Inhibitory activities of novel pyrimidine derivatives on the contact hypersensitivity reaction.

Article abstract of DOI:10.1248/cpb.51.309

In order to obtain novel topically applied anti-inflammatory compounds containing an inexpensive anti-oxidative moiety without chirality, we synthesized compound 2c derivatives having a di-tert-butylphenol moiety, and evaluated by topical administration their anti-inflammatory potentials on picryl chloride-(PC) induced contact hypersensitivity reaction (CHR) in mice. In the course of our structure-activity relationship (SAR) studies on the pyrimidine or the anti-oxidative moiety and the linker between them, the most potent compounds (10, 11) were obtained by the insertion of a C2 unit in compound 2c. The potencies of these compounds were 2-fold greater than that of 1. Compounds 10 and 11 were considered to be useful lead compounds having inexpensive anti-oxidative moieties without chirality.

Full text of DOI:10.1248/cpb.51.309

March 2003
Notes
Chem. Pharm. Bull. 51(3) 309—312 (2003)
309
Inhibitory Activities of Novel Pyrimidine Derivatives on the Contact
Hypersensitivity Reaction
Yoshiaki ISOBE,1) Masanori TOBE,1) Yoshifumi INOUE,1) Yuso GOTO,1) Fumihiro OBARA,
,
1)
1)
*
Masakazu ISOBE, and Hideya HAYASHI
Pharmaceuticals and Biotechnology Laboratory, Japan Energy Corporation; Toda, Saitama 335–8502, Japan.
Received August 7, 2002; accepted December 4, 2002
In order to obtain novel topically applied anti-inflammatory compounds containing an inexpensive anti-ox-
idative moiety without chirality, we synthesized compound 2c derivatives having a di-tert-butylphenol moiety,
and evaluated by topical administration their anti-inflammatory potentials on picryl chloride-(PC) induced con-
tact hypersensitivity reaction (CHR) in mice. In the course of our structure–activity relationship (SAR) studies
on the pyrimidine or the anti-oxidative moiety and the linker between them, the most potent compounds (10, 11)
were obtained by the insertion of a C2 unit in compound 2c. The potencies of these compounds were 2-fold
greater than that of 1. Compounds 10 and 11 were considered to be useful lead compounds having inexpensive
anti-oxidative moieties without chirality.
Key words pyrimidine derivative; CX-659S; contact hypersensitivity reaction (CHR); anti-oxidative activity
We recently found that a novel pyrimidine derivative, CX- ity. In the present study, we report and discuss our prelimi-
659S (1) [(S)-6-amino-5-(6-hydroxy-2,5,7,8-tetramethylchro- nary structure–activity relationship (SAR) study on 2c deriv-
man-2-carboxamido)-3-methyl-1-phenyl-2,4(1H,3H)-pyrim- atives as a new type anti-inflammatory drug.
idinedione] (Fig. 1), had inhibitory activities against a variety
of acute and chronic inflammation models when topically ap- Chemistry
plied.2—4) This compound, having a tocopherol-related moi-
The compounds described in this study are shown in Table
ety with a non-steroidal structure, exerted a potent radical 1, and their synthetic methods are outlined in Charts 1, 2.
scavenging activity and inhibitory effects on lipid peroxida- The syntheses of analogues were accomplished by the fol-
tion both in vitro and in vivo.5)
lowing pathway using the reported methods. Nitrosation of
Generally, a chiral compound such as CX-659S has many 14 with sodium nitrite followed by catalytic hydrogenation of
difficulties for the development of it as a new drug candidate. the nitroso group furnished 15.9,10) Compounds (2—5, 12,
For example, we must establish an efficient, low cost, syn- 13) were obtained by the coupling of 15 with the correspond-
thetic method to prepare it, and conduct a comparative study ing carboxylic acids by using diphenylphosphoryl chloride
on its pharmacology and toxicology and so on by using each (DPP-Cl) as a condensation reagent. Compound 8 was af-
isomer (R or S) and racemate. Moreover, from the viewpoint forded by the reaction of 14 with 2,6-di-tert-butyl-4-hydroxy-
of pharmacokinetics, we must confirm whether one isomer benzyl chloride in the presence of Et3N. The reduction of 2c
converts into the other one in the body or not.
