Synthesis and biological activities of novel antiallergic agents with 5- lipoxygenase inhibiting action

Article abstract of DOI:10.1016/S0968-0896(99)00291-6

Novel benzimidazole derivatives were synthesized and their pharmacological activities were examined. These compounds showed a good suppressive action on histamine release from rat peritoneal mast cells produced by antigen-antibody reaction, an antagonistic action on guinea pig ileum contraction caused by histamine, an inhibitory action on 5-lipoxygenase in rat basophilic leukemia-1 (RBL-1) cells, and a preventive action on NADPH dependent lipid peroxidation induced by Fe³⁺-ADP in rat liver microsomes. In addition, 1-[2-[2-(4-Hydroxy-2,3,5-trimethylphenoxy)ethoxy]-ethyl]-2-(4- methyl-1-homopiperazino)-1H-benzimidazole difumarate (BOM1006) exhibited a dose dependent suppressive action on 48 h homologous passive cutaneous anaphylaxis (PCA) reaction in rats orally administered the drug. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(99)00291-6

Bioorganic & Medicinal Chemistry 8 (2000) 373±380
Synthesis and Biological Activities of Novel Antiallergic Agents
with 5-Lipoxygenase Inhibiting Action
Hiroyuki Nakano, a Tsutomu Inoue, a Nobuhide Kawasaki, b Hideki Miyataka, b
Hitoshi Matsumoto, b Takeo Taguchi, c Naoki Inagaki, d Hiroichi Nagai d
and Toshio Satoh b,*
aOrganic Chemistry Department, Research Laboratories, Fujiyakuhin Co., Ltd., 3936-2, Sashiogi, Ohmiya, Saitama 331-0047, Japan
bFaculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan
cSchool of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo 192-0392, Japan
dDepartment of Pharmacology, Gifu Pharmaceutical University, Gifu 502-8585, Japan
Received 3 September 1999; accepted 29 October 1999
AbstractÐNovel benzimidazole derivatives were synthesized and their pharmacological activities were examined. These compounds
showed a good suppressive action on histamine release from rat peritoneal mast cells produced by antigen±antibody reaction, an
antagonistic action on guinea pig ileum contraction caused by histamine, an inhibitory action on 5-lipoxygenase in rat basophilic
leukemia-1 (RBL-1) cells, and a preventive action on NADPH dependent lipid peroxidation induced by Fe3+-ADP in rat liver
microsomes. In addition, 1-[2-[2-(4-Hydroxy-2,3,5-trimethylphenoxy)ethoxy]-ethyl]-2-(4-methyl-1-homopiperazino)-1H-benzimid-
azole difumarate (BOM1006) exhibited a dose dependent suppressive action on 48 h homologous passive cutaneous anaphylaxis
(PCA) reaction in rats orally administered the drug. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
receptor antagonists have been clinically used success-
fully.4,5 Involvement of 5-lipoxygenase is known in the
biosynthesis of leukotrienes, and some agents that inhi-
bit the synthesis have been developed.6,7 Active oxygen
is released from in¯ammatory cells such as eosinophils
in allergic in¯ammation and this has been regarded as a
cause of worsening symptoms.8±10
In recent years, a rapid increase in the number of
patients with some allergic symptoms has become our
grave concern. Degranulation of mast cell induced by
antigen±antibody reaction triggers allergic disease of
type I including bronchial asthma, allergic rhinitis,
atopic dermatitis, and pollenosis. Histamine is one of
the chemical mediators released in this reaction and
combines with H1 receptors of tissues to cause smooth
muscle contraction, vasodilatation, and vascular per-
meability rising, thereby leading to allergic symptoms.1
The action mechanism for most of the conventional
antiallergic agents was to suppress the release of chemi-
cal mediators, mainly histamine, and to antagonize
them. However, these actions alone were unable to pre-
vent the diseases from worsening.
We previously synthesized trimethylhydroquinone deri-
vatives with an antihistaminic action, an inhibitory
action on 5-lipoxygenase, and an antioxidative action
and reported their e€ectiveness in asthma model ani-
mals.11,12 We then synthesized novel antiallergic agents
with multiple pharmacological activities through hybrid-
ization of emedastine, known as an antiallergic agent
that possesses a suppressive action on histamine release
from mast cells and an antagonistic action on H1 recep-
tor13±15 as the core, benzimidazole derivatives, and tri-
methylhydroquinone derivatives that have an inhibitory
action on 5-lipoxygenase and an antioxidative action.16
In this article, we report synthesis and the pharmaco-
logical activities of the three novel benzimidazole deriva-
tives which were considered most promising in the
previous paper. The activities studied are suppressive
action on histamine release from rat peritoneal mast
cells caused by antigen±antibody reaction, antagonistic
action on contraction of guinea pig ileum produced by
Leukotrienes are chemical mediators deeply involved in
allergic in¯ammation and have activities to accumulate
in¯ammatory cells and contract bronchial smooth
muscles.2,3 Some drugs developed as peptide leukotrienes
Keywords: allergies; enzyme inhibitors; antagonists; antioxidants.
