Fluorine-18-labeled benzamide analogues for imaging the σ2 receptor status of solid tumors with positron emission tomography

Article abstract of DOI:10.1021/jm0614883

A series of fluorine-containing benzamide analogs was synthesized and evaluated as candidate ligands for positron emission tomography (PET) imaging of the sigma-2 (σ₂) receptor status of solid tumors. Four compounds having a moderate to high af

Full text of DOI:10.1021/jm0614883

3194
J. Med. Chem. 2007, 50, 3194-3204
Fluorine-18-Labeled Benzamide Analogues for Imaging the σ2 Receptor Status of Solid Tumors
with Positron Emission Tomography
Zhude Tu,† Jinbin Xu,† Lynne A. Jones,† Shihong Li,† Craig Dumstorff,† Suwanna Vangveravong,† Delphine L. Chen,†
Kenneth T. Wheeler,‡ Michael J. Welch,† and Robert H. Mach*,†
DiVision of Radiological Sciences, Washington UniVersity School of Medicine, 510 South Kingshighway BouleVard, St. Louis, Missouri 63110,
and Department of Radiology, Wake Forest UniVersity School of Medicine, Winston-Salem, North Carolina 27157
ReceiVed December 29, 2006
A series of fluorine-containing benzamide analogs was synthesized and evaluated as candidate ligands for
positron emission tomography (PET) imaging of the sigma-2 (σ2) receptor status of solid tumors. Four
compounds having a moderate to high affinity for σ2 receptors and a moderate to low affinity for sigma-1
(σ1) receptors were radiolabeled with fluorine-18 via displacement of the corresponding mesylate precursor
with [18F]fluoride. Biodistribution studies in female Balb/c mice bearing EMT-6 tumor allografts demonstrated
that all four F-18-labeled compounds had a high tumor uptake (2.5-3.7% ID/g) and acceptable tumor/
normal tissue ratios at 1 and 2 h post-i.v. injection. An analysis of the chemistry and biodistribution data
suggested that N-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2-(2-[18F]-fluoroethoxy)-5-me-
thylbenzamide ([18F]3c) and N-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2-(2-[18F]-fluo-
roethoxy)-5-iodo-3-methoxybenzamide ([18F]3f) are acceptable compounds for imaging the σ2 receptor status
of solid tumors.
Introduction
The search for σ2 selective ligands has led to the identification
of a number compounds having modest to high selectivity for
σ2 versus σ1 receptors (Figure 1). These include CB-184 (10),
CB-64D (11), BIMU-1 (12),13,14 and PB-167 (13)15-17, as well
as the conformationally flexible benzamide analogs developed
in our laboratory (14-16).18,19 We previously reported the
evaluation of several 11C, 76Br, and 125/123I radiolabeled con-
formationally flexible benzamide analogs using EMT-6 tumor-
bearing female Balb/c mice.20-22 The initial in vivo studies of
5-methyl-2-[11C]-methoxy-N-[2-(6,7-dimethoxy-3,4-dihydro-1H-
isoquinolin-2-yl)-butyl]-benzamide and 5-[76Br]-bromo-2,3-
dimethoxy-N-[2-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-
yl)-butyl]-benzamide indicated that these compounds were
potential radiopharmaceuticals for imaging solid tumors and
their proliferative status with PET. However, the radionuclide
properties of 76Br and 11C make them less than ideal for PET
imaging when compared to the radionuclide properties of 18F.
For example, studies comparing the nuclide properties of 18F
and 76Br indicate that radiotracers containing 18F give higher
quality PET images due to the relatively high energy of the
positron emitted by 76Br that often produces “blurred” images.23
Although this is not an issue with 11C-lableled radiotracers, the
longer half-life of 18F (t1/2 ) 109.8 min) compared to 11C (t1/2
) 20.4 min) places fewer time constraints on tracer synthesis
and permits longer scan sessions that usually result in higher
tumor/normal tissue ratios for the 18F-labeled radiotracers.
Therefore, the development of an 18F-labeled radiotracer is
important for imaging the σ2 receptor status of human solid
tumors by PET.
Sigma receptors were originally thought to be a subtype
of the opioid receptors. Subsequent studies revealed that
sigma receptors were a distinct class of receptors that are
expressed in many normal tissues, including liver, kidneys,
endocrine glands, and the central nervous system (CNS).1 It
has been well-established that there are at least two types of
sigma receptors, sigma-1 (σ1) and sigma-2 (σ2).2,3 The σ1
receptor has been cloned from tissues of guinea pig, rat,
mouse, and man4 and displays a 30% sequence homology with
a yeast sterol enzyme isomer.5 The σ2 receptor has not been
cloned, but evidence suggests that this receptor is linked to
potassium channels and intracellular calcium release in NCB-
20 cells.1,6,7
An overexpression of σ2 receptors has been reported in a
variety of human and murine tumors.8-10 The observation that
the density of σ2 receptors is greater than that of σ1 receptors
in a wide panel of tumor cells grown under various cell culture
conditions5 suggested that the σ2 receptor may be a biomarker
of tumor cell proliferation. In a series of studies designed to
test this hypothesis, the density of σ2 receptors was found to be
10-fold higher in proliferating versus quiescent mouse mammary
adenocarcinoma cells, both in vitro8,11 and in vivo.12 This higher
σ2 receptor density in proliferating tumor cells did not depend
on other biological or physiological factors.11,12 Consequently,
these data suggest that radioligands possessing a high affinity
and selectivity for σ2 receptors have the potential to not only
detect solid tumors, but also to measure their proliferative status
using noninvasive imaging techniques such as positron emission
tomography (PET) and single photon emission computed
tomography (SPECT).13
In this paper, we report the synthesis and in vitro/in vivo
characterization of several fluorinated conformationally flexible
benzamide analogs having a moderate to high affinity and
selectivity for σ2 receptors. These 18F-labeled compounds were
prepared and evaluated in Balb/c female mice bearing EMT-6
mammary tumor allografts to assess their potential as PET
radioligands for imaging the σ2 receptor status of human solid
tumors.
* To whom correspondence should be addressed. Robert H. Mach, Ph.D.,
Division of Radiological Sciences, Washington University School of
Medicine, Campus Box 8225, 510 S. Kingshighway Blvd., St. Louis,
Missouri 63110. Tel.: 314-362-8538. Fax: 314-362-0039. E-mail:
rhmach@mir.wustl.edu.
† Washington University School of Medicine.
‡ Wake Forest University School of Medicine.
10.1021/jm0614883 CCC: $37.00 © 2007 American Chemical Society
Published on Web 06/19/2007

18F-Labeled Benzamide Analogues for Imaging
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14 3195
Figure 1. Structure and properties of several σ2 selective ligands.
Results and Discussion
Synthesis of the precursor for the corresponding 5-iodo
analog, 5f, is shown in Scheme 3. Esterification of 5-bromo-
2-methoxy salicylic acid followed by alkylation of the ortho
hydroxyl group with 1-bromoethyl acetate, then hydrolysis of
the acetate and benzoate esters produced the corresponding
2-hydroxyethyl analog, 9. Condensation of 9 with the amine,
1b, gave the amide, 4f, which was converted to the correspond-
ing mesylate, 5f, using the conditions described above for the
analogs 5c-e.
The synthesis of [18F]3c, [18F]3d, [18F]3e, and [18F]3f was
accomplished by treating the mesylate precursors, 5c-f, with
[18F]fluoride/potassium carbonate and Kryptofix 222, using
dimethyl sulfoxide (DMSO) as the solvent. The reaction mixture
was irradiated for 30-40 s in a microwave oven, and the crude
product was separated from the unreacted [18F]fluoride using a
C-18 reverse phase Sep-Pak cartridge and methanol as the
eluent. The crude product was then purified by high-performance
liquid chromatography (HPLC) using a C-18 reverse phase
column. The entire procedure required ∼2 h, and the radio-
chemical yield, corrected for decay to the start of synthesis,
was 20∼30%. The specific activities ranged from 1500 to 2500
Ci/mmol.
