Synthesis of 2-, 4- And 5-(2-alkylcarbamoyl-l-methylvinyl)-7- alkyloxybenzo[b]furans and their leukotriene 64 receptor antagonistic activity

Article abstract of DOI:10.1039/b503615a

Variable benzo[6]furan derivatives having (E)- and (Z)-2-alkylcarbamoyl-l- methylvinyl groups at the 2-, 4- and 5-positions and a carboxylpropoxy or (1-phenyl)ethoxy group at the 7-position were prepared to find novel and selective leukotriene B4 (LTB4) receptor antagonists. (E)-2-(2-Diethylcarbamoyl- l-methylvinyl)-7-(1-phenylethoxy)benzo[b]furan (4v) showed selective inhibition to the human BLT₂ receptor (hBLT₂). On the other hand, (E)-2-acetyl-4-(2-diethylcarbamoyl-l-methylvinyl)-7-(l-phenylethoxy)benzo[b] furan (7v) inhibited both human BLTi receptor (hBLT¹) and hBLT ₂. The (E)-2-(2-diethylcarbamoyl-l-methylvinyl) group lay on approximately the same plane as the benzo[e]furan ring, whereas the (E)-4-(2-diethylcarbamoyl-l-methylvinyl) group had the torsion angle (45.7°) from the benzo[e]furan ring plane. However, the (Z)-(2-alkylcarbamoyl-l- methylvinyl)benzo[b]furans were inactive. The inhibitory activity depended on the conformation of the 2-diethylcarbamoyl-l-methylvinyl group. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b503615a

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A R T I C L E
Synthesis of 2-, 4- and 5-(2-alkylcarbamoyl-1-methylvinyl)-
7-alkyloxybenzo[b]furans and their leukotriene B4 receptor
antagonistic activity†‡
Kumiko Ando,a Eriko Tsuji,a Yuko Ando,a Jun-ichi Kunitomo,a Reina Kobayashi,b
Takehiko Yokomizo,b,c Takao Shimizu,b Masayuki Yamashita,d Shunsaku Ohta,d
Takeshi Nabe,d Shigekatsu Kohnod and Yoshitaka Ohishi*a
a School of Pharmaceutical Sciences, Mukogawa Women’s University, 11-68 Koshien
Kyuban-cho, Nishinomiya 663-8179, Japan. E-mail: yohishi@mwu.mukogawa-u.ac.jp;
Fax: +798 41 2792; Tel: +798 45 9953
b Department of Biochemistry and Molecular Biology, Faculty of Medicine, The University of
Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo, 113-0033, Japan
c PRESTO, 7-3-1 Hongo Bunkyo-ku, Tokyo 113-0033, Japan
d Kyoto Pharmaceutical University, Misasagi, Yamashinaku, Kyoto 607-8412, Japan
Received 10th March 2005, Accepted 21st April 2005
First published as an Advance Article on the web 16th May 2005
Variable benzo[b]furan derivatives having (E)- and (Z)-2-alkylcarbamoyl-1-methylvinyl groups at the 2-, 4- and
5-positions and a carboxylpropoxy or (1-phenyl)ethoxy group at the 7-position were prepared to find novel and
selective leukotriene B4 (LTB4) receptor antagonists. (E)-2-(2-Diethylcarbamoyl-1-methylvinyl)-7-
(1-phenylethoxy)benzo[b]furan (4v) showed selective inhibition to the human BLT2 receptor (hBLT2). On the other
hand, (E)-2-acetyl-4-(2-diethylcarbamoyl-1-methylvinyl)-7-(1-phenylethoxy)benzo[b]furan (7v) inhibited both human
BLT1 receptor (hBLT1) and hBLT2. The (E)-2-(2-diethylcarbamoyl-1-methylvinyl) group lay on approximately the
same plane as the benzo[b]furan ring, whereas the (E)-4-(2-diethylcarbamoyl-1-methylvinyl) group had the torsion
angle (45.7◦) from the benzo[b]furan ring plane. However, the (Z)-(2-alkylcarbamoyl-1-methylvinyl)benzo[b]furans
were inactive. The inhibitory activity depended on the conformation of the 2-diethylcarbamoyl-1-methylvinyl group.
structure were previously reported to be finding antagonistic
Introduction
active compounds.10h This study supported partially our design
Leukotriene B4 is a dihydroxy fatty acid produced mainly by
macrophages and neutrophils. LTB4 plays important physiolog-
ical roles on leukocytes trafficking to the site of infection and
clearance of invaded microorganisms. However, overproduction
using a benzo[b]furan skeleton to find novel LTB4 antagonists.
We recently found the a,b-unsaturated carbamoyl group11 to
be an interesting bioactive functional group.12 This led us to
design and prepare benzo[b]furan derivatives with various 2-
of LTB4 is reported to cause inflammatory diseases including
alkylcarbamoyl-1-methylvinyl groups13 devised from the a,b-
bronchial asthma and inflammatory bowel diseases.2 Thus,
unsaturated carbamoyl group at C-2, C-4 and C-5. (Designed
compounds I, II, III in Fig. 2.)
We describe here the synthesis of the designed compounds
and their antagonistic potency and selectivity for hBLT1 and/or
much work has been done to prepare LTB4 antagonists for
clinical use as anti-inflammatory drugs. The structures of several
antagonistic active compounds are presented in Fig. 1.3
The particularly interesting antagonists are grouped into
hBLT2.
two classes, aliphatic carbon chain types (LY-255283, ONO-
4057, ZK-158252) and ether types (BIIL315, LY-29311). Un-
fortunately, no antagonist has yet been developed for clinical
medicinal applications. Recently, a novel cell surface receptor
Results and discussion
Chemistry
(BLT2) for LTB4 has been isolated and its molecular cloning has
also been established.4 Current studies on LTB4 antagonists and
its receptors (BLT1, BLT2) suggest the possibility of the develop-
ment of new clinical drugs for treating arteriosclerosis,5 immuno-
suppression of allograft rejection in organ transplantation,6
psoriasis,7 cancer,8 and rheumatoid arthritis.9 Stable com-
pounds with definite conformations should be favorable as
antagonists. Thus, simpler heteroaromatic skeleton compounds
should be better than aliphatic carbon chain type and ether
type compounds which may have plural conformations.10a–10g
Cyclization of three partial structures (conjugated triene, single
Bromination of 2-acetyl-7-hydroxybenzo[b]furan (1a)14 with
NBS in an equivalent amount and 2 equivalent amounts in
the presence of AlCl3 gave the 4-bromo compound (1b)15 and
the 4,6-dibromo compound (1c), respectively. Alkylation of
1 with various alkyl halides afforded the corresponding 7-
alkyloxybenzo[b]furans (2a–2q) (Table S1, ESI†). Reactions
of the 2-acetyl-7-alkyloxybenzo[b]furans (2a, 2c–2e, 2g–2j, 2n,
2p, 2q) with N-monoalkyl or N,N-dialkyl diethylphospho-
noacetamides (3a–3g)16 prepared in our laboratories in the
presence of NaH under Horner–Wadsworth–Emmons (HWE)
reaction conditions17 afforded the corresponding (E/Z)-2-(2-
alkylcarbamoyl-1-methylvinyl)-7-alkyloxybenzo[b]furans (4a–4z,
4a) (Scheme 1).
