Indium-labeled macrocyclic conjugates of naltrindole: High-affinity radioligands for in vivo studies of peripheral δ opioid receptors

Article abstract of DOI:10.1021/jm0700013

We have identified a series of hydrophilic indium-labeled DOTA and DO3A conjugates of naltrindole (NTI) that are suited to in vivo studies of peripheral δ opioid receptors. Indium(III) complexes, linked to the indole nitrogen of NTI by six- to nine-atom s

Full text of DOI:10.1021/jm0700013

2144
J. Med. Chem. 2007, 50, 2144-2156
Indium-Labeled Macrocyclic Conjugates of Naltrindole: High-Affinity Radioligands for In Vivo
Studies of Peripheral δ Opioid Receptors
Romain A. Duval,†,‡ Rachel L. Allmon,†,‡ and John R. Lever*,†,‡,§
Departments of Radiology and the Radiopharmaceutical Sciences Institute, and Medical Pharmacology and Physiology, UniVersity of
MissourisColumbia, Columbia, Missouri 65212, and Research SerVice, Harry S. Truman Veterans Administration Medical Center,
Columbia, Missouri 65201
ReceiVed January 1, 2007
We have identified a series of hydrophilic indium-labeled DOTA and DO3A conjugates of naltrindole (NTI)
that are suited to in vivo studies of peripheral δ opioid receptors. Indium(III) complexes, linked to the
indole nitrogen of NTI by six- to nine-atom spacers, display high affinities (0.1-0.2 nM) and excellent
selectivities for binding to δ sites in vitro. The [111In]-labeled complexes can be prepared in good isolated
yields (∼65%) with high specific radioactivities (>3300 mCi/µmol). The spacers serve as pharmacokinetic
modifiers, and log D7.4 values range from -2.74 to -1.79. These radioligands exhibit a high level of specific
binding (75-94%) to δ opioid receptors in mouse gut, heart, spleen, and pancreas in vivo. Uptakes of
radioactivity are saturable by the non-radioactive complexes, inhibited by naltrexone, and blocked by NTI.
Thus, these radiometal-labeled NTI analogues warrant further study by single-photon emission computed
tomography.
Introduction
The development of selective radioligands for molecular
imaging of the three classical types (µ, κ, and δ) of cerebral
opioid receptors has been an active research arena for over two
decades.1 For instance, N1′-([11C]methyl)naltrindole2 (Figure 1)
allows quantitative positron emission tomography (PET) studies
of δ opioid receptors in the brain of healthy persons3 and patient
populations.4 Naltrindole (NTI, 1) was designed by Portoghese
and colleagues using the message-address concept.5 The
benzene ring of the indole provides a δ “address” by mimicking
Phe4 of enkephalin, while the 4,5-epoxymorphinan skeleton
provides a strong, general opioid binding “message.” The pyrrole
serves as a spacer where N1′-substitutions preserve δ opioid
receptor affinity and selectivity. Accordingly, NTI has been a
useful scaffold for the construction of additional radioligands
(Figure 1). Those with δ affinities in the picomolar range include
[18F]fluoroethyl6 and p-[18F]fluorobenzyl7 derivatives for PET,
and an [123I]iodoallyl8 derivative for single-photon emission
computed tomography (SPECT). Bulk tolerance at the indole
nitrogen is high, and even N1′-benzylfluorescein congeners
display appreciable δ opioid receptor affinities (Kis 12-24 nM)
and modest selectivities (4- to 40-fold) over µ and κ sites in
vitro.9
Most radioligands for in vivo studies of opioid receptors have
been designed for brain imaging and are lipophilic to permit
passage across the blood-brain barrier.1,10 However, there is
an increasing need for in vivo imaging studies of peripheral
opioid receptors to help assess the roles they may play in
cancer,11 cardiovascular disease,12 gastrointestinal disorders,13
and newer paradigms for pain relief that use peripherally
restricted opioids.14 The feasibility of imaging δ opioid receptors
of normal human heart,15 and δ sites overexpressed by the
Figure 1. Structures of NTI, radiolabeled analogues, and a hypothetical
radiometal-labeled N1′-conjugate.
primary tumors of lung16 and breast17 cancer patients, has been
demonstrated in limited studies using N1′-([11C]methyl)naltrin-
dole and PET. Hydrophilic opioid radioligands, designed for
peripheral studies, might show lower nonspecific uptake, higher
specific binding, faster blood clearance, and favorable radiation
dosimetry. Further, a focus on hydrophilic ligands permits the
use of chelated radiometals as the marker elements. Depending
upon nuclear properties,18 radiometal-labeled opioids might be
employed for SPECT (e.g., 99mTc, 111In) or PET (e.g., 68Ga,
64Cu) imaging or perhaps for radiotherapy (e.g., 90Y, 177Lu).
The development of radiometal-labeled conjugates of small
molecules that target receptors is a challenge for reasons
including the large size of the metal coordination sphere with
respect to the pharmacophore (cf. Figure 1), and considerations
of the thermodynamic and kinetic stability of the chelated
metal.18,19 The polyaminocarboxylates DOTAa and DO3A are
versatile chelators that form a variety of stable radiometal
* To whom correspondence should be addressed. Phone: (573) 814-
6000 ext. 53686. Fax: (573) 814-6551. E-mail: leverj@health.missouri.edu.
† Department of Radiology and the Radiopharmaceutical Sciences
Institute, University of MissourisColumbia.
a Abbreviations: DOTA, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tet-
raacetic acid; DO3A, 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid;
EDTA, ethylenediaminetetraacetic acid; DIBAL-H, diisobutylaluminum
hydride; NTI, naltrindole; DAMGO, [D-Ala2, N-MePhe4, Gly5-ol]enkephalin;
DPDPE, [D-Pen2, D-Pen5]enkephalin; TIPP(ψ), H-Tyr-Tic[CH2NH]-Phe-
Phe-OH, Tic ) 1,2,3,4-tetrahydroisoquinoline; U69,593, (+)-(5R,7R,8â)-
N-methyl-N-[7-(pyrrolidin-1-yl)-1-oxaspiro[4,5]dec-8-yl] benzeneacetamide.
‡ Harry S. Truman Veterans Administration Medical Center.
§ Department of Medical Pharmacology and Physiology, University of
MissourisColumbia.
10.1021/jm0700013 CCC: $37.00 © 2007 American Chemical Society
Published on Web 04/03/2007

Indium-Labeled Macrocyclic Conjugates of Naltrindole
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9 2145
Scheme 1a
a Reagents and conditions: (a) 3-bromopropionaldehyde dimethylacetal, NaH, DMF, 2 h, 77%; (b) NaOH, i-PrOH, reflux, 15 h, 83%; (c) p-TsOH,
acetone, 3 h, 74%; (d) 6-bromohexanenitrile, NaH, DMF, 1 h, 96%; (e) DIBAL-H, CH2Cl2, toluene, 15 min, 40%; (f) NaOH (1.0 N), reflux, 16 h, 64%.
Scheme 2a
complexes.18,19 They are often used with indium-111, a radio-
nuclide that has appropriate half-life (2.8 days) and γ-emission
characteristics (171 and 245 keV) for SPECT imaging. These
favorable properties, coupled with the acceptance of bulk at the
indole nitrogen, prompted us to investigate a series of indium-
(III) and [111In]-labeled DOTA and DO3A conjugates of NTI.
Here we report their synthesis, lipophilicities, and opioid
receptor binding properties in vitro, along with data showing
high levels of specific binding in vivo to the peripheral δ opioid
receptors of mice.20
Results and Discussion
Organic Synthesis. Two approaches were employed for
connecting NTI to DO3A or DOTA. In the first, conjugates
were prepared by reductive amination of N1′-aldehyde deriva-
tives with DO3A to afford three- and six-carbon alkyl linkers
(Scheme 1). NTI, with the phenol protected by a tosyl moiety
(2),6a was selectively alkylated at the indole nitrogen with
3-bromopropionaldehyde dimethylacetal to give 3 (77%), or with
6-bromohexanenitrile to give 7 (96%). Excess alkylating agent
was required for good conversion due to competing â-elimina-
tion. Hydrolysis of 3 with warm base afforded 4 (83%), and
the dimethylacetal protecting group was removed by acid-
catalyzed transacetalization to give N1′-propionaldehyde 5
(74%). The complementary N1′-hexanaldehyde 8 was prepared
in moderate yield (40%) by partial nitrile reduction of 7 using
diisobutylaluminum hydride (DIBAL-H). These intermediates
were coupled with DO3A, liberated from the tris(t-butyl ester)
by neat trifluoroacetic acid (TFA),21 using reductive amination
mediated by NaBH(OAc)3 and Et3N to provide 6 (68%) and 9
(87%). With this selective borohydride reagent,22 no alcohol
was detectable from aldehyde reduction. Hydrolysis of 9
produced phenol 10 (64%) to complete the sequence.
In the second approach, N1′-aminoalkyl derivatives of NTI
were coupled with the activated N-hydroxysuccinimidyl ester
of DOTA (DOTA-NHS) to afford conjugates linked by six- and
nine-atom alkylcarboxamide chains (Scheme 2). Alkylation of
2 with N-(3-bromopropyl)phthalimide gave 11 in good yield
(84%). Hydrolysis of both protecting groups was accomplished
in one step to give N1′-aminopropyl derivative 12 (93%).
Treatment of N1′-hexanenitrile 7, a common intermediate from
a Reagents and conditions: (a) N-(3-bromopropyl)phthalimide, NaH,
DMF, 2 h, 84%; (b) NaOH, i-PrOH, reflux, 15 h, 93%; (c) 6-bromohex-
anenitrile, NaH, DMF, 1 h, 96%; (d) n-Bu4NOH, CH3OH, 1,4-dioxane,
reflux, 8 h, 68%; (e) DIBAL-H, CH2Cl2, toluene, 3 h, 64%.
the first approach, with methanolic n-Bu4NOH provided phenol
13 (68%). Complete nitrile reduction using excess DIBAL-H
gave N1′-aminohexyl derivative 14 (64%). Amines 12 and 14
were coupled with DOTA-NHS using Et3N and catalytic 4-N,
N′-dimethylaminopyridine (DMAP) to furnish a good yield of
15 (74%) and a modest yield of 16 (34%) as the TFA salts.