*Corresponding author. Fax: +81-88-655-3051; e-mail: tosato@
ph.bunri-u.ac.jp
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00291-6

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H. Nakano et al. / Bioorg. Med. Chem. 8 (2000) 373±380
histamine, inhibitory action on 5-lipoxygenase in rat
basophilic leukemia-1 (RBL-1) cells, and preventive
action on NADPH dependent lipid peroxidation
induced by Fe3+-ADP in rat liver microsomes. More-
over, descriptions are given on the suppressive action of
1-[2-[2-(4-hydroxy-2,3,5-trimethylphenoxy)ethoxy]ethyl]-
2-(4-methyl-1-homopiperazino)-1H-benzimidazole difu-
marate (BOM1006) on rat 48 h homologous passive
cutaneous anaphylaxis (PCA) reaction.
benzimidazole derivatives so that the resultant hybrids
bear several antiallergic pharmacological activities, the
site of hybridization is restricted and only BOM1006
exhibits an antihistaminic activity which has two ether
groups in the spacer between benzimidazole ring and
trimethylhydroquinone, thus suggesting that this is a
most promising hybrid form.16 Hence, we newly
designed benzimidazole derivatives (BOM1011±1013)
with di€erent alkyl chain lengths between ether groups
in the spacer. The synthetic route is shown in Scheme 1.
Compounds 1a±1c were obtained by stirring a mix-
ture of ethyleneglycol and 4-(bromoalkyloxy)-2,3,6-tri-
methylphenol in the presence of NaOH at 45±60 ꢀC for
3±6 h (Method A). The products were brominated with
CBr4 and Ph3P to give compounds 2a±2c (Method B),
Chemistry
It was shown in the previous paper, that when tri-
methylhydroquinone derivatives are hybridized with
Scheme 1.

H. Nakano et al. / Bioorg. Med. Chem. 8 (2000) 373±380
375
which were then introduced into 2-chloro-1H-benzimid-
azole according to the method described in the pre-
vious paper, to yield compounds 3a±3c (Method C).
BOM1011±1013 free bases were obtained through the
condensation reaction of 3a±3c with 1-methylhomopiper-
azine (Method D). Finally, the products were converted
to their difumarates (BOM1011±1013), which were then
used in pharmacological examinations.
Table 1. E€ect of benzimidazole derivatives and emedastine on his-
tamine induced contraction of isolated ileum from guinea pig
a
Compd.
R
pA2
Emedastine
9.09
Results and Discussion
BOM1006
BOM1011
7.49
7.17
The suppressive action of the benzimidazole derivatives
(BOM1006, 1011±1013) was examined on histamine
release from rat peritoneal mast cells induced by anti-
gen±antibody reaction. Mast cells were withdrawn from
rat peritoneal cavity passively sensitized with rat anti-
2,4-dinitrophenyl-bovine serum albumin (DNP-BSA)
serum and the cells were incubated with each of the test
compounds at 37 ꢀC, 15 min. Degranulation of the cells
was then induced through their antigen±antibody reac-
tion with DNP-BSA added as the antigen during 20 min
incubation at 37 ꢀC. The amounts of histamine in mast
cells and released from the cells were determined ¯uor-
ophotometrically using o-phthalaldehyde as the reagent,
and the rate of suppression for the test compounds was
calculated against the control on the basis of the release
rate data of histamine thus obtained. All of the benzi-
midazole derivatives synthesized and emedastine
showed histamine release suppressing action at their
concentration of 10 5 M (Fig. 1). The suppressive action
of these benzimidazole derivatives was as strong as that
of emedastine. This suggests little or no e€ect of the
introduction of trimethylhydroquinone moiety and its
spacer length on the histamine release suppressing
action of benzimidazole derivatives.
BOM1012
BOM1013
6.87
7.11
aTest compound concentration, 10 6±10 9 M. Results are the means
of triplicate determinations.
exhibited a good H1 receptor antagonizing action,
though the action was somewhat weaker than that of
emedastine. Little or no e€ect of the length of spacer
was also shown on this action.
Next, the H1 receptor antagonizing action of the benzi-
midazole derivatives synthesized was examined through
contraction of guinea pig ileum by histamine. Guinea
pig ileum was incubated with each of the test com-
pounds for 5 min and then given histamine in an accu-
mulative way. The values of pA2 obtained were shown
in Table 1. The benzimidazole derivatives synthesized,
The inhibitory action of the synthesized derivatives was
then examined on 5-lipoxygenase in RBL-1 cells. Ara-
chidonic acid was added to a homogenate of the cells
and the amount of 5-hydroxy-6,8,11,14-eicosatetraenoic
Figure 1. E€ect of benzimidazole derivatives and emedastine on histamine release from sensitized rat peritoneal mast cell caused by antigen. Test
compound concentration, 10 5 M. Results are the means of duplicate determinations.

