Antioxidative and 5-lipoxygenase inhibiting activities of novel bis(4-hydroxy-2,3,5-trimethylphenoxy)alkyl derivatives

Article abstract of DOI:10.1248/cpb.49.225

Novel bis(4-hydroxy-2,3,5-trimethylphenoxy)alkyl derivatives were synthesized and evaluations were made of their inhibiting action on Fe³⁺-ADP induced lipid peroxidation in rat liver microsome and reducing action on α,α-diphenyl-β-picrilhydrazy

Full text of DOI:10.1248/cpb.49.225

February 2001
Chem. Pharm. Bull. 49(2) 225—229 (2001)
225
Antioxidative and 5-Lipoxygenase Inhibiting Activities of Novel
Bis(4-hydroxy-2,3,5-trimethylphenoxy)alkyl Derivatives
Takashi HIRANO, Nobuhide KAWASAKI, Hideki MIYATAKA, Mayumi NISHIKI, and Toshio SATOH*
Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770–8514, Japan.
Received September 27, 2000; accepted October 23, 2000
Novel bis(4-hydroxy-2,3,5-trimethylphenoxy)alkyl derivatives were synthesized and evaluations were made
of their inhibiting action on Fe3؉–ADP induced lipid peroxidation in rat liver microsome and reducing action on
a,a-diphenyl-b-picrilhydrazyl (DPPH), a stable radical, in addition to their inhibiting action on 5-lipoxygenase
(5-LO), an enzyme that synthesizes leukotrienes. We performed a structure–activity correlation study on these
derivatives. A strong Fe3؉–ADP induced lipid peroxidation preventing activity was observed for the derivatives
with an odd number of methylene groups including 1,3-bis(4-hydroxy-2,3,5-trimethylphenoxy)propane (3b) and
3a. No change in the DPPH reducing activity was found with change in the number of methylene groups. 5-LO
inhibiting activity among the derivatives was the highest for 1,6-bis(4-hydroxy-2,3,5-trimethylphenoxy)hexane
(3e). MM2 calculations were performed to find a stable steric structure for the derivatives, and 1,5-bis(4-hy-
droxy-2,3,5-trimethylphenoxy)pentane (3d) showed a strong activity in both antioxidative action and 5-LO
inhibiting action.
Key words antioxidative action; 5-lipoxygenase; a,a-diphenyl-b-picrilhydrazyl; lipid peroxidation; trimethylhydroquinone;
anti-allergic agent
When oxygen taken into the living body is utilized in the diseases and cancers.
cells, active oxygen species are formed as by-products in-
cluding superoxide anion radical (·OϪ2 ), hydrogen peroxide Chemistry
(H2O2), hydroxy radical (·OH), and singlet oxygen (1O2) and
We have synthesized various trimethylhydroquinone deriv-
free radicals of lipids such as alkoxy radical (RO·) and per- atives and reported their pharmacological activities. Among
oxide radical (ROO·). These active oxygen species and free the derivatives synthesized, the compound shown in Fig. 1
radicals raise oxygen stress to produce lipid peroxide in the was found to have a strong antioxidative activity.12—14) In this
body, thereby causing inflammation or lesion on various or- work, we synthesize compounds that have two trimethylhy-
gans. This triggers life-style related diseases as represented droquinone moieties in their molecules by the method shown
by cancer, ageing, arteriosclerosis, and allergy.1)
in Chart 1 in order to raise the antioxidative activity. Com-
The involvement of leukotrienes (LTs) as a cause is indi- pounds 3a—i were obtained by refluxing trimethylhydro-
cated in the development of allergic diseases such as polleno- quinone and a straight chain alkyldiol in toluene for 3—20 h
sis and atopic dermatitis, the case numbers of which are now at 130 °C in the presence of phosphomolybdic acid as the
tending to increase now. LT is a chemical mediator that is catalyst according to the method of Taniguchi.15) The reac-
produced by mast cells and eosinophils and is a 5-lipoxyge- tion also gave compounds 4a—i.
nase (5-LO) metabolite of arachidonic acid.2) LTC4, LTD4,
and LTE4 are deeply involved in the aggravation of allergic Results and Discussion
symptoms with their activities like bronchial contraction.3) A
Symmetrical 4-hydroxy-2,3,5-trimethylphenoxyalkanes
number of effective 5-LO inhibiting drugs have so far been exhibited an interesting pharmacological activity. These
reported.4—7) There have also been reports that suggest an compounds were suggested to have an antioxidative activity
important role for the arachidonic acid cascade in the initia- because of the presence of phenolic hydroxyl groups and side
tion and promotion processes of carcinogenesis8) and the
suppression of prostate cancer by chemicals with 5-LO in-
hibiting activity.9—11) Since the arachidonic acid cascade is
known to generate active oxygen through a free radical reac-
tion, compounds with an inhibitory action on both 5-LO and
Fig. 1. Structure of 4-O-Hexyloxy-2,3,6-trimethylphenol
the cascade are expected to be effective in treating allergic
Chart 1
To whom correspondence should be addressed. e-mail: tosato@ph.bunri-u.ac.jp
© 2001 Pharmaceutical Society of Japan

226
Vol. 49, No. 2
chains in their molecules. The side chain is important be- rings face each other in 3b and 3d which have an odd num-
cause it acts mutually with a lipid and appropriate hydropho- ber of methylene groups, while they are located apart from
bic is necessary. Action of these compounds on Fe3ϩ–ADP each other and have the most stable state in 3c and 3e that
induced lipid peroxidation reaction was then examined using have an even number of methylene groups.
