Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6721
NMR (D2O) δ 1.47 (3H, s), 6.39 (1H, d, J ) 14 Hz), 6.75 (1H, d,
J ) 14 Hz). Anal. (C5H8INO2) C, H, N.
Acknowledgment. We are grateful to Dr. Bing Wang of
the NMR Center of Emory University for his assistance with
NMR studies and Zhaobin Zhang and Eugene Malveaux for
their assistance with the in vivo studies. We acknowledge the
use of Shared Instrumentation provided by grants from the NIH
and the NMP for the mass spectroscopy data.
(S)- and (R)-2-[N-(tert-Butoxycarbonyl)amino]-2-methyl-4-
trimethylstannyl-3-(E)-butenoic Acid Methyl Ester (6). The
protected amino acid (S)- or (R)-4 (40 mg, 0.113 mmol) was
dissolved in 2 mL of tetrahydrofuran and degassed with argon for
15 min. Hexamethylditin (74 mg, 0.225 mmol) and tetrakis(triph-
enylphosphine)palladium(0) (13 mg, 0.011 mmol) were added, and
the solution was degassed for another 15 min. The mixture was
heated to 50–60 °C for 2 h and cooled to room temperature. The
solvent was removed under reduced pressure, and the crude product
was purified by silica gel chromatography (ethyl acetate/hexane/
triethylamine ) 1:7:0.1%) to yield 6 as oil. (S)-2-[N-(tert-
butoxycarbonyl)amino]-2-methyl-4-trimethylstannyl-3-(E)-buteno-
References
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1
ic acid methyl ester ((S)-6): 30.9 mg (70%); H NMR (CDCl3) δ
0.151 (9H, s), 1.44 (9H, s), 1.58 (3H, s), 3.75 (3H, s), 5.18 (1H,
broad s), 6.05, 6.10 (1H, d, J ) 18.8 Hz), 6.28, 6.33 (1H, d, J )
18.8 Hz). HRMS, m/z, calcd for C14H28O4N120Sn [M + H]+,
394.103 48; found, 394.103 71. (R)-2-[N-(tert-butoxycarbonyl)amino]-
2-methyl-4-trimethylstannyl-3-(E)-butenoic acid methyl ester ((R)-
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1
6): 22.8 mg (52%); H NMR (CDCl3) δ 0.149 (9H, s), 1.43 (9H,
s), 1.60 (3H, s), 3.75 (3H, s), 5.19 (1H, broad s), 6.05, 6.10 (1H,
d, J ) 18.8 Hz), 6.28, 6.33 (1H, d, J ) 18.8 Hz). HRMS, m/z,
calcd for C14H28O4N120Sn [M + H]+, 394.103 48; found, 394.104 72.
Radiosynthesis. The preparation of radioiodinated (S)-5 and
(R)-5 was carried out with no-carrier-added [123I]sodium iodide or
[
131I]sodium iodide and the appropriate stannyl precursor according
to the method of Van Dort.17 Typically, 80 µg (204 nmol) of (S)-6
or (R)-6 in 200 µL of ethanol was added to [123I]NaI (in 20–50 µL
of 0.1 N NaOH, 1–3 mCi) or [131I]NaI (in 2–3 µL of 0.1 N NaOH,
1–3 mCi) in a 1.5 mL Wheaton V-vial. Then 50 µL of 0.4 N HCl
(aq) and 50 µL of 3% H2O2 (aq, w/v) were added to the V-vial.
The vial was shaken occasionally at room temperature for 20 min.
The reaction was quenched by the addition of aqueous sodium
metabisulfite (50 µL, 250 mg/mL) followed by the addition of
saturated aqueous sodium bicarbonate (500 µL). After extraction
with dichloromethane (3 × 0.5 mL), the combined organic layers
were dried by blowing argon through at 65 °C. The residue was
cooled to room temperature, 400 µL of 4 N HCl was added, and
the mixture was heated at 110 °C for 20 min. The product (S)- or
(R)-[123/131I]5 was obtained by purification through a 7 mm × 120
mm column of ion-retardation resin (AG11A8 50–100 mesh) in
series with an Alumina N SepPak and a C-18 SepPak eluted with
saline (0.9% sodium chloride in water). The eluting fractions
containing radioactivity were collected and used in cell and rodent
studies. The identity of the radiolabeled product was confirmed by
comparing the Rf of the radioactive product visualized with
radiometric TLC with the Rf of the authentic nonradioactive form
of the compound visualized with ninhydrin stain (Rf ) 0.6, 4:1:1
CH3CN/H2O/MeOH). In all radiosyntheses, the only peak present
on radiometric TLC analysis corresponded to (S)-5 or (R)-5, and
the radiochemical purity of the product exceeded 99%. The isolated
radiochemical yields were determined using a dose calibrator.
Amino Acid Uptake and Inhibition Assays. The amino acid
uptake assays were performed with cultured rat 9L gliosarcoma
cells as described previously.12,13 The data from these studies were
normalized as percent uptake relative to standard per 1 × 106 cells.
The cell uptake data were analyzed with one- and two-way
ANOVAs using GraphPad Prism software (San Diego, CA).
Rodent Biodistribution in Tumor Bearing Rats. Cultured 9L
cells were implanted into the brains of male Fischer 344 rats
(125–175 g) for biodistribution studies as described previously.12,13,18
A total of 15 animals were used in the (S)-[ 123I]5 study (5 at each
time point), and 13 animals were used in the (R)-[ 131I]5 study (5
at 30 min, 4 at both 60 and 120 min) in separate biodistribution
studies. Intravenous doses of 15–30 µCi (S)-[123I]5 or (R)-[131I]5
were administered in 0.1–0.3 mL of sterile saline. The biodistri-
bution studies were performed at 10–12 days after implantation,
and the data were analyzed with two-way ANOVAs using GraphPad
Prism software.
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