Design and synthesis of 4,6-di-tert-butyl-2,3-dihydro-5-benzofuranols as a novel series of antiatherogenic antioxidants

Article abstract of DOI:10.1021/jm030062a

Antioxidants have been considered as potential antiatherogenic agents by inhibiting oxidation of low-density lipoprotein (LDL), albeit vitamin E, a natural antioxidant, has failed to show reduction on atherosclerosis in clinical trials. We have rationally

Full text of DOI:10.1021/jm030062a

J . Med. Chem. 2003, 46, 3083-3093
3083
Design a n d Syn th esis of 4,6-Di-ter t-bu tyl-2,3-d ih yd r o-5-ben zofu r a n ols a s a Novel
Ser ies of An tia th er ogen ic An tioxid a n ts
Kunio Tamura,†,‡, Yoshiaki Kato,‡ Akira Ishikawa,‡ Yasuharu Kato,‡ Motomu Himori,‡ Mitsutaka Yoshida,‡
Yoshiaki Takashima,‡ Tsukasa Suzuki,‡ Yoshiki Kawabe,‡ Osamu Cynshi,*,‡ Tatsuhiko Kodama,§
Etsuo Niki,| and Makoto Shimizu
Synthetic Technology Research Department, Chugai Pharmaceutical Co., Ltd., 5-5-1 Ukima, Kita-ku, Tokyo 115-8543, J apan,
Fuji Gotemba Research Laboratories, Chugai Pharmaceutical Co., Ltd., 1-135 Komakado, Gotemba-shi, Shizuoka 412-8513,
J apan, Laboratory for Systems Biology and Medicine, RCAST, The University of Tokyo, 4-6-1 Komaba, Meguro-ku,
Tokyo 153-8904, J apan, Human Stress Signal Research Center, AIST, 1-8-31 Midorigaoka, Ikeda-shi, Osaka 563-8577,
J apan, and Department of Chemistry for Materials, Faculty of Engineering, Mie University, 1515 Kamihama-cho,
Tsu-shi, Mie 514-8507, J apan
Received February 11, 2003
Antioxidants have been considered as potential antiatherogenic agents by inhibiting oxidation
of low-density lipoprotein (LDL), albeit vitamin E, a natural antioxidant, has failed to show
reduction on atherosclerosis in clinical trials. We have rationally designed and synthesized a
novel series of antioxidants, 4,6-di-tert-butyl-2,3-dihydro-5-benzofuranols, to overcome the
clinical limitation of vitamin E. In vitro, the compounds showed a potent inhibitory effect on
lipid peroxidation detected as 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-
3-one (MCLA)-dependent chemiluminescence in linoleic acid autoxidation. They also inhibited
the LDL oxidation induced by Cu2+, and the inhibition is more potent than that of vitamin E
and probucol. In vivo, 4,6-di-tert-butyl-2,3-dihydro-2,2-dipentyl-5-benzofuranol (BO-653, 1f),
an optimal compound, showed the highest concentration in plasma and LDL fraction in
Watanabe heritable hyperlipidemic rabbits, due to its high affinity to LDL. The isolated LDL
samples from the 1f-treated rabbits showed potent resistibility to LDL oxidation. Compound
1f has been taken into clinical trials.
Ch a r t 1. Structures of Probucol and R-Tocopherol
In tr od u ction
Many lines of evidence, based on atherosclerosis
models, suggest that the oxidative modification of low-
density lipoprotein (LDL) has a critical role in the
development of atherosclerosis.1-3 As a consequence of
the oxidation hypothesis, antioxidants have been pro-
posed as antiatherogenic agents, and lipophilic ones in
many kinds of antioxidants have an advantage to
suppress the LDL oxidation due to their affinity to lipid
particles. For the last two decades, various lipophilic
antioxidants have been intensively investigated to
reduce the progression of atherosclerosis in animal
models.4-7
Probucol, a lipophilic antioxidant, is well-known as a
lipid-lowering agent (Chart 1), and its antiatherogenic
effects in various animal models has been reported.8-12
However, there was no demonstration of its antiathero-
genic effect in a clinical trial.13 Probucol has the
untoward effects of lowering serum high-density lipo-
protein (HDL) levels14 and causing arrythmias.15 The
reduction of HDL has a contrary effect on atheroscle-
rosis; that is, the concentration of HDL has been shown
to correlate inversely with the risk of atherosclerosis,16
and this leads to the controversial conclusion that
lipophilic antioxidants do not reduce human atheroscle-
rosis. To demonstrate the oxidation hypothesis, we need
further study using the ideal antioxidant without such
untoward effects.
R-Tocopherol, the most active species of vitamin E, is
a potent natural lipophilic antioxidant and is considered
to play an important role in maintaining life processes
against lipid peroxidation in the body. In general,
R-tocopherol reduces the LDL oxidation 10- to 100-fold
more potently than probucol.17 It has neither of the
untoward effects of HDL reduction and the causing of
arrhythmias; however, the clinical intervention studies
with vitamin E have afforded conflicting results. Rimm
et al. reported that high intake of vitamin E reduces
the incidence of coronary heart disease.18 Meanwhile,
three large-scale trials failed to demonstrate the effect
of vitamin E on cardiovascular disease or cerebrovas-
cular mortality.19-21 We hypothesized that this discrep-
ancy between high reactivity of vitamin E and the
conflicting results in clinical trials would be based on
the inability to exert its antioxidant action in the
hydrophobic core of LDL particle where probucol is
active. Furthermore, the properties of R-tocopherol that
is not only an antioxidant but also a prooxidant may
* To whom correspondence should be addressed. Tel: 81-550-87-
6786, fax: 81-550-87-3637, e-mail: shinshiosm@chugai-pharm.co.jp.
† Synthetic Technology Research Department, Chugai Pharmaceuti-
cal Co.
‡ Fuji Gotemba Research Laboratories, Chugai Pharmaceutical Co.
§ The University of Tokyo.
| Human Stress Signal Research Center, AIST.
Mie University.
10.1021/jm030062a CCC: $25.00 © 2003 American Chemical Society
Published on Web 05/30/2003

3084 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
Tamura et al.
Ch a r t 2. Characteristics Required for Potent LDL Antioxidants
Sch em e 1a
contribute to this discrepancy.22,23 The evidence, that
atherosclerotic lesions contain a certain amount of
R-tocopherol even where the oxidized lipid was gener-
ated,24,25 may support our consideration.
On the basis of the oxidation hypothesis, we com-
menced to design new antioxidants to overcome the
clinical limitation of vitamin E. The points for the design
are as follows:26
1. To have potent antioxidative reactivity that is equal
to or greater than that of R-tocopherol.
2. To exert antioxidant action in the core of LDL
particle.
3. To compensate for the prooxidant action of R-toco-
pherol by a synergic antioxidant action.
Our goal is to furnish a promising candidate for
clinical usage. In this paper, we report novel antioxi-
dants, which have been rationally designed to possess
high reactivity and efficacy in LDL, which must be
required for effective inhibition against LDL oxidation.
a
Reagents: (a) H2SO4, Ac2O; (b) TMSI, CH2Cl2; (c) K2CO3, allyl
Resu lts a n d Discu ssion
bromide, acetone; (d) N,N-dimethylaniline reflux; (e) K2CO3, MeI,
acetone; (f) OsO4, NaIO4, THF, H2O; (g) LiAlH4, THF; (h) H2, 10%
Pd/C, AcOH.
Dr u g Design . Lipid peroxidation is considered to
play a central role in the LDL oxidation.27 Although
endogenous antioxidants such as vitamin E and plasma
antioxidative enzymes and substances act to prevent the
surface of LDL from oxidation, the relevant oxidation
for atherogenesis may happen in the hydrophobic core
of LDL where the chain reaction of lipid peroxidation
can be caused easily. First, we speculated that the point
of the drug design was to realize a high reactivity like
that of R-tocopherol compatible with the existence in the
core of LDL like probucol. Next, we considered that the
new antioxidants possessing high affinity to LDL may
be delivered efficiently to LDL and then the vessel wall.
Finally, we supposed that relative stability of the
intermediate radicals of the new antioxidants would not
lead to the prooxidant action such as R-tocopherol. The
last property might make up for the disadvantage of
R-tocopherol. Therefore, the drug design consists of the
following characteristics: (i) high hydrogen-donating
activity and low prooxidant activity of its intermediate,
(ii) localization inside the core of the LDL particle, and
(iii) efficient delivery to LDL and vascular vessels.
