High specific activity tritium-labeled N-(2-methoxybenzyl)-2,5-dimethoxy-4-iodophenethylamine (INBMeO): A high-affinity 5-HT2A receptor-selective agonist radioligand

Article abstract of DOI:10.1016/j.bmc.2008.04.050

The title compound ([³H]INBMeO) was prepared by an O,O-dimethylation reaction of a t-BOC protected diphenolic precursor using no carrier added tritiated iodomethane in DMF with K₂CO₃. Removal of the t-BOC protecting group and purification by HPLC afforded an overall yield of 43%, with a radiochemical purity of 99% and specific activity of 164 Ci/mmol. The new radioligand was suitable for labeling human 5-HT_2A receptors in two heterologous cell lines and had about 20-fold higher affinity than [³H]ketanserin.

Full text of DOI:10.1016/j.bmc.2008.04.050

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 6116–6123
High specific activity tritium-labeled N-(2-methoxybenzyl)-
2,5-dimethoxy-4-iodophenethylamine (INBMeO): A high-affinity
5-HT2A receptor-selective agonist radioligand
David E. Nichols,a,* Stewart P. Frescas,a Benjamin R. Chemel,a
Kenneth S. Rehder,b Desong Zhongb and Anita H. Lewinb
aDepartment of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmaceutical Sciences,
Purdue University, 1333 Robert E. Heine Pharmacy Building, West Lafayette, IN 47907, USA
bCenter for Organic and Medicinal Chemistry, Research Triangle Institute, Durham, NC 27709, USA
Received 28 February 2008; revised 16 April 2008; accepted 17 April 2008
Available online 25 April 2008
Abstract—The title compound ([3H]INBMeO) was prepared by an O,O-dimethylation reaction of a t-BOC protected diphenolic pre-
cursor using no carrier added tritiated iodomethane in DMF with K2CO3. Removal of the t-BOC protecting group and purification
by HPLC afforded an overall yield of 43%, with a radiochemical purity of 99% and specific activity of 164 Ci/mmol. The new radi-
oligand was suitable for labeling human 5-HT2A receptors in two heterologous cell lines and had about 20-fold higher affinity than
[3H]ketanserin.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
available a radiolabeled agonist ligand. Furthermore, it
is typical that agonist ligands label only a high-affinity
subset of the total receptor population, so any useful
radioligand should have high specific activity. One
ligand that is widely used for this purpose is [125I]-
The serotonin 2A (5-HT2A) receptor is a member of the
G protein-coupled receptor (GPCR) superfamily. Of the
seven different subtypes of serotonin receptors, the 5-
HT2A receptor has been closely linked to complex
behaviors. It is expressed at high density within the fron-
tal cortex, but at lower levels in many other brain
areas1–3 It has been implicated in working memory, cog-
nitive processes, affective disorders such as schizophre-
nia, and mediates the primary effects of hallucinogenic
drugs4–7 In addition, many peripheral tissues also ex-
press the 5-HT2A receptor. For example, 5-HT2A recep-
tors are involved in platelet function, and induce
contraction within the vasculature8,9 Thus, radiolabeled
agonist and antagonist ligands are of high importance in
the pharmacology community to measure density and
changes in density of these receptors.
2,5-dimethoxy-4-iodoamphetamine
([125I]DOI)11,12
Although this ligand does not discriminate between
the 5-HT2A, 5-HT2B, and 5-HT2C receptors13 the high
specific activity of 125I makes it very useful for rapid as-
says of low density receptor populations. This radioli-
gand is presently commercially available, but major
drawbacks to the use of [125I]DOI are its short half-life
and high cost. In our own laboratory, for example,
experiments have often been postponed until the need
for results is sufficiently compelling, or until we have de-
layed a sufficient number of experiments to justify the
cost of [125I]DOI. Therefore, a high specific activity radi-
oligand with a longer half-life is very desirable.
Recently, we reported on a series of high-affinity N-ben-
zyl phenethylamine ligands for the 5-HT2A receptor14
The most potent of the ones reported, N-(2-methoxy-
benzyl)-2,5-dimethoxy-4-iodophenethylamine (INB-
MeO, 1), had exceptionally high affinity for the
5-HT2A receptor, and was obviously suitable for the
introduction of 3–9 tritium atoms by O-methylation.
Based on preliminary work, we had concluded that six
tritium atoms should provide sufficient radioactivity
Unfortunately, few ligands are highly specific for 5-
HT2A receptors, and those that are, such as
M10090710 antagonists. It is desirable, therefore, to have
Keywords: Radioligand; 5-HT2A receptor; Receptor binding.
*
Corresponding author. Tel.: +1 765 494 1461; fax: +1 765 494
1414; e-mail: drdave@pharmacy.purdue.edu
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.04.050

D. E. Nichols et al. / Bioorg. Med. Chem. 16 (2008) 6116–6123
6117
for a useful ligand. This report describes the synthesis and
preliminary characterization of N-(2-[3H-OCH3]benzyl)-
2-[(3H-OCH3]-5-methoxy-4-iodophenyl)ethylamine (1).
to distinguish between 1 and 9–14. Table 1 shows the
retention parameters for the desired product 1, the N-
Boc protected bisphenol 9, the O,O-dimethylated-N-
Boc protected intermediate 10, the partially O-methyl-
ated N-Boc protected potential byproducts 11 and 12,
and the deprotected analogs 13 and 14.
