Syntheses of 3-acetoacetylaminobenzo[b]furan derivatives having cysteinyl leukotriene 2 receptor antagonistic activity

Article abstract of DOI:10.1039/b312682j

Novel 3-acetoacetylaminobenzo[b]furan derivatives having a modified triene system at the 3-position were synthesized starting with 3-aminobenzo[b]furans. The enol isomers, 3-[(3-hydroxybul-2-enonyl)amino]benzo[b]furans (1), of the 3-acetoacetylaminobenzo[b]furans were obtained as stable isomers owing to formation of a hydrogen bonding between the enol hydroxyl group and the amidocarbonyl group. The planarity of the C-2 substituent through the C-3 side chain suggested the existence of a modified conjugational triene system in the enol compound. Cysteinyl leukotriene 1 and 2 receptor antagonistic activities for these compounds were evaluated. 2-(4-Cyanobenzoyl or ethoxycarbonyl)-3-[(2-cyano-3-hydroxybut-2-enonyl)amino]benzo[e]furans (15g, 15o, 15u) were moderately active.

Full text of DOI:10.1039/b312682j

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Syntheses of 3-acetoacetylaminobenzo[b]furan derivatives having
cysteinyl leukotriene 2 receptor antagonistic activity†‡
Kumiko Ando,a Eriko Tsuji,a Yuko Ando,a Noriko Kuwata,a Jun-ichi Kunitomo,a
Masayuki Yamashita,b Shunsaku Ohta,b Shigekatsu Kohnob 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: ϩ 798 41 2797; Tel: ϩ798 45 9953
b Kyoto Pharmaceutical University, Misasagi, Yamashinaku, Kyoto 607-8412, Japan.
E-mail: sohta@mb.kyoto-phu.ac.jp; kohno@mb.kyoto-phu.ac.jp; Fax: ϩ75 595 4795;
Tel: ϩ 75 595 4703
Received 13th October 2003, Accepted 17th December 2003
First published as an Advance Article on the web 28th January 2004
Novel 3-acetoacetylaminobenzo[b]furan derivatives having a modified triene system at the 3-position were
synthesized starting with 3-aminobenzo[b]furans. The enol isomers, 3-[(3-hydroxybut-2-enonyl)amino]benzo[b]furans
(1), of the 3-acetoacetylaminobenzo[b]furans were obtained as stable isomers owing to formation of a hydrogen
bonding between the enol hydroxyl group and the amidocarbonyl group. The planarity of the C-2 substituent
through the C-3 side chain suggested the existence of a modified conjugational triene system in the enol compound.
Cysteinyl leukotriene 1 and 2 receptor antagonistic activities for these compounds were evaluated. 2-(4-Cyanobenzoyl
or ethoxycarbonyl)-3-[(2-cyano-3-hydroxybut-2-enonyl)amino]benzo[b]furans (15g, 15o, 15u) were moderately active.
Introduction
Results and discussion
The cysteinyl leukotrienes (cysLTs) are potent biological medi-
ators in the pathophysiology of inflammatory diseases, particu-
larly of airway obstruction in asthma. Cysteinyl leukotriene 1
receptor (cysLT1) selective antagonists such as pranlukast,
montelukast and zafirlukast, have been shown to be clinically
beneficial against chronic asthma. Recently a second human
cysLT receptor (cysLT2) was molecularly cloned and character-
ized.2
The synthesis of cysLT1 and/or cysLT2 antagonist remains a
challenge. BAY u9773 (5) was reported to be a dual cysLT1 and
cysLT2 antagonist2a,3 and a partial agonist of cysLT2.2d DUO-
LT 4 (6) was also shown to be a dual antagonist. Discovery of
the dual antagonist and/or selective cysLT2 antagonist should
be of therapeutic value.
The importance of the benzo[b]furan ring system in natural
substances and synthetic products with various bioactivities
is evident from the large number of papers published. The
electron density of both C-2 and C-3 on the benzo[b]furan ring
has been reported to be higher than that of other positions.5
We found enamine reactivity6 of the 2-ethyl-3,5-diamino-
benzo[b]furan in the course of our studies,7 which suggested
olefinic as well as aromatic properties of the double bond
between C-2 and C-3. As the starting point for our own investi-
gation, we focused on the double bond between C-2 and C-3
on the benzo[b]furan ring. It was used to construct a modified
conjugational triene system. The designed compound (1), the
enol isomer of 3-acetoacetylaminobenzo[b]furans, may have
the π-conjugation and planarity shown by the bold-line
portions in Fig. 1. The C-7–C-12 portion of cysLTs (4) might be
simulated as shown by the bold-line portions of 1. Two other
series compounds (2, 3) were also prepared for comparison of
potencies. Here, we report the synthesis of new benzo[b]furan
derivatives (1, 2, 3) and their antagonistic activities on cysLT1
and cysLT2.
The designed compound (1) was synthesized starting with
3-aminobenzo[b]furans (9) as the key intermediate. Ring
closure reactions of 2-cyanophenols (7) with several α-halogen-
ated compounds (XCH2COR, ClCH2CN, BrCH2NO2) are con-
venient for preparing 9 having an electron withdrawing group
at the 2-position.
Several 2-cyanophenols (7b, 7c, 7d, 7e, 7f ) were prepared
from the corresponding salicylaldehydes according to the
procedure reported by Astles et al.8
2-Acetyl-3-aminobenzo[b]furans (9a, 9b, 9d, 9e) were pre-
pared by treatment of the 2-cyanophenols (7a, 7b, 7d, 7e) with
chloroacetone under condition c9 (Scheme 1). On the other
hand, treatment of 7a with chloroacetone under condition a
afforded only the intermediate acetyl methyl ether (8a) which
gave the bromide (8b). Reaction of 8b with Et3N (condition d)
easily afforded 2-acetyl-3-amino-5-bromobenzo[b]furan (9b).
Treatment of 4Ј-chloro-, 4Ј-cyano- and 3Ј,4Ј-methylenedioxy-2-
bromoacetophenone with 2-cyanophenols (7b, 7c, 7e) under
condition b or c afforded the 3-amino-2-benzoylbenzo[b]furans
(9g, 9i, 9j, 9k, 9l, 9m).10 Under the mild condition a, reactions
of 2-cyanophenols (7a, 7c) with the 2-bromoacetophenones
gave the intermediate benzoyl methyl ethers (8c, 8e), respect-
ively. Nitration of 8c gave the nitro intermediate (8d). The
intermediates (8c, 8d) were also converted to 3-amino-2-
benzoylbenzo[b]furans (9f, 9h), respectively, by treatment with
Et3N (condition d).
Reactions of 2-cyanophenols (7a, 7b) with ClCH2CN formed
mixtures [(8f, 9n) and (8g, 9o)] of the intermediate cyanomethyl
ethers (8) and the 3-amino-2-cyanobenzo[b]furans (9), respect-
ively, under condition b. The intermediates (8f, 8g) were con-
verted to the corresponding 3-amino-2-cyanobenzo[b]furans
(9n, 9o) under condition e.11 2-Cyanophenol (7a) was treated
with chloroacetamide under condition b to give the stable
intermediate (8h) which was converted to 3-aminobenzo[b]-
furan-2-carboxamide (9p) under condition f.11,12
Treatment of 2-cyanophenol (7a) with ethyl bromoacetate
under condition b gave the intermediate ethoxy carbomethyl
ether (8i) alone without any cyclization product.12 Bromination
of 8i gave bromide (8j). Ring closures of the ethoxy carbo-
† See ref. 1.
‡ Electronic supplementary information (ESI) available: Tables S1–S10
containing the physical data of compounds 8–10, 12, 14–16 and 18–27.
