Synthesis of a potent and selective 18F-labeled δ-opioid receptor antagonist derived from the Dmt-Tic pharmacophore for positron emission tomography imaging

Article abstract of DOI:10.1021/jm7014765

Identification and pharmacological characterization of two new selective δ-opioid receptor antagonists, derived from the Dmt-Tic pharmacophore, of potential utility in positron emission tomography (PET) imaging are described. On the basis of its high δ se

Full text of DOI:10.1021/jm7014765

J. Med. Chem. 2008, 51, 1817–1823
1817
Synthesis of a Potent and Selective 18F-Labeled δ-Opioid Receptor Antagonist Derived from the
Dmt-Tic Pharmacophore for Positron Emission Tomography Imaging
Eun Kyoung Ryu,‡,† Zhanhong Wu,‡ Kai Chen,‡ Lawrence H. Lazarus,§ Ewa, D. Marczak,§ Yusuke Sasaki,# Akihiro Ambo,#
,|
Severo Salvadori,| Chuancheng Ren,× Heng Zhao,× Gianfranco Balboni,*, and Xiaoyuan Chen*,‡
Molecular Imaging Program at Stanford (MIPS), Department of Radiology and Bio-X Program, Stanford UniVersity School of Medicine,
Stanford, California 94305-5484, Korea Basic Science Institute, Chuncheon Center, Korea, Medicinal Chemistry Group, Laboratory of
Pharmacology and Chemistry, National Institute of EnVironmental Health Sciences, Research Triangle Park, North Carolina 27709, Tohoku
Pharmaceutical UniVersity, 4-1, Komatsushima 4-chome, Aoba-Ku, Sendai 981-8558, Japan, Department of Pharmaceutical Science and
Biotechnology Center, UniVersity of Ferrara, I-44100, Ferrara, Italy, Department of Neurosurgery, Stanford UniVersity,
Stanford, California 94305-5327, and Department of Toxicology, UniVersity of Cagliari, I-09124, Cagliari, Italy
ReceiVed NoVember 27, 2007
Identification and pharmacological characterization of two new selective δ-opioid receptor antagonists, derived
from the Dmt-Tic pharmacophore, of potential utility in positron emission tomography (PET) imaging are
described. On the basis of its high δ selectivity, H-Dmt-Tic-ꢀ-Lys(Z)-OH (reference compound 1) is a useful
starting point for the synthesis of 18F-labeled compounds prepared by the coupling of N-succinimidyl
4-[18F]fluorobenzoate ([18F]SFB) with Boc-Dmt-Tic-ꢀ-Lys(Z)-OH under slightly basic conditions at 37 °C
for 15 min, deprotection with TFA, and HPLC purification. The total synthesis time was 120 min, and the
decay-corrected radiochemical yield of [18F]-1 was about 25–30% (n ) 5) starting from [18F]SFB (n ) 5)
with an effective specific activity about 46 GBq/µmol. In vitro autoradiography studies showed prominent
uptake of [18F]-1 in the striatum and cortex with significant blocking by 1 and UFP-501 (selective δ-opioid
receptor antagonist), suggesting high specific binding of [18F]-1 to δ-opioid receptors. Noninvasive microPET
imaging studies revealed the absence of [18F]-1 in rat brain, since it fails to cross the blood-brain barrier.
This study demonstrates the suitability of [18F]-1 for imaging peripheral δ-opioid receptors.
