Design and Investigation of a [18F]-Labeled Benzamide Derivative as a High Affinity Dual Sigma Receptor Subtype Radioligand for Prostate Tumor Imaging

Article abstract of DOI:10.1021/acs.molpharmaceut.6b01020

High overexpression of sigma (σ) receptors (σ₁ and σ₂ subtypes) in a variety of human solid tumors has prompted the development of σ receptor-targeting radioligands, as imaging agents for tumor detection. A majority of these radioligands to date target the σ₂ receptor, a potential marker of tumor proliferative status. The identification of approximately equal proportions of both σ receptor subtypes in prostate tumors suggests that a high affinity, dual σ receptor-targeting radioligand could potentially provide enhanced tumor targeting efficacy in prostate cancer. To accomplish this goal, we designed a series of ligands which bind to both σ receptor subtypes with high affinity. Ligand 3a in this series, displaying optimal dual σ receptor subtype affinity (σ₁, 6.3 nM; σ₂, 10.2 nM) was radiolabeled with fluorine-18 (¹⁸F) to give [¹⁸F]3a and evaluated as a σ receptor-targeting radioligand in the mouse PC-3 prostate tumor model. Cellular assays with PC-3 cells demonstrated that a major proportion of [¹⁸F]3a was localized to cell surface σ receptors, while ～10% of [¹⁸F]3a was internalized within cells after incubation for 3.5 h. Serial PET imaging in mice bearing PC-3 tumors revealed that uptake of [¹⁸F]3a was 1.6 ± 0.8, 4.4 ± 0.3, and 3.6 ± 0.6% ID/g (% injection dose per gram) in σ receptor-positive prostate tumors at 15 min, 1.5 h, and 3.5 h postinjection, respectively (n = 3) resulting in clear tumor visualization. Blocking studies conducted with haloperidol (a nonselective inhibitor for both σ receptor subtypes) confirmed that the uptake of [¹⁸F]3a was σ receptor-mediated. Histology analysis confirmed similar expression of σ₁ and σ₂ in PC-3 tumors which was significantly greater than its expression in normal organs/tissues such as liver, kidney, and muscle. Metabolite studies revealed that >50% of radioactivity in PC-3 tumors at 30 min postinjection represented intact [¹⁸F]3a. Prominent σ receptor-specific uptake of [¹⁸F]3a in prostate tumors and its subsequent clear visualization with PET imaging indicate potential utility for the diagnosis of prostate carcinoma.

Full text of DOI:10.1021/acs.molpharmaceut.6b01020

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Article
Design and Investigation of a [18F]-labeled Benzamide Derivative as a High
Affinity Dual Sigma Receptor Subtype Radioligand for Prostate Tumor Imaging
Dongzhi Yang, Anthony Comeau, Wayne D. Bowen, Robert
H. Mach, Brian D Ross, Hao Hong, and Marcian E. Van Dort
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b01020 • Publication Date (Web): 30 Jan 2017
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Design and Investigation of a [18F]-labeled Benzamide Derivative as a High Affinity Dual
Sigma Receptor Subtype Radioligand for Prostate Tumor Imaging
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Dongzhi Yang1,2, Anthony Comeau3, Wayne D. Bowen3, Robert H. Mach4, Brian D. Ross1, Hao
Hong1, Marcian E. Van Dort1
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1 Center for Molecular Imaging, Department of Radiology, University of Michigan – Ann Arbor,
Michigan 48109ꢀ2200, United States
2 Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical
University, Xuzhou, Jiangsu 221004, China
3 Department of Molecular Pharmacology, Physiology & Biotechnology, Brown University,
Providence, RI 02912, United States
4 Department of Radiology, University of Pennsylvania, Philadelphia, PA 19104, United States
* (H.H.) Fax: 1ꢀ734ꢀ763ꢀ5447; Tel: 1ꢀ734ꢀ615ꢀ4634; Eꢀmail: hahong@med.umich.edu
(M.E.V) Fax: 1ꢀ734ꢀ763ꢀ5447; Tel: 1ꢀ734ꢀ615ꢀ2280; Eꢀmail: mvandort@med.umich.edu
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ABSTRACT: High overexpression of sigma (σ) receptors (σ1 and σ2 subtypes) in a variety of
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human solid tumors has prompted the development of σ receptorꢀtargeting radioligands, as
imaging agents for tumor detection. A majority of these radioligands to date, target the σ2
receptor, a potential marker of tumor proliferative status. The identification of approximately
equal proportions of both σ receptor subtypes in prostate tumors, suggests that a high affinity,
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dual σ receptorꢀtargeting radioligand could potentially provide enhanced tumor targeting efficacy
in prostate cancer. To accomplish this goal, we designed a series of ligands which bind to both σ
receptor subtypes with high affinity. Ligand 3a in this series, displaying optimal dual σ receptor
subtype affinity (σ1: 6.3 nM, σ2: 10.2 nM) was radiolabeled with fluorineꢀ18 (18F) to give [18F]3a
and evaluated as a σ receptorꢀtargeting radioligand in the mouse PCꢀ3 prostate tumor model.
Cellular assays with PCꢀ3 cells demonstrated that a major proportion of [18F]3a was localized to
cell surface σ receptors, while ~10% of [18F]3a was internalized within cells after incubation for
3.5 h. Serial PET imaging in mice bearing PCꢀ3 tumors revealed that uptake of [18F]3a was 1.6 ±
0.8, 4.4 ± 0.3, and 3.6 ± 0.6 %ID/g (% injection dose per gram) in σ receptorꢀpositive prostate
tumors at 15 min, 1.5 h, and 3.5 h postꢀinjection, respectively (n = 3) resulting in clear tumor
visualization. Blocking studies conducted with haloperidol (a nonꢀselective inhibitor for both σ
receptor subtypes) confirmed that the uptake of [18F]3a was σ receptorꢀmediated. Histology
analysis confirmed similar expression of σ1 and σ2 in PCꢀ3 tumors which was significantly
greater than its expression in normal organs/tissues such as liver, kidney, and muscle. Metabolite
studies revealed that >50% of radioactivity in PCꢀ3 tumors at 30 min postꢀinjection represented
intact [18F]3a. Prominent σ receptorꢀspecific uptake of [18F]3a in prostate tumors and its
subsequent clear visualization with PET imaging indicate potential utility for the diagnosis of
prostate carcinoma.