=
C C bond and OH group) originating from possible LTB4
stable conformers (A, B) formed a benzo[b]furan ring with
a O(CH2)3COOH group. Cyclized aliphatic compounds as
LTB4 antagonists designed on the basis of the LTB4 molecular
Most of the (E/Z)-mixtures (4) could be separated into E-
and Z-isomers (Table S2, ESI†). The E-isomers (4) showed
only a nuclear Overhauser enhancement (NOE) correlation
between CH3 on the vinyl group (olefinic CH3) and 3-H, and
the olefinic CH3 of Z-isomers (4) showed NOE correlations
† Electronic supplementary information (ESI) available: Tables S1–S6.
See http://www.rsc.org/suppdata/ob/b5/b503615a/
‡ See ref. 1
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 1 2 9 – 2 1 3 9
2 1 2 9
©

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Fig. 1 Structures of reported LTB4 receptor antagonists.
Fig. 2 Designed benzo[b]furan compounds (I, II, III) and possible conformers (A, B) of LTB4.
1
with both 3-H and olefinic H in their H-NMR. These NOE
data showed that the carbon–carbon double bond of both the
(E)- and (Z)-2-(2-alkylcarbamoyl-1-methylvinyl) group had an
s-trans configuration with a double bond between C-2 and
C-3. The structures of (E)-4a and (Z)-4a, as representative
compounds, and their NOE correlations are shown in Fig. 3.
It was interesting that (Z)-isomers (4) were easily converted
into a mixture (1 : 1) of the (E)-isomer and the (Z)-isomer
at room temperature when exposed to light. Estimation of the
torsion angle between the (E)- and (Z)-2-(2-alkylcarbamoyl-
1-methylvinyl) groups, and the benzo[b]furan ring plane was
found to be 0–4◦ for the (E)-isomers and 25–27◦ for the (Z)-
isomers.18 The stereostructure of (E)-4a was confirmed by X-
ray analysis (Fig. 4).19 The torsion angle (6.9◦) found by X-ray
2 1 3 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 1 2 9 – 2 1 3 9

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Scheme 1
4m) more selectively (E selectivity: 67–100%) (Table S3, ESI†).21
The reactions of several 2-acetylbenzo[b]furans (2a, 2c, 2d, 2e,
2g, 2j, 2m, 2p) with ethyl diethylphosphonoacetate22 instead of
N-monoalkyl or N,N-dialkyl diethylphosphonoacetamides (3)
in the presence of NaH or LiCl–DBU under HWE reaction
conditions also resulted predominantly in the formation of E-
isomers (5a–5h) (Scheme 1) (Tables S3 and S4, ESI†).
2-Acetyl-7-alkyloxy-4-bromobenzo[b]furans (2j–2m, 2o) were
treated with N-alkylcrotonamides (6a–6e) prepared in our
laboratories in the presence of palladium acetate, tri-o-tolylphos-
phine and triethylamine under Heck coupling conditions
selectively affording only (E)-2-acetyl-4-(2-alkylcarbamoyl-1-
methylvinyl)-7-alkyloxybenzo[b]furans (7a–7o).23 The 2-acetyl
compound (7a) was derived to similar cinnamic acid compounds
(8b) via ester (8a) (Scheme 2) (Table S5, ESI†).
Fig. 3 Structures of (E)- and (Z)-4a, and NOE correlations.
analysis was close to the estimated value (4.4◦). Thus, the 2-(2-
alkylcarbamoyl-1-methylvinyl) group of (E)-isomer (4) lay on
nearly the same plane as the benzo[b]furan ring.
E-isomers (4) were obtained preferentially under HWE re-
action conditions using NaH (E selectivity: 51–100%) except
for several sulfonylamide compounds (4q–4s) (E selectivity: 11–
34%).17a,20 LiCl and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)
used in the place of NaH gave (E)-isomers (4a, 4c, 4e, 4i–
Unlike (E)-4, the olefinic CH3 and H of (E)-7 showed
particular NOE correlations with both 3-H and 5-H, respec-
tively (Fig. 5). Some examination of the conformation of the
4-(2-alkylcarbamoyl-1-methylvinyl) group using MM218 and
¨
Dreiding Stereomodels (BUCHI) revealed the great difficulty
of free rotation of the vinyl carbon and C-4 because of
Fig. 4 X-Ray structures of (E)-4a and 7i.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 1 2 9 – 2 1 3 9
2 1 3 1

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Scheme 2
Fig. 5 NOE correlation and structure of 7i, and structure of molecular dissymmetrical b-chloro-b-(2,4,6-trimethyl-3-bromophenyl)-a-methylacrylic
acid (IV) and o-(b, b-dimethyl-a-isopropylvinyl)phenyltrimethylammonium iodide (V).
steric repulsion between the 4-(2-alkylcarbamoyl-1-methylvinyl)
group and both 3-H and 5-H. Similar olefinic molecu-
lar dissymmetrical compounds, b-chloro-b-(2,4,6-trimethyl-3-
bromophenyl)-a-methylacrylic acid (IV)24 and o-(b,b-dimethyl-
a-isopropylvinyl)phenyltrimethylammonium iodide (V)25 were
resolved readily through the appropriate salt because of the
restriction of free rotation (Fig. 5).
approximately the same plane as the benzo[b]furan ring, while
the (E)-4-(2-alkylcarbamoyl-1-methylvinyl) group of 7 was
favorably located at 45◦ from the benzo[b]furan ring plane.
5-Bromobenzo[b]furans (11a, 11b, 11c) were prepared from
1012 and 9.28 Heck coupling of 11 using N-alkylcrotonamides
(6a, 6b, 6c) selectively afforded (E)-5-(2-alkylcarbamoyl-1-
methylvinyl)benzo[b]furans (12a–12d, 12g, 12h). The 2-acetyl
compound (12c) was subjected to the HWE reaction to give 12e,
which was converted into 12f by hydrolysis (Scheme 3) (Table
S6, ESI†). In the 1H-NMR spectrum of (E)-12, olefinic CH3 and
H showed NOE correlations with both 4-H and 6-H, respec-
tively. The torsion angle between the (E)-5-(2-alkylcarbamoyl-
1-methylvinyl group) and the benzo[b]furan ring of 12 was
estimated as being 31.3–38.9◦ by MM2.
Our previous X-ray analysis of 2-(4-cyanobenzoyl)-3-(Z)-
(2-cyano-3-hydroxybuto-2-enonyl)amino-5-(E)-(2-diethylcar-
bamoyl-1- methylvinyl)benzo[b]furan (torsion angle: 33.5◦)12b
supported these estimated values. These results suggested that
the (E)-5-(2-alkylcarbamoyl-1-methylvinyl) group was less
sterically hindered than that at C-4.
