Preparation of a technetium-99m SPECT agent for imaging the sigma-2 receptor status of solid tumors

Article abstract of DOI:10.1002/jlcr.516

The synthesis and in vitro binding of a novel ^99mTc-labeled radiotracer for imaging the sigma-2 (σ₂) receptor status of breast tumors is described. Structural characterization and in vitro binding studies were conducted using the cor

Full text of DOI:10.1002/jlcr.516

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Labelled Cpd Radiopharm 2001; 44: 899–908.
DOI: 10.1002/jlcr.516
Research Article
Preparation of a technetium-99m SPECT
agent for imaging the sigma-2 receptor status
of solid tumors
Robert H. Mach1,2,3,*, Kenneth T. Wheeler1, Suwanna Blair1,
Biao Yang3, Cynthia S. Day4, and Joseph B. Blair1, Seok-Rye Choi5,
Hank F. Kung5,6
1 Department of Radiology, Wake Forest University School of Medicine,
Winston-Salem, North Carolina 27157, USA
2 Department of Physiology and Pharmacology, Wake Forest University School
of Medicine, Winston-Salem, North Carolina 27157, USA
3 Anasazi Biomedical Research, Inc., Winston-Salem, NC 27101, USA
4 Chemistry Department, Wake Forest University, Winston-Salem,
NC 27106, USA
5 Department of Radiology, University of Pennsylvania, Philadelphia,
PA 19104, USA
6 Department of Pharmacology, University of Pennsylvania, Philadelphia,
PA 19104, USA
Summary
The synthesis and in vitro binding of a novel 99mTc-labeled radiotracer for
imaging the sigma-2 (s2) receptor status of breast tumors is described.
Structural characterization and in vitro binding studies were conducted using
the corresponding rhenium surrogate, Re-2. X-ray crystallographic studies
revealed that the complexation reaction gave exclusively the syn isomer. In vitro
binding studies indicated that this complex has a high affinity for s2
(Ki ¼ 13:7 nM) versus s1 receptors (Ki ¼ 1125 nM). These data indicate that
*Correspondence to: R. H. Mach, Department of Radiology, Div Radiologic Sciences, Wake
Forest University School of Medicine, Bowman Gray Campus, Medical Center Boulevard,
Winston-Salem, NC 27157-1088, USA. E-mail: mach@wfubmc.edu
Contract/grant sponsor: National Institute of Health; Contract/grant number: CA 77951;
Contract/grant sponsor: Department of Energy; Contract/grant number: ER 616567
Copyright # 2001 John Wiley & Sons, Ltd.
Received 21 May 2001
Revised 9 July 2001
Accepted 2August 2001

900
R. H. MACH ET AL.
[
99mTc]2 may be a promising agent for imaging the s2 receptor status of tumors
in vivo with the functional imaging technique, single photon emission
computed tomography (SPECT). Copyright # 2001 John Wiley & Sons, Ltd.
Key Words: sigma receptors; breast cancer; SPECT
Introduction
Sigma receptors represent a class of proteins that were initially classified
as a subtype of the opiate receptor.1 Subsequent studies revealed that
sigma receptors are a distinct class of proteins having a widespread
distribution in the CNS and peripheral tissues.1,2 Radioligand
binding and biochemical studies have revealed that there are two
types of sigma receptors, s1 and s2 receptors. The s1 receptor has
been cloned and displays a 30% sequence homology with the enzyme,
yeast sterol isomerase.3 The s2 receptor has not been cloned, but
evidence suggests that this receptor is linked to potassium channels in
NCB-20 cells.1,2
A number of studies have reported an overexpression of sigma
receptors in a variety of human and murine tumors.4,5 The observation
that the density of s2 receptors is greater than that of s1 receptors
in a wide variety of tumor cells grown under cell culture conditions5
suggests that the s2 receptor is a suitable target for developing
receptor-based radiotracers for noninvasive imaging procedures
such as single photon emission computed tomography (SPECT) and
positron emission tomography (PET). In addition, the recent observa-
tion that the density of s2 receptors is 10-fold higher in proliferating
versus quiescent mouse mammary adenocarcinoma cells both in vitro6,7
and in vivo8 suggests that radioligands possessing a high affinity and
selectivity for s2 receptors have the potential to measure the
proliferative status of breast tumors using noninvasive imaging
procedures.