See http://www.rsc.org/suppdata/ob/b3/b312682j/
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 4
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 6 2 5 – 6 3 5
625

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Scheme 1
the corresponding 3-aminobenzo[b]furans compared to
cyclizations of the amide (8h) and the esters (8i, 8j).
The 3-amino-2-nitrobenzo[b]furans (9s, 9t) were obtained by
reactions of 7b and 7f with BrCH2NO2 under condition b with
low yield13 (Scheme 1).
The 2-acetyl-3-aminobenzo[b]furans (12a–12f ) having the 2-
alkyl- or 2-dialkylcarbamoyl-1-methylvinyl functional group14
at the 5-position were also prepared in one step by reaction of
the acetyl methyl ether (8b) with six N-alkyl or N,N-dialkyl-
crotonamides (10a–10f )15 in Et3N under modified Heck
coupling conditions14b,16 (Scheme 2). Consideration of nuclear
Overhauser enhancement spectroscopy (NOESY) spectra of
12a–12f (typical compound, 12a shown in Fig. 2) led to the
conclusion that 12 had an (E)-[(2-alkyl- or 2-dialkyl)carb-
amoyl-1-methylvinyl] group. 3-Amino-2-benzoylbenzo[b]furans
(9g and 9l) were also subjected to the Heck reaction to obtain
the corresponding alkylcarbamoylvinyl derivatives (12g, 12h,
12i and 13). 3-Amino-2-ethoxycarbonyl-5-(2-diethylcarbamoyl-
1-methyl)vinylbenzo[b]furan (12j) was obtained by cyclization
of 11 prepared from 8j (Scheme 2).
2-Acetyl-(14a–14e, 14q–14t), 2-benzoyl-(14f–14j, 14u–14w,
14y), 2-cyano-(14k, 14l), 2-ethoxycarbonyl-3-(5-methylisox-
azole-4-carbonyl)aminobenzo[b]furans (14n, 14o, 14x) and
3-(5-methylisoxazole-4-carboxyl)aminobenzo[b]furan-2-carbox-
amide (14m) were obtained from the corresponding 3-amino-
benzo[b]furans (9, 12, 13) by treatment with 5-methylisox-
azole-4-carboxylic acid chloride17 (Scheme 3).
In 1H-NMR studies of the 3-(5-methylisoxazole-4-carbonyl)-
aminobenzo[b]furans, except for the 2-cyano compounds (14k,
14l), a significant downfield shift (∆ppm: 1.11–1.21) of the
H-4 signals compared to those of the corresponding 3-amino-
benzo[b]furans was observed as shown in Table 1 [∆ppm (1.21)
= [{chemical shift (9.16) of 14c} — {chemical shift (7.95) of
9c}]], and a correlation of the NH with the only isoxazole ring
H (typical compounds, 14b and 14q shown in Fig. 2) was
detected in the NOESY spectra. The magnetic anisotropic
effect caused by the amidocarbonyl and the NOE data
supported the proposed conformation of these 3-(5-methyl-
isoxazole-4-carbonyl)aminobenzo[b]furans as shown in Fig. 2.
In contrast, only the 2-cyano compounds (14k, 14l) showed
characteristic correlation between NH and both isoxazole ring
H and H-4 in the NOESY spectra (14l in Fig. 2). This revealed
that the 2-cyano compounds had the opposite conformation
from the others shown in Fig. 2.
Fig. 1
methyl ethers (8i, 8j) to the 3-amino-2-ethoxycarbonylbenzo-
[b]furans (9q, 9r) were accomplished under condition g.
In the ring closure reactions of 8, the acetyl- (8a, 8b),
benzoyl- (8c, 8d, 8e) and cyanomethyl ethers (8f, 8g) easily gave
The 3-(5-methylisoxazole-4-carbonyl)aminobenzo[b]furans
(14a–14e, 14g, 14h, 14j–14o, 14q–14u, 14y) were rapidly con-
vertible to the respective 2-substituted-3-(2-cyano-3-hydroxy-
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 6 2 5 – 6 3 5
626

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Scheme 2
[{chemical shift (8.63) of 15b} — {chemical shift (7.70) of 9b}]
in Table 1].
The assignments of proton and carbon signals of 15 were
completed by extensive two-dimensional NMR (2D-NMR)
analysis [heteronuclear multiple bond connectivity (HMBC)
and heteronuclear multiple quantum coherence (HMQC)]. The
possible conformations, 1H-NMR and 13C-NMR data of 15q as
a typical compound, are shown in Fig. 3. The stable enol isomer,
3-(2-cyano-3-hydroxybut-2-enonyl)aminobenzo[b]furan, due to
formation of hydrogen bonds, was predominantly formed, in
contrast to the keto isomer, acetocyanoacetylaminobenzo-
[b]furan, which could not be obtained by this preparation
method.
The stereostructures of representative 3-(2-cyano-3-hydroxy-
but-2-enonyl)aminobenzo[b]furans (15b and 15u) were deter-
mined by X-ray analysis as shown in Fig. 4,19 which clearly
shows the conjugation and planarity of the C-2 substituent
through the C-3 side chain that were as we had expected.
3-(But-2-enonyl)amino-(16a–16e) and 3-(3-phenylprop-2-
enoyl)aminobenzo[b]furans (16f–16k), not enol analogues, were
prepared from the 3-aminobenzo[b]furans (9) by treatment with
crotonyl chloride or trans-cinnamic acid chloride (Scheme 3).
In addition, 2-acetyl-4-(2-cyano-3-hydroxybut-2-enonyl)-
amino-7-methoxybenzo[b]furan (22) was prepared, starting
with 2-acetyl-4-amino-7-methoxybenzo[b]furan (20) via 21.
2-[1-[(2-Cyano-3-hydroxybut-2-enonyl)amino]ethyl]benzo[b]-
furans (26a–26e), 2-[1-[(but-2-enonyl)amino]ethyl]benzo[b]-
furans (27a, 27b) and 2-[1-[(3-phenylprop-2-enonyl)amino]-
ethyl]benzo[b]furans (27c, 27d) were prepared starting with
2-(1-amino)ethylbenzo[b]furans (24) (Scheme 4). A significant
NOE between the NH and the isoxazole ring H was observed in
21 and 25, similar to that observed with 14.
Biological evaluation
Fig. 2 NOESY correlation and proposed conformations of 12a, 14b,
14q and 14l.
Most of the compounds reported in this paper were first
evaluated for cysLT1 and cysLT2 antagonistic activity by
measurement of inhibition of agonist-induced calcium release
according to the method reported by Nothacker.2d,20 The
compounds selected on the basis of calcium assay were next
evaluated for their cysLT2 antagonistic activity by radioligand
binding studies. In the calcium assay, three compounds (15g,
15o, 15u) showed more than 50% inhibition of the calcium
release for both cysLT1 and cysLT2 at a concentration of
10 µM. The IC50 values (µM) of the calcium assay indicated that
15g and 15u were more potent than 15o, and 15g displayed
selective antagonistic activity for cysLT2 (IC50 for cysLT2,
but-2-enonyl)aminobenzo[b]furans (15a–15e, 15g, 15h, 15j–
15o, 15q–15u, 15y), the enol isomer of the 3-acetocyanoacetyl-
aminobenzo[b]furan, by treatment with Et3N (Scheme 3).18 In
their 1H-NMR spectra, the 3-(2-cyano-3-hydroxybut-2-enonyl)-
aminobenzo[b]furans (15) showed characteristic hydroxyl
signals (15.2 ppm) caused by strong hydrogen bonding with
amidocarbonyl, and H-4 signals were also shifted downfield
(∆ppm: 0.90–1.00) owing to the magnetic anisotropic effect of
the amidocarbonyl from the chemical shift of the H-4 signals of
the corresponding 3-aminobenzo[b]furans [∆ppm (0.93) =
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 6 2 5 – 6 3 5
627

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Scheme 3
Table 2). The selective activity for cysLT2 of 15g was also sup-
ported by tentative examination using a radioligand binding
assay (Table 3).
the planarity of the C-2 substituent through the C-3 side chain,
which suggested the existence of a modified conjugational
triene system in the designed compound (1), as we had
expected.