KEYWORDS: Sigma receptors, positron emission tomography, PCꢀ3 tumors, prostate cancer
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1. INTRODUCTION:
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Prostate cancer (PCa), the most diagnosed cancer in men, continues to present major
challenges in its effective treatment, in part, due to staging inaccuracies and the inability to
identify clinically important tumors. Noninvasive imaging modalities such as positron emission
tomography (PET) and single photon emission computed tomography (SPECT) could provide
fundamentally important information for early diagnosis, accurate staging, selection of optimum
treatment and the monitoring of treatment efficacy of PCa. Consequently, there has been intense
development in the past decade of new radioligands for the diagnostic imaging of PCa. PET
imaging with [18F]fluoromethylcholine (a choline analog involved in cell membrane
phospholipid biosynthesis) is currently widely used for prostate tumor imaging. However, recent
reports indicate that it has limited sensitivity and specificity for initial staging especially at low
prostate specific antigen (PSA) levels.1 Radioligands targeting the gastrinꢀreleasing peptide
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receptor (GRPR), a member of the bombesin receptor family, radiolabeled with Ga, Cu, and
18F have also been investigated as imaging radioligands for PCa. Among these, 68GaꢀRM2, 64Cuꢀ
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CBꢀTE2AꢀAR06, and Fꢀlabeled BAY 864367 have shown promise in the detection of primary
or recurrent PCa in early clinical trials.2ꢀ5 More recently, prostate specific membrane antigen
(PSMA) which displays high prostate tumor cell overexpression compared to normal tissues, has
been reported as an important imaging biomarker for the diagnosis of PCa.6 To date, smallꢀ
molecule PSMA inhibitors, e.g. [68Ga]HBEDꢀCCꢀPSMA and [18F]DCFPyl, show the most
promise for diagnostic PET application.7
Radioligands which target sigma (σ) receptors present an alternative approach towards
PCa imaging. Οverexpression of σ receptors has been shown in many human solid tumors
including melanoma, breast, lung, pancreas, prostate and those of neural origin.8, 9 σ receptors
comprise a distinct class of nonꢀopiate, membraneꢀbound proteins which display high affinity
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towards the small molecule ligands: 1,3ꢀdiꢀ(2ꢀtolyl) guanidine [DTG], (+)ꢀ3ꢀ(3ꢀhydoxyphenyl)ꢀ
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Nꢀ(1ꢀpropyl)piperidine [(+)ꢀ3ꢀPPP] or haloperidol.10 The endogenous ligands for σ receptors and
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its exact function are yet unknown, however, two distinct σ receptor subtypes [sigmaꢀ1 (σ1) and
sigmaꢀ2 (σ2)] have been identified based on their apparent molecular weights (σ1 = 25 kDa; σ2 =
21.5 kDa), tissue distribution patterns and ligand selectivity profiles.11, 12 σ receptors are also
normally present in many peripheral tissues (including liver, kidney, lung, endocrine glands) and
the central nervous system.12 The σ1 receptor which has been cloned from a variety of tissues
shows no sequence homology to any known mammalian proteins and has been identified as a
unique ligandꢀoperated chaperone protein localized primarily at the endoplasmic reticulum
mitochondrion interface.13 Progesterone receptor membrane component 1 (PGRMC1), a known
biomarker of cell proliferation, has been suggested as a putative σ2 receptor binding site14
although recent reports have suggested that PGRMC1 and σ2 receptors are two separate
molecular entities.15, 16 Tumor σ2 expression has also been recognized as a potential receptorꢀ
based marker of the proliferative status of solid tumors.14, 17 The pharmacology of σ receptor
ligands has been extensively reviewed in the recent literature.18ꢀ22
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Encouraging preꢀclinical studies with σ receptor radioligands labeled with PET and
SPECT radioisotopes have been reported for many cancers including melanoma,23, 24 breast
cancer,25ꢀ27 and nonꢀsmall cell lung cancer.23 In contrast, there has been limited exploration of σ
receptor radioligands for imaging prostate tumors with most reported studies to date focused on
SPECT radioligand development.28 A few examples of [11C]ꢀlabeled σ receptor radioligands for
prostate tumor imaging with PET have appeared in the literature. In one study, Colabufo and
colleagues reported on a σ receptor ligand ([11C]PB183) having good affinity towards both σ1
and σ2; however, in vivo studies with this radioligand have not been reported.29 More recently, a
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structurally similar σ receptor ligand ([11C]PB28) with high affinity against σ2 (Ki = 5.92 nM)
was shown to display poor tumor uptake in mice bearing PCꢀ3 prostate tumors.30
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Typically, σ2 subtype density overexpression exceeds that of σ1 in most tumor types with
the exception of neuroblastoma and prostate tumors where both subtypes are equally
overexpressed.8, 11, 31 This observation has been further confirmed with in vitro binding studies in
a variety of prostate tumor cell lines including PCꢀ3, LNCaP and DUꢀ145.8, 10, 11 The high density
of total σ receptor tumor overexpression (e.g. approximately 1.8 million receptors per cell for
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DUꢀ145 tumors)10 suggests that σ receptor radioligands which bind to both subtypes could
potentially provide enhanced tumor targeting compared to a radioligand which is selective
towards a single subtype. To address this goal, our initial studies focused on the benzamide class
of σ receptor ligands as a template for the design of a [18F]ꢀlabeled dual σ receptor targeting
radioligand for PET imaging of PCa. Fluorineꢀ18 is an ideal radionuclide for PET imaging as its
low positron emission energy (635 keV),32 affords high quality images due to the short range of
the emitted positrons.33 Furthermore, its decay halfꢀlife (109.8 min) is compatible with the in
vivo circulation time of most smallꢀmolecule ligands. We report herein on a series of (ωꢀ
fluoroalkylether)benzamides which display dual σ1/σ2 subtype binding affinity and initial
biological and PET imaging studies with a highꢀaffinity, [Fꢀ18]ꢀlabeled candidate in a mouse
model of human PCa.
2. MATERIALS AND METHODS
2.1. Chemicals and Radiochemicals. Reagents and solvents were purchased from commercial
suppliers and used without additional purification. 5ꢀBromosalicylic acid and 3ꢀbromoꢀ4ꢀ
hydroxybenzoic acid were purchased from Alfa Aesar (Ward Hill, MA). 2ꢀFluoroethyl tosylate34
and 3ꢀfluoropropyl tosylate35 were synthesized as previously reported. All other chemical
reagents were obtained from Aldrich Chemical Co. (Milwaukee, WI). 2ꢀ[18F]fluoroethyl tosylate
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was purchased from the Cyclotron PET Facility, Department of Radiology, University of
Michigan.
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2.2. Chemical Characterization. Thinꢀlayer chromatography (TLC) was performed using
Analtech silica gel GF Uniplates (250 µm). TLC plates were visualized after development with
either ultraviolet (UV) light or by spraying with phosphomolybdic acid reagent and subsequent
heating. Flash column chromatography was performed on silica gel 60 (0.032 – 0.063 µm)
purchased from EMD Millipore Corporation, Taunton, MA. Melting points were determined
with a ThomasꢀHoover melting point apparatus and are uncorrected. Elemental analyses were
conducted by Atlantic Microlab Inc., Norcross, GA.