rat liver microsome.16) Thus, the amount of lipid peroxide
The following mechanisms are generally possible for the
formed through induction with ADP/Fe3ϩ in rat liver micro- action of antioxidant: 1) free radical capturing action, 2) sin-
some was determined as the amount of malondialdehyde glet oxygen depleting action, and 3) metal chelating action.
using the reaction with 2-thiobarbituric acid (TBA), and the Vitamins C and E develop their antioxidant activity through
inhibition percentage was calculated against the control.17) the first mechanism while the second and third mechanisms
Compounds 3a—i were examined using 2,6-di-tert-butyl-p- work for carotenoids and flavonoids, respectively, when they
cresol (BHT), a widely used antioxidant, as the positive con- develop the activity.
trol. As shown in Fig. 2, the compounds with an odd number
To check the above inference, evaluations were made of
of methylene groups and 3a exhibited a strong activity and the antioxidative activity of the compounds by another
the value of IC50 for 3b that had the highest activity was method using a,a-diphenyl-b-picrilhydrazyl (DPPH), a sta-
3.8ϫ10Ϫ7 M. In contrast, 3c, 3e, 3g, and 3i were found to be ble radical, proposed by Blois.19) This agent is often used in
less active than the positive control.
ESR measurements and is a stable crystalline radical. The
Raison and Standen reported that symmetrical di- color of ethanolic solution of DPPH is purple and it fades
aminophenylalkanes have a schistosomicidal activity, and away when the radical is reduced. Figure 4 shows the reduc-
that there is a difference in the activity between the com- tion percentage 3 min after addition of inhibitor to the
pounds with an odd number of methylene groups and those ethanolic solution for the compounds. No appreciable change
with an even number, as in our case.18)
in the activity with the length of side chain was observed for
Molecular simulations (MM2 calculations) were then per- compounds 3a—i which have two phenolic hydroxyl groups,
formed on 3a—i to determine their steric structures. The re- in contrast to the change shown in Fig. 2, since the reducing
sults obtained for 3b—e are shown in Fig. 3. Two benzene activity against DPPH develops mainly through radical cap-
turing action of the phenolic hydroxyl group.
This suggests that compounds 3a—i develop their antiox-
idative activity by mechanisms 1) and 3). Compounds, 3b
and 3d in which two benzene rings face each other would be
highly active because of their strong chelation with Fe.
Separately, Fe3ϩ–ADP induced lipid peroxidation inhibit-
ing activity was examined for 4-alkoxy-2,3,6-trimethylphe-
nols (4a—i) having one trimethylhydroquinone moiety in
their molecules. The compounds exhibited the activities
shown in Fig. 5 where 4f, 4g, and 4h exhibited the strongest
activity with no remarkable difference among then, in con-
trast to that of 3 in Fig. 2. This would imply that the steric
structure for 3a—i changes with the length of side chain be-
cause they have two phenolic hydroxyl groups and among
them the compounds with two benzene rings facing each
Fig. 2. Effect of 3a—i and BHT on Fe3ϩ–ADP Induced NADPH Depen-
dent Lipid Peroxidation in Rat Liver Microsomes
other (3a, 3b, 3d, 3f, 3h) exhibit a high activity since their
Fig. 3. Steric Structures of 3b—e Obtained for Molecular Simulations (MM2 Caluculations)

February 2001
227
gested to develop their activity as 2) and 3) because they
have phenolic hydroxyl groups.
3d showed a strong activity in both antioxidative action
and 5-LD inhibiting action. Novel 1,5-bis(4-hydroxy-2,3,5-
trimethylphenoxy)-pentane (3d) is expected to be effective
against various symptoms including tissue injury, cancer, and
inflammation.
Experimental
NMR spectra were measured on a Varian Unity (1H-NMR; 200 MHz, 13C-
NMR; 50 MHz) or Varian Gemini (1H, 200 MHz; 13C, 50 MHz) spectrometer
in CDCl3 or dimethyl sulfoxide (DMSO)-d6 solution with tetramethylsilane
(TMS) as an internal standard. IR spectra were measured on a JASCO
FT/IR-5300 or JASCO FT/IR-410. Melting points were measured on a
BÜCHI 510. MS spectra were measured on a JEOL JMS-AX 500. MM2
calculations were used on the iris of Silicon Graphics with biograf.
Synthesis of 1,2-Bis(4-hydroxy-2,3,5-trimethylphenoxy)ethane (3a)
and 4-(2-Hydroxyethoxy)-2,3,6-trimethyphenol (4a) Trimethylhydro-
quinone (1) (5.00 g, 32.85 mmol) and ethyleneglycol (2a) (6.12 g, 98.57
mmol) were dissolved in 35 ml of toluene and the solution was refluxed at
130 °C for 20 h after addition of phosphomolybdic acid (1.00 g). The solvent
was then removed from the reaction mixture by evaporation under reduced
pressure and the residue was treated by silica gel chromatography (CHCl3–
AcOEt, 10 : 1). Compounds 3a (1.03 g, 3.11 mmol, 10%) and 4a (3.88 g,
19.78 mmol, 60%) were obtained by recrystallization from methanol and n-
hexane–ethyl acetate, respectively.