Chemistry. The design strategy for generating σ2-selective
ligands suitable for 18F-labeling involved replacing the ortho
methoxy group of the 11C-labeled benzamide analogs21 with a
2-fluoroethyl group, as shown in Scheme 1. Condensation of
1a and 1b with a substituted salicylic acid gave the correspond-
ing substituted 2-hydroxybenzamide analogs, 2a-e. Alkylation
of the ortho hydroxyl group with 2-bromo-1-fluoroethane using
potassium carbonate as a base produced 3a-e in moderate to
high yield. Compound 3f was prepared by iodination of the
corresponding tin precursor, 3g, which was prepared from 3b
using standard stannylation reaction conditions. Compounds
3a-f were then converted into either the hydrochloride or the
oxalic acid salts for the in vitro σ1 and σ2 receptor binding
assays.
Based on the results of the in vitro binding studies with the
unlabeled compounds, 3c-f were radiolabeled with 18F, as
shown in Schemes 2-4. Scheme 2 outlines the synthesis of the
mesylate precursors required for the radiolabeling procedure.
Alkylation of the ortho hydroxyl group of compounds 2c-e
with 1-bromoethyl acetate followed by hydrolysis of the acetate
group produced the corresponding 2-hydroxyethoxy analogs,
4c-e, in good yield. Compounds 4c-e were then converted to
the corresponding mesylates, 5c-e, by treatment with meth-
anesulfonyl chloride in dichloromethane using triethylamine as
an acid scavenger.
In Vitro Binding Studies. In vitro binding studies were
conducted to measure the affinity of the target compounds for
σ1 and σ2 receptors. The binding assays used [3H](+)-penta-
zocine for the σ1 receptors and [3H]1,3-di(2-tolyl)guanidine ([3H]-

3196 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14
Tu et al.
Scheme 1a
a Reagents and conditions: (a) RCOOH, BOP/CH2Cl2 or DCC/CH2Cl2, rt, 18 hrs; (b) BrCH2CH2F, K2CO3/acetone reflux, over 48 h; (c) [Sn(C4H9)3]2,
Pd(PPh3)4(0)/toluene at 110 °C; (d) I2/CH2Cl2, room temperature.
Table 1. Affinity (Ki) of the Benzamide Analogs 6a-e for the σ1 and
σ2 Receptors Assayed In Vitro
group from two carbons (3a) to four carbons (3c) results in a
69-fold increase in the affinity for σ1 receptors, a 15-fold
increase in the affinity for σ2 receptors, and a 0.5 unit
increase in the log D value; a measure of the lipophilicity of
the compounds (Table 1). Although four of the five compounds
(3c-f) with a four-carbon spacer had higher affinities for
σ1 receptors (their Ki values ranged from 330 to 2150 nM)
than the compound (3a) with a two-carbon spacer (Ki ) 22,-
750 nM), the affinities of 3c-f for σ2 receptors increased
proportionately more (their Ki values ranged from 0.26 to
6.95 nM), leading to substantial increases in their σ2/σ1 ratios
(Table 1).
Ki value (nM)
Log Da
σ1
σ2
σ1/σ2 ratio
(pH ) 7.4)
3a
3b
3c
3d
3e
3f
22 750 ( 3410
15 300 ( 2305
330 ( 25
102 ( 4
222
40
48
1656
1230
8190
2.54
3.56
3.06
3.89
4.13
3.46
386 ( 93
6.95 ( 1.63
0.65 ( 0.22
1.06 ( 0.30
0.26 ( 0.07
1076 ( 88
1300 ( 225
2150 ( 410
a Calculated using the program ACD/log D.
DTG) in the presence of 100 nM (+)-pentazocine for the σ2
receptors. The Ki values were determine from Scatchard
plots. The results of the binding assays for compounds 3a-f
are shown in Table 1. Increasing the length of the spacer
It is not clear why compound 3b had a relatively low affinity
for both the σ1 and σ2 receptors, especially considering
compound 3f was found to have the highest affinity for σ2

18F-Labeled Benzamide Analogues for Imaging
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14 3197
Scheme 2a
a Reagents and conditions: (a) BrCH2CH2OAc/acetone, K2CO3, reflux 18 h; (b) NaOH/H2O, CH3OH, room temperature; (c) methanesulfonyl chloride,
CH2Cl2, triethylamine, room temperature.
Scheme 3a
a Reagents and conditions: (a) methanol/98% H2SO4 reflux, 18 h; (b) BrCH2CH2OAc/acetone, K2CO3, reflux, 18 h; (c) [Sn(C4H9)3]2, Pd(PPh3)4(0)/
toluene at 110 °C; (d) I2/CH2Cl2, room temperature; (e) NaOH/H2O, CH3OH, room temperature; (f) 1b, BOP/CH2Cl2 or DCC/CH2Cl2, room temperature,
18 h; (g) methanesulfonyl chloride, CH2Cl2, triethylamine, room temperature, 18 h.
receptors of the six compounds evaluated. The difference
between 3b and 3f only involves substituting an iodine atom
for a bromine atom in the meta position, and this same
substitution resulted in virtually no change in the σ2 receptor
affinity when it was made in the analogs lacking the 3-methoxy
group (compare 3d to 3e).
high σ2/σ1 ratios for the compounds 3c-f suggests that their
corresponding 18F-labeled analogs may be useful radiotracers
for imaging the σ2 receptor status of solid tumors with PET.
Also, the log D values for these compounds, a measure of their
lipophilicity, are within the range that should lead to a high
uptake in solid tumors.21
Finally, the σ2/σ1 ratios for compounds 3c-f varied from 48
to 8190. The excellent σ2 receptor affinities and moderate to
In Vivo Evaluation. The results of the biodistribution studies
in female Balb/c mice bearing EMT-6 tumors are shown in

3198 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14
Tu et al.
Scheme 4
Figure 2. Tumor/organ ratios for the 18F-labeled σ2 selective ligands
3c-f at 1 h (top) and 2 h (bottom) after i.v. injection into female Balb/c
mice bearing EMT-6 tumors.
Table 2. All four labeled compounds displayed excellent
tumor uptake at 5 min postinjection, with values ranging from
2.5 to 3.7% of the injected dose per gram (%ID/g). Tumor
uptake at 1 h postinjection remained high for each of the
ligands [18F]3c-f (1.14, 2.09, 2.72, and 2.15 %ID/g, respec-
tively) and continued to remain relatively high at 2 h postin-
jection (0.64, 0.96, 1.92, and 1.15 %ID/g, respectively) com-
pared with that of the normal tissues, fat, and muscle. This
resulted in acceptable tumor/normal tissue ratios for the
PET imaging studies. For example, the tumor/muscle ratios
ranged from 3-4 and the tumor/fat ratios ranged from 4.5-8
at 2 h postinjection, respectively. Also, the low bone uptake of
all four labeled compounds, which continued to decrease
between the 30 min and the 1 h time points, suggested that
these compounds do not undergo a significant defluorination
in vivo.
The tumor/fat ratios for [18F]3c and [18F]3d were also high,
reaching ∼8 and ∼6, respectively, at 2 h after i.v. injection
However, the tumor/muscle ratios for [18F]3c and [18F]3d were
much lower than that for [18F]3f. Although the tumor uptake
of [18F]3d and [18F]3e is higher than that of [18F]3c at both 1
and 2 h postinjection, these radiotracers cleared much more
slowly from the blood than [18F]3c (Table 2), making them less
desirable than [18F]3c as PET imaging agents. The moderate to
high tumor/normal tissue ratios and the rapid clearance from
the blood for [18F]3c and [18F]3f suggests that these radiotracers
are likely the best candidates for imaging of solid tumors with
PET. Consequently, these two radiotracers were selected for
further studies to evaluate the suitability for detecting solid
tumors and imaging their σ2 receptor status with PET.
