Serotonin Transporter Imaging Agent
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21 5263
J ) 8.0 Hz, 1H), 4.65 (broad s, 2H), 3.60 (s, 2H), 2.32 (s, 6H);
HRMS (ES+) calcd for C15H18N2OI (MH+) 369.0464, found
369.0462.
15 (26%): 1H NMR (200 MHz, CDCl3): δ 0.92 (t, J ) 7.8 Hz,
9H), 1.26-2.03 (m, 18H), 2.26 (s, 6H), 3.49 (s, 2H), 3.96 (s,
2H), 6.69 (s, 1H), 6.78 (d, J ) 7.1 Hz, 1H), 7.02 (d, J ) 7.0 Hz,
1H), 7.05-7.20 (m, 4H).
([125I]/[123I]2-(2-(Dim e t h yla m in om e t h yl)p h e n oxy)-5-
iod op h en yla m in e, [125I]/[123I]4. The tin compound 10 (50 µg
in 50 µL of ethanol), [I-125]/[I-123]sodium iodide, and 1 N HCl
(100 µL) were placed in a sealed vial. To this mixture was
added 100 µL of H2O2 (3% solution in water) via a syringe at
room temperature. The iodination reaction was terminated
after 10 min by an addition of saturated NaHSO3 and the
resulting solution was neutralized by adding a saturated
NaHCO3 solution. The mixture was purified by a C4 mini-
column method (radiochemical yield > 90%). The purity was
determined by HPLC (PRP-1 column, acetonitrile/5 mM 3,3-
dimethylglutaric acid, pH 7.0: 90/10, flow rate ) 1 mL/min;
retention time ) 9.3 min). Radiochemical purity was 99%.
2-(2-(Dim eth yla m in om eth yl)ben zyl)-5-[125I]iod op h en -
yla m in e ([125I]]5). The tin compound 17 (50 µg in 50 µL of
ethanol), [I-125]/[I-123]sodium iodide, and 1 N HCl (100 µL)
were placed in a sealed vial. To this mixture was added 100
µL of H2O2 (3% solution in water) via a syringe at room
temperature. The iodination reaction was terminated after 10
min by an addition of saturated NaHSO3. The resulting
solution was neutralized by adding a saturated NaHCO3
solution. The mixture was loaded on a C4 minicolumn. The
cartridge was first washed with water. The desired product
was washed off the cartridge by ethanol. Radiochemical yield
>90%. Radiochemical purity after the purification was 99%.
(4-Br om o-2-n it r op h e n yl)(2-d im e t h yla m in om e t h yl-
p h en yl)m eth a n ol (13a ). A solution of 2-bromo-N,N-dimeth-
ylbenzylamine (11) (268 mg, 1.25 mmol) in THF (5 mL) was
added to a solution of BuLi (1.7 mL, 1.6 M in THF, 2.7 mmol)
in THF (5 mL) dropwise at RT. The mixture was stirred at
RT for 10 min. The resulting lithium species was added to a
solution of 4-bromo-2-nitrobenzaldehyde19 (12a ) (311 mg, 1.35
mmol) in THF (5 mL) dropwise at -78 °C. The mixture was
stirred at RT overnight. Ice-water was added, and the mixture
was extracted with CH2Cl2. The organic phase was dried under
Na2SO4 and filtered, and the filtrate was concentrated to give
crude product which was purified by PTLC (hexane/ethyl
acetate (1/1)) to give 60 mg of product (12%): 1H NMR (200
MHz, CDCl3) δ 2.28 (s, 6H), 3.11 (d, J ) 12.5 Hz, 1H), 4.14 (d,
J ) 12.5 Hz, 1H), 6.56 (s, 1H), 6.58 (d, J ) 8.8 Hz, 1H), 7.07-
7.24 (m, 3H), 7.86 (d,d, J ) 8.5, 2.2 Hz, 1H), 8.05 (d, J ) 8.5
Hz, 1H), 8.16 (d, J ) 2.0 Hz, 1H).
(2-Dim eth ylam in om eth ylph en yl)(4-iodo-2-n itr oph en yl)-
m eth a n ol (13b). The same procedure described above for
preparation of 13a was employed to prepare 13b. Product 13b
was obtained (68 mg, 16%) from 4-iodo-2-nitrobenzaldehyde
(12b) (277 mg, 1 mmol): 1H NMR (200 MHz, CDCl3) δ 2.32 (s,
6H), 3.15 (d, J ) 12.5 Hz, 1H), 4.31 (d, J ) 12.5 Hz, 1H), 6.03
(s, 1H), 6.41 (d, J ) 7.2 Hz, 1H), 7.09-7.29 (m, 3H), 8.02 (d, J
) 8.5, Hz, 1H), 8.39 (d,d, J ) 6.8, 2.3 Hz, 1H), 8.66 (d, J ) 2.3
Hz, 1H).
5-Br om o-2-(2-(d im e t h yla m in om e t h yl)b e n zyl)p h e n -
yla m in e (14). To a solution of nitro compound 13a (67 mg,
0.18 mmol) in THF (5 mL) was added a solution of BH3 (2 mL,
1 M in THF) dropwise at RT. The resulting mixture was stirred
under reflux overnight. Water was added by caution to destroy
the excess of BH3. The solvent was removed on the Rotavapor.