376
H. Nakano et al. / Bioorg. Med. Chem. 8 (2000) 373±380
acid (5-HETE) produced was determined by HPLC.
The rate of inhibition for the test compounds against
the control was calculated using the analytical data
obtained. As a result, all of the benzimidazole deriva-
tives synthesized were found to show a concentration
dependent inhibitory action on 5-lipoxygenase at their
concentration 3Â10 6 M±10 7 M and the value of IC50
ranged from 4.38Â10 7 to 9.48Â10 7 M (Fig. 2). Mean-
while, their inhibitory action was slightly weaker than
that of nordihydroguaiaretic acid (NDGA) as a positive
control at 3Â10 7 M. Hence, even though the introduc-
tion of trimethylhydroquinone moiety additionally gave
an inhibitory action on 5-lipoxygenase to benzimidazole
derivatives, this action was also suggested to be hardly
a€ected by the spacer length.
lipid peroxidation induced by Fe3+-ADP in rat liver
microsomes as an index of antioxidative action. The
amount of peroxidized lipids produced in rat liver
microsomes was determined as the amount of mal-
ondialdehyde using 2-thiobarbituric acid (TBA) as the
reagent, and the rate of inhibition against the control
was calculated on the basis of the analytical data
obtained. All synthesized benzimidazole derivatives
exhibited a concentration dependent preventive action
on lipid peroxidation at their concentration 5 Â 10 6 M±
10 6 M and the value of IC50 varied from 1.87 Â 10 6 to
2.58 Â 10 6 M (Fig. 3). Moreover, these synthesized
derivatives had a preventive action stronger than that
of butylated hydroxytoluene (BHT) as a positive control
at 3 Â 10 6 M. Thus, the introduction of trimethyl-
hydroquinone moiety was shown to give an anti-
oxidative activity to benzimidazole derivatives, in addition
to 5-lipoxygenase inhibiting activity. The spacer length
An examination was performed of the preventive action
of the synthesized derivatives on NADPH dependent
Figure 2. E€ect of benzimidazole derivatives and NDGA on RBL-1 cell 5-lipoxygenase activity. Results are the means of duplicate determinations.
Figure 3. E€ect of benzimidazole derivatives and BHT on Fe3+-ADP induced NADPH dependent lipid peroxidation in rat liver microsomes.
Results are the means of duplicate determinations.

H. Nakano et al. / Bioorg. Med. Chem. 8 (2000) 373±380
377
was again suggested to have almost no e€ect on this
activity.
General method A. 4-[3-(2-Hydroxyethoxy)propyloxy]-
2,3,6-trimethylphenol (1a). 4-(3-Bromopropyloxy)-2,3,6-
trimethylphenol11 (5.9 g, 21mmol), ethyleneglycol (13.4 g,
216 mmol) and NaOH (867 mg, 21 mmol) were mixed
and stirred at 45 ꢀC for 3 h. The mixture was added to
water, extracted with CHCl3, washed with water and
brine, dried over anhydrous Na2SO4 and evaporated to
Finally, an examination was conducted to check if
BOM1006 has an in vivo antiallergic activity through its
action on 48 h homologous PCA reaction in rat dorsal
skin. Rats were subcutaneously administered rat anti-
DNP-BSA serum in their back and 48 h later intra-
venously administered saline solution containing Evans
blue and DNP-BSA. The animals were sacri®ed half an
hour later by bloodletting, the part of their dorsal skin
that received the antibody was withdrawn, and the rate
of suppression for the agent was calculated against the
control, using the spectrophotometrically determined
concentrations of the dye in the solutions obtained by
extracting it from the withdrawn skin part.
1
a€ord 1a (4.3 g, 16.9 mmol, 80%) as a brown oil, H
NMR d: 1.94 (1H, t, J=5.61 Hz), 2.00±2.08 (2H, m),
2.13 (3H, s), 2.15 (3H, s), 2.22 (3H, s), 3.82±3.86 (2H,
m), 3.91 (2H, q, J=5.61 Hz), 4.01±4.06 (4H, m), 4.34
(1H, s), 6.34 (1H, s). FABMS m/z: 255 (M++1).
Compounds 1b and 1c were prepared in the same man-
ner as 1a (General method A).
4-[4-(2-Hydroxyethoxy)butoxy]-2,3,6-trimethylphenol
(1b). 4-(4-Bromobutoxy)-2,3,6-trimethylphenol (8.0 g,
28 mmol), ethyleneglycol (17.3 g, 279 mmol) and NaOH
(1.1 g, 28 mmol) were mixed and stirred at 45 ꢀC for 5 h.