Complex Formation and Radiolabeling. Preparation of the
non-radioactive and radioactive indium complexes proved
straightforward (Scheme 3). Treatment of the DO3A (6, 10)

2146 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9
DuVal et al.
Scheme 3. Preparation of Indium(III) and [111In]-Labeled Complexes
and DOTA (15, 16) conjugates with InCl3 at 100 °C for 30-
45 min, followed by reversed-phase C18 chromatography and
lyophilization, provided good isolated yields (69-95%) of 17-
20 as TFA salts. The structures depicted in Scheme 3 represent
the typical18 seven-coordinate In3+ complexes shown by NMR
spectroscopy to predominate in solution.23 In the solid state,
however, crystallographic data indicate that the carboxamide
oxygen of DOTA complexes like 19 and 20 can participate in
bonding, leading to an eight-coordinate, inverted square anti-
prism geometry.23
Similarly, treatment of the polydentate chelators with no-
carrier-added [111In]Cl3 (0.5-3.8 mCi) in NH4OAc buffer at
95-100 °C for 30 min gave 83-93% incorporations of
radioactivity. Reversed-phase HPLC purification, followed by
solid-phase extraction, provided good isolated yields (55-83%)
of [111In]17-[111In]20. Average yields, over multiple prepara-
tions, were about 65%. All radioligands were obtained with high
chemical and radiochemical purities, and coeluted with their
non-radioactive counterparts under HPLC conditions that fully
resolved the precursors from the complexes. Representative
analytical traces are shown in Figure 2. Specific radioactivities
exceeded 3300 mCi/µmol, and the radioligands were stable
during overnight storage.
negative. Differences noted between radiolabeled complexes
were consistent with substituent constant additivity,24 with the
most lipophilic complex [111In]18 having a n-hexyl linker, and
the most hydrophilic complex [111In]19 having an acetami-
dopropyl linker. Calculated log Pow values for the parent DO3A
and DOTA chelators showed Spearman rank order (r ) 0.98,
p ) 0.02) correlation with log D7.4 values measured directly
for the radiolabeled complexes, but they were 1.1 to 1.8 log
units higher due to computational treatment as neutral entities.
Competition Binding Assays. Opioid receptor binding assays
(Table 1) were performed using membranes from fresh CD1
mouse (δ, [3H]NTI)26 or frozen guinea pig (µ, [3H]DAMGO;
κ, [3H]U69,593) brains.27 Although guinea pig brain has a
significant population of δ sites,6b,27 we chose to use mouse
brain for these primary site assays because δ receptors, and their
interactions with [3H]NTI, are well defined in this tissue.26a
Thus, the data represents binding of the novel ligands in vitro
in one of the few tissues where δ opioid receptors have been
characterized from the species to be used for in vivo studies
Lipophilicity. Relative lipophilicity is a key physicochemical
parameter that influences pharmacokinetics.10,24,25 Distribution
coefficients were determined for [3H]NTI and for complexes
[
111In]17-[111In]20, using n-octanol and pH 7.4 phosphate buffer
(Table 1). For the DO3A (6, 10) and DOTA (15, 16) chelating
agents, log Pow values were calculated using ClogP software.
All of the macrocyclic analogues of NTI were quite hydrophilic,
and measured log D7.4 values for the [111In]complexes were
Figure 3. Panel A: Saturation binding isotherm, Rosenthal plot insert,
for N1′-([111In]DOTA-propylamido)NTI ([111In]19) in mouse brain
homogenates. Panel B: Inhibition of [111In]19 (0.15 nM, 1500 mCi/
µmol) binding by δ receptor ligands.
Figure 2. Reversed-phase HPLC chromatograms showing coelution
of purified [111In]17 with a standard sample of 17 (top panel) and degree
of separation between these complexes and precursor 6 (bottom panel).

Indium-Labeled Macrocyclic Conjugates of Naltrindole
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9 2147
Table 1. Opioid Receptor Binding Properties and Lipophilicity Measures for NTI, Its DOTA and DO3A Conjugates, and Their Indium Complexes
Ki (nM)a
selectivityb
c
log D7.4
or
compd
δ
µ
κ
µ/δ
κ/δ
99
410
130
131
131
ClogPd
1 (NTI)
6
17
10
18
15
19
16
20
0.15 ( 0.02
4.9 ( 0.31
9.7 ( 0.82
0.20 ( 0.01
0.11 ( 0.01
0.46 ( 0.03
0.21 ( 0.03
0.18 ( 0.01
0.19 ( 0.01
33.9 ( 0.76
1748 ( 248
230 ( 24
48.2 ( 9.0
34.4 ( 1.4
130 ( 25
67.5 ( 6.0
96.6 ( 9.8
35.3 ( 7.5
14.9 ( 0.75
2010 ( 268
1259 ( 175
26.2 ( 2.8
14.4 ( 1.0
367 ( 74
510 ( 143
674 ( 28
130 ( 12
226
357
24
241
313
283
321
537
186
2.41 ( 0.08c
-1.20d
-2.47 ( 0.011c
0.038d
-1.79 ( 0.010c
-1.65d
798
2428
3744
684
-2.74 ( 0.012c
-0.64d
-1.97 ( 0.004c
a Means ( SEM, n ) 3-6. δ, [3H]NTI, mouse brain; µ, [3H]DAMGO, guinea pig brain; κ, [3H]U69,593, guinea pig brain. Hill coefficients near unity
(0.80-1.24). b Ki ratios. c Measured values ( SD, n ) 4, for [3H]NTI and [111In]17-[111In]20 using n-octanol and pH 7.4 phosphate buffer. d Calculated
using ClogP software.
Figure 4. Pseudocolor images from in vitro autoradiography of δ sites in horizontal sections of mouse brain using N1′-([111In]DOTA-propylamido)-
NTI ([111In]19) at 0.1 nM (1500 mCi/µmol) in the presence and absence of opioid receptor ligands naltriben (NTB), DAMGO, and U69,593.
(vide infra). The overall opioid receptor binding profile obtained
for NTI agreed well with determinations by others using similar
radioligands and tissues.6b,26,28 The major finding for the novel
compounds is the use of linkers having six to nine atoms
preserves the high δ opioid receptor affinity of NTI and
maintains or improves δ selectivity. These chelators (10, 15,
16) and their indium(III) complexes (18, 19, 20) give pico-
molar apparent affinities (Kis) for δ opioid receptors ac-
companied by >100-fold selectivities against secondary interac-
tions at µ and κ sites. By contrast, 6 and 17 having three-
atom linkers are far less potent binders at all opioid receptors.
Nonetheless, their δ affinities are in the low nanomolar range,
and δ selectivities are good. Examination of molecular models
suggests that the shorter linker allows closer approach of the
pendant moiety to the phenol. This would hinder binding
to all opioid receptor types since the phenol contributes to the
general binding message of 4,5-epoxymorphinans29 through
hydrogen bonding with histidine residue 17 of transmem-
brane domain (TM) VI.30a As a spot check for other interac-
tions, NTI and the novel macrocyclic analogues were tested in
σ1 and σ2 receptor binding assays,31 and they showed no
displacement of radioligand binding at 10 µM concentrations
(data not shown).
The δ receptor affinities between pairs of polydentate
chelators and their indium complexes (i.e., 6/17; 10/18; 15/19;
16/20) were similar, consistent with a lack of significant
interactions with the hydrophobic address locus of the δ opioid
receptor at TMs VI and VII.27,30 However, the chelators
generally exhibited lower affinities than the metal complexes
for µ and κ sites. This signifies that structural features of the
pendant macrocycle do influence these interactions, with free
charged carboxylate groups detrimental to binding. Complexes
18 and 19 each have six-atom linkers, but they differ by design
in the n-hexyl vs acetamidopropyl chains. Complex 18 was more
potent than 19 for all three opioid receptors, but with lower δ
over κ selectivity. The same trends were noted for the respective
chelators 10 and 15, indicating an effect of linker on binding.
Together, the data show that subtle structural influences are felt
some distance from the primary NTI pharmacophore. Interest-
ingly, affinities tended to become higher with increasing
lipophilicity for complexes 18-20, with the most prominent
effect at κ receptors (Table 1). The profile of 18, having the
highest lipophilicity, most closely resembled that of NTI. One
might speculate that more lipophilic ligands present a greater
effective concentration to the receptors by differential sequestra-
tion in membrane fragments.32 This could enhance competition

2148 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9
Table 2. Biodistribution of [111In]-Labeled Complexes 19 (log D7.4 - 2.74), 20 (log D7.4 - 1.97), and 18 (log D7.4 - 1.79) in Male CD1 Micea
111In]19
DuVal et al.