Thus, several 7-(1-phenylethoxy) derivatives (7c–7g) having
an asymmetric carbon in the substituent group at C-7 were
prepared to examine molecular dissymmetry due to restriction
of free rotation between the vinyl carbon and C-4. However, no
diastereoisomers of compounds (7c–7g) have been discovered
1
through their H-NMR. This suggested that restriction of the
rotation was not enough to increase the molecular dissymmetry
of 7. It was presumably due to insufficient overlap of the 4-
(2-alkylcarbamoyl-1-methylvinyl) group with both 3-H and 5-
H.26 The stereostructure of representative compound 7i was
determined by X-ray analysis as shown in Fig. 4.19 The
torsion angle between the 4-(2-diethylcarbamoyl-1-methylvinyl)
group and the benzo[b]furan ring of 7i was 45.7◦. The tor-
sion angle of several (E)-4-(2-alkylcarbamoyl-1-methylvinyl)-7-
alkyloxybenzo[b]furans (7) was estimated as being 38.8–47.9◦
by MM2.18,27 The conformation of the 4-(2-alkylcarbamoyl-1-
methylvinyl) group considered from the torsion angle could
account for the NOE relations of (E)-4-(2-alkylcarbamoyl-1-
methylvinyl) compounds (7). These results demonstrated that
the (E)-2-(2-alkylcarbamoyl-1-methylvinyl) group of 4 lay on
Bioactivity
Most of the compounds prepared were first evaluated for
their LTB4 antagonistic activity by Method A29 examining the
inhibition of LTB4-induced TXB2 release from bronchoalveolar
eosinophils of guinea pigs. Fourteen compounds (4bꢀ, 4c, 4d,
2 1 3 2
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 1 2 9 – 2 1 3 9

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Scheme 3
Table 1 LTB4 antagonistic activity (ratio of amount of TXB2 released
4c, 4f) and (E)-4-(2-alkylcarbamoyl-1-methylvinyl) compound
(7c) were found to be active at these concentrations. The
concentration-dependent activity for these four compounds
(4bꢀ, 4c, 4f, 7c) was evaluated at concentrations of 0.1 nM,
1 nM, 10 nM, 100 nM and 1 lM. The most active compound
4c completely inhibited LTB4-dependent TXB2 release at
1 lM, and showed 96.2% inhibition at 100 nM and 26.8%
inhibition at 10 nM. Other compounds, 4bꢀ, 4f and 7c, showed
almost complete inhibition at 1 lM, but were less potent than
4c at 100 nM (Fig. 6). These studies showed that (E)-2-(2-
alkylcarbamoyl-1-methylvinyl) compounds (4) and (E)-4-(2-
alkylcarbamoyl-1-methylvinyl) compound (7) were potent and
(E)-5-(2-alkylcarbamoyl-1-methylvinyl) compounds (12) were
inactive.30
Subsequently, a detailed study was achieved by evaluation
of the inhibition activity for hBLT1 and/or hBLT2 (Method B,
inhibition of calcium mobilization in both CHO cells overex-
pressing human BLT1 (CHO–hBLT1) and human BLT2 (CHO–
hBLT2).4e,31 At first, (E)-2-(2-alkylcarbamoyl-1-methylvinyl)
compounds (4a, 4bꢀ, 4c, 4d, 4dꢀ, 4e, 4f, 4g, 4nꢀ, 4u, 4v, 4w, 4y,
4z), (Z)-2-(2-alkylcarbamoy-1-methylvinyl) compounds ((Z)-
4a, (Z)-4v), (E)-4-(2-alkylcarbamoyl-1-methylvinyl) compounds
(7a, 7c, 7d, 7g, 7i, 7j, 7n, 8b) and (E)-5-(2-alkylcarbamoyl-1-
methylvinyl) compound (12c) were evaluated at a concentration
of 10 lM, and ten (E)-compounds (4a, 4bꢀ, 4d, 4dꢀ, 4e, 4f,
4g, 4v, 4y, 7c) showed more than 70% inhibition of calcium
mobilization in CHO–hBLT1 and/or CHO–hBLT2 cells. How-
ever, (Z)-2-(2-alkylcarbamoyl-1-methylvinyl) compounds ((Z)-
4a, (Z)-4v) were inactive to both hBLT1 and hBLT2. Nine (E)-
2-(2-alkylcarbamoyl-1-methylvinyl) compounds (4a, 4bꢀ, 4d, 4dꢀ,
4e, 4f, 4g, 4v, 4y) were more potent for hBLT2 than hBLT1. On the
other hand, the (E)-4-(2-alkylcarbamoyl-1-methylvinyl) com-
pound (7c) inhibited both hBLT1 and hBLT2. A combination
of the (E)-2- or (E)-4-(2-alkylcarbamoyl-1-methylvinyl) group
having a diethyl or morpholino on the nitrogen atom, and 1-
phenylethoxy or 3-carboxypropoxy group at the 7-position was
favorable for increasing the inhibitory activity. The potent com-
pounds (4a, 4bꢀ, 4d, 4f, 4v, 4y, 7c) were also evaluated for their an-
tagonistic activity on cysteinyl leukotriene receptors and found
to be inactive.32 Unfortunately, the (E)-5-(2-alkylcarbamoyl-1-
methylvinyl) compound (12c) was also found to be inactive by
Method B, similar to the results of Method A (Table 2).
against control (%))
Concentration
Compounda
100 lM
1 lMb
10 nMb
4bꢀ
4c
0.2
0.1
2.7
45.8
0.5
0.2
0.3
0.2
40.9
—
0.3
56.6
84.5
—
—
—
—
—
—
—
—
64.7
44.9
60.7
—
—
—
69.1
—
3.7
—
—
—
65.9
—
16.2
—
—
—
—
—
77.8
—
—
47.9
77.8
57.1
—
48.6
90.1
94.1
—
—
—
—
—
—
—
—
87.2
96.3
93.1
—
—
—
97.4
—
89.1
—
—
—
96.3
—
83.6
—
—
—
—
—
97.4
—
—
4d
4e
4f
4g
(Z)-4g
4h
0.3
16.0
91.8
118.6
115.4
115.4
127.3
112.4
14.1
1.4
0.07
n. d.c
54.0
39.0
60.7
0.2
45.0
0.8
41.9
23.7
81.7
1.0
4i
4j
4k
(Z)-4k
4l
4m
(Z)-4m
4n
4nꢀ
4o
(Z)-4q
4r
(Z)-4r
4u
7a
7c
7d
7e
7f
7g
7h
32.1
n. d.c
10.2
105.2
122.0
39.1
101.3
6.1
7i
7j
7k
7l
7m
8a
8b
12a
12b
56.7
113.9
a Compounds without indication of (Z) are (E)-isomers. b Fourteen
potent compounds were further evaluated at concentrations of 1 lM
and 10 nM. c n. d.: no TXB2 release was detected.