In order to prepare 1 with six tritium atoms it was
planned to bis-methylate the diphenolic precursor 9
using no carrier-added tritiated iodomethane. The over-
all synthesis of this precursor was carried out as shown
in Scheme 1.
Conditions for the deprotection of 10 and for isolation
of pure INBMeO (1) were also developed. Thus, treat-
ment of a mixture comprised of 10 and small amounts
of 11 and 12, with trifluoroacetic acid (TFA) for
15 min provided 1, along with 13 and 14 (derived from
11 and 12). Base treatment failed to remove the phenols
13 and 14. Surprisingly, INBMeO was found to decom-
pose in the presence of 1 N sodium hydroxide.
H3CO
OCH3
NH
I
Unfortunately, when the O-methylation reaction condi-
tions were applied on the scale that would be used in the
radiosynthesis, only minute amounts of 10a, 11a, and
12a were produced. Further experiments revealed that
the starting material 9 and the monomethylated inter-
mediate 11a decomposed when a solution of 9–12a in
DMF was treated with sodium hydride for 15 h at room
temperature. A similar experiment using potassium car-
bonate was more promising, showing the disappearance
of intermediate 11a only. Therefore, the methylation
reaction using potassium carbonate to replace sodium
hydride was examined. These conditions gave 10a,
accompanied by small amounts of 11a and 12a, and
OCH3
1
Preliminary studies of the methylation reaction, utilizing
unlabeled iodomethane, demonstrated that treatment of
the N-tert-BOC protected bisphenol 9 with sodium hy-
dride in DMF, followed by the addition of a slight ex-
cess of iodomethane, afforded intermediate 10a
(Scheme 2). Partially methylated intermediates were
likely by-products, so HPLC conditions were developed
O
O
O
CHO
NO2
NH2
c.
a.
b.
OCH3
OCH3
OCH3
2
3
O
CF3
O
CF3
O
O
O
NH
NH2
NH
d.
e.
f.
I
I
OCH3
OCH3
OCH3
5
6
4
O
HO
HO
N
O
OH
OH
NH
NH
g.
h.
BOC
I
I
I
OCH3
OCH3
OCH3
7
8
9
Scheme 1. Reagents and conditions: (a) EtNO2, AcOH, reflux; (b) LiAlH4; (c) CF3COOEt, heat; (d) ICl, AcOH; (e) HCl; (f) o-isopropyloxybenz-
aldehyde, NaCNBH3; (g) BCl3; (h) (tert-BOC)2O.

6118
D. E. Nichols et al. / Bioorg. Med. Chem. 16 (2008) 6116–6123
HO
OH
N
BOC
I
OCH3
9
NaH
CX3I: a X = H, b X = T
X3CO
HO
N
X3CO
N
OCX3
OCX3
OH
N
BOC
BOC
BOC
I
I
I
OCH3
OCH3
OCH3
10
11
12
TFA
TFA
TFA
X3CO
HO
NH
X3CO
NH
OCX3
OCX3
OH
NH
I
I
I
OCH3
OCH3
OCH3
1
13
14
Scheme 2. Methylation and deprotection of precursor 9 to produce 1, as well as partially methylated byproducts 13 and 14.
Table 1. HPLC Retention times for 1 and byproducts obtained in the
radiochemical synthesis
Compound
activity, which was found to be 164 1 Ci/mmol. Be-
cause high specific activity compounds are known to
undergo radiolytically promoted decomposition, the
purity of [3H]INBMeO was monitored over a nine-
month period. As shown in Figure 1, [3H]INBMeO is
usable for a minimum of 60 days (ꢀ95% purity) and
we have carried out successful experiments beyond
150 days (ꢀ90% purity).
9
10
11
12
13
14
INBMeO
Retention time 26.3 31.7 27.7 27.7 14.7 15.5 17.5
(min)
unreacted 9. Deprotection gave INBMeO (1b) in 54%
yield.
We then used the new radioligand [3H]INBMeO 1b to
carry out receptor saturation isotherm experiments in
two cells lines stably expressing human 5-HT2A recep-
tors. For a comparison, we used [3H]ketanserin (67 Ci/
mmol, Perkin-Elmer). The results of that comparison
are presented in Figure 2 and Table 2. The affinity of
[3H]INBMeO was about 20-fold higher than that of
ketanserin, and it labeled about 60% of the sites labeled
by ketanserin. Use of [3H]INBMeO as the radioligand in
competition binding assays with four ligands known to
have high affinity for the 5-HT2A receptor demonstrated
that all of the ligands had comparable affinity, whether
[3H]ketanserin or [3H]INBMeO was used (Table 3).
