Synthesis and 124I-labeling of m-iodophenylpyrrolomorphinan as a potential PET imaging agent for delta opioid (DOP) receptors

Article abstract of DOI:10.1002/jlcr.1216

Condensation of phenylazo-β-ketoamide 4 with oxymorphone 5 afforded an m-iodophenylpyrrolomorphinan (m-IPPM) 6 mediated by elemental zinc in acetic acid/sodium acetate buffer. m-IPPM 6 is a novel opioid receptor agonist (K _i = 4.53 nM for DOP)

Full text of DOI:10.1002/jlcr.1216

Journal of Labelled Compounds and Radiopharmaceuticals
J Label Compd Radiopharm 2007; 50: 165–170.
Published online in Wiley InterScience
JLCR
(www.interscience.wiley.com). DOI: 10.1002/jlcr.1216
Research Article
Synthesis and 124I-labeling of m-iodophenylpyrrolomorphinan
as a potential PET imaging agent for delta opioid (DOP)
receptors
1,
1
2
2
¨
EYUP AKGUN *, PHILIP S. PORTOGHESE , MUNAWWAR SAJJAD and HANI A. NABI
1 Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, MN 55455, USA
2 Department of Nuclear Medicine, State University of New York, Buffalo, NY 14214, USA
Received 17 October 2006; Revised 6 December 2006; Accepted 7 December 2006
Abstract: Condensation of phenylazo-b-ketoamide 4 with oxymorphone 5 afforded an m-iodophenylpyrrolomorphi-
nan (m-IPPM) 6 mediated by elemental zinc in acetic acid/sodium acetate buffer. m-IPPM 6 is a novel opioid receptor
agonist (Ki ¼ 4:53 nM for DOP) with high selectivity for DOP receptors. m-IPPM 6 was converted into the positron
emitter m-[124I]PPM 8 via the stannylated intermediate 7. The final yield was 24.5 ꢀ 1.9 % (n ¼ 6) with a specific
activity of 2.5 ꢀ 1.2 Ci/mmol. Copyright # 2007 John Wiley & Sons, Ltd.
Keywords: m-IPPM; DOP receptor agonist; iodine-124; positron emission tomography
Introduction
receptors are present in the central nervous system
and in the periphery. In the central nervous system
DOP receptors are found in the neocortex, striatum,
olfactory areas, substantia nigra, amygdala and the
nucleus accumbens.10 Activation of these receptors
results in many physiological and behavioral effects
ranging, from modulation of antinociception, mood,
sensory system, motor integration and cognitive func-
tions. In addition, DOP receptors are also present in
several immunocompetent cells, which suggest they
may play an important role in regulating the immune
system.11
The delta opioid (DOP) receptor is a member of the
opioid (ORs) receptor family that comprises of MOP,
DOP, KOP and NOP.1–3 Opioid drugs which exert their
effects predominantly via mu opioid (MOP) receptors4
are the therapeutic of choice for treatment of acute and
chronic pain. However, the long-term use of these
drugs is limited due to severe side effects (tolerance,
dependence, etc.). The development of kappa opioid
(KOP) receptor agonists has waned because of dyspho-
ric effects associated with KOP receptors.5,6 Moreover,
MOP and KOP receptor agonists have limited effective-
ness in suppressing the allodynia and hyperalgesia
that occurs during neuropathic pain in humans and in
animal models. More recently, the focus has shifted to
DOP receptors. Several literature reports suggest that
stimulation of DOP receptors by selective agonists may
be a promising way to treat pain in general and the
neuropathic pain in particular.7–9 In this regards DOP
receptor agonists would have lower potential for abuse
and fewer unwanted side effects associated in compar-
ison with MOP and KOP receptor agonists. DOP
Pharmacological studies of DOP receptors have
suggested the existence of at least two putative receptor
subtypes (delta1 and delta2) which have not been
substantiated by cloning.12,13 These pharmacological
results can be explained on the basis that DOP
receptors form heterodimers14–17 with other G pro-
tein-coupled receptors (GPCRs) and these heterodimers
have different phenotypical behaviors.18,19 The physio-
logical and/or pathological consequence of DOP recep-
tor heterodimerization in vivo remains to be explored. It
is possible that these investigations can contribute to
the development of new drugs with unique proper-
ties.20 Since the relative expression of different GPCRs
in various cell types differs, it may be possible to
develop drugs that are devoid of abuse potential and
¨
*Correspondence to: E. Akgun, Department of Medicinal Chemistry,
College of Pharmacy, University of Minnesota, Minneapolis, MN 55455,
USA. E-mail: akgun001@umn.edu
Contract/grant sponsor: NIDA; contract/grant number: DA01533
Copyright # 2007 John Wiley & Sons, Ltd.