(E)-2-(2-Alkylcarbamoyl-1-methylvinyl) compounds (4a, 4bꢀ,
4d, 4f, 4v, 4y) and (E)-4-(2-alkylcarbamoyl-1-methylvinyl) com-
pound (7c) selected based on the results of the above screening
were evaluated at concentrations of 1 nM, 10 nM, 100 nM,
1 lM and 10 lM. All of the compounds evaluated exhibited
dose-dependent inhibition of hBLT1 and/or hBLT2. The IC50
values of these compounds are shown in Table 3.
4f, 4g, (Z)-4g, 4n, 4nꢀ, 4o, 4u, 7c, 7g, 7i, 8b) at a concentration
of 100 lM completely inhibited LTB4 (100 nM)-induced TXB2
release from the bronchoalveolar eosinophiles (Table 1). Next,
these active compounds were evaluated at 1 lM and 10 nM and
the (E)-2-(2-alkylcarbamoyl-1-methylvinyl) compounds (4bꢀ,
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 1 2 9 – 2 1 3 9
2 1 3 3

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Fig. 6 Effect of (E)-4c, (E)-4bꢀ, (E)-4f, 7c on LTB4-induced TXB2 release from bronchoalveolar eosinophils harvested from Sephadex G-200-treated
guinea pigs (mean S. E., n = 3). (E)-4c, (E)-4bꢀ, (E)-4f, 7c were added 5 min before eosinophil stimulation by LTB4 (100 nM). Statistically significant
differences from the control are indicated (*P < 0.05, ***P < 0.001, Bonferroni’s multiple test). Spon: spontaneous, Cont: control, a) inhibition (%).
Table 2 Inhibition of calcium mobilization in CHO–hBLT1 and CHO–
hBLT2 cells at 300 nM LTB4
Table 3 IC50 for CHO-hBLT1 and CHO-hBLT2
IC50/lM
Inhibition (%)
Compound
CHO-hBLT1
CHO-hBLT2
Compounde (10 lM)
CHO-hBLT1
CHO-hBLT2
(E)-4aa
—
—
4.84
1.78
7.97
0.68
1.18
6.41
0.83
0.48
0.031
4a
21.9
35.8
1.7
41.3
16.5
22.1
24.1
9.0
7.4
7.0
69.9
n. i.c
53.0
8.6
88.1
71.7
6.6
89.3
71.8
83.9
72.1
70.2
4.1
(E)-4da
4bꢀa
(E)-4ya
>10−5
2.88
1.70
>10−5
>10−5
0.42
0.054
4ca
(E)-4va
4d
ZK158252a
4dꢀ
(E)-4bꢀ
b
4e
(E)-4fb
7cb
4fa
4g
ZK158252b
4nꢀ
a Stimulated by LTB4 at 300 nM. b Stimulated by LTB4 at 100 nM.
4u
4v
4w
4y
4z
34.6
com.b
5.0
84.5
53.6
34.4
10.8d
33.3
92.8
29.7
21.0
9.3
53.8
33.3
4.4
4.8
92.7
61.3
To prove that inhibition of calcium mobilization was not
due to a simple cytotoxic effect to the CHO cells, the effects
of these compounds on ATP-dependent calcium mobilization
were examined. Weak inhibition of calcium mobilization was
observed only at a concentration of 10 lM, with no inhibition
being detected at 1 lM.33,34
(Z)-4a
9.2
(Z)-4v
8.4d
8.1
7a
7ca
92.6
10.8
8.4
12.6
20.9
19.2
5.7
8.0
92.3
56.6
7d
7g
7i
7j
7n
8b
Conclusions
This study revealed a significant relationship between stere-
ostructure and hBLT1 and/or hBLT2 inhibitory activity. (E)-
2-(2-Alkylcarbamoyl-1-methylvinyl) compounds (4a, 4bꢀ, 4d, 4f,
4v, 4y) showed selective hBLT2 inhibition, and their (E)-2-(2-
alkylcarbamoyl-1-methylvinyl) groups lay on nearly the same
plane as the benzo[b]furan ring. The (E)-4-(2-alkylcarbamoyl-1-
methylvinyl) compound (7c) inhibited both hBLT1 and hBLT2,
and its (E)-4-(2-alkylcarbamoyl-1-methylvinyl) group had the
torsion angle (ca. 45◦) from the benzo[b]furan ring plane. Both 4v
and 7c had the same (E)-2-(2-diethylcarbamoyl-1-methylvinyl)
group at C-2 or C-4, respectively, and the 1-phenylethoxy group
at C-7. Thus, the difference of conformation of the (E)-(2-
diethylcarbamoyl-1-methylvinyl) group may significantly affect
the selectivity of the antagonistic activity to hBLT1 and/or
hBLT2. To examine the relationship between the conformation
of the (E)-(2-alkylcarbamoyl-1-methylvinyl) group and antag-
onistic activity, some novel benzo[b]furan derivatives having
12c
ZK158252a
ZK158252
a Concentration of LTB4: 100 nM. b Calcium mobilization was com-
pletely inhibited. c Not inhibited. d Concentration of (Z)-4v: 5 lM.
e Compounds without indication of (Z) are (E)-isomers.
All (E)-2-(2-alkylcarbamoyl-1-methylvinyl) compounds (4a,
4bꢀ, 4d, 4f, 4v, 4y) showed BLT2 selective inhibition.
The most potent (E)-2-(2-diethylcarbamoyl-1-methylvinyl)-7-
(1-phenylethoxy)benzo[b]furan (4v) inhibited hBLT2 more than
hBLT1. The inhibition for hBLT2 was superior to ZK-158252 but
the inhibition for hBLT1 was below ZK-158252. Interestingly,
(E)-4-(2-alkylcarbamoyl-1-methylvinyl) compound (7c) showed
high activity for both hBLT1 and hBLT2 (Table 3 and Fig. 7).
2 1 3 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 1 2 9 – 2 1 3 9

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filtration and washed with water, and recrystallized from ethyl
acetate to give 1b (9.8 g, 93.3%) as yellow needles: mp 224.3–
226.6 ◦C; dH (500 MHz; DMSO; Me3Si) 2.60 (3H, s, COCH3),
6.91 (1H, d, J 8.6, 6-H), 7.35 (1H, d, J 8.6, 5-H), 7.74 (1H, s,
3-H), 10.7 (1H, br s, OH); m/z (EI) 253.9585 (M+. C10H7BrO3
requires 253.9579), 254 (M+, 100.00%), 239 (89.88), 211 (17.03),
183 (27.65).