Introduction
techniques for in vivo assessment of drug distribution and
interaction with biochemical targets systems.12 Among opiate
radioligands already available for PET imaging of δ-opioid
receptors, only the antagonist [11C]methylnaltrindole ([11C]MeN-
TI) allows visualization of these receptors in the human
brain.13,14 Although it displayed high affinity for δ-opioid
Opioid receptors are classified into µ, δ, and κ subtypes and
belong to the superfamily of G-protein-coupled receptors
(GPCRs) that produce their effects by activation of intracellular
Gi/Go proteins. The δ-opioid receptors play an important role
in the modulation of nociceptive signaling encountered in animal
models of pain.1 Moreover, δ agonist administration or use of
δ receptor-deficient mice confirmed that δ-opioid receptors are
involved in emotional responses, such as depression-like
behavior and anxiety.2–4 These receptors are implicated in some
aspects of morphine tolerance and dependence5 with limited
contribution compared to µ-opioid receptors.1 Despite of the
δ-opioid receptor involvement in several clinically relevant
diseases and syndromes, such as analgesia, addiction, Parkin-
son’s, Alzheimer’s, and seizure disorders, their precise role in
humans is incompletely understood.6,7 Moreover, peripheral
δ-opioid receptors seem to be involved in cancer,8 cardiovas-
cular disease,9 gastrointestinal disorders,10 and newer paradigms
for pain relief that use peripherally restricted opioids.11
receptor (Kiδ ) 0.49 nM) and moderate affinity for µ (Kiµ
)
39.2 nM) and κ (Kiκ ) 8.33 nM) receptors, the selectivity was
quite low (Kiµ/ Kiδ ) 80; Kiκ/ Kiδ ) 17; Kiµ/ Kiκ ) 4.71).15 The
development of tracers for PET or SPECT requires the presence
of atoms (C, F, I) that can be substituted for their positron (11C,
18F) or γ (123I)-emitting isotope. Considering the usefulness of
the reactant N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB)16
in the synthesis of 18F labeled peptides and the versatility of
the Dmt-Tica as a δ-opioid pharmacophore,3,4,17–21 we selected
two δ-opioid reference compounds, H-Dmt-Tic-ꢀ-Lys(Z)-OH22
and H-Dmt-Tic-Phe-Lys(Z)-OH,23 as potential tools for PET
Radionuclide imaging techniques, such as positron emission
tomography (PET) and single photon emission computed
tomography (SPECT), are unique and complementary imaging
a Abbreviations. In addition to the IUPAC-IUB Commission on Bio-
chemical Nomenclature (J. Biol. Chem. 1985, 260, 14–42), this paper uses
the following additional symbols and abbreviations: AcOEt, ethyl acetate;
AcOH, acetic acid; Bid, 1H-benzimidazole-2-yl; Boc, tert-butyloxycarbonyl;
DAMGO, [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin; DEL C, deltorphin II (H-
Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2); DMF, N,N-dimethylformamide; DM-
SO-d6, hexadeuteriodimethyl sulfoxide; Dmt, 2′,6′-dimethyl-L-tyrosine;
DPDPE, (D-Pen2,D-Pen5)-enkephalin; Et2O, diethyl ether; GPI, guinea pig
ileum; HOBt, 1-hydroxybenzotriazole; HPLC, high-performance liquid
chromatography; MALDI-TOF, matrix assisted laser desorption ionization
time-of-flight; MVD, mouse vas deferens; NMM, 4-methylmorpholine; pA2,
negative log of the molar concentration required to double the agonist
concentration to achieve the original response; Pe, petroleum ether; TEA
triethylamine; TFA, trifluoroacetic acid; Tic, 1,2,3,4-tetrahydroisoquinoline-
3-carboxylic acid; TLC, thin-layer chromatography; WSC, 1-ethyl-3-[3′-
dimethyl)aminopropyl]carbodiimide hydrochloride; Z, benzyloxycarbonyl.
* To whom correspondence should be addressed. For G.B.: phone, (+39)
532-455-275; fax, (+39) 532-455-953; e-mail, gbalboni@unica.it, bbg@
unife.it. For X.C.: phone, (+1) 650-725-0950; fax, (+1) 650-736-7925;
e-mail, shawchen@stanford.edu.
‡
Stanford University School of Medicine.
Korea Basic Science Institute.
National Institute of Environmental Health Sciences.
Tohoku Pharmaceutical University.
University of Ferrara.
Stanford University.
†
§
#
|
×
University of Cagliari.
10.1021/jm7014765 CCC: $40.75
2008 American Chemical Society
Published on Web 03/01/2008

1818 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Scheme 1. Synthesis of Compound 1
Ryu et al.
imaging on the basis of the similarity between the Z protecting
group and the 4-fluorobenzoyl substituent.
[18F]-1 was prepared by coupling N-succinimidyl 4-[18F]fluo-
robenzoate ([18F]SFB) with Boc-Dmt-Tic-ꢀ-Lys-OH (7) under
slightly basic conditions at 37 °C for 15 min and deprotected
with TFA, followed by HPLC purification (Scheme 3). The
radiochemical yield was 25–30% (n ) 5) from [18F]SFB with
high radiochemical purity (>99%). The effective specific
activity was about 46 GBq/µmol.
