Preparation of 2- And 4-(2-alkylcarbamoyl-1-methylvinyl)-7-alkyloxybenzo[b]furans having potent antagonistic activity against human leukotriene B4 BLT1 and/or BLT2 receptors

Article abstract of DOI:10.1039/b411286e

(E)-2- Acetyl-4-(2-diethylcarbamoyl-1-methylvinyl)-7-(1-phenylethoxy)benzo[b]furan (4b) with a characteristic conformation and (E)-2-(2-morpholinocarbo-1-methyl-vinyl)-7-ethoxycarbopropoxybenzo[b]furan ((E)-3b) were prepared and evaluated for their leukot

Full text of DOI:10.1039/b411286e

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C O M M U N I C A T I O N
Preparation of 2- and 4-(2-alkylcarbamoyl-1-methylvinyl)-
7-alkyloxybenzo[b]furans having potent antagonistic activity against
human leukotriene B4 BLT1 and/or BLT2 receptors†
Kumiko Ando,a Eriko Tsuji,a Yuko Ando,a Jun-ichi Kunitomo,a Masayuki Yamashita,b
Shunsaku Ohta,b Takeshi Nabe,b Shigekatsu Kohno,b Takehiko Yokomizo,c,d,e
Takao Shimizuc,e 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 Kyoto Pharmaceutical University, Misasagi, Yamashinaku, Kyoto, 607-8412, Japan
c Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Tokyo,
7-3-1 Hongo Bunkyo-ku, Tokyo, 113-0033, Japan
d PRESTO, 7-3-1 Hongo Bunkyo-ku, Tokyo, 113-0033, Japan
e CREST of JST, 7-3-1 Hongo Bunkyo-ku, Tokyo, 113-0033, Japan
Received 26th July 2004, Accepted 4th October 2004
First published as an Advance Article on the web 1st November 2004
(E)-2-Acetyl-4-(2-diethylcarbamoyl-1-methylvinyl)-7-(1-
phenylethoxy)benzo[b]furan (4b) with a characteristic con-
formation and (E)-2-(2-morpholinocarbo-1-methyl-vinyl)-
7-ethoxycarbopropoxybenzo[b]furan ((E)-3b) were pre-
pared and evaluated for their leukotriene B4 (LTB4) anta-
gonistic activity. Compound 4b showed potent antagonistic
activity against human BLT1 and BLT2 receptors. Com-
pound (E)-3b displayed selective BLT2 receptor antagonistic
activity. Both compounds were inactive to cysteinyl LT
receptors.
suggest that LTB4 selective antagonists may be applicable
for treatment of arteriosclerosis,4 rheumatoid arthritis5 and
pancreatic cancer,6 as well as for immunosuppression
of
2c,7
allograft rejection in organ transplantation. This encouraged
us to find novel BLT2 selective and dual BLT1/BLT2 selective
receptor antagonists. BLT2 selective antagonists may be also
useful for clarifying the bioactive roles of the BLT2 receptor in
the body.
Simple heteroaromatic skeleton compounds of stable confor-
mation may well be favored over the aliphatic carbon chain
type and the ether type compounds.8 Benzopyran
and
8b,8c
8d
dibenzofuran derivatives have been reported previously as
LTB4 antagonists. Here we used the benzo[b]furan derivative
to search for novel LTB4 antagonists. The combination of
Leukotriene B4 (LTB4) plays important roles in the host defence
system against infection and invasion of foreign bodies, however
overproduction of LTB4 is involved in various inflammatory
diseases.1 Thus, many attempts have been made to develop
antagonists of LTB4 as clinical drugs. Of particular interest are
=
conjugated triene and a single C C bond near one OH group
of possible LTB4 conformers (A, B) was based on consid-
eration of the benzo[b]furan ring having an O(CH2)3COOR
group originating from the partial structure of LTB4. The a,b-
unsaturated carbamoyl group9 modified from cinnamic acid
proved to be an interesting functional group showing cysteinyl
LTs antagonistic activity in our recent study.10 Several 2-
alkylcarbamoyl-1-methylvinyl groups11 devised from the a,b-
unsaturated carbamoyl group were introduced at C-2 or C-4
of the benzo[b]furan ring. Introduction of functional groups
at C-2 or C-4 is preferable to other positions to examine the
relationship between the substituent position and bioactivity
because the C-2 and C-4 positions have significantly different
stereochemical and electronic environments. We report here the
2a
2b
2c
2d
ONO-4057, ZK-158252, BIIL315 and LY 293111 (Fig. 1).
These antagonists may be grouped into two classes, aliphatic
carbon chain types (ONO-4057 and ZK-158252) and ether types
(BIIL315 and LY293111). No LTB4 antagonist has yet been
marketed in spite of the clinical application of an LTD4 receptor
antagonist (cysteinyl LT1 receptor antagonist). Recently, a
second LTB4 receptor (BLT2) was discovered and its molecular
cloning was reported.3 Current work on LTB4 and its receptors
† Electronic supplementary information (ESI) available: Experi-
mental details for compounds 3a, 3b, 3f, 4b and 4c. See
http://www.rsc.org/suppdata/ob/b4/b411286e/
Fig. 1
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 4
T h i s j o u r n a l i s
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 4 2 7 – 3 4 3 1
3 4 2 7
©

