M. B. Mallia et al. / Bioorg. Med. Chem. 14 (2006) 7666–7670
7669
1580 dual pump HPLC system, Japan, with a JASCO
1575 tunable absorption detector and a radiometric
detector system, using a C18 reversed phase HiQ Sil
(5 lm, 4 · 250 mm) column. The solvents used for
HPLC were filtered through Millipore filter paper and
dissolved in 1 mL of methanol and 7 mL of 6 N HCl.
The reaction mixture was then stirred at room tempera-
ture for 2 h, after which the solvent was removed under
vacuum to yield the iminodiacetic acid derivative 3
1
(183 mg, 95%); MS (ESI) m/z: 286.9 (M++1); H NMR
1
contained 0.1% trifluoroacetic acid. H NMR spectra
(D2O, d ppm) 2.3 (s, 3H, 5-nitroimidazole-CH3); 3.6 (t,
2H, J = 7.2 Hz, nitroimidazole-CH2CH2N); 4.1 (s, 4H,
[(HO2C–CH2)2N–]); 4.4 (t, 2H, J = 7.2 Hz, nitroimidaz-
ole-CH2CH2N); 8.06 (s, 1H, imidazole H). C, H, N: Ob-
served (calcd) 41.91 (41.96); 5.21 (4.93); 19.63 (19.57).
were recorded on a 300 MHz Varian VXR 300S spectro-
photometer, USA. The mass spectra were recorded on a
QTOF Micromass instrument using electron spray ioni-
zation (ESI) in positive mode. Elemental analysis was
performed on C, H, N, S elemental analyzer, Thermo-
finnigan, Flash EA 1112 series.
4.2. Radiolabeling
4.1. Synthesis
4.2.1. Preparation of [99mTc(CO)3(H2O)3]+ core. A typi-
cal procedure involves dissolution of NaBH4 (5.5 mg),
Na2CO3 (4 mg), and Na/K tartrate (15 mg) in 0.5 mL
double-distilled water in a glass serum vial. The vial
was sealed and carbon monoxide gas was purged
through the solution for 5 min. After the addition of
1 mL of the generator eluate containing 37–74 MBq of
99mTcO4À, the vial was heated at 80 ꢁC for 20 min. After
cooling of the vial and re-equilibration with atmospheric
pressure, pH of the reaction mixture was adjusted to 7
using a mixture of 0.5 M phosphate buffer (pH
7.5):1 M HCl (1:3). The intermediate thus prepared
was characterized by HPLC.
4.1.1. Synthesis of N,N-bis[(tert-butoxycarbonyl)methyl]-
2-bromoethylamine (1). To tert-butylbromoacetate
(3.3 g, 17.3 mmol) dissolved in 15 mL DMF was added
powdered KHCO3 (1.92 g, 19.2 mmol). The suspension
was cooled to 0 ꢁC, and ethanolamine (0.46 mL,
7.5 mmol) was added dropwise over a period of five min-
utes. The reaction mixture was stirred at 0 ꢁC for 30 min
and then for 24 h at room temperature. The semisolid
residue obtained after removing the solvent was dis-
solved in NaHCO3 solution and extracted with ether.
The ether layer was then washed with brine, dried, and
evaporated to give the crude product (ꢀ1.8 g, 83%) as
oil. To the crude dialkylated product dissolved in
20 mL of dichloromethane, Ph3P (2.2 g, 8.45 mmol)
was added, the solution was cooled to 0 ꢁC, and solid
NBS (1.5 g, 8.45 mmol) was added over a period of
5 min. The reaction was continued for a period of 2 h
after which the solvent was removed to yield a semisolid
residue. The semisolid residue was triturated with ether
and the resulting solid was separated. The ether layer
was concentrated and passed through a small column
of silica using ether as the eluant. Evaporation of ether
yielded a yellow oil, which was then purified by silica
gel chromatography (1.7 g, 80%); Rf 0.28 (5/1 hexane/
ether); 1H NMR (CDCl3, d ppm) 1.46 (s, 18H, tert-butyl
CH3); 3.1 (t, 2H, J = 7.5 Hz, BrCH2CH2N); 3.4 (t, 2H,
J = 7.5 Hz, BrCH2CH2N); 3.5 [s, 4H, (tert-butoxycar-
bonyl-CH2)2N]. C, H, N: Observed (calcd) 47.81
(47.73), 7.97 (7.44), 3.96 (3.98).
4.2.2. Labeling of 3 with [99mTc(CO)3(H2O)3]+. Under
optimized conditions, 0.5 mL of freshly prepared
[
99mTc(CO)3(H2O)3]+ precursor was added to 0.5 mL
of double distilled water containing 0.5–1 mg of the li-
gand 3. The pH of the mixture was maintained between
7 and 8. The mixture was vortexed for a minute and then
incubated at room temperature for 30 min. The complex
was characterized by HPLC.
4.3. Quality control techniques
4.3.1. Serum stability studies. Fifty microliters of the la-
beled compound was added to 0.5 mL of human serum
and this mixture was incubated at 37 ꢁC for up to 3 h.
Aliquots were taken at 1 and 3 h, and analyzed by
HPLC to assess the stability of the complex in serum.
4.3.2. HPLC. The radiochemical purity of the prepared
4.1.2. Synthesis of iminodiacetic acid derivative of 2-
methyl-5-nitroimidazole (3). To 2-methyl-5-nitroimidaz-
ole (100 mg, 0.79 mmol) in 15 mL of acetonitrile was add-
ed anhydrous K2CO3 and the suspension stirred for
15 min. N,N-Bis[(tert-butoxycarbonyl)methyl]-2-bromo-
ethylamine] (277 mg, 0.79 mmol) was added and the reac-
tion mixture refluxed for 12 h following which it was
cooled and filtered free of potassium carbonate. The pure
compound, N,N-bis[(tert-butoxycarbonyl)methyl]-2-(2-
methyl-5-nitroimidazolyl)ethylamine (2), was then ob-
tained by silica gel chromatography using chloroform as
[
99mTc(CO)3(H2O)3]+ core as well as the complex was as-
sessed by HPLC using acetonitrile/water, binary solvent
system, and a gradient elution program (Solvent A–aceto-
nitrile, Solvent B–water; 0 min–10% A, 28 min–90% A,
30 min–90% A). The flow rate was maintained at 1 mL/
min.
4.3.3. Partition coefficient. About 100 lL of the labeled
compound was mixed with 0.9 mL water and 1 mL of oct-
anol on a vortex mixer for about 1 min. The two phases
were allowed to separate. Equal aliquots of the organic
and aqueous layers were withdrawn and measured for
radioactivity to determine the partition coefficient.
1
the eluant (267 mg, 85%). H NMR (CDCl3, d ppm)
1.43 (s, 18H, [(H9C4O2C–CH2)2N–]); 2.43 (s, 3H, 5-nitro-
imidazole-CH3); 3.04 (t, 2H, J = 6 Hz, nitroimidazole-
CH2CH2N); 3.35 (s, 4H, [(H9C4O2C–CH2)2N–]); 4.06 (t,
2H, J = 6 Hz, nitroimidazole-CH2CH2N); 7.96 (s, 1H,
imidazole H). C, H, N: Observed (calcd) 54.29 (54.26);
8.11 (7.59); 14.15 (14.06). The purified ester 2 was then
4.4. Biodistribution studies
Swiss mice were used to perform the in vivo uptake as
well as the distribution study of the labeled complex.