Each of the test compounds was orally given to the
animals an hour before the antigen administration.
BOM1006 was found to suppress PCA reaction in a
dose dependent way at doses from 3 to 30 mg/kg (Fig. 4).
The compound thus manifested itself to be a good anti-
allergic agent in vivo even though its action was slightly
weaker than that of emedastine.
1
1b (3.9 g, 14.6 mmol, 52%); pale brown oil, H NMR d:
1.74±1.90 (4H, m), 1.93±2.00 (2H, m), 2.14 (3H, s), 2.17
(3H, s), 2.22 (3H, s), 3.73 (2H, t, J=6.27 Hz), 3.89 (2H,
t, J=5.61 Hz), 4.02±4.06 (2H, m), 4.36 (1H, s), 6.34 (1H,
s). FABMS m/z: 269 (M++1).
In conclusion, we have demonstrated that the novel
benzimidazole derivatives possess multiple pharmaco-
logical activities and BOM1006 is e€ective in allergic
model animals when it is orally administered. BOM1006
may be a leading compound in the future development
of new antiallergic agents.
4-[5-(2-Hydroxyethoxy)pentyloxy]-2,3,6-trimethylphenol
(1c). 4-(5-Bromopentyloxy)-2,3,6-trimethylphenol (10.0 g,
33 mmol), ethyleneglycol (21.0 g, 338 mmol) and NaOH
(1.4 g, 35 mmol) were mixed and stirred at 60 ꢀC for
1
6 h. 1c (5.6 g, 19.9 mmol, 60%); brown oil, H NMR d:
1.52±1.68 (5H, m), 1.73±1.85 (2H, m), 1.92±1.98 (2H,
m), 2.14 (3H, s), 2.16 (3H, s), 2.22 (3H, s), 3.69 (2H, t,
J=6.27 Hz), 3.88 (2H, t, J=6.27 Hz), 4.00±4.04 (2H,
m), 4.34 (1H, s), 6.34 (1H, s). FABMS m/z: 283
(M++1).
Experimental
Chemistry
1H NMR spectra were measured on a JEOL JNM-
EX270 (270 MHz) spectrometer in CDCl3 solution with
tetramethylsilane as an internal standard. FABMS were
obtained on a JEOL JMS-AX505H spectrometer.
General method B. 4-[3-(2-Bromoethoxy)propyloxy]-
2,3,6-trimethylphenol (2a). 1a (4.0 g, 15 mmol) and CBr4
(6.5 g, 19 mmol) were dissolved in 30 mL of CHCl3.
Ph3P (5.0 g, 19 mmol) was added in the mixture on
ice bath. The mixture was stirred at room tempera-
ture for 40 min and evaporated. The residue was
BOM1006 has been described previously.16
Figure 4. E€ect of BOM1006 and emedastine on 48 h homologous PCA reaction in rats. Each value represents the mean and S. E. of 3 to 5 rats.

378
H. Nakano et al. / Bioorg. Med. Chem. 8 (2000) 373±380
chromatographed on silica gel (hexane:AcOEt, 3:1) to
1
3.82±3.98 (4H, m), 4.34 (2H, t, J=5.62 Hz), 4.46 (1H, s),
6.46 (1H, s), 7.24±7.28 (2H, m), 7.36±7.40 (1H, m),
7.66±7.68 (1H, m). FABMS m/z: 403 (M++1).
a€ord 2a (3.3 g, 10.4 mmol, 69%) as a brown oil, H
NMR d: 2.13 (3H, s), 2.17 (3H, s), 2.22 (3H, s), 2.26±
2.36 (2H, m), 3.63 (2H, t, J=6.58 Hz), 3.80±3.84 (2H,
m), 4.01±4.05 (4H, m), 4.30 (1H, s), 6.80 (1H, s).
FABMS m/z: 318 (M++2).
2-Chloro-1-[2-[5-(4-hydroxy-2,3,5-trimethylphenoxy)pen-
tyloxy]ethyl]-1H-benzimidazole (3c). 2-Chloro-1H-ben-
zimidazole (2.0 g, 13 mmol) was dissolved in 20 mL of
DMF. 60% NaH dispersion in mineral oil (600 mg,
13 mmol) was added to the solution on ice bath and
stirred for 30 min. The mixture was added 2c (4.5 g,
13 mmol) and stirred at 80 ꢀC for 4 h. 3c (1.4 g, 3.4 mmol,
26%); pale brown amorphous powder, 1H NMR d:
1.56±1.76 (2H, m), 1.78±1.97 (4H, m), 2.08 (3H, s), 2.14
(3H, s), 2.21 (3H, s), 3.64±3.74 (2H, m), 3.82±3.96 (4H,
m), 4.32 (2H, t, J=5.61 Hz), 4.44 (1H, s), 6.46 (1H, s),
7.22±7.26 (2H, m), 7.36±7.41 (1H, m), 7.64±7.66 (1H,
m). FABMS m/z: 417 (M++1).