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1H NMR spectra were recorded on a Varian INOVA instrument operating at 400 MHz
using CDCl3, acetoneꢀd6 or DMSOꢀd6 as solvents and tetramethylsilane (TMS) as internal
standard. Chemical shifts (δ) and coupling constants (J) are reported in parts per million (ppm)
and Hertz (Hz), respectively. High resolution mass spectral analyses were performed at the
Department of Chemistry, University of Michigan, using either a VGꢀ70ꢀ250ꢀS mass
spectrometer for electron impact (EI) and chemical ionization (DCI) modes, a Waters Autospec
Ultima instrument with an electrospray interface for electrospray ionization (ESI) mode or a
Waters Tofspecꢀ2E run in reflectron mode. High performance liquid chromatography (HPLC)
was performed using a Waters Breeze HPLC System (Waters Corporation, Milford, MA)
equipped with a Waters 2487 Dual Wavelength Absorbance Detector. Radiochemical synthesis
was performed using a TRACERlab FXFN Reactor module (GE Healthcare). HPLC analysis
was conducted at ambient temperature using a Waters Sunfire Cꢀ18 column (4.6 x 250 mm), 5
µm particle, with 0.1% TFA in H2O (component A) and 0.1% TFA in CH3CN (component B)
solvent mixtures as eluent with UV absorbance monitoring at 254 and 280 nm. HPLC runs were
conducted using a solvent gradient of 10%B to 90%B over 25 min at a flow rate of 1 mL/min.
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Radioactivity was monitored with a Bioscan Flow Count FCꢀ3300 NaI/PMT Radiodetector
(Bioscan, Inc., Washington, DC) equipped with a 1.5” x 1.5” NaI(Tl) crystal. LCꢀMS/MS
analysis was conducted at ambient temperature using a Waters XBridge Cꢀ18 column (4.6 x 50
mm), 5 µm particle, with 0.1% HCOOH in H2O (component A) and 0.1% HCOOH in CH3CN
(component B) solvent mixtures as eluent. HPLC runs for liver microsomal assays were
conducted with 5 ꢀL injections at a flow rate of 1 mL/min using the following solvent gradient
(%B): 0 min: 2%, 0.5 min: 2%, 2.5 min: 90%, 3.5 min: 90%, 3.6 min: 2%, 5.6 min: 2%.
Radioactivity measurements were obtained with a Capintec CRCꢀ15W Radioisotope Dose
Calibrator (Ramsey, NJ).
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2.3. Radioligand Design Strategy
Previously described benzamide compound structures with selectivity towards a single σ
receptor subtype (Figure 1) were utilized as templates for the design of potential dual σ receptor
subtype binding radioligands. Compounds 4ꢀIPAB10 and PIMBA10 display nanomolar σ1 affinity
and moderate selectivity (17ꢀ to 80ꢀfold) over σ2 whereas the 1,2,3,4ꢀtetrahydroisoquinoline
analogs (8, 9) are highꢀaffinity, σ2 selective (σ2/ σ1 ratio >1200) ligands (Figure 1).36
Comparison of these two classes of chemical structures suggested that inclusion of lipophilic
ether substituents at the 2ꢀposition of the aromatic ring in core structures such as 4ꢀIPAB and
PIMBA could lead to an improvement in its σ2 affinity. Accordingly, a series of benzamide
derivatives incorporating either 2ꢀ or 4ꢀfluoroalkylated ether linkers were initially synthesized
for screening of their σ1 and σ2 receptor binding affinities for investigation as potential
radiofluorinated dual SR subtype binding radioligands.
2.4. Synthesis of 5-bromo-2-hydroxy-N-{2-(piperidin-1-yl)ethyl}benzamide (2). A stirred
suspension of 5ꢀbromosalicyclic acid (1) (3.0 g, 13.8 mmol) in anhydrous CHCl3 (120 mL) was
treated with SOCl2 (4.9 g, 41.5 mmol) and DMF (0.2 mL) and refluxed for 3 h. The mixture was
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concentrated under reduced pressure to afford the crude acid chloride as a yellow solid. A
suspension of the acid chloride in a mixture of toluene (70 mL) and DMF (4 mL) was added
dropwise to a solution of Nꢀ(2ꢀaminoethyl)piperidine (2.12 g, 16.6 mmol) and Et3N (3.07 g, 30.4
mmol) in toluene (70 mL) and stirred at ambient temperature for 3 h. The mixture was washed
with brine, H2O and dried (Na2SO4). Purification by silicaꢀgel flash chromatography (5%
CH3OH in CHCl3 with 1% added NH4OH) gave 2.8 g (62% overall yield) of the title compound
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as a cream amorphous solid: H NMR (acetoneꢀd6): δ 8.29 (br s, 1H), 7.91 (d, J = 2.5 Hz, 1H),
7.51 (dd, J = 8.8 Hz , 2.3 Hz, 1H), 6.88 (dd, J = 8.8 Hz , 3.1 Hz, 1H), 3.54 – 3.52 (m, 2H), 2.58 –
2.56 (m, 2H), 2.54 – 2.45 (m, 4H), 1.58 – 1.52 (m, 4H), 1.43 – 1.42 (m, 2H). HRMS [ESI with
Na+ added]: Calculated for C14H20N2O2Br [M + H+]: 327.0708. Observed: 327.0710. Anal.
(C14H19N2O2Br): C, H, N. HPLC: tR (chemical purity) = 11.7 min (99.9%).
2.5. Synthesis of 5-Bromo-2-(2-fluoroethoxy)-N-{2-(piperidin-1-yl)ethyl}benzamide (3a). A
stirred solution of 2 (0.2 g, 0.61 mmol) in anhydrous CH2Cl2 (9 mL) was treated with a solution
of tetrabutylammonium hydroxide (1M in CH3OH, 0.67 mL, 0.67 mmol) and concentrated under
reduced pressure after 15 min. Residual water was removed by treatment of the residue thrice
with 5 mL portions of anhydrous CH3CN and concentration under reduced pressure. The residue
was reconstituted in anhydrous DMF (5 mL), treated with a solution of 2ꢀfluoroethyl tosylate
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(0.16 g, 0.73 mmol) in DMF (1 mL) and heated at 65 – 70 C for 3 h. The crude mixture was
partitioned between EtOAc and brine and the organic layer was washed with brine, H2O and
dried (Na2SO4). Silicaꢀgel flash chromatography (gradient of 3% ꢀ 10% CH3OH in CH2Cl2 with
1% added NH4OH) of the crude product mixture provided 0.13 g (57%) of material which was
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recrystallized from hexane:EtOAc (1:1) to give a white crystalline solid: mp 101 – 102 C; H
NMR (CDCl3): δ 8.33 (d, J = 2.8 Hz, 1H), 8.10 (br s, 1H), 7.51 (dd, J = 8.6 Hz, 2.6 Hz, 1H), 6.83
(d, J = 8.8 Hz, 1H), 4.86 (dt, JHꢀF = 47.4 Hz, 4.1 Hz, 2H), 4.35 (dt, JHꢀF = 27.2 Hz, 4.1 Hz, 2H),
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3.59 – 3.55 (m, 2H), 2.53 (t, J = 6.2 Hz, 2H), 2.42 (br s, 4H), 1.61 – 1.55 (m, 4H), 1.48 – 1.45
(m, 2H). HRMS [ESI with Na+ added]: Calculated for C16H23N2O2BrF [M+H+]: 373.0927.
Observed: 373.0926. Anal. (C16H22N2O2BrF): C, H, N. HPLC: tR (chemical purity) = 12.0 min
(99.9%, Figure S1).