with BH3·Me2S gave 9. Compounds 10 was prepared by the
In the present study, to avoid the problems mentioned condensation of 15 (R4ϭPh) with 2,6-di-tert-butyl-4-hydroxy-
above, we sought to find a new type of compound with a sim- cinnamic acid, and the hydrogenation of 10 gave 11.
ple structure without chirality. Typical antioxidants without
For preparation of the dimethylamino derivative of 2c (7),
chirality are the polyphenols such as pyrogallol6) and nitration of 14 was needed instead of nitrosation. But the ni-
methoxyphenol.7) So, we synthesized compounds 2a2) and tration of 14 gave a di-nitro compound (nitration proceeded
2b, having partial antioxidant structures, and evaluated their at not only the C(5) position of pyrimidine, but also at the
inhibitory activities toward picryl chloride (PC)-induced con- para position of the phenyl group). To avoid this, we used 4-
tact hypersensitivity reaction (CHR) in mice by the topical fluorophenyl compound. The conversion of 16 with phospho-
administration. The anti-inflammatory activities of 2a and 2b rus oxychloride, followed by the treatment with methylamine
were almost equipotent with that of 1 (Table 1). Compounds or dimethylamine, led to 17 or 20, respectively. Treatment of
2a and 2b were evaluated as their acetate form, because their 20 with sodium nitrate in sulfuric acid followed by catalytic
free acids were poorly soluble in application solvents such as hydrogenation provided 22. The condensation of 19 or 22
acetone, ethyl acetate, and EtOH, and could not be used for with 2,6-di-tert-butyl-4-hydroxybenzoic acid afforded 6 or 7,
the evaluation of anti-inflammatory activity in this study.
Moreover, as both compounds were found to be unstable due
to mainly their deacetylation under the moisture condition
(data not shown), we judged that compounds 2a and 2b were
not suitable as lead compounds. On the other hand, 2c,2) hav-
ing a di-tert-butylphenol moiety, showed a moderate in-
hibitory activity (ED50ϭ0.81 mg/ear), which was 3-fold
weaker than that of 1. The di-tert-butylphenol moiety is also
well-known to have an anti-oxidative activity,8) and is widely
used as an inexpensive anti-oxidative moiety without chiral- Fig. 1. The Structures of 1 and 2a—c
* To whom correspondence should be addressed. e-mail: hayashih@sumitomopharm.co.jp
© 2003 Pharmaceutical Society of Japan

310
Vol. 51, No. 3
Table 1. Inhibitory Effects of the Test Compounds on PC-Induced CHR in Mice
a)
No
R4
R5
X
R1
R2
R3
ED50
AlogP
1
2a
2b
2c
3
4
5
6
7
8
9
10
0.29
0.21
0.27
0.81
4%
0.31
0.58
0.25
0.26
nd
20%
0.14
0.13
0%
17%
0.03
Ph
Ph
Ph
NH2
NH2
NH2
NH2
NH2
NH2
NHMe
NMe2
NH2
NH2
NH2
NH2
NH2
NH2
NHCO
NHCO
NHCO
NHCO
NHCO
NHCO
NHCO
NHCO
CH2
OAc
OMe
OAc
OAc
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OAc
OAc
OAc
H
tert-Bu
tert-Bu
tert-Bu
tert-Bu
tert-Bu
tert-Bu
tert-Bu
tert-Bu
tert-Bu
tert-Bu
Me
tert-Bu
tert-Bu
tert-Bu
tert-Bu
tert-Bu
tert-Bu
tert-Bu
tert-Bu
tert-Bu
tert-Bu
Me
4.86
3.28
5.77
5.07
5.27
5.48
6.57
5.52
5.22
5.35
3.07
2.09
Me
1-Napth
4-F-Ph
4-F-Ph
4-F-Ph
Ph
Ph
Ph
Ph
Ph
NHCH2
NHCOCHϭCH
NHCO(CH2)2
NHCO
11
122)
13
Ph
NHCO
H
H
Prednisolone
a) ED50 (mg/ear) or % inhibition at 1 mg/ear. nd; not determined owing to its poor solubility in application solvents.
