Synthesis of styrylbenzofuran derivatives as styrylquinoline analogues for HIV-1 integrase inhibitor

Article abstract of DOI:10.1016/j.farmac.2003.08.001

A series of styrylbenzofuran derivatives (8a-i) as styrylquinoline isosters were efficiently prepared by Wittig reaction and evaluated for inhibitory activity against HIV-1 integrase. In this series, compounds 8g, 8h and 8i containing a free catechol ring showed moderate inhibitory activities (IC₅₀=～40 μM) against HIV-1 integrase, while less than the corresponding styrylquinoline compound (I).

Full text of DOI:10.1016/j.farmac.2003.08.001

Il Farmaco 58 (2003) 1243ꢀ1250
/
www.elsevier.com/locate/farmac
Synthesis of styrylbenzofuran derivatives as styrylquinoline analogues
for HIV-1 integrase inhibitor
Ho Yoo a,b, Jae Yeol Lee a, Jang Hyun Park a, Bong Young Chung b,
Yong Sup Lee a,*
a Medicinal Chemistry Research Center, Life Sciences Division, Korea Institute of Science & Technology, P.O. Box 131 Cheongryang, Seoul 130-650,
South Korea
b Department of Chemistry, Korea University, 1-Anamdong, Seoul 136-701, South Korea
Received 19 June 2003; accepted 11 August 2003
Abstract
A series of styrylbenzofuran derivatives (8aꢀ/i) as styrylquinoline isosters were efficiently prepared by Wittig reaction and
evaluated for inhibitory activity against HIV-1 integrase. In this series, compounds 8g, 8h and 8i containing a free catechol ring
showed moderate inhibitory activities (IC50
compound (I).
ꢁ
/
ꢀ40 mM) against HIV-1 integrase, while less than the corresponding styrylquinoline
/
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: AIDS; HIV; Integrase inhibitor; Styrylbenzofuran; Wittig reaction
1. Introduction
the styrylquinoline derivative (I) as shown in Fig. 1 is a
potent HIV-1 integrase inhibitor in in vitro experiments,
blocks the replication of HIV-1 in cell culture, and is
devoid of cytotoxicity [8].
AIDS is essentially a viral disease and should be
treated by antiretroviral agents [1,2]. From this stand-
point, HIV DNA integration into genomic DNA of the
host cell, a crucial step in the life cycle of the virus,
constitutes a particularly attractive target for AIDS
chemotherapeutics, including potential synergy with
currently available HIV reverse transcriptase and pro-
tease inhibitors [3,4]. HIV-1 IN is currently recognized
as an attractive and safe target against AIDS [5],
particularly because it is essential in the replication of
HIV-1 and there are no similar enzymes involved in
human cellular function.
Therefore, a large number of IN inhibitors have been
described for the past 13 years and many inhibitors
exhibited potent inhibition of integrase in extracellular
assays. However, most of them failed to provide anti-
viral efficacy in HIV-infected cells except the recent
5CITEP [6] and L-708,906 [7], which enter trial in
human volunteers. It was additionally reported that
In our continuous efforts to discover new HIV-1
integrase inhibitor [9], we decided to modify the basic
scaffold of styrylquinoline compound as styrylbenzo-
furan compound in order to investigate the effect of its
core structure on the inhibitory activity against the HIV-
1 integrase. The present paper describes the synthesis
and evaluation of styrylbenzofuran derivatives as styr-
ylquinoline isosters.
2. Results and discussion
2.1. Chemistry
As shown in Scheme 1, 7-methoxy-2-methylbenzo-
furan (2) was prepared by cyclization reaction of 6-allyl-
2-methoxyphenol (1) with iodine in the presence of
DBU as a base [10]. After demethylation of compound 2
with pyridine hydrochloride [11], the obtained com-
pound 3 was subjected to Kolbeꢀ
(K2CO3, CO2 (gas), 285 8C) to afford 7-hydroxy-2-
/
Schmit condition
* Corresponding author.
E-mail address: yslee@kist.re.kr (Y.S. Lee).
´
0014-827X/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2003.08.001

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H. Yoo et al. / Il Farmaco 58 (2003) 1243ꢀ1250
/
Fig. 1. Styrylquinoline (I) and styrylbenzofuran derivatives (8aꢀi).
/
methyl-benzofuran-6-carboxylic acid (4) in 54% yield
[12].
the hydrolysis of 8b by LiOH [19] for the deprotection of
only methyl ester group of compound 8b. The results of
all reactions were summarized in Table 1.
In order to test the inhibitory effect of substituents of
benzofuran ring against HIV-integrase, both functional
groups of compound 4 were protected by two different
procedures: CH3I, K2CO3 and TBDPSCl, imidazole,
respectively [13,14]. Allylic positions of compound 5a
and 5b were halogenated to afford compounds 6a and
6b, respectively in 75 and 91% yields by treating them
with N-bromosuccinimde (NBS) and benzoyl peroxide
[15]. The phosphonium salt 7a for the Wittig reaction
was prepared from the reaction of 6a with triphenylpho-
sphine in acetonitrile [16]. For compound 7b containing
free salicylic moiety, on the other hand, the desilylation
of 6b with TBAF was followed by the subsequent
treatment of Ph3P to provide the compound 7b in 50%
yield [17] (Scheme 1).
