570
Vol. 49, No. 5
(14.5 ml) and refluxed for 2 h. After removal of EtOH in vacuo, the residue
was dissolved in CHCl3 (100 ml) and the solution was washed with aq.
NaHCO3 and dried. After concentration of the solution to ca. 20 ml in vacuo,
to the solution was added (Boc)2O (3.7 g) and triethylamine (1.7 g) followed
by stirring at 60 °C for 3 h. The reaction mixture was washed with 5% aq.
citric acid, water and dried. The solvent was removed in vacuo to give an oil,
which was purified by column chromatography (CHCl3–MeOH) to yield 31
(4.8 g, yield 96%) as an oil. 1H-NMR (CDCl3) d: 1.38 (t, 3H, Jϭ6.3 Hz),
1.57 (s, 9H), 1.6—2.4 (m, 2H), 3.3—4.0 (m, 5H), 4.32 (q, 2H, Jϭ6.3 Hz),
Jϭ8.0 Hz), 4.28 (br s, 4H), 6.40 (d, 1H, Jϭ8.0 Hz), 6.9—7.5 (m, 4H), 7.53
(s, 1H): IR (Nujol): 1700 cmϪ1
.
1-Boc-6-(imidazol-1-yl)methylindoline (35c): Yield 92% as an oil. 1H-
NMR (CDCl3) d: 1.55 (s, 9H), 3.08 (t, 2H, Jϭ7.5 Hz), 3.99 (t, 2H, Jϭ7.5
Hz), 5.08 (s, 2H), 6.65 (d, 1H, Jϭ8.5 Hz), 6.92 (d, 1H, Jϭ8.5 Hz), 6.95 (s,
1H), 7.05 (s, 1H), 7.60 (s, 1H), 7.70 (s, 1H). IR (neat): 1700, 1500 cmϪ1
.
Ethyl 2-[5-(Imidazol-1-yl)methylindolin-1-yl]acetate (36b) To a solu-
tion of 35b (12.0 g) in CHCl3 (300 ml) was added 10 N HCl in EtOH solution
(40 ml). The reaction mixture was stirred for 2 h at 20 °C. After neutraliza-
tion with aq. NaHCO3, the organic layer was washed with water and dried.
The solvent was removed in vacuo to give an oil, which was purified by col-
umn chromatography (CHCl3–MeOH) to give 5-(imidazol-1-yl)methylindo-
line (3.6 g, yield 47%) as a solid. A mixture of 5-(imidazol-1-yl)methylindo-
line (1.0 g), ethyl bromoacetate (1.0 g) and K2CO3 (1.4 g) in DMF (20 ml)
was stirred at 20 °C for 20 h. The reaction mixture was diluted with AcOEt
(200 ml), the solution was washed with water and dried. The solvent was re-
moved in vacuo to give an oil, which was purified by column chromatogra-
phy (CHCl3–MeOH) to give 36b (0.8 g, yield 56%) as an oil. 1H-NMR
(CDCl3) d: 1.25 (t, 3H, Jϭ6.3 Hz), 2.98 (t, 2H, Jϭ7.5 Hz), 3.56 (t, 2H,
Jϭ7.5 Hz), 3.87 (s, 2H), 4.18 (q, 2H, Jϭ6.3 Hz), 4.95 (s, 2H), 6.30 (d, 1H,
Jϭ8.0 Hz), 6.7—6.9 (m, 3H), 6.95 (s, 1H), 7.43 (s, 1H). IR (neat): 1740,
7.5—8.0 (m, 3H). IR (neat): 3350, 1695, 1500 cmϪ1
.