[
tissue
5 min
15 min
30 min
60 min
120 min
240 min
blood
heart
lung
liver
spleen
pancreas
stomach
s. intestine
cecum
l. intestine
kidney
muscle
bone
3.40 ( 0.22
3.58 ( 0.28
2.79 ( 0.10
9.73 ( 2.49
2.00 ( 0.18
1.48 ( 0.23
1.52 ( 0.48
3.06 ( 0.36
1.01 ( 0.19
1.94 ( 0.21
6.82 ( 0.81
0.78 ( 0.10
0.85 ( 0.21
0.16 ( 0.09
5.5
1.19 ( 0.10
4.01 ( 0.60
2.11 ( 1.23
20.96 ( 1.92
2.30 ( 0.30
1.45 ( 0.31
2.03 ( 0.38
6.09 ( 0.69
1.72 ( 0.22
3.67 ( 0.84
3.30 ( 1.34
0.33 ( 0.03
0.33 ( 0.06
0.06 ( 0.02
14.6
0.58 ( 0.07
4.61 ( 0.64
1.08 ( 0.07
18.27 ( 2.42
1.89 ( 0.72
1.84 ( 0.52
3.65 ( 0.22
9.45 ( 1.92
1.74 ( 0.50
3.58 ( 1.48
1.55 ( 0.20
0.19 ( 0.01
0.16 ( 0.06
0.04 ( 0.01
13.9
0.32 ( 0.10
3.65 ( 1.92
0.82 ( 0.18
13.08 ( 2.27
2.34 ( 0.20
1.46 ( 0.81
3.24 ( 1.81
10.73 (2.25
1.77 ( 0.20
3.32 ( 0.40
1.75 ( 0.42
0.16 ( 0.02
0.10 ( 0.03
0.02 ( 0.00
22.9
0.32 ( 0.20
3.70 ( 0.29
0.71 ( 0.14
8.03 ( 1.52
1.75 ( 0.16
1.67 ( 0.13
5.08 ( 1.39
13.21 (3.24
5.55 ( 2.66
5.31 ( 2.08
1.61 ( 0.39
0.12 ( 0.03
0.11 ( 0.02
0.02 ( 0.01
31.5
0.43 ( 0.35
2.46 ( 0.23
0.52 ( 0.10
4.40 ( 0.98
1.36 ( 1.21
1.25 ( 0.47
4.72 ( 3.08
8.87 ( 2.40
28.31 ( 14.18
12.33 ( 3.52
1.12 ( 0.01
0.08 ( 0.02
0.06 ( 0.02
0.01 ( 0.00
28.0
brain
urine
[
111In]20
tissue
5 min
15 min
30 min
60 min
120 min
240 min
blood
heart
lung
liver
spleen
pancreas
stomach
s. intestine
cecum
l. intestine
kidney
muscle
bone
6.66 ( 1.15
6.29 ( 1.79
4.63 ( 0.90
3.91 ( 0.46
2.88 ( 0.44
2.03 ( 0.30
1.43 ( 0.28
6.22 ( 0.66
1.86 ( 0.06
4.36 ( 0.71
5.85 ( 1.01
1.04 ( 0.17
1.15 ( 0.52
0.21 ( 0.06
3.6
3.98 ( 0.05
8.68 ( 1.67
3.64 ( 0.21
4.94 ( 0.06
3.70 ( 0.69
2.83 ( 1.67
2.40 ( 0.20
11.74 ( 1.03
2.86 ( 0.39
7.84 ( 0.75
4.45 ( 0.31
0.66 ( 0.05
0.64 ( 0.18
0.14 ( 0.01
14.8
2.18 ( 0.32
7.12 ( 1.47
2.45 ( 0.07
4.37 ( 0.33
3.91 ( 0.86
3.57 ( 0.69
3.27 ( 1.07
14.95 ( 0.78
3.50 ( 0.72
8.93 ( 0.54
5.04 ( 2.87
0.52 ( 0.09
0.39 ( 0.05
0.08 ( 0.01
11.4
0.95 ( 0.11
6.14 ( 0.17
1.72 ( 0.15
4.44 ( 0.34
4.22 ( 0.89
3.32 ( 1.30
2.92 ( 0.72
12.55 ( 0.81
3.45 ( 0.49
9.22 ( 1.37
3.32 ( 0.59
0.35 ( 0.03
0.33 ( 0.08
0.05 ( 0.00
25.3
0.47 ( 0.02
4.08 ( 0.22
1.32 ( 0.07
4.51 ( 0.47
2.45 ( 0.05
2.35 ( 1.35
4.48 ( 0.84
10.81 ( 2.18
5.12 ( 0.62
9.43 ( 1.31
2.66 ( 0.26
0.22 ( 0.01
0.16 ( 0.06
0.03 ( 0.01
33.1
0.34 ( 0.07
3.09 ( 0.54
0.99 ( 0.20
4.32 ( 0.25
2.09 ( 0.78
2.02 ( 1.12
2.78 ( 0.34
9.36 ( 3.08
6.66 ( 0.72
7.54 ( 1.12
2.40 ( 0.21
0.18 ( 0.02
0.18 ( 0.03
0.03 ( 0.00
31.2
brain
urine
[
111In]18
tissue
5 min
15 min
30 min
60 min
120 min
240 min
blood
heart
lung
liver
spleen
pancreas
stomach
s. intestine
cecum
l. intestine
kidney
muscle
bone
8.09 ( 0.45
7.22 ( 0.74
6.47 ( 0.80
6.24 ( 0.47
3.42 ( 1.69
2.24 ( 0.34
1.73 ( 0.24
9.09 ( 1.32
2.06 ( 0.25
6.28 ( 1.30
8.86 ( 1.23
1.55 ( 0.33
1.24 ( 0.23
0.28 ( 0.11
6.4
3.84 ( 0.53
7.38 ( 0.94
3.47 ( 0.43
7.17 ( 0.31
3.15 ( 0.73
2.19 ( 0.37
2.78 ( 0.88
11.07 ( 0.70
2.86 ( 0.32
7.40 ( 0.60
5.45 ( 1.55
0.69 ( 0.13
0.50 ( 0.20
0.12 ( 0.03
15.8
2.17 ( 0.19
7.41 ( 2.08
2.40 ( 0.11
7.07 ( 0.40
2.92 ( 0.69
3.12 ( 0.17
3.22 ( 0.39
15.07 ( 1.20
4.13 ( 0.27
9.18 ( 0.78
3.66 ( 0.44
0.53 ( 0.13
0.41 ( 0.09
0.08 ( 0.01
24.6
0.98 ( 0.24
7.92 ( 1.75
1.87 ( 0.45
6.29 ( 1.19
2.82 ( 0.56
4.81 ( 1.65
4.47 ( 1.12
16.81 ( 0.67
4.13 ( 0.87
12.28 ( 1.95
2.87 ( 0.59
0.37 ( 0.05
0.23 ( 0.13
0.04 ( 0.00
29.2
0.54 ( 0.08
5.94 ( 0.18
1.45 ( 0.34
3.89 ( 0.48
3.39 ( 0.43
3.02 ( 1.20
3.99 ( 0.71
16.73 ( 1.38
5.29 ( 2.20
9.80 ( 1.39
2.64 ( 0.66
0.25 ( 0.04
0.18 ( 0.03
0.04 ( 0.01
30.7
0.25 ( 0.12
4.56 ( 0.41
1.02 ( 0.14
3.15 ( 0.71
3.32 ( 1.60
2.75 ( 1.09
3.91 ( 0.97
15.48 ( 1.97
11.80 ( 3.59
10.43 ( 2.28
2.28 ( 0.19
0.18 ( 0.02
0.13 ( 0.03
0.03 ( 0.00
35.0
brain
urine
a Values are %ID/g (means ( SD, n ) 3), except for urine which is %ID.
during assays, and it may be one contributing factor to the
observed affinities and selectivities.
state for [111In]17 and [111In]19, respectively (data not shown).
The Kd values are in good agreement with the Ki values from
the δ opioid receptor competition binding assays (Table 1), and
the Bmax values are near the 83.9 fmol/mg protein previously
obtained in whole mouse brain using [3H]NTI.26a
Direct Binding Assays. Findings from the competition
experiments were confirmed by direct determination of binding
parameters for [111In]17 and [111In]19 in mouse brain mem-
branes. “Cold” saturation studies using a low, fixed amount of
The specific binding of both [111In]17 and [111In]19 was
inhibited potently by δ, but not by µ or κ, opioid receptor
ligands. Ki values for δ selective ligands against [111In]17 were
0.28 ( 0.09 nM for NTI and 3.68 ( 1.10 nM for the peptide
DPDPE (data not shown). Against [111In]19, Ki values were 0.16
( 0.01 nM for NTI and 4.19 ( 0.24 nM for DPDPE (Figure
3B). Hill coefficients were near unity in each case, ranging from
0.95 to 1.10. Neither the µ selective peptide DAMGO, nor the
κ selective arylacetamide U69,593, displaced the specific
binding of [111In]17 or [111In]19 at 1.0-2.0 µM (data not
shown). Further, specific binding of the complexes was not
[
111In]17 in the presence of increasing concentrations of 17 gave
a measured Kd of 7.21 ( 0.94 nM and a site density (Bmax) of
82.7 ( 8.1 fmol/mg protein (data not shown). “Hot” saturation
studies using increasing concentrations of [111In]19, intentionally
lowered to 1500 mCi/µmol by addition of 19, gave a Kd of 0.16
( 0.01 nM and a Bmax of 122 ( 1.3 fmol/mg protein (Figure
3A). Rosenthal transformations of both data sets were linear,
consistent with labeling of a single binding site. Incubation times
at 37 °C for these assays were based upon association studies
showing that 45 min and 2 h were necessary to achieve steady

Indium-Labeled Macrocyclic Conjugates of Naltrindole
Table 3. Effects of Pretreatments on In Vivo Uptake of [111In]-Labeled Complexes 19, 20, and 18 in Male CD1 Micea
111In]19
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9 2149
[
tissue
saline controls
naltrexone
complex 19
NTI
stomach
s. intestine
cecum
l. intestine
heart
spleen
pancreas
lung
muscle
brain
liver
kidney
urine
5.24 ( 1.85
9.18 ( 1.22
2.16 ( 0.51
5.65 ( 0.88
3.10 ( 0.60
2.43 ( 0.32
1.53 ( 0.12
0.80 ( 0.10
0.11 ( 0.02
0.02 ( 0.00
14.79 ( 1.18
1.71 ( 0.36
22.2
4.62 ( 2.24
2.67 ( 0.65
0.45 ( 0.14
1.17 ( 0.15
0.52 ( 0.12
0.58 ( 0.10
0.33 ( 0.10
0.61 ( 0.08
0.09 ( 0.01
0.03 ( 0.00
18.58 ( 1.34
1.93 ( 0.31
31.6
2.48 ( 1.11
1.52 ( 0.76
0.13 ( 0.02
0.21 ( 0.02
0.26 ( 0.03
0.43 ( 0.11
0.22 ( 0.06
0.56 ( 0.03
0.08 ( 0.02
0.03 ( 0.01
17.70 ( 1.78
3.68 ( 2.50
24.7
2.45 ( 3.08
1.53 ( 0.68
0.25 ( 0.19
0.29 ( 0.04
0.30 ( 0.04
0.61 ( 0.08
0.20 ( 0.08
0.64 ( 0.10
0.11 ( 0.03
0.03 ( 0.01
16.25 ( 2.21
3.38 ( 2.51
35.3
blood
0.28 ( 0.05
0.78 ( 0.14
0.61 ( 0.05
1.45 ( 1.01
[
111In]20
tissue
saline controls
naltrexone
complex 20
NTI
stomach
s. intestine
cecum
l. intestine
heart
spleen
pancreas
lung
muscle
brain
liver
kidney
urine
blood
3.89 ( 0.67
13.91 ( 2.18
4.28 ( 0.73
10.23 ( 1.10
7.28 ( 0.99
5.01 ( 1.22
3.76 ( 1.46
1.92 ( 0.05
0.40 ( 0.07
0.07 ( 0.03
4.36 ( 0.37
5.05 ( 1.63
22.0
2.63 ( 0.84
4.65 ( 0.16
1.48 ( 0.10
4.35 ( 0.66
2.30 ( 0.53
1.57 ( 0.24
1.35 ( 0.62
2.11 ( 0.31
0.38 ( 0.04
0.07 ( 0.01
7.07 ( 0.62
4.08 ( 0.73
0.68 ( 0.17
1.50 ( 0.27
0.56 ( 0.14
1.22 ( 0.29
1.77 ( 0.34
1.34 ( 0.32
1.18 ( 0.19
3.47 ( 1.04
0.72 ( 0.18
0.13 ( 0.04
4.73 ( 0.71
9.12 ( 3.96
23.2
0.63 ( 0.06
1.10 ( 0.07
0.40 ( 0.04
0.99 ( 0.04
1.00 ( 0.07
0.81 ( 0.10
0.74 ( 0.14
2.01 ( 0.13
0.37 ( 0.02
0.08 ( 0.00
6.99 ( 0.23
4.29 ( 0.589
34.0
2.10 ( 0.25
41.4
1.22 ( 0.12
3.88 ( 0.65
2.23 ( 0.20
[