Technetium-99m (99mTc) is the radionuclide of choice for nuclear
medicine imaging procedures. The popularity of this radionuclide stems
from the low cost and widespread availability of the 99Mo/99mTc
generator, its medium gamma ray energy (140 keV) which is suitable for
gamma camera detection, and its physical half-life (t1=2 ¼ 6 h) which is
convenient forimage acquisition. A numberof ercent studies have
indicated that it is possible to prepare 99mTc-labeled imaging agents
possessing a high affinity for a receptor. For example, [99mTc]TRO-
Copyright # 2001 John Wiley & Sons, Ltd.
J Labelled Cpd Radiopharm 2001; 44: 899–908

TECHNETIUM-99m SIGMA-2 RECEPTOR LIGAND
901
O
S
S
N
O
H3CO
N
C =O
Tc
H3C
N
O
Tc
N
S
N
N
F
S
C l
99mTc]TRODAT-1
[
99mTc]Technepine
[
CH3
N
H
O
O
N
H
OCH3
1
CH3
O
N
H
N
O
O
Tc
N
S
O
N
S
H
OCH3
[
99mTc]2
Figure 1. Structures of [99mTc]TRODAT-1, [99mTc]technepine, lead compound 1,
and [99mTc]2
DAT-19 and [99mTc]technepine10 have been shown to possess a high
affinity for the dopamine transporter (Figure 1). These studies suggest
the feasibility of preparing a small molecule based 99mTc-labeled
radiotracer for imaging s2 receptors provided that there is a suitable
lead compound for radiotracer development.
We previously reported that O-(9-benzyl-9-azabicyclo[3.3.1]nonan-
3a-yl)-N-(2-methoxy-4-methylphenyl)carbamate, 1, has a high affinity
and moderate selectivity for s2 versus s1 receptors.11 The goal of
the current study was to determine if it is possible to prepare
a
99mTc-labeled analogue of 1 using the strategy described pre-
viously forthe development of [ 99mTc] technepine (Figure 1). In
this communication, we describe the synthesis, in vitro binding,
and preliminary in vivo evaluation of [99mTc] 2, a potential radiotracer
formeasuirng the
SPECT.
s2 receptor status of solid tumors in vivo with
Copyright # 2001 John Wiley & Sons, Ltd.
J Labelled Cpd Radiopharm 2001; 44: 899–908

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R. H. MACH ET AL.
Results
Chemistry
The first step in the development of [99mTc]2 involved the synthesis of
the rhenium complex, Re-2. Since the chemistry of technetium and
rhenium are similar, the corresponding rhenium complex is generally
used in the structural characterization and in vitro binding studies of
receptor-based radiotracers using technetium as the radionuclide.
Reduction of 9-benzyl-9-azabicyclo[3.3.1]nonan-3-one, 3, with L-
Selectride gave the corresponding 3-a hydroxy granatane, 4, in 90%
yield.11 The NMR signal confirming the formation of the 3a-hydroxy
granatane product, 4, involved the interaction between the C1, C5
bridgehead protons and the equatorial protons on C2 and C4, which
produced the characteristic doublet with a large coupling constant
(Jꢀ10 Hz). This splitting pattern is similar to that observed with the 3a-
amino granatane analogues.12,13 Treatment of 4 with 2-methoxy-5-
methylisocyanate gave the corresponding carbamate derivative, 1, in
70% yield. Removal of the N-benzyl group via catalytic dehydrogena-
tion afforded the secondary amine, 5, in quantitative yield. Treatment of
amine 5 with [N-[2-[(30-chloropropyl)(2-mercaptoethyl)-amino]acetyl]2-
aminoethanethiolato]rhenium (V) oxide10 afforded the corresponding
rhenium complex, Re-2, in an overall yield of 45% that was exclusively
the syn isomer. This is consistent with the results obtained by Meltzer
et al. forthe synthesis of the rhenium-complex of technepine. 10 The syn
relationship between the Re¼O core and the N-alkyl substituent was
confirmed by X-ray crystallography (Figure 2).