Fig. 4 a,a-Diphenyl-b-picrilhydrazyl (DPPH) Reducing Activity of 3a—i
and DL-a-Tocopherol
3a: White powder, mp 192—193 °C. IR (neat) cmϪ1: 3355, 2934, 1595,
1
1132. H-NMR (200 MHz, DMSO-d6) d: 2.04 (3H, s), 2.07 (3H, s), 2.13
(3H, s), 4.12 (2H, s), 6.60 (1H, s), 7.60 (1H, s). 13C-NMR (50 MHz, DMSO-
d6) d: 11.8 (q), 12.6 (q), 16.8 (q), 68.0 (t), 112.7 (d), 121.9 (s), 122.9 (s),
124.7 (s), 146.8 (s), 149.4 (s). HR-EI-MS: 330.1857 (Mϩ) (Calcd for
C20H26O4: 330.1831).
4a: White powder, mp 124—126 °C. IR (neat) cmϪ1: 3374, 2920, 1588,
Fig. 5. Effect of 4a—i and BHT on Fe3ϩ–ADP Induced NADPH Depen-
dent Lipid Peroxidation in Rat Liver Microsomes
1
1123. H-NMR (200 MHz, CDCl3) d: 2.16 (3H, s), 2.18 (3H, s), 2.22 (3H,
s), 3.98 (4H, m), 4.35 (1H, s), 6.55 (1H, s). 13C-NMR (50 MHz, CDCl3) d:
11.9 (q), 12.7 (q), 16.9 (q), 60.0 (t), 70.9 (t), 112.6 (d), 122.0 (s), 123.0 (s),
124.8 (s), 146.8 (s), 149.8 (s). HR-EI-MS: 196.1074 (Mϩ) (Calcd for
C11H16O3: 196.1099).
Synthesis of 1,3-Bis(4-hydroxy-2,3,5-trimethylphenoxy)propane (3b)
and 4-(3-Hydroxypropyloxy)-2,3,6-trimethyphenol (4b) Trimethyhydro-
quinone (1) (5.00 g, 32.85 mmol) and 1,3-propanediol (2b) (7.50 g, 98.57
mmol) were dissolved in 35 ml of toluene and the solution was refluxed at
130 °C for 5 h after addition of phosphomolybdic acid (1.00 g). The solvent
was then removed from the reaction mixture by evaporation under reduced
pressure and the residue was treated by silica gel chromatography (CHCl3–
AcOEt, 10 : 1). Compounds 3b (1.08 g, 3.14 mmol, 10%) and 4b (4.61 g,
21.94 mmol, 67%) were obtained by recrystallization from methanol and n-
hexane–ethyl acetate, respectively.
3b; White powder, mp 124 °C. IR (neat) cmϪ1: 3437, 2917, 1595, 1120.
1H-NMR (200 MHz, DMSO-d6) d: 2.02 (3H, s), 2.06 (3H, s), 2.11 (3H, s),
4.00 (2H, t, Jϭ6.2 Hz), 6.54 (1H, s), 7.58 (1H, s). 13C-NMR (50 MHz,
DMSO-d6) d: 11.8 (q), 12.7 (q), 16.8 (q), 29.4 (t), 65.3 (t), 112.0 (d), 121.9
(s), 122.6 (s), 124.7 (s), 146.6 (s), 149.5 (s). HR-EI-MS: 344.1975 (Mϩ)
(Calcd for C21H28O4: 344.1987).
Fig. 6. Effect of 3a—i and NDGA on RBL-1 Cell 5-Lipoxygenase Activ-
ity
4b: White powder, mp 90—91 °C. IR (neat) cmϪ1: 3283, 2949, 1589,
1123. 1H-NMR (200 MHz, CDCl3) d: 2.04 (2H, tt, Jϭ5.5, 5.5 Hz), 2.13 (3H,
s), 2.17 (3H, s), 2.22 (3H, s), 3.89 (2H, t, Jϭ5.9 Hz), 4.04 (2H, t, Jϭ5.7 Hz),
4.39 (1H, s), 6.55 (1H, s). 13C-NMR (50 MHz, CDCl3) d: 12.7 (q), 12.9 (q),
16.9 (q), 32.9 (t), 61.9 (t), 68.3 (t), 113.0 (d), 120.9 (s), 124.3 (s), 124.8 (s),
147.0 (s), 151.1 (s). HR-EI-MS: 210.1240 (Mϩ) (Calcd for C12H18O3:
210.1256).
chelation with Fe is strong.
From what has been mentioned so far, Fe3ϩ in liver micro-
some is considered to be localized to a certain extent, instead
of being uniformly distributed over a wide area, since the
compounds with a structure in which Fe3ϩ is enclosed by
benzene rings had a strong activity.