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structural characteristics of I and II and their LTB4 antagonistic
activity characterized by the potency and selectivity for human
BLT1 and/or BLT2 receptors (designed compounds I and II,
Fig. 2).
a representative compound of (E)-3, was determined by X-◦ray
analysis as shown in Fig. 3,16 with the torsion angle being 6.9 .15
Conformation and NOE correlation of (E)-3b are shown in
Fig. 4. The main isomer of (E/Z)-3d was isolated and identified
as s-trans-(Z)-isomer, (Z)-3d, on the basis of its 1H-NMR data.
Compounds 2c, 2d and 2e were treated with 2-butenamides (6)
in the presence of (CH3COO)2Pd, (2-CH3C6H4)3P and (C2H5)3N
11b,17
under the Heck coupling conditions
to afford (E)-2-
acetyl-4-(2-alkylcarbamoyl-1-methylvinyl)-7-alkyloxybenzo[b]
furans (4a–4d) on the basis of their NOE analyses (Scheme 1).
In the NOE of compounds 4, it was very interesting that both
the olefinic CH3 and olefinic H had NOE correlations with 3-H
and 5-H, respectively. Consideration of the NOE correlations
led to speculation concerning the molecular dissymmetry of 4
arising from restriction of the rotation about the single bond at
the C-4.18 However, this speculation was rejected by the absence
of the diastereoisomer of 4b which contained one asymmetric
carbon atom in the substituent group at C-7 on the basis of 1H-
NMR studies. The torsion angle of representative compound 4c
between the benzo[b]furan plane and the 2-alkylcarbamoyl-1-
methylvinyl group at C-4 estimated using MM215 was 44.7◦.
The X-ray structure of 4c is shown in Fig. 3,16 with the
torsion angle being 45.7◦. Conformation and NOE correlation
of 4c are shown in Fig. 4. The 2-alkylcarbamoyl-1-methylvinyl
groups of compounds (E)-3 and 4 showed distinctly different
conformations from each other, because the 2-alkylcarbamoyl-
1-methylvinyl group of compound 4 was subjected to significant
steric hindrance from 3-H and 5-H, while there was little
hindrance from 3-H on compound (E)-3 (Fig. 4).
Compounds ((E)-3, 4) were evaluated for their LTB4 antago-
nistic activity by two in vitro methods: Method A,19 inhibition of
LTB4-induced TXB2 release from bronchoalveolar eosinophils
of guinea pigs and Method B, inhibition of calcium mobilization
in CHO-humanBLT1 (CHO-hBLT1) and CHO-humanBLT2
(CHO-hBLT2) cells by LTB4.
Method A. Compounds (E)-3a, -3b, -3c, -3d, -3e, 4a, 4b, 4c and
4d at 100 lM completely inhibited LTB4 (100 nM)-induced TXB2
release from the bronchoalveolar eosinophils. Compounds (E)-
3a, -3b and 4b even at 1 lM produced complete inhibition, but 4c
showed 84% inhibition at the same concentration. Furthermore,
the concentration-dependent antagonistic activity for (E)-3a,
-3b and 4b was evaluated at the concentrations of 0.1 nM, 1 nM,
10 nM, 100 nM and 1 lM. The most potent compound (E)-3a
Fig. 2 Possible conformers (A, B) of LTB4 and designed benzo[b]furan
derivatives (I, II).
Alkylation of 2-acetyl-7-hydroxybenzo[b]furans (1a, 1b) with
alkyl halides gave the 7-alkyloxybenzo[b]furans (2a, 2c–2e).
Treatment of 2a with phosphonoamides (5)12 under Horner–
Wadsworth–Emmons (HWE) reaction13 conditions afforded
(E/Z)-2-(2-alkylcarbamoyl-1-methylvinyl)benzo[b]furans ((E/
Z)-3a, -3b, -3c). Compounds 2b, 2c and 2f14 were also subjected
to HWE reaction with the phosphonoamide (5) to give (E/Z)-2-
(2-alkylcarbamoyl-1-methylvinyl)benzo[b]furans ((E/Z)-3d, -3e
and -3f), respectively. Yields and ratios of E/Z of 3 are shown in
Scheme 1. (E)-Isomers ((E)-3a, -3b, -3c, -3e, -3f) were isolated
from the corresponding E/Z mixtures ((E/Z)-3a, -3b, -3c, -3e,
-3f). The (E)-isomers (E)-3 showed only a nuclear Overhauser
enhancement (NOE) correlation between the olefinic CH3 and
3-H. This could be explained reasonably, since the double bond
of the (E)-2-(2-alkylcarbamoyl-1-methylvinyl) group had an s-
trans configuration with a double bond between C-2 and C-
3, and also this group lay on approximately the same plane
as the benzo[b]furan ring.15 The stereostructure of (E)-3f, as
Scheme 1
3 4 2 8
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 4 2 7 – 3 4 3 1

