, m), 6.68
(2 H, d, J = 15.6 Hz), 7.42 (2H, d, J = 16.2 Hz), 7.57 (2H, dd, J = 8.3,
2.0 Hz), 7.67 (2H, d, J = 8.3 Hz), 7.85 (2H, d, J = 1.9 Hz), 8.20–8.27
(2H, m). FABMS m/z: 459 (M+1)+. Anal. Calcd for C20H16Cl4N2O2: C,
52.43; H, 3.52; N, 6.11. Found: C, 52.35; H, 3.42; N, 6.20.
5. Experimental
5.1. Chemistry
5.1.4. (2E,20E)-N,N0-Propane-1,3-diylbis[3-(3,4-dichlorophenyl)-
acrylamide] (6b)
1H NMR spectra were recorded on a JEOL JNM-LA300 or JEOL
JNM-EX400 spectrometer, with the chemical shifts expressed in d
(ppm) using tetramethylsilane as an internal standard (NMR
abbreviations: s = singlet, d = doublet, dd = double doublet, t = trip-
let, q = quartet, m = multiplet, and br = broad peak). Mass spectra
were recorded on a JEOL JMS-700T or the micromass Q-T of an Ul-
tima API spectrometer. The elemental analyses were performed
with a Yanaco MT-5 microanalyzer (C, H, N), and were within
0.4% of theoretical values. During the work-up, organic solutions
were dried over anhydrous MgSO4 or Na2SO4.
This compound was prepared in the same manner as 1 using
1,3-propylenediamine instead of 1,4-butylenediamine (76% yield).
1H NMR (400 MHz, DMSO-d6) d: 1.60–1.70 (2H, m), 3.23 (4H, q,
J = 6.8 Hz), 6.69 (2H, d, J = 15.6 Hz), 7.40 (2H, d, J = 16.1 Hz), 7.56
(2H, dd, J = 8.3, 1.9 Hz), 7.67 (2H, d, J = 8.8 Hz), 7.85 (2H, d,
J = 1.9 Hz), 8.15 (2H, t, J = 5.3 Hz). FABMS m/z: 473 (M+1)+. Anal.
Calcd for C21H18Cl4N2O2: C, 53.42; H, 3.84; N, 5.93. Found: C,
53.26; H, 3.81; N, 6.11.
5.1.5. (2E,20E)-N,N0-Pentane-1,5-diylbis[3-(3,4-dichlorophenyl)-
acrylamide] (6c)
5.1.1. (2E,20E)-N,N0-Butane-1,4-diylbis[3-(3,4-dichlorophenyl)-
acrylamide] (1)
This compound was prepared in the same manner as 1 using
1,5-pentylenediamine instead of 1,4-butylenediamine (42% yield).
1H NMR (400 MHz, DMSO-d6) d: 1.30–1.40 (2H, m), 1.43–1.55
(4H, m), 3.18 (4H, q, J = 6.3 Hz), 6.68 (2H, d, J = 15.6 Hz), 7.38 (2H,
d, J = 15.6 Hz), 7.54 (2H, dd, J = 8.3, 1.9 Hz), 7.65 (2H, d,
J = 8.3 Hz), 7.82 (2H, d, J = 1.5 Hz), 8.10 (2H, t, J = 5.4 Hz). FABMS
m/z: 501 (M+1)+. Anal. Calcd for C23H22Cl4N2O2: C, 55.22; H, 4.43;
N, 5.60. Found: C, 54.99; H, 4.32; N, 5.54.