Reagents and conditions: (a) NaNO2, 12 N HCl, H2O, (b) 5% Pd/C, H2, MeOH, (c) R–COOH, DPP–Cl, Et3N, CH2Cl2, then 15, Et3N, (d)
BH3·Me2S, THF, reflux, (e) 3,5-di-tert-butyl-4-hydroxybenzyl chloride, Et3N, 2-PrOH, reflux.
Chart 1
Reagents and conditions : (f) POCl3, reflux, (g) MeNH2, EtOH, reflux, (h) Me2NH, EtOH, reflux, (i) NaNO3/H2SO4.
Chart 2

March 2003
311
respectively.
Results and Discussions
We examined the inhibitory effects of topically applied test
compounds on PC-induced CHR in mice,11) and investigated
the relationship between the anti-inflammatory activity and
the lipophilicity (AlogP) with regard to the skin tissue per-
meability. AlogP values were computed by using a Cerius 4.6
AlogP98 descriptor.12)
We first examined the substituent at the N(1)-position of
the pyrimidine ring of 2c. As shown in Table 1, the inhibitory
activity was diminished when a methyl group (3) replaced
the phenyl one (2c) at this position, whereas the inhibitory
effect with the 1-naphthyl group (4) or 4-fluorophenyl one
(5) was increased. We could not observe the relationship be-
tween the anti-oxidative activity and the inhibitory activity
on PC-induced CHR, because anti-oxidative activities (Fen-
ton reaction) of these compounds were nearly the same (data
not shown). On the other hand, the order of potency of these
Fig. 2. The Inhibitory Activities of 1, 10 and 11 on PC-Induced CHR at a
Dose of 0.1 mg/ear
*** pϽ0.001 versus vehicle (Dunnet’s test).
Compact MALDI 3 V4.0.0 spectrometer. Elemental analyses were per-
formed at the Toray Research Center. Wakogel C-200 (Wako; 70—150 mm)
was used for column chromatography. Monitoring of reactions was carried
out by using Merck 60 F254 silica gel, glass-supported TLC plates, and visu-
alization with UV light (254, 365 nm). The following abbreviations are used
compounds was well consistent with that of the calculated for reagents and solvents: DMF (N,N-dimethylformamide), DPP-Cl
(diphenylphosphoryl chloride), EDC·HCl (N-ethyl-NЈ-[3-(dimethylamino)-
propyl] carbodiimide hydrochloride), EtOAc (ethyl acetate), and HOBt (1-
hydroxybenzotriazole).
5-(4-Acetoxy-3-methoxyphenylcarboxamido)-6-amino-3-methyl-1-
AlogP value, suggesting that the increase in lipophilicity of
the compounds may be contributed to enhanced skin tissue
permeability. Next, to further elevate the lipophilicity of 5,
we changed the amino group at the C(6)-position to the
methylamino group (6) or the dimethylamino one (7). The
activities of 6 and 7 were 2-fold more potent than that of 5 at
the most. These results show the limitation of enhancing the
inhibitory activity by improving the AlogP value. In addition,
to improve the lipophilicity, we changed the linker moiety
between the pyrimidine ring and di-tert-butylphenol. Espe-
cially, the hint of the distance between the hydroxy group at
the chroman and the pyrimidine moiety of 1 prompted us to
synthesize 10 and 11. Both 10 and 11 showed the most po-
tent inhibitory activities, with an ED50 value of 0.14 mg/ear
for 10 and that of 0.13 mg/ear for 11. As shown in Fig. 2, the
inhibitory activities of 10 and 11 were about 3-fold greater
than that of 1 at a dose of 0.1 mg/ear. In spite of compound 9,
having a high lipophilicity, its inhibitory activity was signifi-
phenyl-2,4-(1H,3H)-pyrimidinedione (2b) To a solution of 4-acetoxy-3-
methoxybenzoic acid (200 mg, 1.0 mmol) and Et3N (0.16 ml, 1.2 mmol) in
EtOAc (10 ml), DPP-Cl (0.24 ml, 1.2 mmol) was added. After 1 h of stirring
at room temperature, 15 (190 mg, 0.9 mmol) was added, and the mixture was
then stirred for 12 h at room temperature. After partitioning with water
(10 ml), the organic layer was dried over MgSO4 and concentrated. The
residue was sonicated in EtOAc to give 2b as a white solid (230 mg, 66%).