Compound [18F]3f had the highest tumor/muscle ratio (∼8)
To demonstrate that the in vivo binding of [18F]3c and [18F]-
3f was specific for σ2 receptors, a no-carrier-added dose of these
Table 2. [18F]3c-f Biodistribution in Female Balb/c Mice Bearing EMT-6 Tumors
and a tumor/fat ratio of ∼7 at 2 h after i.v. injection (Figure 2).
5 min
30 min
[18F]3c
60 min
120 min
5 min
30 min
[18F]3d
60 min
120 min
blood
lung
liver
kidney
muscle
fat
heart
brain
bone
tumor
2.49 ( 0.49
10.26 ( 0.71
23.33 ( 4.22
29.18 ( 1.92
1.86 ( 0.08
1.95 ( 0.33
3.73 ( 0.15
0.76 ( 0.06
2.49 ( 0.19
3.67 ( 0.45
1.16 ( 0.10
2.36 ( 0.19
10.51 ( 0.87
6.86 ( 0.45
0.70 ( 0.19
0.59 ( 0.13
1.15 ( 0.06
0.27 ( 0.05
0.96 ( 0.15
2.54 ( 0.27
[18F]3e
0.56 ( 0.08
0.88 ( 0.12
4.14 ( 0.55
2.51 ( 0.51
0.34 ( 0.05
0.22 ( 0.04
0.55 ( 0.07
0.18 ( 0.03
0.55 ( 0.07
1.14 ( 0.10
0.35 ( 0.05
0.43 ( 0.07
2.05 ( 0.43
0.87 ( 0.13
0.24 ( 0.08
0.08 ( 0.02
0.27 ( 0.04
0.12 ( 0.02
0.45 ( 0.11
0.64 ( 0.10
3.57 ( 0.43
12.08 ( 1.98
32.60 ( 3.96
42.94 ( 3.34
1.95 ( 0.19
2.85 ( 0.47
3.74 ( 0.37
1.09 ( 0.11
2.90 ( 0.39
3.28 ( 0.41
2.81 ( 0.32
3.08 ( 0.23
13.12 ( 1.39
17.55 ( 2.75
0.98 ( 0.18
0.96 ( 0.13
1.55 ( 0.11
0.40 ( 0.03
1.17 ( 0.06
2.59 ( 0.19
[18F]3f
1.69 ( 0.63
1.60 ( 0.27
5.69 ( 0.54
6.92 ( 1.61
0.58 ( 0.11
0.38 ( 0.05
1.04 ( 0.21
0.32 ( 0.05
1.12 ( 0.16
2.09 ( 0.28
0.52 ( 0.10
0.51 ( 0.08
2.30 ( 0.33
1.12 ( 0.16
0.28 ( 0.10
0.15 ( 0.06
0.40 ( 0.07
0.20 ( 0.03
1.28 ( 0.28
0.96 ( 0.24
blood
lung
liver
kidney
muscle
fat
heart
brain
bone
tumor
4.60 ( 0.44
9.71 ( 0.83
37.26 ( 4.88
36.07 ( 2.28
1.52 ( 0.10
2.32 ( 0.46
3.22 ( 0.27
0.55 ( 0.04
2.59 ( 0.28
2.54 ( 0.62
4.30 ( 0.59
4.07 ( 0.46
17.35 ( 2.72
17.43 ( 1.95
1.12 ( 0.05
1.04 ( 0.06
2.37 ( 0.29
0.44 ( 0.04
1.31 ( 0.14
2.81 ( 0.62
3.39 ( 0.29
2.34 ( 0.12
7.31 ( 0.98
9.36 ( 0.90
0.83 ( 0.04
0.62 ( 0.10
1.61 ( 0.11
0.36 ( 0.02
0.99 ( 0.07
2.72 ( 0.13
1.92 ( 0.59
1.36 ( 0.24
4.25 ( 1.56
3.92 ( 0.98
0.60 ( 0.11
0.44 ( 0.08
1.00 ( 0.23
0.37 ( 0.06
1.67 ( 0.27
1.92 ( 0.10
1.82 ( 0.25
18.47 ( 3.07
15.21 ( 2.21
19.98 ( 1.66
2.50 ( 0.33
4.13 ( 0.86
5.60 ( 0.45
0.71 ( 0.09
2.23 ( 0.56
3.05 ( 0.43
1.23 ( 0.28
3.75 ( 0.58
10.73 ( 2.98
7.73 ( 2.25
0.73 ( 0.14
1.36 ( 0.50
1.54 ( 0.31
0.27 ( 0.04
2.01 ( 0.53
3.11 ( 0.16
0.65 ( 0.09
1.51 ( 0.13
5.57 ( 0.31
3.50 ( 0.80
0.40 ( 0.07
0.47 ( 0.07
0.69 ( 0.06
0.14 ( 0.02
0.93 ( 0.16
2.15 ( 0.25
0.28 ( 0.01
0.74 ( 0.03
2.61 ( 0.69
1.34 ( 0.10
0.15 ( 0.01
0.17 ( 0.05
0.39 ( 0.04
0.08 ( 0.01
0.59 ( 0.09
1.15 ( 0.23

18F-Labeled Benzamide Analogues for Imaging
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14 3199
Conclusion
In the present study, we synthesized several novel confor-
mationally flexible benzamide analogues having a moderate to
high binding affinity and selectivity for σ2 receptors (Table 1).
Four of these compounds were selected as candidates for
developing 18F-labeled PET probes to image the σ2 receptor
status of solid tumors. [18F]3c, [18F]3d, [18F]3e, and [18F]3f were
successfully synthesized and evaluated as potential radiotracers
for imaging EMT-6 tumors in female Balb/c mice. Of the four
18F-labeled analogues, [18F]3c and [18F]3f had the best biodis-
tribution kinetics and tumor/normal tissue ratios. Blocking
studies confirmed that the uptake of [18F]3c and [18F]3f was σ2
receptor mediated. Our initial CT/PET studies indicate that [18F]-
3c and [18F]3f are acceptable agents for detecting solid tumors
and imaging their σ2 receptor status with PET. Further evaluation
of these two radioligands is still required before selecting one
of them for translation to the clinic.
Figure 3. Comparison of the tumor/fat and tumor/muscle ratios for
[18F]3c or [18F]3f when there is no carrier added and when the σ1 and
σ2 receptors are blocked with 1 mg/kg of YUN-143. All values were
obtained 1 h after injection of the radiotracer.
Experimental Section
Materials. All reagents were purchased from commercial
suppliers and used without further purification unless otherwise
stated. Tetrahydrofuran (THF) was distilled from sodium hydride
immediately prior to use. Anhydrous toluene was distilled from
sodium/toluene shortly before use.
General. All anhydrous reactions were carried out in oven-dried
glassware under an inert nitrogen atmosphere unless otherwise
stated. When the reactions involved extraction with dichloromethane
(CH2Cl2), chloroform (CHCl3), ethyl acetate (EtOAc), or ethyl ether
(Et2O), the organic solutions were dried with anhydrous Na2SO4
and concentrated with a rotary evaporator under reduced pressure.
Flash column chromatography was conducted using silica gel 60a,
“40 Micron Flash” (32-63um; Scientific Adsorbents, Inc.). Melting
points were determined using the MEL-TEMP 3.0 apparatus and
left uncorrected. 1H NMR spectra were recorded at 300 MHz on a
Varian Mercury-VX spectrometer with CDCl3 as solvent and
tetramethylsilane (TMS) as the internal standard. All chemical shift
values are reported in ppm (δ). Elemental analyses (C, H, N) were
determined by Atlantic Microlab, Inc.
Figure 4. MicroPET and microCT images of EMT-6 tumors in female
Balb/c mice. All MicroPET images were acquired 1 h after i.v. injection
of either [18F]3c or [18F]3f.