The first characteristic (i) is rationally achieved by
lowering the dissociation energy of the phenolic O-H
bond, which is determined in part by the resonance
stabilization of the resulting phenoxyl radical. Ingold
et al. reported that the p-type lone-pair orbital of the
oxygen atom located in the para position of the phenolic
hydroxyl group can stabilize the phenoxyl radical opti-
mally when the oxygen atom belongs to a five-membered
ring,28,29 although vitamin E has a six-membered ring.
Thus, we designed a 2,3-dihydro-5-benzofuranol struc-
ture to achieve the former characteristic (Chart 2).
To achieve the second characteristic (ii), we intended
to utilize two tert-butyl groups on the ortho position of
the hydroxyl group instead of the methyl groups of
R-tocopherol. The two tert-butyl groups are expected to
obstruct the hydrophilicity of the hydroxyl group and
to result in the preferential localization to the hydro-
phobic region. Moreover, they make the resulting
phenoxyl intermediate kinetically persistent due to their
steric hindrance, and this persistence leads to a reduc-
tion in the prooxidant action of its phenoxyl intermedi-
ate. Thus, we realized the latter half of the first
characteristic (i). Furthermore, accompanying its high
reactivity, the relative stability of the intermediate also
leads to a reduction in the coexisting prooxidant action
of R-tocopherol.
The third characteristic (iii) is an essential point for
LDL antioxidants, and we examined a series of com-
pounds concerning their distribution to lipoproteins. We
synthesized 2,3-dihydro-5-benzofuranol derivatives bear-
ing different alkyl substituents on the 2-position and
evaluated their distribution using Watanabe heritable
hyperlipidemic (WHHL) rabbits in order to achieve this
characteristic.
Ch em istr y. The synthesis of dihydrobenzofuranol 1a
was carried out as shown in Scheme 1. 3,5-Di-tert-butyl-
4-hydroxyanisole 2 was acetylated with acetic anhydride

Synthesis of Antiatherogenic Antioxidants
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14 3085
Sch em e 2a
intermediate 12 was accomplished according to a pro-
cedure similar to that used by Stokker et al.30 Treat-
ment of phenol 3 with 2-chloro-N-(hydroxymethyl)-
acetamide31 in the presence of sulfuric acid afforded a
mixture of 9 and 10, which was hydrolyzed to give
benzylamine 11. Subsequently, 11 was treated with
hexamethylenetetramine in aqueous acetic acid to yield
12.32 Next, conversion of 12 into 1c-o was carried out
as follows: Treatment of 12 with more than 2 equiv of
Grignard reagents and following neutralization with an
aqueous ammonium chloride solution gave intermedi-
ates 13c-o (Method A). Subsequently, 13c-o were
converted to benzofurans 15c-o via dehydrated com-
pounds 14c-o by treatment with boron trifluoride
diethyl etherate. Alternatively, compounds 14c-o were
obtained by treatment with diluted hydrochloric acid
after the Grignard reaction (Method B). Deprotection
of 15c-o afforded 1c-o.
a
Reagents: (a) NaH, DMF, 3-chloro-2-methylpropene; (b) N,N-
dimethylaniline reflux; (c) 1N HCl aq, Et2O; (d) LiAlH4, THF.
and sulfuric acid, followed by demethylation with io-
dotrimethylsilane to give phenol 3 (71%). Treatment of
3 with allyl bromide in the presence of potassium
carbonate yielded ether 4 (96%) which was converted
into 5 via Claisen rearrangement in N,N-dimethyl-
aniline and subsequent methylation (79% for two steps).
Oxidation of 5 with sodium periodate and a catalytic
amount of osmium tetroxide, followed by demethylation,
afforded benzofuran 6 (83%). Benzofuran 6 was depro-
tected using lithium aluminum hydride in THF and
then reduced catalytically to yield 1a (71% for two
steps).
Compound 1b was prepared as shown in Scheme 2.
Phenol 3 was alkylated with 3-chloro-2-methylpropene
in the presence of sodium hydride to give ether 7 (90%).
Claisen rearrangement of 7 and subsequent treatment
with hydrochloric acid gave benzofuran 8, which was
then deprotected to yield 1b (45% for three steps).
Dihydrobenzofuranols 1c-o were prepared via addi-
tion of Grignard reagents to benzaldehyde 12 as a key
reaction (Scheme 3). First, the synthesis of the key
In the case of dihydrobenzofuranol 1p , preparation
of the corresponding Grignard reagent was not success-
ful. However, the combination of lithium metal and
alkylhalide 16 with aldehyde 12 (Barbier reaction) gave
the addition compound 13p (46%, Scheme 4). Compound
13p was cyclized with boron trifluoride diethyl etherate
into compound 15p , which was deprotected with lithium
aluminum hydride to afford 1p (54% for two steps).
In the preparation of dihydrobenzofuranol 1q, ben-
zofuranol 1733 was tert-butylated with 2-methyl-2-
propanol in the presence of methanesulfonic acid to give
tri-tert-butylbenzofuranol 18. Furthermore, 18 was
reduced with triethylsilane in trifluoroacetic acid to
yield 1q (Scheme 5).
An tioxid a n t Activity. The antioxidant activity was
measured as the inhibition of a Cypridina luciferin
analogue, 2-methyl-6- (p-methoxyphenyl)-3,7-dihydro-
imidazo[1,2-a]pyrazin-3-one (MCLA)34-dependent chemi-
luminescence generated in linoleic acid autoxidation in
which the scavenging activity of 1f against linoleate
peroxyl radicals was previously reported.26 As shown
Sch em e 3a
a
Reagents: (a) N-hydroxymethyl-2-chloroacetamide, H2SO4, AcOH; (b) concentrated HCl, EtOH reflux; (c) (CH2)6N4, AcOH, H2O, then
HCl aq; (d) R1R2CHMgBr, THF; (e) NH4Cl aq.; (f) HCl aq; (g) BF3‚OEt2, CH2Cl2; (h) LiAlH4, THF.

3086 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
Tamura et al.
Ta ble 1. Effects of Dihydrobenzofuranols on Chemiluminescence Generated by Linoleic Acid Autoxidation and LDL Oxidation
Induced by Soybean Lipoxygenase or CuSO4
LDL oxidation (% of control)c
SLO
CuSO4
10µM
chemiluminescenceb
compd
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
R1
R2
cLogPa
IC50 (µM)
0.5 µM
5 µM
1 µM
H
CH3
C2H5
H
5.21
6.25
7.31
8.37
9.42
10.48
11.54
12.60
13.66
9.09
6.64
7.20
7.76
8.32
4.73
10.22
7.06
7.2
6.7
4.3
4.0
7.9
36.0
39.8
9.4
6.9
1.8
1.3
0
0.5
4.6
2.7
18.5
11.3
3.6
0.4
0
2.3
9.0
4.7
7.1
1.8
14.0
53
56
77
87
82
96
86
89
85
93
85
77
91
92
87
78
74
87
11
10
10
13
12
42
23
83
72
11
12
16
9
CH3
C2H5
n-C3H7
n-C4H9
n-C3H7
n-C4H9
n-C5H11
n-C6H13
n-C7H15
n-C8H17
CH2C6H5
33.3
51.5
60.9
75.5
88.4
89.8
63.5
46.2
34.1
93.4
97.2
71.8
91.5
99.0
73.9
n-C5H11
n-C6H13
n-C7H15
n-C8H17
CH2C6H5
(CH2)4
(CH2)5
(CH2)6
(CH2)7
4.6
n.d.d
n.d.d
5.6
10.6
6.6
9.3
7.9
9.4
8.2
5.7
8.4
165
22
22
23
11
28
(CH2)2O(CH2)2
(CH2)2i-Pr
tert-C4H9
(CH2)2i-Pr
1q
probucol
H
10.75
a
b
cLogP for each compound was calculated with CLOGP v.3.54 (Daylight C.I.S.Inc., Claremont, CA). Inhibition of MCLA chemi-
luminescence generated by autoxidation of linoleic acid. c Inhibitory activities against LDL oxidation induced by SLO or CuSO4. Not
d
determined.
Sch em e 4a
Sch em e 5a
a
Reagents: (a) t-BuOH, MsOH, CHCl3; (b) Et3SiH, TFA.
a
Reagents: (a) Li, THF, then NH4Cl aq; (b) BF3‚OEt2, CH2Cl2;
determined by gel-permeation chromatography, respec-
tively. The TBARS represents the oxidation of lipids,
and the fluorescence represents the oxidation of pro-
teins. As shown in Table 1, the compounds inhibited the
LDL oxidation induced by either CuSO4 or SLO. Com-
pounds possessing long alkyl groups (1h and 1i) and
probucol at 5 µM showed less potent inhibition in
oxidation by SLO.