mp 160—162 °C. 1H-NMR (DMSO-d6) d: 9.01 (1H, s), 7.72 (1H, s), 7.53—
7.72 (4H, m), 7.34—7.38 (2H, m), 7.19 (1H, d, Jϭ8.1 Hz), 6.08 (2H, s),
3.85 (3H, s), 3.16 (3H, s), 2.28 (3H, s). MS (TOF): m/z 425 (MϩH)ϩ. Anal.
Calcd for C21H20N4O6·H2O: C, 57.01; H, 5.01; N, 12.66. Found: C, 57.24;
H, 4.73; N, 12.66.
Compounds 3—5, 10, 13 were also prepared by using the same procedure
as for 2b.
6-Amino-5-(3,5-di-tert-butyl-4-hydroxyphenylcarboxamido)-1,3-di-
methyl-2,4-(1H,3H)-pyrimidinedione (3) Yield 77%. mp 234—236 °C.
1H-NMR (DMSO-d6) d: 8.74 (1H, s), 7.74 (2H, s), 7.38 (1H, s), 6.62 (2H,
s), 3.31 (3H, s), 3.13 (3H, s), 1.42 (18H, s). MS (TOF): m/z 403 (MϩH)ϩ.
cantly diminished compared with 2c. We considered that an Anal. Calcd for C21H30N4O4·1.5H2O: C, 58.72; H, 7.74; N, 13.04. Found: C,
58.59; H, 7.34; N, 12.81.
6-Amino-5-(3,5-di-tert-butyl-4-hydroxyphenylcarboxamido)-3-methyl-
active NH group at the C(5) of 9 might react with some pro-
teins before reaching to inflamed tissues, therefore, causing 9
1-(1-naphthyl)-2,4-(1H,3H)-pyrimidinedione (4) Yield 53%. mp 172—
to have poor inhibitory activity. These findings suggest that
the presence of the carbonyl moiety in the 2c-type com-
pounds may be needed to exhibit the inhibitory activity and
that a favorable AlogP value (around 5.0—5.5) would poten-
tiate the inhibitory activity.
In conclusion, we conducted a preliminary SAR study on
topically applied 2c derivatives with respect to anti-inflam-
matory activity, and found that the inhibitory activities were
improved with an increase in the AlogP value. Among the
compounds, 10 and 11 showed 2-fold more potent activity
than 1. They could be considered to be useful lead com-
pounds, having inexpensive anti-oxidative moieties without
chirality. A study for further optimization is still underway.
174 °C. 1H-NMR (DMSO-d6) d: 8.85 (1H, s), 8.08—8.14 (2H, m), 7.77 (2H,
s), 7.57—7.71 (5H, m), 7.39 (1H, s), 6.03 (2H, s), 3.19 (3H, s), 1.42 (18H,
s). MS (TOF): m/z 515 (MϩH)ϩ. Anal. Calcd for C30H34N4O4·0.7H2O: C,
68.53; H, 6.65; N, 10.66. Found: C, 68.20; H, 6.77; N, 10.66.
6-Amino-5-(3,5-di-tert-butyl-4-hydroxyphenylcarboxamido)-1-(4-fluo-
rophenyl)-3-methyl-2,4-(1H,3H)-pyrimidinedione (5) Yield 71%. mp
231—233 °C. 1H-NMR (CDCl3) d: 8.75 (1H, s), 7.73 (2H, s), 7.37 (2H, m),
7.41 (2H, m), 6.03 (2H, s), 3.15 (3H, s), 1.41 (18H, s). MS (TOF): m/z 483
(MϩH)ϩ. Anal. Calcd for C26H31FN4O3·0.6H2O: C, 63.30; H, 6.58; N,
11.36. Found: C, 63.09; H, 6.40; N, 11.31.
6-Amino-5-(3,5-di-tert-butyl-4-hydroxycinnamoylamido)-3-methyl-1-
phenyl-2,4-(1H,3H)-pyrimidinedione (10) Yield 36%. mp 178—180 °C.