2.2. Biological activity
The prepared styrylbenzofuran derivatives (8aꢀi)
/
were in vitro screened for inhibitory activity against
HIV-1 IN in 3?-processing reaction and the resultant
anti-HIV integrase activity was summarized in Table 1.
For the comparison of biological activity, styrylquino-
line compound (I) as a standard compound was
synthesized and tested for its inhibitory activity under
our assay conditions, and its data (IC50ꢁ
mM) was inserted in Table 1.
A shown in Table 1, at first glance, these compounds
can be roughly classified as inactive (8aꢀf) and active
(8gꢀi). As a structural characteristic based on the
inhibitory activity, all compounds (8gꢀi) bearing a free
/
18.99
/
10.8
/
Styrylbenzofuran derivatives (8aꢀ
/
8c, 8f, 8hꢀ8i) with
/
/
E geometry were prepared through Wittig reaction
between triphenylphosphonium salts (7a or 7b) and
various benzaldehydes under the basic condition, as
shown in Scheme 2 [16]. Compounds 8e and 8g were also
prepared by demethylation from 8b and 8h, respectively,
with 1 M BBr3 [18]. Finally, 8d was prepared through
/
cathecol ring again have moderate effects on anti-HIV-1
IN activity with IC50 values ranging from 42.9 to 44.1
mM, though two fold less than the standard compound
(I, IC 50ꢁ
containing monohydroxy- (8dꢀ
/
18.99
/
10.8 mM). However, other compounds
/
f), monomethoxy- (8a)
Scheme 1. Synthesis of intermediates (7a and 7b) for the Wittig reaction.

H. Yoo et al. / Il Farmaco 58 (2003) 1243ꢀ
/
1250
1245
Scheme 2. Synthesis of styrylbenzofurans (8aꢀi).
/
Table 1
HIV-1 Inhibitory activities of styrylbenzofurans (8aꢀ
/i)
or dimethoxy (8c)-substituented phenyl ring showed the
complete lack of inhibitory potency (IC50ꢁ 100 mM),
icylic moiety is not always required for the biological
activity.
In conclusion, a series of styrylbenzofuran derivatives
/
ꢁ
/
irrespective of the presence of a free salicylic moiety
(ortho hydroxyl and carboxyl pattern of the parent
benzofuran ring). This result is very remarkable in that
in the case of standard compound, styrylquinoline (I),
both of a salicylic pattern in the quinoline and a cathecol
in the subunit are required for the inhibitory activity
against anti-HIV-1 IN [8].
When compared the activities of compound 8g and 8h
with that of 8i, it seems that the free cathcol moiety in
subunit of benzofuran derivatives plays more important
role than its salicylic moiety for 3?-processing inhibition
against HIV-1 IN. This result implies that in the case of
styrylbenzofuran derivatives, the presence of free sal-
(8aꢀ
inhibitory activities as styrylquinoline isosters. Among
them, styrylbenzofuran derivatives (8gꢀi) bearing free
/i) were prepared and evaluated for the HIV-1 IN
/
cathechol ring showed comparable inhibitory activities
to styrylquinoline (I) against HIV-1 IN in 3?-processing
reaction. Also, it were concluded that the salicylic
moiety in benzofuran derivatives don’t have any im-
portant role in binding mode for the inhibitory activity
against HIV-1 IN in 3?-processing reaction, but the free
catechol moiety at phenyl ring is required for the
inhibitory activity. This results would show the possibi-
lity that styrylbenzofuran derivatives have different

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H. Yoo et al. / Il Farmaco 58 (2003) 1243ꢀ1250
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binding modes from that of styrylquinoline in the active
site of the HIV-1 IN [8].
NMR (CD3OD) d 156.2, 144.6, 142.8, 132.3, 124.0,
112.4, 110.5, 103.8, 14.3.
3.1.3. 7-Hydroxy-2-methylbenzofuran-6-carboxylic acid
(4)
3. Experimental
The mixture of compound 3 (5 g, 33.7 mmol),
potassium carbonate (9.3 g, 67.5 mmol) and CO2 gas
(10 atm) in the autoclave was heated to 285 8C (15 atm).
This condition was kept for 6 h. After cooling to r.t., the
reaction mixture was diluted with water and washed out
with EtOAc. The aqueous layer was acidified by 3 N
HCl to pH 3 and extracted with diethyl ether. The
organic layer was dried over MgSO4, filtered and
concentrated to give 4 (3.5 g, 54%) as a yellow solid:
3.1. Chemistry: general
1H- and 13C NMR spectra were recorded on a Gemini
Varian-300 (300 and 75 MHz, respectively). Mass
spectra (EI) were determined on HP GC 5972 and HP
MS 5988A system at 70 eV. Melting points (m.p.) were
determined on a ThomasꢀHoover capillary melting
/
apparatus and are uncorrected. Infrared (IR) spectra
were recorded on Perkin Elmer 16F-PC FT-IR and
MIDAC 101025 using a potassium bromide pellet.