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Fig. 3 X-Ray structures of (E)-3f and 4c.
in comparison with standard LTB4 antagonists (ZK-158252
and ONO-4057). Compound 4b showed the most potent and
concentration-dependent inhibition of calcium mobilization in
both CHO-hBLT1 and CHO-hBLT2 cells and its potency lay
between ZK-158252 and ONO-4057. Characteristically, (E)-
3b showed selective and concentration-dependent inhibition in
CHO-hBLT2 cells (Fig. 6).21 In contrast, (E)-3a was found to be
inactive by Method B.
Fig. 4 Conformation and NOE correlations of (E)-3b and 4c.
showed almost complete inhibition at 100 nM and 1 lM, and
26.8% inhibition at 10 nM. Compounds (E)-3b and 4b were less
active than (E)-3a: 18.7 and 17.1% inhibition at 10 nM, and 37.7
and 31.7% inhibition at 100 nM, respectively (Fig. 5).20
Fig. 5 Effect of (E)-3a, (E)-3b, 4b on LTB4-induced TXB2 release from
bronchoalveolar eosinophils harvested from Sephadex G-200-treated
with guinea pigs (mean S.E., n = 3). (E)-3a, (E)-3b and 4b was 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 (%).
Fig. 6 Effect of (E)-3a, (E)-3b, 4b on calcium mobilization by LTB4
(100 nM) in CHO-hBLT1 and CHO-hBLT2 cells (mean S.D., n = 3).
Statistically significant differences from the control are indicated (*P <
0.05, **P < 0.01, unpaired t-test).
Method B. Three compounds ((E)-3a, -3b, 4b) selected from
the findings with Method A were evaluated by Method B
at concentrations of 10 nM, 100 nM, 1 lM and 10 lM,
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 4 2 7 – 3 4 3 1
3 4 2 9