A similar reaction mixture was produced in the master
radiosynthesis. Thus, treatment of a DMF solution of
the bisphenol 9 with an 11-fold excess of iodomethane
(100 mCi, specific activity 80 Ci/mmol) in the presence
of potassium carbonate gave primarily 10b
(10.6 mCi), accompanied by small amounts of 9, 11b,
and 12b. Because we had found that 10a was more sta-
ble than 1a, only a portion of 10b (3.7 mCi) was depro-
tected. The product was purified by preparative HPLC
to afford 1.8 mCi (49% yield) of [3H]INBMeO (1b) of
nominal specific activity 160 Ci/mmol. A portion of
10b was used to determine experimentally the specific

D. E. Nichols et al. / Bioorg. Med. Chem. 16 (2008) 6116–6123
6119
Table 3. A comparison of competition binding for selected 5-HT2A
ligands using [3H]ketanserin and [3H]INBMeO
100
95
90
85
80
75
70
Displacing liganda
A20 Cells
[3H]ketb [3H]INBc [3H]ketb
120 21 160 17 130 65 150 20
Hh2A Cells
[3H]INBc
y = -0.0617x + 99.624
R2 = 0.954
5-HT
Cinanserin
DOI
LSD
17 2.8
29 5.2
24 2.4
28
13 5.1
13 2.0
2.6 0.9 4.0 0.5 3.1 0.7 3.5 0.4
22 5.9
14 1.2
8
Ki values given are nM SEM.
a (n = 3).
b [3H]ketanserin.
c [3H]INBMeO.
0
50
100
150
200
250
300
Days stored at -70 ºC
Figure 1. Stability of [3H]INBMeO when stored at À70 ꢁC in EtOH.
2. Conclusions
We have prepared a new high specific activity tritiated
ligand for the 5-HT2A/2C receptors. It appears to have
utility similar to ketanserin, but has about 20-fold high-
er affinity for 5-HT2A/2C receptors. It may be an eco-
nomical alternative to the use of [125I]DOI as a
radioligand for these receptors.
3. Experimental
3.1. Chemistry—general
The chemicals, reagents, and solvents used in the synthe-
sis were inspected and released for use based on visual
inspection and conformance of the individual lot with
the manufacturer’s Certificate of Analysis. Nuclear mag-
netic resonance (NMR) spectra were determined on a
Bruker Avance 300 MHz NMR spectrometer. HPLC
analyses were performed on a dual pump system (Ranin
HPXL solvent delivery system) equipped with a Rheo-
dyne injector, a Varian ProStar 325 UV–vis detector,
and a b-RAM radiodetector, (IN/US Systems, Inc.) con-
nected after the UV detector, controlled by Varian Star
Workstation software. The columns and solvent systems
are given below. The water used in HPLC solvent sys-
tems was obtained from a Millipore Milli-Q Plus Ul-
tra-Pure Water System.
Radioactive samples were counted on Tri-Carb Liquid
Scintillation Analyzer (Packard Bioscience Company,
model: 2100TR) using IN-FLOW 3 as liquid scintilla-
tion counting cocktail (IN/US Systems, Inc.).
Figure 2. Representative receptor isotherm using (A) [3H]ketanserin
and (B) [3H]INBMeo to label 5-HT2A receptors in Hh2A (HEK) and
A20 (A549) cells.
HPLC conditions for analysis: Phenomenex Synergy
Fusion-RP column (3 · 150 mm, 4 lm), solvent A:
[20 mM NaH2PO4–H2O], solvent B: CH3CN, gradient,
0–30 min, solvent B: 20–90%; 30–40 min, solvent B:
90%; flow rate: 0.6 mL/min, UV detector wavelength:
210 and 215 nm, b-RAM detector (for analyzing radio-
chemical purity of samples).
Table 2. A comparison of receptor saturation isotherm data for
[3H]ketanserin and [3H]INBMeO
Cells
[3H]ketanserin
[3H]INBMeO
Kd
Bmax
Kd
Bmax
(nM)
(fmol/mg)
(nM)
(fmol/mg)
A20
Hh2A
2.2 0.3
2.7 0.2
890 160
3260 360
0.097 0.02
0.15 0.01
480 70
2210 100
HPLC conditions for purification of the final radioligand:
Phenomenex Synergy Polar-RP column (3 · 150 mm,
4 lm), solvent A: 80% [20 mM NaH2PO4–H2O]/20%
EtOH, solvent B: EtOH, gradient, 0–30 min, solvent B:
30–80%; 30–40 min, solvent B: 80%; flow rate: 0.4 mL/
min, UV detector wavelength: 210 and 215 nm.
A20cells n = 10, Hh2A cells n = 4.
Curiously, although INBMeO is an agonist, it labeled
approximately 60% of the sites that were labeled by
ketanserin.

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D. E. Nichols et al. / Bioorg. Med. Chem. 16 (2008) 6116–6123
3.1.1. 2-(2-Isopropoxy-5-methoxyphenyl)nitroethene (2).