Ab sor p t ion in WH H L R a b b it s. The pharmaco-
kinetic profile is important for antioxidants to achieve
the clinical benefits. As our design for LDL antioxidants
leads to highly lipophilic compounds, the design auto-
matically leads to the realization that low solubility in
water might cause low absorption. Furthermore, ex-
perimental animals show low plasma cholesterol levels
in general and most of the plasma cholesterol in rodents
comes from HDL. The animals fed normal diet show low
absorption for lipophilic compounds. Normal animals
should not be appropriate for evaluating the absorption
(c) LiAlH4, THF.
in Table 1, the chemiluminescence was effectively
quenched more than 10-fold by the compounds including
1f, compared with probucol. That is, they have much
more potent hydrogen donating activities compared with
probucol. In addition to the chemical reactivity in the
homogeneous solution, these compounds were also
examined for their antioxidant activities on LDL oxida-
tion induced by cupric ions (CuSO4) or soybean lipoxy-
genase (SLO). The reason for using two kinds of
initiators is to remove the initiator specific inhibition
from the inhibition of lipid peroxidation. Thus, we could
select the inhibitors against chain propagation process
of lipid peroxidation. Rabbit’s LDL accompanying each
compound was oxidized with CuSO4 and SLO for 24 h,
and the antioxidant activity was evaluated as the
resulting thiobarbituric acid reactive substances (TBARS)
and the fluorescence generation from LDL fraction

Synthesis of Antiatherogenic Antioxidants
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14 3087
F igu r e 1. Plasma concentration of dihydrobenzofuranols in WHHL rabbits. Two rabbits were orally administered once a day
with the test compound (250 mg/kg) or probucol (500 mg/kg) for 9 days and the plasma concentrations were evaluated at 24 h
after daily administration. The results are shown as Mean ( Range: (left panel) 1b, open squares; 1d , closed squares; 1e, open
circles; 1f, closed circles; 1i, open triangles; probucol, closed triangles; (right panel) 1j, open squares; 1m , closed squares; 1p ,
open circles; 1q, closed circles; probucol, closed triangles.
in hyperlipidemia patients who will use the drugs
against atherosclerosis. To overcome the discrepancy
between patients and normal animals, we used WHHL
rabbits of which the high LDL level allows us to assess
the absorption in hyperlipidemia patients. WHHL rab-
bits were orally administered once a day with the
dihydrobenzofuranols (250 mg/kg) or probucol (500 mg/
kg) for 9 days, and the concentrations in plasma were
measured. Figure 1 shows the serial changes in the
plasma concentrations at 24 h after daily oral admin-
istration, in which the left panel shows the result of the
dihydrobenzofuranols bearing two straight alkyl side
chains, and the right panel shows the result of the other
dihydrobenzofuranols. The compounds that possess the
moderate length alkyl side chain (1e and 1f in the left
panel, and 1p in the right panel) showed superior
absorption in WHHL rabbits than probucol.
F igu r e 2. Relationship between cLogP and plasma concen-
tration of dihydrobenzofuranols in WHHL rabbits. Two rabbits
were orally administered once a day with test compound (250
mg/kg) for 9 days and the plasma concentrations were evalu-
ated at 24 h after the final administration. Closed squares
indicate the compounds bearing straight side chains and open
circles indicate the other structures. The results are shown
as mean ( range.
The above result suggests a correlation between the
lipophilicity of the dihydrobenzofuranols and their
absorption profiles in WHHL rabbits. Figure 2 shows
the relationship between the calculated distribution
coefficient (cLogP) of dihydrobenzofuranols and their
plasma concentration in WHHL rabbits at 24 h after
the final administration. The shape of the graph sug-
gested that the dihydrobenzofuranols had an optimal
cLogP value for the absorption and that the optimal
length as a straight side chain could be determined from
the graph (refer to closed squares in Figure 2). Thus,
we selected as an optimal compound, compound 1f,
which has two pentyl groups as moderate length side
chains from the straight chain type.
Com p a r ison of 1f w ith r-Tocop h er ol. To confirm
the antioxidant activity of the compound 1f, we com-
pared it with R-tocopherol and probucol. In addition to
TBARS and fluorescence generation that was previously
reported,26 electrophoretic mobility is an important
parameter in evaluating LDL oxidation as it reflects the
surface charge of LDL that is relevant to binding with
both LDL and scavenger receptors. We compared the
changes of the electrophoretic mobility induced by
CuSO4 and SLO oxidation, as shown in Figure 3. The
change in the LDL treated with 1f was smallest for both
oxidations. In the case of CuSO4 oxidation, the mobility
of the LDL incubated with 1 µM of 1f was 15.6 ( 0.8
mm (mean ( SD) where the control was 22.7 ( 0.8 mm
and was similar to that of native LDL (13.3 ( 0.4 mm).
The change with 1 µM of 1f was much smaller than that
with 10 µM of either probucol or R-tocopherol. Similar
results were obtained for both parameters of TBARS
and fluorescence generation (data not shown). The
results confirm the antioxidant properties of these
designed compounds that might be useful in their
intended activity.
Oxid iza bilit y of Isola t ed LDL fr om 1f-Tr ea t ed
Ra bbits. If the progress of atherosclerosis is closely
related to oxidative modification of LDL, the practical
target of antioxidants is to deliver them to LDL.
Therefore, we examined the distribution to lipoproteins
when a series of dihydrobenzofuranols were orally
administered in WHHL rabbits. Table 2 shows the
distribution of the compounds to the very low-density

3088 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
Tamura et al.
F igu r e 3. Inhibitory effects of 1f, probucol and R-tocopherol on electrophoretic mobility change during LDL oxidation. Rabbit
LDL at a concentration of 200 µg/mL was oxidized by incubating with either CuSO4 (Cu2+) or lipoxygenase (SLO) at 37 °C for 24
h in the presence of 1f, probucol, or R-tocopherol (1 or 10 µM). The electrophoretic mobility after the LDL oxidation was evaluated
by one-dimensional agarose gel electrophoresis. The results are shown as mean ( SD (triplicate).
Ta ble 2. Distribution of Dihydrobenzofuranols into Lipoprotein
Fractions of WHHL Rabbit’s Plasmaa
plasma
VLDL
LDL
HDL
compd
1e
1f
1i
1p
1q
(µg/mL)
(µg/mg)
(µg/mg)
(µg/mg)
0.89 ( 0.05 57.07 ( 1.69 11.04 ( 0.64 3.22 ( 0.78
2.22 ( 0.41 119.25 ( 14.58 28.60 ( 6.43 2.98 ( 1.33
0.01 ( 0.00
0.59 ( 0.12 25.64 ( 2.53
0.44 ( 0.16 27.69 ( 9.28
1.16 ( 0.26
0.36 ( 0.06 n.d.b
9.34 ( 0.30 n.d.b
5.08 ( 1.49 n.d.b
2.57 ( 1.20 0.44 ( 0.01
probucol 0.14 ( 0.07
5.48 ( 2.12
a
Each group is composed of two rabbits. The data are expressed
b
as mean ( range. Not detected.
lipoprotein (VLDL), LDL, and HDL fractions in plasma.
The results indicated that the orally administered
dihydrobenzofuranols were distributed to the VLDL and
LDL fractions rather than the HDL fraction, and that
the concentration of 1f in the VLDL and LDL fractions
was higher than that of probucol. In general, lipophilic
compounds such as 1f would be absorbed via the
pathway of lipid absorption. Therefore, we speculated
that the absorption occurred at the intestine via chylo-
micron, and it was carried through the lymph to be
utilized for the synthesis of VLDL and LDL. VLDL has
the fate of being converted to LDL by lipoprotein lipase.
Thus, the lipophilic compounds seem to possess a self-
organized drug-delivery system, targeting by themselves
the practical targets of the VLDL and LDL.
Finally, we assessed the inhibitory activity of 1f by
evaluating the oxidizability of isolated LDL from the 1f-
treated WHHL rabbits. The isolated LDL was oxidized
by CuSO4 or SLO, and the oxidation was evaluated as
the fluorescence generation of LDL. As shown in Figure
4, the fluorescence increased due to oxidation by either
CuSO4 or SLO, and the LDL from the 1f-treated
animals showed significant resistance to oxidation more
than that from probucol-treated animals. Together with
the in vivo study,26 the results described here imply that
1f possesses potent inhibitory activity against LDL
oxidation.