In vitro binding studies. In vitro binding studies were conducted in
order to measure the affinity of the Re-2 for s1 and s2 receptors using
established assays for these receptors.14,15 The affinity of Re-2 forthe s2
receptor was 13.7 Æ 0.9 nM. The affinity of Re-2 for s1 receptors was
1125 Æ 35 nM, indicating that this compound has ꢀ80-fold selectivity
for s2 versus s1 receptors.
Radiolabeling studies. The N2S2 chelate, 6, was prepared using the
procedure outlined in Scheme 1.10 Radiolabeling with 99mTc was
accomplished using acidic conditions with Sn-glucoheptonate and
[
99mTc]sodium pertechnetate at 1208C for30 min. The porlonged
heating at this temperature was required to remove the trityl protecting
Copyright # 2001 John Wiley & Sons, Ltd.
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TECHNETIUM-99m SIGMA-2 RECEPTOR LIGAND
903
Figure 2. Crystal structure for the rhenium complex, Re-2
groups prior to complexation with 99mTc. The reaction mixture was
diluted with saturated aqueous sodium bicarbonate and the product was
extracted into ethyl acetate. The yield of the radiolabeling reaction
ranged from 10 to 57%. Reversed-phase HPLC analysis showed that
the syn isomer was obtained in >99% radiochemical purity (Figure 3).
The labeled compound, [99mTc]2, was stable at room temperature for at
least 18 h (data not shown).
In conclusion, we report in this communication the synthesis and
structural characterization of a Re–N2S2 complex possessing a
nanomolaraffinity and high (80-fold) selectivity for s2 versus s1
receptors. The high density of s2 receptors in a wide variety of human
tumors grown under cell culture conditions4,5 indicates that [99mTc]2
may be a useful radiotracer for imaging a wide variety of solid tumors.
Furthermore, in vivo biodistribution studies indicate that there is a high
uptake of [99mTc]2 in mouse mammary adenocarcinoma xenografts.16
Additional studies are currently being conducted in order to determine
if this radiotracer is suitable for SPECT studies aimed at determining
the s2 receptor status of solid tumors.
Copyright # 2001 John Wiley & Sons, Ltd.
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R. H. MACH ET AL.
H
N
N
O
a
OH
4
3
CH3
N
H
O
b
c
O
N
H
OCH3
1
O
CH3
CH3
H
N
H
O
O
d
N
H
O
N
N
S
M
O
N
O
N
H
H
S
OCH3
OCH3
5
2
; M = 99mTc: [99mTc]
Re-2
2
M = Re:
d
O
CH3
N
H
O
NH
N
O
N
H
Ph3CS
CPh3S
OCH3
6
Scheme 1. Reagents:(a) L-Selectride/THF/-788C; (b) 2-methoxy-5-methyliso-
cynate/CH2Cl2/dibutyltin diacetate; (c) H2/Pd-charcoal/methanol; (d)10
Figure 3. Reversed-phase HPLC chromatogram of Re-2 and [99mTc]2. Condi-
tions:PRP-1 column, 250 Â 4.1 mm; CH3CN/3,3-dimethylglutarate buffer,
5 mM, pH 7, volume ratio 9 :1; flow rate 1 ml/min. Re-2 was monitored at 254 nm
Copyright # 2001 John Wiley & Sons, Ltd.
