2650 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16
Wakisaka et al.
21.8 mmol) in dry chloroform (120 mL) at 0 °C. The mixture
was stirred vigorously at the same temperature for 1 h. After
filtration of the precipitate, the filtrate was evaporated in
vacuo, and the residue was chromatographed on silica gel
using a mixture of chloroform-methanol-28% aqueous am-
monia solution (80:16:3) as an eluent to produce compound
6c (3.00 g, 68%) as a pale yellow oil. 1H-NMR (CDCl3): δ
1.21-1.55 (6H, m, (CH2)3), 1.37 (2H, t, NH2), 1.41 (9H, s, Boc),
2.69 (2H, t, NH2CH2), 3.11 (2H, q, NHCH2), 4.57 (1H, br, NH).
Anal. (C15H23N2O6‚1/10CHCl3) C, H, N.
Compound 7c was then synthesized by reaction of compound
6c with compound 5 as described above in 87% yield. Mp: 58-
59 °C. 1H-NMR (CDCl3): δ 1.20-1.66 (6H, m, (CH2)3), 1.44
(9H, s, Boc), 3.10 (2H, q, NHCH2), 3.52 (2H, t, NCH2), 4.54
(1H, br, NH), 6.70 (2H, s, maleimide). FAB-MS calcd for
C14H23N2O4(MH+): m/z 283. Found: 283. Anal. (C14H22N2O4‚
1/12CHCl3) C, H, N.
aromatic), 7.91 (1H, d, aromatic), 8.24 (1H, t, aromatic). FAB-
MS calcd for C19H21N3O4I (MH+): m/z 514. Found: 514. Anal.
(C19H20N3O6I‚2/5CHCl3) C, H, N.
Syn th esis of N-(5-Ma leim id op en tyl) 3′-Iod oh ip p u r ic
Acid Am id e. A mixture of N-succinimidyl 3-iodohippurate
(82 mg, 0.15 mmol), 5′-aminopentylmaleimide TFA salt (43 mg,
0.15 mmol), and N,N-diisopropylethylamine (30.2 µL, 0.17
mmol) in dry THF (5 mL) was stirred at room temperature
for 1.5 h. After filtration of the precipitate, the solvent was
removed in vacuo, and the residue was chromatographed on
silica gel using ethyl acetate as an eluent to produce N-(5-
maleimidopentyl) 3′-iodohippuric acid amide (80 mg, 85%) as
a colorless oil. 1H-NMR (CDCl3): δ 1.25-1.66 (6H, m, (CH2)3),
3.29 (2H, q, NHCH2), 3.51 (2H, t, NCH2), 4.19 (2H, d, NHCH2),
6.45 (1H, t, NH), 6.69 (2H, s, maleimide), 7.18 (1H, t, aromatic),
7.32 (1H, t, NH), 7.79 (1H, d, aromatic), 7.84 (1H, d, aromatic),
8.19 (1H, t, aromatic). FAB-MS calcd for C18H20N3O4I (MH+):
m/z 470. Found: 470. Anal. (C18H20N3O4I) C, H, N.
Syn th esis of 3′-(Tr i-n -bu tylsta n n yl)h ip p u r yl NE-Ma le-
oyl-L-lysin e (8a ). Compound 7a (0.56 g) was treated with 4
M HCl in dry ethyl acetate (5 mL) for 30 min at room
temperature before addition of 15 mL of dry ether at 0 °C.
The HCl salt of Nꢀ-maleoyl-L-lysine (0.3 g) precipitated as
white crystals. A mixture of compound 3 (215 mg, 0.38 mmol),
the HCl salt of Nꢀ-maleoyl-L-lysine (100 mg, 0.38 mmol), and
N,N-diisopropylethylamine (69.5 µL, 0.40 mmol) in dry aceto-
nitrile (4 mL) was stirred at room temperature for 1 h. After
filtration, the filtrate was evaporated in vacuo, and the residue
was chromatographed on silica gel using a mixture of chloro-
form-methanol-acetic acid (250:50:1) as an eluent to produce
compound 8a (198 mg, 78%) as a colorless oil. 1H-NMR
(CDCl3): δ 0.85-1.94 (33H, m, SnBu3, (CH2)3), 3.49 (2H, t,
NCH2), 4.24 (2H, d, NHCH2), 4.29 (1H, q, CH), 6.86 (2H, s,
maleimide), 7.38 (1H, t, aromatic), 7.60 (1H, d, aromatic), 7.72
(1H, d, aromatic), 7.84 (1H, t, aromatic). FAB-MS calcd for
C31H46N3O6Sn (M-): m/z 676. Found: 676.