F igu r e 4. Oxidizability of LDL isolated from antioxidant-
treated WHHL rabbits. Two rabbits were orally administered
once a day with test compound for 9 days, and blood samples
were collected at 24 h after the final administration. The
isolated LDL was oxidized by incubating with either CuSO4
(Cu) or lipoxygenase (SLO) at 37 °C for 24 h. The oxidizability
was assessed as the generation of fluorescence induced by the
LDL oxidation, integrating from the void to 150 kDa on gel-
permeation chromatography. The results are shown as mean
( range.
ously, Noguchi et al. described the radical scavenging
activities of 1f against lipid peroxidation35 and the
inhibitory action on LDL oxidation.36 Furthermore, we
demonstrated the antiatherogenic effects of 1f in three
different animal models including the WHHL rabbit.26
Here, we have achieved the constructing of a strategy
for screening LDL antioxidants to select the optimal
dihydrobenzofuranol.
The western style of foods and the exposure to
pollutants in the environment are increasing the risk
of oxidative stress and the progression of atherosclero-
sis. The drugs that possess preferable antioxidative
action could be beneficial to various diseases relevant
to oxidative stress. Such drugs could prevent the oxida-
tive modification of LDL and so could prevent coronary
heart diseases such as angina and myocardial infarc-
tion. We concluded in this study that the rationally
designed antioxidant 1f (BO-653)37 would be a promis-
ing candidate as a new antiatherogenic agent. The
remaining question is the clinical efficacy of BO-653 that
will be answered in coming clinical trials that will reveal
the potential of antioxidants for cardiovascular diseases
as atherosclerosis.
Con clu sion s
In this study, we demonstrated the design and
synthesis of 4,6-di-tert-butyl-2, 3-dihydro-5-benzofura-
nols as a novel series of antioxidants. Furthermore, we
demonstrated that the selected compound 1f possesses
a preferable distribution to LDL, the target molecule of
our drug design for an antiatherogenic agent. Previ-

Synthesis of Antiatherogenic Antioxidants
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14 3089
layer was washed with water and saturated brine, dried over
anhydrous MgSO4, and concentrated. The residue was purified
by silica gel column chromatography (10% EtOAc in n-hexane)
to yield 5 (31 g, 99%) as a colorless oil: 1H NMR (270 MHz,
CDCl3) δ 1.34 (s, 9H), 1.34 (s, 9H), 2.30 (s, 3H), 3.63-3.68 (m,
2H), 3.78 (s, 3H), 4.88-5.02 (m, 2H), 5.89-6.02 (m, 1H), 6.83
(s, 1H); MS m/e 318 (M+).
4-Acetoxy-3,5-di-ter t-bu tyl-2-for m ylm eth ylan isole. Com-
pound 5 (19 g, 60 mmol) was dissolved in a 1:1 mixture (200
mL) of THF and water. OsO4 (0.12 g, 0.5 mmol) and NaIO4
(26.9 g, 126 mmol) were added to the solution, and the
resulting mixture was stirred at room temperature for 48 h.
After addition of saturated aqueous Na2S2O3, the mixture was
extracted with EtOAc. The organic layer was washed with
water and saturated brine, dried over anhydrous MgSO4, and
concentrated. The residue was purified by silica gel column
chromatography (25% EtOAc in n-hexane) to afford the title
compound (16 g, 84%) as a colorless oil: 1H NMR (60 MHz,
CDCl3) δ 1.36 (s, 9H), 1.40 (s, 9H), 2.30 (s, 3H), 3.77 (s, 3H),
3.87 (bs, 2H), 6.89 (s, 1H), 9.63 (bs, 1H); MS m/e 320 (M+).
Exp er im en ta l Section
In str u m en ts a n d An a lyses. Column chromatography was
carried out on Wako-gel C-200 (70-230 mesh) purchased from
Wako Pure Chemical Industries (Osaka, J apan). Thin-layer
chromatography was performed on Kieselgel F254 plates pur-
chased from E. Merck and Co. HPLC analyses were performed
on a Hitachi L-4000 UV detector with a Hitachi L-6200 pump
using a YMC-Pack C8 A-212 column eluted with CH3CN-i-
PrOH-water (8:1:1) at a flow rate of 1.0 mL/min. Melting
points were recorded on a Yanagimoto micro melting point
apparatus or a METTLER FP62 melting point apparatus and
1
are uncorrected. H NMR spectra were recorded on a Hitachi
R-24B spectrometer (60 MHz) or on a J EOL EX-270 spectrom-
eter (270 MHz) with tetramethylsilane as an internal stan-
dard. Mass spectra were recorded on a Shimadzu GCMS-
QP1000 spectrometer. Infrared spectra were recorded on a
Hitachi 270-30 infrared spectrophotometer. High resonance
mass spectra (HRMS) were measured at the Toray Research
Center (Tokyo, J apan). All elemental analyses are within 0.4%
of the calculated values unless otherwise specified.
5-Acetoxy-4,6-d i-ter t-bu tylben zofu r a n (6). To a solution
of 4-acetoxy-3,5-di-tert-butyl-2-formylmethylanisole (16 g, 50
mmol) in CH2Cl2 (100 mL) was added dropwise at 0 °C
iodotrimethylsilane (7.1 mL, 50 mmol). The reaction mixture
was stirred at room temperature for 1 h, and then a saturated
aqueous Na2S2O3 solution was added. The mixture was
extracted with CHCl3, and the organic layer was washed with
water and saturated brine, dried over anhydrous MgSO4, and
concentrated. The residue was purified by silica gel column
chromatography (10% EtOAc in n-hexane) to yield 6 (14.3 g,
99%) as a white solid, mp 88 °C: 1H NMR (270 MHz, CDCl3)
δ 1.39 (s, 9H), 1.51 (s, 9H), 2.35 (s, 3H), 6.98 (d, J ) 2.3 Hz,
1H), 7.46 (s, 1H), 7.55 (d, J ) 2.3 Hz, 1H); MS m/e 288 (M+).
4-Acetoxy-3,5-d i-ter t-bu tyla n isole. A mixture of 3,5-di-
tert-butyl-4-hydroxyanisole 2 (23.6 g, 0.1 mol) and concentrated
H2SO4 (0.5 mL) in acetic anhydride (150 mL) was stirred at
70 °C for 2 h. The reaction mixture was concentrated under
reduced pressure, and saturated aqueous NaHCO3 was added
to the concentrate. After the mixture was extracted with
EtOAc, the extract was dried over anhydrous MgSO4 and
concentrated. The residue was recrystallized from MeOH-
water (2:1) to give the title compound (24.5 g, 88%) as a white
solid, mp 97 °C: 1H NMR (60 MHz, CDCl3) δ1.06 (s, 18H),
2.02 (s, 3H), 3.47 (s, 3H), 6.53 (s, 2H); MS m/e 278 (M+).
4-Acetoxy-3,5-d i-ter t-bu tylp h en ol (3). To a solution of
4-acetoxy-3,5-di-tert-butylanisole (0.50 g, 1.8 mmol) in CH2-
Cl2 (2 mL) was added dropwise at 0 °C iodotrimethylsilane
(0.31 mL, 2.2 mmol). The mixture was allowed to warm to room
temperature and was stirred for 2 days. A saturated aqueous
solution of NaHCO3 was added to the reaction mixture, and
the mixture was extracted with Et2O. The extract was washed
with saturated brine, dried over anhydrous MgSO4, and
concentrated. The residue was purified by silica gel column
chromatography (15% EtOAc in n-hexane) to give 3 (0.38 g,
80%) as a white solid, mp 157 °C: 1H NMR (60 MHz, CDCl3)
δ 1.27 (s, 18H), 2.27 (s, 3H), 5.22 (br, 1H), 6.67 (s, 2H); MS
m/e 264 (M+).
4-Ac e t o x y -3,5-d i-t er t -b u t y l-1-(2-p r o p e n y lo x y )b e n -
zen e (4). To a mixture of 3 (10 g, 37.8 mmol) and K2CO3 (15.6
g, 0.11 mol) in acetone (300 mL) was added allyl bromide (6.55
mL, 75.6 mmol). The mixture was heated to reflux for 24 h
and then cooled to room temperature and concentrated under
reduced pressure. After addition of water, the mixture was
extracted with Et2O. The extract was washed with water and
saturated brine, dried over anhydrous MgSO4, and concen-
trated. The residue was purified by silica gel column chroma-
tography (10% EtOAc in n-hexane) to give 4 (11 g, 96%) as a
colorless oil: 1H NMR (60 MHz, CDCl3) δ 1.30 (s, 18H), 2.27
(s, 3H), 4.47 (d, J ) 5.0 Hz, 2H), 5.05-5.57 (m, 2H), 5.68-
6.37 (m, 1H), 6.81 (s, 2H); MS m/e 304 (M+).