111In]18
tissue
saline controls
naltrexone
complex 18
NTI
stomach
s. intestine
cecum
l. intestine
heart
spleen
pancreas
lung
muscle
brain
liver
kidney
urine
blood
3.43 ( 0.64
15.62 ( 1.48
4.15 ( 0.76
11.97 ( 1.46
6.08 ( 0.65
3.12 ( 1.06
3.35 ( 0.56
1.79 ( 0.09
0.36 ( 0.03
0.05 ( 0.01
6.63 ( 0.94
2.75 ( 0.18
27.3
1.57 ( 0.30
4.20 ( 0.43
1.32 ( 0.18
4.06 ( 0.23
1.83 ( 0.30
1.19 ( 0.19
0.90 ( 0.36
1.68 ( 0.18
0.28 ( 0.02
0.07 ( 0.02
8.81 ( 1.33
3.62 ( 0.40
1.69 ( 0.78
2.10 ( 0.54
0.44 ( 0.10
0.98 ( 0.15
1.28 ( 0.24
0.98 ( 0.13
1.00 ( 0.19
2.70 ( 0.49
0.61 ( 0.18
0.09 ( 0.01
9.87 ( 1.46
9.00 ( 2.90
21.8
1.01 ( 0.29
2.26 ( 0.51
0.27 ( 0.04
0.79 ( 0.08
0.85 ( 0.06
0.61 ( 0.10
0.76 ( 0.29
1.65 ( 0.08
0.29 ( 0.05
0.06 ( 0.01
8.77 ( 0.56
4.02 ( 0.67
33.3
35.7
1.44 ( 0.09
0.79 ( 0.10
3.72 ( 1.28
1.69 ( 0.09
a Values are %ID/g (means ( SD, n ) 4) 60 min after radioligand administration. Urine is %ID. Naltrexone (10 µmol/kg), indium(III) complexes (5
µmol/kg), and NTI (5 µmol/kg) were given 5 min prior to radioligand.
inhibited by haloperidol at 1.0-2.0 µM, concentrations that
would block significant contributions from serotonin 5-HT2,
dopamine D1-4, σ1-2, or R1-adrenergic receptors.31,33 Thus, these
NTI.35 The δ opioid receptor distribution of [111In]19 was
abolished by the δ selective naltriben (5 nM), but not by 50-
fold higher concentrations of µ selective DAMGO or κ selective
U69,593.
[
111In]complexes selectively label δ opioid receptors in vitro
when used near their Kd values.
In Vivo Pharmacokinetics. Pharmacokinetic profiles for the
Autoradiography. The δ opioid receptor binding of [111In]-
19 was investigated by autoradiography in order to extend the
findings from homogenate assays. Although the [111In]com-
plexes were not designed for brain studies in vivo, we used
sections from mouse brain since the populations of the multiple
opioid receptors are well-defined. Appropriate topography for
selective labeling of δ sites was observed (Figure 4), with the
levels of radioactivity being high in striatal and cortical
regions, intermediate in the hippocampal formation, low in
thalamus and colliculi, and barely detectable in cerebellum.
This rank order is in full accord with δ opioid receptor den-
sities known from autoradiographic studies of mouse brain
using [125I]-[D-Ala2]deltorphin-I34 and of rat brain using [3H]-
[
111In]complexes were determined after iv administration to male
CD1 mice. Data for high-affinity complexes [111In]18-[111In]-
20 is given in Table 2, and a graphical representation for [111
-
In]18 is shown in Figure 5. Biodistributions reflect the effect
of relative lipophilicities on first-pass extraction, coupled with
clearance from tissues having few δ opioid receptors and
prolonged retention in peripheral organs that are rich in δ opioid
receptors (vide infra). None of the [111In]complexes penetrated
the brain in accord with their high molecular weights and
hydrogen bonding ability. Bone uptakes were low, a finding
consistent with good metabolic stability. Negligible radioactivity,
<0.2% of the injected dose (ID), was noted in feces during the
studies. Cumulative urinary excretion of radioactivity was nearly

2150 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9
DuVal et al.
able, presumably due to lower affinity for δ opioid receptors.
Radioactivity cleared quickly from blood and most peripheral
organs (data not shown), with retention for the intestines (ca.
15% ID/g, 60 min).
In Vivo Pharmacology. The pharmacology of the [111In]-
complexes was assessed by pretreatment of mice with various
opioids and determination of tissue radioactivity 60 min later.
As shown in Table 3 and Figure 6, uptakes of the high-affinity
complexes [111In]18-[111In]20 in heart, spleen, pancreas, and
all regions of the gastrointestinal tract were blocked by the
“universal” opioid receptor antagonist naltrexone (10 µmol/kg),
saturated by the corresponding cold complex (5 µmol/kg), and
fully inhibited by the δ selective NTI (5 µmol/kg). The only
exception was the stomach of [111In]19 where coefficients of
variation were high, and inhibition did not reach statistical
significance. Using NTI blockade to define nonspecific binding,
levels of specific binding to δ opioid receptors were 86-90%
in heart, 77-87% in pancreas, 75-84% in spleen, and 83-
Figure 5. Temporal biodistribution after intravenous administration
of [111In]18 (3.5 µCi) to male CD1 mice. Values are %ID/g (means (
SEM, n ) 3).
94% across the three intestinal regions. For low-affinity [111
-
complete for all radioligands within the first 60 min and ranged
from 22.9% to 29.2% ID.
In]17, gut levels were reduced 22-52% by naltrexone, cold
complex, and NTI, but reached significance only for naltrexone
and cold complex (data not shown). By contrast, the inhibitors
had either little effect on, or in some cases increased, radioactiv-
ity levels in blood, lung, kidney, and skeletal muscle (Table 3,
Figure 6). We attribute these elevations to greater radiotracer
availability for nonspecific partitioning as a consequence of
blockade of specific δ opioid receptor binding in other organs.
The cold complexes exerted pronounced effects compared to
NTI or naltrexone. We conclude that the [111In]complexes
exhibit a component of binding in vivo that is saturable, but
nonspecific to classical opioid receptors, in as yet unidentified
peripheral regions.
There are few radioligand binding studies of peripheral δ
opioid receptors in vitro or in vivo that can be used for direct
comparison to the data obtained with these [111In]complexes in
mouse. However, significant populations of δ sites are known
for organ systems where the [111In]complexes showed ap-
preciable specific binding. Radioligand binding studies have
demonstrated δ and κ, but not µ, opioid receptors in membranes
from adult rat heart.36 Likewise, δ and κ mRNA expression has
been found for human heart,12b and cardioprotective activation
of δ sites is of clinical interest.12 Binding studies have shown
δ opioid receptors in islets of Langerhans from rat pancreas37
and in rat splenocytes.38 The significance of opioid receptors
to gastrointestinal function is well recognized, and multiple
opioid receptors are distributed throughout the stomach and
intestines of mice, rats, guinea pigs, dogs, and human beings.13,39
The most detailed studies are in porcine gut, where δ sites
having picomolar affinity for [3H]NTI are located in high
densities (∼60 fmol/mg protein) in all intestinal segments.39d
Initial blood levels of radioactivity, as well as the 5 min
uptakes by lung, heart, spleen, pancreas, intestines, and skeletal
muscle, increased with radioligand lipophilicity (cf. Tables 1
and 2). The most lipophilic complex, [111In]18 (log D7.4 -1.79),
displayed 2- to 3-fold higher levels of uptake than the least
lipophilic complex [111In]19 (log D7.4 -2.74). Correlation of
these early volumes of distribution with hydrophobicity is pre-
sumably due to differential membrane permeability and binding
to serum proteins. After extraction, radioactivity for heart,
spleen, pancreas, and the gastrointestinal tract, organs with
appreciable δ opioid receptors,12,13,36-39 remained stable or
increased over time (Table 2, Figure 5). By contrast, all three
radioligands showed rapid clearance from blood, lung, skeletal
muscle, and kidney in accord with low levels of δ opioid
receptors.11a,b,37,40 These data were fitted by monoexponential
curves (r ) 0.89-0.99; data not shown; cf. Figure 5).