Compounds 2b and 2c were prepared in the same man-
ner as 2a (General method B).
4-[4-(2-Bromoethoxy)butoxy]-2,3,6-trimethylphenol (2b).
Compounds 1b (4.4 g, 16 mmol) and CBr4 (6.5 g,
19 mmol) were dissolved in 30 mL of CHCl3. Ph3P
(5.0 g, 19 mmol) was added in the mixture on ice bath.
The mixture was stirred at room temperature for
40 min. 2b (3.8 g, 11.5 mmol, 72%); brown oil, 1H NMR
d: 1.90±1.97 (2H, m), 2.03±2.13 (2H, m), 2.12 (3H, s), 2.17
(3H, s), 2.21 (3H, s), 3.49 (2H, t, J=6.60 Hz), 3.82±3.86
(2H, m), 3.91 (2H, t, J=5.94 Hz), 4.03±4.06 (2H, m), 4.30
(1H, s), 6.80 (1H, s). FABMS m/z: 333 (M++2).
General method D. 1-[2-[3-(1-Hydroxy-2,3,6-trimethyl-
phenoxy)propyloxy]ethyl]-2-(4-methyl-1-homopiperazino)-
1H-benzimidazole (BOM1011 (free base)). Compound
3a (1.2 g, 3.1 mmol) and 1-methylhomopiperazine (927 mg,
9.2 mmol) were mixed at 130 ꢀC for 3 h. The mixture
was chromatographed on silica gel (CHCl3:MeOH,
50:1) to a€ord BOM1011 (1.2 g, 2.6 mmol, 84%) as a
4-[5-(2-Bromoethoxy)pentyloxy]-2,3,6-trimethylphenol
(2c). 1c (4.9 g, 17 mmol) and CBr4 (7.0 g, 21 mmol)
were dissolved in 30 mL of CHCl3. Ph3P (5.5 g,
21 mmol) was added in the mixture on an ice bath. The
mixture was stirred at room temperature for 1 h. 2c
1
brown oil, H NMR d: 1.74±1.92 (2H, m), 2.00±2.14
1
(3.7 g, 10.7 mmol, 63%); brown oil, H NMR d: 1.62±
(2H, m), 2.10 (3H, s), 2.14 (3H, s), 2.20 (3H, s), 2.38
(3H, s), 2.70±2.88 (4H, m), 3.62±3.68 (4H, m), 3.74±3.79
(2H, m), 3.82±3.97 (4H, m), 4.23 (2H, t, J=6.28 Hz),
6.44 (1H, s), 7.08±7.17 (2H, m), 7.23±7.28 (1H, m), 7.54
(1H, dd, J=1.21, 6.95 Hz). FABMS m/z: 467 (M++1).
1.68 (2H, m), 1.75±1.86 (2H, m), 1.88±1.99 (2H, m),
2.10 (3H, s), 2.14 (3H, s), 2.22 (3H, s), 3.45 (2H, t,
J=6.93 Hz), 3.81±3.86 (4H, m), 4.00±4.06 (2H, m), 4.26
(1H, s), 6.51 (1H, s). FABMS m/z: 347 (M++2).
General method C. 2-Chloro-1-[2-[3-(4-hydroxy-2,3,5-tri-
methylphenoxy)-propyloxy]ethyl]-1H-benzimidazole (3a).
2-Chloro-1H-benzimidazole (2.0 g, 13 mmol) was dis-
solved in 20 mL of N,N-dimethylformamide (DMF).
60%NaH dispersion in mineral oil (600 mg, 13 mmol)
was added to the solution on ice bath and stirred for
30 min. To the mixture was added 2a (4.2 g, 13 mmol)
and stirred at 80 ꢀC for 4 h. The mixture was added to
water, extracted with AcOEt, washed with water and
brine, dried over anhydrous Na2SO4 and evaporated.
The residue was washed with hexane:AcOEt (1:1) to
a€ord 3a (2.4 g, 6.2 mmol, 48%) as a pale brown amor-
BOM1012 and BOM1013 were prepared in the same
manner as BOM1011 (General method D).
1-[2-[4-(1-Hydroxy-2,3,6-trimethylphenoxy)butoxy]ethyl]-
2-(4-methyl-1-homopiperazino)-1H-benzimidazole (BOM
1012 (free base)). Compound 3b (1.2 g, 2.9 mmol)
and 1-methylhomopiperazine (880 mg, 8.8 mmol) were
mixed at 130 ꢀC for 3 h. BOM1012 (1.0 g, 2.1 mmol,
1
72%); brown oil, H NMR d: 1.36±1.42 (2H, m), 1.64±
1.83 (2H, m), 2.00±2.04 (2H, m), 2.11 (3H, s), 2.14 (3H,
s), 2.21 (3H, s), 2.40 (3H, s), 2.68±2.82 (4H, m), 3.62±
3.67 (4H, m), 3.78±3.90 (2H, m), 3.92±3.98 (4H, m), 4.26
(2H, t, J=6.25 Hz), 6.42 (1H, s), 7.06±7.14 (2H, m),
7.22±7.26 (1H, m), 7.52 (1H, dd, J=1.32, 6.72 Hz).
