Synthesis and in vivo evaluation of [11C]MPTQ: A potential PET tracer for alpha2A-adrenergic receptors

Article abstract of DOI:10.1016/j.bmcl.2010.04.099

Radiosynthesis and in vivo evaluation of [N-methyl-¹¹C] 5-methyl-3-[4-(3-phenylallyl)-piperazin-1-ylmethyl]-3,3a,4,5- tetrahydroisoxazolo[4,3-c]quinoline (1), a potential PET tracer for alpha2-adrenergic receptors is described. Syntheses of nonradioactive standard 1 and corresponding desmethyl precursor 2 were achieved from 2-aminobenzaldehyde in 40% and 65% yields, respectively. Methylation using [¹¹C]CH ₃I in presence of aqueous potassium hydroxide in DMSO afforded [ ¹¹C]1 in 25% yield (EOS) with >99% chemical and radiochemical purities with a specific activity ranged from 3-4 Ci/μmol (n = 6). The total synthesis time was 30 min from EOB. PET studies in anesthetized baboon show that [¹¹C]1 penetrates BBB and accumulates in alpha2A-AR enriched brain areas.

Full text of DOI:10.1016/j.bmcl.2010.04.099

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3654–3657
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and in vivo evaluation of [11C]MPTQ: A potential PET tracer
for alpha2A-adrenergic receptors
Jaya Prabhakaran a,b, Vattoly J. Majo a,b, Matthew S. Milak a,b, Pratap Mali b, Lyudmila Savenkova a,b
,
J. John Mann a,b,c, Ramin V. Parsey a,b, J. S. Dileep Kumar a,b,c,
*
a Division of Molecular Imaging and Neuropathology, Department of Psychiatry, Columbia University, New York, United States
b New York State Psychiatric Institute, New York, United States
c Department of Radiology, Columbia University, New York, NY 10032, United States
a r t i c l e i n f o
a b s t r a c t
Radiosynthesis and in vivo evaluation of [N-methyl-11C] 5-methyl-3-[4-(3-phenylallyl)-piperazin-1-
ylmethyl]-3,3a,4,5-tetrahydroisoxazolo[4,3-c]quinoline (1), a potential PET tracer for alpha2-adrenergic
receptors is described. Syntheses of nonradioactive standard 1 and corresponding desmethyl precursor
2 were achieved from 2-aminobenzaldehyde in 40% and 65% yields, respectively. Methylation using
Article history:
Received 10 March 2010
Revised 20 April 2010
Accepted 21 April 2010
Available online 22 May 2010
[
11C]CH3I in presence of aqueous potassium hydroxide in DMSO afforded [11C]1 in 25% yield (EOS) with
>99% chemical and radiochemical purities with a specific activity ranged from 3–4 Ci/lmol (n = 6). The
Keywords:
Alpha2-AR
Positron emission tomography
Biological imaging
Baboon
total synthesis time was 30 min from EOB. PET studies in anesthetized baboon show that [11C]1 pene-
trates BBB and accumulates in alpha2A-AR enriched brain areas.
Ó 2010 Elsevier Ltd. All rights reserved.
Radiotracer
Adrenergic receptors or adrenoceptors are G-protein coupled
receptors (GPCR) that mediate the central and peripheral actions
of the endogenous adrenergic amines, adrenaline and noradrena-
line. Abnormalities in adrenaline and noradrenaline function have
been implicated in anxiety, depression and degenerative disor-
ders.1–5 Based on the amino acid sequences, signaling mechanism
and pharmacologic properties, these receptors are divided into
three major types: alpha1, alpha2, and beta receptors.6–9 Alpha2-
adrenergic receptors are autoreceptors on noradrenergic neurons
(alpha2-ARs) and are localized in both central and peripheral tis-
sues. They have been implicated in a number of different central
and peripheral pathologies. Many studies have also postulated
the role of alpha2-AR in depression, mood disorder, stimulant
withdrawal, social stress, alcoholic suicides, Parkinson’s disease,
Alzheimer’s disease, and attention deficit hyper activity syn-
drome.2,10–12 Alpha2-AR consists of three subtypes, alpha2A, al-
pha2B, and alpha2C, respectively. Postmortem human studies
show that alpha2A-AR subtype is abundant in most of the brain re-
gions with a Bmax ranging from 34 to 90 fmol/mg of protein,
whereas, only small population of alpha2B/2C was detected (8
and 5 fmol per mg of protein, respectively).13,14 The concentration
of alpha2B and 2C receptors are too small in brain to detect by PET.
These studies also indicate that in the human brain the predomi-
nant and functionally relevant alpha2 receptor subtype is al-
pha2A-AR and its expression is not heterogeneous in brain.14
Several PET ligands for this receptor have been synthesized and
evaluated in animals and human (Fig. 1) with limited promise.
Among these [11C]MK-912 and [11C]WY-26703 have been evalu-
ated in rodents and monkeys and the tracers show a fast washout
of radioactivity without significant retention in alpha2-AR
enriched brain regions.15,16 Similarly, [18F]fluoroatipamezole was
evaluated in rat,17 [N-methyl-11C]mianserine in swine,18 and [O-
methyl-11C]RS-15385-197 in rat and human.19 However, these
ligands did not show the potential to be used as a PET ligand for
routine use to quantify alpha2-AR due to limited degree of regional
specificity and poor heterogeneity in biodistribution of radioactiv-
ity. The most recently developed candidate, [11C]R107474, has
been tested in rats and showed poor in vivo specificity and hence
will not be useful for the quantification of alpha2-AR.20 18F]-RS-
[
15385-FP, a fluoropropyl analogue of RS-16385197 has been
evaluated in rats and monkeys, however, additional studies are
required to prove the usefulness of this ligand to image alpha2A-
AR.21 Mirtazapine, the antidepressant drug, has been labeled and
evaluated in animals and human to study the actions of antide-
pressant drugs in vivo. However, it is non-selective for alpha2-
AR22 and hence cannot be used to quantify this receptor.23,24 Hence
currently there is no adequate PET tracer to quantify alpha2A-AR
and monitor its changes in vivo.
* Corresponding author. Tel.: +1 212 543 1163; fax: 212 543 1054.
E-mail address: dk2038@columbia.edu (J.S. Dileep Kumar).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.099