A solution of 2-isopropoxy-5-methoxybenzaldehyde15,16
(15.0 g, 72 mmol) and n-butylamine (6.84 g, 94 mmol) in
200 mL of toluene was heated at reflux for 45 min with
H2O removal using a Dean-Stark apparatus. The sol-
vents were removed by rotary evaporation and the resi-
due was dissolved in nitromethane (75 mL). This
solution was stirred efficiently while glacial acetic acid
(13.0 g, 216 mmol) was added dropwise. After stirring
at room temperature for 12 h, the solvents were removed
by rotary evaporation. The residue was dissolved in
dichloromethane, washed with H2O (2· 100 mL) and
brine (100 mL), and dried over MgSO4. After filtration
to remove the drying agent, the filtrate was concentrated
by rotary evaporation to afford a yellow solid that was
recrystallized from methanol to provide 2-(2-isopro-
poxy-5-methoxyphenyl)-nitroethene (14.22 g, 83.3%) as
(100 mL), dried over MgSO4, and filtered to remove
the drying agent. The filtrate was concentrated by rotary
evaporation to afford 23.42 g (80.3%) of amide 4 as a
waxy white solid. This material could be recrystallized
from methanol, distilled (155 ꢁC at 0.2 torr), or, most
advantageously, sublimed (30 ꢁC at 0.2 torr), resulting
in a crystalline product with a sharp melting point of
35 ꢁC. 1H NMR (300 MHz, CDCl3, d ppm): 1.32 (s,
3H), 1.34 (s, 3H), 2.87 (t, J = 6.15, 2H), 3.56 (q,
J = 8.40, 2H), 3.71 (s, 3H), 4.46 (m, 1H), 6.74 (m, 2H),
6.87 (d, 1H), 7.32 (br s, 1H), CIMS m/z 306 (M+1).
Anal. (C14H18F3NO3) C, H, N.
3.1.4. N-(2,2,2-Trifluoroacetyl)-2-(4-iodo-2-isopropoxy-
5-methoxyphenyl)ethylamine (5). Acetic anhydride
(25 mL) was added to a solution of trifluoroacetamide
4 (5.0 g, 16.4 mmol) dissolved in glacial acetic acid
(25 mL), and the reaction was cooled to 10 ꢁC. A solu-
tion of iodine monochloride (2.92 g, 18 mmol) in acetic
acid (5 mL) was added dropwise over 30 min to the vig-
orously stirred solution. The reaction mixture was stir-
red for 6 h, and then poured onto ice. Excess saturated
aqueous Na2SO3 was added to neutralize any free halo-
gen. A white precipitate was collected from the aqueous
mixture by vacuum filtration and washed on the filter
with H2O. The aqueous filtrate was extracted with
CH2Cl2 (3· 75 mL). The collected solid was dissolved
in CH2Cl2 (125 mL), combined with the organic ex-
tracts, washed with H2O (2· 75 mL) and brine
(75 mL) and dried over MgSO4. After filtration, the sol-
vent was removed by rotary evaporation and the residue
was recrystallized from methanol to provide the iodin-
ated trifluoroacetamide 5 (5.59 g, 79.1%) as a white crys-
1
bright yellow crystals, mp 65 ꢁC. H NMR (300 MHz,
CDCl3, d ppm): 1.41 (s, 6H), 3.81 (s, 3H), 4.60 (m,
1H), 6.94 (m, 3H), 7.86 (d, J = 4.29 Hz, 1H), 8.14 (d,
J = 4.29 Hz, 1H); CIMS m/z 238 (M+1). Anal.
(C12H15NO4) C, H, N.
3.1.2. 2-(2-Isopropoxy-5-methoxyphenyl)ethylamine (3).
To lithium aluminum hydride (9.07 g, 238 mmol) in a
flame-dried 2 L reaction vessel was added 600 mL of
anhydrous tetrahydrofuran. A solution of 2-(2-isopro-
poxy-5-methoxyphenyl)-nitroethene (20.0 g, 84 mmol)
in 150 mL of THF was then added dropwise over
45 min. The mixture was heated at 65 ꢁC for 4 h, after
which TLC analysis showed absence of starting mate-
rial. After cooling to room temperature the reaction
was quenched by the stepwise addition of 50% THF/
H2O (10 mL), 15% NaOH solution (10 mL), and H2O
(30 mL). The precipitated solids were removed by vac-
uum filtration, and the filtrate was reduced under vac-
uum to afford a colorless oil. The oil was dissolved in
Et2O, washed with H2O (2· 100 mL), and then extracted
into 1 M HCl (2· 200 mL). The acidic extract was
washed with Et2O (2· 75 mL), and then made strongly
basic with 5 M NaOH. The basic solution was extracted
with Et2O (3· 100 mL), the ether extract was washed
with H2O (2· 75 mL) and brine (75 mL), dried over
MgSO4, and filtered. The filtrate was concentrated by
rotary evaporation and the residue was distilled at
97 ꢁC and 0.17 torr, providing 13.32 g (75.5%) of the
1
talline solid; mp 93 ꢁC. H NMR (300 MHz, CDCl3, d
ppm): 1.32 (s, 3H), 1.34 (s, 3H), 2.86 (t, J = 5.89 Hz,
2H), 3.56 (q, J = 9.06 Hz, 2H), 3.85 (s, 3H), 4.49 (m,
1H), 6.60 (s, 1H), 7.06 (br s, 1H), 7.33 (s, 1H, ArH),
CIMS m/s 432 (M+1). Anal. (C14H17F3INO3) C, H, N.
3.1.5. 2-(4-Iodo-2-isopropoxy-5-methoxyphenyl)ethyla-
mine (6). A solution of the trifluoroacetamide 5
(2.50 g, 58 mmol) in EtOH (50 mL) was cooled to
10 ꢁC and aqueous 5 M NaOH (10 mL) was added.
After stirring overnight at RT H2O (50 mL) was added
and the solution was extracted with Et2O (3· 75 mL).