Ethyl 1-Boc-3-[2-(imidazol-1-yl)ethyl]indoline-5-carboxylate (32) To
a solution of 31 (4.8 g) and CBr4 (9.9 g) in CH3CN (100 ml) was added Ph3P
(4.7 g). The reaction mixture was stirred for 2 h at 20 °C. After removal of
the solvent in vacuo, the residue was diluted with acetone (200 ml). To the
solution was added imidazole (6.6 g) and K2CO3 (5.3 g) followed by reflux-
ing for 4 h. After removal of the solvent in vacuo, the residue was dissolved
in AcOEt (300 ml). The solution was washed with water and dried. The sol-
vent was removed in vacuo to give an oil, which was purified by column
chromatography (CHCl3–MeOH) to give 32 (3.2 g, yield 58%) as an oil. 1H-
NMR (CDCl3) d: 1.39 (t, 3H, Jϭ6.3 Hz), 1.59 (s, 9H), 1.9—2.4 (m, 2H),
3.0—4.1 (m, 5H), 4.34 (q, 2H, Jϭ6.3 Hz), 6.78 (s, 1H), 7.03 (s, 1H), 7.43 (s,
1H), 7.5—8.0 (m, 3H). IR (neat): 1705, 1495 cmϪ1
.
1500 cmϪ1
.
3-[2-(Imidazol-1-yl)ethyl]indoline-5-carboxylic Acid Dihydrochloride
(3) To a solution of 32 (3.2 g) in 90% EtOH (30 ml) was added NaOH (1.5
g) followed by stirring at 60 °C for 6 h. After neutralization with 5% aq. cit-
ric acid, 1-Boc-3-[2-(imidazol-1-yl)ethyl]indoline-5-carboxylic acid was ex-
tracted with CHCl3 (200 ml). The extracts were washed with water and
dried. The solvent was removed in vacuo to give an oil, which was purified
by column chromatography (CHCl3–MeOH) to give the precursor of 3 (1.4
g, yield 47%) as an oil. To a solution of this oil (1.4 g) in CHCl3 (30 ml) was
added 10 N HCl in EtOH solution (20 ml) followed by stirring at 20 °C for 2
h. Crystals from the reaction mixture were recrystallized from EtOH to give
Ethyl 2-{4-[2-(Imidazol-1-yl)ethoxy]indolin-1-yl}acetate (36a): Yield
13% as a solid. 1H-NMR (CDCl3) d: 1.27 (t, 3H, Jϭ6.5 Hz), 2.93 (t, 2H,
Jϭ8.0 Hz), 3.55 (t, 2H, Jϭ8.0 Hz), 3.85 (s, 2H), 4.07 (q, 2H, Jϭ6.5 Hz),
4.10 (br s, 4H), 6.07 (d, 1H, Jϭ8.5 Hz), 6.19 (d, 1H, Jϭ8.5 Hz), 6.7—7.2
(m, 3H), 7.55 (s, 1H). IR (Nujol): 1730, 1620, 1600 cmϪ1
.
Ethyl 3-[6-(Imidazol-1-yl)methylindolin-1-yl]propionate (36c): Yield
58% as an oil. 1H-NMR (CDCl3) d: 1.23 (t, 3H, Jϭ6.3 Hz), 2.55 (t, 2H,
Jϭ6.3 Hz), 2.92 (t, 2H, Jϭ7.5 Hz), 3.2—3.6 (m, 4H), 4.15 (q, 2H, Jϭ6.3
Hz), 5.00 (s, 2H), 6.27 (s, 1H), 6.40 (d, 1H, Jϭ8.5 Hz), 6.90 (s, 1H), 6.97 (d,
1H, Jϭ8.5 Hz), 7.05 (s, 1H), 7.53 (s, 1H). IR (neat): 1735, 1505 cmϪ1
.