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TECHNETIUM-99m SIGMA-2 RECEPTOR LIGAND
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Materials and methods
Experimental section
1H NMR spectra were recorded on a Bruker 300 MHz NMR spectro-
meter. Chemical shifts are reported in d values (parts per million, ppm)
relative to an internal standard of tetramethylsilane (TMS). The
following abbreviations are used for multiplicity of NMR signals:
brs=borad singlet, d=doublet, dd=doublet of doublets, m=multi-
plet, q=quartet, s=singlet, t=triplet, td=triplet of doublets. Melting
points were determined on a Fischer-Johns melting point apparatus and
are uncorrected. Elemental analyses were performed by Atlantic
Microlab, Inc., Norcross, GA and were within Æ 0.4% of the
calculated values. All reactions were carried out under an inert
atmosphere of nitrogen. Mass spectrometry studies were conducted by
the Washington University Resource for Biomedical and Bio-organic
Mass Spectrometry, St. Louis, MO. The radiosynthesis of [99mTc]2, in
vitro stability studies, and detailed in vivo biodistribution studies will be
published separately.16
[N-[2[-[30-N0-Propyl-300a(2-methoxy-5-methylphenylcarbamate)-
granatane]-(2-mercaptoethyl)amino]acetyl]-2-aminoethanethiolato]-
rhenium(V) oxide (Re-2). To a solution of amine 5 (340 mg,
1.13 mmol) in dry acetonitrile (10 ml) was added [N-[2-[(30-chloropro-
pyl)(2-mercaptoethyl)amino]acetyl]-2-aminoethane-thiolato]rhenium(V)
oxide10 (530 mg, 1.13 mmol), potassium iodide (187 mg, 1.13 mmol), and
potassium carbonate (1.6 g, 11.3 mmol). The reaction mixture was
refluxed overnight. Upon completion of reaction (as shown by TLC),
the mixture was cooled to room temperature. Silica gel (3 g) was added,
and the solvent was evaporated. The resulting solid was layered onto a
silica gel column and eluted with ethyl acetate : methanol : ammonium
hydroxide (200 : 4 : 1). Compound Re-2 was isolated in 45% yield
1
(375 mg): mp 193–1948C (dec); H NMR (300 MHz, CDCl3) d 7.93
(s, 1H), 7.14 (s, 1H), 6.73–6.80 (m, 2H), 5.05–5.14 (m, 1H), 4.69 (d, 1H,
J=16 Hz), 4.55–4.61 (m, 1H), 3.91–4.13 (m, 3H), 3.85 (s, 3H), 3.61–3.67
(m, 1H), 3.38–3.46 (m, 1H), 3.18–3.27 (m, 1H), 3.03 (brs, 2H), 2.85–
2.91 (m, 1H), 2.62–2.73 (m, 2H), 2.38–2.48 (m, 2H), 2.30 (s, 3H), 2.10–
2.17 (m, 1H), 1.78–1.87 (m, 4H), 1.51–1.67 (m, 4H), 1.24–1.33 (m, 2H);
HRFAB calculated forC 26H39N4O5S2Re [M]+ 738.1920, found
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R. H. MACH ET AL.
738.1944. Anal. (C26H39N4O5S2Re) calcd. C, 42.27; H, 5.32; N, 7.59.
Found C, 42.63; H, 5.40; N, 7.44.
O-(9-Azabicyclo[3.3.1]nonan-3a-yl)-N-(2-methoxy-5-methylphenyl)
carbamate (5). The solution of N-benzylcarbamate 1 (3.0 g) in
methanol (200 ml) was added to 10% Pd/C (0.3 g). The suspension
was connected to a hydrogenation apparatus at 50 p.s.i. overnight. The
catalyst was filtered through a pad of celite and the solvent was removed
1
to give the carbamate 5 in a quantitative yield: H NMR (300 MHz,
CDCl3) d 7.93 (s, 1H), 7.16 (s, 1H), 6.73–6.80 (m, 2H), 4.95–5.05 (m,
1H), 3.85 (m, 3H), 3.34–3.37 (m, 2H), 2.34–2.41 (m, 2H), 2.30 (s, 3H),
2.01–2.19 (m, 1H), 1.63–1.75 (m, 2H), 1.43–1.54 (m, 6H). Compound 5
was converted to the hydrochloride for elemental analysis. Anal.
(C16H24N2O3.HCl) calcd. C, 59.97; H, 7.41; N, 8.23. Found C, 59.71; H,
7.46; N, 8.15.
30-a(2-methoxy-5-methylphenylcarbamate)-9-[3-[N-2-[[[2-[[triphenyl-
methyl-thio]ethyl]amino]carbonyl]methyl-N-[S-(triphenylmethyl)-thio]
ethyl]amino]-propyl]granatane (6). To a solution of amine 5 (304 mg,
1.0 mmol) in dry acetonitrile (20 ml) was added [N-[[[2-[(triphenyl-
methyl)-thio]ethyl]amino]carbonyl]methyl]-N-(30-chloropropyl)-S-tri-
phenylmethyl)-2-aminoethanethiol10 (755 mg, 1.0 mmol), potassium
iodide (166 mg, 1.0 mmol), and potassium carbonate (1.38 g, 10 mmol).