The organic extracts were washed with H2O (3·
75 mL) and brine (75 mL), dried with MgSO4, and fil-
tered. The filtrate was concentrated by rotary evapora-
tion and the resulting golden oil (1.74 g, 89.3%) was
used without further purification. An analytical sample
was purified by radial TLC (Chromatotron, silica plate,
CH2Cl2 as eluent, with nitrogen bubbled through concd
NH4OH as the atmosphere). 1H NMR (300 MHz,
CDCl3, d ppm): 1.29 (s, 3H), 1.31 (s, 3H), 1.65 (br s,
2H), 2.72 (t, J = 7.16 Hz, 1H), 2.92 (t, J = 7.16 Hz,
1H), 3.71 (q, J = 10.27 Hz, 2H), 3.82 (s, 3H), 4.42 (m,
1H), 6.65 (s, 1H), 7.25 (s, 1H), CIMS m/z 336 (M+1).
Anal. (C12H18INO2) C, H, N.
1
product as a clear oil. H NMR (300 MHz, CDCl3, d
ppm): 1.28 (s, 3H), 1.32 (s, 3H), 1.70 (br s, 2H), 2.73
(t, J = 6.67 Hz, 2H), 2.92 (t, J = 6.67 Hz, 2H), 3.75 (s,
3H), 4.42 (m, 1H), 6.74 (m, 3H); CIMS m/z 210
(M+1). Anal. (C12H19NO2) C, H, N.
3.1.3.
methoxyphenyl)ethylamine (4). To a solution of the
amine (20.0 g, 96 mmol) in anhydrous THF
N-(2,2,2-Trifluoroacetyl)-2-(2-isopropoxy-5-
3
(100 mL) and triethylamine (9.65 g, 96 mmol) in a dried
250 mL round-bottomed flask was added ethyl trifluoro-
acetate (17.73 g, 125 mmol), and the solution was stirred
efficiently at room temperature overnight. TLC showed
complete consumption of the starting material. The sol-
vents were then removed by rotary evaporation and the
residue was dissolved in 150 mL of CH2Cl2. The organic
solution was washed with H2O (2· 100 mL), brine
3.1.6. N-(2-Isopropoxybenzyl)-2-(4-iodo-2-isopropoxy-5-
methoxyphenyl)ethylamine (7). To a solution of the
crude amine 6 obtained above (0.99 g, 2.95 mmol) in tol-
uene (30 mL) in a 100-mL round-bottomed flask was

D. E. Nichols et al. / Bioorg. Med. Chem. 16 (2008) 6116–6123
6121
added 0.629 g (3.83 mmol) of 2-isopropoxybenzalde-
hyde17 and the reaction was heated at reflux for 1 h with
H2O removal using a Dean-Stark trap. When H2O pro-
duction ceased, the toluene was removed by rotary evap-
oration and the residue was dissolved in anhydrous
EtOH (50 mL). Sodium borohydride (0.56 g, 15 mmol)
was added portion-wise and the reaction was stirred at
room temperature for 4 h. The excess NaBH4 was
decomposed by transferring the reaction mixture to a
250 mL flask and adding methanol (50 mL). The solu-
tion was then heated at reflux until gas evolution ceased.
The solvent was removed by rotary evaporation and the
residue was dissolved in Et2O (50 mL). The ether solu-
tion was extracted with 1 M HCl (3· 75 mL). The acidic
aqueous extract was washed with Et2O (3· 25 mL), fol-
lowed by basification with 2 M NaOH. The liberated
free base was extracted into Et2O (3· 75 mL), the organ-
ic layer was washed with H2O (2· 50 mL) and brine
(50 mL), dried over MgSO4, and filtered. The filtrate
was reduced to dryness by rotary evaporation and the
resulting golden oil was purified using radial TLC
(Chromatotron, 4 mm silica plate, CH2Cl2 as eluent,
and nitrogen bubbled through concd NH4OH as the
atmosphere), providing 0.86 g (60.1%) of the pure amine
as a clear oil. 1H NMR (300 MHz, CDCl3, d ppm): 1.24
(s, 3H), 1.25 (s, 3H), 1.26 (s, 3H), 1.27 (s, 3H), 1.80 (br s,
1H), 2.80 (m, 4H), 3.73 (s, 2H), 3.80 (s, 3H), 6.65 (s, 1H),
6.67–6.90 (m, 3H), 7.13–7.24 (m, 2H), HRMS (ESI): cal-
culated for C22H30INO3: 484.1349. Found: 484.1346.
to 0 ꢁC and a solution of di-tert-butyl dicarbonate
(0.784 g, 3.59 mmol) in dry THF (2 mL) was added
dropwise using a syringe. The reaction mixture was al-
lowed to warm to room temperature and was stirred
for a total of 2 h. The THF was removed by rotary evap-
oration and the residue was dissolved in 20 mL CH2Cl2.
This organic solution was washed with H2O (2· 25 mL)
and brine (25 mL), dried with MgSO4, and filtered. The
filtrate was concentrated by rotary evaporation, and the
residual dark brown oil was purified using radial TLC
(Chromatotron, 4 mm silica plate, CH2Cl2 as eluent,
and nitrogen bubbled through NH4OH as the atmo-
sphere) to provide 0.984 g (54.9%) of the t-Boc protected
amine 9 as a light golden oil. After drying under high
1
vacuum the oil solidified to an easily handled glass. H
NMR (300 MHz, CDCl3, d ppm): 1.33 (s, 9H), 2.67 (t,
J = 7.05 Hz, 2H), 3.38 (m, 2H), 3.70 (s, 3H), 4.25 (s,
2H), 4.45 (br s, 1H), 6.30 (s, 1H), 6.65–6.90 (m, 2H),
7.00–7.22 (m, 3H), 9.25 (br s, 1H), CIMS m/z 500
(M+1). Anal. C21H26INO5 C, H, N.