1H-NMR (CDCl3) d: 7.57—7.62 (4H, m), 7.49 (1H, br s), 7.37—7.40 (4H,
m), 6.50 (1H, d, Jϭ15.7 Hz), 5.53 (2H, br s), 5.50 (1H, s), 3.40 (3H, s), 1.46
(18H, s). MS (TOF): m/z 491 (MϩH)ϩ. Anal. Calcd for C28H38N4O4·H2O:
C, 66.12; H, 7.13; N, 11.02. Found: C, 66.33; H, 6.84; N, 11.01.
Experimental
5-(4-Acetoxyphenylcarboxamido)-6-amino-3-methyl-1-phenyl-2,4-
General All reagents and solvents were obtained from commercial sup- (1H,3H)-pyrimidinedione (13) Yield 63%. mp 156—157 °C. 1H-NMR
pliers and were used without further purification. Melting points were mea- (DMSO-d6) d: 8.99 (1H, s), 8.02 (2H, dd, Jϭ7.2, 1.8 Hz), 7.53—7.58 (3H,
sured with a BÜCHI 535 melting point apparatus and were uncorrected. m), 7.36 (2H, m), 7.23 (2H, d, Jϭ7.2 Hz), 6.09 (2H, s), 3.16 (3H, s), 2.30
Proton NMR spectra were recorded on a JEOL GSX270 FT NMR spectrom- (3H, s). MS (TOF): m/z 395 (MϩH)ϩ. Anal. Calcd for C20H18N4O5·H2O: C,
eter. Chemical shifts were given in parts per million (ppm) using tetra- 58.25; H, 4.89; N, 13.59. Found: C, 58.15; H, 4.76; N, 13.52.
methylsilane as the internal standard for spectra obtained in DMSO-d6 and
1-(4-Fluorophenyl)-3-methyl-6-methylamino-2,4-(1H,3H)-pyrimidine-
CDCl3. TOF MS (time-of-flight mass spectrometry) were recorded on a dione (17) A solution of 16 (3.1 g, 13.1 mmol) in POCl3 (30 ml) was re-

312
Vol. 51, No. 3
fluxed for 1 h. The solvent was concentrated and then partitioned with solid. The solid was washed with water and recrystallized from EtOH/water
CH2Cl2 (100 ml) and water (100 ml). And the organic layer was washed with to give 8 (1.31 g, 59%) as a white solid. mp 280—282 °C. 1H-NMR
brine, dried over MgSO4, and concentrated to give 6-chloro compound as (DMSO-d6) d: 7.39—7.54 (5H, m), 7.00 (2H, s), 6.63 (1H, s), 5.77 (2H, s),
a pale yellow solid (3.37 g, 100%). A suspension of this solid (0.95 g,
3.7 mmol) and 40% methylamine (40% in H2O, 4 ml) in 2-PrOH (40 ml) was
3.58 (2H, s), 3.13 (3H, s), 1.32 (18H, s). MS (TOF): m/z 436 (MϩH)ϩ. Anal.
Calcd for C26H33N3O3: C, 71.49; H, 7.58; N, 9.61. Found: C, 71.70; H, 7.64;
refluxed for 3 h. After cooling, the resulting precipitate was collected and N, 9.65.
washed with EtOH to give 17 as a white solid (0.82 g, 88%).