1-Ethyl-3-(30-dimethylaminopropyl)carbodiimide hydrochlo-
ride (210 mg, 1.10 mmol) and 1-hydroxybenzotriazole hydrate
(170 mg, 1.11 mmol) were added to a solution of (2E)-3-(3,4-
dichlorophenyl)acrylic acid (217 mg, 1.00 mmol) and 1,4-butylen-
ediamine (44 mg, 0.50 mmol) in DMF (5 mL), and this mixture was
stirred at room temperature for 18 h. H2O (20 mL) was added to
the reaction mixture, and the resulting precipitate was collected
and washed with H2O (20 mL) and EtOH/AcOEt (1:1, 10 mL) to
yield 1 (206 mg, 85%) as a colorless solid: 1H NMR (400 MHz,
DMSO-d6) d: 1.90–2.10 (4 H, m), 3.15–3.25 (4H, m), 6.68 (2H, d,
J = 15.6 Hz), 7.37 (2H, d, J = 16.1 Hz), 7.55 (2H, dd, J = 8.3, 1.9 Hz),
7.66 (2H, d, J = 8.3 Hz), 7.83 (2H, d, J = 1.9 Hz), 8.13 (2H, t,
J = 5.3 Hz). FABMS m/z: 487 (M+1)+. Anal. Calcd for C22H20Cl4N2O2:
C, 54.35; H, 4.15; N, 5.76. Found: C, 54.21; H, 3.95; N, 6.08.
5.1.6. (2E,20E)-N,N0-Hexane-1,6-diylbis[3-(3,4-dichlorophenyl)-
acrylamide] (6d)
This compound was prepared in the same manner as 1 using
1,6-hexylenediamine instead of 1,4-butylenediamine (86% yield).
1H NMR (400 MHz, DMSO-d6) d: 1.25–1.37 (4H, m), 1.40–1.53
(4H, m), 3.10–3.22 (4H, m), 6.68 (2H, d, J = 15.6 Hz), 7.38 (2H, d,
J = 15.6 Hz), 7.55 (2H, dd, J = 8.3, 1.5 Hz), 7.66 (2H, d, J = 8.3 Hz),
7.83 (2H, d, J = 1.5 Hz), 8.10 (2H, t, J = 5.3 Hz). FABMS m/z: 515
(M+1)+. Anal. Calcd for C24H24Cl4N2O2: C, 56.05; H, 4.70; N, 5.45.
Found: C, 55.96; H, 4.50; N, 5.72.
5.1.2. (2E)-3-(3,4-Dichlorophenyl)-N-propylacrylamide (5)
This compound was prepared in the same manner as 1 using 1-
propylamine instead of 1,4-butylenediamine (93% yield). 1H NMR
(400 MHz, DMSO-d6) d: 0.88 (3H, t, J = 7.4 Hz), 1.42–1.52 (2H, m),
3.14 (2H, q, J = 6.8 Hz), 6.69 (1H, d, J = 15.6 Hz), 7.39 (1H, d,
J = 15.6 Hz), 7.56 (1H, dd, J = 8.8, 2.0 Hz), 7.67 (1H, d, J = 8.3 Hz),
7.84 (1H, d, J = 2.0 Hz), 8.10 (1H, t, J = 5.4 Hz). FABMS m/z: 258
5.1.7. (2E,20E)-N,N0-Heptane-1,7-diylbis[3-(3,4-dichlorophenyl)-
acrylamide] (6e)
This compound was prepared in the same manner as 1 using
1,7-heptylenediamine instead of 1,4-butylenediamine (72% yield).

8632
K. Onda et al. / Bioorg. Med. Chem. 16 (2008) 8627–8634
1H NMR (400 MHz, DMSO-d6) d: 1.25–1.35 (6H, m), 1.35–1.50 (4H,
m), 3.17 (4H, q, J = 6.3 Hz), 6.68 (2H, d, J = 15.6 Hz), 7.38 (2H, d,
J = 15.6 Hz), 7.55 (2H, dd, J = 8.3, 2.0 Hz), 7.68 (2H, d, J = 8.3 Hz),
7.83 (2H, d, J = 2.0 Hz), 8.09 (2H, t, J = 5.9 Hz). FABMS m/z: 529
(M+1)+. Anal. Calcd for C25H26Cl4N2O2: C, 56.84; H, 4.96; N, 5.30.