¨
166 E. AKGUN ET AL.
other side-effects. A second possibility for existence of
putative subtypes of DOP receptors is that they
represent different affinity states of the same receptors
due to dimerization (heterodimerization/or oligomer-
ization).21
Results and discussion
Reaction of diketene 1 with m-iodoaniline 2 in anhy-
drous benzene proceeded smoothly at 08C affording b-
ketone 3 in nearly quantitative yield. The phenyl
diazonium chloride was prepared in situ and reacted
with b-ketone 3 mediated by pyridine (Scheme 1).
The b-keto phenyl diazonium amide 4 was obtained
in 41% yield after the gravity column (SiO2) separation
using a solvent mixture of dichloromethane(D)/metha-
nol(M)/ammonium hydroxide(A). The condensation of
4 with oxymorphone 5 was effected by elemental zinc in
glacial acetic acid affording the pyrrolomorphinan 6.
The morphinan 6 was separated on a gravity column
(SiO2) using solvent mixture of dichloromethane/
methanol/ammonium hydroxide. The average yield
was ꢁ40%. The proton NMR and mass spectra
confirmed the formation of the product 6. In its
biological evaluation, the in vitro characteristic of 6
showed good affinity for DOP receptor (Ki ¼ 4:53 nM for
DOP). The binding assay (inhibition of 3H-diprenor-
phine in HEK 293 cells) showed Ki selectivity of m=d ¼
24 and k=d ¼ 98.
The characterization of DOP receptors will help to
understand DOP receptors role at the functional level.
One useful tool for this is positron emission tomogra-
phy (PET) which is a non-invasive technique.22 PET
utilizes a pharmacophore labeled with positron emit-
ters. We were interested in developing a DOP receptor
agonist ligand labeled with
a positron emitter of
relatively long half-life such as 124I (t1=2 ¼ 4:2 days).
For DOP receptors studies, the DOP receptor agonist 6
([8R-(4bS, 8a, 8a´b, 12bb)-7,10-dimethyl-5,6,7,8,12,
12b,-hexahydro-4,8-methanobenzofuro [3,2-e] pyrrolo
[2,3q] isoquinoline-1,8a-dihydroxy-11-(3-iodo-phenyl)-
carboxamide)23–26 has been prepared and evaluated
in vitro. The synthesis of the corresponding positron
emitting ligand 8 has been prepared in two steps from
the pyrrolomorphinan 6 as it is illustrated in Scheme 1.
The radioligand 8 (t1=2 ¼ 4:2 days) complements exist-
ing PET imaging agents such as N10-([11C] methyl)
naltrindole27,28 (t1=2 ¼ 20 min) and N10-([18F] fluor-
oethyl) naltrindole29 (t1=2 ¼ 110 min). The present re-
port summarizes our results.
The product
6 was converted into stannylated
derivative 7 via replacement of iodine using hexam-
ethydistannane mediated by bis-(triphenylphosphine)-
I
I
O
O
a
b
O
NH2
N
H
O
1
2
3
N
OH
O
N
O
I
OH
O
I
H
N
O
O
5
N
OH
N
H
O
H
N
c
N
OH
N
4
6
N
OH
OH
O
124I
H
Sn(CH3)3
H
d
e
N
N
N
N
H
O
H
O
O
OH
OH
7
8
Scheme 1 (a) Benzene 08C; (b) phenylhydrozonium chloride, 08C, then pyridine, rt; (c) Zn, aceticacid/sodium acetate, 608C, then
reflux. (d) hexamethyldistannane, bis-(triphenylphosphine)-palladium (II)-dichloride; (e) [124I]I2.
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 165–170
DOI: 10.1002.jlcr