6-Amino-5-(3,5-di-tert-butyl-4-hydroxybenzylamino)-3-methyl-1-
5-(3,5-Di-tert-butyl-4-hydroxyphenylcarboxamido)-1-(4-fluorophenyl)-
3-methyl-6-methylamino-2,4-(1H,3H)-pyrimidinedione (6) To a suspen-
phenyl-2,4-(1H,3H)-pyrimidinedione (9) To a solution of 2c (700 mg,
1.5 mmol) in THF (10 ml), borane–methylsulfide complex (2 M solution in
sion of 17 (0.72 g, 2.9 mmol) in H2O (20 ml), sodium nitrate (0.24 g, THF, 3 ml) was added, and the mixture was refluxed for 3 h under a N2 at-
3.5 mmol) and 12 N HCl (0.7 ml) was added, and the mixture was stirred for mosphere. The solvent was concentrated, partitioned with CH2Cl2 (20 ml)
2 h at room temperature. The resulting solid was collected and washed with and water (20 ml), the organic layer was dried over MgSO4 and concen-
H2O to give 18 as a violet solid (0.54 g, 68%). A suspension of 18 (0.5 g, trated. The residue was purified by column chromatography on silica gel
1.8 mmol) and 5% Pd/C (25 mg) in MeOH (30 ml) was stirred under a H2 at- (CH2Cl2/MeOHϭ100 : 1) to give 9 (450 mg, 67%) as a white solid. mp
mosphere for 12 h. The catalyst was removed by the filtration, and the solu- 153—155 °C. 1H-NMR (DMSO-d6) d: 7.50—7.54 (3H, m), 7.23—7.26 (2H,
tion was concentrated to give crude 19. Compound 19 was used for the next m), 7.07 (2H, s), 6.78 (1H, s), 5.52 (2H, s), 6.08 (2H, s), 3.76 (2H, s), 3.15
step without further purification. A solution of 3,5-di-tert-butyl-4-hydroxy-
(3H, s), 1.36 (18H, s). MS (TOF): m/z 451 (MϩH)ϩ. Anal. Calcd for
benzoic acid (60 mg, 0.24 mmol) and HOBt (36 mg, 0.26 mmol) in DMF C26H34N4O3: C, 69.31; H, 7.61; N, 12.43. Found: C, 69.22; H, 7.71; N,
(5 ml) was stirred at room temperature for 1 h. To this solution, 19 (100 mg, 12.45.
0.37 mmol) and EDC·HCl (51 mg, 0.26 mmol) were added, and this mixture
was stirred for 12 h. The solvent was concentrated and partitioned with 3-methyl-1-phenyl-2,4-(1H,3H)-pyrimidinedione (11) A solution of 10
CH2Cl2 and 1 N HCl, then the organic layer was washed with 5% NaHCO3 (140 mg, 0.28 mmol) and 5% Pd/C (7 mg) in MeOH (6 ml) was stirred under
6-Amino-5-[2-(3,5-di-tert-butyl-4-hydroxyphenyl)-ethylcarboxamido]-
solution and water, dried over MgSO4, and concentrated. The residue was a H2 atmosphere for 12 h. The catalyst was removed by the filtration, and the
purified by column chromatography on silica gel (CH2Cl2/MeOHϭ100 : 1), solution was concentrated. Recrystallized from CH2Cl2/hexane to give 11 as
and recrystallized from EtOAc/hexane to give 6 (36 mg, 19%) as a white a white solid (110 mg, 79%). mp 130—132 °C. 1H-NMR (CDCl3) d: 7.55—
1
solid. mp 154—157 °C. H-NMR (CDCl3) d: 7.75 (2H, s), 7.38—7.43 (3H, 7.69 (3H, m), 7.33—7.36 (2H, m), 7.22 (1H, br s), 7.01 (2H, s), 5.08 (1H, s),
m), 7.22 (2H, d, Jϭ7.3 Hz), 5.63 (1H, s), 4.49 (1H, br s), 3.36 (3H, s), 2.67 5.02 (2H, br s), 3.36 (3H, s), 2.94 (2H, t, Jϭ7.8 Hz), 2.70 (2H, q, Jϭ7.8 Hz),
(3H, d, Jϭ5.4 Hz), 1.47 (18H, s). MS (TOF): m/z 497 (MϩH)ϩ. High reso- 1.41 (18H, s). MS (TOF): m/z 493 (MϩH)ϩ. Anal. Calcd for C28H36N4O4·
lution (HR)-MS. Calcd for C27H33FN4O4: 496.2486; Found: 496.2489.
6-Dimethylamino-1-(4-fluorophenyl)-3-methyl-2,4-(1H,3H)-pyrim-
idinedione (20) A suspension of 6-chloro compound prepared from 16
0.4H2O: C, 67.28; H, 7.42; N, 11.21. Found: C, 67.46; H, 7.56; N, 10.92.