Analytical thin layer chromatographies (TLC) were
carried out by precoated silica gel (E. Merck Kiesegel
60F254 layer thickness 0.25 mm). Flash column chroma-
tographies were performed with Merck Kiesegel 60 Art
m.p. 225ꢀ227 8C; IR (KBr) 2986, 2572, 1662, 1438, 1298
/
1
cmꢂ1; H NMR (CD3OD) d 7.64 (1H, d, Jꢁ
benzofuranꢀH5), 6.95 (1H, d, Jꢁ9.0 Hz, benzofuranꢀ
H4), 6.44 (1H, s, benzofuranꢀH3), 2.46 (3H, s,
benzofuranꢀ 174.4,
CH3); 13C NMR (CD3OD)
/
9.0 Hz,
/
/
/
/
/
d
160.3, 149.2, 143.7, 137.7, 125.3, 111.5, 108.4, 104.6,
14.0.
9385 (230ꢀ400 mesh). All solvents used were purified
/
according to standard procedures.
3.1.4. Methyl 7-methoxy-2-methylbenzofuran-6-
carboxylate (5a)
3.1.1. 7-Methoxy 2-methylbenzofuran (2)
To a solution of 6-allyl-2-methoxyphenol (1, 15 g,
91.4 mmol) in 100 ml of acetonitrile was added iodine
(23.2 g, 182.8 mmol) in the dark. The mixture was
stirred for 27 h at room temperature (r.t.). The reaction
mixture was diluted with 100 ml of CH2Cl2 and treated
with DBU (68.0 ml, 457.0 mmol). After being stirred for
20 h at r.t., the reaction mixture was extracted with
CHCl3. The organic layer was washed with aqueous
Na2S2O3, dried over MgSO4, filtered and concentrated.
The residue was purified by flash column chromatogra-
To a solution of compound 4 (280 mg, 1.4 mmol) in
30 ml of acetone was added potassium carbonate (667
mg, 4.9 mmol) and iodomethane (0.2 ml, 3.5 mmol). The
reaction mixture was heated under reflux for 2 h. The
mixture was concentrated and washed with CH2Cl2. The
organic layer was concentrated and purified by flash
column chromatography (EtOAc:n-hexaneꢁ1:6) to
/
give 5a (250 mg, 99%) as an oil; IR (KBr) 2922, 1726,
1602, 1486, 1238, 1080 cmꢂ1; 1H NMR (CDCl3) d 7.56
(1H, d, Jꢁ
8.4 Hz, benzofuranꢀ
4.12 (3H, s, benzofuranꢀ
/
8.4 Hz, benzofuranꢀ
H4), 6.25 (1H, s, benzofuranꢀ
CO2CH3), 3.81 (3H, s,
/
H5), 7.02 (1H, d, Jꢁ
/
phy (EtOAc:n-hexaneꢁ
an oil: IR (KBr) 2950, 1614, 1492, 1272, 1096 cmꢂ1; 1H
NMR (CD3OD) d 7.00ꢀ6.98 (2H, m, benzofuranꢀ
H4,H6), 6.66ꢀ6.63 (1H, m, benzofuranꢀH5), 6.27
(1H, s, benzofuranꢀH3), 3.83 (3H, s, benzofuranꢀ
OCH3), 2.31 (3H, s, benzofuranꢀ
CH3); 13C NMR
/1:20) to give 2 (8.4 g, 57%) as
/
/H3),
/
/
/
benzofuranꢀ
/
OCH3), 2.35 (3H, s, benzofuranꢀCH3);
/
/
/
13C NMR (CDCl3) d 167.0, 158.4, 146.2, 145.5, 135.6,
/
/
125.8, 117.3, 114.0, 103.5, 61.5, 52.0, 14.2.
/
(CD3OD) d 159.4, 149.1, 148.0, 135.1, 127.1, 116.5,
109.7, 106.8, 59.2, 16.8.
3.1.5. Methyl 2-bromomethyl-7-methoxybenzofuran-6-
carboxylate (6a)
To a solution of compound 5a (100 mg, 0.5 mmol) in
5 ml of boiling benzene was added benzoyl peroxide (50
mg, 0.3 mmol) and NBS (120 mg, 0.7 mmol). The
reaction mixture was heated under reflux for 2 h. After
cooling to r.t., the mixture was concentrated and
purified by flash column chromatography (EtOAc:n-
3.1.2. 7-Hydroxy-2-methylbenzofuran (3)
The mixture of compound 2 (3.2 g, 19.7 mmol) and
pyridine hydrochloride (6.8 g, 59.7 mmol) was heated
under reflux for 2 h at 210 8C. After cooling to r.t., the
reaction mixture was neutralized with 3 N HCl and
extracted with diethyl ether. The organic layer was dried
over MgSO4, filtered and concentrated. The residue was
purified by flash column chromatography (EtOAc:n-
hexaneꢁ
(KBr) 2924, 1722, 1614, 1456, 1292, 1236 cmꢂ1
NMR (CDCl3) d 7.71 (1H, d, Jꢁ8.1 Hz, benzofuranꢀ
H5), 7.24 (1H, d, Jꢁ 8.1 Hz, benzofuranꢀH4), 6.79
(1H, s, benzofuranꢀH3), 4.61 (2H, s, benzofuranꢀ
CH2Br), 4.27 (3H, s, benzofuranꢀCO2CH3), 3.95 (3H,
s, benzofuranꢀ
OCH3); 13C NMR (CDCl3) d 167.7,
159.6, 146.9, 127.1, 126.8, 117.5, 101.8, 60.9, 51.8, 22.0.