P r ep a r a tion of [131I]-L-HML-NGA. The stannyl precursor
of L-HML (L-SHML) was radioiodinated with Na[131I]I in the
presence of N-chlorosuccinimide (NCS) as previously de-
scribed.17 Briefly, L-SHML was dissolved in methanol contain-
ing 1% acetic acid (0.64 mg/mL), and 16.3 µL of this solution
was mixed with 2 µL of Na[131I]I. After addition of 4.44 µL of
NCS in methanol (0.5 mg/mL), the reaction mixture was
incubated at room temperature for 25 min. Aqueous sodium
bisulfite (2.22 µL, 0.72 mg/mL) was then added to quench the
reaction. The radiochemical yield of [131I]-L-HML was deter-
mined by TLC developed with a mixture of chloroform-
methanol-acetic acid (40:5:2). Methanol was removed by an
N2 flow prior to subsequent conjugation reaction with NGA.
NGA was pretreated with 5 molar excess of DTT to expose
1 thiol group/molecule of the protein. Under these conditions,
none of the 17 disulfide bonds of HSA were accessible to DTT
reduction.26 Briefly, 50 µL of freshly prepared DTT (0.45 mg/
mL) in well-degassed 0.1 M PB (pH 6.8) containing 0.3 M NaCl
was added to 50 µL of NGA (40 mg/mL) in the same buffer.
After incubation for 30 min at room temperature, a 100 µL
solution of 0.1 M PB (pH 6.0) containing 2 mM ethylene-
diaminetetraacetic acid (EDTA) was added. Excess DTT was
removed by the centrifuged column procedure28 using a Sepha-
dex G-50 column (Pharmacia Biotech, Tokyo, J apan) equili-
brated with well-degassed 0.1 M PB (pH 6.0) containing 2 mM
EDTA. A small portion of this mixture was sampled, and the
number of thiol groups was estimated with 2,2′-dithiopyri-
dine.27 The filtrate (100 µL) was then added to the reaction
vial containing crude [131I]-L-HML. After gentle agitation of
the reaction mixture for 1.5 h at room temperature, crude [131I]-
L-HML-NGA was purified by the centrifuged column procedure
using a Sephadex G-50 column equilibrated with 0.1 M PB
(pH 7.4).
Syn th esis of 3′-(Tr i-n -bu tylsta n n yl)h ip p u r yl NE-Ma le-
oyl-D-lysin e (8b). This compound was synthesized according
to the procedure as described above except using NR-Boc-D-
lysine in place of NR-Boc-L-lysine as the starting material in
70% yield. 1H-NMR (CDCl3): δ 0.85-1.94 (33H, m, SnBu3,
(CH2)3), 3.49 (2H, t, NCH2), 4.27 (2H, d, NHCH2), 4.64 (1H, q,
CH), 6.64 (2H, s, maleimide), 7.36 (1H, t, aromatic), 7.60 (1H,
d, aromatic), 7.72 (1H, d, aromatic), 7.91 (1H, t, aromatic).
FAB-MS calcd for C31H46N3O6Sn (M-): m/z 676. Found: 676.