Picryl Chloride-Induced Contact Hypersensitivity Reaction Male
ICR mice were sensitized by applying 100 ml of 7% (w/v) PC solution in
(0.95 g, 3.7 mmol) and dimethylamine (50% in H2O, 1.64 ml) in 2-PrOH acetone to the shaved abdomen. Seven days later, the mice were challenged
(30 ml) was refluxed for 8 h. After concentration, water (20 ml) was added, by applying 20 ml of 1% (w/v) PC solution in acetone to the left ear. The ear
and the resulting solid was collected to give 20 as a white solid (0.87 g,
89%).
5-(3,5-Di-tert-butyl-4-hydroxyphenylcarboxamido)-6-dimethylamino-
1-(4-fluorophenyl)-3-methyl-2,4-(1H,3H)-pyrimidinedione (7) To a so-
lution of 20 (0.77 g, 2.9 mmol) in sulfuric acid (17 ml), sodium nitrite
thickness was measured with a digital thickness gauge before and 24 h after
the challenge, and the difference in thickness between the left and right ear
was calculated. Test compounds were dissolved in acetone and were admin-
istered 5 min after the challenge.
(0.26 g, 3.1 mmol) was added and the mixture was stirred on ice for 1 h. The References and Notes
resulting solid was collected and washed with water to give 21 as a yellow
solid (0.78 g, 87%). A suspension of 21 (0.7 g, 2.3 mmol) and 5% Pd/C
(35 mg) in MeOH (30 ml) was stirred under a H2 atmosphere for 12 h. The
catalyst was removed by the filtration, and the solution was concentrated to
give crude 22. Compound 22 was used for the next step without further pu-
rification. A solution of 3,5-di-tert-butyl-4-hydroxybenzoic acid (58 mg,
0.23 mmol) and HOBt (34 mg, 0.25 mmol) in DMF (10 ml) was stirred at
room temperature for 1 h. To this solution, 22 (100 mg, 0.35 mmol) and
EDC·HCl (49 mg, 0.25 mmol) were added, and this mixture was stirred for
12 h. The solvent was concentrated and partitioned with CH2Cl2 and 1 N
HCl, then the organic layer was washed with 5% NaHCO3 solution and
water, dried over MgSO4, and concentrated. The residue was purified by col-
umn chromatography on silica gel (CH2Cl2/MeOHϭ100 : 1) to give 7 as a
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Nishiyama K., Sugano M., In Vitro Cell. Dev. Biol., 35A, 169 (1999).
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1
white solid (38 mg, 21%). mp 164—165 °C. H-NMR (CDCl3) d: 7.70 (2H,
s), 7.31 (2H, d, Jϭ8.1 Hz), 7.18 (2H, d, Jϭ8.1 Hz), 5.61 (1H, s), 3.36 (3H,
s), 2.50 (6H, s), 1.47 (18H, s). MS (TOF): m/z 511 (MϩH)ϩ. HR-MS. Calcd
for C28H35FN4O4: 510.2642; Found: 510.2649.
8) Burton G. W., Ingold K. U., J. Am. Chem. Soc., 103, 6472—6477
(1981).
9) Papesch V., Schroeder E. F., J. Org. Chem., 16, 1879—1890 (1951).
6-Amino-5-(3,5-di-tert-butyl-4-hydroxybenzyl)-3-methyl-1-phenyl-2,4- 10) Ohtsuka Y., Bull. Chem. Soc. Jpn., 46, 506—509 (1973).
(1H,3H)-pyrimidinedione (8) To a solution of 14 (1.1 g, 5.1 mmol) and
Et3N (10 ml) in 2-PrOH (100 ml), 2,6-di-tert-butyl-4-hydroxybenzyl chlo-
ride (1.9 g, 7.5 mmol) was added, and the mixture was refluxed for 30 h. The
solvent was concentrated and then triturated with hexane to give a crude
11) Asherson G. L., Ptak W., Immunology, 15, 405—416 (1968).
12) Ghose A. K., Viswanadhan R. A., Wendoloski J. J., J. Phys. Chem.,
102, 3762—3772 (1998).