4-Acetoxy-3,5-d i-ter t-bu tyl-2-(2-p r op en yl)p h en ol. A so-
lution of 4 (11 g, 36 mmol) in N,N-dimethylaniline (50 mL)
was heated to reflux for 18 h under a N2 atmosphere. After
being cooled to room temperature, the reaction mixture was
concentrated under reduced pressure. Purification of the
concentrate by silica gel column chromatography (15% EtOAc
in n-hexane) gave the title compound (8.84 g, 80%) as a white
solid, mp 104 °C: 1H NMR (60 MHz, CDCl3) δ 1.30 (s, 9H),
1.42 (s, 9H), 2.28 (s, 3H), 3.52-3.84 (m, 2H), 4.88-5.42 (m,
3H), 5.68-6.45 (m, 1H), 6.79 (s, 1H); MS m/e 304 (M+).
4-Acet oxy-3,5-d i-ter t-b u t yl-2-(2-p r op en yl)a n isole (5).
To a mixture of 4-acetoxy-3,5-di-tert-butyl-2-(2-propenyl)phenol
(30 g, 99 mmol) and K2CO3 (13.8 g, 0.1 mol) in acetone (300
mL) was added iodomethane (28 g, 0.2 mol). The mixture was
heated to reflux for 24 h and then cooled to room temperature
and concentrated under reduced pressure. After addition of
water, the mixture was extracted with EtOAc. The organic
4,6-Di-ter t-bu tyl-5-ben zofu r a n ol. Lithium aluminum hy-
dride (LiAlH4) (1.31 g, 34.5 mmol) was suspended in dry THF
(150 mL) under a N2 atmosphere. A solution of 6 (10 g, 34.7
mmol) in dry THF (100 mL) was added dropwise to the
suspension at 0 °C, and then the mixture was heated to reflux
for 3 h. After the mixture was cooled to room temperature,
water was added dropwise to quench the excess LiAlH4. After
addition of 10% aqueous HCl (100 mL), the mixture was
extracted with EtOAc. The extract was washed with saturated
brine, dried over anhydrous MgSO4, and concentrated. The
residue was purified by silica gel column chromatography (10%
EtOAc in n-hexane) to give the title compound (8.3 g, 97%) as
a fine-grained pale yellow crystal, mp 60 °C: IR (KBr) 3648,
1
2960 cm-1; H NMR (60 MHz, CDCl3) δ 1.49 (s, 9H), 1.62 (s,
9H), 5.11 (s, 1H), 7.02 (d, J ) 2.4 Hz, 1H), 7.35 (s, 1H), 7.43
(d, J ) 2.4 Hz, 1H); MS m/e 246 (M+), 231, 57.
4,6-Di-ter t-bu tyl-2,3-d ih yd r o-5-ben zofu r a n ol (1a ). 4,6-
Di-tert-butyl-5-benzofuranol (6.0 g, 24 mmol) was dissolved in
acetic acid (50 mL). After addition of 10% Pd/C (5.0 g), the
solution was stirred under a H2 atmosphere (4 atm.) for 15 h.
The Pd catalyst was removed by filtration, and the filtrate was
concentrated in vacuo. The residue was purified by silica gel
column chromatography (10% EtOAc in n-hexane) to yield 1a
(4.4 g, 73%) as a fine-grained white crystal, which was
recrystallized from n-hexane, mp 117-118 °C: IR (KBr) 3623,
2969 cm-1; 1H NMR (270 MHz, CDCl3) δ 1.41 (s, 9H), 1.51 (s,
9H), 3.42 (t, J ) 8.6 Hz, 2H), 4.39 (t, J ) 8.6 Hz, 2H), 4.75 (s,
1H), 6.70 (s, 1H); MS m/e 248 (M+), 233, 191, 57. Anal.
(C16H24O2) C, H.
4-Ace t oxy-3,5-d i-t er t -b u t yl-1-(2-m e t h yl-2-p r op e n yl-
oxy)ben zen e (7). To a suspension of 60% oily NaH (0.18 g,
4.5 mmol) in dry DMF (10 mL) was added dropwise at 0 °C a
solution of 3 (1.0 g, 3.8 mmol) in dry DMF (5 mL). The mixture
was stirred for 30 min at the same temperature. Subsequently,
the mixture was allowed to warm to room temperature, and
3-chloro-2-methylpropene (0.45 mL, 4.5 mmol) was added.
After stirring at room temperature for 2 h, saturated aqueous
NH4Cl (15 mL) was added to the reaction mixture, and the
mixture was extracted with Et2O. The extract was washed
with saturated brine, dried over anhydrous MgSO4, and
concentrated. The residue was purified by silica gel column

3090 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
Tamura et al.
chromatography (10% EtOAc in n-hexane) to afford 7 (1.08 g,
90%) as a colorless oil: 1H NMR (60 MHz, CDCl3) δ 1.30 (s,
18H), 1.83 (s, 3H), 2.30 (s, 3H), 4.37 (br, J ) 6.6 Hz, 2H), 6.83
(s, 2H); MS m/e 318 (M+).
mixture was heated to reflux for additional 20 min. After being
cooled, the reaction mixture was poured into water, neutralized
with 1 N NaOH, and extracted with EtOAc. The organic layer
was dried over anhydrous MgSO4 and concentrated. The
concentrate was purified by silica gel column chromatography
(CHCl3) to afford 12 (19.0 g, 59% for three steps) as a fine-
grained pale yellow crystal, mp 79 °C: IR (KBr) 2976, 1758
5-Acet oxy-4,6-d i-ter t-b u t yl-2,3-d ih yd r o-2,2-d im et h yl-
ben zofu r a n (8). Compound 7 (2.22 g, 7.0 mmol) was dissolved
in N,N-dimethylaniline (8 mL), and the solution was refluxed
under a N2 atmosphere for 36 h. After being cooled to room
temperature, the reaction solution was concentrated under
reduced pressure. To the concentrate were added 1 N HCl (5
mL) and Et2O (10 mL), and the mixture was stirred for 15
min. The organic layer was separated, and the aqueous layer
was extracted with Et2O. The combined extracts were washed
with saturated brine, dried over anhydrous MgSO4, and
concentrated. The residue was purified by silica gel column
chromatography (5% EtOAc in n-hexane) to give 8 (1.19 g,
54%) as a white solid, mp 98 °C: 1H NMR (60 MHz, CDCl3) δ
1.16-1.60 (m, 24H), 2.25 (s, 3H), 3.18 (s, 2H), 6.63 (s, 1H);
MS m/e 318 (M+).
1
cm-1; H NMR (60 MHz, CDCl3) δ 1.35 (s, 9H), 1.54 (s, 9H),
2.35 (s, 3H), 6.92 (s, 1H), 10.67 (s, 1H), 12.32 (s, 1H); MS m/e
292 (M+), 250, 235, 217, 57.
4-Acet oxy-3,5-d i-ter t-b u t yl-2-(2-et h yl-1-b u t en yl)p h e-
n ol (14c). To a mixture of Mg (0.21 g, 8.6 mmol) and dry THF
(10 mL) was added dropwise under a N2 atmosphere a solution
of 3-bromopentane (1.3 g, 8.6 mmol) in dry THF (10 mL) to
prepare a Grignard reagent. A solution of 12 (1.0 g, 3.4 mmol)
in dry THF (5 mL) was added dropwise to the Grignard
reagent, and the mixture was stirred for 30 min at room
temperature. A 5:2 mixed solution (7 mL) of water and
concentrated HCl was added to the reaction mixture, followed
by stirring at room temperature for 30 min and subsequent
extraction with EtOAc. The organic layer was dried over
anhydrous MgSO4 and concentrated. The concentrate was
purified by silica gel column chromatography (10% EtOAc in
n-hexane) to give 14c (0.85 g, 72%) as a yellow oil: 1H NMR
(60 MHz, CDCl3) δ 0.78-1.57 (m, 6H), 1.33 (s, 9H), 1.37 (s,
9H), 1.73-2.48 (m, 4H), 2.27 (s, 3H), 5.38 (d, J ) 9.6 Hz, 1H),
6.17 (s, 1H), 6.87 (s, 1H); MS m/e 346 (M+), 304, 289, 57.
5-Acetoxy-4,6-d i-ter t-bu tyl-2,2-d ieth yl-2,3-d ih yd r oben -
zofu r a n (15c). To a solution 14c (0.85 g, 2.5 mmol) in CH2-
Cl2 (10 mL) was added dropwise under a N2 atmosphere BF3
diethyl etherate (0.4 mL, 3.2 mmol). After the mixture was
stirred at room temperature for 3 h, water was added to the
reaction mixture, and the mixture was extracted with EtOAc.