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2.6. 5-Bromo-2-(3-fluoropropoxy)-N-{2-(piperidin-1-yl)ethyl}benzamide (3b). A similar
procedure to that shown for 3a above provided 0.18 g (61%) of the title compound 3b which
was recrystallized from hexane:EtOAc (1:1) to give a white crystalline solid: mp 64 – 66 0C; 1H
NMR (CDCl3): δ 8.31 (d, J = 2.5 Hz, 1H), 8.09 (br s, 1H), 7.50 (dd, J = 8.8 Hz, 2.7 Hz, 1H),
6.86 (d, J = 8.8 Hz, 1H), 4.66 (dt, JHꢀF = 47.1 Hz, 5.5 Hz, 2H), 4.27 (t, J = 6.1 Hz, 2H), 3.58 –
3.54 (m, 2H), 2.51 (t, J = 6.1 Hz, 2H), 2.41 – 2.24 (overlapping m, 6H), 1.60 – 1.54 (m, 4H),
1.48 – 1.45 (m, 2H). HRMS [ESI with Na+ added]: Calculated for C17H25N2O2BrF [M+H+]:
387.1078. Observed: 387.1078. Anal. (C17H24N2O2BrF): C, H, N. HPLC: tR (chemical purity) =
12.9 min (99.9%, Figure S1).
2.7. Synthesis of 3-Bromo-4-hydroxybenzoic acid succinimidyl ester (5). A stirred suspension
of 3ꢀbromoꢀ4ꢀhydroxybenzoic acid (4) (2.36 g, 10.9 mmol) and N,Nꢀdisuccinimidyl carbonate
(3.1 g, 12 mmol) in anhydrous CH3CN (100 mL) was treated dropwise with pyridine (0.97 mL,
0.95 g, 12 mmol) and stirred at ambient temperature for 18 h. The mixture was concentrated
under reduced pressure and purified by silicaꢀgel flash chromatography (gradient of 30% ꢀ 60%
EtOAc in hexane) to give 3 g (88%) of material which was recrystallized from hexanes:EtOAc
(1:1) to give a white crystalline solid: mp 171 – 172 0C; 1H NMR (DMSOꢀd6): δ 11.82 (br s, 1H),
8.15 (d, J = 2.1 Hz, 1H), 7.96 (dd, J = 8.6 Hz, 2.1 Hz, 1H), 7.15 (d, J = 8.6 Hz, 1H), 2.88 (s, 4H).
HRMS [ESI with Na+ added]: Calculated for C11H8NO5BrNa [M + Na+]: 335.9478. Observed:
335.9472. Anal. (C11H8NO5Br): C, H, N.
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2.8. Synthesis of 3-bromo-4-hydroxy-N-{2-(piperidin-1-yl)ethyl}benzamide (6). A stirred
suspension of 5 (2.31 g, 7.35 mmol) in anhydrous CH2Cl2 (80 mL) was treated by dropwise
addition with a solution of Nꢀ(2ꢀaminoethyl)piperidine (0.94 g, 7.35 mmol) in CH2Cl2 (5 mL)
and stirred for 3 h. The crude product was purified by silicaꢀgel flash chromatography (20%
CH3OH in CH2Cl2 with 1% added NH4OH) to give 1.7 g (71%) of the title compound 6 as a
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white amorphous solid: H NMR (acetoneꢀd6): δ 8.03 (d, J = 2.1 Hz, 1H), 7.73 (dd, J = 8.4 Hz,
2.1 Hz, 1H), 7.59 (br s, 1H), 7.03 (d, J = 8.5 Hz, 1H), 3.49 (doublet, J = 6.4 Hz, 1H), 3.46
(doublet, J = 6.4 Hz, 1H), 2.53 (t, J = 6.5 Hz, 2H), 2.45 (m, 4H), 1.58 – 1.53 (m, 4H), 1.45 –
1.41 (m, 2H). HRMS [ESI]: Calculated for C14H20N2O2Br [M + H+]: 327.0703. Observed:
327.0712. Anal. (C14H19N2O2Br): C, H, N. HPLC: tR (chemical purity) = 8.7 min (99.9%).
2.9. 3-Bromo-4-(2-fluoroethoxy)-N-{2-(piperidin-1-yl)ethyl}benzamide (7a). A similar
procedure to that shown for 3a above provided the title compound in 90% yield as a white
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crystalline solid: mp 108 – 109 C [hexane:EtOAc (1:1)]; H NMR (CDCl3): δ 8.01 (d, J = 2.2
Hz, 1H), 7.74 (dd, J = 8.6 Hz, 2.1 Hz, 1H), 6.96 (br s, 1H), 6.94 (d, J = 8.6 Hz, 1H), 4.82 (dt, JHꢀ
F = 47.2 Hz, 4.3 Hz, 2H), 4.33 (dt, JHꢀF = 27.0 Hz, 4.1 Hz, 2H), 3.54 – 3.50 (m, 2H), 2.58 (t, J =
5.9 Hz, 2H), 2.47 (br s, 4H), 1.65 – 1.60 (m, 4H), 1.51 – 1.48 (m, 2H). HRMS [ESI+]: Calculated
for C16H23N2O2BrF [M+H+]: 373.0921. Observed: 373.0919. Anal. (C16H22N2O2BrF): C, H, N.
HPLC: tR (chemical purity) = 11.7 min (99.3%, Figure S1).
2.10. 3-Bromo-4-(3-fluoropropoxy)-N-{2-(piperidin-1-yl)ethyl}benzamide (7b). A similar
procedure to that shown for 3b above provided the title compound in 89% yield as a colorless
viscous oil; 1H NMR (CDCl3): δ 8.00 (d, J = 2.1 Hz, 1H), 7.71 (dd, J = 8.6 Hz, 2.4 Hz, 1H), 6.93
(d, J = 8.6 Hz, 1H), 6.85 (br s, 1H), 4.71 (dt, JHꢀF = 46.9 Hz, 5.7 Hz, 2H), 4.21 (t, J = 6.0 Hz,
2H), 3.52 – 3.48 (m, 2H), 2.54 (t, J = 6.0 Hz, 2H), 2.43 (br s, 4H), 2.31 – 2.18 (overlapping m,
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2H), 1.63 – 1.57 (m, 4H), 1.50 – 1.47 (m, 2H). HRMS [ESI+]: Calculated for C17H25N2O2BrF
[M+H+]: 387.1078. Observed: 387.1080. Anal. (C17H24N2O2BrF): C, H, N. HPLC: tR (chemical
purity) = 12.9 min (98.8%, Figure S1).
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2.11. In vitro σ1 Binding Assays. These assays were conducted as previously reported.10 In
brief, guinea pig brain membrane homogenates (300 – 500 µg of total protein) were incubated
with 3 nM [3H]ꢀ(+)ꢀpentazocine (51.7 Ci/mmol) in 0.5 mL of 50 mM TrisꢀHCl, pH 8.0, at 25 0C
for 120 min. Test ligands were added in concentrations ranging from 10ꢀ4 to 10ꢀ12 M. Assays
were terminated by the addition of 5 mL of iceꢀcold 10 mM TrisꢀHCl (pH 8.0) and filtered
through glass fiber filters and filters washed twice with 5 mL of iceꢀcold 10 mM Tris HCl (pH
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8.0). Filters were soaked in 0.5% polyethyleneimine for at least 30 min at 25 C prior to use.