2-Acetyl-4,6-dibromo-7-hydroxybenzo[b]furan (1c)
To a solution of 1 (22.0 g, 0.125 mol) in CH2Cl2 (1200 ml) was
added AlCl3 (66.7 g, 0.5 mol) and the mixture was stirred for 1 h
at 40 ◦C. Br2 (40.0 g, 0.25 mol) was added dropwise over 45 min
to the reaction mixture at 26 ◦C. The mixture was stirred for
16 h at 26 ◦C, and poured into ice water, and treated with 6 N
HCl solution (500 ml). The resulting precipitate was collected by
filtration and washed with water, then recrystallized from ethyl
acetate to give 1c (33.9 g, 81.8%) as colorless needles: mp 214.6–
216.5 ◦C; dH (500 MHz; DMSO; Me3Si) 2.60 (3H, s, COCH3),
7.65 (1H, s, 5-H), 7.75 (1H, s, 3-H); m/z (EI) 331.8682 (M+.
C10H6Br2O3 requires 331.8684), 334 (100.00%), 332 (M+, 60.78),
319 (89.98), 291 (10.79), 263 (21.07), 211 (17.03), 183 (27.65)
2-Acetyl-7-ethoxycarbonylmethoxybenzo[b]furan (2b)
General procedure for 2a, 2c–2f, 2q, 2j–2o, 2p. A mixture of
1a (5.7 g, 32.4 mmol), K2CO3 (13.0 g, 93.9 mmol), and ethyl
bromoacetate (3.8 ml, 34.1 mmol) in dry acetone (120 ml) was
stirred at 54 ◦C for 2 h. After an insoluble portion was filtered off,
the filtrate was distilled under reduced pressure. The ethyl acetate
solution of the resulting residue was washed with 5% NaOH
solution and brine, and dried. Concentration of the solution
gave 2b (5.1 g, 60.1%) as pale yellow needles.
In a similar manner to that described above, 1a gave 2a, 2c–2f,
2q, 1b gave 2j–2o, and 1c gave 2p.
2-Acetyl-7-(ethoxycarbonylmethoxy)-4-
ethylsulfamoylbenzo[b]furan (2g)
General procedure for 2h, 2i. The fine powder 2b (5.0 g,
0.019 mol) was carefully added to chlorosulfonic acid (10.1 ml,
0.152 mol) in portions at 26–30 ◦C over 3 h under vigorous
stirring. After the mixture was stirred at the same temperature
for 5 min, it was poured into ice water and extracted with ethyl
acetate. The organic layer was washed with brine, then dried,
and evaporated quickly to give the intermediate sulfonyl chloride
as a yellow solid, Rf = 0.74, benzene–methanol (2 : 1), which
was suitable for use in subsequent reactions. To a mixture of
ethylamine hydrochloride (2.0 g, 0.082 mol) and triethylamine
(4.5 g, 0.044 mol) in CH2Cl2 (30 ml) was added dropwise the
sulfonyl chloride in dry CHCl3 (30 ml) at 27–30 ◦C with stirring.
After the mixture was stirred at 25 ◦C for 1 h, it was poured
into ice water and made acidic with 5% HCl solution, then
extracted with ethyl acetate. The extract was washed with brine,
then dried. The solvent was evaporated off, giving a residue
which was recrystallized from ethyl acetate to give 2g (1.9 g,
27.0%) as yellow prisms.
Fig. 7 Effect of (E)-4v, 7c, ZK158252 on calcium mobilization by LTB4
(300 nM) in CHO–hBLT1 and CHO–hBLT2 cells (mean S. D., n =
3,4). a) Stimulated by LTB4 at 100 nM. **p <0.001 (two-way ANOVA).
more characteristic conformations are being prepared and will
be evaluated for their antagonistic activity.
Experimental
All melting points were determined using a Yanako microscopic
hot-stage apparatus and are uncorrected. 1H-NMR, 13C-NMR,
HMBC and HMQC spectra were obtained on a JEOL GSX-500
and a JNM-PMX 60 spectrometer with tetramethylsilane as an
internal standard. MS spectra (MS, HRMS) were obtained using
a JEOL JMS DX-303 EIMS spectrometer. Elemental analyses
were performed on a CHN CORDER MT-3 (Yanako). All
organic extracts were dried over anhydrous MgSO4. Column
chromatography was carried out on Wakogel C-200 (100–200).
Thin layer chromatography was performed on an E. Merck silica
gel plate (0.5 mm, 60F-254).
In a similar manner to that described above, 2c gave 2h, and
2f gave 2i.
N, N-Diethyl diethylphosphonoacetamide (3a)
A mixture of N,N-diethylchloroacetamide (6.9 ml, 50.0 mmol)
and triethyl phosphite (8.8 ml, 50.0 mmol) was stirred at 180 ◦C
for 8 h. The reaction mixture was cooled to room temperature
and distilled to give 3a (8.8 g, 70.0%) as a colorless oil: bp3
136–140 ◦C; dH (60 MHz; CDCl3; Me3Si) 0.90–1.40 (12H, m,
CH2CH3 × 4), 3.00 (2H, d, J 20.2, PCH2), 3.10–3.60 (4H, m,
NCH2CH3 × 2), 3.92–4.50 (4H, m, OCH2CH3 × 2); m/z (EI)
251 (M+, 24.24%), 179 (21.28), 79 (100.00).
2-Acetyl-4-bromo-7-hydroxybenzo[b]furan (1b)
To a solution of 1 (7.3 g, 0.041 mol) in CH2Cl2 (300 ml) was
added AlCl3 (21.9 g, 0.16 mol) and the mixture was stirred for
◦
1 h at 40 C. Br2 (8.0 g, 0.05 mol) was added dropwise to the
reaction mixture over 1 h at 26 ◦C. The mixture was stirred for
◦
3 h at 26 C, poured into ice water, and treated with 6 N HCl
solution (300 ml). The resulting precipitate was collected by
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 1 2 9 – 2 1 3 9
2 1 3 5

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Diethyl [2-(3-methoxyphenylamino)-2-oxoethyl]phosphonate (3b)
added dropwise a solution of N,N-diethyl phosphonoacetamide
(1.6 g, 6.2 mmol) in anhydrous THF (10 ml) under an N2
atmosphere at −3 ◦C with stirring. The solution was then stirred
at 25 ◦C until it became clear. A solution of 2c (1.5 g, 5.2 mmol)
in anhydrous THF (10 ml) was added dropwise to the clear
solution at 25 ◦C, and the mixture stirred at 25 ◦C for 4 h.
The mixture was then quenched with H2O, and evaporated. The
residue was added to saturated NH4Cl solution and extracted
with ethyl acetate. The organic layer was washed with brine, then
dried. The solvent was evaporated off, giving a residue which
was purified with silica gel column chromatography [CHCl3–
ethyl acetate (5 : 2)] to give E-4a (0.62 g, 30.8%) as a colorless
oil, and Z-4a (0.16 g, 8.0%) as a colorless oil.
General procedure for 3c–3g. To a solution of m-anisidine
(15.0 g, 0.12 mol) in dry THF (200 ml) was added dropwise
chloroacetyl chloride (10.0 ml, 0.13 mol) in dry THF (20 ml)
over 15 min at −5–0 ◦C. The reaction mixture was stirred at
0–10 ◦C for 3 h, poured into ice water, and extracted with ethyl
acetate. The organic layer was washed with brine and dried.