Our designed compound (1) may be a novel lead compound
as a cysLT2 antagonist, which is still moderately active. Opti-
mization of 15g to find more potent and selective cysLT2
antagonistic active compounds is in progress.
In contrast, 2-acetyl-4-(2-cyano-3-hydroxybut-2-enonyl)-
amino-7-methoxybenzo[b]furan (22), 2-[1-[(2-cyano-3-hydroxy-
but-2-enonyl)amino]ethyl]benzo[b]furans (26) and 4-tri-
fluoromethyl-(2-cyano-3-hydroxybut-2-enonyl)aminobenzene
(29)18,21, the simple control compounds, were inactive in the
calcium assay (Table 2). The (2-cyano-3-hydroxybut-2-enonyl)-
amino functional groups of 22 and 26 displayed no conju-
gation with the double bond between C-2 and C-3. Also the
functional group of 29 was not conjugated with any olefinic
double bonds. These findings showed that the functional group
required a conjugated double bond to display activity. These
results demonstrated, as we had hypothesized, a significant
contribution of the double bond between C-2 and C-3 for
the appearance of activity. In addition, the enol form of the
3-acetoacetylamino functional group may play a crucial role in
the antagonistic activities for cysLT1 and cysLT2 because of the
very poor activities of 3-(but-2-enonyl)amino-(16a, 16b, 16d,
16e) and 3-(3-phenylprop-2-enonyl)aminobenzo[b]furans (16j,
16k). X-ray crystallography studies of 15b and 15u indicated
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 spectrometer with tetramethylsilane as an internal stand-
ard. 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 chromato-
graphy 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).
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 6 2 5 – 6 3 5
628

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Table 1 Downfield shift of H-4 of 14 and 15
Chemical shift (ppm)
∆ppm (14 ↔ 9)
Chemical shift (ppm)
∆ppm (15 ↔ 9)
Chemical shift (ppm)
9b
7.70
14b
8.84
15b
8.63
1.14
0.93
9c
7.95
14c
9.16
15c
8.95
1.21
1.00
Chemical shift (ppm)
∆ppm (14 ↔ 12)
Chemical shift (ppm)
∆ppm (15 ↔ 12)
Chemical shift (ppm)
12a
14q
15q
7.62
1.14
1.11
8.76
0.92
0.90
8.54
12e
7.66
14t
8.77
15t
8.56
Fig. 3 Conformation, 1H-NMR and 13C-NMR of 15q.
Scheme 4
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 6 2 5 – 6 3 5
629

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Table 2 Inhibition of agonist-induced calcium release
Inhibition (%) (10 µM)
IC50 (µM)
cysLT1
Inhibition (%) (10 µM)
Comp.
cysLT1
cysLT2
cysLT2
Comp.
cysLT1
cysLT2
15a
15b
15e
15g
15h
15j
6.5
36.3
20.1
52.1
23.1
24.5
6.3
15.7
8.4
49.8
3.3
11.5
25.7
35.0
51.0
18.1
16.1
24.1
7.5
0.7
67.1
29.1
1.6
22.1
Ϫ11.2
7.8
14a
14b
14c
14m
14n
14o
14s
14t
16a
16b
16d
16e
16j
11.9
11.1
0.2
Ϫ11.2
8.4
2.4
5.5
Ϫ11.8
13.5
2.7
Ϫ3.4
27.5
4.3
3.1
21.1
6.5
Ϫ13.0
16.3
11.0
7.5
>10.0
>10.0
6.0 1.6
5.7
15k
15l
35.5
13.0
2.8
24.6
17.0
11.8
6.8
37.5
13.3
15.1
6.5
14.7
5.0
15m
15o
15p
15q
15r
61.4
3.5
>10.0
15.0
16.9
13.3
57.6
10.1
2.3
15t
16k
21
22
15u
28
9.7
>10.0
>10.0
7.7
>10.0
>10.0
5.7
Ϫ8.5
5.4
29
25b
25c
26b
26c
27a
27b
27c
27d
BAY u97732d
0.44 0.182
0.30 0.092
6.6
0.1
12.7
Ϫ14.6
1.3
17.9
7.7
Ϫ10.0
20.4
14.8
18.4
Table 3 Radioligand binding assay at CysLT2
Comp.
Inhibition (%) (10µM)
IC50 (nM)
15g
85.0
9.5
3700
>10.0
597 279
15o
BAY u97732c
dryness to give a residue which was recrystallized from ethyl
acetate to give 8a (6.4 g) as colorless needles.
4-Bromo-2-(2-oxopropoxy)benzonitrile (8b)
AlCl3 (18.3 g, 0.14 mol) and N-bromosuccinimide (24.4 g, 0.14
mol) were added to a solution of 8a (20.0 g, 0.11 mol) in
acetonitrile (150 ml). The reaction mixture was stirred at 24 ЊC
for 1 h and poured into ice water to give a colorless powder. The
powder was recrystallized from ethanol to give 8b (29.0 g) as
colorless needles.
2-[2-(4-Cyanophenyl)-2-oxoethoxy]-4-nitrobenzonitrile (8d)
To a solution of 8c (0.71 g, 2.7 mmol) in acetic anhydride (135
ml) was added concentrated H2SO4 (0.20 ml) and HNO3 (0.21
ml, 4.6 mmol) at 6 ЊC. The reaction mixture was stirred for 0.5 h
and poured into ice water to give 8d (0.77 g) as colorless prisms.
2-Cyanomethoxybenzonitrile (8f) General procedure for 8g–8i,
9j, 9l–9m, 9s–9t from 7 (condition b)
To a suspension of 7a (1.0 g, 8.4 mmol) and K2CO3 (1.5 g,
10.9 mmol) in dry acetone (100 ml) was added chloroacetone
(0.69 ml, 10.9 mmol) at 20 ЊC with stirring. The reaction mix-
ture was heated at 56 ЊC, and an insoluble portion was filtered
off. The filtrate was evaporated in vacuo to give a residue which
was recrystallized from ethyl acetate to afford 8f (0.60 g) as pale
yellow needles.
Fig. 4 X-Ray structures of 15b and 15u.
2-(2-Oxopropoxy)benzonitrile (8a) General procedure for 8c, 8e
(condition a)
(4-Bromo-2-cyanophenoxy)acetic acid ethyl ester (8j)
Br2 (0.90 g, 5.6 mmol) was added to a solution of 8i (1.0 g,
4.9 mmol) in acetic anhydride (10 ml). The reaction mixture was
stirred at 26 ЊC for 5 h, and poured into ice water to give a
colorless powder. The powder was washed with hexane to give
8j (0.80 g).
To a solution of 7a (5.0 g, 4.2 mmol) in dry acetone (500 ml)
was added K2CO3 (11.6 g, 84.0 mmol) and chloroacetone
(4.4 ml, 54.6 mmol). The reaction mixture was stirred at 25 ЊC
for 6 h. After filtration, the filtrate was concentrated in vacuo to
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 6 2 5 – 6 3 5
630

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2-Acetyl-3-aminobenzo[b]furan (9a) General procedure for
9d–9e, 9g, 9i, 9k (condition c)
dropwise over 30 min at 0 ЊC. The reaction mixture was stirred
at 3–5 ЊC for 1 h. The solvent was evaporated off. The residue
was recrystallized from ethyl acetate to give 10b (9.6 g) as color-
less needles.