The organic layer was washed with saturated aqueous NaH-
CO3, dried over anhydrous MgSO4, and concentrated. The
concentrate was purified by silica gel column chromatography
(10% EtOAc in n-hexane) to afford 15c (0.45 g, 53%) as a pale
yellow oil: 1H NMR (60 MHz, CDCl3) δ 0.80-1.79 (m, 10H),
1.29 (s, 9H), 1.37 (s, 9H), 2.26 (s, 3H), 3.10 (s, 2H), 6.71 (s,
1H); MS m/e 346 (M+), 304, 57.
4,6-Di-ter t-bu tyl-2,3-d ih yd r o-2,2-d im eth yl-5-ben zofu r a -
n ol (1b). LiAlH4 (0.10 g, 2.7 mmol) was suspended in dry THF
(5 mL) under a N2 atmosphere. A solution of 8 (0.86 g, 2.7
mmol) in dry THF (6 mL) was added dropwise to the suspen-
sion, and the mixture was refluxed for 4 h. After the mixture
was cooled to room temperature, water was added dropwise
to quench the excess LiAlH4. After addition of 1 N NaOH (5
mL), the mixture was extracted with Et2O. The extract was
washed with saturated brine, dried over anhydrous MgSO4,
and concentrated. The residue was purified by silica gel column
chromatography (5% EtOAc in n-hexane) to give 1b (0.62 g,
83%) as a white solid, mp 109-110 °C: IR (KBr) 3632, 2964,
1
1404, 1386, 1134 cm-1; H NMR (270 MHz, CDCl3) δ 1.41 (s,
9H), 1.42 (s, 6H), 1.49 (s, 9H), 3.24 (s, 2H), 4.70 (s, 1H), 6.64
(s, 1H); MS m/e 276 (M+). Anal. (C18H28O2) C, H.
4-Acetoxy-3,5-d i-ter t-bu tyl-2-(ch lor oa cetyla m in om eth -
yl)p h en ol (9) a n d 6-Acetoxy-5,7-d i-ter t-bu tyl-3-ch lor o-
a cetyl-2,3-d ih yd r o-4H-1,3-ben zoxa zin e (10). Phenol 3 (29.0
g, 0.11 mol) was dissolved in a 9:1 mixed solution (200 mL) of
acetic acid and H2SO4. After addition of 2-chloro-N-(hydroxym-
ethyl)acetamide31 (34.0 g, 0.28 mol), the mixture was stirred
at room temperature for 48 h. Subsequently, the reaction
mixture was poured into water, neutralized with 1 N NaOH,
and extracted with EtOAc. The organic layer was dried over
anhydrous MgSO4 and concentrated. The concentrate was used
in the subsequent reaction without further purification. When
a portion of the concentrate was purified by silica gel column
chromatography (20% EtOAc in n-hexane), the title compounds
were obtained: 9 as a colorless oil; 1H NMR (60 MHz, CDCl3)
δ 1.30 (s, 9H), 1.43 (s, 9H), 2.28 (s, 3H), 4.00 (s, 2H), 4.73 (d,
J ) 6.0 Hz, 2H), 6.88 (s, 1H), 7.54 (t, J ) 6.0 Hz, 1H); MS m/e
4,6-Di-ter t-b u t yl-2,2-d iet h yl-2,3-d ih yd r o-5-b en zofu r a -
n ol (1c). LiAlH4 (76 mg, 2.0 mmol) was suspended in dry THF
(5 mL) under a N2 atmosphere. A solution of 15c (0.17 g, 0.5
mmol) in dry THF (5 mL) was added dropwise to the suspen-
sion at 0 °C. After heated to reflux for 3 h, the reaction mixture
was allowed to cool to room temperature, and water was added
dropwise to quench the excess LiAlH4. After addition of 1 N
aqueous NaOH (5 mL), the mixture was extracted with Et2O.
The extract was washed with saturated brine, dried over
anhydrous MgSO4, and concentrated. The residue was purified
by silica gel column chromatography (10% EtOAc in n-hexane)
to afford 1c (0.13 g, 87%) as a pale yellow oil: IR (film) 3663,
1
369 (M+), 327, 234, 57; and 10 as a colorless oil; H NMR (60
MHz, CDCl3) δ 1.30 (s, 9H), 1.47 (s, 9H), 2.30 (s, 3H), 4.17 (s,
2H), 5.00 (s, 2H), 5.33 (s, 2H), 6.83 (s, 1H); MS m/e 381 (M+),
339, 304, 57.
1
2975 cm-1; H NMR (270 MHz, CDCl3) δ 0.92 (t, J ) 7.4 Hz,
6H), 1.40 (s, 9H), 1.49 (s, 9H), 1.65-1.75 (m, 4H), 3.18 (s, 2H),
4.66 (s, 1H), 6.63 (s, 1H); MS m/e 304 (M+), 289, 163, 57; HPLC
analysis (CH3CN-i-PrOH-water (8:1:1)) tR ) 6.9 min (90.7%).
HRMS Calcd for C20H32O2: 304.2402. Found: 304.2419.
4-Acetoxy-2-a m in om eth yl-3,5-d i-ter t-bu tylp h en ol (11).
The concentrate obtained in the above reaction was dissolved
in a 10:3 mixed solution (550 mL) of EtOH and concentrated
HCl, and the reaction solution was heated to reflux for 2 h.
After being cooled, the solution was poured into water, and
the mixture was neutralized with 1 N NaOH, followed by
extraction with EtOAc. The organic layer was dried over
anhydrous MgSO4 and concentrated. The concentrate was used
in the subsequent reaction without further purification. When
a portion of the concentrate was purified by silica gel column
chromatography (20% MeOH in CHCl3), phenol 11 was
obtained: 1H NMR (60 MHz, CDCl3) δ 1.27 (s, 9H), 1.37 (s,
9H), 2.25 (s, 3H), 4.22 (s, 2H), 5.18 (bs, 3H), 6.85 (s, 1H); MS
m/e 293 (M+), 234, 191, 57.
5-Acetoxy-4,6-di-ter t-bu tyl-2-h ydr oxyben zaldeh yde (12).
The concentrate obtained in the above reaction was dissolved
in a 11:3 mixed solution (636 mL) of acetic acid and water.
After addition of hexamethylenetetramine (19.3 g, 0.14 mol),
the mixture was heated to reflux for 4 h. Subsequently, 4.5 N
HCl (85 mL) was added to the mixture, and the resulting
The procedure used for the preparation of 1c (Method B)
was repeated with the following compounds using compound
12 and the corresponding Grignard reagents.
4,6-Di-ter t-bu tyl-2,3-d ih yd r o-2,2-d ip r op yl-5-ben zofu r a -
1
n ol (1d ). 23% yield from 12: IR (film) 3662, 2971 cm-1; H
NMR (270 MHz, CDCl3) δ 0.92 (t, J ) 7.3 Hz, 6H), 1.20-1.35
(m, 4H), 1.40 (s, 9H), 1.49 (s, 9H), 1.60-1.70 (m, 4H), 3.19 (s,
2H), 4.66 (s, 1H), 6.61 (s, 1H); MS m/e 332 (M+); HPLC analysis
(CH3CN-i-PrOH-water (8:1:1)) tR ) 8.9 min (97.5%). HRMS
Calcd for C22H36O2: 332.2715. Found: 332.2730.
2,2-Dibu tyl-4,6-d i-ter t-bu tyl-2,3-d ih yd r o-5-ben zofu r a -
n ol (1e). 15% yield from 12: IR (film) 3663, 2964 cm-1 1H
;
NMR (270 MHz, CDCl3) δ 0.90 (t, J ) 6.9 Hz, 6H), 1.30-1.38
(m, 8H), 1.40 (s, 9H), 1.49 (s, 9H), 1.60-1.71 (m, 4H), 3.18 (s,
2H), 4.66 (s, 1H), 6.62 (s, 1H); MS m/e 360 (M+); HPLC analysis
(CH3CN-i-PrOH-water (8:1:1)) tR ) 11.7 min (95.8%). HRMS
Calcd C24H40O2: 360.3028. Found: 360.3005.

Synthesis of Antiatherogenic Antioxidants
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14 3091
4,6-Di-ter t-bu tyl-2,3-d ih yd r o-5-ben zofu r a n ol-2-sp ir o-1′-
cyclop en ta n e (1k ). 23% yield from 12: mp 105-106 °C; IR
3.17 (s, 2H), 4.65 (s, 1H), 6.61 (s, 1H); MS m/e 472 (M+); HPLC
analysis (CH3CN-i-PrOH-water (8:1:1)) tR ) 52.3 min (90.5%).