Nonꢀspecific binding was determined in the presence of 10 µM haloperidol. IC50 values were
determined using the computerized iterative curveꢀfitting program GraphPad. Ki values were
calculated from the IC50 values using the ChengꢀPrussoff equation. Protein concentration was
determined by the Lowry method.
2.12. In vitro σ2 Binding Assays. These assays were conducted by incubation of rat liver
membrane homogenates (150 – 200 µg of total protein) with 3 nM [3H]DTG (39.4 Ci/mmol)in
the presence of 1 µM dextrallorphan (to mask σ1 sites). Nonꢀspecific binding was determined in
the presence of 10 µM haloperidol. All other procedures were identical to those described for the
σ1 receptor binding assay described above.
2.13. Mouse Liver Microsomal Assay. This assay was conducted by incubation of 3a with a
mouse liver microsomal preparation as follows: 10 ꢀL of microsomal mixture (20 mg/mL) was
diluted with 366 ꢀL of 0.1M phosphate buffer (3.3 mM MgCl2) followed by the addition of 4 ꢀL
of 100 ꢀM stock solution of 3a and Verapamil (positive control). The incubation mixture was
preꢀwarmed to 37 0C for 3 min. The reaction was initiated by treatment with 20 ꢀL of a NADPHꢀ
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generating system consisting of 4 mg of NADPH in 240 ꢀL of 0.1M phosphate buffer (3.3 mM
MgCl2). The final concentration of test compounds in the reaction system was 1 ꢀM. At
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designated time intervals (0, 5, 10, 15, 30, 45 and 60 min), 30 ꢀL aliquots of the reaction
solution were removed by pipette and quenched by the addition of 9 volumes of cold CH3CN
containing 10 nM of CE302 as internal standard. The incubation solution was centrifuged at
3500 g for 15 min to precipitate protein and the supernatant was used for LCꢀMS/MS analysis.
2.14. Radiosynthesis of [18F]3a. The radiosynthesis of [18F]3a was conducted by reaction of the
tetrabutylammonium phenolate salt of 2 with 2ꢀ[18F]fluoroethyl tosylate (Scheme 3). In brief, a
solution of 2 (2.3 mg, 6.7 ꢁmoles) in 1 mL of CH3CN and tetrabutylammonium hydroxide
solution (1M in CH3OH, 6.7 ꢁl, 6.7 ꢁmoles) were added sequentially to the FXFN reactor
module and evaporated to dryness at 45 0C with a helium stream under vacuum. [18F]fluoroethyl
tosylate was eluted from an Oasis Cꢀ18 cartridge using anhydrous CH3CN (1 mL) into the
reactor containing the anhydrous tetrabutylammonium salt precursor. The mixture was heated at
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90 C for 10 min, then cooled to 45 C, diluted with water (3 mL) and injected onto a semiꢀ
preparative reversed phase HPLC column (Phenomenex Gemini Cꢀ18, 5 ꢁm (10 mm × 250
mm)). Product purification was conducted under isocratic conditions with a mobile phase of 28%
acetonitrile in water with 0.1% added TFA at a flow rate of 4.0 mL/min with UV absorbance
(254 nm) and radioactivity detection. The radioactive fraction corresponding to [18F]3a (11.2 <
tR < 12.4 min) was collected in a flask containing sterile water (25 mL) and eluted through a preꢀ
activated (methanol, H2O) Empore C18 XDBꢀXC cartridge. The C18 cartridge was rinsed with
additional water (5 mL) and the trapped, purified radiolabeled product was eluted from the
cartridge with ethanol (0.3 mL) followed by 0.9% saline (3 mL). The formulated solution was
filtered using a sterile Millipore GV 0.22 ꢁm filter into a 10 mL vial. Product formulations in
saline containing 5% ethanol by volume were subsequently used for animal studies.
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Confirmation of product identity was conducted using HPLC analysis by coꢀinjection with the
authentic unlabeled standard 3a (tR = 11.6 ± 0.3 min; n = 8).
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2.15. Determination of Radiochemical Purity and Specific Activity. Chemical and
radiochemical purity estimates were obtained on the same sample by HPLC analysis. Specific
activity estimates were determined by comparison of the UV absorbance peak area
corresponding to the radioligand on the HPLC chromatogram with a standard calibration curve
relating mass (0.5 – 20 µg) to UV absorbance peak area (254 nm).
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2.16. Cell Internalization/Efflux Studies. Cell uptake/internalization assays were conducted
with [18F]3a using PCꢀ3 cells to further characterize its cell interaction kinetics during the time
frame of PET imaging (5 min – 3.5 h). PCꢀ3 cells were washed thrice with cold PBS, detached
by a cell dissociation buffer (Life Technologies) for 15 min, and reꢀsuspended in PBS at a final
concentration of 2 × 106/mL. The cells were then divided into two portions. One portion of cells
was incubated with 105 cpm/mL (cpm: counts per minute) of [18F]3a. At serial time points (5
min, 15 min, 0.5, 1.0, 1.5, 2.0, 2.5, and 3 h) an aliquot (0.3 mL) of cell suspension was removed
and centrifuged for pellet isolation. After repeated washing with PBS, cell pellets were reꢀ
suspended in 0.01 M sodium citrate, 0.14 M NaCl buffer (pH = 2) for 2 min at room temperature
and then centrifuged. The amount of internalized [18F]3a was calculated by assay of the
radioactivity that remained associated with the cells after the wash with pH 2 buffer, while the
radioactivity in the supernatant represented cell surfaceꢀassociated [18F]3a. An additional portion
of PCꢀ3 cells was incubated with 105 cpm/mL of [18F]3a for 0.5 h (when maximal internalization
was achieved). The cell suspension was then centrifuged and washed with PBS repeatedly. PCꢀ3
cells were reꢀsuspended in PBS at a density of 2 × 106/mL, and 0.3 mL of the cell suspension
was removed from the culture and centrifuged at serial time points (5 min, 15 min, 0.5, 1.0, 1.5,
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2.0, 2.5, and 3 h). Radioactivity measured in the supernatant represented the efflux of [18F]3a
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2.17. Tumor Model and PET Imaging. All animal studies were conducted under an animal use
protocol approved by the University Committee on Use and Care of Animals (UCUCA) at the
University of Michigan. Tumors were established by subcutaneous injection of 2 × 106 of PCꢀ3
cells suspended in 100 µL of a 1:1 mixture of PBS and Matrigel (BD Biosciences, Franklin
Lakes, NJ) into the front flank of male nude mice (6ꢀ10 weeks old, 20 – 25 g) purchased from
Charles River Laboratories. Tumor size was monitored every other day and mice were subjected
to in vivo experiments when the tumor diameter reached 5ꢀ8 mm (typically 4ꢀ6 weeks after
inoculation).