Liver uptakes for [111In]18 and [111In]20, the most lipophilic
complexes, were similar and low (4 - 7% ID/g; Table 2).
Higher uptakes were shown by [111In]19 (Table 2) and [111In]-
17, the least lipophilic complexes, with particularly high values
for [111In]17 (24.0% ID/g, 5 min; 12.1% ID/g, 240 min; data
not shown). Such differences might rise from substituent effects
on liver metabolism or carrier-mediated transport.41 However,
a simple explanation is higher binding of [111In]18/[111In]20 to
serum proteins leads to reduced availability for hepatocellular
uptake compared to [111In]17/[111In]19 as previously shown for
doxorubicin analogues.42 Thus, an optimal range of lipophilicity
exists that reduces liver uptake and might improve SPECT image
quality. Other biodistribution data for [111In]17 were unremark-
Figure 6. Inhibitor effects on in vivo uptake of [111In]18 at 60 min in male CD1 mice. Values are %ID/g (means ( SD, n ) 4). Naltrexone (10
µmol/kg), 18 (5 µmol/ kg), and NTI (5 µmol/kg) were given 5 min prior to radioligand. *Tissues showing significant inhibition of radioligand
binding.

Indium-Labeled Macrocyclic Conjugates of Naltrindole
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9 2151
Mallinckrodt, Inc., [3H]NTI was from Perkin-Elmer, and [3H]-
DAMGO and [3H]U69,593 were from Amersham Biosciences.
High-resolution mass spectroscopy (HRMS) by electron impact (EI)
or electrospray (ESI) mode was done at the University of Minnesota.
1H NMR spectra were obtained at the University of Missouri,
Department of Chemistry, on a Bruker DRX 300 MHz spectrometer.
Chemical shifts are parts per million (δ) relative to residual solvent
(CHCl3, 7.24 ppm; HOD, 4.80 ppm; CD2HCN, 1.94 ppm), with
coupling constants (J) in hertz (Hz). Elemental analyses (Atlantic
Microlab, Inc.) agreed with calculated values ((0.4%). Normal-
phase silica gel (<230 mesh; Merck 7729) and reversed-phase C18
(35-75 µm; Analtech) column chromatography were done under
N2. TLC was done on Macherey-Nagel silica gel 60 UV254 (250
µm) or Whatman reversed-phase MKC18F 60 Å plates. Animal
studies were done humanely in compliance with National Institutes
of Health guidelines and with Institutional Animal Care and Use
Committee approvals.
17-(Cyclopropylmethyl)-6,7-didehydro-4,5r-epoxy-3-(p-tosy-
loxy)-14-hydroxy-1′-(3′′,3′′-dimethoxypropyl)indolo[6,7:2′,3′]-
morphinan (3). An ice-cold solution of 26a (0.70 g, 1.2 mmol)
and 3-bromopropionaldehyde dimethylacetal (0.94 mL, 6.9 mmol)
in anhydrous DMF (7 mL) under argon was treated with powdered
NaH (0.13 g, 5.0 mmol) under stirring. The mixture was warmed
to room temperature, stirred 2 h, and partitioned between saturated
sodium carbonate buffer (55 mL, pH 9.5) and EtOAc (30 mL).
The organic layer was washed with buffer (4 × 20 mL), dried (Na2-
SO4), filtered, and concentrated. Silica gel chromatography (EtOAc/
cyclohexane, 1:1; 1% Et3N) gave 3 (0.64 g, 77%) as a white foam.
HRMS-EI m/z calcd for C38H42N2O7S [M + H]+ 671.2785; found
671.2783. Anal. (C38H42N2O7S‚0.5H2O) C, H, N.
17-(Cyclopropylmethyl)-6,7-didehydro-4,5r-epoxy-3,14-dihy-
droxy-1′-(3′′,3′′-dimethoxypropyl)indolo[6,7:2′,3′]morphinan (4).
A stirred solution of 3 (0.76 g, 1.1 mmol) in i-PrOH (50 mL) was
treated with 1 N NaOH (25 mL) and brought to reflux under argon.
After 15 h the mixture was cooled, brought to pH 9.5 with 1 N
HCl (20 mL), diluted with brine (100 mL), and extracted with CH2-
Cl2 (5 × 20 mL). Combined extracts were dried (Na2SO4), filtered,
and concentrated. Silica gel chromatography (EtOAc/cyclohexane,
1:1; 1% Et3N) gave 4 (0.50 g, 83%) as an off-white solid. HRMS-
EI m/z calcd for C31H36N2O5 [M + H]+ 517.2697; found 517.2728.
Anal. (C31H36N2O5‚1.5H2O) C, H, N.
17-(Cyclopropylmethyl)-6,7-didehydro-4,5r-epoxy-3,14-dihy-
droxy-1′-(2′′-formylethyl)indolo[6,7:2′,3′]morphinan (5). A solu-
tion of 4 (0.34 g, 0.64 mmol) and p-TsOH‚H2O (0.26 g, 1.3 mmol)
in acetone (9 mL) was refluxed 3 h under argon. The mixture was
cooled, concentrated, and partitioned between sodium carbonate
buffer (10 mL) and EtOAc (20 mL). The organic layer was washed
with carbonate buffer, dried (Na2SO4), filtered, and concentrated
to give 5 (0.32 g, ∼74%) of 88% purity. This material was typically
used without further purification due to losses (40%) upon
chromatography. For characterization, 5 was purified by C18
chromatography (CH3CN:H2O, 30:70, 0.1% TFA) and partitioned
between EtOAc and carbonate buffer. The organic extracts were
dried (Na2SO4), filtered, and concentrated to give the free base.
HRMS-ESI m/z calcd for C29H30N2O4 [M + H]+ 471.2284; found
471.2293. Anal. (C29H30N2O4‚0.5H2O) C, H, N.
In keeping with the pharmacokinetic and pharmacologic data
for the [111In]complexes, δ opioid receptors are not thought to
be highly expressed by lung, skeletal muscle, liver, or kidney.
Detection of δ sites was not possible in normal mouse lung
homogenates by radioligand binding,11a and gene expression was
not observed in normal human lung.11b Radioligand binding to
δ sites in rat lung has been reported,43 raising the possibility of
species differences. Skeletal muscle is typically considered an
opioid “blank,”37 though low levels of functional δ sites can be
localized on mouse muscle fibers by autoradiography.40 Opioid
peptides are present in rat liver extracts, but classical opioid
receptors are not found in liver preparations.37 Opioid drugs
influence renal function,44 and δ receptor agonists increase
urinary flow and sodium excretion in rats as long as renal nerves
are intact.45 Functional δ opioid receptors must be present, but
so far only transcripts have been reported in rat kidney.46
Similarly, the low levels of functional opioid receptors expressed
by cells of the immune system are difficult to detect by
conventional radioligand binding assays.47 The high specific
activity, affinity, and selectivity of the [111In]-labeled conjugates
of NTI may present an opportunity for sensitive in vitro assays
of δ opioid receptors in such systems, although the 2.8 day half-
life imposes constraints on routine usage.
Overall, the in vivo data for complexes [111In]18-[111In]20
is consistent with a high level of specific labeling of δ opioid
receptors throughout the periphery of mice. Other in vivo studies
of radioligand binding to peripheral opioid receptors have been
more limited. The δ opioid peptide [3H]DPDPE showed
persistent uptake (∼30% ID) in the small intestines of mice for
1-2 h.48 However, blocking studies were not performed to
differentiate hepatobiliary clearance from specific receptor
binding. An in vivo study of the δ pseudopeptide, [125I]ITIPP-
(ψ), in nude mice bearing small cell lung cancer xenografts
showed uptake in tumor, intestines, and liver that was inhibited
(40-63%) by non-radioactive TIPP(ψ).49 The data suggests
specific binding to δ sites. Yet, as noted by the authors, only a
single inhibitor, closely related in structure to the radiotracer,
was employed. A radioiodinated diprenorphine showed in vivo
uptake by breast cancer xenografts, intestines, and heart of
immunodeficient mice that could be blocked by naltrexone (23-
59%).50 This radioligand, however, is not selective for a single
type of opioid receptor, a limited inhibition profile was reported,
and significant metabolic degradation was observed.
Conclusions. We have identified a series of hydrophilic
[
111In]-labeled DOTA and DO3A conjugates of NTI that exhibit
picomolar affinities and excellent selectivity for δ opioid
receptors in vitro and show high levels of specific binding to
peripheral δ opioid receptors in vivo. These [111In]complexes
seem well suited for studies of cardiac, intestinal, and other
peripheral δ opioid receptors by SPECT, have the potential for
noninvasive characterization of cancers that express these sites,
and might aid in certain new drug development efforts.
Extensions to include ligands labeled with a variety of other
radiometals also should be possible.
17-(Cyclopropylmethyl)-6,7-didehydro-4,5r-epoxy-3,14-dihy-
droxy-1′-(10′′′-[3′′-propyl]-[1′′′,4′′′,7′′′,10′′′-tetraazacyclododecane-
1′′′,4′′′,7′′′-triacetic acid])indolo[6,7:2′,3′]morphinan; N1′-(DO3A-
propyl)NTI (6). A suspension of crude 5 (66 mg, 98 µmol), DO3A‚
2TFA (0.15 g, 0.26 mmol) and Et3N (265 µL, 1.89 mmol) in
CH3CN (1.3 mL) under argon was treated with NaBH(OAc)3 (46
mg, 0.21 mmol) and stirred 5 h at room temperature. The reaction
was quenched with H2O (0.25 mL) and concentrated. The residue
was dissolved (CH3CN:H2O, 50:50, 1% TFA; 3 mL) and taken to
dryness three times. A solution (0.1% TFA) was then loaded on a
C18 column (1 × 8 cm) and eluted with CH3CN:H2O (30:70, 0.1%
TFA). Similar fractions were combined, concentrated, diluted with
H2O, and lyophilized to give 6 (102 mg, 87%) as a white powder.