FABMS m/z: 481 (M++1).
1
phous powder, H NMR d: 2.08 (3H, s), 2.16 (3H, s),
2.18±2.24 (2H, m), 2.21 (3H, s), 3.70±3.75 (2H, m),
3.82±3.98 (4H, m), 4.32 (2H, t, J=5.62 Hz), 4.46 (1H, s),
6.48 (1H, s), 7.23±7.27 (2H, m), 7.37±7.41 (1H, m),
7.66±7.69 (1H, m). FABMS m/z: 389 (M++1).
1-[2-[5-(1-Hydroxy-2,3,6-trimethylphenoxy)pentyloxy]-
ethyl]-2-(4-methyl-1-homopiperazino)-1H-benzimidazole
(BOM1013 (free base)). Compound 3c (1.2 g, 2.8 mmol)
and 1-methylhomopiperazine (860 mg, 8.6 mmol) were
mixed at 130 ꢀC for 5 h. BOM1013 (1.1 g, 2.2 mmol,
Compounds 3b and 3c were prepared in the same man-
ner as 3a (General method C).
1
2-Chloro-1-[2-[4-(4-hydroxy-2,3,5-trimethylphenoxy)but-
oxy]ethyl]-1H-benzimidazole (3b). 2-Chloro-1H-benzi-
midazole (2.0 g, 13 mmol) was dissolved in 20 mL of
DMF. 60% NaH dispersion in mineral oil (600 mg,
13 mmol) was added to the solution on ice bath and
stirred for 30 min. The mixture was added 2b (4.2 g,
13 mmol) and stirred at 80 ꢀC for 4 h. 3b (1.9 g,
4.7 mmol, 36%); pale brown amorphous powder, 1H
NMR d: 1.82±1.88 (2H, m), 1.97±2.10 (2H, m), 2.10
(3H, s), 2.14 (3H, s), 2.22 (3H, s), 3.69±3.74 (2H, m),
79%); brown oil, H NMR d: 1.41±1.58 (2H, m), 1.72±
1.86 (4H, m), 2.01±2.11 (2H, m), 2.13 (3H, s), 2.16 (3H,
s), 2.22 (3H, s), 2.41 (3H, s), 2.68±2.84 (4H, m), 3.63±
3.66 (4H, m), 3.76±3.90 (2H, m), 3.94±3.98 (4H, m), 4.24
(2H, t, J=6.24 Hz), 6.44 (1H, s), 7.04±7.13 (2H, m),
7.20±7.24 (1H, m), 7.51 (1H, dd, J=1.28, 6.68 Hz).
FABMS m/z: 495 (M++1).
Synthesis of benzimidazole derivative difumarate (BOM
1011±1013). Free base of benzimidazole derivative

H. Nakano et al. / Bioorg. Med. Chem. 8 (2000) 373±380
379
was dissolved in EtOH. Fumaric acid (2.2 eq) in hot
EtOH solution was added. The mixture was cooled,
evaporated, washed with hexane and dried to a€ord
the benzimidazole derivative difumarate (BOM1011±
1013).
Measurement of histamine induced contraction of iso-
lated ileum from guinea pig. Male Hartley guinea pigs
weighing 300±400 g were sacri®ced and the ilea were
prepared. The segments (about 1 cm) of ileum were sus-
pended in a Magnus tube (35 ꢀC, 95% O2/5% CO2)
containing 10 mL of Tyrode solution. After 30 min, test
compound was added and incubated for 5 min. Hista-
mine was cumulatively added to the tube to obtain a
concentration-response curve. The contractile responses
were measured using an isotonic transducer (TD-112S;
Nihon Kohden Co., Ltd.). pA2 values of test com-
pounds were calculated by the method of Takayanagi.19
Pharmacology
Materials. Male Wistar rats and male Hartley guinea
pigs were obtained from Japan SLC Inc. DNP-BSA and
rat anti-DNP-BSA serum were prepared according to
the method described in a previous paper.17 RBL-1 cell
was obtained from Dainippon Pharmaceutical Co., Ltd.
5-HETE and l-a-phosphatidyl-l-serine were obtained
from Sigma Chemical Co. ADP, histamine dihy-
drochloride and b-NADPH were obtained from Wako
Pure Chemical Industries, Ltd. Arachidonic acid, BHT
and NDGA were obtained from Nacalai Tesque, Inc.