Results and Discussion
Chemistry and Radiochemistry. Compounds 1 [H-Dmt-Tic-
ꢀ-Lys(Z)-OH] and 2 [H-Dmt-Tic-Phe-Lys(Z)-OH] were pre-
pared stepwise by solution peptide synthetic methods, outlined
in Schemes 1 and 2, respectively. Boc-Tic-ꢀ-Lys(Z)-OMe (3)22
was deprotected at its N-terminus by TFA treatment and
condensed with Boc-Dmt-OH via WSC/HOBt. The resulting
fully protected intermediate Boc-Dmt-Tic-ꢀ-Lys(Z)-OMe (5)
was Z deprotected by catalytic hydrogenation (H2, 10% Pd/C)
and condensed with 4-fluorobenzoic acid via WSC/HOBt.
Hydrolysis of C-terminal methyl ester (NaOH) and Boc removal
(TFA) at N-terminus gave the final compound 1. Compound 2
was synthesized starting from the fully protected intermediate
Boc-Dmt-Tic-Phe-Lys(Z)-OMe (10)23 (Scheme 2). After Z
removal (H2, 10% Pd/C), it was coupled with 4-fluorobenzoic
acid via WSC/HOBt. Hydrolysis of C-terminal methyl ester
(NaOH) and N-terminal Boc deprotection (TFA) gave the final
compound 2 (Scheme 2).
Receptor Affinity Analysis. Receptor binding and functional
bioactivities are reported in Table 1. Like the majority of the
compounds of general formula H-Dmt-Tic-ꢀ-Lys(R)-R′ (R )
-NH2, -NH-Ac, -NH-Z; R′ ) -CONH-Ph, CONH-
1
CH2-Ph, -Bid [Bid, H-benzimidazole-2-yl]),22 1 exhibited
subnanomolar affinity for δ-opioid receptors (Kiδ ) 0.17 nM).
As expected, the presence of a free carboxylic function in
compounds containing the Dmt-Tic pharmacophore increases
δ selectivity (Kiµ/ Kiδ ) 240) by suppressing µ-opioid receptor
affinity (Kiµ ) 41.2 nM). Compound 2 arose from the second
reference compound [H-Dmt-Tic-Phe-Lys(Z)-OH] by substitu-
tion of the Z protecting group in the Lys4 side chain with a
4-fluorobenzoyl function, causing a slight decrease in δ affinity

Synthesis of δ-Opioid Receptor Antagonist
Scheme 2. Synthesis of Compound 2
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1819
Scheme 3. Synthesis of [18F]-1
(Kiδ ) 0.25 nM) and µ affinity 3-fold (Kiµ ) 13.2 nM) and
resulting in a 4.5-fold loss in δ selectivity (Kiµ/ Kiδ ) 53).
Functional Bioactivity. Analogues 1 and 2 were tested in
the electrically stimulated MVD and GPI assays for intrinsic
functional bioactivity (Table 1). Similar to the reference
compound containing Lys linked to the Dmt-Tic pharmacophore
through its ꢀ-amine group, 1 maintained the same δ antagonism
(MVD, pA2 ) 8.25) and weak µ agonism (GPI, IC50 ) 1916
nM). For the second reference [H-Dmt-Tic-Phe-Lys(Z)-OH],
in which the replacement of the Z protecting group with the

1820 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Ryu et al.
Table 1. Receptor Binding Affinities and Functional Bioactivities of Compounds 1 and 2 and δ Selectivity Kiµ/Kiδa
a The Ki values (nM) were determined according to Cheng and Prusoff.33 The mean ( SE (n repetitions in parentheses) is based on independent duplicate
binding assays with five to eight peptide doses using several different synaptosomal preparations. b Agonist activity was expressed as IC50 obtained from
dose–response curves representing the mean ( SE for at least five to six fresh GPI tissue samples. Deltorphin II and endomorphin-2 were the internal
standards for MVD (δ-opioid receptor bioactivity) and GPI (µ-opioid receptor bioactivity) in the tissue preparations, respectively. c The pA2 values of opioid
antagonists were determined against the agonists deltorphin II and endomorphin-2 according to the method of Kosterlitz and Watt.36 d Data taken from
Balboni et al.22 e Data taken from Balboni et al.23
MicroPET Imaging. Static microPET scans were performed
on a Sprague–Dawley rat and selected coronal, sagittal, and
transaxial images at 15 min after femoral vein injection of [18F]-
1 (Figure 2). Despite the fact that [18F]-1 exhibited prominent
accumulation in δ-opioid receptor positive regions of the brain
by in vitro autoradiography, noninvasive PET imaging clearly
demonstrated the absence of uptake into intact rat brain in vivo,
indicating that this compound does not cross the blood-brain
Figure 1. Autoradiography of rat brain slices incubated with [18F]-1.
barrier (BBB).