1
3 (0.8 g, yield 62%) as white crystals. mp 209—212 °C: H-NMR (DMSO-
Sodium 2-[5-(Imidazol-1-yl)methylindolin-1-yl]acetate (4) To a solu-
tion of 36b (0.8 g) in 90% EtOH (40 ml) was added NaOH (0.2 g) followed
by stirring at 20 °C for 2 h. After removal of EtOH in vacuo, the residue was
dissolved in water (300 ml) and washed with AcOEt (100 ml). The solvent
was concentrated to ca. 100 ml in vacuo and subjected to Diaion HP-21
column chromatography. The fraction eluted with MeOH–water was
lyophilized to give 4 (0.25 g, yield 30%) as a solid. 1H-NMR (DMSO-d6) d:
2.87 (t, 2H, Jϭ7.6 Hz), 3.47 (t, 2H, Jϭ7.6 Hz), 3.85 (s, 2H), 4.97 (s, 2H),
6.35 (d, 1H, Jϭ8.5 Hz), 6.7—7.1 (m, 3H), 7.12 (s, 1H), 7.70 (s, 1H). IR
d6) d: 1.8—3.0 (m, 2H), 3.2—4.0 (m, 3H), 4.40 (t, 2H, Jϭ6.8 Hz), 7.08 (d,
1H, Jϭ9.0 Hz), 7.6—8.0 (m, 4H), 9.25 (s, 1H), 10.4 (br, 4H). IR (Nujol):
1710, 1490 cmϪ1. Anal. Calcd for C14H15N3O2·2HCl·0.25H2O: C, 50.23; H,
5.27; N, 12.55. Found: C, 49.94; H, 5.18; N, 12.54.
1-Boc-4-(2-hydroxyethoxy)indoline (34a) A mixture of 33a (15.2 g),
K2CO3 (22.0 g) and ethylene bromohydrin (20.7 g) in N,N-dimethylfor-
mamide (DMF) (100 ml) was stirred for 10 h at 60 °C. The reaction mixture
was diluted with AcOEt (500 ml), washed with water and dried. The solvent
was removed in vacuo to give an oil, which was purified by column chro-
(Nujol): 1600, 1500 cmϪ1
.
1
matography (CHCl3–MeOH) to give 34a (10.2 g, yield 56%) as a solid. H-
Sodium 2-{4-[2-(Imidazol-1-yl)ethoxy]indolin-1-yl}acetate (5): Yield
52% as a solid. 1H-NMR (DMSO-d6) d: 2.80 (t, 2H, Jϭ7.8 Hz), 3.2—3.8
(m, 4H), 3.9—4.6 (m, 4H), 6.05 (d, 1H, Jϭ8.5 Hz), 6.20 (d, 1H, Jϭ8.5 Hz),
NMR (CDCl3) d: 1.55 (s, 9H), 2.99 (t, 2H, Jϭ7.5 Hz), 3.7—4.3 (m, 6H),
6.52 (d, 1H, Jϭ8.5 Hz), 6.9—7.5 (m, 2H). IR (Nujol): 3320, 1710, 1605
cmϪ1
.
6.7—7.1 (m, 2H), 7.28 (s, 1H), 7.70 (s, 1H). IR (Nujol): 1600 cmϪ1
.
1-Boc-5-hydroxymethylindoline (34b) To a solution of ethyl indoline-
5-carboxylate 33b (19.0 g) in dry benzene (200 ml) was added 0.1 M DIBAL-
H in toluene solution (200 ml) dropwise at 20 °C. The reaction mixture was
stirred for 30 min at 40 °C, then quenched with water and the product was
extracted with CHCl3 (500 ml). The extract was concentrated to ca. 100 ml
in vacuo and to the solution was added (Boc)2O (43.2 g) followed by stirring
for 10 h at 20 °C. The solvent was removed in vacuo to give an oil, which
was purified by column chromatography (benzene–AcOEt) to give 34b (10
g, yield 40%) as an oil. 1H-NMR (CDCl3) d: 1.58 (s, 9H), 3.05 (t, 2H, Jϭ7.5
Hz), 3.97 (t, 2H, Jϭ7.5 Hz), 4.58 (s, 2H), 7.05 (br d, 1H), 7.12 (s, 1H), 7.55
Sodium 3-[6-(Imidazol-1-yl)methylindolin-1-yl]propionate (6): Yield
42% as a solid. 1H-NMR (DMSO-d6) d: 2.22 (t, 2H, Jϭ6.5 Hz), 2.82 (t, 2H,
Jϭ7.5 Hz), 3.0—3.7 (m, 4H), 5.01 (s, 2H), 6.35 (s, 1H), 6.38 (d, 1H, Jϭ8.5
Hz), 6.85 (s, 1H), 6.92 (d, 1H, Jϭ8.5 Hz), 7.05 (s, 1H), 7.62 (s, 1H). IR
(Nujol): 1560 cmϪ1
.