HRMS Calcd for C32H56O2: 472.4280. Found: 472.4301.
2,2-Diben zyl-4,6-d i-ter t-bu tyl-2,3-d ih yd r o-5-ben zofu r a -
n ol (1j). 9% yield from 12: mp 132-133 °C; IR (KBr) 3661,
2970 cm-1; 1H NMR (270 MHz, CDCl3) δ 1.37 (s, 9H), 1.39 (s,
9H), 2.97 (s, 4H), 3.22 (s, 2H), 4.59 (s, 1H), 6.63 (s, 1H), 7.23
(s, 10H); MS m/e 328 (M+). Anal. (C30H36O2) C, H.
1
(KBr) 3644, 2979 cm-1; H NMR (270 MHz, CDCl3) δ 1.41 (s,
9H), 1.49 (s, 9H), 1.62-2.08 (m, 8H), 3.40 (s, 2H), 4.69 (s, 1H),
6.64 (s, 1H); MS m/e 302 (M+). Anal. (C20H30O2) C, H.
4-Ac e t o x y -3,5-d i-t er t -b u t y l-2-(1-h y d r o x y -2-p e n t y l-
h ep tyl)p h en ol (13f). To a Grignard reagent, which was
prepared with Mg (30 g, 1.23 mol), 6-bromoundecane (290 g,
1.23 mol) prepared in a usual manner, and dry THF (1.2 L)
under a N2 atmosphere, was added dropwise a solution of 12
(120 g, 0.41 mol) in dry THF (400 mL). After the reaction
mixture was stirred at room temperature for 2 h, a saturated
aqueous NH4Cl solution was added to the mixture, followed
by extraction with EtOAc. The organic layer was dried over
anhydrous MgSO4 and concentrated. The concentrate was
purified by silica gel column chromatography (5% EtOAc in
n-hexane) to give 13f (74.3 g, 40%) as a white solid, mp 126
°C: IR (KBr) 3493, 1761, 1369, 1190, 908 cm-1; 1H NMR (270
MHz, CDCl3) δ 0.74 (t, J ) 6.8 Hz, 3H), 0.91 (t, J ) 6.6 Hz,
3H), 0.95-1.63 (m, 16H), 1.29 (s, 9H), 1.40 (s, 9H), 2.12 (m,
1H), 2.28 (s, 3H), 2.50 (d, J ) 2.6 Hz, 1H), 5.22 (dd, J ) 2.6,
9.9 Hz, 1H), 6.77 (s, 1H), 7.89 (s, 1H); MS m/e 448 (M+).
5-Acet oxy-4,6-d i-ter t-b u t yl-2,3-d ih yd r o-2,2-d ip en t yl-
ben zofu r a n (15f). To a solution 13f (74.3 g, 0.166 mol) in CH2-
Cl2 (500 mL) was added dropwise under a N2 atmosphere BF3
diethyl etherate (20 mL, 0.16 mol). After being stirred at room
temperature for 4.5 h, the reaction mixture was added to ice
water, neutralized with 2 N KOH, and extracted with CH2-
Cl2. The extract was washed with saturated aqueous NaCl,
dried over anhydrous MgSO4, and concentrated to give 15f as
a pale yellow oil. The concentrate was used in the subsequent
reaction without further purification: IR (film) 1760, 1567,
1365, 1214, 1172, 943 cm-1; 1H NMR (270 MHz, CDCl3) δ 0.88
(t, J ) 6.6 Hz, 6H), 1.22-1.39 (m, 12H), 1.30 (s, 9H), 1.37 (s,
9H), 1.55-1.75 (m, 4H), 2.29 (s, 3H), 3.10 (d, J ) 15.3 Hz,
1H), 3.21 (d, J ) 15.3 Hz, 1H), 6.72 (s, 1H); MS m/e 430 (M+).
4,6-Di-ter t-bu tyl-2,3-d ih yd r o-2,2-d ip en tyl-5-ben zofu r a -
n ol (1f). LiAlH4 (6.3 g, 0.166 mol) was suspended in dry THF
(200 mL) under a N2 atmosphere. A solution of 15f in dry THF
(400 mL) was added dropwise to the suspension at 0 °C. After
being heated to reflux for 3 h, the reaction mixture was allowed
to cool to room temperature, and EtOAc was added dropwise
to quench the excess LiAlH4. After addition of 10% HCl, the
mixture was extracted with EtOAc. The extract was washed
with saturated brine, dried over anhydrous MgSO4, and
concentrated. The residue was purified by silica gel column
chromatography (n-hexane) to afford 1f (55.0 g, 85% for two
4,6-Di-ter t-bu tyl-2,3-d ih yd r o-5-ben zofu r a n ol-2-sp ir o-1′-
cycloh exa n e (1l). 56% yield from 12: mp 129-131 °C; IR
1
(KBr) 3650, 2934 cm-1; H NMR (270 MHz, CDCl3) δ 1.41 (s,
9H), 1.49 (s, 9H), 1.37-1.63 (m, 6H), 1.75 (m, 4H), 3.18 (s, 2H),
4.68 (s, 1H), 6.65 (s, 1H); MS m/e 316 (M+). Anal. (C21H32O2)
C, H.
4,6-Di-ter t-bu tyl-2,3-d ih yd r o-5-ben zofu r a n ol-2-sp ir o-1′-
cycloh ep ta n e (1m ). 30% yield from 12: mp 94-95 °C; IR
1
(KBr) 3646, 2927 cm-1; H NMR (270 MHz, CDCl3) δ 1.40 (s,
9H), 1.49 (s, 9H), 1.57-2.02 (m, 12H), 3.22 (s, 2H), 4.67 (s,
1H), 6.63 (s, 1H); MS m/e 330 (M+). Anal. (C22H34O2) C, H.
4,6-Di-ter t-bu tyl-2,3-d ih yd r o-5-ben zofu r a n ol-2-sp ir o-1′-
cycloocta n e (1n ). 13% yield from 12: mp 96.8 °C; IR (film)
1
3660, 2933 cm-1; H NMR (270 MHz, CDCl3) δ 1.40 (s, 9H),
1.49 (s, 9H), 1.56-1.76 (m, 10H), 2.03 (m, 4H), 3.18 (s, 2H),
4.67 (s, 1H), 6.63 (s, 1H); MS m/e 344 (M+). Anal. (C23H36O2)
C, H.
4,6-Di-ter t-bu tyl-2,3-d ih yd r o-5-ben zofu r a n ol-2-sp ir o-4′-
tetr a h yd r op yr a n (1o). 52% yield from 12: mp 185-186 °C;
1
IR (KBr) 3365, 2972 cm-1; H NMR (270 MHz, CDCl3) δ 1.41
(s, 9H), 1.49 (s, 9H), 1.71-1.89 (m, 4H), 3.24 (s, 2H), 3.72-
3.80 (m, 2H), 3.86-3.94 (m, 2H), 4.73 (s, 1H), 6.67 (s, 1H); MS
m/e 318 (M+). Anal. (C20H30O3‚0.25H2O) C, H.
4-Acetoxy-3,5-d i-ter t-bu tyl-2-[1-h yd r oxy-5-m eth yl-2-(3-
m eth ylbu tyl)h exyl]p h en ol (13p ). Lithium (2.8 g, 0.40 mol)
was added portionwise to a solution of 12 (24.3 g, 83 mmol)
and 5-bromo-2,8-dimethylnonane (47.0 g, 0.20 mol) in dry THF
(200 mL) at 0 °C under a nitrogen atmosphere, and the
mixture was stirred for 24 h. The reaction mixture was
carefully poured into ice water, neutralized with saturated
aqueous NH4Cl, and extracted with Et2O. The extract was
washed with saturated brine, dried over anhydrous MgSO4,
and concentrated. The residue was purified by silica gel column
chromatography (10% EtOAc in n-hexane) to afford 13p (17.0
g, 46%) as a colorless oil: 1H NMR (60 MHz, CDCl3) δ 0.80-
1.81 (m, 11H), 0.87 (d, J ) 7.0 Hz, 12H), 1.25 (s, 9H), 1.37 (s,
9H), 2.24 (s, 3H), 3.51 (br, 1H), 5.19 (d, J ) 10.0 Hz, 1H), 6.72
(s, 1H), 7.92 (s, 1H); MS m/e 448 (M+).
