Synthesis and in vitro autoradiographic evaluation of a novel high-affinity radioiodinated ligand for imaging brain cannabinoid subtype-1 receptors

Article abstract of DOI:10.1016/j.bmcl.2009.08.092

There is strong interest to study the involvement of brain cannabinoid subtype-1 (CB₁) receptors in neuropsychiatric disorders with single photon emission computed tomography (SPECT) and a suitable radioligand. Here we report the synthesis of a

Full text of DOI:10.1016/j.bmcl.2009.08.092

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6209–6212
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and in vitro autoradiographic evaluation of a novel high-affinity
radioiodinated ligand for imaging brain cannabinoid subtype-1 receptors
b
b
b
a
b
Sean R. Donohue a,b, , Katarina Varnäs , Zhisheng Jia , Balázs Gulyás , Victor W. Pike , Christer Halldin
*
a Molecular Imaging Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892, USA
b Karolinska Institutet, Department of Clinical Neuroscience, Psychiatry Section, Karolinska Hospital, S-17176 Stockholm, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
There is strong interest to study the involvement of brain cannabinoid subtype-1 (CB1) receptors in
neuropsychiatric disorders with single photon emission computed tomography (SPECT) and a suitable
radioligand. Here we report the synthesis of a novel high-affinity radioiodinated CB1 receptor ligand
Received 27 July 2009
Revised 26 August 2009
Accepted 31 August 2009
Available online 3 September 2009
([125I]8,
[
125I]1-(2-iodophenyl)-4-cyano-5-(4-methoxyphenyl)-N-(piperidin-1-yl)-1H-pyrazole-3-car-
boxylate, [125I]SD7015). By autoradiography in vitro, [125I]8 showed selective binding to CB1 receptors
on human brain postmortem cryosections and now merits labeling with iodine-123 for further evalu-
ation as a SPECT radioligand in non-human primate.
Keywords:
SPECT
PET
Ó 2009 Elsevier Ltd. All rights reserved.
CB1 receptors
Radioiodination
Radioligand
Cannabis sativa (marijuana) is one of the oldest known plant de-
rived-therapeutics, owing mainly to its anti-nociceptive proper-
ties.1 Other beneficial effects of cannabis intake may include
anti-emesis and appetite stimulation. Conversely, the accompany-
ing psychological ‘high’ and memory impairment have limited its
therapeutic value. The most abundant and psychoactive cannabi-
the development of radioligands for more widespread single-pho-
ton computed tomography (SPECT) is much less advanced. Due to
the longer half-life of radioiodine for SPECT imaging (e.g., 123I,
t1/2 = 13.2 h), an on-site cyclotron is not required for radiolabeling,
as with PET, which allows for more widespread use in pharmaceu-
tical, academic, and hospital settings. Here we report the synthesis,
receptor screening, radioiodination, and in vitro autoradiographic
evaluation of a novel radioiodinated CB1 receptor radioligand
([125I]4-cyano-1-(2-iodophenyl)-5-(4-methoxyphenyl) -N-(piperi-
din-1-yl)-1H-pyrazole-3-carboxamide, [125I]8, [125I]SD 7015). Our
results strongly suggest that [123I]8 would merit evaluation as a
SPECT radioligand in vivo.
Compound 8 was synthesized according to the procedure de-
picted in Scheme 1. Conversion of 2-iodoaniline into a diazonium
salt, followed by treatment with ethyl 2-chloroacetoacetate in
ethanol–water solution under basic conditions gave chloro[(2-
iodophenyl)hydrazono]ethyl acetate (9). Heating of a solution of
9, 4-methoxybenzoylacetonitrile and N,N-diisopropylethylamine
in tert-butanol to reflux gave 10 in low but adequate yield. Hydro-
lysis of 10 with LiOH in aq-tetrahydrofuran gave the corresponding
carboxylic acid, which was then converted into the acyl chloride
with oxalyl chloride and a catalytic amount of N,N-dimethylform-
amide. The synthesis of 8 was completed by coupling the acyl
chloride with 1-aminopiperidine under basic conditions.
noid,
D D
9-tetrahydrocannabinol ( 9-THC, 1), binds with high affin-
ity to two known body receptor systems. These receptors have
been named cannabinoid subtype-1 (CB1) and cannabinoid sub-
type-2 (CB2).2–4 CB1 receptors have high-densities in brain and
are implicated in a number of neuropsychiatric disorders5 such
as, schizophrenia6,7 and depression.8,9 CB2 receptors are located
mainly in the periphery and are of less interest to neuropsychiatric
research.10,11
Ability to image and measure brain CB1 receptors non-inva-
sively with radiation computed tomography would assist neuro-
psychiatric research and drug development. The development of
radioligands suitable for imaging brain CB1 receptors is therefore
of strong importance. Recently, there has been a great deal of
progress in the development of useable radioligands for positron
emission tomography (PET). Some of the more notable PET radioli-
gands (Fig. 1) are [11C]MePPEP ([11C]2),12,13 18F]PipISB ([18F]3),14,15
[
[
18F]MK-9470 ([18F]4),16,17 (–)-[11C]SD5014 ((À)-[11C]5),18
[
11C]-
OMAR ([11C]6),19,20 and [11C]JHU75575 ([11C]7).20,21 In comparison,
The trimethylstannylated precursor, 11, required for radiolabel-
ing, was synthesized by refluxing a solution of the known bromo
compound 720 and hexamethylditin in the presence of palladium
catalyst (Scheme 2).
* Corresponding author at present address: Johns Hopkins PET Center, Division of
Nuclear Medicine, Radiology, Johns Hopkins Medicine, Nelson B1-122, 600 North
Wolfe Street, Baltimore, MD 21287-0816, USA. Tel./fax: +1 410 614 0110.
E-mail address: Sean_Donohue@tracer.nm.jhu.edu (S.R. Donohue).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.08.092