HCl solution (20 mL, 2% solution) was added, and the mixture
was refluxed for 30 min NH4OH (concentrated) was added
after cooling to adjust the pH of the solution to basic. The
mixture was extracted with mixed solvent (CH2Cl2/MeOH (95/
5)). The organic phase was dried under Na2SO4 and filtered,
and the filtrate was concentrated to give crude product which
was purified by PTLC (hexane/ethyl acetate (1/1)) to give 34
mg of product (58%): 1H NMR (200 MHz, CDCl3) δ 2.25(s, 6H),
3.47 (s, 2H), 3.90 (s, 2H), 6.71 (d, J ) 2.0 Hz, 1H), 6.80 (d,d, J
) 8.0, 2.0 Hz, 1H), 6.96 (d, J ) 7.9 Hz, 1H), 7.03-7.08 (m,
1H), 7.12-7.20 (m, 3H).
2-(2-(Dim e t h yla m in om e t h yl)b e n zyl)-5-iod op h e n yl-
a m in e (5). The same procedure described above for prepara-
tion of 14 was employed to prepare 5. Product 5 (13 mg, 21%)
was obtained from 13b (68 mg, 0.16 mmol). The HCl salt was
prepared for analytical purposes: 1H NMR (200 MHz, CDCl3)
δ 2.23 (s, 6H), 3.34 (s, 2H), 4.00 (s, 2H), 6.54 (d,d, J ) 8.2, 2.3
Hz, 1H), 6.62 (d, J ) 8.2 Hz, 1H), 6.97 (m, 1H), 7.13-7.25 (m,
3H), 7.32-7.40 (m, 1H); HRMS (CI+) calcd for C16H19N2I (M+)
366.0593, found 366.0587.
P r ep a r a tion of Mem br a n e Hom ogen a tes. LLC-PK1 cells
from pig kidney permanently expressing DAT, NET and SERT,
respectively (LLC-DAT, LLC-NET and LLC-SERT) were kindly
provided by Dr. G. Rudnick at Yale University. These cells
were grown to confluence as a monolayer on 15 cm Petri dish
as reported previously.25 Cells were then washed once with
phosphate buffer saline (w/Ca2+ and Mg2+) (Bio-Whittaker) and
harvested with 10 mL of PBS by scraping. The cell suspensions
were homogenized on ice for 20 strokes and centrifuged at
17 000 rpm for 20 min at 4 °C. The pellets were resuspended
in PBS, frozen quickly in liquid N2 and stored in a -70 °C
freezer.
Bin d in g Assa ys. Binding assays were performed in a final
volume of 0.2 mL. Aliquots of membrane suspensions (100 µL,
corresponding to 30-40 µg of protein) were mixed with 50 mM
Tris-HCl, pH 7.4, 120 mM NaCl and 0.1% BSA (all from Sigma,
St. Louis, MO), 0.4 nM [125I]IPT or [125I]IDAM, and 8-10
concentrations (10-10 to 10-5 M) of competing drugs. Nonspe-
cific binding was defined with 10 µM CFT for [125I]IPT assays
and 1 µM IDAM for [125I]IDAM assays. Incubation was carried
out for 1 h at room temperature, and the bound ligand was
collected on glass fiber filters (Schleicher & Schuell No. 25,
Keene, NH) presoaked with 1% polyethylenimine (Sigma, St.
Louis, MO) and counted in a gamma counter (Packard 5000).
Results of competition experiments were subjected to nonlinear
regression analysis using EBDA (Elsevier-BIOSOFT, Cam-
bridge, UK).
P a r tition Coefficien ts. Partition coefficients were mea-
sured by mixing [125I] compound with 3 g each of 1-octanol and
buffer (pH 7.4, 0.1 M phosphate) in a test tube. The test tube
was vortexed for 3 min at room temperature, then centrifuged
for 5 min Two weighed samples (2 g each) from the 1-octanol
and buffer layers were counted in a well counter. The partition
coefficient was determined by calculating the ratio of cpm/g
of 1-octanol to that of buffer. Samples from the 1-octanol layer
were repartitioned until consistent partition coefficient values
were obtained. The measurement was repeated three times.
Biod istr ibu tion in Ra ts. Three rats per group were used
for each biodistribution study. While under ether anesthesia,
0.2 mL of a saline solution containing 10 µCi of radioactive
tracer was injected into the femoral vein. The rats were
sacrificed at the time indicated by cardiac excision while under
ether anesthesia. Organs of interest were removed and weighed,
and the radioactivity was counted. The percent dose per organ
was calculated by a comparison of the tissue counts to counts
of 1% of the initial dose (100 times diluted aliquots of the
injected material) measured at the same time.
Regional brain distribution in rats was measured after an
iv injection of the radioactive tracer. Samples from different
brain regions: cortex (frontal plus occipital), striatum, hip-
pocampus, cerebellum and hypothalamus were dissected,
weighed and counted. The percentage dose/g of each sample
was calculated by comparing sample counts with the counts
of the diluted initial dose described above. The specific binding
ratio in each region was obtained by dividing the %dose/gram
values between region by that in the cerebellum ([region-
2-(2-(Dim et h yla m in om et h yl)b en zyl)-5-(t r ib u t ylt in )-
p h en yla m in e (15). The mixture of bromide 14 (14 mg, 0.04
mmol), bistributyltin (0.1 mL), (PPh3)4Pd (10 mg) and triethyl-
amine (0.1 mL) in dioxane (2 mL) was stirred at 90 °C
overnight. Solvent was removed, and the residue was purified
by PTLC (hexane/ethyl acetate (1/1)) to give 6 mg of product
Kung, Hank F.