3.1.9. N-(tert-Butoxycarbonyl)-N-(2-methoxybenzyl)-2-
(2,5-dimethoxy-4-iodophenyl)ethylamine (10a). A mix-
ture of the t-Boc protected amine
9 (123 mg,
0.246 mmol) in anhyd DMF (20 mL) and NaH (60%
in mineral oil, 275 mg, 6.89 mmol) was stirred under ar-
gon for 45 min. A solution of CH3I (87 mg, 0.617 mmol)
in toluene (4 mL) was added and the reaction mixture
was stirred overnight. TLC analysis showed total con-
sumption of the starting material. The reaction mixture
was carefully quenched with H2O and the solvent was
removed by rotary evaporation under high vacuum.
The resulting syrupy residue was dissolved in Et2O
(50 mL), washed with H2O (2· 20 mL), brine (20 mL),
dried over MgSO4, and filtered. The filtrate was concen-
trated by rotary evaporation and the residue was puri-
fied by radial TLC (Chromatotron, 2 mm silica plate,
CH2Cl2 as eluent, with nitrogen bubbled through concd
NH4OH as the atmosphere) to provide 81 mg (63.0%) of
chromatographically pure O,O-dimethylated t-Boc pro-
3.1.7.
N-(2-Hydroxybenzyl)-2-(4-iodo-2-hydroxy-5-
methoxyphenyl)ethylamine (8). A solution of amine 7 ob-
tained in the previous step (0.75 g, 1.55 mmol) in anhy-
drous CH2Cl2 (30 mL) in a 100-mL single-neck round-
bottomed flask was cooled to À78 ꢁC and a solution
of 1 M BCl3 in CH2Cl2 (4.65 mL, 4.65 mmol) was added
dropwise using a syringe. The reaction mixture was al-
lowed to warm to room temperature and was stirred
overnight. The flask was cooled in an ice bath and meth-
anol (20 mL) was added dropwise. The solvent was then
removed by rotary evaporation. Methanol (30 mL) was
added to the residue, the solvent was removed by rotary
evaporation, and the procedure was repeated using
25 mL of toluene. The resulting salt was basified with
excess 1 M NH4OH, and the aqueous solution was ex-
tracted with CH2Cl2 (3· 25 mL). The combined organic
extract was washed with H2O (25 mL) and brine
(25 mL), dried with MgSO4, filtered, and the solvent
was evaporated under vacuum. The residual free base
was purified by radial TLC (Chromatotron, 4 mm silica
plate, CH2Cl2 as eluent, with nitrogen bubbled through
NH4OH as the atmosphere) to provide 0.442 g (71.4%)
of the O,O-dealkylated amine 8 as a golden oil. 1H
NMR (300 MHz, CDCl3, d ppm): 2.80 (t, J = 8.4 Hz,
2H), 2.95 (t, J = 8.4 Hz, 2H), 3.80 (s, 3H), 3.95 (s,
2H), 6.55 (s, 1H), 6.60–6.90 (m, 3H), 7.00–7.25 (m,
2H), HRMS (ESI): Calculated for C16H18INO3:
400.0410. Found: 400.0412.
1
tected amine 10a as a clear oil. H NMR (300 MHz,
CDCl3, d ppm): 1.33 (s, 9H), 2.66 (t, J = 6.93 Hz, 1H),
2.76 (t, J = 6.93 Hz, 1H), 3.25 (t, J = 6.93 Hz, 1H),
3.35 (t, J = 6.93 Hz, 1H), 3.65 (s, 3H), 3.72 (s, 3H),
3.74 (s, 3H), 4.28 (1, 1H), 4.40 (s, 1H), 6.44, 6.60 (2s,
1H), 6.77 (d, 1H), 6.83 (t, 3H), 7.00–7.20 (m, 3H).
HRMS (ESI): Calculated for C23H30INO5: 550.1066
(M+Na). Found: 550.1075.
3.1.10. N-(2-Methoxybenzyl)-2-(2,5-dimethoxy-4-iodo-
phenyl)ethylamine hydrochloride (1a). The t-BOC pro-
tected amine 10a from the previous step (50 mg,
94.8 mmol) was added to 2 M HCl in methanol
(3 mL), followed by stirring at room temperature for
12 h, a procedure that completely removed the t-BOC
protecting group. The solvent was removed by rotary
evaporation and the resulting white powder was recrys-
tallized from acetonitrile to provide 43 mg (98%) of the
desired deprotected amine (1a) hydrochloride; melting
point 167 ꢁC. 1H NMR (300 MHz, CDCl3, d ppm):
3.11 (br s, 4H), 3.63 (s, 3H), 3.76 (s, 3H), 3.84 (s, 3H),
4.16 (t, J = 4.90 Hz, 2H), 6.79 (s, 1H), 6.84 (d, 1H),
3.1.8. N-(tert-Butoxycarbonyl)-N-(2-hydroxybenzyl)-2-
(4-iodo-2-hydroxy-5-methoxyphenyl)ethylamine (9).