Synthesis of 1,4-Bis(4-hydroxy-2,3,5-trimethylphenoxy)butane (3c)
and 4-(4-Hydroxybutoxy)-2,3,6-trimethylphenol (4c) Trimethylhydro-
quinone (1) (1.00 g, 6.57 mmol) and 1,4-butanediol (2c) (1.78 g, 19.75
mmol) were dissolved in 25 ml of toluene and the solution was refluxed at
130 °C for 3 h after addition of phosphomolybdic acid (0.25 g). The solvent
was then removed from the reaction mixture by evaporation under reduced
pressure and the residue was treated by silica gel chromatography (CHCl3–
AcOEt, 10 : 1). Compounds 3c (0.38 g, 1.05 mmol, 16%) and 4c (0.16 g,
0.71 mmol, 43%) were obtained by recrystallization from methanol and n-
hexane–ethyl acetate, respectively.
5-LO inhibiting activity was then examined in the com-
pounds.20) As shown in Fig. 6, they exhibited an activity
stronger than that of nordihydroguaiaretic acid (NDGA),4)
the positive control, with that of 3e being the strongest. Bell
et al. classified compounds that inhibit 5-lipoxygenase into
three types, 1) substrate analogues, 2) compounds with radi-
cal scavenging activity, and 3) chelating agents of Fe, the ac-
tive center metal of 5-LO.21) Here, compounds 3a—i are sug-
3c: White powder, mp 188—189 °C. IR (neat) cmϪ1: 3362, 2924, 1593,

228
Vol. 49, No. 2
1
(s), 124.7 (s), 146.5 (s), 149.6 (s). HR-EI-MS: 400.2627 (Mϩ) (Calcd for
C25H36O4: 400.2613).
1117. H-NMR (200 MHz, DMSO-d6) d: 1.81—1.87 (2H, m), 2.02 (3H, s),
2.06 (3H, s), 2.12 (3H, s), 3.86—3.92 (2H, m), 6.53 (1H, s), 7.56 (1H, s).
13C-NMR (50 MHz, DMSO-d6) d: 11.8 (q), 12.7 (q), 16.9 (q), 26.0 (t), 68.1
(t), 112.0 (d), 121.8 (s), 122.5 (s), 124.7 (s), 146.5 (s), 149.6 (s). HR-EI-MS:
358.2157 (Mϩ) (Calcd for C22H30O4: 358.2114).
4f: White powder, mp 73—75 °C. IR (neat) cmϪ1: 3347, 2934, 1590,
1119. 1H-NMR (200 MHz, CDCl3) d: 1.33–1.79 (10H, m), 2.13 (3H, s),
2.16 (3H, s), 2.20 (3H, s), 3.63 (2H, t, Jϭ6.4 Hz), 3.86 (2H, t, Jϭ6.4 Hz),
4.62 (1H, s), 6.51 (1H, s). 13C-NMR (50 MHz, CDCl3) d: 11.9 (q), 12.2 (q),
16.2 (q), 25.6 (t), 26.1 (t), 29.1 (t), 29.5 (t), 32.6 (t), 62.9 (t), 69.2 (t), 112.3
(d), 120.3 (s), 123.7 (s), 124.2 (s), 145.9 (s), 150.7 (s). HR-EI-MS: 266.1890
(Mϩ) (Calcd for C16H26O3: 266.1882).
4c: White powder, mp 70—71 °C. IR (neat) cmϪ1: 3395, 2944, 1557,
1119. 1H-NMR (200 MHz, CDCl3) d: 1.68—1.98 (4H, m), 1.78 (2H, tt,
Jϭ5.5, 5.5 Hz), 2.13 (3H, s), 2.16 (3H, s), 2.20 (3H, s), 3.68 (2H, t, Jϭ5.9
Hz), 3.86 (2H, t, Jϭ6.2 Hz), 4.76 (1H, s), 6.51 (1H, s). 13C-NMR (50 MHz,
CDCl3) d: 11.9 (q), 12.2 (q), 16.2 (q), 26.2 (t), 29.7 (t), 62.9 (t), 69.2 (t),
113.0 (d), 120.9 (s), 124.3 (s), 124.8 (s), 147.0 (s), 151.1 (s). HR-EI-MS:
224.1404 (Mϩ) (Calcd for C13H20O3: 224.1412).
Synthesis of 1,8-Bis(4-hydroxy-2,3,5-trimethylphenoxy)octane (3g)
and 4-(8-Hydroxyoctyloxy)-2,3,6-trimethylphenol (4g) Trimethylhy-
droquinone (1) (5.00 g, 32.85 mmol) and 1,8-octanediol (2g) (14.41 g, 98.56
mmol) were dissolved in 35 ml of toluene and the solution was refluxed at
130 °C for 3 h after addition of phosphomolybdic acid (1.00 g). The solvent
was removed from the reaction mixture by evaporation under reduced pres-
sure and the residue was treated by silica gel chromatography (CHCl3–
AcOEt, 10 : 1). Compounds 3g (1.09 g, 2.63 mmol, 8%) and 4g (6.57 g,
23.47 mmol, 72%) were obtained by recrystallization from n-hexane and n-
hexane–ethyl acetate, respectively.