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PET imaging was performed on a Siemens Inveon PET/CT small animal scanner
(Siemens Medical Solutions, Knoxville, TN) equipped with a computer controlled bed and
operated in threeꢀdimensional list mode. Each tumorꢀbearing mouse was injected with 5ꢀ10 MBq
of [18F]3a via the tail vein and subjected to static PET scans (40 million events per scan, 350 –
750 keV energy window) at various time points postꢀinjection (p.i.). Mice were anesthetized
prior to scanning with 2% (v/v) isoflurane and maintained thereafter with 1 – 2% isoflurane
(v/v). PET images were reconstructed with 3ꢀDimensional OrderedꢀSubsets Expectation
Maximization (3DꢀOSEM) algorithms without CT attenuation correction and analyzed using
Siemens IRW software. Circular regions of interest (ROI) were drawn around tumor/tissue (or
regions of interest of the highest uptake within tumor/tissue). Regional uptake of radioactivity
was decayꢀcorrected to the time of injection and tumor uptake (units of % injected dose per gram
[%ID/g]) was calculated as the activity concentration within an ROI normalized by the ratio of
the injected dose and the animal weight. Radioactivity tissue concentration data obtained from
these studies were combined to generate composite timeꢀactivity curves for selected tissues.
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Separate groups of three mice were subjected to dynamic PET scans (10 frames with 2 min per
time frame) immediately after intravenous injection of [18F]3a to determine its in vivo circulation
time.
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2.18. Tissue Biodistribution Studies. Biodistribution studies were carried out to correlate the
radiotracer uptake based on PET imaging with the quantitative radioactivity distribution data
obtained in PCꢀ3 tumorꢀbearing mice. Following PET imaging at 3.5 h p.i., mice were
euthanized and blood, tumor, and major organs/tissues were collected and wetꢀweighed. The
radioactivity in each sample was measured using a WIZARD2 automatic gammaꢀcounter
(PerkinꢀElmer) and recorded as %ID/g (mean ± SD). Tumor, liver, spleen, and muscle were
also frozen for further histological analysis. A separate group of PCꢀ3 tumorꢀbearing mice (n =
3) were injected via the tail vein with 0.5 – 1 MBq of [18F]3a and sacrificed at 1.5 h after
radioligand injection to compare its early organ distribution profile with the data acquired in PET
studies. All radioactivity measurements were corrected for radioisotope decay.
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2.19. Tissue Radiometabolite Assays. Tumor, liver and blood were analyzed for
radiometabolites of [18F]3a at 5 min and 30 min after intravenous injection. Mice (n = 2 per time
point) were injected with [18F]3a (6 – 10 MBq) and euthanized by decapitation under isoflurane
anesthesia at selected time intervals. Tissues were minced, homogenized in 400 ꢁL of iceꢀcold
CH3CN:H2O (95:5) and centrifuged at 12,000 g for 10 min. The supernatant and pellet were
separated and assayed with a gammaꢀcounter to determine the extraction efficiency (extraction
efficiency >95%). The supernatant was filtered, concentrated by evaporation with a nitrogen
stream and aliquots were spiked with nonradioactive 3a prior to HPLC analysis. The percentage
of intact radioligand and metabolites were calculated as the % ratio of their respective HPLC
radiochromatogram peak areas to the total peak area.
2.20. Statistical Analysis
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Data is reported as mean ± standard deviation (SD). Differences in tissue uptake in blocking
studies were evaluated for statistical significance with a paired 2ꢀtailed Students t test using
Microsoft Excel. P values < 0.05 were considered as statistically significant.
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3.1. Synthetic Chemistry. Synthesis of the 2ꢀ(ωꢀfluoroalkyl)ether substituted compounds (3a,
3b) were conducted as shown in Scheme 1 by initial conversion of 5ꢀbromosalicylic acid (1) to
the acid chloride using thionyl chloride followed by treatment with Nꢀ(2ꢀaminoethyl)piperidine
to give the 2ꢀhydroxybenzamide analog 2. Treatment of the tetrabutylammonium phenolate salt
of 2 with the appropriate ωꢀfluoroalkyl tosylate in DMF at 65 0C provided compounds 3a and 3b
in 57 – 61% yields. Synthesis of the 4ꢀ(ωꢀfluoroalkyl)ether substituted compounds (7a, 7b) were
conducted using a different approach (Scheme 2) as we were unsuccessful in our attempts to
convert 3ꢀbromoꢀ4ꢀhydroxybenzoic acid (4) to its corresponding 4ꢀhydroxybenzamide derivative
(6) by the previous method. Compound 6 was subsequently synthesized by initial conversion of
4 to the corresponding succinimidyl ester (5) followed by treatment with Nꢀ(2ꢀ
aminoethyl)piperidine (Scheme 2). Alkylation of the tetrabutylammonium phenolate salt of 6
with the appropriate ωꢀfluoroalkyl tosylate as described previously gave the final compounds 7a
and 7b in 89 – 90% yields.
3.2. σ Receptor Binding
The binding affinities of new benzamide ligands for σ receptor subtypes were determined using
in vitro competitive binding assays. These data (expressed as inhibition constants, Ki) are
presented in Table 1. All four fluoroalkylether substituted benzamide derivatives displayed
nanomolar binding affinities towards both σ receptor subtypes. Analog 3a bearing a 2ꢀ
fluoroethoxy substituent at the 2ꢀposition of the aromatic ring showed the highest σ receptor
binding affinity in the series (σ1 = 6.26 nM; σ2 = 10.2 nM). However, shifting the 2ꢀfluoroethoxy
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substituent to the 4 position (analog 7a) resulted in a 6ꢀ and 4ꢀfold reduction in its σ1 and σ2
affinity, respectively. In contrast, the 3ꢀfluoropropylether substituent was moderately well
tolerated at the 4ꢀposition (analog 7b: σ1 = 23.3 nM; σ2 = 28.3 nM) but relatively less tolerated at
the 2ꢀposition (analog 3b) resulting in a 9ꢀfold reduction in its σ1 and σ2 affinity. The phenol
intermediates 2 and 6 were devoid of σ receptor binding affinity.
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3.3. Mouse Liver Microsomal Metabolism
The rate of metabolism of 3a in the presence of mouse liver microsomes was determined by
quantification of intact 3a using LCꢀMS/MS analysis. The halfꢀlife (T1/2) of metabolic
degradation of 3a was determined to be 5.94 min (Table S2).
3.4. Radiochemistry
Radiosynthesis was completed in approximately 55 min and afforded decayꢀcorrected
radiochemical yields of [18F]3a of 35 ꢀ 50% (n = 12) based on initial activity of 2ꢀ
[18F]fluoroethyl tosylate, with an average specific activity of 226 GBq/ꢀmol (n = 6). The
radiochemical and chemical purity of the final product was >99% by HPLC analysis (tR = 11.6 ±
0.3 min; n = 8).