In vitro autoradiograms of coronal sections from rat brain in the
presence of (A) [18F]-1, (B) [18F]-1 and 1 (10 µM), and (C) [18F]-1
Conclusions
and UPF-501 (10 µM).
This study showed that 1 is a potent and selective δ-opioid
receptor antagonist, and although [18F]-1 specifically binds to
4-fluorobenzoyl substituent was detrimental for functional
bioactivity (2), δ antagonism fell 95-fold from pA2 ) 11.43 to
δ-opioid receptor in brain slices in vitro, there was an absence
of uptake in brain because of limited penetration of the BBB.
pA2 ) 9.45 accompanied by increased µ agonism (GPI, IC50
)
At present, there is an increasing demand of radioligands for in
vivo imaging studies of peripheral opioid receptors to help assess
the roles they may play in cancer, cardiovascular disease,
gastrointestinal disorders, and pain relief.24 The feasibility of
imaging δ-opioid receptors in normal human heart25 and δ sites
overexpressed in primary tumors of lung26 and breast cancer
patients27 was initially demonstrated in limited studies using
N1′-([11C]methyl)naltrindole and PET; however, because of the
higher affinity and selectivity, [18F]-1 could represent a new
useful tracer for PET imaging of peripheral δ-opioid receptors
as a marker in various disease states.
50.6 nM), which was absent in the corresponding reference
compound. Compound 2 also displayed weak δ agonism at
concentrations greater than 5 µM, which was not observed by
the reference compound. Thus, on the basis of the better
selectivity for the δ-opioid receptor and a high level of δ
antagonism, 1 was exploited for labeling with 18F.
Autoradiography Studies. In vitro autoradiography revealed
that [18F]-1 exhibited high uptake in the caudate putamen
(striatum) and cortex of rat brain slices, regions known to have
a high expression of opioid receptors. The radiotracer uptake
into both the striatum and the cortex was significantly blocked
by 1 or UPF-501 [N,N(Me)2-Dmt-Tic-OH, a potent and selective
δ-pioid antagonist],17 indicating that [18F]-1 binds specifically
and selectively to δ-opioid receptors (Figure 1).
Experimental Section
Chemistry. TFA ·H-Tic-E-Lys(Z)-OMe (4). Boc-Tic-ꢀ-Lys(Z)-
OMe (3)22 (1.67 g, 3.02 mmol) was treated with TFA (2 mL) for

Synthesis of δ-Opioid Receptor Antagonist
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1821
Figure 2. In vivo PET analysis of the distribution of [18F]-1 in rats. Representative coronal, transaxial, and sagittal microPET images of [18F]-1
in the normal Sprague–Dawley rat brain 15 min after femoral vein injection as detailed in the Experimental Section.
0.5 h at room temperature. Et2O/Pe (1:1, v/v) were added to the
solution until the product precipitated: yield 1.58 g (92%); Rf(A)
) 0.49; HPLC K′ ) 4.96; mp 118–120 °C; [R]20D -19.6; m/z 454
(M + H)+.
temperature, 1 N NaOH (0.23 mL, 0.23 mmol) was added. The
reaction mixture was stirred for 4 h at room temperature. After
ethanol was evaporated, the residue was dissolved in solvent B and
directly purified by preparative HPLC as reported above in general
methods: yield 0.1 g (92%); Rf(B) ) 0.67; HPLC K′ ) 4.73; mp
146–148 °C; [R]20D -18.4; m/z 598 (M + H)+; 1H NMR (DMSO-
d6) δ 1.29–1.78 (m, 15H), 2.35 (s, 6H), 2.92–3.49 (m, 7H),
4.41–4.92 (m, 4H), 6.29 (s, 2H), 6.96–7.02 (m, 4H).