Synthesis of 1,5-Bis(4-hydroxy-2,3,5-trimethylphenoxy)pentane (3d)
and 4-(5-Hydroxypentyloxy)-2,3,6-trimethylphenol (4d) Trimethylhy-
droquinone (1) (5.00 g, 32.85 mmol) and 1,5-pentanediol (2d) (10.27 g,
98.57 mmol) were dissolved in 35 ml of toluene and the solution was re-
fluxed at 130 °C for 6 h after addition of phosphomolybdic acid (1.00 g). The
solvent was then removed from the reaction mixture by evaporation under
reduced pressure and the residue was treated by silica gel chromatography
(CHCl3–AcOEt, 10 : 1). Compounds 3d (0.97 g, 2.62 mmol, 8%) and 4d
(5.33 g, 22.38 mmol, 68%) were obtained by recrystallization from n-hexane
and n-hexane–ethyl acetate, respectively.
3g: White powder, mp 136—137 °C. IR (neat) cmϪ1: 3358, 2913, 1595,
1
1121. H-NMR (200 MHz, DMSO-d6) d: 1.36—1.42 (4H, m), 1.64—1.68
(2H, m), 2.01 (3H, s), 2.06 (3H, s), 2.12 (3H, s), 3.81 (2H, t, Jϭ6.4 Hz),
6.51 (1H, s), 7.55 (1H, s). 13C-NMR (50 MHz, DMSO-d6) d: 11.8 (q), 12.7
(q), 16.9 (q), 25.6 (t), 28.7 (t), 29.0 (t), 68.3 (t), 112.0 (d), 121.8 (s), 122.5
(s), 124.7 (s), 146.5 (s), 149.6 (s). HR-EI-MS: 414.2783 (Mϩ) (Calcd for
C26H38O4: 414.2770).
3d: White powder, mp 124—125 °C. IR (neat) cmϪ1: 3335, 2940, 1593,
1
1121. H-NMR (200 MHz, DMSO-d6) d: 1.57—1.77 (3H, m), 2.02 (3H, s),
2.06 (3H, s), 2.12 (3H, s), 3.84 (2H, t, Jϭ6.2 Hz), 6.52 (1H, s), 7.56 (1H, s).
13C-NMR (50 MHz, DMSO-d6) d: 11.8 (q), 12.7 (q), 16.9 (q), 22.5 (t), 28.8
(t), 68.3 (t), 112.0 (d), 121.8 (s), 122.5 (s), 124.7 (s), 146.5 (s), 149.6 (s).
HR-EI-MS: 372.2286 (Mϩ) (Calcd for C23H32O4: 372.2300).
4g: White powder, mp 70—71 °C. IR (neat) cmϪ1: 3385, 2934, 1581,
1119. 1H-NMR (200 MHz, CDCl3) d: 1.28–1.83 (12H, m), 2.14 (3H, s),
2.16 (3H, s), 2.21 (3H, s), 3.63 (2H, t, Jϭ6.9 Hz), 3.86 (2H, t, Jϭ6.7 Hz),
6.51 (1H, s). 13C-NMR (50 MHz, CDCl3) d: 11.9 (q), 12.2 (q), 16.2 (q), 25.6
(t), 26.1 (t), 29.3 (t), 29.3 (t), 29.3 (t), 29.5 (t), 32.7 (t), 63.0 (t), 69.3 (t),
112.4 (d), 120.2 (s), 123.6 (s), 124.2 (s), 145.9 (s), 150.7 (s). HR-EI-MS:
280.2048 (Mϩ) (Calcd for C17H28O3: 280.2038).
4d: White powder, mp 79—81 °C. IR (neat) cmϪ1: 3277, 2940, 1588,
1124. 1H-NMR (200 MHz, CDCl3) d: 1.52—1.66 (4H, m), 1.78 (2H, tt,
Jϭ6.7, 6.7 Hz), 2.13 (3H, s), 2.16 (3H, s), 2.20 (3H, s), 3.68 (2H, t, Jϭ5.9
Hz), 3.86 (2H, t, Jϭ6.2 Hz), 4.76 (1H, s), 6.51 (1H, s). 13C-NMR (50 MHz,
CDCl3) d: 11.9 (q), 12.2 (q), 16.2 (q), 22.4 (t), 29.3 (t), 32.3 (t), 62.7 (t),
69.1 (t), 112.4 (d), 120.4 (s), 123.8 (s), 124.2 (s), 145.9 (s), 150.6 (s). HR-
EI-MS: 238.1567 (Mϩ) (Calcd for C14H22O3: 238.1569).
Synthesis of 1,9-Bis(4-hydroxy-2,3,5-trimethylphenoxy)nonane (3h)
and 4-(9-Hydroxynonyloxy)-2,3,6-trimethylphenol (4h) Trimethylhydro-
quinone (1) (1.00 g, 6.57 mmol) and 1,9-nonanediol (2h) (3.16 g, 19.70
mmol) were dissolved in 10 ml of toluene and the solution was refluxed at
130 °C for 19 h after addition of phosphomolybdic acid (0.50 g). The solvent
was removed from the reaction mixture by evaporation under reduced pres-
sure and the residue was treated by silica gel chromatography (CHCl3–
AcOEt, 10 : 1). Compounds 3h (0.18 g, 0.42 mmol, 6%) and 4h (0.81 g, 2.75
mmol, 42%) were obtained by recrystallization from n-hexane and n-
hexane–ethyl acetate, respectively.