/
1:8) to give 6a (39 mg, 75%) as an oil; IR
;
1H
/
/
hexaneꢁ
3394, 2924, 1608, 1452, 1196, 1046 cmꢂ1
(CD3OD) d 6.94ꢀ6.91 (2H, m, benzofuranꢀ
6.63ꢀ6.61 (1H, m, benzofuranꢀH5), 6.35 (1H, s,
benzofuranꢀH3), 2.42 (3H, s, benzofuranꢀ
CH3); 13C
/
1:3) to give 3 (2.5 g, 86%) as an oil: IR (KBr)
1H NMR
H4,H6),
/
/
;
/
/
/
/
/
/
/
/
/
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H. Yoo et al. / Il Farmaco 58 (2003) 1243ꢀ
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3.1.6. Methyl 7-methoxy-2-methylbenzofuran-6-
135.8, 135.7, 135.6, 134.1, 133.4, 132.6, 130.1, 129.6,
127.9, 127.5, 126.6, 113.2, 106.3, 27.5, 26.8, 22.1, 20.2,
19.7.
carboxylate triphenylphosphonium bromide salt (7a)
To a solution of compound 6a (250 mg, 0.8 mmol) in
15 ml of acetonitrile was added triphenylphosphine (438
mg, 1.7 mmol). The reaction mixture was heated under
reflux for 2 h, cooled to r.t., and concentrated. The
resultant residue was solidified with diethyl ether to give
3.1.9. 7-Hydroxy-2-methylbenzofuran-6-carboxylic acid
triphenylphosphonium bromide salt (7b)
To a solution of compound 6b (500 mg, 0.7 mmol) in
10 ml of THF was added a solution of TBAF (1M
solution, 2.0 ml) in THF at 0 8C. The reaction mixture
was stirred for 40 min at 0 8C and concentrated in
vacuo. The resulting oily residue was diluted in 20 ml of
acetonitrile and treated with triphenylphosphine (702
mg, 2.6 mmol) at r.t. The reaction mixture was heated
under reflux for 5 h, treated with 10 ml of water, and
extracted with CH2Cl2. The organic layer was washed
with brine, dried over MgSO4, filtered and concentrated.
The residue was solidified with n-hexane to give 7b (180
7a (390 mg, 83%) as a solid: m.p. 201ꢀ
2920, 1718, 1614, 1444, 1288, 1130 cmꢂ1
(CDCl3) d 7.88ꢀ7.60 (16H, m, PPh3ꢀH), 7.57 (1H, d,
Jꢁ8.1 Hz, benzofuranꢀ
H3), 7.06 (1H, d, Jꢁ8.1 Hz, benzofuranꢀ
(2H, d, Jꢁ14.4 Hz, benzofuranꢀ
benzofuranꢀ
/
203 8C; IR (KBr)
;
1H NMR
/
/
/
/
H5), 7.22 (1H, s, benzofuranꢀ
/
/
/
H4), 6.13
/
/CH2PPh3), 3.88 (3H, s,
/
CO2CH3) 3.72 (3H, s, benzofuranꢀ
/
OCH3);
13C NMR (CDCl3) d 167.9, 149.3, 149.2, 147.1, 146.3,
136.6, 135.2, 133.7, 133.0, 132.9, 131.5, 130.0, 129.9,
127.2, 119.7, 119.4, 118.3, 115.8, 111.1, 111.0, 61.7, 52.7.
mg, 50%) as a solid: m.p. 223ꢀ225 8C; IR (KBr) 3414,
/
1
3.1.7. t-Butyldiphenylsilyl 7-t-butyldiphenylsilyloxy-2-
methylbenzofuran-6-carboxylate (5b)
1660, 1444, 1292, 1024 cmꢂ1; H NMR (CD3OD) d
7.8ꢀ7.69 (16H, m, benzofuranꢀH5,
triphenylphosphineꢀ15H), 6.81 (1H, d, Jꢁ8.4 Hz,
benzofuranꢀH4), 6.68 (1H, s, benzofuranꢀH3), 5.37
(2H, d, Jꢁ14.4 Hz, benzofuranꢀCH2ꢀ
PPh3); 13C
/
/
To a solution of compound 4 (500 mg, 2.6 mmol) in
10 ml of DMF was added TBDPSCl (2.0 ml, 7.8 mmol)
and imidazole (780 mg, 10.4 mmol). The reaction
mixture was stirred for 24 hr at r.t., diluted with a
mixture of 100 ml of EtOAc and 100 ml of water, and
extracted with EtOAc. The organic layer was washed
with aqueous NaHCO3. The organic layer was dried
over MgSO4, filtered and concentrated. The residue was
solidified with diethyl ether to give 5b (1.3 g, 72%) as a
/
/
/
/
/
/
/
NMR (CD3OD) d 176.0, 153.9, 149.3, 148.2, 148.0,
144.8, 136.6, 135.2, 135.0, 134.2, 133.0, 131.5, 131.4,
130.0, 126.7, 119.6, 118.5, 114.4, 111.1, 111.0, 110.8,
53.8.