To a suspension of 7a (1.0 g, 8.4 mmol) and K2CO3 (2.9 g,
21.0 mmol) in dry methyl ethyl ketone (50 ml) was added
chloroacetone (0.64 ml, 10.1 mmol) at 25 ЊC with stirring. The
reaction mixture was heated at 82 ЊC for 1 h. After an insoluble
portion was filtered off, the filtrate was evaporated in vacuo. The
residue was recrystallized from ethanol to give 9a (1.1 g) as a
pale yellow powder.
(E )-[2-Cyano-4-(2-diethylcarbamoyl-1-methylvinyl)phenoxy]-
acetic acid ethyl ester (11)
A mixture of 8j (1.0 g, 3.5 mmol), 10a (0.99 g, 7.0 mmol), Et3N
(7.0 ml, 50.3 mmol), palladium acetate (0.59 g, 0.26 mmol) and
tri-o-tolylphosphine (0.11 g, 0.35 mmol) was heated at 100 ЊC
for 3 h, and the resulting precipitate was collected by filtration.
The precipitate was dissolved with ethyl acetate, and the in-
soluble portion was filtrated off. The filtrate was evaporated
to dryness. The residue was recrystallized from ethyl acetate
to give 11 (0.22 g, 18%) as pale brown needles. mp: 100 ЊC,
δH (CDCl3) 1.18 (6H, t, J 7.0, NCH2CH3 × 2), 1.30 (3H, t, J 7.0,
2-Acetyl-3-amino-5-bromobenzo[b]furan (9b) General procedure
for 9f, 9h (condition d)
A solution of 8b (8.0 g, 31.5 mmol) in Et3N (350 ml) was heated
at 90 ЊC for 5 h. Evaporation of excess reagent left a yellow
residue, which was recrystallized from ethanol to give 9b (7.2 g)
as pale yellow prisms.
OCH CH ), 2.25 (3H, bs, C᎐C–CH ), 3.37 (2H, q, J 7.3,
᎐
2
3
3
NCH2CH3), 3.47 (2H, q, J 7.0, NCH2CH3), 4.27 (2H, q, J 7.0,
OCH CH ), 4.77 (2H, s, OCH ), 6.24 (1H, bs, C᎐C–H), 6.83
(1H, d, J 8.8, 6-H), 7.58 (1H, dd, J 8.8 and 2.6, 5-H), 7.66 (1H,
d, J 2.6, 3-H). m/z: (ES) Found: 344.1736 (Mϩ). [C19H24O4N2]
requires 344.1736. m/z: 344 (Mϩ, 72.0%), 329 (100).
2-Acetyl-3-amino-5-cyanobenzo[b]furan (9c)
᎐
2
3
2
A mixture of 9b (1.0 g, 3.9 mmol), CuI (1.0 g, 5.3 mmol) and
CuCN (0.95 g, 10.6 mmol) in DMF (25 ml) was heated at
153 ЊC for 8 h. After an insoluble portion was filtered off, the
filtrate was poured into ice water and extracted with ethyl acet-
ate. The organic layer was washed with brine, then dried, and
evaporated to give a brown powder. The powder was recrystal-
lized from methanol to give 9c (70.0 mg) as yellow needles.
(E )-3-(2-Acetyl-3-aminobenzo[b]furan-5-yl)but-2-enoic acid
diethylamide (12a) General procedure for 12b–12f
To a solution of 8b (3.0 g, 11.8 mmol) in acetonitrile (20 ml)
was added 10a (3.3 g, 23.6 mmol), Et3N (7.1 ml, 50.7 mmol),
palladium acetate (0.13 g, 0.59 mmol) and tri-o-tolylphosphine
(0.35 g, 1.2 mmol) in a tube. The tube was sealed and heated at
130 ЊC for 10 h. The reaction mixture was cooled to room tem-
perature and an insoluble portion was filtered off. The filtrate
was evaporated to dryness. The residue was poured into ice
water and extracted with CH2Cl2, the extract was washed with
brine and dried. The solvent was evaporated off. The residue
was recrystallized from ethanol to give 12a (1.9 g) as yellow
needles.
3-Amino-2-cyanobenzo[b]furan (9n) Procedure for 9o
(condition e)
A suspension of 8f (1.0 g, 6.3 mmol) and K2CO3 (2.2 g,
15.8 mmol) in acetonitrile (50 ml) was heated at 82 ЊC for 3 h.
After an insoluble portion was filtered off, the filtrate was evap-
orated off in vacuo. The residue was poured into ice water, and
extracted with ethyl acetate. The organic layer was washed with
brine, then dried, and evaporated to give a pale brown powder
which was recrystallized from ethyl acetate to give 9n (0.65 g) as
pale yellow needles.
( E )-3-[3-Amino-2-(4-cyanobenzoyl)benzo[b]furan-5-yl]but-2-
enoic acid diethyl amide (12g) General procedure for 12h–12i
3-Aminobenzo[b]furan-2-carboxamide (9p) (condition f )
A suspension of 8h (6.0 g, 31.1 mmol) and KOH (4.0 g, 71.4
mmol) in ethanol (80 ml) was heated at 80 ЊC for 3 h. The
reaction mixture was poured into ice water to give 9p (4.6 g) as
colorless needles.
To a solution of 9g (2.5 g, 7.0 mmol) in THF (30 ml) was added
10a (2.0 g, 14.0 mmol), Et3N (7.0 ml, 70.2 mmol), palladium
acetate (0.16 g, 0.70 mmol) and tri-o-tolylphosphine (0.22 g,
0.70 mmol) in a tube. The tube was sealed and heated at 160 ЊC
for 7 h. The mixture was worked up in a similar manner as 12a,
and crude 12g was recrystallized from ethanol as yellow needles
(1.2 g).
3-Amino-2-ethoxycarbonylbenzo[b]furan (9q) Procedure for 9r
(condition g)
A suspension of 8i (5.0 g, 24.0 mmol) and NaH (1.0 g,
27.0 mmol) in dry DMSO (11 ml) was stirred at 25–27 ЊC for
10 h. The reaction mixture was poured into ice water, and
extracted with ether. The ether layer was washed with brine,
then dried, and evaporated to give 9q (3.7 g) as yellow needles.
(E )-3-Amino-5-(2-diethylcarbamoyl-1-methylvinyl)benzo[b]-
furan-2-carboxylic acid ethyl ester (12j)
A mixture of 11 (0.66 g, 1.9 mmol) and NaH (78.0 mg,
32.5 mmol) in dry DMSO (10 ml) was stirred at 25 ЊC for 2 h,
then poured into ice water and extracted with ether. The extract
was washed with brine and dried. The solvent was evaporated
off, and the residue was recrystallized from ethyl acetate to give
12j (0.24 g) as colorless needles.
N,N-Diethyl crotonamide (10a)
A solution of crotonyl chloride (60.0 ml, 0.63 mol) in dry ben-
zene (160 ml) was added to a solution of diethylamine (75.0 ml,
0.73 mol) and ethydiisopropylamine (76.0 ml, 0.42 mol) in dry
benzene (420 ml) at 0 ЊC with stirring. The mixture was allowed
to stand for 4 h at room temperature, then was washed succes-
sively with 10% Na2CO3, 10% HCl solution, and brine and
dried. This was fractionated under reduced pressure to give 10a
(50.8 g) as colorless oil, b.p.8 88 ЊC.