Synthesis of 1,6-Bis(4-hydroxy-2,3,5-trimethylphenoxy)hexane (3e)
and 4-(6-Hydroxyhexyloxy)-2,3,6-trimethyphenol (4e) Trimethylhy-
droquinone (1) (1.00 g, 6.57 mmol) and 1,6-hexanediol (2e) (2.33 g, 19.72
mmol) were dissolved in 10 ml of toluene and the solution was refluxed at
130 °C for 3 h after addition of phosphomolybdic acid (0.40 g). The solvent
was then removed from the reaction mixture by evaporation under reduced
pressure and the residue was treated by silica gel chromatography (CHCl3–
AcOEt, 10 : 1). Compounds 3e (0.20 g, 0.52 mmol, 8%) and 4e (1.00 g, 3.99
mmol, 61%) were obtained by recrystallization from n-hexane and n-
hexane–ethyl acetate, respectively.
3h: White powder, mp 103—104 °C. IR (neat) cmϪ1: 3355, 2919, 1595,
1
1128. H-NMR (200 MHz, DMSO-d6) d: 1.32—1.41 (6H, m), 1.64—1.68
3e: White powder, mp 175—177 °C. IR (neat) cmϪ1: 3295, 2915, 1591,
(2H, m), 2.02 (3H, s), 2.06 (3H, s), 2.12 (3H, s), 3.81 (2H, t, J ϭ6.3 Hz),
6.51 (1H, s), 7.55 (1H, s). 13C-NMR (50 MHz, DMSO-d6) d: 11.8 (q), 12.6
(q), 16.8 (q), 25.6 (t), 28.6 (t), 29.0 (t), 68.3 (t), 112.0 (d), 121.8 (s), 122.5
(s), 124.6 (s), 146.5 (s), 149.6 (s). HR-EI-MS: 428.2932 (Mϩ) (Calcd for
C27H40O4: 428.2926).
1
1119. H-NMR (200 MHz, DMSO-d6) d: 1.48—1.51 (2H, m), 1.67—1.74
(2H, m), 2.01 (3H, s), 2.06 (3H, s), 2.12 (3H, s), 3.82 (2H, t, Jϭ6.3 Hz),
6.51 (1H, s), 7.56 (1H, s). 13C-NMR (50 MHz, DMSO-d6) d: 11.8 (q), 12.7
(q), 16.8 (q), 29.0 (t), 68.3 (t), 112.0 (d), 121.8 (s), 122.5 (s), 124.7 (s),
146.5 (s), 149.6 (s). HR-EI-MS: 386.2432 (Mϩ) (Calcd for C24H34O4:
386.2457).
4h: White powder, mp 72—74 °C. IR (neat) cmϪ1: 3341, 2932, 1593,
1
1121. H-NMR (200 MHz, CDCl3) d: 1.21—1.79 (14H, m), 2.14 (3H, s),
4e: White powder, mp 77—78 °C. IR (neat) cmϪ1: 3387, 2938, 1591,
1119. 1H-NMR (200 MHz, CDCl3) d: 1.41—1.81 (8H, m), 2.14 (3H, s),
2.16 (3H, s), 2.21 (3H, s), 3.65 (2H, t, Jϭ6.4 Hz), 3.87 (2H, t, Jϭ6.3 Hz),
4.51 (1H, s), 6.51 (1H, s). 13C-NMR (50 MHz, CDCl3) d: 11.9 (q), 12.2 (q),
16.2 (q), 25.5 (t), 26.0 (t), 29.5 (t), 32.7 (t), 62.9 (t), 69.2 (t), 112.4 (d), 120.2
(s), 123.6 (s), 124.2 (s), 145.9 (s), 150.7 (s). HR-EI-MS: 252.1698 (Mϩ)
(Calcd for C15H24O3: 252.1726).
2.16 (3H, s), 2.21 (3H, s), 3.62 (2H, t, Jϭ6.5 Hz), 3.86 (2H, t, Jϭ6.4 Hz),
4.61 (1H, s), 6.51 (1H, s). 13C-NMR (50 MHz, CDCl3) d: 11.9 (q), 12.2 (q),
16.2 (q), 25.7 (t), 26.1 (t), 29.3 (t), 29.3 (t), 29.5 (t), 29.5 (t), 32.7 (t), 63.0
(t), 69.3 (t), 112.4 (d), 120.3 (s), 123.7 (s), 124.2 (s), 145.9 (s), 150.7 (s).
HR-EI-MS: 294.2202 (Mϩ) (Calcd for C18H30O3: 294.2195).
Synthesis of 1,10-Bis(4-hydroxy-2,3,5-trimethylphenoxy)decane (3i)
and 4-(10-Hydroxydecanyloxy)-2,3,6-trimethylphenol (4i) Trimethylhy-
droquinone (1) (5.00 g, 32.85 mmol) and 1,10-decanediol (2i) (17.18 g,
98.56 mmol) were dissolved in 35 ml of toluene and the solution was re-
fluxed at 130 °C for 4 h after addition of phosphomolybdic acid (1.00 g). The
solvent was removed from the reaction mixture by evaporation under re-
duced pressure and the residue was treated by silica gel chromatography
(CHCl3–AcOEt, 10 : 1). Compounds 3i (0.96 g, 2.18 mmol, 7%) and 4i
(7.99 g, 25.93 mmol, 79%) were obtained by recrystallization from n-hexane
and n-hexane–ethyl acetate, respectively.