Syn th esis of N-(5-Ma leim id op en tyl) 3′-(Tr i-n -bu tyl-
sta n n yl)h ip p u r ic Acid Am id e (8c). To a solution of com-
pound 7c (120 mg, 42.5 mmol) in dry chloroform (1.5 mL) was
added 1.5 mL of TFA, and the mixture was stirred for 30 min
at room temperature. Volatile components were removed in
vacuo to yield 5′-aminopentylmaleimide as the TFA salt. A
mixture of compound 3 (80 mg, 0.20 mmol), 5′-aminopentyl-
maleimide TFA salt (59 mg, 0.20 mmol), and N,N-diisopropyl-
ethylamine (41.7 µL, 0.24 mmol) in dry tetrahydrofuran (TFA;
7 mL) was stirred at room temperature for 1.5 h. After
filtration of the precipitate, the solvent was removed in vacuo,
and the residue was chromatographed on silica gel using a
mixture of ethyl acetate-hexane (4:1) as an eluent to produce
compound 8c (58 mg, 63%) as a colorless oil. 1H-NMR
(CDCl3): δ 0.85-1.63 (33H, m, SnBu3, (CH2)3), 3.27 (2H, q,
NHCH2), 3.51 (2H, t, NCH2), 4.21 (2H, d, NHCH2), 4.29 (1H,
q, CH), 6.26 (1H, t, NH), 6.67 (2H, s, maleimide), 7.01 (1H, t,
NH), 7.36 (1H, t, aromatic), 7.61 (1H, d, aromatic), 7.71 (1H,
d, aromatic), 7.90 (1H, t, aromatic). FAB-MS calcd for
C30H47N3O4Sn (M-): m/z 632. Found: 632.
P r ep a r a tion of [125I]-L-HML-IT-NGA. Radioiodination of
L-SHML with Na[125I]I was performed as described above using
Na[125I]I in place of Na[131I]I. Before conjugation with [125I]-
L-HML, NGA was treated with a 30 molar excess of 2-IT, as
previously reported.30 Briefly, 177 µL of freshly prepared 2-IT
(1.0 mg/mL) in well-degassed 0.16 M borate buffer (BB; pH
8.0) containing 2 mM EDTA was added to 120 µL of NGA (25
mg/mL) in the same buffer. After gentle stirring at room
temperature for 1 h, excess 2-IT was removed by the centri-
fuged column procedure as described above. The number of
thiol groups introduced by this treatment was determined with
2,2′-dithiopyridine. The filtrate solution (100 µL) was then
added to the reaction vial containing 9.8 MBq (265 µCi) of
crude [125I]-L-HML. After gentle agitation of the reaction
mixture for 1.5 h at room temperature, the radiolabeled NGA
was purified in a manner similar to [131I]-L-HML-NGA.
P r ep a r a tion of [125I]-D-HML-NGA. Radioiodination of
compound 8c with Na[125I]I and subsequent conjugation with
NGA were performed by the procedures similar to those used
for [131I]-L-HML-NGA.
Syn th esis of 3′-Iod oh ip p u r yl NE-Ma leoyl-L-lysin e.
A
mixture of N-succinimidyl 3-iodohippurate (138 mg, 0.34
mmol), the HCl salt of Nꢀ-maleoyl-L-lysine (90 mg, 0.34 mmol),
and N,N-diisopropylethylamine (62.0 µL, 0.36 mmol) in dry
acetonitrile (4 mL) was stirred at room temperature for 24 h.
After filtration, the filtrate was evaporated in vacuo, and the
residue was chromatographed on silica gel using a mixture of
chloroform-methanol-acetic acid (40:5:2) as an eluent to
produce 3′-iodohippuryl Nꢀ-maleoyl-L-lysine (145 mg, 76%) as
a colorless oil. 1H-NMR (CD3OD): δ 0.87-1.95 (6H, m, (CH2)3),
3.49 (2H, t, NCH2), 4.08 (2H, d, NHCH2), 4.37 (1H, q, CH),
6.77 (2H, s, maleimide), 7.25 (1H, t, aromatic), 7.87 (1H, d,
P r ep a r a tion of [125I]MP H-NGA. MPH was radioiodinated
by the procedures similar to those used for [125I]-D-HML-NGA
except that 0.60 mg/mL compound 8b dissolved in methanol
containing 1% acetic acid was used in place of D-SHML.
Radiochemical purity of [125I]MPH was determined by TLC