6194
K. Kikuchi et al. / Bioorg. Med. Chem. 14 (2006) 6189–6196
NMR (CDCl3): isomer I: d 7.84 (d, 1H, JAB = 8.5 Hz),
7.33 (d, 1H, J = 2.2 Hz), 6.70 (dd, 1H, JAB = 8.5 Hz,
Jm = 2.2 Hz), 3.7 (br, 2H, exchanges with D2O), 1.63
(s, 9H), 1.39 (s, 18H). Isomer II: d 7.74 (d, 1H,
JAB = 8.55 Hz), 7.00 (d, 1H, J = 2.4 Hz), 6.78 (dd, 1H,
JAB = 8.55 Hz, Jm = 2.4 Hz), 3.7 (b, 2H), 1.63 (s, 9H),
1.39 (s, 18H).
4.8. Substrates
5-Nitro, 6-nitro-, 5,7-dinitro-, 5-chloro-, and 6-chloro-
benzisoxazole were prepared according to the litera-
ture.8,26 4-Cyano- and 4-fluorosalicylaldehyde were
prepared from the corresponding phenols by a Duff
reaction.27 4,5-Dichlorosalicylaldehyde was prepared
by a Reimer–Tiemann reaction.28 The general literature
was followed to prepare benzisoxazoles from the corre-
sponding salicylaldehydes.8
4.5. 1-tert-Butoxycarbonyl-2-(N,N-bis-tert-butoxycar-
bonyl)-5-glutaramidebenzimidazole (8a) and 1-tert-bu-
toxycarbonyl-2-(N,N-bis-tert-butoxycarbonyl)-6-gluta-
ramidebenzimidazole (8b)
4.9. 5-Cyanobenzisoxazole
Glutaric anhydride (1 g) was added to a mixture of 7a
and 7b (2.74 g, 6.1 mmol) dissolved in 80 ml dry
dichloromethane, and the resulting solution was stirred
at room temperature overnight. The clear solution be-
came cloudy after a few minutes as a precipitate began
to form. The precipitate was collected by filtration,
washed with dichloromethane, and dried under vacu-
um to give the glutaric acid half-amide as a mixture
1H NMR (300 MHz, CDCl3): d 8.83 (d, J = 0.9,
1H), 8.16 (dd, J1 = 0.9, J2 = 1.8, 1H), 7.84 (dd,
J1 = 1.8, J2 = 8.6, 1H), 7.76 (dd, J1 = 1.1, J2 = 8.6,
1H); MS (FAB-NBA/NaI) 145 (M+H)+; mp 245 ꢁC
(dec).
4.10. 5,6-Dichlorobenzisoxazole
1
of two isomers (2.9 g, 84.5%). H NMR (DMSO-d6):
1H NMR (300 MHz, CDCl3): d 8.68 (d, J = 1.0, 1H),
7.85 (s, 1H), 7.80 (d, J = 1.0, 1H); MS (FAB-NBA/
NaI) 189 (M+H)+, 211 (M+Na)+; mp 92 ꢁC (sub).
d 12.2 (bs, 1H, exchanges with D2O), 10.1 (s, 1H,
exchanges with D2O), 8.64 (d, 0.5H), 8.11 (d, 0.5H),
7.86 (d, 0.5H), 7.65 (d, 0.5H), 7.56 (dd, 0.5H), 7.42
(dd, 0.5H), 2.40 (t, 2H, J = 7.3 Hz), 2.30 (t, 2H,
J = 7.3 Hz), 1.84 (quintet, 2H), 1.6 (s, 9H), 1.35 (s,
18H). MS-FAB ꢀve: 346.1 (30%), 461.3 (50%), 561.4
(100%, Mꢀ1).
4.11. 5-Fluorobenzisoxazole
1H NMR (300 MHz, CDCl3): d 8.7 (d, J = 0.8, 1H), 7.59
(m, 1H), 7.38 (m, 1H), 7.32 (m, 1H); MS (FAB-NBA/
NaI) 139 (M+H)+; mp 68 ꢁC (sub).