Procedure A: General Method for Synthesis of the Substi-
tuted 2-Hydroxybenzoic Acid Amides, 2a-e. N-[2-(6,7-Dimethoxy-
3,4-dihydro-1H-isoquinolin-2-yl)-ethyl]-2-hydroxy-5-methyl-
benzamide (2a). 1,3-dicyclohexycarbodimide (432.6 mg, 2.1 mmol)
and 1-hydroxybenzotriazole (283.8 mg, 2.10 mmol) were added to
an ice-water bath cooled solution of 1a21 (472.0 mg, 2.0 mmol)
and 2-hydroxy-5-methyl-benzoic acid (152 mg, 2.0 mmol) in 30
mL of dichloromethane. After the reaction mixture was stirred
overnight, analysis of the products using thin layer chromatography
with 20% methanol and 80% ethyl ether as the mobile phase
indicated that the reaction was complete. After completion of the
reaction, another 50 mL of dichloromethane was added to the
mixture. The organic solution was then washed with an aqueous
saturated NaHCO3 solution and brine, sequentially. The organic
solution was dried with anhydrous sodium sulfate. After removal
of the solvent, the crude product was purified by column chroma-
tography using 20% methanol and 80% ethyl ether as the mobile
radiotracers was coinjected into EMT-6 tumor-bearing mice with
N-(4-fluorobenzyl)piperidinyl-4-(3-bromophenyl) acetamide
(YUN-143a), a sigma ligand displaying a high affinity for both
σ1 and σ2 receptors. Previous studies from our group have shown
that 1 mg/kg YUN-143 is highly effective at blocking σ1 and
σ2 receptors in vivo.21,23,26,27 Coinjection of YUN-143 with either
[18F]3c or [18F]3f resulted in a significant decrease (∼50%) in
the tumor/muscle and tumor/fat ratios at 1 h postinjection (Figure
3). These data indicate that both [18F]3c and [18F]3f bind
selectively to σ2 receptors in vivo.
To confirm the feasibility of using these radioligands as PET
imaging agents for determining the σ2 receptor status of solid
tumors, a CT/PET study using either [18F]3c or [18F]3f in female
Balb/c mice bearing EMT-6 tumors was performed on a
microPET-F220 (CTI-Concorde Microsystems, Inc.) and a
MicroCAT-II system. All mice were imaged at 1.0 h after i.v.
injection of the radiotracer. The EMT-6 tumors were readily
identifiable using either radioligand, indicating that they are both
acceptable agents for detecting solid tumors and imaging their
σ2 receptor status with PET (Figure 4). Nevertheless, further
studies will still be required before selecting one of these
compounds for translation to the clinic.
1
phase. The yield of 2a was 37.1%. The H NMR spectrum (300
MHz, CDCl3) of the purified product was 2.25 (s, 3H), 2.75-2.95
(m, 6H), 3.58-3.65 (m, 4H), 3.82-3.83 (s, 6H), 6.48-6.51 (s, 1H),
6.62-6.63 (s, 1H), 6.82-6.85 (d, 1H), 7.02 (s, 1H), 7.08 (s, 1H),
7.20 (d, 1H). LCMS m/z 371.2 (M + H).
5-Bromo-N-[4-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-
yl)-butyl]-2-hydroxy-3-methoxy-benzamide (2b). Compound 2b
was prepared from 5-bromo-2-hydroxy-3-methoxy-benzoic acid and
1b as described above for 2a. The yield of 2b was 16.7%. The 1H
NMR spectrum (300 MHz, CDCl3) of the purified product was
1.73-1.76 (m, 4H), 2.57-2.59 (m, 2H), 2.76-2.81 (m, 4H), 3.45-
3.47 (m, 2H), 3.58-3.61 (m, 2H), 3.82 (s, 3H), 3.86 (s, 3H), 3.88
(s, 3H), 6.48-6.51 (t, 1H), 6.56-6.59 (t, 1H), 6.97-7.00 (m, 1H),
7.07-7.10 (m, 1H). LCMS m/z 493.10 (M + H).
a Abbreviations: YUN-143, N-(4-fluorobenzyl)piperidinyl-4-(3-bro-
mophenyl) acetamide; CT, computed tomography.

3200 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14
Tu et al.
N-[4-(6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl]-
2-hydroxy-5-methyl-benzamide (2c). Compound 2c was prepared
from 2-hydroxy-5-methyl-benzoic acid and 1b as described above
experiments, the free base was converted into the oxalic acid salt;
mp 131-133 °C. Anal. (C26H34FN2O2) C, H, N.
N-[4-(6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl]-
2-(2-fluoro-ethoxy)-5-bromo-benzamide (3d). Compound 3d was
prepared from 2d as described above for 3a. The yield of 3d was
38.90%. The 1H NMR spectrum (300 MHz, CDCl3) of the purified
product was 1.57-1.80 (m, 4H), 2.62-2.66 (m, 3H), 2.78-2.82
(m, 3H), 3.48-3.51 (m, 2H), 3.60-3.64 (m, 2H), 3.82 (s, 3H),
3.83 (s, 3H), 4.21-4.25 (t, 1H), 4.31-4.35 (t, 1H), 4.70-4.74 (t,
1H), 4.86-4.90 (t, 1H), 6.49 (s, 1H), 6.57 (s, 1H), 6.78-6.82 (d,
1H), 7.48-7.52 (d, 1H), 7.96 (s, 1H), 8.26 (d, 1H). LCMS m/z
509.1 (M + H). For the in vitro binding experiments, the free base
was converted into the oxalic acid salt; mp 119-121 °C. Anal.
(C25H31BrFN2O6) C, H, N.
N-[4-(6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl]-
2-(2-fluoro-ethoxy)-5-iodo-benzamide (3e). Compound 3e was
prepared from 2e as described above for 3a. The yield of 3e was
41.4%. The 1H NMR spectrum (300 MHz, CDCl3) of the purified
product was 1.61-1.72 (m, 4H), 2.46-2.52 (m, 2H), 2.67-2.71
(m, 2H), 2.76-2.78 (m, 2H), 3.41-3.47 (m, 2H), 3.52 (t, 2H),
3.80 (s, 3H), 3.83 (s, 3H), 4.19-4.21 (m, 1H), 4.27-4.31 (m, 1H),
4.67-4.71 (m, 1H), 4.83-4.86 (m, 1H), 6.46 (s, 1H), 6.55 (s, 1H),
6.62-6.66 (d, 1H), 7.62-7.67 (m, 1H), 7.87 (s, 1H), 8.40-8.41
(d, 1H). LCMS m/z 557.13 (M + H). For the in vitro binding
experiments, the free base was converted into the oxalic acid salt;
mp 121-123 °C. Anal. (C25H31FIN2O6) C, H, N.
1
for 2a. The yield of 2c was 45%. The H NMR spectrum (300
MHz, CDCl3) of the purified product was 1.75 (m, 4H), 2.12 (s,
3H), 2.58 (m, 2H), 2.75-2.77 (m, 2H), 2.82-2.84 (m, 2H), 3.40-
3.50 (m, 2H), 3.58 (s, 2H), 3.83 (s, 3H), 3.85 (s, 3H), 6.50 (s, 1H),
6.60 (s, 1H), 6.85 - 6.88 (d, 1H), 7.06 (s, 1H), 7.13-7.16 (d, 2H),
7.61 (s, 1H). LCMS m/z 399.20 (M + H).
N-[4-(6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl]-
2-hydroxy-5-bromo-benzamide (2d). Compound 2d was prepared
from 5-bromo-2-hydroxy-benzoic acid and 1b as described above
1
for 2a. The yield of 2d was 28.0%. The H NMR spectrum (300
MHz, CDCl3) of the purified product was 1.71-1.81 (m, 4H),
2.55-2.85 (m, 6H), 3.44-3.48 (m, 2H), 3.58-3.60 (m, 2H), 3.82
(s, 3H), 3.89 (s, 3H), 6.50 (s, 1H), 6.59 (s, 1H), 6.82-6.84 (d,
1H), 7.30-7.40 (d, 1H), 7.52 (d, 1H), 8.30 (s,1H). Anal. (C22H27-
BrN2O4‚1.25H2O) C, H, N.