Found: C, 56.62; H, 4.98; N, 5.38.
red at room temperature for 4 h. The resulting mixture was con-
centrated in vacuo and the residue was washed with AcOEt to
yield 8b (700 mg, 76%) as a colorless solid. 1H NMR (400 MHz,
DMSO-d6) d: 3.05–3.17 (4H, m), 3.51 (4H, q, J = 5.9 Hz)), 6.71 (2H,
d, J = 15.6 Hz), 7.43 (2H, d, J = 15.6 Hz), 7.55 (2H, dd, J = 8.3,
1.9 Hz), 7.66 (2H, d, J = 8.3 Hz), 7.83 (2H, d, J = 1.9 Hz), 8.54 (2H,
t, J = 5.9 Hz), 8.94 (2H, br s). FABMS m/z: 502 (M+1)+. As free base.
Anal. Calcd for C22H21Cl4N3O2ꢀHCl: C, 49.14; H, 4.12; N, 7.81.
Found: C, 48.99; H, 4.15; N, 7.64.
5.1.8. (2E,20E)-N,N0-Octane-1,8-diylbis[3-(3,4-dichlorophenyl)-
acrylamide] (6f)
This compound was prepared in the same manner as 1 using
1,8-octylenediamine instead of 1,4-butylenediamine (81% yield).
1H NMR (400 MHz, DMSO-d6) d: 1.25–1.32 (8H, m), 1.40–1.50
(4H, m), 3.16 (4H, q, J = 6.3 Hz), 6.68 (2H, d, J = 16.1 Hz), 7.38 (2H,
d, J = 15.6 Hz), 7.55 (2H, dd, J = 8.3, 1.9 Hz), 7.66 (2H, d,
J = 8.3 Hz), 7.83 (2H, d, J = 1.9 Hz), 8.09 (2H, t, J = 5.9 Hz). FABMS
m/z: 543 (M+1)+. Anal. Calcd for C25H26Cl4N2O2: C, 57.58; H, 5.20;
N, 5.17. Found: C, 57.47; H, 5.24; N, 5.17.
5.1.14. (2E,20E)-N,N0-(Oxydiethane-2,1-diyl)bis[3-(3,4-dichloro-
phenyl)acrylamide] (8c)
This compound was prepared in the same manner as 1 using
2,20-oxydiethanamine instead of 1,4-butylenediamine (87% yield).
1H NMR (400 MHz, DMSO-d6) d: 3.36 (4H, q, J = 5.9 Hz), 3.51 (4H,
t, J = 5.8 Hz), 6.74 (2H, d, J = 15.6 Hz), 7.39 (2H, d, J = 16.1 Hz),
7.53 (2H, dd, J = 8.3, 2.0 Hz), 7.63 (2H, d, J = 8.3 Hz), 7.81 (2H, d,
J = 1.5 Hz), 8.15 (2H, t, J = 5.4 Hz). FABMS m/z: 503 (M+1)+. Anal.
Calcd for C22H20Cl4N2O3ꢀ0.25H2O: C, 52.15; H, 4.08; N, 5.53. Found:
C, 52.08; H, 3.87; N, 5.64.
5.1.9. N,N0-Pentane-1,5-diylbis[3-(3,4-dichlorophenyl)-
propanamide] (7a)
This compound was prepared in the same manner as 1 using 3-
(3,4-dichlorophenyl)propionic acid instead of (2E)-3-(3,4-dichloro-
phenyl)acrylic acid (84% yield). 1H NMR (400 MHz, DMSO-d6) d:
1.05–1.14 (2H, m), 1.25–1.35 (4 H, m), 2.36 (4H, t, J = 7.4 Hz),
2.81 (4H, t, J = 7.4 Hz), 2.97 (4H, q, J = 6.8 Hz), 7.18 (2H, dd,
J = 8.3, 1.9 Hz), 7.44–7.53 (4H, m), 7.76 (2H, t, J = 5.4 Hz). FABMS
m/z: 505 (M+1)+. Anal. Calcd for C23H26Cl4N2O2: C, 54.78; H, 5.20;
N, 5.56. Found: C, 55.14; H, 5.22; N, 5.65.