Concentration of the extract gave a brown solid. The solid was
recrystallized from ethyl acetate to give a white solid (21.2 g,
92.0%, mp 83.0–85.0 ◦C). The solid and triethyl phosphite
(18.8 ml, 0.12 mol) were stirred at 130 ◦C for 15 h. The reaction
mixture was cooled to room temperature, and the resulting
precipitate was recrystallized from ethyl acetate to give3b (21.9 g,
65.8%) as colorless prisms: mp 86.5–88.8 ◦C; dH (500 MHz;
CDCl3; Me3Si) 1.36 (6H, t, J 7.3, CH2CH3 × 2), 2.99 (2H, d, J
20.6, PCH2), 3.79 (3H, s, OCH3), 4.14–4.21 (4H, m, CH2CH3 ×
2), 6.65–7.26 (4H, m, 2-, 4-, 5-, 6-H); m/z (EI) 301 (M+, 82.37%),
123 (100.00).
In a similar manner to that described above, 2c gave 4b–4f,
4u, 2e gave 4g–4m, 2j gave 4n, 2n gave 4o, 2g gave 4p, 2h gave 4q,
2i gave 4r, 4s, 2p gave 4t, 2q gave 4v–4z, and 4a gave 4a.
The acids 4bꢀ, 4dꢀ, 4nꢀ, 4tꢀ were obtained according to the usual
manner from 4b, 4d, 4n, 4t, respectively.
(E/Z)-2-(2-Ethoxycarbonyl-1-methylvinyl)-7-(3-
ethoxycarbopropoxy)benzo[b]furan (5b)
Diethyl [2-(4-methoxyphenylamino)-2-oxoethyl]phosphonate (3c)
Yield: 4.1 g (33.0%), white needles; mp 79.5–82.5 ◦C; dH
(500 MHz; CDCl3; Me3Si) 1.35 (3H, t, J 6.9, CH2CH3), 1.35
(3H, t, J 6.9, CH2CH3), 2.98 (2H, d, J 20.6, PCH2), 3.78 (3H, s,
OCH3), 4.18 (4H, m, CH2CH3 × 2), 6.84 (2H, d, J 8.9, 2-, 6-H
or 3-, 5-H), 7.43 (2H, d, J 8.9, 2-, 6-H or 3-, 5-H), 8.68 (1H, br s,
NH); m/z (EI) 301 (M+, 48.00%), 152 (18.67), 123 (100.00), 108
(36.59).
General procedure for 5a, 5c–5h. To a suspension of NaH
(60% in oil, 0.08 g, 3.5 mmol) in anhydrous THF (3 ml) was
added dropwise a solution of ethyl diethylphosphonoacetamide
(0.77 g, 3.5 mmol) in anhydrous THF (10 ml) under an N2
atmosphere at 5 ◦C with stirring. The solution was then stirred
at 25 ◦C until it became clear. A solution of 2d (0.2 g, 0.69 mmol)
in anhydrous THF (4 ml) was added dropwise at 25 ◦C, and
◦
the mixture stirred at 25 C for 30 min. The mixture was then
Diethyl [2-(3,4-dimethoxyphenylamino)-2-oxoethyl]phosphonate
(3d)
quenched with saturated NH4Cl solution and extracted with
ethyl acetate. The organic layer was washed with brine, then
dried. The solvent was evaporated off, giving a residue which
was purified with silica gel column chromatography [hexane–
ethyl acetate (10 : 1)] to give a mixture of E-5b and Z-5b (0.13 g,
52.0%) as a colorless oil.
In a similar manner to that described above, 2a gave 5a, 2d
gave 5c, 2e gave 5d, 2j gave 5e, 2m gave 5f, 2g gave 5g, and 2p
gave 5h.
Yield: 4.8 g (60.0%), pale yellow needles; mp 117.8–119.9 ◦C;
dH (500 MHz; CDCl3; Me3Si) 1.30 (6H, t, J 7.0, CH2CH3 × 2),
3.0 (2H, d, J 24.0, PCH2), 3.80 (6H, s, OCH3 × 2), 4.10 (2H, q,
CH2CH3), 4.25 (2H, q, CH2CH3), 6.75 (1H, d, J 8.0, 5-H), 6.98
(1H, d, J 2.0, 2-H), 7.25 (1H, dd, J 8.0 and 2.0, 6-H), 9.1 (1H,
br s, NH); m/z (EI) 331 (M+, 100.00%), 179 (4.42), 153 (56.15),
138 (39.55), 125 (26.99).
Diethyl [2-(2-(3,4-dimethoxyphenyl)ethylamino)-
2-oxoethyl]phosphonate (3e)
(E/Z)-2-(Diethylcarbamoyl-1-methylvinyl)-7-
diphenylmethoxybenzo[b]furan (4h) (procedure using LiCl–DBU
instead of NaH)
Yield: 0.7 g (96.9%), white needles; mp 84.2–85.9 ◦C; dH
(500 MHz; CDCl3; Me3Si) 0.95–1.43 (6H, m, CH2CH3 × 2), 2.82
(2H, d, J 20.0, PCH2), 2.60–3.00 (2H, m, NCH2CH2), 3.35–3.70
(2H, m, NCH2CH2), 3.80 (3H, s, OCH3), 3.93 (3H, s, OCH3),
3.90–4.35 (4H, m, CH2CH3 × 2), 6.80–6.90 (3H, m, arom-H);
m/z (EI) 359 (M+, 12.37%), 164 (100.00), 151 (15.04).
General procedure for 4a, 4c, 4e, 4i-4m. To a solution of
LiCl (0.19 g, 4.5 mmol) in acetonitrile (10 ml) was added
dropwise N,N-diethyl phosphonoacetamide (0.47 g, 1.9 mmol)
in acetonitrile (5 ml) at 25 ◦C. After the solution was stirred for
5 min, DBU (0.34 g, 2.3 mmol) and 2e (0.32 g, 0.94 mmol) in
acetonitrile (5 ml) were added to the solution. The mixture was
heated at 83 ◦C for 5.5 h, and the solvent was evaporated. The
residue was added to saturated NH4Cl solution and extracted
with ethyl acetate. The organic layer was washed with brine, then
dried. The solvent was evaporated off, giving a residue which was
purified with silica gel column chromatography [hexane–ethyl
acetate (20 : 1)] to give E-4h (0.12 g, 29.3%) and Z-4h (0.09 g,
22.0%) as a colorless oil.
Diethyl [2-(morpholino)-2-oxoethyl]phosphonate (3f)
Yield: 3.8 g (87.8%), yellow oil; dH (500 MHz; CDCl3; Me3Si)
1.32 (6H, m, CH2CH3 × 2), 2.95 (2H, d, J 22.0, PCH2), 3.50 (8H,
br s, NCH2CH2O × 2), 3.80–4.45 (4H, m, CH2CH3 × 2); m/z
(EI) 265 (M+, 30.61%), 235 (70.43), 197 (40.07), 179 (100.00),
151 (48.13).