3.5. Cell Uptake Studies
Prior to initiating in vivo experiments, we examined the cell interaction kinetics
(internalized, surfaceꢀbound, and efflux) between [18F]3a and σ receptorꢀpositive PCꢀ3 cells and
these results are shown in Figure 2. As seen from this data, the majority of [18F]3a was
associated with the cell membrane (Panel A) as compared to the cellꢀinternalized component
(peak values at 15 min postꢀincubation, 13033.0 ± 593.1 Bq/106 cells). In comparison, the
internalized fraction of [18F]3a (Panel B) was significantly lower (peak values at 30 min postꢀ
incubation, 1058.8 ± 593.1 Bq/106 cells). We estimate that ~ 10% of [18F]3a was internalized
within PCꢀ3 cells, possibly mediated via σ receptor internalization. Cell efflux studies (Panel C)
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also revealed that ~33% of [18F]3a was effluxed from PCꢀ3 cells after cell internalization (at 3 h
postꢀincubation).
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S2). Based on the short circulation time, PET imaging was conducted at 15 min, 1.5 h, and 3.5 h
following intravenous injection of [18F]3a into PCꢀ3 tumor bearing mice. Reconstructed coronal
slices containing PCꢀ3 tumors are shown in Figure 3A. As seen from the mouse PET image
data, [18F]3a undergoes rapid excretion via both renal and hepatobiliary pathways (Figure 3B),
while also displaying increasing tumor accumulation with time compared to nonꢀtarget organs
such as liver, kidney and spleen which resulted in clear visualization of PCꢀ3 tumors. [18F]3a
showed a maximum tumor uptake of 4.4 ± 0.3 %ID/g at 1.5 h postꢀinjection with corresponding
tumor uptake values of 1.6 ± 0.8, and 3.6 ± 0.6 %ID/g at 15 min and 3.5 h p.i., respectively
(Figure 3A & B). To further investigate the specificity of [18F]3a towards σ
receptors, “blocking” experiments were conducted by coꢀinjection of the highꢀaffinity, nonꢀ
selective σ receptor ligand haloperidol (10 ꢁmol/kg37) with [18F]3a into tumorꢀbearing mice (n =
3). Haloperidol administration reduced the tumor uptake of [18F]3a at both 1.5 h and 3.5 h p.i. (p
< 0.05) – the uptake was 1.2 ± 0.2, 3.3 ± 0.3, and 2.0 ± 0.2 %ID/g at 15 min, 1.5 h, and 3.5 h p.i.,
respectively (Figure 3B, C & D) confirming that tumor uptake was σ receptorꢀmediated.
3.7. Ex Vivo Studies (Organ Distribution & Histology)
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studies to corroborate the PET imaging data. These studies demonstrate that accumulation of
[18F]3a in PCꢀ3 tumors was higher compared to other organs/tissues (e.g. liver, kidney, and
spleen) (Figure 4A), affording optimal contrast for tumor detection with PET. In the haloperidol
“blocking” group, a faster clearance of [18F]3a was detected (statistical difference in blood,
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heart, lung, liver, tumor, and brain), an observation likely attributable to decreased availability
of σ1 and σ2 receptors due to occupancy by haloperidol. Overall, [18F]3a demonstrates good
tumor contrast in PCꢀ3 tumorꢀbearing mice with a tumorꢀtoꢀmuscle ratio of 4.0 ± 0.3 at 3.5 h p.i.
(n = 3), compared with 1.8 ± 0.2 for the haloperidol “blocking” group (n = 3). Additionally,
biodistribution studies conducted on a separate batch of mice (n = 3) at 1.5 h p.i. (at peak tumor
uptake) after radioligand administration revealed an organ distribution pattern consistent with
that observed in the PET images (Figure 4A), and the tumorꢀtoꢀmuscle ratio at this time was 3.1
± 0.6 (n = 3).
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Immunofluorescence staining for σ1 and σ2 receptor expression was conducted in mouse
PCꢀ3 tumor sections as well as selected organs/tissues. These studies revealed prominent
expression of both σ receptor subtypes in PCꢀ3 tumor sections (Figure 4B). Furthermore,
substantially weaker fluorescence was observed in liver, kidney and muscle.
3.8. Radiometabolite Analysis
HPLC analysis of tumor and blood radioactivity at 5 min postꢀinjection showed that >99% of the
total extracted radioactivity was associated with intact [18F]3a (tR = 11.8 min). However, at 30
min postꢀinjection the corresponding values for tumor and blood had declined to 52% and 24%,
respectively, and the remaining radioactivity was associated with a single polar metabolite
eluting at tR = 3.8 min. In the case of liver, intact [18F]3a comprised 46% and 67% of the total
radioactivity at 5 min and 30 min postꢀinjection, respectively, and the remaining radioactivity
was a single polar metabolite (tR = 3.8 min) as seen previously.
4. DISCUSSION
Our design strategy based on the benzamide compound structure as template led to the
discovery of several ωꢀfluoroalkylether derivatized benzamides displaying potent affinity
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towards both σ receptor subtypes. An important advantage of the benzamide core structure is its
conformational flexibility, low to moderate lipophilic properties and ease of structural
modification for radiolabel incorporation. Radioligand lipophilicity as measured by its log P
value (or log D for ionizable compounds) impacts its plasma protein bound fraction, ability to
cross cell membranes and nonꢀspecific binding. In general, high radioligand lipophilicity leads to
increased nonꢀspecific binding resulting in a lowered signalꢀtoꢀbackground contrast ratio. Log P
or (log D) values within the 1 ꢀ 3 range have been proposed as desirable for candidate
radioligands.38, 39 The fluorinated benzamides in our series display clog P(D) values (Table 1)
within this optimal range for in vivo radioligands that demonstrate reduced nonꢀspecific binding.
In vitro receptor binding studies showed that the four fluorinated benzamide derivatives in the
series displayed high to moderate dual σ receptor binding affinity (6.3 nM < Ki < 92.9 nM)
confirming our design hypothesis (Table 1). We subsequently selected analog 3a for [18F]ꢀ
labeling and additional in vivo evaluation based on its optimal dual SR binding affinity (σ1 =
6.26 nM; σ2 = 10.2 nM).
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PET imaging studies with [18F]3a showed increasing tumor accumulation of radioactivity
with time with a peak tumor uptake of 4.4 %ID/g at 1.5 h postꢀinjection. Moreover, rapid
excretion of radioactivity via both renal and hepatobiliary pathways from 1 – 3.5 h postꢀinjection
resulted in clear tumor visualization in the PET images during this time interval. Additionally,
biodistribution studies conducted on PCꢀ3 tumor bearing mice at 1.5 h (at peak tumor uptake)
and 3.5 h after radioligand administration revealed an organ distribution pattern consistent with
that seen with the PET image data. We used haloperidol which displays high affinity towards
both σ1 (Ki = 4.95 nM) and σ2 (Ki = 20.7 nM)40 receptor subtypes for pharmacological blockade
studies to demonstrate in vivo σ receptor specificity of [18F]3a. Coꢀinjection of [18F]3a with
haloperidol (1 mg/Kg) resulted in a significant reduction (45%) in tumor radioactivity at 3.5 h
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postꢀinjection demonstrating that tumor uptake was σ receptor mediated. These results were also
confirmed by the biodistribution data wherein haloperidol administration showed a 55%
reduction in the tumorꢀtoꢀmuscle binding ratio of [18F]3a from 4.0 to 1.8 at this time interval and
also reduced radioligand accumulation in organs known to contain σ receptors such as liver,
spleen and lung. Blockade of tumor radioactivity was however less pronounced at 1.5 h (25%)
which may be related to its lower specific binding fraction. A possible explanation for the
improved blockade observed at 3.5 h versus 1.5 h could be due to improved σ receptor specific
binding and higher blood clearance of nonꢀspecific binding at later time intervals resulting in
improved specific to nonꢀspecific binding ratios. It is also conceivable that haloperidol
pretreatment (instead of coꢀinjection as in this study) may be more effective at σ receptor
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blockade at earlier time intervals as demonstrated in other σ receptor radioligand studies.40ꢀ42
The cell binding studies conducted with [18F]3a showed that cell membrane associated
radioactivity peaked at 15 min while the internalized component peaked at 30 min postꢀ
incubation. The efflux of [18F]3a from PCꢀ3 cells was continuous over the 3 h monitoring time
(~25% at 3 h post incubation). These results may explain the slight decrease in tumor uptake
observed with PET imaging at 3.5 h postꢀinjection. The observation that the decrease in tumor
uptake is lower than that seen in the cell studies could be due to the higher cell density present in
tumors compared to that in cell suspensions.