5-Acetoxy-4,6-d i-ter t-bu tyl-2,3-d ih yd r o-2,2-d i-(3-m eth -
ylbu tyl)ben zofu r a n (15p ). To a solution of 13p (17.0 g, 38
mmol) in CH2Cl2 (200 mL) was added BF3 etherate (4.7 mL,
37 mmol) dropwise at 0 °C under a nitrogen atmosphere. After
stirring the reaction mixture overnight at room temperature,
a saturated aqueous NaHCO3 was added to the mixture. The
mixture was extracted with EtOAc, and the organic layer was
washed with saturated aqueous NaCl, and dried over anhy-
drous MgSO4. The solvent was distilled off to afford 15p (15.5
g, 95%) as a colorless oil: IR (film) 2956, 1764 cm-1; 1H NMR
(60 MHz, CDCl3) δ 0.87 (d, J ) 5.8 Hz, 12H), 1.04-1.93 (m,
10H), 1.28 (s, 9H), 1.35 (s, 9H), 2.25 (s, 3H), 3.14 (s, 2H), 6.67
(s, 1H); MS m/e 430 (M+).
1
steps) as a colorless oil: IR (film) 3652, 2956 cm-1; H NMR
(270 MHz, CDCl3) δ 0.88 (t, J ) 6.9 Hz, 6H), 1.30 (br, 12H),
1.40 (s, 9H), 1.49 (s, 9H), 1.58-1.70 (m, 4H), 3.18 (s, 2H), 4.66
(s, 1H), 6.62 (s, 1H); MS m/e 388 (M+); HPLC analysis (CH3-
CN-i-PrOH-water (8:1:1)) tR ) 15.7 min (95.1%). HRMS
Calcd for C26H44O2: 388.3341. Found: 388.3339.
The procedure used for the preparation of 1f (Method A)
was repeated with the following compounds using compound
12 and the corresponding Grignard reagents.
4,6-Di-ter t-bu tyl-2,2-d ih exyl-2,3-d ih yd r o-5-ben zofu r a -
n ol (1g). 16% yield from 12: IR (film) 3650, 2920 cm-1 1H
;
NMR (270 MHz, CDCl3) δ 0.87 (t, J ) 6.9 Hz, 6H), 1.28 (br,
16H), 1.40 (s, 9H), 1.49 (s, 9H), 1.60-1.70 (m, 4H), 3.18 (s,
2H), 4.66 (s, 1H), 6.61 (s, 1H); MS m/e 416 (M+); HPLC analysis
(CH3CN-i-PrOH-water (8:1:1)) tR ) 23.0 min (94.6%). HRMS
Calcd for C28H48O2: 416.3654. Found: 416.3637.
4,6-Di-ter t-bu tyl-2,2-d ih ep tyl-2,3-d ih yd r o-5-ben zofu r a -
n ol (1h ). 3% yield from 12: IR (film) 3656, 2928 cm-1; 1H NMR
(270 MHz, CDCl3) δ 0.87 (t, J ) 7.3 Hz, 6H), 1.27 (br, 20H),
1.40 (s, 9H), 1.49 (s, 9H), 1.57-1.70 (m, 4H), 3.18 (s, 2H), 4.66
(s, 1H), 6.62 (s, 1H); MS m/e 444 (M+); HPLC analysis (CH3-
CN-i-PrOH-water (8:1:1)) tR ) 34.5 min (98.3%). HRMS.
Calcd for C30H52O2: 444.3967C. Found: 444.3958.
4,6-Di-ter t-bu tyl-2,3-d ih yd r o-2,2-d i-(3-m eth ylbu tyl)-5-
ben zofu r a n ol (1p ). LiAlH4 (1.90 g, 50 mmol) was suspended
in dry THF (200 mL) under a nitrogen atmosphere. A solution
of 15p (17.4 g, 40 mmol) in dry THF (60 mL) was added
dropwise to the suspension at 0 °C. After being heated to reflux
overnight, the reaction mixture was allowed to cool to room
temperature. To the mixture were added water and a satu-
rated aqueous NH4Cl dropwise to quench the excess LiAlH4.
The resulting insolubles were filtered off on Celite, and the
filtrate was extracted with Et2O. The extract was washed with
saturated brine, dried over anhydrous MgSO4, and concen-
trated. The residue was purified by silica gel column chroma-
tography (n-hexane) to afford 1p (9.0 g, 57%) as a colorless
4,6-Di-ter t-b u t yl-2,3-d ih yd r o-2,2-d ioct yl-5-b en zofu r a -
n ol (1i). 2% yield from 12: IR (film) 3652, 2928, 2856, 1412
1
1
cm-1; H NMR (270 MHz, CDCl3) δ 0.88 (t, J ) 6.9 Hz, 6H),
oil: IR (film) 3652, 2956 cm-1; H NMR (270 MHz, CDCl3) δ
1.26 (br, 24H), 1.40 (s, 9H), 1.49 (s, 9H), 1.59-1.70 (m, 4H),
0.89 (d, J ) 6.6 Hz, 12H), 1.19-1.69 (m, 10H), 1.41 (s, 9H),

3092 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
Tamura et al.
1.49 (s, 9H), 3.17 (s, 2H), 4.66 (s, 1H), 6.62 (s, 1H); MS m/e
388 (M+); HPLC analysis (CH3CN-i-PrOH-water (8:1:1)) tR
) 14.6 min (92.9%). HRMS Calcd for C26H44O2: 388.3341.
Found: 388.3342.
plasma was prepared and extracted using EtOAc, and then
plasma concentrations of compounds were determined by
reverse-phase HPLC. Plasma lipoproteins were isolated ac-
cording to the procedure of Havel et al.38 from the WHHL
rabbits treated with compounds at 24 h after the final
administration. The concentrations in the lipoproteins were
determined as mentioned above. The data are expressed as
mean ( range (n ) 2).
2,4,6-Tr i-ter t-bu tyl-5-ben zofu r a n ol (18). To a mixture of
4-tert-butyl-5-benzofuranol 1733 (35 g, 0.18 mol) and 2-methyl-
2-propanol (70 g, 0.94 mol) in CHCl3 (50 mL) at 0 °C was added
methanesulfonic acid (14 mL, 0.22 mol) dropwise. After being
stirred at 0 °C for 20 min, the mixture was poured into ice-
water. Subsequently, the mixture was neutralized with 1 N
NaOH and extracted with EtOAc. The extract was washed
with saturated aqueous NaHCO3, dried over anhydrous
MgSO4, and concentrated. The residue was purified by silica
gel column chromatography (n-hexane) to give 18 (10.87 g,
20%) as a fine-grained, pale yellow crystal, mp 116 °C: IR
Oxid iza bility of th e Isola ted LDL fr om An tioxid a n t
Tr ea ted WHHL Ra bbits. The LDL (200 µg/mL) isolated from
WHHL rabbits treated with 1f or probucol was incubated at
37 °C for 24 h with 10 µM CuSO
4 or 40 µg/mL SLO. The extent
of LDL oxidation was measured by the fluorescence intensity
as mentioned above. The data are expressed as mean ( range
(n ) 2).
1
(KBr) 3641, 2969 cm-1; H NMR (60 MHz, CDCl3) δ 1.33 (s,
9H), 1.47 (s, 9H), 1.62 (s, 9H), 5.06 (s, 1H), 6.58 (s, 1H), 7.29
Ack n ow led gm en t. We gratefully thank Drs. Yasu-
hiro Ohba and J un-ichiro Aono for their help with the
in vivo experiments and valuable discussions and Mrs.
Atsuko Higashida for her help on the determination of
the compound’s plasma concentrations. We also ac-
knowledge Dr. Paul Langman for his useful advice in
the preparation and language editing of this paper.
(s, 1H); MS m/e 302 (M+), 287, 57.
2,4,6-Tr i-ter t-bu tyl-2,3-d ih yd r o-5-ben zofu r a n ol (1q). To
a mixture of 18 (24.7 g, 81.7 mmol) and triethylsilane (76 mL,
0.48 mol) at 0 °C was added trifluoroacetic acid (38 mL)
dropwise. The mixture was stirred first at 0 °C for 15 min,
then at room temperature for 15 min. After being poured into
ice water, the mixture was neutralized with 1 N NaOH and
extracted with EtOAc. The extract was washed with saturated
aqueous NaHCO3, dried over anhydrous MgSO4, and concen-
trated. The residue was purified by silica gel column chroma-
tography (CHCl3) to afford 1q (19.0 g, 76%) as a white needle,
which was recrystallized from n-hexane, mp 90-92 °C: IR
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(KBr) 3669, 2973 cm-1; H NMR (270 MHz, CDCl3) δ 0.97 (s,
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Synthesis of Antiatherogenic Antioxidants
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