The reaction mixture was refluxed overnight. The reaction mixture was
cooled to room temperature, filtered, and concentrated in vacuo. The
resulting solid was dissolved in ethyl acetate and purified by silica gel
column chromatography (5% triethylamine in ethyl acetate). Com-
1
pound 6 was isolated in 68% yield (740 mg): mp 83–848C (dec); H
NMR (300 MHz, CDCl3) d 7.96 (s, 1H), 7.13–7.48 (m, 31H), 6.76 (m,
2H), 5.07 (m, 1H), 3.85 (s, 3H), 2.97–3.01 (m, 6H), 2.25–2.85 (m, 11H),
1.12–1.83 (m, 15H).
X-ray crystallography. Single crystals of purified Re-2 were obtained by
slow growth of the interface of a hexane/anhydrous ethanol suspension
maintained at room temperature. The crystals are, at 228 K, triclinic,
(
(
space group P-1 (No. 2) with a ¼ 7:166ð2Þ A, b ¼ 11:812ð3Þ A,
(
c ¼ 17:241ð4Þ A, a ¼ 103:297ð9Þ8, b ¼ 91:82ð2Þ8, g ¼ 93:33ð3Þ8, V ¼
1416:4ð6Þ A , and Z=2{dcalcd=1.730 g cmꢁ3; ma(MoKa)=4.480 mmꢁ1}.
3
(
A total of 5919 psi-scan corrected reflections having 2Y (MoKa)550.768
(the equivalent of 0.8 limiting CuKa sphere) were collected on a
Copyright # 2001 John Wiley & Sons, Ltd.
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TECHNETIUM-99m SIGMA-2 RECEPTOR LIGAND
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computer-controlled Bruker P4 autodiffractometer using o scans and
graphite-monochromated MoKa radiation of which 4721 were inde-
pendent. The structure was solved using ‘Direct Methods’ techniques
with the Bruker SHELXTL-PC (vers 5.1) software package. The
resulting structural parameters have been refined to convergence {R1
(unweighted, based on F)=0.0477 for3596 independent erflections
having 2Y (MoKa)550.768 and F2>2s(F2)} {R1 (unweighted, based
on F)=0.0802 and wR2 (weighted, based on F2)=0.0906 forall 4721
reflections} using counter-weighted full-matrix least-squares techniques
and a structural model which incorporated anisotropic thermal
parameters for all nonhydrogen atoms. Hydrogen atom H24 was
located from a difference Fourier map and was refined as an
independent isotropic atom. The two methyl groups (C25, C26 and their
hydrogens) were refined as rigid rotors with idealized sp3-hybridized
(
geometry and a C–H bond length of 0.97 A. The remaining hydrogen
atoms were included in the structure factor calculations as idealized
atoms (assuming sp2-orsp 3-hybridization of the carbon atoms and C–H
(
bond lengths of 0.94–0.99 A) ‘riding’ on their respective carbon atoms.
The isotropic thermal parameter for H24 refined to a final value of
2
0.06(4) A . The isotropic thermal parameters of the remaining hydrogen
(
atoms were fixed at values 1.2 (nonmethyl) or 1.5 (methyl) times the
equivalent isotropic thermal parameters of the carbon atoms to which
they are covalently bonded.
All calculations were performed using the SHELXTL-PC (Version 5.1)
interactive software package (G. Sheldrick, Bruker-AXS, Madison, WI).
(
(
Crystal data for Re-2: triclinic, P-1, a ¼ 7:166ð2Þ A, b ¼ 11:812ð3Þ A,
(
and c ¼ 17:241ð4Þ A, a ¼ 103:297ð9Þ8, b ¼ 91:82ð2Þ8, g ¼ 93:33ð3Þ8,
V ¼ 1416:4ð6Þ A , Z ¼ 2, Dx ¼ 1:730 g/cm , T ¼ 228ð2Þ K. y range
2.37–25.388; reflections collected/unique: 5919/4721.
3
3
(
Acknowledgements
This work was supported by grants awarded by the National Institutes of
Health (CA 77951) and the Department of Energy (ER-61657).
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