3.2. General procedure A for Wittig reaction between 7a
or 7b and the various aromatic aldehydes
solid: m.p. 146ꢀ
1476, 1282, 1118 cmꢂ1; H NMR (CDCl3) d 7.99ꢀ
(9H, m, benzofuranꢀH5, TBDPSꢀphenyl), 7.52ꢀ
(12H, m, TBDPSꢀphenyl), 7.11 (1H, d, Jꢁ
benzofuranꢀH4), 6.18 (1H, s, benzofuranꢀH3), 1.89
(3H, s, benzofuranꢀCH3), 1.33 (9H, s, TBDPSꢀCH3)
1.15 (9H, s, TBDPSꢀ
CH3); 13C NMR (CDCl3) d 164.2,
/
147 8C; IR (KBr) 2936, 1708, 1606,
1
/
7.84
/
/
/
7.32
To a solution of 7a or 7b in toluene was added
triethylamine (2.5 equiv.) and the various aromatic
aldehydes (2.0 equiv.). The reaction mixture was heated
/
/
8.2 Hz
/
/
/
/
under reflux for 2ꢀ4 h. The mixture was diluted with
/
/
H2O and extracted with CH2Cl2. The organic layer was
dried over MgSO4, filtered and concentrated. The
residue was purified by flash column chromatography
157.7, 135.9, 135.6, 135.1, 134.9, 134.5, 132.8, 130.1,
129.5, 127.9, 127.4, 126.2, 112.2, 103.0, 27.5, 26.9, 20.3,
19.7, 13.7.
(EtOAc:n-hexaneꢁ
/
1:1ꢀ1:7) to give various styrylben-
/
zofurans (8aꢀ8c, 8f, 8h, 8i)
/
3.1.8. t-Butyldiphenylsilyl 7-t-butyldiphenylsilyloxy-2-
bromomethylbenzofuran-6-carboxylate (6b)
3.2.1. Methyl (E)-7-methoxy-2-[2-(4-
To a solution of compound 5b (1.1 g, 1.7 mmol) in
150 ml of boiling benzene was added benzoyl peroxide
(205.9 mg, 0.8 mmol) and NBS (450.3 mg, 2.5 mmol).
The reaction mixture was heated under reflux for 2 h
and cooled to r.t. The mixture was concentrated and the
resultant residue was solidified with n-hexane to give 6b
methoxyphenyl)ethenyl]-6-benzofurancarboxylate (8a)
Procedure A using 7a (200 mg, 0.36 mmol), triethy-
lamine (0.12 ml, 0.89 mmol) and 4-methoxybenzalde-
hyde (92 mg, 0.72 mmol) in toluene (5 ml) afforded 8a
(63 mg, 52%) as a solid: m.p. 75ꢀ
1720, 1602, 1418, 1260 cmꢂ1; 1H NMR (CDCl3) d 7.69
(1H, d, Jꢁ8.4 Hz, benzofuranꢀH5), 7.49 (2H, d, Jꢁ
6.9 Hz, phneylꢀH2, H6), 7.34 (1H, d, Jꢁ 16.5 Hz, ꢀ
CH ÄCHꢀphenyl), 7.19 (1H, d, Jꢁ8.4 Hz, benzofuranꢀ
H4), 6.93 (2H, d, Jꢁ6.9 Hz, phneylꢀH3, H5), 6.88
(1H, d, Jꢁ16.5 Hz, ꢀCHÄCH ꢀphenyl), 6.60 (1H, s,
benzofuranꢀH3), 4.31 (3H, s, CO2CH3), 3.93 (3H, s,
benzofuranꢀOCH3), 3.83 (3H, s, phenylꢀ
OCH3); 13C
NMR (DMSO) d 166.0,159.8, 157.7, 145.3, 144.5, 134.9,
/
77 8C; IR (KBr) 2940,
(1.2 g, 91%) as a solid: m.p. 154ꢀ
1712, 1610, 1476, 1278, 1124 cmꢂ1; 1H NMR (CDCl3) d
7.93 (1H, d, Jꢁ8.1 Hz, benzofuranꢀH5), 7.87ꢀ7.78
(8H, m, TBDPSꢀphenyl), 7.48ꢀ7.28 (12H, m, TBDPSꢀ
phenyl), 7.13 (1H, d, Jꢁ8.1 Hz, benzofuranꢀH4), 6.52
(1H, s, benzofuranꢀH3), 3.90 (2H, s, benzofuranꢀ
CH2), 1.25 (9H, s, TBDPSꢀCH3), 1.07 (9H, s,
TBDPSꢀ
CH3); 13C NMR (CDCl3) d 162.8, 157.0,
/
155 8C; IR (KBr) 2936,
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/

1248
H. Yoo et al. / Il Farmaco 58 (2003) 1243ꢀ1250
/
131.5, 128.6, 128.5, 125.5, 117.9, 114.8, 114.3, 113.7,
104.6, 61.4, 55.2, 52.0.