Synthesis of 1,7-Bis(4-hydroxy-2,3,5-trimethylphenoxy)heptane (3f)
and 4-(7-Hydroxyheptyloxy)-2,3,6-trimethylphenol (4f) Trimethylhy-
droquinone (1) (1.00 g, 6.67 mmol) and 1,7-heptanediol (2f) (2.61 g, 13.14
mmol) were dissolved in 10 ml of toluene and the solution was refluxed at
130 °C for 4 h after addition of phosphomolybdic acid (0.25 g). The solvent
was removed from the reaction mixture by evaporation under reduced pres-
sure and the residue was treated by silica gel chromatography (CHC3–
AcOEt, 10 : 1). Compounds 3f (0.22 g, 0.55 mmol, 8%) and 4f (1.43 g,
5.38 mmol. 82%) were obtained by recrystallization from n-hexane and n-
hexane–ethyl acetate, respectively.
3i: White powder, mp 111—113 °C. IR (neat) cmϪ1: 3331, 2932, 1616,
1
1125. H-NMR (200 MHz, DMSO-d6) d: 1.30—1.40 (6H, m), 1.63—1.67
3f: White powder, mp 110—112 °C. IR (neat) cmϪ1: 3293, 2936, 1593,
(2H, m), 2.01 (3H, s), 2.06 (3H, s), 2.11 (3H, s), 3.80 (2H, t, Jϭ6.3 Hz),
6.51 (1H, s), 7.55 (1H, s). 13C-NMR (50 MHz, DMSO-d6) d: 11.8 (q), 12.6
(q), 16.8 (q), 25.6 (t), 28.7 (t), 28.9 (t), 29.0 (t), 68.3 (t), 112.0 (d), 121.8 (s),
122.4 (s), 124.6 (s), 146.5 (s), 149.6 (s). HR-EI-MS: 442.3087 (Mϩ) (Calcd
for C28H42O4: 442.3082).
1
1123. H-NMR (200 MHz, DMSO-d6) d: 1.40—1.48 (4H, m), 1.65—1.72
(2H, m), 2.02 (3H, s), 2.06 (3H, s), 2.12 (3H, s), 3.82 (2H, t, Jϭ6.3 Hz),
6.51 (1H, s), 7.55 (1H, s). 13C-NMR (50 MHz, DMSO-d6) d: 11.8 (q), 12.7
(q), 16.8 (q), 25.6 (t), 28.4 (t), 28.9 (t), 68.3 (t), 112.0 (d), 121.8 (s), 122.5

February 2001
229
4i: White powder, mp 58—61 °C. IR (neat) cmϪ1: 3389, 2928, 1587,
1117. H-NMR (200 MHz, CDCl3) d: 1.23—1.82 (16H, m), 2.14 (3H, s),
centrifuged (2000ϫg, 15 min), 5-HETE in the supernatant was analyzed by
HPLC (column; COSMOSIL 5C18-MS Waters 4.6ϫ150 mm (Nacalai
Tesque, Inc.), mobile phase; CH3CN:0.1% AcOH aq. (6:4), flow rate;
1 ml/min, temperature; r.t., absorbance; 235 nm).
1
2.17 (3H, s), 2.21 (3H, s), 3.63 (2H, t, Jϭ6.5 Hz), 3.86 (2H, t, Jϭ6.3 Hz),
4.43 (1H, s), 6.52 (1H, s). 13C-NMR (50 MHz, CDCl3) d: 11.9 (q), 12.2 (q),
16.2 (q), 25.7 (t), 26.1 (t), 29.3 (t), 29.4 (t), 29.5 (t), 29.5 (t), 29.6 (t), 32.7
(t), 63.0 (t), 69.3 (t), 112.4 (d), 120.1 (s), 123.6 (s), 124.3 (s), 145.9 (s),
150.8 (s). HR-EI-MS: 308.2354 (Mϩ) (Calcd for C19H32O3: 308.2357).
Pharmacology Materials Male Wistar rats were obtained from Japan
SLC Inc. RBL-1 cells were obtained from Dainippon Pharmaceutical Co.,
Ltd. 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) was obtained from
Sigma Chemical Co. Adenosine 5Ј-diphosphate disodium salt (ADP), nicoti-
namide adenine dinucleotide phosphate reduced form (b-NADPH), and
sodium dodecyl sulfate (SDS) were purchased from Wako Pure Chemical
Industries, Ltd. BHT, TBA, D,L-a-tocopherol, L-ascorbic acid, NDGA, and
arachidonic acid were purchased from Nacalai Tesque, Inc.
References
1) Slater T. F., Biochem. J., 222, 1—15 (1984).
2) Samuelsson B., Hammarstrom S., Murphy R. C., Borgeat P., Allergy,
35, 375—381 (1980).
3) Dahlen S. E., Hedqvist P., Hammarstrom S., Samuelsson B., Nature
(London), 288, 484—486 (1980).
4) Naito Y., Sugiura M., Yamaura Y., Fukaya C., Yokoyama K., Naka-
gawa Y., Ikeda T., Senda M., Fujita T., Chem. Pharm. Bull., 39,
1736—1745 (1991).
5) Carter G. W., Young P. R., Albert D. H., Bouska J., Dyer R., Bell R. L.,
Summers J. B., Brooks D. W., J. Pharmcol. Exp. Ther., 256, 929—937
(1991).