N-[4-(6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl]-
2-hydroxy-5-iodo-benzamide (2e). Compound 2e was prepared
form 2-hydroxy-5-iodo-benzoic acid and 1b as described above for
2a. The yield of 2e was 27.0%. The 1H NMR spectrum (300 MHz,
CDCl3) of the purified product was 1.69-1.81 (m, 4H), 2.54-
2.65 (m, 2H), 2.75-2.83 (m, 2H), 3.44-3.48 (m, 2H), 3.58 (s,
2H), 3.82 (s, 3H), 3.85 (s, 3H), 6.50 (s, 1H), 6.58 (s, 1H), 6.70-
6.74 (d, 1H), 7.54-7.55 (d, 1H), 7.65-7.67 (d, 1H), 8.20 (s, 1H).
Anal. (C22H27IN2O4‚0.75H2O) C, H, N.
Procedure C: General Method for Converting the Substi-
tuted 5-Bromo-benzoic Acid Derivatives into their Substituted
5-Tributylstannanyl Benzoic Acid Derivatives. N-[4-(6,7-
Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl]-2-(2-fluoro-
ethoxy)-3-methoxy-5-tributylstannanyl-benzamide (3g). Nitrogen
was bubbled for 5-10 min through a solution of 3b (200 mg, 0.371
mmol) in 20 mL of fresh distilled toluene. The whole system was
covered with aluminum foil. Tetrakis(triphenylphosphine palladium-
(0) [(PPh3)4Pd(0); 42 mg, 0.036 mmol] and bis(tributytin) ([Sn-
(C4H9)3]2; 575 mg, 0.99 mmol) was added to the reaction mixture
and heated overnight at 110 °C with an oil bath. Thin layer
chromatography with 45% hexane, 45% ethyl ether, and 10%
methanol as the mobile phase was used to assess when the reaction
was complete. After quenching the reaction, the crude product was
purified on a silica gel column to isolate the tin intermediate, 3g.
Procedure B: General Method for the Synthesis of the
Substituted 2-(2-Fluoroethoxy) Benzoic Acid Amides, 3a-e.
[N-(6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-ethyl]-2-(2-
fluoro-ethoxy)-5-methyl-benzamide (3a). Potassium carbonate
(792.5 mg, 4.88 mmol) was added to a solution of 2a (278 mg,
0.75 mmol) and 2-bromo-1-fluoroethane (620 mg, 4.88 mmol) in
acetone (60 mL). The reaction mixture was refluxed for 48 h until
the reaction was complete as determined by thin layer chromatog-
raphy with 5% methanol and 95% ethyl ether as the mobile phase.
The solvent was evaporated, 30 mL of water was added to the flask,
and then the mixture was extracted with dichloromethane (25 mL
× 3). After the organic layer was dried with anhydrous sodium
sulfate, the crude product was purified by column chromatography
using 5% methanol and 95% ethyl ether as the mobile phase. The
1
1
yield of 3a was 90%. The H NMR spectrum (300 MHz, CDCl3)
The yield of 3g was 64%. The H NMR spectrum (300 MHz,
of the purified product was 2.33 (s, 3H), 2.64-2.80 (m, 6H), 3.63-
3.75 (m, 4H), 3.84 (s, 3H), 3.86 (s, 3H), 4.16 (m, 1H), 4.21 (m,
1H), 4.41 (m, 1H), 4.60 (m, 1H), 6.55 (s, 1H), 6.61 (s, 1H), 6.78-
6.81 (d, 1H), 7.20 (d, 1H), 8.00 (s, 1H), 8.28 (s, 1H). LCMS m/z
417.22 (M + H). For the in vitro binding experiments, the free
base was converted into the hydrochloride salt; mp 159-161 °C.
Anal. (C23H30ClFN2O4) C, H, N.
CDCl3) of the purified product was 0.87-1.58 (m, 27H), 1.61-
1.69 (m, 4H), 2.56 (s, 2H), 2.72 (s, 2H), 2.81-2.82 (s, 2H), 3.47-
3.49 (d, 2H), 3.57 (s, 2H), 3.83 (s, 6H), 3.88 (s, 3H), 4.25 (d, 1H),
4.37 (s, 1H), 4.62-4.65 (s, 1H), 4.78-4.81 (s, 1H), 6.51 (s, 1H),
6.58 (s, 1H), 7.81 (s, 1H), 8.07 (s, 1H). LCMS m/z 747.60 (M -
H).
Procedure D: General Method for Converting the Tin
Precursor of the Benzoic Acid Derivatives into their Corre-
sponding Iodine Substituted Benzoic Acid Derivatives. (N-[4-
(6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl]-2-(2-
fluoro-ethoxy)-3-methoxy-5-iodo-benzamide (3f). A solution of
iodine in CHCl3 (5 mL, 0.5 M) was added dropwise to a solution
of the tin precursor, 3g (258 mg, 0.34 mmol), in 20 mL of CH2Cl2
until the color of the solution persisted. The reaction was stirred at
room temperature for 30 min, and a solution of 5% aqueous
NaHSO3 was added until the solution was colorless. The mixture
was extracted with CH2Cl2, and the organic layers were washed
with brine before being dried with Na2SO4. The organic layers were
then concentrated under vacuum and purified using a silica gel
column with 15% methanol and 85% ether as the mobile phase to
5-Bromo-N-[4-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-
yl)-butyl]-2-(2-fluoro-ethoxy)-3-methoxy-benzamide (3b). Com-
pound 3b was prepared from 2b as described for 3a above. The
1
yield of 3a was 50%. The H NMR spectrum (300 MHz, CDCl3)
of the purified product was 1.64 (m, 4H), 2.48-2.52 (t, 3H), 2.64-
2.66 (t, 3H), 2.74-2.78 (t, 3H), 3.42-3.55 (m, 4H), 3.79-3.87
(m, 9H), 4.19-4.22 (t, 2H), 4.29-4.32 (t, 2H), 4.58-4.61 (t, 2H),
4.74-4.77 (t, 2H), 6.47 (s, 1H), 6.54 (s, 1H), 7.07 (d, 1H), 7.78
(d, 1H), 8.10 (s, 1H). For the in vitro binding experiments, the free
base was converted into the oxalic acid salt; mp 127-129 °C.
LCMS m/z 590.30 (M + Li). Anal. (C26H33BrFN2O7) C, H, N.
N-[6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl-butyl]-2-
(2-fluoro-ethoxy)-5-methyl-benzamide (3c). Compound 3c was
prepared from 2c as described above for 3a. The yield of 3c was
1
isolate 3f. The yield of 3f was 36%. The H NMR spectrum (300
1
MHz, CDCl3) of the purified product was 1.60-1.80 (m, 2H),
1.80-2.10 (m, 4H), 3.19-3.2 (m, 2H), 3.40-3.50 (m, 2H), 3.68
(m, 2H), 3.83 (m, 2H), 3.92 (s, 3H), 3.95 (s, 3H), 3.99 (s, 3H),
4.26 (t, 1H), 4.37 (s, 1H), 4.65 (s, 1H), 4.80 (s, 1H), 6.50 (s, 1H),
6.58 (s, 1H), 7.28 (d, 1H), 8.02 (d, 1H), 8.21 (s, 1H). LCMS m/z
587.14 (M + H). For the in vitro binding experiments, the free
base was converted into the oxalic acid salt; mp 125-127 °C.