3.2.5. Methyl (E)-7-methoxy-2-[2-(3,4-
dihydroxyphenyl)ethenyl]-6-benzofurancarboxylate (8h)
Procedure A using 7a (200 mg, 0.36 mmol), triethy-
lamine (0.12 ml, 0.89 mmol) and 3,4-dihydroxybenzal-
dehyde (99 mg, 0.72 mmol) in toluene (7 ml) afforded 8h
3.2.2. Methyl (E)-7-methoxy-2-[2-(4-
hydroxyphenyl)ethenyl]-6-benzofurancarboxylate (8b)
Procedure A using 7a (200 mg, 0.36 mmol), triethy-
lamine (0.12 ml, 0.89 mmol) and 4-hydroxybenzalde-
hyde (88 mg, 0.72 mmol) in toluene (5 ml) afforded 8b
(90 mg, 73%) as a solid: m.p. 193ꢀ
/
194 8C; IR (KBr)
3396, 1588, 1442, 1230 cmꢂ1; H NMR (CD3OD) d
7.67 (1H, d, Jꢁ8.2 Hz, benzofuranꢀH5), 7.30 (1H, d,
Jꢁ16.3 Hz, ꢀCH ÄCHꢀphenyl), 7.27 (1H, d, Jꢁ8.1
Hz, phneylꢀH5), 7.11 (1H, s, phneylꢀH2), 7.00 (1H, d,
Jꢁ8.1 Hz, phneylꢀH6), 6.97 (1H, d, Jꢁ16.3 Hz, ꢀ
CHÄCH ꢀphenyl), 6.85 (1H, d, Jꢁ8.2 Hz, benzofuranꢀ
H4), 6.77 (1H, s, benzofuranꢀH3), 4.28 (3H, s,
CO2CH3), 3.94 (3H, s, benzofuranꢀ
OCH3); 13C NMR
1
/
/
/
/
/
/
/
(100 mg, 86%) as a solid: m.p. 185ꢀ
3356, 2948, 1694, 1432, 1272 cmꢂ1; 1H NMR (CD3OD)
d 7.63 (1H, d, Jꢁ8.1 Hz, benzofuranꢀH5), 7.48 (2H, d,
Jꢁ6.9 Hz, phneylꢀH2, H6), 7.34 (1H, d, Jꢁ15.9 Hz,
CH ÄCHꢀphenyl), 7.24 (1H, d, Jꢁ8.1 Hz,
benzofuranꢀH4), 6.99 (1H, d, Jꢁ15.9 Hz, ꢀCHÄ
CH ꢀphenyl), 6.83 (2H, d, Jꢁ6.9 Hz, phneylꢀH3,
H5), 6.74 (1H, s, benzofuranꢀH3), 4.26 (3H, s,
CO2CH3), 3.91 (3H, s, benzofuranꢀ
OCH3); 13C NMR
/
186 8C; IR (KBr)
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
ꢀ
/
/
/
/
/
/
/
/
/
(DMSO) d 166.0, 158.0, 146.8, 145.6, 145.3, 144.5,
135.1, 132.4, 127.4, 125.6, 119.7, 117.8, 115.8, 114.7,
113.7, 112.6, 104.1, 61.4, 52.0.
/
/
/
/
/
(DMSO) d 166.7, 159.1, 158.7, 146.0, 145.2, 135.8,
132.7, 129.4, 127.6, 126.3, 118.6, 116.5, 115.4, 113.4,
104.8, 62.1, 52.7.
3.2.6. (E)-7-Hydroxy-2-[2-(3,4-
dihydroxyphenyl)ethenyl]-6-benzofurancarboxylic acid
(8i)
Procedure A using 7b (80 mg, 0.15 mmol), triethyla-
mine (0.04 ml, 0.30 mmol) and 3,4-dihydroxybenzalde-
hyde (41 mg, 0.30 mmol) in toluene (4 ml) afforded 8i
(10 mg, 20%) as a solid: m.p. 250 8C (dec.); IR (KBr)
3376, 1620, 1590, 1444, 1298 cmꢂ1; 1H NMR (CD3OD)
3.2.3. Methyl (E)-7-methoxy-2-[2-(3,4-
dimethoxyphenyl)ethenyl]-6-benzofurancarboxylate (8c)
Procedure A using 7a (200 mg, 0.36 mmol), triethy-
lamine (0.12 ml, 0.89 mmol) and 3,4-dimethoxybenzal-
dehyde (119.6 mg, 0.72 mmol) in toluene (7 ml) afforded
d 7.62 (1H, d, Jꢁ
Jꢁ16.5 Hz, ꢀCH Ä
H2), 6.87 (3H, m, benzofuranꢀ
CH ꢀphenyl), 6.72 (1H, d, Jꢁ8.1 Hz, benzofuranꢀ
6.62 (1H, s, benzofuranꢀ
H3); 13C NMR (CD3OD,
/
8.1 Hz, benzofuranꢀ
CHꢀphenyl), 6.98 (1H, s, phneylꢀ
H4, phenylꢀH6, ꢀCHÄ
H4),
/
H5), 7.15 (1H, d,
8c (100 mg, 75%) as a solid: m.p. 117ꢀ
2950, 1716, 1456, 1268, 1006 cmꢂ1; 1H NMR (CDCl3) d
7.65 (1H, d, Jꢁ8.1 Hz, benzofuranꢀH5), 7.27 (1H, d,
Jꢁ16.2 Hz, ꢀCH ÄCHꢀphenyl), 7.14 (1H, d, Jꢁ8.4
Hz, phneylꢀH5), 7.06 (1H, d, Jꢁ8.4 Hz, phneylꢀH6),
7.03 (1H, s, phneylꢀH2), 6.84 (1H, d, Jꢁ16.2 Hz, ꢀ
CHÄCH ꢀphenyl), 6.83 (1H, d, Jꢁ8.1 Hz, benzofuranꢀ
H4), 6.57 (1H, s, benzofuranꢀH3), 4.27 (3H, s,
CO2CH3), 3.91 (3H, s, benzofuranꢀOCH3), 3.89 (3H,
s, phenylꢀOCH3), 3.87 (3H, s, phenylꢀ
OCH3); 13C
/
118 8C; IR (KBr)
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
DMSO) d 174.7, 156.7, 145.8, 145.0, 142.4, 133.4,
130.6, 128.0, 124.7, 119.1, 115.2, 113.9, 112.9, 112.7,
108.4, 104.0.