(E )-3-[4-(3-Amino-7-methoxybenzo[b]furan-2-carbonyl)-
phenyl]but-2-enyl acid [2-(3,4-dimethoxyphenyl)ethyl]amide (13)
To a solution of 9l (3.0 g, 8.7 mmol) in THF (50 ml), was added
Et3N (10 ml), 10b (4.3 g, 17.4 mmol), tri-o-tolylphosphine (0.27
g, 8.7 mmol) and palladium acetate (0.19 g, 0.87 mmol) in a
tube. The tube was sealed and heated at 160 ЊC for 15 h. The
mixture was cooled to room temperature and an insoluble por-
tion was filtered off. The filtrate was dried up. The residue was
poured into ice water and extracted with CHCl3. The extract
was washed with brine and dried. The solvent was evaporated,
and the residue was recrystallized from ethyl acetate to give 13
(E )-N-[2-(3,4-Dimethoxyphenyl)ethyl]-2-butenamide (10b)
General procedure for 10c–10f
To a mixture of 3,4-dimethoxyphenethylamine (6.9 ml, 40.0
mmol) and Et3N (5.5 ml) in dry benzene (180 ml) was added
crotonyl chloride (4.2 ml, 44.0 mol) in dry benzene (40 ml)
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 6 2 5 – 6 3 5
631

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(3.6 g) as yellow prisms. mp: 198 ЊC, δH(CDCl3) 2.59 (3H, d,
J 1.5, C᎐C–CH ), 2.84 (2H, t, J 7.0, NHCH CH ), 3.61 (2H, q,
J 7.0, NHCH2CH2), 3.87 (6H, s, 3Љ-, 4Љ-OCH3), 4.01 (3H, s,
7-OCH3), 5.58 (1H, t, J 5.9, CONH), 5.98 (2H, s, NH2), 6.02
6 h, and then the solvent was evaporated. The residue was
poured into ice water, and made acid with 5% HCl solution and
extracted with CHCl3. The extract was washed with brine and
dried. The solvent was evaporated. The residue was recrystal-
lized from acetonitrile to give 15a (0.23 g) as colorless needles.
᎐
3
2
2
(1H, d, J 1.4, C᎐C–H), 6.75 (1H, d, J 1.8, 2Љ-H), 6.77 (1H, dd,
᎐
J 8.9 and 1.8, 6Љ-H), 6.83 (1H, d, J 8.9, 5Љ-H), 6.99 (1H, m, 6-H),
7.19 (1H, dd, J 11.3, 5-H), 7.19 (1H, d, J 6.2, 4-H), 7.55 (2H,
dd, J 7.0 and 1.8, 3Ј-, 5Ј-H), 8.27 (2H, dd, J 6.6 and 1.8, 2Ј-,
6Ј-H). m/z: (ES) Found: 514.2103 (Mϩ). [C30H30O6N2] requires
514.2104. MS m/z: 514 (Mϩ,17.19%), 164 (100.00).
5-Bromo-3-(2-cyano-3-hydroxybut-2-enonyl)aminobenzo[b]-
furan-2-carboxylic acid ethyl ester (15o) Procedure for 15n
A solution of 14o (0.47 g, 1.1 mmol) and Et3N (1.5 ml, 11.1
mmol) in anhydrous THF (20 ml) was stirred at 23 ЊC for 1 h,
then the solvent was evaporated. The residue was poured into
ice water, and made acid with 5% HCl solution and extracted
with ethyl acetate. The extract was washed with brine and dried.
The solvent was evaporated. The residue was recrystallized
from methanol to give 15o (0.37 g) as colorless needles.
5-Methylisoxazole-4-carboxylic acid chloride
Thionyl chloride (4.3 ml, 39.5 mmol) was added dropwise
at 26 ЊC to 5-methylisoxazole-4-carboxylic acid (0.44 g, 3.4
mmol). The solution was heated at 89 ЊC for 1 h, and excess
thionyl chloride was removed under reduced pressure to pro-
vide acid chloride as a yellow oil, suitable for use in subsequent
reactions.
3-(2-Cyano-3-hydroxybut-2-enonyl)aminobenzo[b]furan-2-
carboxylic acid (15p)
To a solution of 14n (0.84 g, 2.7 mmol) in ethanol (30 ml) and
DMSO (20 ml), was added dropwise 10% NaOH aqueous solu-
tion (17 ml) at 15 ЊC with stirring. The mixture was stirred at
19 ЊC for 1 h, and poured into ice water, and made acid with 5%
HCl solution and extracted with ethyl acetate. The extract was
washed with brine and dried. The solvent was evaporated. The
residue was recrystallized from ethyl acetate to give 15p (0.46 g)
as colorless needles.
2-Acetyl-5-bromo-3-(5-methylisoxazole-4-carbonyl)amino-
benzo[b]furan (14b) General procedure for 14a, 14c–14e,
14k–14m, 14q–14t
To a solution of 9b (0.20 g, 0.79 mmol) in anhydrous THF (10
ml), was added dropwise 5-methylisoxazole-4-carboxylic acid
chloride (0.10 g, 0.79 mmol) under N2 atmosphere with vigor-
ous stirring at 26 ЊC. The solution was heated at 65 ЊC for 2 h.
The reaction mixture was cooled to room temperature and the
resulting precipitate was collected by filtration. The precipitate
was recrystallized from ethyl acetate to give 14b (0.19 g) as pale
yellow needles.
2-Cyano-3-hydroxybut-2-enyl acid [2-[4-[2-{2-(3,4-dimethoxy-
phenyl)ethylcarbamoyl}-1-methylvinyl]benzoyl]-7-methoxy-
benzo[b]furan-3-yl]amide (15y)
A suspension of 14y (0.50 g, 0.80 mmol) and KOH (90.0 mg,
1.6 mmol) in THF (270 ml) was heated at 65 ЊC for 5 h. The
solvent was evaporated in vacuo. The residue was poured into
ice water, and made acid with 5% HCl solution, and the result-
ing precipitate was collected. The precipitate was recrystallized
from ethyl acetate to give 15y (0.10 g) as colorless needles.
5-Bromo-2-(4-chlorobenzoyl)-3-(5-methylisoxazole-4-carbonyl)-
aminobenzo[b]furan (14h) General procedure for 14f–14g,
14i–14j, 14u–14w, 14y
To a solution of 9i (0.20 g, 0.57 mmol) in anhydrous THF (6 ml)
was added dropwise 5-methylisoxazole-4-carboxylic acid
chloride (0.14 g, 1.1 mmol) in anhydrous THF (3 ml) under N2
atmosphere with vigorous stirring at 26 ЊC. The solution was
heated at 65 ЊC for 7 h. After usual work up, the precipitate was
recrystallized from methanol to give 14h (0.99 g) as yellow
needles.
2-Acetyl-5-cyano-3-(Z )-(2-cyano-3-hydroxybut-2-enonyl)-
aminobenzo[b]furan (15c)
To a solution of 9c (0.90 g, 4.5 mmol) in anhydrous THF
(100 ml) was added dropwise 5-methylisoxazole-4-carboxylic
acid chloride (1.9 g, 14.0 mmol) under N2 atmosphere with
vigorous stirring at 28 ЊC. The solution was heated at 66 ЊC for
7 h and treated with pyridine (10 ml). A resulting precipitate
was collected by filtration, and recrystallized from ethanol to
give 15c (0.28 g, 20%) as colorless needles. Treatment of the
filtrate gave 14c (0.19 g, 14%) as colorless needles.
2-Ethoxycarbonyl-3-(5-methylisoxazole-4-carbonyl)amino-
benzo[b]furan (14n) Procedure for 14o
To a solution of 9q (3.0 g, 14.6 mmol) in acetonitrile (18 ml),
was added dropwise 5-methylisoxazole-4-carboxylic acid chlor-
ide (2.2 g, 17.5 mmol) in acetonitrile (2 ml) under N2 atmos-
phere with vigorous stirring at 25 ЊC. The reaction mixture was
stirred at 27 ЊC for 3 h, and the resulting precipitate was
collected by filtration. The precipitate was recrystallized from
ether to give 14n (3.3 g) as colorless needles.