5.1.15. (2E,20E)-N,N0-(Thiodiethane-2,1-diyl)bis[3-(3,4-dichloro-
phenyl)acrylamide] (8d)
This compound was prepared in the same manner as 1 using
2,20-thiodiethanamine instead of 1,4-butylenediamine (92% yield).
1H NMR (400 MHz, DMSO-d6) d: 2.67 (4H, t, J = 6.8 Hz), 3.38 (4H, q,
J = 6.4 Hz), 6.71 (2H, d, J = 15.6 Hz), 7.40 (2H, d, J = 15.6 Hz), 7.56
(2H, dd, J = 8.3, 2.0 Hz), 7.66 (2H, d, J = 8.3 Hz), 7.84 (2H, d,
J = 2.0 Hz), 8.28 (2H, t, J = 5.8 Hz). FABMS m/z: 519 (M+1)+. Anal.
Calcd for C22H20Cl4N2O2S: C, 50.98; H, 3.89; N, 5.41. Found: C,
50.77; H, 3.83; N, 5.69.
5.1.10. (2Z,20Z)-N,N0-Pentane-1,5-diylbis[3-(3,4-dichloro-
phenyl)acrylamide] (7b)
This compound was prepared in the same manner as 1 using
(2Z)-3-(3,4-dichlorophenyl)acrylic acid instead of (2E)-3-(3,4-
dichlorophenyl)acrylic acid (30% yield). 1H NMR (400 MHz,
DMSO-d6) d: 1.20–1.27 (2H, m), 1.40–1.45 (4H, m), 3.08 (4H, q,
J = 6.3 Hz), 6.09 (2H, d, J = 12.7 Hz), 6.65 (2H, d, J = 12.7 Hz), 7.56–
7.59 (4H, m), 8.00 (2 H, d, J = 1.5 Hz), 8.21 (2H, t, J = 5.8 Hz). FABMS
m/z: 501 (M+1)+. Anal. Calcd for C23H22Cl4N2O2: C, 55.22; H, 4.43;
N, 5.60. Found: C, 55.08; H, 4.36; N, 5.59.
5.1.16. (2E,20E)-N,N0-(Sulfinyldiethane-2,1-diyl)bis[3-(3,4-
dichlorophenyl)acrylamide] (8e)
3-Chloroperbenzoic acid (70%, 123 mg, 0.50 mmol) was added
to a stirred solution of 8d (259 mg, 0.50 mmol) in CH2Cl2 (50 mL)
at 0 °C, and this mixture was stirred at 0 °C for 50 min. The result-
ing solids were collected and washed with CH2Cl2 to yield 8e
(177 mg, 66%) as a colorless solid. 1H NMR (400 MHz, DMSO-d6)
d: 2.85–2.93 (2H, m), 3.00–3.06 (2H, m), 3.48–3.66 (4H, m), 6.68
(2H, d, J = 15.6 Hz), 7.41 (2H, d, J = 15.6 Hz), 7.55 (2H, dd, J = 8.3,
1.9 Hz), 7.66 (2H, d, J = 8.3 Hz), 7.84 (2H, d, J = 2.0 Hz), 8.43 (2H,
t, J = 5.9 Hz). FABMS m/z: 535 (M+1)+. Anal. Calcd for
5.1.11. N,N0-Pentane-1,5-diylbis[3-(3,4-dichlorophenyl)prop-2-
ynamide] (7c)
This compound was prepared in the same manner as 1 using 3-
(3,4-dichlorophenyl)prop-2-ynoic acid instead of (2E)-3-(3,4-
dichlorophenyl)acrylic acid (8.3% yield). 1H NMR (400 MHz,
DMSO-d6) d: 1.25–1.35 (2H, m), 1.40–1.55 (4H, m), 3.14 (4H, q,
J = 6.4 Hz), 7.56 (2H, dd, J = 6.4, 1.9 Hz), 7.72 (2H, d, J = 8.3 Hz),
7.87 (2H, d, J = 1.9 Hz), 8.83 (2H, t, J = 5.3 Hz). FABMS m/z: 497
(M+1)+. Anal. Calcd for C23H18Cl4N2O2: C, 55.67; H, 3.66; N, 5.65.