/
/
/
/
/
/
/
/
/
3.3. General procedure B for demethylation
/
/
NMR (DMSO) d 166.7, 158.5, 150.4, 149.7, 146.1,
145.2, 135.7, 132.7, 129.5, 126.3, 121.7, 118.7, 115.5,
114.6, 112.4, 110.3, 105.2, 62.1, 56.2, 56.1, 52.7.
To a solution of 8b or 8h in CH2Cl2 was slowly added
1 M BBr3 at ꢂ78 8C. The reaction mixture was stirred
/
for 5 h at r.t. The mixture was quenched by an addition
of MeOH, concentrated and purified by flash column
3.2.4. (E)-7-Hydroxy-2-[2-(4-hydroxyphenyl)ethenyl]-
6-benzofurancarboxylic acid (8f)
chromatography (EtOAc:n-hexaneꢁ1:5 and 2:1) to
give styrylbenzofurans (8e, 8g).
/
Procedure A using 7b (85 mg, 0.16 mmol), triethyla-
mine (0.04 ml, 0.32 mmol) and 4-hydroxybenzaldehyde
(39 mg, 0.32 mmol) in toluene (3 ml) afforded 8f (25 mg,
53%) as a solid: m.p. 227 8C (dec.); IR (KBr) 3482, 3298,
3.3.1. Methyl (E)-7-hydroxy-2-[2-(4-
hydroxyphenyl)ethenyl]-6-benzofurancarboxylate (8e)
Procedure B using 8b (100 mg, 0.31 mmol) and 1 M
BBr3 (0.6 ml) in CH2Cl2 (10 ml) afforded 8e (50 mg,
1
1704, 1604, 1438, 1276 cmꢂ1; H NMR (CD3OD) d
7.72 (1H, d, Jꢁ
Jꢁ8.7 Hz, phneylꢀ
CH ÄCHꢀphenyl), 6.98 (1H, d, Jꢁ
CH ꢀphenyl), 6.93 (1H, d, Jꢁ8.4 Hz, benzofuranꢀ
H4), 6.83 (2H, d, Jꢁ8.7 Hz, phneylꢀH3, H5), 6.66
(1H, s, benzofuranꢀ
H3); 13C NMR (DMSO) d 177.8,
/
8.4 Hz, benzofuranꢀ
H2, H6), 7.36 (1H, d, Jꢁ
15.9 Hz, ꢀ
/
H5), 7.47 (2H, d,
15.9 Hz,
CHÄ
52%) as a solid: m.p. 235ꢀ
/
236 8C; IR (KBr) 3382, 1668,
1436, 1296 cmꢂ1; H NMR (CD3OD) d 7.70 (1H, d,
Jꢁ8.1 Hz, benzofuranꢀH5), 7.49 (2H, d, Jꢁ8.7 Hz,
phneylꢀH2, H6), 7.35 (1H, d, Jꢁ15.9 Hz, ꢀCH ÄCHꢀ
phenyl), 7.26 (1H, d, Jꢁ8.1 Hz, benzofuranꢀH4), 7.01
(1H, d, Jꢁ15.9 Hz, ꢀCHÄCH ꢀphenyl), 6.84 (2H, d,
Jꢁ8.7 Hz, phneylꢀH3, H5), 6.76 (1H, s, benzofuranꢀ
H3), 3.97 (3H, s, benzofuranꢀ
CO2CH3); 13C NMR
1
/
/
/
ꢀ
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
163.4, 161.3, 156.7, 148.9, 137.8,135.1, 133.7, 132.8,
130.3, 121.2, 120.5, 119.1, 111.7, 110.3.
/
/
/
/

H. Yoo et al. / Il Farmaco 58 (2003) 1243ꢀ
/
1250
1249
(DMSO) d 170.9, 159.4, 159.2, 147.2, 142.6, 136.7,
133.0, 129.6, 127.8, 125.0, 116.6, 113.6, 112.4, 108.6,
105.4, 53.3.