6) Bell R. L., Harris R. R., Malo P. E., Bouska J. B., Shaughnessy T. K.,
Hulkower K. I., Brooks C. D. W., Carter G. W., J. Pharmacol. Exp.
Ther., 280, 1366—1373 (1997).
7) Brooks C. D. W., Summers J. B., J. Med. Chem., 39, 2629—2654
(1996).
8) Nakadate T., Yamamoto S., Iseki H., Sonoda S., Takemura S., Ura A.,
Hosoda Y., Kato R., Gann, 73, 841—843 (1982).
9) Hong S. H., Avis I., Vos M. D., Martínez A., Treston A. M., Mulshine
J. L., Cancer Res., 59, 2223—2228 (1999).
10) Ghosh J., Myers C. E., Proc. Natl. Acad. Sci. U.S.A., 95, 13182—
13187 (1998).
11) Ghosh J., Myers C. E., Biochem. Biophy. Res. Commun., 235, 418—
423 (1997).
12) Nihro Y., Furukawa H., Sogawa S., Wang T. C., Miyataka H., Matsu-
moto H., Miki T., Satoh T., Chem. Pharm. Bull., 42, 576—579 (1994).
13) Hino T., Kawanisi S., Yasui H., Oka S., Sakurai H., Biochim. Biophys.
Acta, 1425, 47—60, (1998).
14) Hirose M., Satoh T., Ito N., Drug News Perspective., 7, 167—170
(1994).
15) Taniguchi K., Japan Kokai Tokkyo Kohoo JP60215643 (1985) [Chem.
Abstr., 104, 109228 (1986)].
16) Kiso Y., Tohkin M., Hikino H., Hattori M., Sakamoto T., Namba T.,
Planta Medica, 50, 298—302 (1984).
17) Ohkawa H., Ohishi N., Yagi K., Anal. Biochem., 95, 351—358 (1979).
18) Raison C. G., Standen O. D., Brit. J. Pharmacol., 10, 191—199
(1955).
19) Blois M. S., Nature (London), 181, 1199—1200 (1958).
20) Blackham A., Griffiths R. J., Hallam C., Mann J., Mitchell P. D., Norris
A. A., Simpson W. T., Agents Actions, 30, 432—442 (1990).
21) Bell R. L., Young P. R., Albert D., Lanni C., Summers J. B., Brooks D.
W., Rubin P., Carter G. W., Int. J. Immunopharmac., 14, 505—510
(1992).
Measurement of Fe3؉–ADP Induced NADPH Dependent Lipid Perox-
idation in Rat Liver Microsome.12) The modified method of Kiso et al.16)
was used. Microsomes were prepared from male Wistar rats weighing about
200 g. The rat liver was homogenized in cold 0.25 M sucrose and the ho-
mogenate was centrifuged at 4 °C (8000ϫg, 30 min). The supernatant frac-
tion was then collected and ultracentrifuged at 4 °C (105000ϫg, 30 min).
The pellet obtained was resuspended in 83.5 mM KCl 37.2 mM Tris–HCl
buffer (pH 7.4) and stocked at Ϫ20 °C until use. Protein concentration was
determined by the method of Lowry et al.22) The assay system (1 ml) con-
sisted of 83.5 mM KCl 37.2 mM Tris–HCl buffer (pH 7.4), the test compound
in 1% DMSO, 0.2 mM NADPH, 1 mM ADP, 1 mg protein/ml rat liver micro-
somes and 10 mM FeCl3. The reaction mixture 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.17) Thus, 8.1% SDS (0.2 ml), 20%
AcOH (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 and mixed vigorously. After centrifugation (780ϫg, 10 min), the or-
ganic layer was separated, and the absorbance was measured at 532 nm. The
amount of TBA-positive material was expressed as the corresponding
amount of malondialdehyde.
Measurement of DPPH Reducing Activity19) The modified method of
Nihro et al.23) was used. The assay system (2 ml) consisted of 0.1 mM DPPH
in ethanol and the test compound in 1% DMSO. The reaction mixture was
left at room temperature for 3 min, and the absorbance was measured at
517 nm.
Measurement of RBL-1 Cell 5-Lipoxygenase Activity24) The modi-
fied method of Blackham et al.20) 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 (5ϫ105—106 cells/ml) were collected by centrifugation
(100ϫg, 5 min) and suspended at 3ϫ107 cells/ml in 50 mM phosphate buffer
(0.25 M sucrose, 1 mM EDTA, 2 mM glutathione, pH 7.4), then stored at
Ϫ80 °C until use. The assay system (0.5 ml) consisted of 50 mM phosphate
buffer (0.25 M sucrose, 1 mM EDTA, 2 mM glutathione, pH 7.4), the test com-
pound 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 of
homogenate. The 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
22) Lowry O. H., Rosebrough N. J., Farr A. L., Randall R. J., J. Biol.
Chem., 193, 265—275 (1951).
23) Nihro Y., Sogawa S., Sudo T., Miki T., Matsumoto H., Satoh T., Chem.
Pharm. Bull., 39, 1731—1735 (1991).
24) Sogawa S., Nihro Y., Ueda H., Izumi A., Miki T., Matsumoto H.,
Satoh T., J. Med. Chem., 36, 3904—3909 (1993).