Our in vitro mouse liver microsomal stability data shows a relatively fast metabolic
degradation for 3a (T = 5.94 min) which is in contrast with the tumor uptake for [18F]3a seen in
½
the PET images. For example, [18F]3a associated tumor radioactivity showed a steady
accumulation in tumor with a peak uptake of 4.4% ID/g at 1.5 h postꢀinjection despite the fast
metabolic degradation predicted by the in vitro liver microsomal studies. A possible explanation
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is that in vitro liver microsome studies may not completely reflect their in vivo behavior as
previously noted.43 Furthermore, our plasma analysis data shows that intact [18F]3a accounts for
>99% and 24% of the total radioactivity at 5 min and 30 min p.i. respectively. Hence, the
availability of sufficiently high plasma concentrations of intact [18F]3a and its slow binding
kinetics for tumor accumulation could explain the PET imaging data.
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The studies reported herein describe our initial attempts at dual targeting of σ1 and σ2
receptor overexpression in prostate tumors for diagnostic imaging of PCa. Radioligands that
target PSMA or GPCR overexpression have received increased attention recently as PCa imaging
agents. As an alternate approach, radioligands which target σ receptor overexpression could be
useful in patients that display either PSMAꢀnegative44 or GRPRꢀnegative tumors.45 Further
refinements in this class of σ receptor radioligands include the possibility of substitution of
bromine in 3a with the chemically related iodine atom, to provide either imaging (PET [124I] and
SPECT [123I]) or radiotherapeutic ([131I]) capabilities. Future studies to investigate the detailed σ
receptor binding/interaction mechanisms for these ligands and further evaluate their SAR are
planned. This information could facilitate development of σ receptor ligands with improved
affinity, metabolic stability and internalization into σ receptorꢀpositive tumor cells to further
improve its tumor retention. Additionally, tethering these radioligands with multimeric
structures,46, 47 or coupling them to specific nanomaterials to utilize the “multivalency effect” (i.e.
simultaneous binding to multiple σ receptors to improve binding affinity and tumor targeting
specificity),48, 49 is a possibility to achieve improved σ receptorꢀpositive tumor targeting.
Supporting Information
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The supporting information is available free of charge on http://pubs.acs.org for this manuscript,
which includes the dynamic PET scan measurement of circulation halfꢀlife, HPLC analysis,
elemental analysis, and liver microsome metabolic stability data.
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ACKNOWLEDGMENTS
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This work was supported, in part, by seed grants from the University of Michigan
Comprehensive Cancer Center and The Michigan Memorial Phoenix Project at the University of
Michigan. Additional support from Elsa U. Pardee Foundation (to H.H.), the NIH P01ꢀ
CA085878, Jiangsu Government Scholarship for Overseas Studies (to D.Y.), and the National
Science Foundation of China (No. 81201696) is gratefully acknowledged. The authors would
also like to thank Dr. Christy John (CDER/FDA, Silver Spring, MD) for many helpful
discussions during the course of this work. We thank Dr. Peter Scott and Brian Hockley at the
University of Michigan Cyclotron/PET facility for providing 2ꢀ[18F]fluoroethyl tosylate.
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FIGURE LEGENDS
Scheme 1 Synthesis of compound 3a and 3b.
Scheme 2 Synthesis of compound 7a and 7b.
Scheme 3 Synthesis of [18F]3a.
Figure 1 Design Strategy for Dual SRꢀtargeting Ligands
Figure 2 Interaction of [18F]3a with SRꢀpositive PCꢀ3 cells. Cell surfaceꢀbound (A); Internalized
(B); Efflux rate (C) of [18F]3a after PCꢀ3 cell internalization.
Figure 3 In vivo PET studies using [18F]3a in PCꢀ3 tumor bearing mice. (A) Representative
coronal PET images of PCꢀ3 tumorꢀbearing mice at 15 min, 1.5, and 3.5 h postꢀinjection
of [18F]3a and 18F]3a coꢀinjected with haloperidol (i.e. blocking group). Yellow arrowheads
indicate tumor location. Timeꢀactivity curves of U87MG tumor, liver, blood, and muscle are also
shown upon injection of (B) [18F]3a (n = 3) and (C) [18F]3a with blocking (n = 3). (D)
Comparison of PCꢀ3 tumor uptake in [18F]3a and blocking group. * P<0.05.
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Figure 4 Ex vivo evaluation of [18F]3a. (A) Organ distribution profile of [18F]3a and blocking
group at 1.5 h postꢀinjection and 3.5 h postꢀinjection in PCꢀ3 tumorꢀbearing mice. Blocking data
was only shown at 3.5 h p.i. (B) Immunohistological staining of PCꢀ3 tumor, kidney, liver, and
muscle to confirm the expression level of σ1 + σ2 receptors. Magnitude: 200×. Scale bar: 50 ꢁm.
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[CH2]nF
O
O
OH
Br
O
OH
Br
O
N
N
b
NH
OH
NH
a
Br
3a (n = 1)
3b (n = 2)
1
2
aReagents and conditions: (a) 1. SOCl2, CHCl3, reflux, 2. N-(2-aminoethyl)piperidine, Et3N,
toluene/DMF, rt, 62%; (b) TBAOH, F-[CH2]n-OTos (n = 2, 3), DMF, 65 0C, 57 - 61%.
Scheme 1
Scheme 2
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Scheme 3
Ki (nM)
Compound
4-IPAB
R1
I
R2
σ1
σ2
205 ± 67
206 ± 11
H
I
2.57 ± 0.70
11.82 ± 0.68
PIMBA
CH3O
Ki (nM)
Compound
R1
R2
H
σ1
σ2
8
9
FCH2CH2O
CH3O
1076 ± 88
0.65 ± 0.22
8.2 ± 1.4
CH3O
12,900 ± 111
Figure 1
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15000
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Internalized [18F]3a
Surface-bound [18F]3a
1200
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Figure 2
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