Boc-Dmt-Tic-E-Lys(Z)-OMe (5). To a solution of Boc-Dmt-
OH (0.22 g, 0.71 mmol) and TFA·H-Tic-ꢀ-Lys(Z)-OMe (4) (0.4
g, 0.71 mmol) in DMF (10 mL) at 0 °C, NMM (0.08 mL, 0.71
mmol), HOBt (0.12 g, 0.78 mmol), and WSC (0.15 g, 0.78 mmol)
were added. The reaction mixture was stirred for 3 h at 0 °C and
24 h at room temperature. After DMF was evaporated, the residue
was dissolved in EtOAc and washed with citric acid (10% in H2O),
NaHCO3 (5% in H2O), and brine. The organic phase was dried
(Na2SO4) and evaporated to dryness. The residue was precipitated
from Et2O/Pe (1:9, v/v): yield 0.46 g (87%); Rf(B) ) 0.91; HPLC
K′ ) 7.30; mp 133–135 °C; [R]20D -17.5; m/z 746 (M + H)+; 1H
NMR (DMSO-d6) δ 1.29–1.90 (m, 15H), 2.35 (s, 6H), 2.92–3.20
(m, 6H), 3.67 (s, 3H), 4.41–5.34 (m, 7H), 6.29 (s, 2H), 6.96–7.19
(m, 9H).
Boc-Dmt-Tic-Phe-Lys-OMe (11). To a solution of Boc-Dmt-
Tic-Phe-Lys(Z)-OMe (10)23 (0.71 g, 0.8 mmol) in methanol (30
mL) was added Pd/C (10%, 0.2 g), and H2 was bubbled for 1 h at
room temperature. After filtration, the solution was evaporated to
dryness. The residue was precipitated from Et2O/Pe (1:9, v/v): yield
0.58 g (88%); Rf(B) ) 0.81; HPLC K′ ) 5.24; mp 144–146 °C;
[R]20 +35.2; m/z 759 (M + H)+.
D
Boc-Dmt-Tic-Phe-Lys(4-fluorobenzoyl)-OMe (13). This com-
pound was obtained by condensation of Boc-Dmt-Tic-Phe-Lys-OMe
(11) with 4-fluorobenzoic acid via WSC/HOBt as reported for Boc-
Dmt-Tic-ꢀ-Lys(4-fluorobenzoyl)-OMe: yield 0.23 g (85%); Rf(B)
) 0.78; HPLC K′ ) 5.10; mp 135–137 °C; [R]20D +30.6; m/z 881
Boc-Dmt-Tic-E-Lys-OMe (6). To a solution of Boc-Dmt-Tic-
ꢀ-Lys(Z)-OMe (5) (0.37 g, 0.5 mmol) in methanol (30 mL) was
added Pd/C (10%, 0.1 g), and H2 was bubbled for 1 h at room
temperature. After filtration, the solution was evaporated to dryness.
The residue was precipitated from Et2O/Pe (1:9, v/v): yield 0.27 g
1
(M + H)+; H NMR (DMSO-d6) δ 1.29–1.90 (m, 15H), 2.35 (s,
6H), 2.92–3.20 (m, 8H), 3.67 (s, 3H), 4.41–4.92 (m, 6H), 6.29 (s,
2H), 6.96–7.93 (m, 13H).
(90%); Rf(B) ) 0.74; HPLC K′ ) 5.06; mp 139–141 °C; [R]20
Boc-Dmt-Tic-Phe-Lys(4-fluorobenzoyl)-OH (14). This com-
pound was obtained by hydrolysis of Boc-Dmt-Tic-Phe-Lys(4-
fluorobenzoyl)-OMe (13) with 1 N NaOH as reported for Boc-
Dmt-Tic-ꢀ-Lys(4-fluorobenzoyl)-OH: yield 0.15 g (91%); Rf(B) )
0.75; HPLC K′ ) 5.0; mp 139–141 °C; [R]20D +31.4; m/z 867 (M
+ H)+.
D
-18.1; m/z 612 (M + H)+.
Boc-Dmt-Tic-E-Lys(4-fluorobenzoyl)-OMe (8). To a solution
of Boc-Dmt-Tic-ꢀ-Lys-OMe (6) (0.43 g, 0.7 mmol) and 4-fluo-
robenzoic acid (0.1 g, 0.7 mmol) in DMF (10 mL) at 0 °C, HOBt
(0.12 g, 0.77 mmol) and WSC (0.15 g, 0.77 mmol) were added.