A
solution of amine 8 prepared in the previous step
(1.56 g, 3.59 mmol) in 10 mL of dry THF was cooled

6122
D. E. Nichols et al. / Bioorg. Med. Chem. 16 (2008) 6116–6123
6.95 (t, 1H), 7.28 (s, 1H), 7.33 (t, 1H), 7.39 (d, 1H), 9.45
(br s, 2H), CIMS m/z 428 (M+1).
3.2.3. N-(2-[3H-OCH3]benzyl)-2-(2-[3H-OCH3]-5-meth-
oxy-4-iodophenyl)ethylamine ([3H]INBMeO) (1b). The
crude intermediate 10b (4.8 mCi) was stirred in 1:5
TFA:CH2Cl2 (2 mL) at RT for 1 h. The solvent was
evaporated under a stream of nitrogen and the crude
produce (3.8 mCi) was purified by preparative HPLC
(sample loaded in 0.5 mL of 80% [20 mM NaH2PO4-
H2O]/20% EtOH). The pure final product, [3H]INBMeO
(1b) (1.8 mCi, radiochemical purity: 99%, specific activ-
ity: 160 Ci/mmol) gave a single peak with Rt 18.2 min
(UV detector), when coinjected with standard INBMeO
1a.
3.2. Radiochemical synthesis
3.2.1. N-(tert-butoxycarbonyl)-N-(2-[3H-OCH3]- benzyl)-
2-(2-[3H-OCH3]-5-methoxy-4-iodophenyl)ethylamine (10b).
A 0.2 mL aliquot (28.8 lg, 0.0577 lmol) was withdrawn
from a solution of 3.5 mg (7.2 mmol) of N-(tert-butoxy-
carbonyl)-N-(2-hydroxybenzyl)-2-(4-iodo-2-hydroxy-5-
methoxyphenyl)ethylamine (9) in anhyd DMF (25 mL).
The aliquot was added to DMF (10 mL) containing
potassium carbonate (30 mg, 0.22 mmol) in a pear-
shaped flask and the solution was stirred under nitrogen
for 30 min. The flask was then connected to a vacuum
manifold and the mixture was de-gassed three times by
freezing/evacuating/thawing. A sealed tube containing
[3H]methyl iodide (100 mCi, 80 Ci/mmol, 1.25 lmol,
and 11 equiv, Perkin-Elmer Life Sciences) in toluene
(2 mL) was connected to the vacuum manifold using a
thermometer adapter that was connected to a double-
end female adapter connected to an adapter containing
P2O5 (429 mg, 3 mmol) and a cotton plug. The sealed tube
was evacuated for 5 min and the [3H]methyl iodide/tolu-
ene solution was transferred to the reaction flask through
the P2O5 layer over a 2 h period. The reaction mixture was
stirred at RT for an additional 5 min, cooled with liquid
nitrogen, and then thawed. The stirring/freezing/thawing
process was repeated three times. Finally, the reaction
mixture was stirred at RT for 15 h. The unreacted
[3H]methyl iodide was transferred backto the sealed-tube.
H2O (2 mL) was added to the reaction mixture followed
by stirring at RT for 30 min. The mixture was transferred
to a 15 mL centrifuge tube and extracted with Et2O (5·
2 mL). The combined ether extract was washed with
H2O (3· 2 mL), dried over Na2SO4, then filtered into a
scintillation vial, providing crude intermediate (10b)
(10.6 mCi, radiochemical purity: 77%).
3.3. Pharmacology
3.3.1. Materials. [3H]Ketanserin (67 Ci/mmol) was pur-
chased from Perkin-Elmer (Boston, MA). Cinanserin
HCl and ketanserin tartrate were purchased from Tocris
Bioscience (Ellisville, MO). DOI and LSD were synthe-
sized in our own laboratory. Serotonin HCl and most
other reagents were purchased from Sigma–Aldrich
Chemical Co. (St. Louis, MO). Cell culture reagents
were purchased from Fisher Scientific (Pittsburgh, PA)
or Invitrogen (Carlsbad, CA).
3.3.2. Cell culture. A549 cells, stably expressing the hu-
man 5-HT2A receptor and the ICAM-1 luciferase repor-
ter gene (A20), were a generous gift from Dr. Ulrike
Weyer-Czernilofsky of the Ernst Boehringer Institut,
Bender and Co., Vienna, Austria18 The creation of the
HEK cells stably expressing the human 5-HT2A receptor
(Hh2A) had been previously reported19 Both cell lines
were grown in culture plates at 37 ꢁC with 5% CO2 in
Dulbecco’s modified Eagle’s medium (DMEM) contain-
ing 10% dialyzed fetal bovine serum (dFBS, Hyclone,
Logan, UT), 2 mM L-glutamine, 100 U/mL penicillin,
100 lg/mL streptomycin, and 300 lg/mL G-418 with
75 lg/mL hygromycin B (A20) or 20 lg/mL Zeocin
(Hh2A).