6210
S. R. Donohue et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6209–6212
*
F
N
*
CN
O
O
S
O
N
NH
NH
O
O
O
H2N
N
NH
N
NC
HN
O
N
N
N
R2
O
O
N
S
N
N
R1
*
F3C
O
R3
O
Cl
18F]4
[
11C]2
[
18F]3
[
11C]5
[
[
[
11C]6, R1 = Me*O, R2 = R3 =Cl
11C]7, R1 = Me*O, R2 = Br, R3 = H
125I]8, R1 = MeO, R2 = *I, R3 = H
[
F
Figure 1. Structures of [11C]2, [18F]3, [18F]4, [11C]5–7, and [125I]8. Asterisks denote positions of radiolabels.
O
O
O
Cl
O
NH2
NC
N
a
b
c,d,e
I
N
HN
8
N
I
I
O
9
10
Scheme 1. Synthesis of 8. Reagent, conditions and yields: (a) concentrated HCl, NaNO2, ethyl 2-chloroacetoacetate, NaOAc, EtOH–H2O, 16 h, 72%; (b) 4-methoxybenzoyl-
acetonitrile, DIPEA, tert-BuOH, reflux, 16 h, 6%; (c) aq-LiOH, THF, 65 °C, 4 h; (d) DMF(cat), (COCl)2, DCM; (e) 1-aminopiperidine, DIPEA, DCM, 2 h, 73%.
A radioligand for imaging brain CB1 receptors would ideally
possess adequately high affinity and moderate lipophilicity to pro-
mote rapid development of a high ratio of receptor-specific binding
to non-specific binding in brain in vivo, that might then allow
accurate computation of output measures, such as binding poten-
tial.22,23 CB1 receptors are one of the most abundant G protein-cou-
cal properties of 8 compare well with other successful PET radioli-
gands.20,21 Therefore, ligand 8 presents acceptable CB1 receptor
affinity and lipophilicity for development as a SPECT radioligand
(Table 1).
In addition to acceptable binding affinity and lipophilicity a
candidate SPECT radioligand should be selective for binding to
pled receptors in brain, reaching
a
concentration (Bmax
)
of
the target protein. Ligand 8, at 10
<50% inhibition (n = 4) of radioligand binding to the sites: 5-
HT1B–E, 5-HT2A–C, 5-HT3, 5-HT5A, 5-HT6, 5-HT7, a1A,B 2A,B, b1–3
D1–4, DOR, H1–4, M1–5, NET, SERT, 1,2, V1A,1B,2. Ki values (n = 3) of
93.5 20.4 nM (5-HT1A), >10,000 nM ( 2C), 1477 148 nM (KOR),
lM concentration, showed
1752 fmol/mg protein (175 nM) in rat cerebellum.24 Generally, a
SPECT radioligand should show a substantial Bmax/Kd value (>5)
to be successful. Hence, an effective CB1 receptor radioligand might
require only a moderately high affinity (Ki or Kd <35 nM). Ligand 8
was found to have about 10-fold higher affinity (3.4 nM; Table 1)
,
a
,
r
a
1496 216 nM (MOR), and 3166 586 nM (TSPO) were found. De-
tails of the employed binding assays may be found at the NIMH
PDSP web site: http://pdsp.med.unc.edu. Hence, 8 was found to
have excellent CB1 receptor selectivity for development as a SPECT
radioligand.
than perhaps needed. Calculated ligand lipophilicity (cLog D7.4
)
can be an important predictor of blood–brain barrier penetration
and brain non-specific binding. Moderate lipophilicity (cLog D7.4
in the range 2.0–3.5) is usually preferred for adequate brain entry
without incurring excessive non-specific binding in brain.23 How-
ever, when target binding sites exist in high concentration, higher
lipophilicity may be tolerated. For example, [11C]MePPEP is a suc-
cessful PET radioligand for brain CB1 receptors despite having a
high cLog D7.4 value of 5.42.12,13 The cLog D7.4 value of 8 is 4.14
[
125I]8 was prepared by [125I]iodo-destannylation of the corre-
sponding trimethylstannyl precursor (11) with [125I]NaI, aq-HCl
and chloramine-T (oxidizer) in methanol (Scheme 2). The crude
product was purified with high-performance liquid chromatogra-
phy (HPLC) as described in Supplementary data. The decay-cor-
rected radiochemical yield of [125I]8 ranged from 48% to 59%. The
and appears more favorable for
a
radioligand than that of
[
11C]MePPEP. Furthermore, the physiochemical and pharmacologi-
specific radioactivity of [125I]8 was 81.4 GBq/
lmol and the radio-
N
NH
N
NH
N
NH
O
O
O
NC
NC
NC
b, [125I]NaI
a
N
N
N
N
N
N
125I
Sn
Br
O
O
O
[
125I]8
7
11
Scheme 2. Radiosynthesis of [125I]8. Reagents, conditions and yields: (a) hexamethylditin, Pd(PPh3)4, toluene, 20 h, 32%; (b), chloramine-T, aq-HCl, 5 min, 48–59%.