In a similar manner to that described above, 2c gave 4a, 4c,
4e, and 2e gave 4i–4m.
Diethyl [2-(3,4-dimethoxyphenylamino)-2-oxoethyl]phosphonate
(3g)
Yield: 0.60 g (76.0%), yellow oil; dH (60 MHz; CDCl3; Me3Si)
1.00–1.60 (9H, m, CH2CH3 × 3), 2.95 (2H, d, J 22.0, PCH2),
3.90–4.40 (8H, m, OCH2CH3 × 3, NCH2), 7.80 (1H, br s,
NH); m/z (EI) 281 (M+, 25.71%), 236 (19.45), 208 (52.91), 179
(100.00), 152 (17.48).
(E)-N-[2-(3,4-Dimethoxyphenyl)ethyl]-2-butenamide (6b)
General procedure for 6a, 6c–6e. To a mixture of 3,4-
dimethoxyphenylethylamine (6.9 ml, 40.0 mmol) and Et3N
(5.5 ml) in dry benzene (180 ml) was added dropwise crotonyl
chloride (4.2 ml, 44.0 mmol) in dry benzene (40 ml) over 30 min
at 0 ◦C. The reaction mixture was stirred at 3–5 ◦C for 1 h.
The solvent was evaporated off. The residue was recrystallized
from ethyl acetate to give 6b (9.6 g, 96.0%) as colorless needles:
(E/Z)-2-(Diethylcarbamoyl-1-methylvinyl)-7-(3-
ethoxycarbopropoxy)benzo[b]furan (4a)
◦
General procedure for 4b–4z, 4a. To a suspension of NaH
mp 76.8–79.1 C; dH (500 MHz; CDCl3; Me3Si) 1.82 (3H, dd,
(60% in oil, 0.23 g, 6.2 mmol) in anhydrous THF (10 ml) was
J 8.0 and 2.0, CH3), 2.78 (2H, t, CH2CH2N), 3.50 (2H, m,
2 1 3 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 1 2 9 – 2 1 3 9

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CH2CH2NH), 3.82 (6H, s, OCH3 × 2), 5.93 (1H, dd, J 16.0 and
arom-H); m/z (EI) 249 (M+, 35.46%), 164 (100.00).
diethylphosphonoacetamide (3.1 g, 14.0 mmol) in anhydrous
THF (10 ml) under an N2 atmosphere at 5 ◦C with stirring.
The solution was then stirred at 25 C until it became clear. A
=
=
2.0, CH CHCH3), 6.75 (1H, m, CH CHCH3), 6.85 (3H, s,
◦
solution of 7a (2.0 g, 4.7 mmol) in anhydrous THF (20 ml) was
added dropwise to the clear solution at 25 ◦C, and the mixture
was stirred at 25 ◦C for 40 min. The mixture was then quenched
with saturated NH4Cl solution and extracted with ethyl acetate.
The organic layer was washed with brine, then dried. The solvent
was evaporated off, giving a residue which was purified with
silica gel column chromatography [CHCl3–ethyl acetate (5 : 2)]
to give E-8a (1.3 g, 53.5%) as a yellow oil.
(E)-N,N-Diethyl-2-butenamide (6a)
◦
Yield: 50.8 g (56.8%), colorless oil; bp8 88.0 C; dH (500 MHz;
CDCl3; Me3Si) 1.11–1.20 (6H, m, CH2CH3 × 2), 1.88 (3H, dd,
J 7.2 and 1.7, CH3), 3.31–3.43 (4H, m, CH2CH3 × 2), 6.22 (1H,
=
dq, J 15.0 and 1.7, CH CHCH3), 6.90 (1H, dq, J 15.0 and 6.8,
CH CHCH3); m/z (EI) 141 (M , 100.00%).
+
=
(E)-4-(1-Oxo-2-butenyl)morpholine (6c)
Yield: 18.7 g (93.7%), white needles; mp 55.8–58.0 ◦C; dH
2-(2-Carboxy-1-methylvinyl)-7-(3-carboxypropoxy)-4-(2-
diethylcarbamoyl-1-methylvinyl)benzo[b]furan (8b)
(60 MHz; CDCl3; Me3Si) 1.81 (3H, br s, CH3), 3.55 (8H, br s,
8b was obtained according to the usual manner from 8a. Yield:
0.37 g (46.4%).
+
=
CH2CH2 × 2), 6.20–7.09 (2H, m, CH CH); m/z (EI) 155 (M ,
61.77%), 140 (100.00).
2-Acetyl-5-bromo-3-[2-(4-methoxyphenyl)-
acetylamino]benzo[b]furan (11a)
(E)-1-(1-Oxo-2-butenyl)-4-phenylpiperazine (6d)
Yield: 1.4 g (69.0%), yellow needles; mp 124.3–125.4 ◦C; dH
(60 MHz; CDCl3; Me3Si) 1.90 (3H, dd, J 6.6 and 1.8, CH3), 3.17–
3.19 (4H, m, NCH2 × 2), 3.70–3.86 (4H, m, NCH2 × 2), 6.10–
To a solution of 10 (1.5 g, 5.9 mmol) in anhydrous THF
(80 ml) was added dropwise 4-methoxyphenylacetic acid chlo-
ride [prepared by treatment of 4-methoxyphenylacetic acid
(2.2 g, 11.8 mmol) with SOCl2 (5 ml, 4.8 mmol)] under an N2
atmosphere with stirring at 25 ◦C. The mixture was stirred at
25 ◦C for 20 h. The solvent was evaporated off. An ethyl acetate
solution of the resulting residue was washed with 5% NaOH
solution and brine, and dried. The solvent was evaporated off,
giving a residue which was recrystallized from ethyl acetate to
give 11a (1.3 g, 55.6%) as pale yellow prisms: mp 192.1–194.7 ◦C;
dH (60 MHz; CDCl3; Me3Si) 2.64 (3H, s, COCH3), 7.28–8.10
(6H, m, 5-, 6-H and 2ꢀ-, 3ꢀ-, 5ꢀ-, 6ꢀ-H), 8.93 (1H, d, J 2.4, 4-H),
10.29 (1H, br s, NH); m/z (EI) 390.9607 (M+. C17H11BrClNO3
requires 390.9611), 391 (M+, 32.45%), 139 (100.00).
=
=
6.50 (1H, m, CH CHCH3), 6.80–7.48 (6H, m, CH CHCH3
and phenyl H), m/z (EI) 230 (M+, 91.40%), 161 (29.32), 132
(100.00).