HRMS-ESI m/z calcd for C43H56N6O9 [M + H]+ 801.4182; found
Experimental Section
General Information. Reagents and solvents were the best
grades available and were used as received. NTI was prepared5
from naltrexone‚HCl (Mallinckrodt, Inc.) and converted6a to p-
toluenesulfonate ester 2 as previously reported. DO3A was prepared
from 1,4,7,10-tetraazacyclododecane-1,4,7-tris(t-butyl acetate) (Mac-
rocyclics, Inc.) by treatment with neat TFA,21 and it was character-
ized by elemental analysis as DO3A‚2TFA. DOTA-NHS was from
Macrocyclics, Inc. Naltriben mesylate, NTI‚HCl, DAMGO, DP-
DPE, and U69,593 were from Sigma-RBI. [111In]Cl3 was from

2152 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9
DuVal et al.
801.4191. m/z calcd for [M + Na]+ 823.4006; found 823.4006.
Anal. (C43H56N6O9‚3TFA‚3H2O) C, H, N, F.
between saturated sodium carbonate buffer (35 mL) and EtOAc
(70 mL). The organic layer was washed with carbonate buffer (4
× 30 mL), dried (Na2SO4), filtered, and concentrated. The yellow
syrup was purified by silica gel chromatography (EtOAc/cyclo-
hexane, 2:3; 1% Et3N) to give 11 (1.07 g, 84%) as a white foam.
HRMS-ESI m/z calcd for C44H41N3O7S [M + H]+ 756.2743; found
756.2775. Anal. (C44H41N3O7S) C, H, N.
17-(Cyclopropylmethyl)-6,7-didehydro-4,5r-epoxy-3,14-dihy-
droxy-1′-(3′′-aminopropyl)indolo[6,7:2′,3′]morphinan (12). A
suspension of 11 (0.54 g, 0.71 mmol) in i-PrOH (23 mL) was treated
with 1 N NaOH (17 mL) and brought to reflux under argon. After
15 h, the mixture was cooled in an ice bath and brought to pH 9.5
with 1 N HCl (20 mL). Solid NaCl was added, with swirling, until
phases separated. The aqueous phase was extracted with i-PrOH
(3 × 10 mL), and the extracts were concentrated. Cold CH3CN
(70 mL, 0.3% TFA) was added and then concentrated. The residue
was loaded on a plug of C18, washed with H2O (0.1% TFA), and
then eluted with CH3CN (0.1% TFA). Final purification was
achieved by C18 chromatography (CH3CN:H2O, 25:75, 0.1% TFA)
to give 12 (0.55 g, 93%) as a white powder. HRMS-ESI m/z calcd
for C29H33N3O3 [M + H]+ 472.2600; found 472.2601. Anal.
(C29H33N3O3‚3TFA‚H2O) C, H, N, F.
17-(Cyclopropylmethyl)-6,7-didehydro-4,5r-epoxy-3,14-dihy-
droxy-1′-(5′′-cyanopentyl)indolo[6,7:2′,3′]morphinan (13). A stirred
solution of 7 (1.49 g, 2.25 mmol) in 1,4-dioxane (17 mL) was
treated dropwise with methanolic n-Bu4NOH (7.7 mL, 1 M) and
brought to reflux under argon. After 8 h, the mixture was cooled,
quenched (3 N HCl, 2.5 mL), and partitioned between saturated
sodium carbonate buffer (50 mL) and EtOAc (150 mL). The organic
layer was washed with carbonate buffer (3 × 30 mL), dried (Na2-
SO4), filtered, and concentrated. Silica gel chromatography (EtOAc/
cyclohexane, 2:3; 1% Et3N) gave 13 (0.79 g, 68%) as a pale yellow
powder. HRMS-ESI m/z calcd for C32H35N3O3 [M + H]+ 510.2751;
found 510.2775. Anal. (C32H35N3O3‚0.5H2O) C, H, N.
17-(Cyclopropylmethyl)-6,7-didehydro-4,5r-epoxy-3,14-dihy-
droxy-1′-(6′′-aminohexyl)indolo[6,7:2′,3′]morphinan (14). A so-
lution of DIBAL-H in toluene (10 mL, 1.0 M) was added over 8
min to a stirred solution of 13 (0.50 g, 0.96 mmol) in CH2Cl2 (3.5
mL) at 0 °C under argon. After 3 h, the mixture was quenched by
dropwise addition of 0.1 N HCl (12 mL) and taken to dryness under
reduced pressure. The residue was diluted with CH3CN:H2O (35:
65, 1% 3 N HCl; 20 mL), loaded on a C18 column (2.5 × 12 cm),
and eluted with CH3CN:H2O (35:65, 1% 3 N HCl). Similar
fractions, giving a pink color with vanillin-sulfuric acid spray
reagent, were pooled, concentrated, and then purified again under
the same conditions. Combined fractions were concentrated, diluted
with H2O, and lyophilized to give 14 (387 mg, 64%). HRMS-ESI
m/z calcd for C32H39N3O3 [M + H]+ 514.3070; found 514.3064.
Anal. (C32H39N3O3‚4HCl‚4.5H2O) C, H, N.
17-(Cyclopropylmethyl)-6,7-didehydro-4,5r-epoxy-3-(p-tosy-
loxy)-14-hydroxy-1′-(5′′-cyanopentyl)indolo[6,7:2′,3′]morphi-
nan (7). An ice-cold solution of 2 (1.39 g, 2.44 mmol) and
6-bromohexanenitrile (1.02 mL, 7.31 mmol) in anhydrous DMF
(14 mL) under argon was treated with powdered NaH (0.12 g, 4.9
mmol) under vigorous stirring. The mixture was warmed to room
temperature, stirred 1 h, and then partitioned between sodium
carbonate buffer (40 mL) and EtOAc (100 mL). The organic layer
was washed with carbonate buffer (4 × 30 mL), dried (Na2SO4),
filtered, and concentrated. Silica gel chromatography (EtOAc/
cyclohexane, 2:3; 1% Et3N) gave 7 (1.56 g, 96%) as a white foam.
HRMS-ESI m/z calcd for C39H41N3O5S [M + H]+ 664.2840; found
664.2868. m/z calcd for [M + Na]+ 686.2659; found 686.2644.
Anal. (C39H41N3O5S) C, H, N.
17-(Cyclopropylmethyl)-6,7-didehydro-4,5r-epoxy-3-(p-tosy-
loxy)-14-hydroxy-1′-(5′′-formylpentyl)indolo[6,7:2′,3′]morphi-
nan (8). A solution of DIBAL-H in toluene (3.7 mL, 1.0 M) was
added over 1 min to a stirred solution of 7 (0.41 g, 0.62 mmol) in
CH2Cl2 (8 mL) at room temperature under argon. After 15 min,
the mixture was quenched by dropwise addition of 3 N HCl (10
mL), and diluted with CH3CN:H2O (50:50, 1% 3N HCl) until
homogeneous. This solution was loaded on a C18 column (2.5 ×
12 cm) and eluted with CH3CN:H2O (50:50, 1% 3 N HCl). Similar
fractions, giving a brown color with vanillin-sulfuric acid spray
reagent, were pooled, concentrated, and then purified a second time
using the same conditions. The residue was partitioned between
CH2Cl2 (5 × 25 mL) and saturated sodium carbonate buffer (50
mL). The combined extracts were dried (Na2SO4), filtered, and
concentrated to give 8 (167 mg, 40%) as a white solid. HRMS-
ESI m/z calcd for C39H42N2O6S [M + H]+ 667.2836; found
667.2865. Anal. (C39H42N2O6S‚0.4H2O) C, H, N.
17-(Cyclopropylmethyl)-6,7-didehydro-4,5r-epoxy-3-(p-tosy-
loxy)-14-hydroxy-1′-(10′′′-[6′′-hexyl]-[1′′′,4′′′,7′′′,10′′′-tetraaza-
cyclododecane-1′′′,4′′′,7′′′-triacetic acid])indolo[6,7:2′,3′]mor-
phinan (9). A suspension of 8 (105 mg, 156 µmol), DO3A‚2TFA
(0.12 g, 0.20 mmol), and Et3N (220 µL, 0.83 mmol) in DMF (2.0
mL) under argon was treated with NaBH(OAc)3 (52 mg, 0.24
mmol) and stirred 1 h at room temperature. The reaction was
quenched with H2O (0.10 mL), and concentrated. The residue was
treated with H2O (10 mL), neat TFA (100 µL), and CH3CN (2.5
mL) and then purified by gradient C18 column chromatography
(H2O to CH3CN:H2O, 50:50; 0.1% TFA). Similar fractions were
combined, concentrated, diluted with H2O, and lyophilized to give
9 (140 mg, 68%) as a white powder. HRMS-ESI m/z calcd for
C53H68N6O11S [M + H]+ 997.4740; found 997.4736. m/z calcd for
[M + Na]+ 1019.4559; found 1019.4570. Anal. (C53H68N6O11S‚
2.5TFA‚2H2O) C, H, N, F.
17-(Cyclopropylmethyl)-6,7-didehydro-4,5r-epoxy-3,14-dihy-
droxy-1′-(10′′′-[6′′-hexyl]-[1′′′,4′′′,7′′′,10′′′-tetraazacyclododecane-
1′′′,4′′′,7′′′-triacetic acid])indolo[6,7:2′,3′]morphinan; N1′-(DO3A-
hexyl)NTI (10). A solution of 9 (100 mg, 76 µmol) in 1 N NaOH
(10 mL) was refluxed 16 h under argon. The reaction was chilled,
treated dropwise with 30% TFA (3 mL), and then diluted with water
(5 mL) and CH3CN (4 mL). The mixture was loaded on a C18
column (1 × 8 cm), and eluted with aqueous TFA (0.1%, 10 mL)
followed by CH3CN:H2O (40:60, 0.1% TFA). A second C18
purification (CH3CN:H2O, 25:75, 0.1% TFA) was done, and the
usual isolation procedure followed by lyophilization gave 10 (60
mg, 64%) as white flakes. HRMS-ESI m/z calcd for C46H62N6O9
[M + H]+ 843.4651; found 843.4695. m/z calcd for [M + Na]+
865.4470; found 865.4488. Anal. (C46H62N6O9‚3TFA ‚2.75H2O)
C, H, N, F.