67%. The H NMR spectrum (300 MHz, CDCl3) of the purified
product was 1.67-2.00 (m, 4H), 2.33 (s, 3H), 2.51-2.56
(t, 3H), 2.67-2.72 (t, 3H), 3H), 2.78-2.82 (t, 3H), 3.48-3.54 (m,
4H), 3.82 (s, 3H), 3,83 (s, 3H), 4.21-4.24 (t, 1H), 4.30-4.33 (t,
1H), 4.68-4.71 (t, 1H), 4.84-4.87 (t, 1H), 6.49 (s, 1H), 6.57 (s,
1H), 6.79-6.82 (d, 1H), 7.20 (m, 1H), 7.96 (s, 1H), 7.99-7.80 (d,
1H). LCMS m/z 445.25 (M + H). For the in vitro binding

18F-Labeled Benzamide Analogues for Imaging
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14 3201
Procedure E: General Method for Converting the Substi-
tuted 2-Hydroxy Benzoic Acid Derivatives into their Substituted
2-(2-Hydroxy-ethoxy)-benzoic Acid Amides. N-[4-6,7-Dimethoxy-
3,4-dihydro-1H-isoquinolin-2-yl)-butyl]-2-(2-hydroxy-ethoxy)-
5-methyl-benzamide (4c). Anhydrous potassium carbonate (546.0
mg, 3.26 mmol) was added to a solution of 2c (200.0 mg, 0.5 mmol)
and 2-bromoethyl acetate (547.0 mg, 3.27 mmol) in 60 mL of
acetone. The reaction mixture was refluxed for 48 h under nitrogen.
After 48 h, thin layer chromatography with 15% methanol and 85%
ether as the mobile phase indicated that the reaction was complete.
After evaporating the solvent, the residue was dissolved in 30 mL
of water and extracted with ethyl acetate (20 × 3 mL). Then the
organic component was washed with brine, dried with anhydrous
sodium sulfate, and concentrated, and the final product was purified
on a silica gel column to isolate the 2-{2-[4-(6,7-dimethoxy-3,4-
dihydro-1H-isoquinolin-2-yl)-butylcarbamoyl]-4-methyl-phenoxy}-
ethyl ester. The yield of this intermediate was 82.2%. The 1H NMR
spectrum (300 MHz, CDCl3) of the purified product was 1.70 (m,
4H), 2.01 (s, 3H), 2.33 (s, 3H), 2.56 (m, 2H), 2.71-2.73 (m, 2H),
2.81 (m, 2H), 3.50-3.52 (m, 2H), 3.55 (s, 2H), 3.83 (s, 3H), 3.84
(s, 3H), 4.23 (t, 2H), 4.50 (t, 2H), 6.50 (s, 1H), 6.58 (s, 1H), 6.78-
6.82 (d, 1H), 7.18-7.25 (d, 1H), 7.95 (s, 1H), 8.02 (s, 1H).
NaOH (30 mg, 0.75mmol) was added to a solution of this
intermediate (182 mg, 0.375mmol) in 20 mL of methanol and 10
mL of water. The reaction mixture was stirred overnight until the
reaction was complete. Then 0.375 mL of 2 N HCl was added to
neutralize the solution. After evaporating the solvent, the residue
was dissolved in 60 mL of ethyl acetate. The solution was washed
first with water, then brine, and finally dried with anhydrous sodium
sulfate. After evaporating the solvent, the crude product was purified
4H), 4.20 (s, 3H), 4.22 (s, 3H), 4.60-4.68 (t, 2H), 4.95-4.97 (t,
2H), 6.88 (s, 1H), 6.95 (s, 1H), 7.15-7.18 (d, 1H), 7.50-7.60 (d,
1H), 8.20 (s, 1H), 8.19 (s, 1H). Anal. (C26H36N2O7S) C, H, N.
2-(4-Bromo-2-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-
yl)butylcarbamoyl)phenoxy)ethyl Methanesulfonate (5d). Com-
pound 5d was prepared from 4d as described above for 5c. The
1
yield of 5d was 77%. The H NMR spectrum (300 MHz, CDCl3)
of the purified product was 1.72-1.75 (m, 4H), 2.61-2.68 (m, 2H),
2.85 (s, 3H), 3.05 (s, 2H), 3.42-3.58 (m, 4H), 3.68 (s, 2H), 3.82
(s, 3H), 3.83 (s, 3H), 4.29 (t, 2H), 4.60 (t, 2H), 6.50 (s, 1H), 6.57
(s, 1H), 6.70-6.77 (d, 1H), 7.45-7.55 (d, 1H), 7.95 (s, 1H), 8.20-
8.22 (d, 1H). LCMS m/z 585.10 (M + H).
2-(2-(4-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)bu-
tylcarbamoyl)-4-iodophenoxy)ethyl Methanesulfonate (5e). Com-
pound 5e was prepared from 4e as described above for 5c. The
1
yield of 5e was 80%. The H NMR spectrum (300 MHz, CDCl3)
of the purified product was 1.60-1.80 (m, 4H), 2.52-2.55 (m, 2H),
2.67-2.70 (m, 2H), 2.70-2.78 (m, 2H), 3.02 (s, 3H), 3.46-3.52
(m, 4H), 3.82 (s, 3H), 3.83 (s, 3H), 4.23-4.26 (t, 2H), 4.56-4.60
(t, 2H), 6.48 (s, 1H), 6.55 (s, 1H), 6.60-6.64 (d, 1H), 7.64-7.68
(d, 1H), 7.85 (s, 1H), 8.38-8.39 (d, 1H). LCMS m/z 633.10 (M +
H).
2-(2-Acetoxy-ethoxy)-5-bromo-3-methoxy-benzoic Acid Meth-
yl Ester (7). Initially, 1.0 mL of 98% concentrated sulfuric acid
was added to a solution of 5-bromo-2-hydroxy-3-methoxy-benzoic
acid, 6 (1.0 g, 4.0 mmol), in 50 mL of methanol. The reaction
mixture was refluxed overnight until thin layer chromatography
using 20% ethyl acetate and 80% hexane as the mobile phase
indicated that the reaction was complete. After evaporating the
methanol, the residue was dissolved in 60 mL of ethyl acetate and
washed with a saturated NaHCO3 aqueous solution and then brine.
After drying with anhydrous sodium sulfate, the solution was
concentrated and the crude product was purified on a silica gel
column to isolate the intermediate, 5-bromo-2-hydroxy-3-methoxy-
benzoic acid methyl ester. The yield of this intermediate was 94%.
The 1H NMR spectrum (300 MHz, CDCl3) of the purified product
was 3.88 (s, 3H), 3.95 (s, 3H), 7.09 (d, 1H), 7.55 (t, 1H), 10.96 (d,
1H).
Potassium carbonate (2.90 g, 21.0 mmol) was added to a solution
of the above intermediate (0.84 g, 3.23 mmol) and 2-bromoethyl
acetate (3.5 g, 20.96 mmol) in 60 mL of acetone. The reaction
mixture was refluxed for 72 h until thin layer chromatography using
20% ethyl acetate and 80% hexane as the mobile phase indicated
that the reaction was complete. After evaporating the solvent, the
residue was dissolved in 30 mL of water and then extracted with
ethyl acetate (25 mL × 3). The organic solution was dried with
anhydrous sodium sulfate, resuspended, and purified on a silica
gel column. The yield of 7 was 78%. The 1H NMR spectrum (300
MHz, CDCl3) of the purified product was 2.10 (s, 3H), 3.86 (s,
3H), 3.88 (s, 3H), 4.21-4.24 (t, 2H), 4.38-4.41 (t, 3H), 7.14 (s,
1H), 7.45 (s, 1H).
1
on a silica gel column. The yield of 4c was 96%. The H NMR
spectrum (300 MHz, CDCl3) of the purified product was 1.68-
1.85 (m, 4H), 2.39 (s, 3H), 2.45 (s, 1H), 2.51-2.61 (m, 2H), 2.80-
2.87 (m, 4H), 3.45-3.61 (m, 4H), 3.76-3.80 (t, 2H), 3.83 (s, 3H),
3.85 (s, 3H), 3.83 (s, 3H), 4.05-4.08 (t, 2H), 6.49 (s, 1H), 6.60 (s,
1H), 6.79-6.83 (d, 1H), 7.15-7.19 (d, 2H), 7.93 (s, 1H), 8.30 (s,
1H).