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To prove that inhibition of calcium mobilization was not due
to a simple cytotoxic effect, cytotoxicity studies using ATP
were performed.22 Cysteinyl LT antagonistic activity assay23
for the active compounds ((E)-3a, -3b, 4b) was also carried
out to examine their selectivity for LTB4 antagonistic activity,
and they were found to be inactive. Compound 4b, shown to
be moderately active by Method A was revealed to be the
most potent compound with greater potency than the standard
compound ONO-4057 using both human BLT1 and BLT2 in
Method B. Unfortunately, the most active compound (E)-3a
according to Method A was completely inactive when examined
by Method B. Thus, the substituent position (at C-4) and
the conformation of the 2-alkylcarbamoyl-1-methylvinyl group
described above for the structure of 4 may contribute to its
antagonistic potency against human BLT receptors. On the
other hand, (E)-3b with the 2-morpholinocarbo-1-methylvinyl
group at the C-2, which lies on nearly the same plane as the
benzo[b]furan ring showed selective activity for human BLT2.
Compound (E)-3b is, to the best of our knowledge, the first
antagonist showing selective BLT2 antagonistic activity.
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
as a 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 of 300 nM LTB4 (Cayman Chemicals) to give a
final concentration of 100 nM, and the LTB4-dependent increase
in the fluorescent intensity was measured using FlexStation
(Molecular Devices). CHO cells transfected with empty vector
did not respond to 100 nM LTB4 (data not shown).
Acknowledgements
We thank the staff of the instrumental analysis center of
Mukogawa Women’s University for the 1H-NMR and MS
measurements.
In this study, we found a potent human BLT1 and BLT2
receptor antagonist, (E)-2-acetyl-4-(2-diethylcarbamoyl-1-
methylvinyl)-7-(1-phenylethoxy)benzo[b]furan (4b), and a BLT2
selective antagonist, (E)-2-(2-morpholinocarbo-1-methylvinyl)-
7-ethoxycarbopropoxybenzo[b]furan ((E)-3b). The next step
would be to synthesize new derivatives and evaluate them to
find more potent and selective (2-alkylcarbamoyl-1-methyl-
vinyl)benzo[b]furan derivatives. Such work should clarify the
relationships between the selective antagonist activities and the
stereochemistry of the functional groups in a series of these
derivatives.
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Experimental
(E)-2-(2-Morpholinocarbo-1-methylvinyl)-7-ethoxycarbo-
propoxybenzo[b]furan ((E)-3b)
To a suspension of NaH (60% in oil, 0.24 g, 6.1 mmol) in
anhydrous THF (10 ml) was added dropwise a solution of [2-
(4-morpholinyl)-2-oxoethyl]phosphonic acid diethyl ester (1.6 g,
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−5 ◦C with stirring. The solution was then stirred at 25 ◦C until
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94.8 ◦C.
(E)-2-Acetyl-4-(2-diethylcarbamoyl-1-methylvinyl)-7-(1-phenyl-
ethoxy)benzo[b]furan (4b)
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butenamide (0.47 g, 3.3 mmol), palladium acetate (0.031 g,
0.14 mmol), tri-o-tolylphosphine (0.085 g, 0.28 mmol) and Et3N
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21 These data are also reported as simple IC50 values.
9 H. Nakai, M. Konno, S. Kosuge, S. Sakuyama, M. Toda, Y. Arai,
T. Obata, N. Katsube, T. Miyamoto, T. Okegawa and A. Kawasaki,
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635.
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12 M. Watanabe, S. Hisamatsu, H. Hotokezaka and S. Hurukawa,
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Compound
IC50 for hBLT1/M
IC50 for hBLT2/M
¨
O. Gudmundsson and R. Sigurdardottir, Acta Chem. Scand., 1993,
ZK-158252
ONO-4057
4b
(E)-3a
(E)-3b
5.4×10−8
6.2×10−6
4.2×10−7
>10−5
3.1×10−8
4.7×10−6
4.8×10−7
>10−5
47, 1112–1116.
14 Y. Ohishi, T. Mukai, M. Nagahara, M. Yajima and N. Kajikawa,
Chem. Pharm. Bull., 1989, 37, 2398–2404.
15 The torsion angle between the benzo[b]furan ring and the 2-
alkylcarbamoyl-1-methylvinyl group at the C-2 of (E)-3a, (E)-3b and
(E)-3f was examined using Cambridge Soft, Chem3D 5.0, MM2.
(E)-3a: 0.3◦, (E)-3b: 3.5◦, (E)-3f: 4.4◦.
>10−5
8.3×10−7
16 3f: Formula: C17H19NO4, formula weight: 301.34, crystal system:
22 Inhibition of calcium mobilization in CHO-hBLT2 by (E)-3b was
not due to its cytotoxicity, because this compound at 10 lM did not
affect calcium mobilization in CHO-hBLT1 (Fig. 6). We also have
confirmed that the inhibition by these compounds was not due to
their cytotoxicity because they did not affect ATP-dependent calcium
mobilization through intrinsic ATP receptors in CHO cells at the
concentrations of BLT inhibition (data not shown).
23 H.-P. Nothacker, Z. Wang, Y. Zhu, R. K. Reinscheid, S. H. S. Lin
and O. Civelli, Mol. Pharmacol., 2000, 58, 1601–1608.
24 T. Yokomizo, T. Izumi, K. Chang, Y. Takuwa and T. Shimizu, Nature,
1997, 387, 620–624.
˚
monoclinic, space group: P 21/a (# 14), a =◦ 7.309(1) A, b =
˚
15.546(1) A, c = 13.8909(9), b = 104.340(1) , V = 1529.1(3)
A , Z = 4, Dcalc = 1.309 g cm−3, F000 = 640.0◦0, l(Cu-Ka) =
3
˚
7.68 cm−1, k(Cu-Ka) = 1.54178 A, x–2h scans at 23 C, 2754 unique
˚
reflections (2h < 135.1◦), R1 = 0.066 [2240 reflections to calc. R1].
4c: Formula: C31H31NO4, formula weight: 481.59, crystal system:
¯
˚
˚
triclinic, space group: P1 (# 2),◦a = 10.259(1) A, b = 16.120(2) A,
◦
◦
˚
c = 8.654(2) A, a = 104.45(1) , b = 105.78(1) , c = 96.235(8) ,
V = 1309.5(3) A , Z = 2, Dcalc = 1.221 g cm−3, F000 = 512.00,
3
˚
l(Cu-Ka) = 6.42 cm−1, k(Cu-Ka) = 1.54178 A, x–2h scans at
˚
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 4 2 7 – 3 4 3 1
3 4 3 1