4.6. 2-Amino-5-glutaramidebenzimidazole (4)
4.12. 5,6-Dinitrobenzisoxazole
Boc-protected hapten (0.4 g) was suspended in concen-
trated HCl (10 ml, diluted with 5 ml water and 5 ml
THF). The solution became clear after 5 min. After stir-
ring at room temperature for about 2 h, a precipitate ap-
peared. After stirring for a further 5 h, the solvent was
evaporated. The residue was washed with ether and
dried in vacuo to give hapten 4 (0.18 g, 85.7%). 1H
NMR (DMSO-d6): d 12.45 (br, 2H), 12.2 (br, 1H),
10.22 (s, 1H), 8.51 (s, 2H), 7.91 (d, 1H, J = 1.6 Hz),
7.32 (dd, 1H, JAB = 8.6 Hz, Jm = 1.6 Hz), 7.27 (d, 1H,
JAB = 8.6 Hz), 2.37 (t, 2H, J = 7.5 Hz), 2.28 (t, 2H,
J = 7.5 Hz), 1.80 (quintet, 2H, J = 7.5 Hz); the protons
at 12.45, 12.2, 10.22, and 8.51 all exchange with D2O.
13C NMR (DMSO-d6): d 174.264, 170.849, 150.713,
135.327, 129.800, 125.268, 114.660, 111.355, 102.608,
35.536, 33.183, 20.672. MS-FAB +ve: 263.1 (100%),
(MꢀCl+H). Elemental analysis C12H14N4O3ÆHCl, calcd:
C, 48.25; H, 5.06; Cl, 11.87; N, 18.75. Found: C, 47.44;
H, 5.04; N, 18.97. The pKa value of the protonated benz-
imidazolium ring was determined by standard pH
titration.
6-Nitrobenzisoxazole (200 mg, 1.22 mmol) was dis-
solved in 1.6 ml ice-cooled H2SO4. Two hundred micro-
liters of fuming HNO3 (q 1.5) was added dropwise. The
reaction mixture was heated at 80 ꢁC for 30 min. The
starting material was completely consumed. The reac-
tion mixture was poured on ice and then extracted
immediately with CH2Cl2. The organic layer was dried
over MgSO4 and purified by column chromatography
(silica gel 60, CH2Cl2) to give 142 mg (679 mmol) prod-
uct (56%). 1H NMR (300 MHz, CDCl3): d 9.15 (d, 1H),
8.52 (s, 1H), 8.17 (d, 1H); MS (FAB-NBA/NaI) 210
(M+H)+; mp 112 ꢁC.
4.13. 5-Nitro-6-chlorobenzisoxazole
6-Chlorobenzisoxazole was nitrated at position 5 by the
same method used to prepare 5,6-dinitrobenzisoxazole
in 61.9% yield. 1H NMR (300 MHz, CDCl3): d 8.83
(d, J = 1.1, 1H), 8.36 (dd, J1 = 0.9, J2 = 1.8, 1H), 7.88
(d, J = 1.1, 1H); MS (FAB-NBA/NaI) 199 (M+H)+;
mp 106 ꢁC.
4.7. Hapten conjugates
4.14. 5-Aminobenzisoxazole29 (with modifications)
Compound 4 was coupled to thyroglobulin (TG) via its
carboxyl group as described by Lauer et al.24 The epi-
tope density of the resulting protein conjugate (42 hap-
tens per carrier) was determined by the method of
Habeeb.25 A hapten with bovine serum albumin (BSA)
was prepared analogously for binding studies. It had
an epitope density of 10 haptens per BSA.
5-Nitrobenzisoxazole (70 mg, 0.43 mmol) was mixed
with 914 mg (0.55 lmol) SnCl4 in 400 ll of 12 M HCl
and ice cooled. SnCl2 (291 mg, 1.29 lmol) in 12 M
HCl was added dropwise. The ice bath was removed
and the reaction mixture was stirred for 5 h at rt with
an additional 2.5 ml of 12 M HCl. The mixture was