Measurement of RBL-1 cell 5-lipoxygenase activity.20
The modi®ed method of Blackham et al.21 was used.
RBL-1 cells were grown in Roswell Park Memorial
Institute (RPMI)-1640 medium containing 10% heat-
inactivated newborn calf serum, 100 units/mL penicillin
and 0.1 mg/mL streptomycin. Cells were cultured at
37 ꢀC in 5% CO2/air. Cells in the growth phase (5 Â 105±
106 cells/mL) were collected by centrifugation (100 Â g,
5 min) and suspended at 3 Â 107 cells/mL in 50 mM
phosphate bu€er (0.25 M sucrose, 1 mM EDTA, 2 mM
glutathione, pH 7.4). Cells were stored at 80 ꢀC until
use. The assay system (0.5 mL) consisted of 50 mM
phosphate bu€er (0.25 M sucrose, 1 mM EDTA, 2 mM
glutathione, pH 7.4), the test compound in 1% dimethyl
sulfoxide (DMSO), 2 mM CaCl2, 0.2 mg/mL arachi-
donic acid (10 mg/mL MeOH soln; 10 mL) and 107 cells/
mL RBL-1 cells homogenate. Reaction mixture was
incubated at 37 ꢀC for 3 min, and then 0.5 mL of MeOH
was added to terminate the reaction. The mixture was
centrifuged (2000 Â g, 15 min), 5-HETE in the super-
natant was analyzed by HPLC (column; COSMOSIL
5C18-MS Waters 4.6 Â 150 mm (Nacalai Tesque, Inc.),
mobile phase; CH3CN:0.1% AcOH aq (6: 4), ¯ow rate;
1 mL/min, temperature; rt, absorbance; 235 nm).
Assay of histamine release from sensitized rat peritoneal
exudate cell caused by antigen.17 Male Wistar rats
weighing about 300 g were passively sensitized by an
intraperitoneal injection of 2 mL of physiological saline
containing 10 mL of rat anti-DNP-BSA serum (titer;
1:2000 or greater). After 2 days, rats were exsanguinated
and injected intraperitoneally with 15 mL of Tyrode
solution containing 10 mM HEPES and 0.05% gelatin
(pH 7.4). The abdominal region was gently massaged
for 1.5 min. The peritoneal exudate cells were collected
and recovered by washing the cavity with 15 mL of
Tyrode solution. Cells were washed 3 times with Tyrode
solution by centrifugation (55Âg) at 4 ꢀC for 8 min and
resuspended in a small volume of Tyrode solution. Mast
cells were counted after staining with toluidin blue.
A Tyrode solution containing 10 mM HEPES and
0.05% gelatin (pH 7.4) was added 30 mg/mL l-a-phos-
phatidyl-l-serine and test compound, and preincubated
at 37 ꢀC for 5 min. After adding 105 mast cells/mL peri-
toneal exudate cells to the preincubated solution, the
resultant solution was incubated at 37 ꢀC for 15 min. To
the incubated solution was added 10 mg protein/ml
DNP-BSA and the solution was further incubated at
37 ꢀC for 20 min (®nal volume 1 mL). After ice-cooling
for 10 min, the solution was centrifuged at 4 ꢀC for
10 min (300 Â g). An aliquot (0.5 mL) of the supernatant
liquid was used to determine the amount of released
histamine after adding 0.5 mL of 0.2 N HCl. To the
precipitate was added 2 mL of 0.1 N HCl and the resul-
tant dispersion was treated at 100 ꢀC for 10 min. After
ice-cooling for 10 min, the dispersion was centrifuged at
4 ꢀC for 10min (780 Â g), an aliquot (1 mL) of the super-
natant liquid of which was then used to determine the
amount of histamine associated with the cells. Determi-
nations of the amount of histamine were carried out
according to the method of Shore et al.18 Thus, 0.1 mL
of 2 N NaOH and 0.05 mL of 1% o-phthalaldehyde
MeOH solution were added to 1 mL of test sample,
which was then allowed to stand at room temperature
for 4 min. After adding 0.1 mL of 2 M citrate, the ¯uo-
rescence intensity of the sample was measured (EX 356
nm, EM 440 nm) and the rate of histamine release was
calculated.
Measurement of Fe3+-ADP induced NADPH dependent
lipid peroxidation in rat liver microsome.22 The modi®ed
method of Kiso et al.23 was used. Microsomes were
prepared from male Wistar rats weighing about 200 g.
The rats liver was homogenized in cold 0.25 M sucrose.