The reaction mixture was stirred for 3 h at 0 °C and 24 h at room
temperature. After DMF was evaporated, the residue was dissolved
in EtOAc and washed with citric acid (10% in H2O), NaHCO3 (5%
in H2O), and brine. The organic phase was dried (Na2SO4) and
evaporated to dryness. The residue was precipitated from Et2O/Pe
(1:9, v/v): yield 0.45 g (88%); Rf(B) ) 0.79; HPLC K′ ) 5.25; mp
134–136 °C; [R]20D -17.3; m/z 734 (M + H)+; 1H NMR (DMSO-
d6) δ 1.29–1.94 (m, 15H), 2.35 (s, 6H), 2.92–3.20 (m, 6H), 3.67
(s, 3H), 4.41–4.92 (m, 5H), 6.29 (s, 2H), 6.96–7.93 (m, 8H).
Boc-Dmt-Tic-E-Lys(4-fluorobenzoyl)-OH (9). To a solution of
Boc-Dmt-Tic-ꢀ-Lys(4-fluorobenzoyl)-OMe (8) (0.51 g, 0.7 mmol)
in ethanol (10 mL) at room temperature, 1 N NaOH (1.1 mL, 1.1
mmol) was added. The reaction mixture was stirred for 4 h at room
temperature. After ethanol was evaporated, the residue was dis-
solved in EtOAc and washed with citric acid (10% in H2O) and
brine. The organic phase was dried (Na2SO4) and evaporated to
dryness. The residue was precipitated from Et2O/Pe (1:9, v/v): yield
0.45 g (90%); Rf(B) ) 0.75; HPLC K′ ) 5.14; mp 141–143 °C;
TFA ·H-Dmt-Tic-Phe-Lys(4-fluorobenzoyl)-OH (2). Boc-Dmt-
Tic-Phe-Lys(4-fluorobenzoyl)-OH (14) was treated with TFA as
reported for TFA ·H-Tic-ꢀ-Lys(Z)-OMe: yield 0.08 g (96%); Rf(A)
) 0.40; HPLC K′ ) 3.86; mp 146–149 °C; [R]20D +31.1; m/z 767
1
(M + H)+; H NMR (DMSO-d6) δ 1.29–1.78 (m, 6H), 2.35 (s,
6H), 2.92–3.20 (m, 8H), 3.95–4.92 (m, 5H), 6.29 (s, 2H), 6.96–7.93
(m, 13H). Anal. (C45H49F4N5O9) C, H, N.
Boc-Dmt-Tic-Phe-Lys-OH (12). This compound was obtained
by hydrolysis of Boc-Dmt-Tic-Phe-Lys-OMe (11) with 1N NaOH
as reported for Boc-Dmt-Tic-ꢀ-Lys-OH: yield 0.06 g (86%); Rf(B)
0.65; HPLC K′ 4.75; mp 140–142 °C; [R]20 +36.1; m/z 745 (M
D
+ H)+; 1H NMR (DMSO-d6) δ 1.29–1.78 (m, 15H), 2.35 (s, 6H),
2.92–3.17 (m, 8H), 4.41–4.92 (m, 6H), 6.29 (s, 2H), 6.96–7.21 (m,
9H).
Radiochemistry. For radiochemistry, analytical and semi-
preparative reversed-phase HPLC were performed on a Dionex 680
chromatography system with a UVD 170U absorbance detector and
model 105S single-channel radiation detector (Carroll & Ramsey
Associates). The recorded data were processed using Chromeleon
software (version 6.50). Isolation of peptides and 18F-labeled
peptides was performed using a Vydac protein and peptide column
(218TP510, 5 µm, 250 mm × 10 mm). The flow was set to 5 mL/
min using a gradient system starting from 95% solvent A (0.1%
trifluoroacetic acid [TFA] in water) and 5% solvent B (0.1% TFA
in acetonitrile [ACN]) (0–2 min) and increased to 35% A and 65%
B at 32 min. Analytical HPLC used the same gradient system but
with another Vydac column (218TP54, 5 µm, 250 mm × 4.6 mm)
and with a flow of 1 mL/min. The ultraviolet (UV) absorbance
[R]20 -17.9; m/z 720 (M + H)+.
D
TFA·H-Dmt-Tic-E-Lys(4-fluorobenzoyl)-OH (1). Boc-Dmt-
Tic-ꢀ-Lys(4-fluorobenzoyl)-OH (9) was treated with TFA as
reported for TFA ·H-Tic-ꢀ-Lys(Z)-OMe: yield 0.09 g (94%); Rf(A)
) 0.42; HPLC K′ ) 3.34; mp 149–151 °C; [R]20D -18.5; m/z 620
1
(M + H)+; H NMR (DMSO-d6) δ 1.29–1.82 (m, 6H), 2.35 (s,
6H), 2.92–3.20 (m, 6H), 3.95–4.92 (m, 5H), 6.29 (s, 2H), 6.96–7.93
(m, 8H). Anal. (C36H40F4N4O8) C, H, N.