2-Acetyl-5-bromo-3-(but-2-enonyl)aminobenzo[b]furan (16a)
Procedure for 16b–16e
Crotonyl chloride (0.29 ml, 3.04 mmol) was added to a solution
of 9b (0.7 g, 2.76 mmol) in anhydrous THF (20 ml) at 25 ЊC
with stirring. The mixture was stirred at 54 ЊC for 3 h. The
solvent was evaporated, leaving a residue. The residue was
dissolved in ethyl acetate. This solution was washed with 5%
NaOH aqueous solution and brine, then dried. Concentration
of the solution gave a powder which was recrystallized from
ethyl acetate to afford 16a (0.61 g) as pale yellow needles.
5-(2-Diethylcarbamoyl-1-methylvinyl)-2-ethoxycarbonyl-3-
(5-methylisoxazole-4-carbonyl)aminobenzo[b]furan (14x)
To a solution of 12j (1.0 g, 2.9 mmol) in acetonitrile (200 ml),
was added dropwise 5-methylisoxazole-4-carboxylic acid chlor-
ide (1.9 g, 2.9 mmol) in acetonitrile (10 ml) under N2 atmos-
phere with vigorous stirring at 22 ЊC. The solution was stirred at
the same temperature for 3 h, and heated at 30 ЊC for 2 h. The
solvent was evaporated in vacuo. After usual work up, the
residue was recrystallized from ethanol to give 14x (1.4 g) as
colorless needles.
2-Acetyl-5-bromo-3-(3-phenylprop-2-enonyl)aminobenzo[b]furan
(16f) General procedure for 16g–16j
A mixture of trans-cinnamic acid (1.16 ml, 7.88 mmol) and
thionyl chloride (5 ml) was heated at 55 ЊC for 3 h. The mixture
was concentrated in vacuo to give a yellow oil. The oil was
added to a solution of 9b (1.0 g, 3.94 mmol) in anhydrous THF
(30 ml). The mixture was stirred at 64 ЊC for 4 h and worked up
according to the procedure mentioned above. The product was
recrystallized from ethyl acetate to give 16f (1.0 g) as colorless
2-Acetyl-3-(Z )-(2-cyano-3-hydroxybut-2-enonyl)aminobenzo[b]-
furan (15a) General procedure for 15b, 15d–15m, 15q–15u
A solution of 14a (0.50 g, 1.8 mmol) and Et3N (2.4 ml,
0.18 mmol) in anhydrous THF (30 ml) was heated at 68 ЊC for
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 6 2 5 – 6 3 5
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prisms. The acid (16k) was obtained according to the usual
manner from 16j.
hydrochloride (3.2 g, 46.0 mmol). The reaction mixture was
heated at 65 ЊC for 1 h, and then the solvent was evaporated. A
solution of the residue in ethyl acetate was washed with brine,
then dried, and evaporated to give a yellow powder. The powder
was recrystallized from ethyl acetate to give 23a (6.8 g) as yellow
prisms.
(2-Acetylbenzo[b]furan-7-yloxy)acetic acid ethyl ester (18b)
General procedure for 18a, 18c and 18f
A mixture of 17 (5.7 g, 34.1 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 ЊC for 2 h. After an insoluble portion was
filtered off, the filtrate was dried. Ethyl acetate solution of the
resulting residue was washed with 5% NaOH aqueous solution
and brine, and dried. Concentration of the solution gave 18b
(5.1 g) as pale yellow needles.
2-(1-Aminoethyl)-7-ethoxycarbonylmethyloxybenzo[b]furan
(24a) General procedure for 24b and 24d
To a mixture of 23a (1.5 g, 5.4 mmol) and 37% HCl solution
(2.0 ml) in ethanol (150 ml) was hydrogenated over 10% Pd–C
(0.20 g) at 26 ЊC for 2 h under 5 atmospheres of hydrogen. The
catalyst was removed by filtration and the filtrate was concen-
trated in vacuo. The residue was poured into ice water and made
acid with 5% HCl solution and washed with CHCl3. The acidic
aqueous solution was made alkaline with 10% NaOH aqueous
solution and extracted with CHCl3. The extract was washed
with brine and dried. The solvent was evaporated off to give
24a (1.1 g) as yellow oil, Rf = 0.70, CHCl3–ethyl acetate (5 : 2).
2-Acetyl-7-methoxybenzo[b]furan-4-sulfonic acid ethylamide
(18d) Procedure for 18e from 18b
The fine powder 18a (3.0 g, 15.7 mmol) was carefully added to
chlorosulfonic acid (8.4 ml, 0.13 mol) in portions at 26–30 ЊC
over 1 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 (4.7 g) as a yellow solid, Rf =
0.43, hexane–ethyl acetate (1 : 1), which was suitable for use in
subsequent reactions. To a solution of ethylamine hydrochlor-
ide (1.7 g, 20.5 mmol) and triethylamine (5.1 ml, 36.3 mmol) in
CHCl3 (10 ml), was added dropwise the sulfonyl chloride in dry
CHCl3 (50 ml) at 27–30 ЊC with stirring. After the mixture was
stirred at 30 ЊC for 0.5 h, it was poured into ice water and made
acid with 5% HCl solution, then extracted with CHCl3. The
extract was washed with brine, then dried. The solvent was
evaporated off, giving a residue which was recrystallized from
methanol to give 18d (3.1 g) as yellow needles.
[2-[1-[(5-Methylisoxazole-4-carbonyl)amino]ethyl]benzo[b]-
furan-7-yloxy]acetic acid (25a)
5-Methylisoxazole-4-carboxylic acid chloride (0.50 g, 4.6
mmol) was added to a solution of 24a (2.0 g, 7.6 mmol) in
anhydrous THF (50 ml) at 32 ЊC. After the mixture was stirred
at 63 ЊC for 1 h, the solvent was evaporated. The residue was
poured into ice water and extracted with ethyl acetate. The
extract was washed with 5% HCl solution and brine, then dried.
Concentration of the extract gave the ester as yellow oil 1.1 g,
Rf = 0.72, ethyl acetate—methanol (5 : 1). The oil (ester) was
treated with 37% HCl solution (0.10 ml) in acetonitrile (30 ml)
at 25 ЊC for 2 h, and then the solvent was evaporated. The
residue was poured into ice water and extracted with ethyl
acetate, then washed with brine, and dried. Concentration of
the extract gave an oil. The crude oil was purified on a silica gel
column [CHCl3–ethyl acetate (10 : 1)] to give 25a (0.20 g) as a
colorless powder.
2-Acetyl-7-methoxy-4-nitrobenzo[b]furan (19)
To a solution of 18a (10.0 g, 53.0 mmol) in acetic anhydride
(200 ml) was added HNO3 (16.0 ml, 0.36 mmol) at 6 ЊC. The
reaction mixture was stirred at 26 ЊC for 1 h, and poured into
ice water to give 19 (9.7 g) as pale yellow needles.
4-[2-[1-[(5-Methylisoxazole-4-carbonyl)amino]ethyl]benzo[b]-
furan-7-yloxy)]butyric acid ethyl ester (25b) General procedure
for 25c and 25d
2-Acetyl-4-amino-7-methoxybenzo[b]furan (20)
A mixture of 19 (0.21 g, 0.85 mmol) and 20% titanium trichlor-
ide solution (3.9 ml, 5.1 mmol) was evacuated to approximately
87 mmHg and stirred at 26 ЊC for 2.5 h. The mixture was
poured into ice water and made alkaline with 10% NH4OH and
extracted with CHCl3. The extract was washed with brine and
dried. The solvent was evaporated off to give 20 (77 mg) as pale
brown needles.