S. R. Donohue et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6209–6212
6211
mortem autoradiography in vitro, [125I]8 bound substantially to
human brain regions with high CB1 receptor densities (Fig. 2; pan-
els A, C, E, and G) with highest binding in globus pallidus and sub-
stantia nigra (Fig. 2; panels A and E). Additionally [125I]8 showed
much lower binding in brain regions with low CB1 receptor den-
sity, including thalamus (Fig. 2; panels C and E). Under conditions
in which the CB1 receptors were blocked with the selective CB1 li-
Table 1
Ki values for CB1 and CB2 receptors, selectivities for CB1 versus CB2 receptors and
calculated lipophilicities
N
O
NH
NC
gand, rimonabant (10 l
M), the binding of [125I]8 in CB1 rich regions
N
was greatly reduced and the distribution of radioligand became
more homogeneous (Fig. 2, panels B, D, F, and H). Therefore, the re-
gional selectivity of [125I]8 indicated that 123I-labeled 8 would be
promising for imaging brain CB1 receptors with SPECT.
Ligand 8 demonstrated high affinity and good selectivity for CB1
receptors. [125I]8 was obtained in acceptable radiochemical yield,
specific radioactivity and purity for evaluation in vitro. Autoradio-
graphs of human brain obtained with [125I]8 showed radioactivity
distribution according to known regional CB1 receptor densities.
Future research evaluating [123I]8 with SPECT imaging is therefore
warranted.
N
I
O
b
Ligand
CB1 Kia
(nM)
CB2 Kia
(nM)
CB1 versus CB2
selectivitya
cLog D7.4
Rimonabant 1.38 0.17 927 66
3.40 0.43 548 23
672 94
161 47
6.01
4.14
8
a
Values represent mean SD of three determinations.
cLog D7.4 values were calculated using Advanced Chemistry Development (ACD)
b
9.2.
Acknowledgments
chemical purity >98%. [125I]8 was thus obtained in adequate yield
and purity for further evaluation with sensitive postmortem auto-
radiography in vitro. Furthermore, these conditions would be
applicable to labeling 8 with iodine-123 for evaluation in SPECT
imaging.
CB1 receptors are spread heterogeneously in brain, with high-
densities appearing in substantia nigra, globus pallidus, amygdala,
cortical regions, and striatum.25,26 Brain regions with low CB1
receptor densities are thalamus, pons, and white matter. In post-
S.R.D. and V.W.P. were supported by the Intramural Research
Program of the National Institutes of Health, specifically the Na-
tional Institute of Mental Health (NIMH), project number
ZO1MH002793. We thank the NIMH Psychoactive Drug Screening
Program (PDSP) for performing binding assays and Siv Eriksson
for the excellent technical assistance with autoradiographic exper-
iments. The PDSP is directed by Bryan L. Roth, PhD, with project
officer Jamie Driscoll (NIMH) at the University of Carolina at Chapel
Hill (contract NO1MH32004).
Figure 2. Autoradiographs from whole-hemisphere cryosections (panels A–D; 100
l
m thickness) and cryosections (20
l
m thickness) covering thalamus and brainstem
M; panels B, D, F and H)
(panels E and F) and temporal cortex (panels G and H) incubated with [125I]8 under baseline (panels A, C, E and G) and blocked (rimonabant, 10
l
conditions. Abbreviations: Amg, amygdala; Ca, caudate nucleus; CG, cingulate gyrus; FC, frontal cortex; Hi, hippocampus; IGP, globus pallidus, internal segment; PC, parietal
cortex; Pu, putamen; SN, substantia nigra; TC, temporal cortex; Th, thalamus.

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S. R. Donohue et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6209–6212
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