SYNTHESIS AND 124I-LABELING OF M-IODOPHENYLPYRROLOMORPHINAN 167
palladium (II)-dichloride catalysis. 1H-NMR of the
In summary, m-IPPM 5 was prepared and converted
into m-124IPPM 8 via stannylated precursor 7 that will
be used to study DOP receptors in vivo in mouse brain.
In the future, the mouse brain will be imaged using
micro PET system to map DOP receptors.
product 7 has the characteristic trimethylstannine
peak at 0.25 ppm with Sn-satellite peaks. The electro-
philic aromatic iodination of the m-trimethylstannyl
morphinan 7 with Na124I in 5% acetic acid in methanol
in the presence of an iodogen bead afforded 124I-
labelled m-[124I]IPPM 8. Representative semi-prepara-
tive HPLC traces for the purification [124I]IPPM 8 are
shown in Figure 1. The radioligands were separated
easily. The overall radiochemical yield was 24.5 ꢀ
1.9%(n ¼ 6). The radiochemical purity was greater than
98% and the specific activity was 2.5 ꢀ 1.2 Ci/mmol
(n ¼ 6). The product 8 was dissolved in saline contain-
ing 10% ethanol and the product was stable in vitro.
Figure 2 illustrates HPLC trace of the purified product
8. Figure 3 shows that the purified product 8 and the
cold compound 6 eluted at the same time.
Experimental
All solvents and reagents used in the syntheses were of
the highest quality commercially available and when
required were purified and dried by standard methods.
Melting points were determined with Mel-Temp La-
boratory
Devices
and
uncorrected.
1H-NMR
(300.007 MHz) and 13C-NMR (75.462 MHz) were ob-
tained with a Varian Gemini-300 instrument. Chemical
shifts are reported in ppm (d) relative to internal Me4Si
in CDCl3. High-resolution fast bombardment mass
Figure 1 HPLC trace of the purification of m-[124I]IPPM 8. The peak eluted between 15 and 18 min was 8 and the major UV peak at
43–45 min was trimethyl tin precursor 7.
Figure 2 HPLC analysis of the purified m-[124I]IPPM 8.
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 165–170
DOI: 10.1002.jlcr

¨
168 E. AKGUN ET AL.
Figure 3 HPLC profile of the mixture of purified m-[124I]IPPM 8 and m-IPPM 6. The labeled 8 and the authentic cold compound
m-IPPM 6 are eluted simultaneously.
spectroscopy (HRFABMS) was done at the University of
Minnesota Mass Spectroscopy Facility. Analytical TLC
was done on Baker-flex, silica gel IB2-F plates.
an aniline solution at a rate so that the temperature did
not reach 08C. The entire solution was then transferred
via a double needle to a solution containing amide 3
(2.3 mmol) in pyridine/water (10 ml, 1:1, v/v) cooled
with ice–water. The product precipitated as viscous oil.
The solvent was decanted and the material was
dissolved in methylene chloride. After drying over
sodium sulfate, the solvent was evaporated. The
residue was separated via flash chromatography
(SiO2, D: M: A (97:2.5:0.5, v/v/v)). Yield: 41%; mp
>1308C Decomposed; Rf 0.54 (SiO2, D:M:A, 97:2.5:0.5,
v/v/v); 1H-NMR (d6-DMSO):d ¼ 11:1 (s, 1H), 8.2 (s, 1H),
7.5–7.1 (m, 9H); 13C (d6-DMSO): d ¼ 198:6, (C = O),
162.3 (C = O), 142.4, 139.3, 130.2, 128.9, 127.8,
126.6, 95.5; MS (M+Na): 430.0025; 430.0030 calcu-
lated for C16H14IN3O2Na.
High-performance liquid chromatography was car-
ried out on a system comprised of a Chrom Tech Iso-
2000 pump, Hitachi L-4000 UV detector and a radia-
tion detector. These detectors are in series and are also
connected to a computer with HP Chemstation software
via HP35900E interface. Iodine-124 was produced by
the 124Te (p, n) 124I reaction.30 124TeO2 (tellurium
oxide) target was irradiated by 14.1 MeV protons at
24 mA current for about 4 h in a cyclotron. The radio-
iodine was separated by the dry distillation method.
The distilled iodine-124 was trapped in 0.1 N NaOH.
Synthesis of b-ketoamide 331
m-Iodoaniline (5 g, 22.8 mmol) and diketene (3 ml,
excess) were mixed in anhydrous benzene at 08C and
stirred overnight. The following day, the white pre-
cipitate was filtered and washed with n-hexanes and
the solid was dried in vacuo. Rf: 0.8 in D:M:A
(97:2.5:0.5; v/v/v).
´
[8R-(4bS,8a,8ab,12bb)-7,10-Dimethyl-5,6,7,8,12,12b,-
hexahydro-4,8-methanobenzofuro
[3,2-e]pyrrolo
[2,3q] isoquinoline-1,8a-dihydroxy-11-(3-iodo-phe-
nyl)-carboxamide 6
To diazoamide 4 (0.63 mmol) dissolved in 2 ml glacial
Yield: 6.91 g, White powder, 97.6%; mp 110–1128C.;
1H-NMR (d6-DMSO):d ¼ 10:1 (s, 1H), 7.5–7.0 (m, 4H),
3.5 (s, 2H), 2.1 (s, 3H); 13C (d6-DMSO): d ¼ 203:1(C=O),
165.9 (C=O), 140.8, 132.5, 131.4, 127.8, 118.8, 95.3,
53.0, 31.0.
acetic acid oxymorphone
5 (177.8 mg, 0.52 mmol,
1.2 eq) was added, followed by sodium acetate (52 mg,
0.63 mmol, 1.2 eq). The mixture was warmed to 608C
under N2-flow. At this temperature, elemental zinc
(156 mg, 2.38 mmol, 4.4 eq) was added in small portion
to the mixture. After completion of zinc addition, the
mixture was refluxed for 2 h. Then it was cooled to room
temperature and decanted into ice–water. The reaction
flask was rinsed twice, each with 2 ml glacial acetic
acid. The combined acetic acid solution was adjusted to
pHꢁ9 with 20% NaOH solution. The pyrrolomorphinan
Synthesis of phenylazo-b-ketoamide 432
Aniline (214 mg, 2.3 mmol) was dissolved in 10 ml HCl
(5 N) and cooled with methanol–ice. Sodium nitrite
(159 mg, 2.3 mmol) dissolved in water was dripped into
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 165–170
DOI: 10.1002.jlcr