To a solution of 24b (3.3 g, 11.3 mmol) in anhydrous THF
(15 ml) was added 5-methylisoxazole-4-carboxylic acid chloride
(1.5 g, 13.6 mmol) in anhydrous THF (15 ml) at 32 ЊC. The
mixture was heated 65 ЊC for 1 h. After work up, the powder
was recrystallized from ethanol to give 25b (3.0 g) as pale yellow
needles.
2-Acetyl-7-methoxy-4-(5-methylisoxazole-4-carbonyl)-
aminobenzo[b]furan (21)
[2-[1-(2-Cyano-3-hydroxybut-2-enonyl)amino]ethylbenzo[b]-
furan-7-yloxy]acetic acid (26a) Procedure for 26b from 25b
To a solution of 20 (2.4 g, 8.5 mmol) in acetonitrile (15 ml), was
added 5-methylisoxazole-4-carboxylic acid chloride (1.4 g, 11.0
mmol) in acetonitrile (5 ml) dropwise at 7 ЊC. The mixture was
stirred at 20 ЊC for 1.5 h. After work up, the residue was
recrystallized from benzene to give 21 (0.45 g) as pale brown
prisms.
To a solution of 25a (0.30 g, 0.87 mmol) in ethanol (8 ml) was
added 10% NaOH aqueous solution (12.0 ml). After the mix-
ture was stirred for 5 min at 25 ЊC, the solvent was evaporated.
The residue was poured into ice water and extracted with ethyl
acetate. The extract was washed with 5% HCl solution and
brine, then dried. Concentration of the extract gave 26a (0.19 g)
as colorless prisms.
2-Acetyl-4-(2-cyano-3-hydroxybut-2-enonyl)amino-7-methoxy-
benzo[b]furan (22)
4-[2-[1-[(2-Cyano-3-hydroxybut-2-enonyl)amino]ethyl]-7-
methoxybenzo[b]furanyl]-N-ethylsulfonamide (26c) Procedure
for 26d
A mixture of 21 (0.20 g, 0.63 mmol) and Et3N (0.89 ml, 6.3
mmol) in anhydrous THF (30 ml) was stirred at 72 ЊC for 1 h.
After work up, the powder was recrystallized from methanol to
give 22 (0.16 g) as pale yellow needles.
To a solution of 25c (0.50 g, 1.2 mmol) in anhydrous THF
(100 ml) was added Et3N (1.7 ml, 12.2 mmol). The reaction
mixture was heated at 70 ЊC for 12 h, and then the solvent was
evaporated. The residue was usually treated to afford a pale
yellow oil. The oil was purified on silica gel column chromato-
graphy [CHCl3–ethyl acetate (10 : 1)] and recrystallized from
acetonitrile to give 26c (0.20 g) as colorless prisms.
7-Ethoxycarbonylmethyloxy-2-(1-hydroxyimino)ethylbenzo[b]-
furan (23a) General procedure for 23b–23f
To a solution of 18b (10.0 g, 38.2 mmol) in methanol (100 ml),
was added pyridine (3.7 ml, 46.0 mmol) and hydroxylamine
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4-[2-[1-(2-Cyano-3-hydroxybut-2-enonylamino)ethyl]-4-ethyl-
sulfamoylbenzo[b]furan-7-yloxy]butyric acid (26e)
Acknowledgements
We express our appreciation to the staff of the Instrumental
Analytical Center of Mukogawa Women’s University for the
NMR and MS measurements and elemental analyses.
To a solution of 26d (1.1 g, 2.2 mmol) in ethanol (60 ml) was
added 10% NaOH aqueous solution (7.0 ml). The mixture was
heated at 60 ЊC for 2.5 h, and then the solvent was evaporated.
The residue was treated according the usual manner to give a
yellow powder. The powder was recrystallized from ethanol to
give 26e (0.46 g) as a colorless powder.
Notes and References
1 Part of this work has been published as
a preliminary
communication: E. Tsuji, K. Ando, J. Kunikomo, M. Yamashita,
S. Ohta, S. Kohno and Y. Ohishi, Org. Biomol. Chem., 2003, 1, 3139–
3141.
2-[1-(But-2-enonyl)aminoethyl]-7-methoxybenzo[b]furan (27a)
Procedure for 27b from 23f
2 (a) M. Bäck, Life Sciences, 2002, 71, 611–622; (b) J. F. Evans,
Prostaglandins & Other Lipid Mediators, 2002, 68–69, 587–597;
(c) C. E. Heise, B. F. O’Dowd, D. J. Figueroa, N. Sawyer, T. Nguyen,
D.-S. Im, R. Stocco, J. N. Bellefeuille, M. Abramovitz, R. Cheng,
D. V. Williams Jr., Z. Zeng, Q. Liu, L. Ma, M. K. Clements,
N. Coulombe, Y. Liu, C. P. Austin, S. R. George, G. P. O’Neill, K. M.
Metters, K. R. Lynch and J. F. Evans, J. Biol. Chem., 2000, 275,
30531–30536; (d ) H.-P. Nothacker, Z. Wang, Y. Zhu, R. K.
Reinscheid, S. H. S. Lin and O. Civelli, Mol. Pharmacol., 2000, 58,
1601–1608.
Catalytic reduction of 23e (1.5 g, 7.3 mmol) in a similar manner
as for 23a gave amine (24e, 0.61 g) as a yellow oil. Crotonyl
chloride (0.5 ml, 4.8 mmol) was added to a solution of the oil in
anhydrous THF (30 ml). After stirring at 25 ЊC for 6 h, the
mixture was worked up in the usual way to afford a yellow
powder which was recrystallized from ethyl acetate to give 27a
(0.41 g) as pale yellow prisms.
3 W. Laurence, N. Xavier, B. Magnus, G. Lean Pierre, D. Sven-Erik
and B. Charies, Br. J. Pharm., 2002, 137, 1339–1345.
7-Methoxy-2-[1-[(3-phenylprop-2-enonyl)]aminoethyl]benzo[b]-
furan (27c) Procedure for 27d from 23f
4 H. Galczenski, J. Hutchinson, D. J. Figueroa, J. Scheigetz, R. Young
and J. F. Evans, American Thoracic Society – 98th International
Conference (May 2002, Atlanta) [D38] [Poster F4] Characterization
of DUO-LT : A Novel Dual Cysteinyl Receptor Antagonist.
5 I. Fleming, Frontier Orbitals and Organic Chemical Reactions, Wiley,
New York, 1976, pp. 58.
6 Unstable 2-ethyl-3,5-diaminobenzo[b]furan obtained by reduction
of 2-ethyl-3,5-dinitrobenzo[b]furan with H2 over Pd/C in ethanol
easily gave 5-amino-2-ethyl-3(2H )-benzo[b]furanone by treatment
with HCl. On the other hand, catalytic reduction of 2-ethyl-3,5-
diaminobenzo[b]furan in acetic anhydride gave stable 2-ethyl-3,5-
diacetylaminobenzo[b]furan (unpublished data obtained in our
laboratory).
Catalytic reduction of 23e (0.8 g, 3.9 mmol) in a similar manner
as for 23a gave the crude amine (24e) as a yellow oil. trans-
Cinnamic acid chloride [prepared by treatment of trans-
cinnamic acid (0.76 g, 4.64 mmol) with SOCl2 (5 ml, 4.8 mmol)]
was added to a solution of the oil in anhydrous THF (30 ml).
After stirring at 65 ЊC for 3 h, work up in the usual way gave a
yellow oil which was purified on silica gel column chromato-
graphy [hexane–ethyl acetate (10 : 1)] to give 27c (0.73 g) as
colorless prisms.