17-(Cyclopropylmethyl)-6,7-didehydro-4,5r-epoxy-3-(p-tosy-
loxy)-14-hydroxy-1′-[3′′-(N-phthalimido)propyl]indolo[6,7:2′,3′]-
morphinan (11). An ice-cold solution of 2 (0.96 g, 1.69 mmol)
and N-(3-bromopropyl)phthalimide (3.67 g, 13.4 mmol) in anhy-
drous DMF (10 mL) under argon was treated with powdered NaH
(0.28 g, 11 mmol) under vigorous stirring. The mixture was allowed
to warm to ambient temperature, stirred for 2 h, and then partitioned
17-(Cyclopropylmethyl)-6,7-didehydro-4,5r-epoxy-3,14-dihy-
droxy-1′-(10′′′-[3′′-acetamidopropyl]-[1′′′,4′′′,7′′′,10′′′-tetraaza-
cyclododecane-1′′′,4′′′,7′′′-triacetic acid])indolo[6,7:2′,3′]mor-
phinan; N1′-(DOTA-propylamido)NTI (15). A mixture of 12 (209
mg, 0.251 mmol), DOTA-NHS (347 mg, 0.418 mmol), DMAP (10
mg, 82 µmol), and Et3N (500 µL, 3.56 mmol) in CH3CN (2.2 mL)
was stirred 5 h at room temperature under argon and then
concentrated. Cold H2O (10 mL) and TFA (80 µL) were added
and then concentrated. Gradient C18 chromatography (CH3CN:H2O,
25:75 to 35:65, 0.1% TFA) was performed twice to give 15 (225
mg, 74%) as an off-white powder after concentration, dilution with
H2O, and lyophilization. HRMS-ESI m/2z calcd for C45H59N7O10
[M + 2H]2+ 429.7239; found 429.7259. Anal. (C45H59N7O10‚
2.5TFA‚3.5H2O) C, H, N, F.
17-(Cyclopropylmethyl)-6,7-didehydro-4,5r-epoxy-3,14-dihy-
droxy-1′-(10′′′-[6′′-acetamidohexyl]-[1′′′,4′′′,7′′′,10′′′-tetraazacy-
clododecane-1′′′,4′′′,7′′′-triacetic acid])indolo[6,7:2′,3′]morphi-
nan; N1′-(DOTA-hexylamido)NTI (16). A mixture of 14 (78 mg,
0.15 mmol), DOTA-NHS (190 mg, 0.229 mmol), DMAP (4.0 mg,
33 µmol), and Et3N (210 µL, 1.49 mmol) in DMSO (1.2 mL) was
stirred 1.5 h at room temperature under argon. Workup as described
above, using gradient C18 chromatography (CH3CN:H2O, 10:90

Indium-Labeled Macrocyclic Conjugates of Naltrindole
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9 2153
to 30:70, 0.1% TFA), provided 16 (65 mg, 34%) as a shiny white
powder after lyophilization. HRMS-ESI m/z calcd for C48H65N7O10
[M + Na]+ 922.4685; found 922.4694. Anal. (C48H65N7O10‚
2.75TFA‚3H2O) C, H, N, F.
tetraazacyclododecane-1′′′, 4′′′,7′′′-triacetate]])indolo[6,7:2′,3′]-
morphinan; N1′-(In(III)-DOTA-hexylamido)NTI (20). A solution
of 16 (20 mg, 16 µmol) and InCl3 (10.0 mg, 45 µmol) in H2O (4.5
mL) was refluxed 45 min, cooled to ambient temperature, and
quenched with 0.1 N disodium EDTA (0.75 mL). Workup as above,
using C18 chromatography (CH3CN:H2O, 35:65, 0.1% TFA), gave
20 (20 mg, 95%) as white flakes after lyophilization. 1H NMR (D2O,
300 MHz) δ 7.45 (d, J ) 7.3 Hz, 1H, H7′), 7.43 (d, J ) 7.9 Hz,
1H, H4′), 7.25 (dd, J ) 7.0 Hz, J ) 7.9 Hz, 1H, H5′), 7.09 (dd, J
) 7.0 Hz, J ) 7.3 Hz, 1H, H6′), 6.82 (d, J ) 8.2 Hz, 1H, H1/ H2),
6.77 (d, J ) 8.2 Hz, 1H, H1/H2), 5.90 (s, 1H, H5), 4.24 (m, 3H,
H9, H1′′), 2.86-3.56 (m, 24H, H8R/H8â, H10, H16ax/H16eq, H18,
H6′′, N-CH2CdO, N-CH2), 2.35-2.79 (m, 11H, H8R/H8â,
H15ax/H15eq, H16ax/H16eq, N-CH2), 1.77 (m, 3H, H2′′, H15ax/
H15eq), 1.42 (m, 2H, H5′′), 1.32 (m, 2H, H3′′), 1.27 (m, 2H, H4′′),
1.08 (m, 1H, H19), 0.79 (m, 2H, H20, H21), 0.44 (m, 2H, H20,
H21). HRMS-ESI m/z calcd for C48H62InN7O10 [M + H]+
1012.3670; found 1012.3657. Anal. (C48H62InN7O10‚2.25TFA‚
4H2O) C, H, N, F.
17-(Cyclopropylmethyl)-6,7-didehydro-4,5r-epoxy-3,14-dihy-
droxy-1′-(indium(III)-[10′′′-[3′′-propyl]-[1′′′,4′′′,7′′′,10′′′-tetraaza-
cyclododecane-1′′′,4′′′,7′′′-triacetate]])indolo[6,7:2′,3′]morphi-
nan; N1′-(In(III)-DO3A-propyl)NTI (17). A solution of 6 (35 mg,
29 µmol) and InCl3 (9.6 mg, 43 µmol) in 0.4 M NH4OAc (8.2
mL) was refluxed for 30 min, cooled to ambient temperature, and
quenched with 0.1 N disodium EDTA (0.7 mL). This solution was
loaded on a C18 column (1 × 8 cm) equilibrated with H2O (0.1%
TFA), washed with H2O (0.1% TFA), and then eluted with CH3-
CN:H2O (30:70, 0.1% TFA). Similar fractions were combined,
concentrated under reduced pressure, diluted with H2O, and
lyophilized to give (34 mg, 93%) as white flakes. 1H NMR (D2O,
300 MHz) δ 7.48 (d, J ) 7.8 Hz, 2H, H4′, H7′), 7.31 (dd, J ) 7.5
Hz, J ) 7.8 Hz, 1H, H5′), 7.16 (dd, J ) 7.5 Hz, J ) 7.8 Hz, 1H,
H6′), 6.84 (d, J ) 8.2 Hz, 1H, H1/H2), 6.72 (d, J ) 8.2 Hz, 1H,
H1/H2), 5.93 (s, 1H, H5), 4.34 (m, 2H, H1′′), 4.25 (m, 1H, H9),
3.67 (d, J ) 17.4 Hz, 1H, H8R/H8â), 3.37-3.58 (m, 2H, H8R/
H8â, H10â), 3.33 (m, 1H, H10R), 2.40-3.33 (m, 27H, H16ax/
H16eq, H18, H3′′, N-CH2, N-CH2CdO), 2.31 (m, 2H, H2′′a,
H16ax/ H16eq), 2.01 (m, 1H, H2′′b), 1.81 (m, 2H, H15), 1.09 (m,
1H, H19), 0.82 (m, 2H, H20, H21), 0.48 (m, 2H, H20, H21).
HRMS-ESI m/z calcd for C43H53InN6O9 [M + H]+ 913.2986; found
913.2994. Anal. (C43H53InN6O9‚2.5TFA‚3H2O) C, H, N, F.
17-(Cyclopropylmethyl)-6,7-didehydro-4,5r-epoxy-3,14-dihy-
droxy-1′-(indium(III)-[10′′′-[6′′-hexyl]-[1′′′,4′′′,7′′′,10′′′-tetraaza-
cyclododecane-1′′′,4′′′,7′′′-triacetate]])indolo[6,7:2′,3′]morphi-
nan; N1′-(In(III)-DO3A-hexyl)NTI (18). A solution of 10 (23 mg,
19 µmol) and InCl3 (9.0 mg, 41 µmol) in 0.4 M NH4OAc (3.0
mL) was refluxed 30 min, cooled to ambient temperature, and
quenched with 0.1 N disodium EDTA (0.4 mL). Workup as above,
using C18 chromatography (CH3CN:H2O, 30:70, 0.1% TFA), gave
18 (17 mg, 72%) as white flakes after lyophilization. 1H NMR (D2O,
300 MHz) δ 7.53 (d, J ) 7.6 Hz, 1H, H7′), 7.49 (d, J ) 8.1 Hz,
1H, H4′), 7.31 (dd, J ) 7.2 Hz, J ) 8.1 Hz, 1H, H5′), 7.15 (dd, J
) 7.2 Hz, J ) 7.6 Hz, 1H, H6′), 6.82 (m, 2H, H1, H2), 5.96 (s,
1H, H5), 4.32 (m, 3H, H2′′, H1′′), 3.65 (d, J ) 17.1 Hz, 1H, H10â),
3.62 (d, J ) 17.4 Hz, 1H, H8R/H8â), 3.50 (s, 4H, N-CH2CdO),
3.43 (m, 1H, H10R), 3.32 (d, J ) 17.4 Hz, 1H, H8R/H8â), 2.81-
3.29 (m, 15H, H16ax/H16eq, H18, N-CH2, N-CH2CdO), 2.45-
2.80 (m, 9H, H16ax/H16eq, H6′′, N-CH2), 2.35 (m, 1H, H15ax/
H15eq), 1.98 (m, 1H, H15ax/ H15eq), 1.87 (m, 2H, H2′′), 1.38
(m, 2H, H3′′), 1.12 (m, 3H, H19, H5′′), 0.99 (m, 2H, H4′′), 0.83
(m, 2H, H20, H21), 0.49 (m, 2H, H20, H21). HRMS-ESI m/z calcd
for C46H59InN6O9 [M + H]+ 955.3455; found 955.3464. m/z calcd
for [M + Na]+ 977.3275; found 977.3303. Anal. (C46H59InN6O9‚
2.2TFA‚4H2O) C, H, N, F.