3.2.2. Specific activity determination. A Beer’s Law cali-
bration curve for 10a was generated by injection of ali-
quots (2, 3, 4, 5, 10, and 15 lL) of a standard solution
(6.23 · 10À6 M) of 10a in CH3OH onto a Supelco
Ascentis Express C18 HPLC column (2.1 · 100 mm,
2.7lm), eluting with a gradient of 55–90% B, over
20 min with solvent A: 0.05% NH4OH–H2O and solvent
B: CH3CN, flow rate: 0.4 mL/min, and monitoring at
200 nm. Plotting the amount (lmol, Y) versus the UV
response (integrated area, X) gave a straight line
(Y = 2.0 · 10À11 X, R2 = 0.9984). To determine the
radiochemical purity of 10b the percentage of radioac-
tivity associated with 10b (53.8%) was determined by
integration of the counted (b-RAM) peaks and counting
(scintillation counter) of the collected total effluent from
a separate 8 lCi injection to confirm that no radioactiv-
ity remained on the column. To determine the specific
activity, three injections of 8.0 lCi each (containing
0.538 · 8 lCi = 4.3 lCi) were made. The molar amounts
of 10b, determined from the calibration curve, were
2.65 · 10À5, 2.61 · 10À5, and 2.62 · 10À5 lmol, giving
values of 163, 165, and 164 Ci/mmol, respectively, for
the specific activity of 10b.
3.3.3. Radioligand assays. Methods for membrane prep-
aration and radioligand assays were performed as previ-
ously described20 Briefly, cells were grown to confluency
in 150 mm culture dishes before being lysed on ice with
lysis buffer (1 mM Hepes, 2 mM EDTA, pH 7.4). After
10 min, the cells were scraped and centrifuged for 30 min
at 30,000g at 4 ꢁC. The supernatant was discarded and
the pellets were resuspended in receptor binding buffer
(RBB, 50 mM Tris, 4 mM MgCl2, pH 7.4) by mechani-
cal homogenization. This suspension was transferred to
microcentrifuge tubes and centrifuged for 10 min at
13,000g and 4 ꢁC. The supernatant was discarded and
the pellets were frozen at À80 ꢁC until use.
All radioligand binding experiments were performed in
the presence of 1% bovine serum albumin in receptor
binding buffer, and incubated for 60 minutes at 25 ꢁC.
For saturation binding experiments, 0.3–10.0 nM
[3H]ketanserin and 0.03–4.0 nM [3H]INBMeo were
used. Nonspecific binding was defined by 10 lM cinan-
serin in a total volume of 500 lL. Competition binding
experiments were performed with 1.0–3.0 nM [3H]ketan-
serin or 0.1–0.3 nM [3H]NBMeO in a total volume of

D. E. Nichols et al. / Bioorg. Med. Chem. 16 (2008) 6116–6123
6123
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500 lL. Binding experiments were terminated by rapid
filtration through FB glass fiber harvest plates with ice
cold wash buffer (10 mM Tris, 0.9% NaCl) using a 96-
well Packard FilterMate cell harvester. One well in each
plate was spotted with 10 lL of radioligand stock solu-
tion to quantify total radioactivity, and the plates were
dried overnight before 30 lL of Packard Microscint-O
scintillation fluid was added to each well. Radioactivity
per well was determined using a Packard TopCount
scintillation counter. The saturation and competition
binding curves were analyzed using GraphPad Prism
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3.3.4. Screening at other receptor types. INBMeO was
submitted to the NIMH-sponsored psychoactive drug
screening program. The ligand had low affinity for most
receptors, with the following reported Ki values (nM) for
receptors where it had significant affinity: 5-HT2C (2);
5HT6 (73 12); l opiate (82 14); H1 (189 35); 5-
HT2B (231 73); kappa opiate (288 50); all other Ki
values were greater than 500 nM: 5-HT1A, dopamine
D3, histamine H2, 5-HT1D, a1A adrenergic, d opiate,
serotonin uptake transporter, 5-HT5A, 5-HT1B, dopa-
mine D2, 5-HT7, dopamine D1, 5-HT3, 5-HT1E, dopa-
mine D5, muscarinic M1–M5, histamine H3, and the
dopamine uptake transporter.
10. Kehne, J. H.; Baron, B. M.; Carr, A. A.; Chaney, S. F.;
Elands, J.; Feldman, D. J.; Frank, R. A.; van Giersbergen,
P. L.; McCloskey, T. C.; Johnson, M. P.; McCarty, D. R.;
Poirot, M.; Senyah, Y.; Siegel, B. W.; Widmaier, C. J.
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A. Neuropharmacology 1987, 26, 1803–1806.
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Mol. Pharmacol. 2006, 70, 1956–1964.
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Acknowledgments
This work was supported by NIH Grant DA02189 from
NIDA and by the NIMH under contract NO1-MH-
32005.
17. Kingsbury, J. S.; Harrity, J. P. A.; Bonitatebus, P. J., Jr.;
Hoveyda, A. H. J. Am. Chem. Soc. 1999, 121, 791–799.
18. Weyer, U.; Schafer, R.; Himmler, A.; Mayer, S. K.;
Burger, E.; Czernilofsky, A. P.; Stratowa, C. Receptors
Channels 1993, 1, 193–200.
19. Parrish, J. C.; Braden, M. R.; Gundy, E.; Nichols, D. E. J.
Neurochem. 2005, 95, 1575–1584.
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Orbaugh, D. M.; Marona-Lewicka, D.; Nichols, D. E. J.
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