7 Y. Ohishi and T. Nakanishi, Chem. Pharm. Bull., 1983, 31, 3418–
3423; Y. Ohishi, Y. Doi and T. Nakanishi, Chem. Pharm. Bull., 1984,
32, 4260–4270; Y. Ohishi, Y. Doi and T. Nakanishi, Chem. Pharm.
Bull., 1985, 33, 2598–2601; Y. Ohishi, K. Kuriyama, Y. Doi and
T. Nakanishi, Chem. Pharm. Bull., 1985, 33, 2854–2861; Y. Ohishi,
Y. Doi and T. Nakanishi, Chem. Pharm. Bull., 1988, 36, 1336–1341;
Y. Ohishi, T. Mukai, M. Nagahara, M. Yajima and N. Kajikawa,
Chem. Pharm. Bull., 1989, 37, 2398–2405.
8 C. P. Astles, J. T. Brown, F. Halley, M. C. Handscombe, V. N. Harris,
N. T. Majid, C. McCarthy, M. I. McLay, A. Morley, B. Porter,
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Chem., 2000, 43, 900–910.
9 K. Gewald and H. J. Kaensch, J. Prakt. Chem., 1973, 315, 779–785;
V. P. Baidya, S. B. Mahajam and Y. S. Agasimundin, Indian J. Chem.,
1981, 20B, 391–393.
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C.-J. Schmitges and C. Wilm, Bioorg. Med. Chem. Lett., 1999, 9,
619–622; (b) S. Rádl, P. Hezký, J. Urbánková, P. Váchal and
I. Krejcˇf, Collect. Czech. Chem. Commun., 2000, 65, 280–297;
(c) S. Rádl, P. Hezký, P. Konvicˇka and I. Krejcˇf, Collect. Czech.
Chem. Commun., 2000, 65, 1093–1108.
Calcium release experiments
Calcium mobilization assays were carried out using stably
transfected HEK 293T-cysLT2R or CHO-cysLT1R cells loaded
with Fura 2-AM fluorescent indicator dye (Molecular Probes,
Eugene, OR) in the fluorescent imaging plate reader system
(Hamamatsu Photonics, Japan). Briefly, the cells were seeded
in microtiter plate at 1.0 × 105 cells/well (HEK 293T cell) or
0.4 × 105 cells/well (CHO cell) and incubated at 37 ЊC for 24 h
(5% CO2). The cells were loaded with 7.5 µM Fura 2-AM
in Dulbecco’s modified Eagle’s medium (HEK 293T cell) or
F-12 medium (CHO cell) containing 10% fetal bovine serum,
20 mM HEPES (pH 7.4) and 2.5 mM probenecid at 37 ЊC
for 30 min. The cells were washed with Hanks’ balanced salt
solution containing 20 mM HEPES (pH 7.4). LTD4 (100 nM)
were added the cells, and fluorescence values were taken
at 3-second intervals for 90 seconds. Test compounds were
added 1 min before LTD4 (100 nM) stimulation, and the
LTD4 response was obtained by calculating peak fluorescence
values.
11 S. S. Sangapure and S. Y. Agasimundin, Indian J. Chem., 1976, 14B,
6886–6891.
12 G. Viti, D. Giannotti, R. Nannicini, R. Ricci and V. Pestellini,
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13 G. S. Harwalkar, S. S. Sangapure and Y. S. Agasimundin, Indian
J. Heterocycl. Chem., 1995, 4, 235–236.
Radioligand binding studies
Stably transfected HEK 293T-cysLT2R cells were grown and
harvested, and the membranes were prepared according to the
procedure reported by Obata et al.22 For competition binding
studies, the membranes (125 µg of total membrane protein)
were incubated with 1.0 nM [3H]LTD4 (Perking Elmer, Boston,
MA) and various concentrations of test compounds in assay
buffer (pH 7.4) containing 10 mM HEPES, 20 mM CaCl2 and
20 mM -penicillamine at room temperature for 1 h. The reac-
tions were stopped by ice-cold wash buffer (pH 7.4) containing
10 mM HEPES and 0.01% bovine serum albumin, and the
bound ligand was captured on Whatman GF/B filters. Radio-
activity was quantitated by liquid scintillation counter. Non-
specific binding was determined in the presence of 1 µM
unlabelled LTD4.
14 (a) P. D. Greenspan, A. J. Main, S. S. Bhagwat, L. I. Barsky, R. A.
Doti, A. R. Engle, L. M. Fery, H. Zhou, K. E. Lipson, M. H. Chin,
R. H. Jackson and S. Uziel–Fusi, Bioorg. Med. Chem. Lett., 1997, 7,
949–954; (b) P. D. Greenspan, R. A. Fujimoto, P. J. Marshall,
A. Raychaudhuri, K. F. Lipson, H. Zhou, R. A. Doti, D. E. Coppa,
L. Zhu, R. Pelletier, S. Uziel-Fusi, R. H. Jackson, M. H. Chin,
B. L. Kotyuk and J. J. Fitt, J. Med. Chem., 1999, 42, 164–172.
15 K. Takamura, A. Shioya, T. Yamamoto, S. Takama and Y. Nitta,
Chem. Pharm. Bull., 1965, 13, 211–217.
16 F. R. Heck, Organic Reactions, John Wiley & Sons Publishers, New
York, 1982, 27, pp 345–390.
17 5-Methylisoxazole-4-carboxlic acid was prepared starting with ethyl
acetoacetate in our laboratory [A. Graul and J. Castañer, Drugs of
the Future, 1998, 23, 827–837].
18 E. Kuo, P. Hambleton, D. Kay, P. Evans, S. Matharu, E. Little,
C. McDowall, B. Jones, C. Hedgecock, C. Yea, A. Chan, P. Hairsine,
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 6 2 5 – 6 3 5
634

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I. Ager, W. Tully, R. Williamson and R. Westwood, J. Med. Chem.,
1996, 39, 4608–4621.
V = 2581.5(7) Å3; Dcalc. = 1.341 g cmϪ3; F000 = 1072. CCDC reference
numbers 214287 and 222922. See http://www.rsc.org/suppdata/ob/
b3/b312682j/ for crystallographic data in .cif or other electronic
format.
19 Single crystals of 15b and 15u suitable for X-ray analysis were
obtained by very slow evaporation of the CHCl3 and acetonitrile–
CHCl3 solutions, respectively. All measurements were done on a
RIGAKU AFC7R diffractometer with filtered Cu–Kα (λ = 1.54178
Å) radiation and a rotating anode generator. 15b: C15H11Br1N2O4;
M = 363.17, approximate dimensions = 0.15 × 0.10 × 0.30 mm, space
group P21/n (#14), Z = 8 with a = 7.357(2) Å, b = 9.914(2) Å,
20 Some of compounds here fluoresced and could not be evaluated for
activity.
21 A simple control compound (29) was prepared by cleavage of the
isoxazole ring of 28 in our laboratory. M. Herrmann,
R. Schleyerbach and B. Kirschbaum, Immunopharmacology, 2000,
47, 273–289; F. C. Breedveid and J.-M. Dayer, Ann. Rheum. Dis.,
2000, 59, 841–849.
c = 40.665(1) Å, β = 94.22(1)Њ; V = 2958.0(9) Å3; Dcalc. = 1.631 g cmϪ3
;
F000 = 1456. 15u : C29H26N4O5; M = 510.55, approximate dimensions
= 0.20 × 0.03 × 0.20 mm, space group P21/n (#14), Z = 4 with
a = 17.709(3) Å, b = 7.553(2) Å, c = 19.656(2) Å, β = 100.926(8)Њ;
22 T. Obata, Y. Okada, M. Motoishi, N. Nakagawa, T. Terawaki and
H. Aishita, Jpn. J. Pharmacol., 1992, 60, 227–237.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 6 2 5 – 6 3 5
635