Boc-Dmt-Tic-E-Lys-OH (7). To a solution of Boc-Dmt-Tic-ꢀ-
Lys-OMe (6) (0.09 g, 0.15 mmol) in ethanol (10 mL) at room

1822 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Ryu et al.
was monitored at 218 nm, and the identification of the peptides
was confirmed on the basis of the UV spectrum acquired using a
PDA detector.
anesthesia. Five minute static scans were taken 15 min after
injection, and the images were reconstructed using a two-
dimensional ordered-subsets expectation maximum (OSEM) algo-
rithm. No correction was necessary for attenuation and scattering.
The [18F]SFB was synthesized following a reported procedure.28,29
The [18F]SFB was dissolved in DMSO (100 µL) and added to the
Boc-protected-compound (Boc-Dmt-Tic-ꢀ-Lys-OH, 200 µg, 0.95
µmol) in borate buffer (300 µL, 0.05 M, pH 8.5) at 37 °C for 15
min. The Boc group was removed with anhydrous TFA (500 µL)
for 5 min at room temperature (Scheme 3). The mixture was purified
by HPLC, and the collected fractions (retention time: 17.9–18.1
min) were evaporated. The radioactivity was then reconstituted in
phosphate-buffered saline (PBS) and passed through a 0.22-µm
Millipore filter into a sterile multidose vial for in vivo applications.
Pharmacology. Competitive Binding Assays. Opioid receptor
affinities were determined under equilibrium conditions [2.5 h at
room temperature (23 °C)] in competition assays using brain P2
synaptosomal membranes prepared from Sprague–Dawley rats.30,31
Synaptosomes were preincubated to remove endogenous opioid
peptides and stored at -80 °C in buffered 20% glycerol.30,32 Each
analogue was analyzed in duplicate assays using five to eight
dosages and three to five independent repetitions with different
synaptosomal preparations (n values are listed in Table 1 in
parentheses and results are the mean ( SE). Unlabeled peptide (2
µM) was used to determine nonspecific binding in the presence of
1.9 nM [3H]deltorphin II (45.0 Ci/mmol, Perkin-Elmer, Boston,
MA; KD ) 1.4 nM) for δ-opioid receptors and 3.5 nM [3H]DAMGO
(50.0 Ci/mmol, Amersham Bioscience, Buckinghamshire, U.K.; KD
) 1.5 nM) for µ-opioid receptors. Glass fiber filters (Whatman
GFC) were soaked in 0.1% polyethylenimine in order to enhance
the signal-to-noise ratio of the bound radiolabeled synaptosome
complex, and the filters were washed thrice in ice-cold buffered
BSA.30 The affinity constants (Ki) were calculated according to
Cheng and Prusoff.33
Biological Activity in Isolated Tissue Preparation. The my-
enteric plexus longitudinal muscle preparations (2–3 cm segments)
from the small intestine of male Hartley strain of guinea pigs (GPI)
measured µ-opioid receptor agonism, and a single mouse vas
deferens (MVD) was used to determine δ-opioid receptor agonism
as described previously.34 The isolated tissues were suspended in
organ baths containing balanced salt solutions in a physiological
buffer, pH 7.5. Agonists were tested for the inhibition of electrically
evoked contraction and expressed as IC50 (nM) obtained from the
dose–response curves. The IC50 values represent the mean ( SE
of five or six separate assays, and the δ-antagonist potencies in the
MVD assay were determined against the δ-agonist deltorphin-II,
while µ-antagonism (GPI assay) used the µ-agonist endomorphin-
2. Antagonism is expressed as pA2 determined using the Schild
plot.35
Acknowledgment. This study was supported in part by NCI
Grants R01 CA119053, R21 CA121842, R21 CA102123, and
P50 CA114747 to X.C. and by grants from University of
Cagliari to G.B., from University of Ferrara to S.S., and from
the Intramural Research Program of the NIH and NIEHS to
L.H.L. and E.D.M.
Supporting Information Available: Chemistry general methods,
elemental analysis results, MS and HPLC data. This material is
available free of charge via the Internet at http://pubs.acs.org.
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