SYNTHESIS AND 124I-LABELING OF M-IODOPHENYLPYRROLOMORPHINAN 169
was extracted with EtOAc (4 ꢂ 50 ml). After drying over
was eluted with CH3OH/H2O/NH4OH (70:30:0.1) at a
flow rate of 1 ml/min. The UV detector was set at
254 nm wavelength. The desired product 8 (m-124IPPM)
eluted at 12–14 min (Figure 1) was collected and the
solvent was evaporated to dryness azeotropically with
CH3CN (2 ꢂ 1 ml) at 1108C. The product was dissolved
in saline containing 10% ethanol. A standard curve was
obtained between peak area versus mass by injecting
known mass of carrier 6 (Scheme 1) onto the column.
The mass associated with the labeled product was
calculated by relating the peak area of UV absorbance
peak of 6 in the labeled product to the standard curve.
The specific activity was obtained by dividing the
activity of the labeled product collected by the calcu-
lated mass in micromoles.
sodium sulfate, the solvent was evaporated. The final
purification using flash chromatography (SiO2, D: M: A
(95:4.5:0.5, v/v/v)). Yield: 34.5%; colorless powder, mp
213–2168C.; Rf 0.6 (SiO2, D:M:A, 97:2.5:0.5, v/v/v).;
1H-NMR (d6-DMSO):d ¼ 9:4 (s, 1H), 8.1 (s, 1H), 8.6–
7.05 (m, 4H), 6.5 (AB, JAB ¼ 7:3 Hz, 2H), 5.29 (s, 1H),
3.1–1.4 (m, 8H), 2.4 (s, 3H), 2.2 (s, 3H); MS: (M+1):
584.1064; 584.1048, calculated for C28H26IN3O4.
Synthesis of [8R-(4bS,8a,8ab,12bb)-7,10-dimethyl-
5,6,7,8,12,12b,-hexahydro-4,8-methanobenzofuro
[3,2-e] pyrrolo[2,3q] isoquinoline-1,8a-dihydroxy-11-
(3-trimethylstannyl-phenyl)-carboxamide 733
The product was analyzed for radiochemical purity
on HPLC column (Maxsil 5 C18, 4.6 ꢂ 250 mm, Phe-
nomenex), which was eluted with CH3CN/0.01 M
(NH4)2HPO4 (70:30) at a flow rate of 1 ml/min. Figure
2 shows the chromatogram of the final product. Carrier
6 was added to the labeled product and was analyzed.
Figure 3 shows the chromatogram showing the labeled
and cold compound eluting at the same time.
To a double neck flask a solution of pyrrolomorphinan
6 (100 mg, 0.171 mmol) in degassed 1,4-dioxane (5 ml)
were added 100 ml hexamethyldistannane (0.41 mmol)
and (1.7 mg, 2.4 ꢂ 10ꢃ6 mmol) bis-(triphenylpho-
sphine)-palladium (II)-dichloride. The solution was
stirred at 608C for 90 min. After removal of solvent the
crude mixture was separated by gravity column (SiO2)
using chloroform/methanol in a ratio of 97:3 (v: v). 1H-
NMR (d6-DMSO): d ¼ 11:3 (s, 1H), 9.3 (s, 1H), 8.9(s,
1H), 7.8–7.05 (m, 4H), 6.4 (AB, JAB ¼ 7:2 Hz, 2H), 5.3
(s, 1H), 4.6 (s, 1H), 3.2–1.4 (m, unresolved), 0.25 (s,
with Sn satellites, 9H).
Acknowledgements
We thank Mike Powers for the binding studies. We also
thank Erol Bars (University of New York) for cyclotron
operations. This work was supported by NIDA, under
research grant DA01533.
Biological Studies
Receptor binding assays
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J Label Compd Radiopharm 2007; 50: 165–170
DOI: 10.1002.jlcr