strand), 5?-ACTGCTAGAGATTTTCCACA-3?. The
oligonucleotides were purified by 20% polyacrylamide
gel before use. In order to construct oligonucleotide
substrate, oligonucleotide K16 of 30 pmol was labeled at
the 5? end using [-32P]-ATP of 250 Ci (3000 Ci/mmol; 1
3.3.2. Methyl (E)-7-hydroxy-2-[2-(3,4-
dihydroxyphenyl)ethenyl]-6-benzofurancarboxylate (8g)
Procedure B using 8d (110 mg, 0.32 mmol) and 1M
BBr3 (0.65 ml) in CH2Cl2 (10 ml) afforded 8g (50 mg,
Ciꢁ
(T4 PNK, New England Biolabs) of 10 U in 40 l of
reaction buffer (70 mM TrisꢀHCl [pH 7.6], 10 mM
/
37 GBq; Amersham) and T4 polynucleotide kinase
/
42%) as a solid: m.p. 203ꢀ
1672, 1438, 1298 cmꢂ1; 1H NMR (CD3OD) d 7.60 (1H,
d, Jꢁ8.4 Hz, benzofuranꢀH5), 7.26 (1H, d, Jꢁ16.2
Hz, ꢀCH ÄCHꢀphenyl), 7.04 (1H, s, phneylꢀH2), 6.99
(1H, d, Jꢁ8.4 Hz, phneylꢀH5), 6.92 (1H, d, Jꢁ8.4 Hz,
phneylꢀH6), 6.84 (1H, d, Jꢁ16.2 Hz, ꢀCHÄCH ꢀ
phenyl), 6.78 (1H, d, Jꢁ8.4 Hz, benzofuranꢀH4),
6.62 (1H, s, benzofuranꢀH3), 3.93 (3H, s,
benzofuranꢀ
CO2CH3); 13C NMR (DMSO) d 170.8,
/
205 8C; IR (KBr) 3404, 2952,
MgCl2, 5 mM dithiothreitol) at 37 8C for 15 min. The
labeling reaction was subjected to 10 mM EDTA, and
heated to 85 8C for 15 min to inactivate T4 PNK. After
addition of complementary oligonucleotide K17 of 30
pmol, the reaction mixture was boiled for 3 min and
cooled down slowly. Labeled substrate was separated
from unincorporated nucleotide by passage through a
Biospin 6 (Bio-Rad).
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
159.3, 147.6, 147.0, 146.3, 142.4, 136.6, 133.2, 128.1,
124.9, 120.5, 116.5, 114.4, 113.3, 112.2, 108.5, 105.2,
53.2.
3.4.3. HIV-1 integrase reaction
A standard reaction assay of the endonucleolytic
activity was carried out in the presence of potential
inhibitor containing 0.1 pmol of duplex oligonucleotide
substrate and 15 pmol of HIV-1 integrase in 15 mM
3.3.3. (E)-7-Methoxy-2-[2-(4-hydroxyphenyl)ethenyl]-
6-benzofurancarboxylic acid (8d)
To a solution of 8b (100 mg, 0.31 mmol) in mixed
TrisꢀHCl (pH 7.4), 100 mM NaCl, 1 mM MnCl2, 2 mM
/
2-mercaptoethanol, 2.5 mM CHAPS, 0.1 mM EDTA,
0.1 mM PMSF, 1% glycerol, and 10 mM imidazole in a
total volume of 10 ml. Inhibitors or drugs were dissolved
in 100% DMSO and added to the reaction to be 5%
DMSO in the final volume. Reaction mixtures were
incubated at 33 8C for 90 min and stopped by addition
of 4 ml of 95% formamide, 20 mM EDTA, 0.05%
bromophenol blue, 0.05% xylene cyanol FF. The reac-
tions were heated to 90 8C for 3 min and electrophoresed
on a 20% denaturing polyacrylamide gel. Reaction
products were visualized by autoradiography of the
wet gel. IC50 was calculated by scanning bands on
Kodak-5 film (Image Master VDS, Pharmacia Bio-
tech.).
solvent (THF:H2Oꢁ7 ml:7 ml) was added LiOH (129
/
mg, 3.08 mmol). The reaction mixture was heated for 12
h to 40 8C. After cooling to r.t., the mixture was
extracted with EtOAc. The organic layer was dried
over MgSO4, filtered and concentrated. The residue was
solidified by diethyl etherꢀ
89%) as a solid: m.p. 236ꢀ237 8C; IR (KBr) 3172, 1700,
1600, 1430, 1286 cmꢂ1; 1H NMR (CD3OD) d 7.70 (1H,
d, Jꢁ8.1 Hz, benzofuranꢀH5), 7.49 (2H, d, Jꢁ8.7 Hz,
phneylꢀH2, H6), 7.35 (1H, d, Jꢁ15.9 Hz, ꢀCH ÄCHꢀ
phenyl), 7.26 (1H, d, Jꢁ 8.1 Hz, benzofuranꢀH4), 7.01
(1H, d, Jꢁ15.9 Hz, ꢀCHÄCH ꢀphenyl), 6.84 (2H, d,
Jꢁ8.7 Hz, phneylꢀH3, H5), 6.76 (1H, s, benzofuranꢀ
H3), 3.97 (3H, s, benzofuranꢀ
CO2CH3); 13C NMR
/n-hexane to give 8d (85 mg,
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
(CD3OD) d 168.4, 158.8, 158.7, 145.9, 145.5, 136.3,
132.4, 128.1, 126.4, 118.2, 115.9, 114.6, 112.9, 104.1,
61.2.
Acknowledgements
These investigations were financially supported by
Ministry of Science and Technology.
3.4. Biological test
3.4.1. HIV-1 integrase [20]
Recombinant human immunodeficiency virus type 1
(HIV-1) integrase was expressed in Escherichia coli and
purified using nickel-chelated column in an one-step
manner. Aliquots of HIV-1 integrase of 0.5 mg/ml as
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