Rabbit Platelet Aggregation (in Vitro) Male Japanese white rabbits
(approximately 3 kg) were used. Blood was taken from the carotid artery
under anesthesia with sodium pentobarbital (20 mg/kg, i.v.). Platelet-rich
plasma (PRP) and platelet-poor plasma (PPP) were obtained by centrifuga-
tion at 1000 rpm for 10 min and at 3000 rpm for 10 min at 4 °C, respectively.
The platelet density of PRP was adjusted to 4ϫ105 cells/ml. Platelet aggrega-
tion was measured with an NKK Hema Tracer (Niko Bioscience). PRP was
incubated with test compounds or ozagrel at 37 °C for 1 min, followed by
addition of arachidonic acid or ADP.
TXA2 Synthesis in Washed Rabbit Platelets Rabbit blood was col-
lected from the carotid artery under anesthesia with sodium pentobarbital
and mixed with acid–citrate–dextrose (ACD) solution (sodium citrate 85
mM, citric acid 64 mM and dextrose 100 mM). PRP was obtained and the
platelets were washed three times with Tyrode’s solution. The platelets were
finally suspended in Tyrode’s solution at 105 cells/ml. The platelet suspension
was incubated at 37 °C for 10 min in the presence of aspirin (10Ϫ5 M) and
then further incubated with PGH2 for 30 min. TXA2 produced was deter-
mined as TXB2 using a TXB2[3H]RIA kit.
(br d, 1H). IR (neat): 3420, 1700, 1495 cmϪ1
.
N-Boc-6-hydroxymethylindoline (34c): Yield 55% as an oil. 1H-NMR
(CDCl3) d: 1.53 (s, 9H), 3.02 (t, 2H, Jϭ7.5 Hz), 3.95 (t, 2H, Jϭ7.5 Hz),
4.62 (s, 2H), 6.92 (d, 1H, Jϭ8.5 Hz), 7.06 (d, 1H, Jϭ8.5 Hz), 7.70 (s, 1H).
IR (neat): 3400, 1700, 1500 cmϪ1
.
1-Boc-5-(imidazol-1-yl)methylindoline (35b) To a solution of 34b
(10.0 g) and CBr4 (26.7 g) in CH3CN (200 ml) was added Ph3P (10.5 g) at
10 °C. The reaction mixture was stirred for 10 min at 20 °C. To the reaction
mixture was added imidazole (68.0 g) followed by stirring for 2 h at 20 °C.
After removal of the solvent in vacuo, the residue was dissolved in AcOEt
(500 ml), the solution was washed with water and dried. The solvent was re-
moved in vacuo to give an oil, which was purified by column chromatogra-
phy (CHCl3–MeOH) to give 35b (12.0 g, yield 95%) as an oil. 1H-NMR
(CDCl3) d: 1.55 (s, 9H), 3.02 (t, 2H, Jϭ7.5 Hz), 3.96 (t, 2H, Jϭ7.5 Hz),
5.00 (s, 2H), 6.7—7.1 (m, 4H), 6.95 (s, 1H), 7.15 (br d, 1Hz). IR (neat):
Free Radical Scavenging DPPH, a stable free radical was dissolved at
10Ϫ4 M in ethanol and incubated at 25 °C for 20 min in the absence or pres-
ence of the synthesized compounds (10Ϫ4 M) according to the method previ-
ously reported.8) Remaining DPPH in the absence and presence of the test
compounds was determined spectrophotometrically at a wavelength of 517
1700, 1500 cmϪ1
.
1-Boc-4-[2-(imidazol-1-yl)ethoxy]indoline (35a): Yield 37% as a solid.
1H-NMR (CDCl3) d: 1.57 (s, 9H), 2.96 (t, 2H, Jϭ8.0 Hz), 3.99 (t, 2H,