Found: C, 55.39; H, 3.71; N, 5.60.
C
22H20Cl4N2O3Sꢀ0.2H2O: C, 49.12; H, 3.82; N, 5.21. Found: C,
48.97; H, 3.61; N, 5.25.
5.1.17. (2E,20E)-N,N0-(Sulfonyldiethane-2,1-diyl)bis[3-(3,4-
dichlorophenyl)acrylamide] (8f)
3-Chloroperbenzoic acid (70%, 247 mg, 1.00 mmol) was added
to a stirred solution of 8d (259 mg, 0.50 mmol) in CH2Cl2 (50 mL)
at 0 °C, and this mixture was stirred at room temperature for 2 h.
The resulting solid was collected and washed with CH2Cl2. m-CPBA
(70%, 247 mg, 1.00 mmol) was added to a suspension of this solid
in CH2Cl2 (100 mL), and this mixture was stirred at room temper-
ature for 1 h. The resulting solids were collected and washed with
CH2Cl2 followed by heated EtOH/AcOEt to yield 8f (33 mg, 12%) as
a colorless solid. 1H NMR (400 MHz, DMSO-d6) d: 3.37 (4H, t,
J = 6.3 Hz), 3.60 (4H, t, J = 6.3 Hz), 6.68 (2H, d, J = 15.7 Hz), 7.39
(2H, d, J = 16.1 Hz), 7.53 (2H, dd, J = 8.3, 1.5 Hz), 7.65 (2H, d,
J = 8.3 Hz), 7.81 (2H, d, J = 1.5 Hz), 8.38 (2H, t, J = 5.9 Hz). FABMS
m/z: 551 (M+1)+. Anal. Calcd for C22H20Cl4N2O4S: C, 48.02; H,
3.66; N, 5.09. Found: C, 48.06; H, 3.76; N, 5.16.
5.1.12. tert-Butyl bis(2-{[(2E)-3-(3,4-dichlorophenyl)prop-2-
enoyl]amino}ethyl)carbamate (8a)
This compound was prepared in the same manner as 1 using
tert-butyl bis(2-aminoethyl)carbamate instead of 1,4-butylenedi-
amine (42% yield). 1H NMR (400 MHz, DMSO-d6) d: 1.37 (9H, s),
3.24–3.36 (8H, m), 6.68 (2H, d, J = 15.6 Hz), 7.40 (2H, d,
J = 15.6 Hz), 7.55 (2H, dd, J = 8.3, 2.0 Hz), 7.66 (2H, d, J = 8.3 Hz),
7.83 (2H, d, J = 2.0 Hz), 8.14–8.30 (2H, m). FABMS m/z: 602
(M+1)+. HRMS Calcd for C27H29Cl4N3O4 (M+1)+: 600.0985. Found:
600.0992.
5.1.13. (2E,20E)-N,N0-(Iminodiethane-2,1-diyl)bis[3-(3,4-
dichlorophenyl)acrylamide] hydrochloride (8b)
5.1.18. [(3,4-Dichlorophenyl)ethynyl](trimethyl)silane (10)
A HCl solution in AcOEt (4 M, 5 mL) was added to a solution of
8a (1.03 g, 1.71 mmol) in EtOH (20 mL), and this mixture was stir-
Bis(triphenylphosphine)palladium
0.684 mmol) and copper iodide (33 mg, 0.173 mmol) were added
dichloride
(480 mg,

K. Onda et al. / Bioorg. Med. Chem. 16 (2008) 8627–8634
8633
to a mixture of 1,2-dichloro-4-iodobenzene (9.34 g, 34.2 mmol)
and trimethylsilylacetylene (4.03 g, 41.0 mmol) in diethylamine
(100 mL), and this mixture was stirred at 45 °C for 2 h. The result-
ing mixture was concentrated in vacuo and the residue was parti-
tioned between ether (300 mL) and H2O (300 mL), and the ether
layer was washed with saturated NaCl solution, and then dried
and concentrated in vacuo. The residue was purified via column
chromatography on silica gel (n-hexane) to yield 10 (8.26 g, 99%)
as a slightly yellow oil. 1H NMR (300 MHz, CDCl3) d: 0.24 (9H, s),
7.27 (1H, dd, J = 8.3, 2.0 Hz), 7.37 (1 H, d, J = 8.3 Hz), 7.54 (1H, d,
J = 1.8 Hz). EIMS m/z: 242 (M)+.