17-(Cyclopropylmethyl)-6,7-didehydro-4,5r-epoxy-3,14-dihy-
droxy-1′-(indium(III)-[10′′′-[3′′-acetamidopropyl]-[1′′′,4′′′,7′′′,-
10′′′-tetraazacyclododecane-1′′′,4′′′,7′′′-triacetate]])indolo[6,7:
2′,3′]morphinan; N1′-(In(III)-DOTA-propylamido)NTI (19). A
solution of 15 (60 mg, 50 µmol) in 0.4 M NH4OAc (6 mL) was
treated with InCl3 (13.2 mg, 60 µmol) in 0.05 N HCl (3.3 mL) at
reflux for 30 min, cooled to ambient temperature, and quenched
with 0.1 N disodium EDTA (0.9 mL). Workup as above, using
C18 chromatography (CH3CN:H2O, 40:60, 0.1% TFA), gave 19
(43 mg, 69%) as white flakes after lyophilization. 1H NMR (D2O,
300 MHz) δ 7.48 (d, J ) 7.8 Hz, 2H, H4′, H7′), 7.33 (dd, J ) 7.3
Hz, J ) 8.0 Hz, 1H, H5′), 7.13 (dd, J ) 7.3 Hz, J ) 7.6 Hz, 1H,
H6′), 6.85 (m, 2H, H1, H2), 5.98 (s, 1H, H5), 4.40 (m, 2H, H1′′),
4.30 (m, 1H, H9), 2.61-3.38 (m, 33H, H8, H10, H16ax/H16eq, H18,
H3′′, N-CH2, N-CH2CdO), 2.50 (m, 2H, H15ax/H15eq, H16ax/
H16eq), 2.14 (m, 2H, H2′′), 1.88 (m, 1H, H15ax/H15eq), 1.11 (m,
1H, H19), 0.83 (m, 2H, H20, H21), 0.48 (m, 2H, H20, H21).
HRMS-ESI m/z calcd for C45H56InN7O10 [M + H]+ 970.3206; found
970.3206. Anal. (C45H56InN7O10‚2TFA‚3H2O) C, H, N, F.
17-(Cyclopropylmethyl)-6,7-didehydro-4,5r-epoxy-3,14-dihy-
droxy-1′-(indium(III)-[10′′′-[6′′-acetamidohexyl]-[1′′′,4′′′,7′′′,10′′′-
General Radiolabeling Procedure. [111In]17-[111In]20 were
prepared by treating a water solution (50 µL) containing 50 µg of
chelator (6, 10, 15, 16) with 0.4 M NH4OAc (30 µL) and aliquots
of [111In]InCl3 (100-300 µL, 0.05 N HCl; 0.5 - 3.8 mCi) at 95 -
100 °C for 30 min in a septum-sealed glass vessel. Mixtures were
cooled to room temperature, quenched with disodium EDTA (20
µL, 1.0 mM), and taken up in a syringe along with rinses (3 × 50
µL) of the vessel with the ternary mobile phase used for ion-pair
HPLC purifications. Separations were run on a radial compression
module (Nova-Pak C18, 8 × 100 mm, 6 µm) at a flow rate of 4
mL/min using a Waters system with UV (254 nm) detection
interfaced with a flow-through NaI(Tl) scintillation detector (EE&G/
Ortec). Conditions were used where complexes were resolved from
precursors and from minor side products. Radioligand fractions were
collected, diluted with an equal volume of water, and passed through
an activated solid-phase extraction cartridge (Sep-Pak Light, t-C18)
that was flushed with water (2.0 mL) and then with air. Elution
with 70% EtOH (0.75-1.5 mL) furnished [111In]17-[111In]20 in
55-83% yields. Radioligands were pure (>98%) by HPLC and
coeluted with their respective non-radioactive complexes. Specific
radioactivities were >3300 mCi/µmol based on minimum detectable
mass (HPLC, 254 nm) in samples of known radioactivity (Capintec
CRC-15W). Radioligands were stable for 24-48 h when stored in
70% EtOH at -20 °C in the dark, and nominal decomposition
(∼5%) was noted during overnight storage at room temperature in
saline (2-5% EtOH). Representative HPLC mobile phase composi-
tions, retention times (tR) and capacity factors (k′) used during
synthesis and analysis follow.
[
111In]17: tR ) 15.9 min, k′ ) 31; 6: tR ) 12.4 min, k′ ) 24;
mobile phase: MeOH (9.0%), MeCN (9.0%); H2O (82%) with Et3N
(2.1%), HOAc (2.8%).
[
111In]18: tR ) 23.2 min, k′ ) 45; 10: tR ) 18.0 min, k′ ) 35;
mobile phase: MeOH (11.5%), MeCN (11.5%); H2O (77%) with
Et3N (2.1%), HOAc (2.8%).
[
111In]19: tR ) 11.7 min, k′ ) 22; 15: tR ) 16.8 min, k′ ) 32;
mobile phase: MeOH (10.0%), MeCN (10.0%); H2O (80%) with
Et3N (2.1%), HOAc (2.8%).
[
mobile phase: MeOH (11.0%), MeCN (11.0%); H2O (78%) with
Et3N (2.1%), HOAc (2.8%).
111In]20: tR ) 21.3 min, k′ ) 42; 16: tR ) 26.0 min, k′ ) 51;
Log D Determinations. Log D values for freshly purified
[
111In]complexes (3-5 µCi) were determined, in one session, by
measuring distribution coefficients (D) between equal volumes (3.5
mL) of n-octanol and phosphate buffered saline (0.1 M, pH 7.4;
PBS) where each phase had been saturated with the other. Mixtures
were vortexed (30 s) and then centrifuged (2500 rpm, 5 min).
Triplicate samples from the n-octanol (0.5 mL) and PBS (0.1 mL)
phases were counted in an automated gamma counter, and D was
determined as the ratio of mean CPM/mL for n-octanol compared
to PBS. The process was repeated four times for each complex.
Log D for [3H]NTI, purified by HPLC, was determined similarly
except the initial n-octanol solution was washed three times with

2154 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9
DuVal et al.
PBS to remove any hydrophilic contaminants. The n-octanol phase
was partitioned, four times in sequence, with an equal volume of
fresh PBS. After each partition, duplicate samples (0.1 mL) from
each phase were counted by liquid scintillation to obtain the DPM/
mL ratio.
tail vein injections of saline (0.15 mL), cold complex (5 µmol/kg),
naltrexone (10 µmol/kg), or NTI (5 µmol/kg) in saline (0.15 mL).
Differences between control and treatment groups at 1 h were
analyzed by ANOVA with a post hoc Dunnett’s test.
Acknowledgment. We thank Ms. Lisa D. Watkinson and
Mr. Terry L. Carmack for their capable work on biodistribution
experiments and Ms. Emily A. Fergason for assistance with
binding assays. We thank the National Cancer Institute (P50
CA 103130: Center for Single Photon-Emitting Cancer Imaging
Agents) for support of this research and for a postdoctoral
fellowship (R.A.D.) through the Career Development Core. We
also acknowledge resources and facilities provided by the Harry
S. Truman Memorial Veterans Hospital, the University of
Missouri Life Sciences Mission Enhancement Program, and NSF
CHE-95-31247 and NIH 1S10RR11962-01 grant awards for
NMR instrumentation.
Radioligand Binding Assays. Assays were conducted in mem-
branes from fresh mouse (δ) or frozen guinea pig (µ, κ) brains
using modifications of published procedures.26,27 Nonspecific
binding was defined for [3H]NTI (δ) by NTI (1.0 µM), for [3H]-
U69,593 by U69,593 (10 µM), and for [3H]DAMGO (µ) by
DAMGO (5 µM). [3H]DAMGO was used at 0.6 nM for 75 min at
25 °C, [3H]U69,593 at 0.6 nM for 90 min at 25 °C, and [3H]NTI
at 0.1 nM for 90 min at 37 °C. Assays were run in duplicate glass
test tubes using 10 inhibitor concentrations spaced equally on log
scale and centered on the estimated IC50. Protein concentrations of
0.3-0.5 mg/tube were used and were determined using the
bicinchoninic acid assay against a bovine serum albumin (BSA)
standard curve. Incubation buffer for µ and κ assays was 50 mM
Tris-HCl (pH 7.4 at 25 °C). For δ assays, this buffer was
supplemented with protease/peptidase inhibitors (50 µg/mL baci-
tracin, 30 µΜ bestatin, 10 µΜ captopril, 0.1 mM phenylmethyl-
sulfonylfluoride) and 0.1% BSA. Assays were terminated by
addition of ice-cold 50 mM Tris-HCl and filtered using a 48-well
harvester (Brandel, Inc.) through glass fiber papers (GF/B) pre-
treated with polyethyleneimine (0.5%). Tubes and filter papers were
washed with cold buffer (3 × 5 mL), and the papers were dried
under vacuum. Scintillation counting (47% efficiency) was carried
out after incubation of the discs with cocktail for 120 h. Data were
analyzed using Radlig 6.0 (Biosoft, Inc.) and Prism 4.0 (GraphPad
Software, Inc.). Kis were calculated from IC50s by the Cheng-
Prusoff equation using measured Kds for [3H]DAMGO (0.60 nM),
[3H]U69,593 (0.60 nM), and [3H]NTI (0.061 nM) under these
conditions. Association, saturation, and competition binding using
Supporting Information Available: Elemental analysis results
1
for 3-20, H NMR data for 3-16, and chart of the numbering
conventions used in spectral descriptions. This material is available
free of charge via the Internet at http://pubs.acs.org.
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