The homogenate was centrifuged at 4 ꢀC (8000 Â g,
10 min). The supernatant fraction was then collected
and ultracentrifuged at 4 ꢀC (105,000 Â g, 30 min). The
pellet obtained was resuspended in 83.5 mM KCl±
37.2 mM Tris±HCl bu€er (pH 7.4) and stocked at
20 ꢀC until use. Protein concentration was determined
by the method of Lowry et al.24 The assay system
(1 mL) consisted of 83.5 mM KCl±37.2 mM Tris±HCl
bu€er (pH 7.4), the test compound in 1% DMSO,
0.2 mM NADPH, 1 mM ADP, 1 mg protein/mL rat
liver microsomes and 10 mM FeCl3. The reaction mix-
ture was incubated at 37 ꢀC for 20 min, and then cooled
on ice to terminate the reaction. Lipid peroxide was
measured by the method of Ohkawa et al.25 Thus, 8.1%
sodium dodecyl sulfate (0.2 mL), 20% AcOH containing
0.27 M HCl adjusted to pH 3.5 with NaOH (1.5 mL)
and 0.8% TBA (1.5 mL) were added to the reaction
mixture. The mixture was then boiled at 100 ꢀC for
20 min and the reaction was stopped by cooling on ice.
Thereafter, n-BuOH-pyridine (15:1, 4 mL) was added,

380
H. Nakano et al. / Bioorg. Med. Chem. 8 (2000) 373±380
and vigorous mixing was performed. After centrifuga-
tion (780 Â g, 10 min), the organic layer was separated,
and the absorbance was measured at 532 nm. The
amount of TBA-positive material was expressed as a
corresponding amount of malondialdehyde.
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6. Samuelsson, B.; Hammarstrom, S.; Murphy, R. C.; Bor-
geat, P. Allergy 1980, 35, 375.
E€ects on 48 h homologous PCA reaction in rats.26
DNP-BSA and rat anti-DNP-BSA serum were used as
antigen and antiserum, respectively. The dorsum of a
male Wistar rat weighting about 200 g, was shaved and
0.1 mL of an antiserum, with a 48 h homologous PCA
strength of 1:128±1:256 diluted with physiological sal-
ine, was injected into the dorsum intracutaneously.
After 48 h, 1 mL of 0.5% Evans blue physiological sal-
ine, containing DNP-BSA equivalent to 1 mg protein,
was injected into the caudal vein. After 30 min, the rat
was killed by exsanguination and the pigment freckle
generated on the back skin was cut out and the amount
of transudate pigment was determined. To do this, the
cut out skin was put in a test tube, 1 mL of 1 N KOH
was added and allowed to stand overnight at 37 ꢀC to
elute the pigment and then 9 mL of a mixture of acetone
and 0.6 N phosphoric acid (mixed at 13:5) was added
with shaking. The insoluble substances were removed by
centrifugation at 1500 Â g for 10 min, and the absorb-
ence of the supernatant was measured at 620 nm to
determine the amount of pigment. The specimen was
suspended in 0.2% carboxymethyl cellulose sodium salt,
and given orally to the rat, in dose of 0.5 mL per 100 g
body weight, 1 h before antigen administration.
7. Bell, R. L.; Young, P. R.; Albert, D.; Lanni, C.; Summers,
J. B.; Brooks, D. W.; Rubin, P.; Carter, G. W. Int. J. Immuno-
pharmac. 1992, 14, 505.
8. Miyachi, Y. Pharma Medica 1991, 11, 193.
9. Yoshikuni, H. Jpn. J. Clin. Immun. 1991, 14, 166.
10. Akagi, M.; Katakuse, Y.; Fukuishi, N.; Kan, T.; Akagi, R.
Biol. Pharm. Bull. 1994, 17, 732.
11. Kawasaki, N.; Miyataka, H.; Nishiki, M.; Matsumoto, H.;
Inagaki, N.; Nagai, H.; Satoh, T. Chem. Pharm. Bull. 1999, 47,
177.
12. Kawasaki, N.; Miyataka, H.; Matsumoto, H.; Yamashita,
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13. Fukuda, T.; Morimoto, Y.; Iemura, R.; Kawashima, T.;
Tsukamoto, G.; Ito, K. Arzneim.-Forsch. Drug Res. 1984, 34,
801.
14. Fukuda, T.; Saito, T.; Tajima, S.; Shinohara, K.; Ito, K.
Arzneim.-Forsch. Drug Res. 1984, 34, 805.
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16. Nakano H.; Inoue T.; Kawasaki N.; Miyataka H.; Mat-
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17. Inagaki, N.; Kawasaki, H.; Nagai, H. In¯amm. Res. 1995,
44, 541.
18. Shore, P. A.; Burkhalter, A.; Cohn, V. H., Jr. J. Pharma-
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Acknowledgements
The authors express their thanks to Mr. Tomonori
Takahashi and Ms. Mariko Fujimoto for skilful tech-
nical assistance.
23. Kiso, Y.; Tohkin, M.; Hikino, H.; Hattori, M.; Sakamoto,
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