NADP to reduced b-NADPH was measured at 340 nm for 2 h. The
hLGPa inhibition IC50 values were calculated using the logistic
regression method with SAS software.
5.3. Crystallization and data collection
The crystal of hLGP complexed with 6c was obtained at 4 °C
using the hanging drop vapor diffusion method. The hanging drop
consisted of an equal volume of the concentrated protein solution
(20 mg/mL) and a reservoir solution containing 100 mM NaMES
(pH 6), 22.5% MPD, 60 mM D-glucose, and 0.9 mM 6c. The crystal
belongs to the trigonal space group P31, with cell dimensions of
a = b = 124.1 Å and c = 123.6 Å. The diffraction data set was 99.8%
complete to 2.43 Å. The molecular replacement method was used
to solve the structure, using the published hLGP/CP-403700 com-
plex structure (PDB code 1EXV) as the starting model.10 The
CNX28 program was used for refinement to 2.45 Å resolution with
a final R-factor of 0.241 (Rfree: 0.279).
5.1.19. 1,2-Dichloro-4-ethynylbenzene (11)
1 M KOH (18 mL) was added to a solution of 10 (4.00 g,
16.4 mmol) in MeOH (40 mL), and this mixture was stirred at room
temperature for 3 h. The resulting mixture was concentrated in va-
cuo, and the residue was partitioned between n-hexane (150 mL)
and 0.5 M HCl (100 mL). The n-hexane layer was washed with sat-
urated NaCl solution, and then dried and concentrated in vacuo to
yield 11 (2.55 g, 91%) as a colorless solid. 1H NMR (300 MHz, CDCl3)
d: 3.14 (1H, s), 7.30 (1H, dd, J = 8.3, 1.8 Hz), 7.40 (1H, d, J = 8.3 Hz),
7.57 (1H, d, J = 1.8 Hz). EIMS m/z: 170 (M)+.
Acknowledgments
The authors thank Ms. Fumie Narazaki and Ms. Akiko Matsuy-
ama-Yokono for their support during the preparation of this man-
uscript, and the staff of the Division of Analytical Science
Laboratories for the elemental analysis and spectral measure-
ments. 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.
5.1.20. 3-(3,4-Dichlorophenyl)prop-2-ynoic acid (12)
n-BuLi (1.56 M solution in n-hexane, 17 mL, 26.5 mmol) was
added to a solution of 11 (3.95 g, 23.1 mmol) in THF (50 mL) at
0 °C in an argon atmosphere. This mixture was stirred at room
temperature for 1 h, then CO2 (solid, 5.00 g) was added at 0 °C.
The resulting mixture was stirred at room temperature for 1 h,
then concentrated in vacuo and the residue was partitioned be-
tween AcOEt (200 mL) and 0.5 M HCl (200 mL). The AcOEt layer
was washed with saturated NaCl solution, and then dried and con-
centrated in vacuo. The residue was washed with n-hexane to yield
12 (2.06 g, 41%) as a slightly yellow solid. 1H NMR (300 MHz,
DMSO-d6) d: 7.63 (1H, dd, J = 8.4, 2.0 Hz), 7.75 (1H, d, J = 8.4 Hz),
7.97 (1H, d, J = 1.8 Hz). FABMS m/z: 213 (Mꢁ1)+.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.08.010.
References and notes
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lM),
a
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l
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8634
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