5-Bromo-N-[4-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-
yl)-butyl]-2-(2-hydroxy-ethoxy)-benzamide (4d). Compound 4d
was prepared from 2d as described above for 4c. The yield of 4d
was 70%. The 1H NMR spectrum (300 MHz, CDCl3) of the purified
product was 1.65-1.90 (m, 4H), 2.66-2.70 (m, 2H), 2.92 (m, 4H),
3.51-3.54 (m, 2H), 3.72 (m, 2H), 3.72-3.81 (t, 2H), 3.83 (s, 3H),
3.85 (s, 3H), 4.05-4.09 (t, 2H), 6.51 (s, 1H), 6.60 (s, 1H), 6.77-
6.81 (d, 1H), 7.44-7.48 (d, 2H), 8.17 (s, 1H), 8.30 (s, 1H).
N-[4-(6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl]-
2-(2-hydroxy-ethoxy)-5-iodo-benzamide (4e). Compound 4e was
prepared from 2e as described above for 4c. The yield of 4e was
1
77%. The H NMR spectrum (300 MHz, CDCl3) of the purified
product was 1.60-1.90 (m, 4H), 2.63-2.66 (m, 3H), 2.90 (s, 2H),
3.52-3.55 (m, 2H), 3.70 (m, 2H), 3.79-3.82 (t, 2H), 3.83 (s, 3H),
3.85 (s, 3H), 4.08-4.10 (t, 2H), 6.50 (s, 1H), 6.60 (s, 1H), 6.67-
6.70 (d, 1H), 7.60-7.70 (d, 1H), 8.30 (s, 1H), 8.40-8.41 (d, 1H).
Procedure F: General Method for Converting the Sub-
stituted 2-Hydroxy-ethoxy Benzoic Acid Amides to their
Methanesulfonic Acid Esters. 2-(2-(4-(6,7-Dimethoxy-3,4-dihy-
droisoquinolin-2(1H)-yl)butylcarbamoyl)-4-methylphenoxy)-
ethyl Methanesulfonate (5c). Methanesulfonic chloride (120 mg,
1.04 mmol) was added to an ice-water cooled solution of 4c (354
mg, 0.8 mmol) and triethylamine (242 mg, 2.4 mmol) in 30 mL of
dichloromethane. The reaction mixture was stirred for 3 h until
thin layer chromatography using 5% methanol and 95% dichlo-
romethane as the mobile phase indicated that the reaction was
complete. After 3 h, 20 mL of dichloromethane was added, the
solution was washed with first a saturated sodium carbonate aqueous
solution (20 mL × 3) and then brine, and finally dried with
anhydrous sodium sulfate. After evaporating the solvent, the crude
product was purified on a silica gel column to isolate 5c. The yield
2-(2-Acetoxy-ethoxy)-5-iodo-3-methoxy-benzoic Acid Methyl
Ester (8). Nitrogen was bubbled for 5-10 min through a solution
of of 2-(2-acetoxy-ethoxy)-5-bromo-3-methoxy-benzoic acid methyl
ester, 7 (270 mg, 0.778 mmol), in 20 mL of freshly distilled toluene.
The reaction system was covered with aluminum foil. Tetrakis-
(triphenylphosphine) palladium(0) [(PPh3)4Pd(0); 100 mg, 0.087
mmol] and bis(tributlytin) ([Sn(C4H9)3]2; 899 mg, 1.55 mmol) were
added to the reaction mixture and heated overnight at 110 °C in an
oil-bath while stirring. After quenching, thin layer chromatography
using 15% ethyl acetate and 85% hexane as the mobile phase
indicated that the reaction was complete. The product was then
purified on a silica gel column to isolate the tin precursor, 2-(2-
acetoxy-ethoxy)-3-methoxy-5-tributylstannanyl-benzoic acid methyl
1
ester. The yield of the tin precursor was 37.3%. The H NMR
spectrum (300 MHz, CDCl3) of the purified product was 0.8-1.75
(m, 27H), 2.10 (s, 3H), 3.87 (s, 3H), 3.89 (s, 3H), 4.24-4.27 (t,
2H), 4.38-4.41 (t, 2H), 7.09-7.30 (s 1H), 7.35 (s 1H).
A solution of iodine in CHCl3 (5 mL, 0.5 M) was added dropwise
to a solution of above tin precursor (680 mg, 1.22 mmol) in 20
mL of CH2Cl2 until the color of the solution persisted. Then the
1
of 5c was 81.6%. The H NMR spectrum (300 MHz, CDCl3) of
the purified product was 2.07-2.10 (m, 4H), 2.71 (s, 3H), 2.80-
3.00 (m, 2H), 3.08-3.20 (m, 4H), 3.40 (m, 3H), 3.80-4.00 (m,

3202 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14
Tu et al.
reaction was stirred at room temperature for 30 min, and a quench
solution of 5% aqueous NaHSO3 was added until the solution
became colorless. The mixture was extracted with CH2Cl2, and the
organic layers were washed with brine and dried by Na2SO4. The
organic layers were then condensed under vacuum and purified
using a silica gel column with 15% ethyl acetate and 85% hexane
as the mobile phase. The yield of 8 was 90%. The 1H NMR
spectrum (300 MHz, CDCl3) of the purified product was 2.10 (s,
3H), 3.85 (s, 3H), 3.88 (s, 3H), 4.21-4.25 (t, 2H), 4.37-4.41 (t,
2H), 7.29-7.30 (s 1H), 7.63 (s 1H).
2-(2-Hydroxy-ethoxy)-5-iodo-3-methoxy-benzoic Acid (9). Com-
pound 9 was prepared from 8 as described in Procedure E. The
yield of 9 was 81%. The 1H NMR spectrum (300 MHz, CDCl3) of
the purified product was 3.89 (s, 3H), 3.93-3.96 (t, 2H), 4.33-
4.36 (t, 2H), 7.08 (s, 1H), 7.62 (s, 1H).
the biodistribution and imaging studies. The entire procedure
required ∼2 h.
Quality control analysis was performed on an analytical HPLC
system that consisted of an Alltech econosil reversed phase C-18
column (250 × 4.6 mm) with a mobile phase of 35% acetonitrile
and 65% 0.1 M ammonium formate buffer at pH 4.0-4.5. At a
flow rate of 1.2 mL/min, the [18F]3f eluted at 13.2 min with a
radiochemical purity of >99%. The labeling yield was ∼30%
(decay corrected), and the specific activity was >2000 Ci/mmol.
[18F](N-[4-(6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-
butyl]-2-(2-fluoro-ethoxy)-5-methyl-benzamide ([18F]3c). Com-
pound [18F]3c was prepared from 5c as described above for [18F]3f,
with the following exceptions. The semipreparative HPLC mobile
phase was 39% methanol and 61% 0.1 M formate buffer. At a flow
rate of 3.5 mL/min, the [18F]3c eluted at ∼33 min with a
radiochemical purity of >99%. The labeling yield was ∼35%
(decay corrected), and the specific activity was >1500 Ci/mmol.
The entire procedure took ∼2 h.
N-[4-(6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl]-
2-(2-hydroxy-ethoxy)-5-iodo-3-methoxy-benzamide (4f). Com-
pound 4f was prepared from 9 and 1b as described in Procedure
To check that the chemical characteristics of [18F]3c were
identical to the cold standard, 3c, both compounds were run on the
analytical HPLC system with a mobile phase of 52% methanol and
48% 0.1 M formate buffer. At a flow rate of 1.5 mL/min, the two
compounds coeluted with a retention time of 4.7 min.
1
A. The yield of 4f was 29%. The H NMR spectrum (300 MHz,
CDCl3) of the purified product was 1.72-1.75 (m, 4H), 2.56 (m,
2H), 2.75-2.77 (m, 2H), 2.81-2.83 (m, 2H), 3.49-3.51 (m, 2H),
3.55 (s, 2H), 3.56-3.60 (t, 2H), 3.82 (s, 3H), 3.83 (s, 3H), 3.85 (s,
3H), 4.06-4.10 (t, 2H), 6.47 (s, 1H), 6.57 (s, 1H), 6.90 (s, 1H),
7.57 (s, 1H), 7.70-7.80 (s, 1H).