(E)-1-(1-Oxo-2-butenyl)-4-(phenylmethyl)piperazine (6e)
Yield: 18.0 g (90.0%), white needles; mp 79.6–81.2 ◦C; dH
(60 MHz; CDCl3; Me3Si) 1.81 (3H, d, J 6.0, CH3), 2.40 (4H,
t, J 5.0, NCH2 × 2), 3.53 (2H, s, CH2), 3.59 (4H, t, J 5.0,
=
NCH2 × 2), 6.50–6.90 (2H, m, CH CH), 7.40 (5H, m, phenyl
H); m/z (EI) 244 (M+, 48.83%), 91 (100.00).
(E)-2-Acetyl-4-(2-diethylcarbamoyl-1-methylvinyl)-7-
diphenylmethoxybenzo[b]furan (7i)
2-(2-Carboxy-1-methylvinyl)-5-(2-morpholinocarbo-1-
methylvinyl)benzo[b]furan (12f)
General procedure for 7a, 7c–7h, 7j–7n, 12a–12d, 12g, 12h.
A mixture of 2m (2.0 g, 4.8 mmol), 6a (1.4 g, 9.9 mmol),
palladium acetate (0.054 g, 0.24 mmol), tri-o-tolylphosphine
(0.42 g, 1.4 mmol) and Et3N (4.0 ml, 0.028 mol) was heated at 90–
100 ◦C for 8 h. The mixture was treated with ethyl acetate, and
the insoluble portion was removed by filtration. The filtrate was
evaporated to dryness. The residue was poured into ice water,
made acidic with 1 N HCl solution and extracted with ethyl
acetate. The organic layer was washed with brine and dried. The
solvent was evaporated off. The residue was purified with silica
gel column chromatography [CHCl3–ethyl acetate (5 : 2)] to give
7i (0.73 g, 31.7%) as a yellow solid.
To a suspension of NaH (60% in oil, 0.07 g, 1.76 mmol) in
anhydrous THF (8 ml) was added dropwise a solution of ethyl
diethylphosphonoacetamide (0.39 g, 1.8 mmol) in anhydrous
THF (10 ml) under an N2 atmosphere at 5 ◦C with stirring.
◦
The solution was then stirred at 10 C until it became clear. A
solution of 12c (0.5 g, 1.6 mmol) in anhydrous THF (10 ml) was
added dropwise to the clear solution at 10 ◦C, and the mixture
◦
was stirred at 20 C for 1.5 h. The mixture was then quenched
with saturated NH4Cl solution and extracted with ethyl acetate.
The organic layer was washed with brine, then dried. The solvent
was evaporated off, and the crude product (12e) was used for the
next step without further purification. A mixture of 12e (0.46 g),
NaOH (1.0 g, 0.025 mol), methanol (40 ml) and H2O (10 ml) was
In a similar manner to that described above, 2j gave 7a, 2k
gave 7c–7g, 2l gave 7h, 2m gave 7i–7m, 2o gave 7n, 11a gave 12a,
12b, 11b gave 12c, 12d, and 11c gave 12g, 12h. 7b was obtained
according to the usual manner from 7a.
◦
heated at 75 C for 2.5 h. The solvent was evaporated off, and
made acidic with 5% HCl solution. The resulting precipitate was
collected by filtration and washed with water, and recrystallized
from ethyl acetate to give 12f (0.068 g, 11.9% from 12c) as white
needles.
(E)-2-Acetyl-7-[3-(4-chlorophenylsulfanyl)propoxy]-4-
(diethylcarbamoyl-1-methylvinyl)benzo[b]furan (7o)
A mixture of 7n (0.24 g, 0.61 mmol), K2CO3 (0.67 g, 4.8 mmol)
and 4-chlorobenzenethiol (0.26 g, 1.8 mmol) in dry acetone
(50 ml) was stirred at 54 ◦C for 48 h. After an insoluble portion
was filtered off, the filtrate was distilled under reduced pressure.
The residue was purified with silica gel column chromatography
[CHCl3–ethyl acetate (10 : 1)] to give a pale yellow solid. The
solid was washed with hexane to give 7o (0.12 g, 38.7%) as a
white solid.
Measurement of LTB4-induced TXB2 release from
bronchoalveolar eosinophils
Bronchoalveolar eosinophils were harvested from guine◦a pigs
treated with Sephadex G-200.29 After preincubation at 37 C for
5 min, the test compound was added to the purified eosinophils
(1.1 × 106 cells per ml), and the mixture was incubated for 5 min.
Next, LTB4 (100 nM) was adde◦d to the mixture, and the reaction
was allowed to proceed at 37 C for 15 min. The reaction was
stopped by cooling in ice–water, followed by centrifugation at
1700 × g for 10 min at 4 ◦C. The resultant supernatant was stored
at −80 ◦C until assay of TXB2. The TXB2 in the supernatant was
measured by a TXB2 enzyme immunoassay (EIA) kit (Cayman
Chem.) and expressed as pg/106 eosinophils.
4-(2-Diethylcarbamoyl-1-methylvinyl)-7-ethoxycarbopropoxy-2-
(2-ethoxycarbonyl-1-methylvinyl)benzo[b]furan (8a)
To a suspension of NaH (60% in oil, 0.56 g, 14.0 mmol) in
anhydrous THF (10 ml) was added dropwise a solution of ethyl
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Measurement of calcium mobilization in CHO cells
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CHO cells stably expressing human BLT131 or BLT22f, seeded on
a 96-well glass-bottom plate (Coster 3603) at 4 × 104 cells per
well, were loaded with 4 lM Fluo-3 (Dojin, Kumamoto, Japan)
in 1 × HBSS (Hanks balanced salt solution, Sigma) containing
◦
0.04% pluoronic acid and 1% FCS at 37 C for 1 h. The cells
were washed twice with 1 × HBSS and pretreated with various
concentrations of antagonists diluted in 100 ll of 1 × HBSS,
1% FCS for 30 min. A stock of BLT antagonists was prepared
in DMSO solution, and the final concentration of DMSO in
the assay was adjusted to 0.1% in all wells. To each well was
added 50 ll (or 150 ll) of 300 nM LTB4 (Cayman Chem.) to
give a final concentration of 100 nM (or 300 nM), and the LTB4-
dependent increase in fluorescent intensity was measured using
FlexStation (Molecular Devices). CHO cells transfected with an
empty vector did not respond to 100 nM LTB4 (data not shown).
Acknowledgements
This work was supported in part by a grant from the Ministry
of Education, Culture, Sports, Science and Technology for the
“University–Industry Joint Research” Project (2004–2008). We
thank the staff of the instrumental analysis center of Mukogawa
1
Women’s University for the H-NMR and MS measurements
and elemental analyses.
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2 1 3 8
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18 The torsion angle between the 2-(2-alkylcarbamoyl-1-methylvinyl)
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34 Inhibition of calcium mobilization in the CHO–hBLT2 cell by
compounds (4a, 4bꢀ, 4d, 4f, 4y) was not due to its cytotoxicity, because
these compounds did not affect calcium mobilization in the CHO–
hBLT1 cell at 10 lM.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 1 2 9 – 2 1 3 9
2 1 3 9