[18F](N-[4-(6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-
butyl]-2-(2-fluoro-ethoxy)-5-bromo-benzamide ([18F]3d). Com-
pound [18F]3d was prepared from 5d as described above for [18F]3f
with the following exceptions. The semipreparative HPLC mobile
phase was 13% of THF and 87% 0.1 M formate buffer. At a flow
rate of 3.5 mL/min, the [18F]3d eluted at ∼20 min with a
radiochemical purity of >98%. The labeling yield was ∼30%
(decay corrected), and the specific activity was >1500 Ci/mmol.
The entire procedure took ∼2 h.
2-(2-(4-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)bu-
tylcarbamoyl)-4-iodo-6-methoxyphenoxy)ethyl Methanesulfonate
(5f). Compound 5f was prepared from 4f as described in Procedure
1
F. The yield of 5f was 61%. The H NMR spectrum (300 MHz,
CDCl3) of the purified product was 1.72 (m, 4H), 2.55 (s, 2H),
2.70 (d, 2H), 22.76 (d, 2H), 3.05 (s, 3H), 3.85 (m, 9H), 4.26 (m,
2H), 4.49 (m, 2H), 6.49 (s, 1H), 6.55 (s, 1H), 6.93 (d, 1H), 7.51
(m, 1H), 8.02 (s, 1H). LCMS m/z 663.20 (M + H). Anal. (C25H32-
FIN2O5) Calcd: C, 51.20; H, 5.50; N, 4.78. Found: C, 36.61; H,
4.34; N, 3.12.
Radiochemistry. Production of [18F]Fluoride. [18F]Fluoride
was produced in our institution by proton irradiation of enriched
18O water (95%) [reaction: 18O(p, n)18F] using either a JSW BC-
16/8 (Japan Steel Works) or a CS-15 cyclotron (Cyclotron Corp)
To check that the chemical characteristics of [18F]3d were
identical to the cold standard, 3d, both compounds were run on
the analytical HPLC system with a mobile phase of 38% acetonitile
and 62% 0.1 M formate buffer. At a flow rate of 1.5 mL/min, the
two compounds coeluted with a retention time of ∼8 min.
[18F](N-[4-(6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-
butyl]-2-(2-fluoro-ethoxy)-5-Iodo-benzamide ([18F]3e). Com-
pound [18F]3e was prepared from 5e as described above for [18F]3f,
with the following exceptions. The semipreparative HPLC mobile
phase was 15% THF and 85% 0.1 M formate buffer. At a flow
rate of 6.0 mL/min, the [18F]3e eluted at ∼35 min with a
radiochemical purity of >99%. The labeling yield was ∼30%
(decay corrected), and the specific activity was >1500 Ci/mmol.
The entire procedure took ∼2 h.
Procedure G: General Method for Labeling the Substituted
2-(2-Fluoroethoxy) Benzoic Acid Amide Analogs with 18F. [18F]-
(N-[4-(6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl]-
2-(2-fluoro-ethoxy)-3-methoxy-5-iodo-benzamide ([18F]3f). [18F]-
fluoride (100-150 mCi) was added to a 10-mL Pyrex screw cap
tube containing 5-6 mg of Kryptofix 222 and 0.75 mg of K2CO3.
Using HPLC grade acetonitrile (3 × 1.0 mL), the water was
azeotropically evaporated from this mixture at 110 °C under a
stream of argon. After all of the water was removed, a solution of
the precursor, 5f (1.5-2.0 mg), in DMSO (0.2 mL) was added to
the reaction vessel containing the 18F/Kryptofix mixture. A 3 mm
glass bead was added to the reaction vessel to ensure a more
homogeneous heat distribution when the sample was irradiated with
microwaves, and the vessel was capped firmly on a specially
designed remotely operated capping station. After vortexing, the
reaction mixture was irradiated with microwaves for 30-40 s at
medium power (60 W) until the thin layer chromatography scanner
with a 25% of methanol and 75% dichloromethane mobile phase
indicated that the incorporation yield was 40-60%.
After adding 6 mL of water and shaking, the solution was loaded
on a C-18 reverse phase Waters Oasis cartridge (HLB-6 cm3) that
had previously been rinsed with a solution of 5% methanol in water
(5-8 mL). The sample was then rinsed three times with 6 mL of
water to eliminate the unreacted fluoride. The retained activity was
eluted with 5-8 mL of acetonitrile. After evaporating the aceto-
nitrile to a volume of <0.5 mL, the sample was loaded on a C-18
Alltech econosil semipreparative HPLC column (250 × 10 um).
The product was eluted with 29% acetonitrile and 71% 0.1 M
ammonium formate buffer at a flow rate of 4.5 mL/min. The
retention time of the [18F]3f was ∼33 min. The solution containing
the [18F]3f was concentrated and resuspended in saline, and a 100
µL aliquot was sent for quality control analysis before using it in
To check that the chemical characteristics of [18F]3e were
identical to the cold standard, 3e, both compounds were run on the
analytical HPLC system with a mobile phase of 17% tetrahydro-
furan and 83% 0.1 M ammonium formate buffer. At a flow rate of
2.0 mL/min, the two compounds coeluted with a retention time of
15.2 min.
Characterization of the Potential σ2 Selective Ligands In
Vitro and In Vivo. Sigma Receptor Binding Assays. The novel
sigma ligands were dissolved in N,N-dimethylformamide (DMF),
DMSO, or ethanol and then diluted in 50 mM Tris-HCl buffer
containing 150 mM NaCl and 100 mM EDTA at pH 7.4 prior to
performing the σ1 and σ2 receptor binding assays The procedures
for isolating the membrane homogenates and performing the σ1
and σ2 receptor binding assays have been described in detail
previously.21
Briefly, the σ1 receptor binding assays were conducted in 96-
well plates using guinea pig brain membrane homogenates (∼300
µg protein) and ∼5 nM [3H](+)-pentazocine (34.9 Ci/mmol, Perkin-
Elmer, Boston, MA). The total incubation time was 90 min at room
temperature. Nonspecific binding was determined from samples that
contained 10 µM of cold haloperidol. After 90 min, the reaction
was terminated by the addition of 150 µL of ice-cold wash buffer
(10 mM Tris-HCl, 150 mM NaCl, pH 7.4) using a 96 channel
transfer pipet (Fisher Scientific, Pittsburgh, PA). The samples were
harvested and filtered rapidly through a 96-well fiberglass filter

18F-Labeled Benzamide Analogues for Imaging
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14 3203
References
plate (Millipore, Billerica, MA) that had been presoaked with 100
µL of 50 mM Tris-HCl buffer at pH 8.0 for 1 h. Each filter was
washed three times with 200 µL of ice-cold wash buffer, and the
filter was counted in a Wallac 1450 MicroBeta liquid scintillation
counter (Perkin-Elmer, Boston, MA).
The σ2 receptor binding assays were conducted using rat liver
membrane homogenates (∼300 µg protein) and ∼5 nM [3H]DTG
(58.1 Ci/mmol, Perkin-Elmer, Boston, MA) in the presence of 1
µM (+)-pentazocine to block σ1 sites. The incubation time was
120 min at room temperature. Nonspecific binding was determined
from samples that contained 10 µM of cold haloperidol. All other
procedures were identical to those described for the σ1 receptor
binding assay above.
Data from the competitive inhibition experiments were modeled
using nonlinear regression analysis to determine the concentration
that inhibits 50% of the specific binding of the radioligand (IC50
value). Competitive curves were best fit to a one-site fit and gave
pseudo-Hill coefficients of 0.6-1.0. Ki values were calculated using
the method of Cheng and Prusoff 24 and are presented as the mean
( 1 SEM. For these calculations, we used a Kd value of 7.89 nM
for [3H](+)-pentazocine and guinea pig brain; for [3H]DTG and
rat liver, we used 30.73 nM20
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Acknowledgment. This work was supported by Grant
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Grant DAMD17-01-1-0446 awarded by the Department of
Defense Breast Cancer Research Program of the U.S. Army
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for their excellent technical assistance. Mass spectrometry
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Supporting Information Available: Analytical data on all new
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