J. Prabhakaran et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3654–3657
3655
O
S
O
11CH3
N
N
N
O
O
N
N
N
N
11CH3
11CH3
[
11C]MK912
[
11C]WY2673
[N-methyl-11C]mianserin
O
N
N
18F
H311C
N
O
H
11C
N
H
O
S
N
O
N
HN
O
[O-methyl-11C]RS15385197
[
18F]Fluoroatipamezole
[
11C]R107474
Figure 1. Alpha2A-AR CNS imaging agents.
We have selected the piperazine compound 1 as a candidate
PET ligand for alpha2A-AR imaging owing to its nanomolar affinity,
good selectivity and demonstrated alpha2AR blocking activity
in vivo indicating CNS penetration.25,26 Compound 1 is subtype
selective towards the predominant subtype alpha2A-AR (alpha2A
Ki = 1.6 nM, alpha2C Ki = 4.5 nM; 5-HTT Ki = 16 nM) as evident
from competition binding studies with [3H]rauwolscine and
[3H]paroxetine, respectively.25 Compound 1 is 10-fold selective to
alpha2A-AR over 5-HTT. In addition the lipophilicity was adequate
for BBB penetration (Clog P value 2.86 0.78 measured with
ACD log PDB program). As indicated above alpha2B/2C-ARs are
less abundant in brain and therefore we anticipate little or no
interference of these receptors in the binding of compound 1 in tra-
cer dose to alpha2A-AR.14 Since the Bmax of alpha2A-AR and 5-HTT
are comparable in brain,14,27 we anticipate a 10-fold affinity of
compound 1 to alpha2A-AR over 5-HTT is advantageous as an
imaging agent with PET. Additionally, the availability of suitable
sites for incorporating C-11 isotope encouraged us to test com-
pound 1 for its ability to image alpha2A-AR in vivo by PET. Synthe-
sis of nonradioactive standard 1 and desmethyl precursor 2 were
achieved in moderate yield starting from 2-amino benzaldehyde
3 (25, Scheme 1). The trifluoroacetyl compound 4 was obtained
from 2-aminobenzaldehyde 3 by treating it with trifluoroacetic
anhydride in presence of triethyl amine, which was then con-
densed with methyl-4-bromocrotonate to afford ester 5. Com-
pound
5
was subsequently treated with hydroxylamine
CHO
CHO
NH2
CHO
ii
i
iii
NHCOCF3
CO2Me
N
3
4
O
5
CF3
HO
N
N
O
N
O
N
iv
OH
CO2Me
v
CO2Me
N
N
H
O
CF3
CF3
O
6
8
7
O
N
N
Cl
O
N
N
vi
vii
N
N
H
N
H
9
2
O
N
viii
N
N
1
Scheme 1. Syntheses of compounds 1 and 2. Reagents and conditions: (i) TFAA, Et3N, ether, À10 to 0 °C, 96%; (ii) methyl-4-bromocrotonate, NaH, 18-crown-6, DMF, 60%; (iii)
NH2OHÁHCl, pyridine, ethanol, 65%; (iv) chloramine-T, ethanol, 48%; (v) NaBH4, THF/H2O, 90%; (vi) CCl4, Ph3P, THF, 62%; (vii) 1-cinnamylpiperazine, KI, dioxane, 65%; (viii)
HCHO, NaCNBH3, ZnBr2, CH3OH 40%.

3656
J. Prabhakaran et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3654–3657
hydrochloride and chloramine-T, respectively, to obtain the isoxaz-
ole 7. Ester group in 7 was reduced to obtain alcohol 8, which was
then converted to the chloride 9 in moderate yield using carbon
tetrachloride and triphenylphosphine. The chloride 9 was con-
densed with 1-cinnamyl piperazine to obtain desmethyl precursor
2 in 65% yield. The nonradioactive standard 1 was obtained by
methylation of amine 2 using formaldehyde and sodium cyano-
borohydride (40%).
Radiolabeling was initially attempted by treating precursor 2 in
DMF (0.5 mg in 0.5 mL) with [11C]CH3I using potassium tert-butox-
ide and heating at 80 °C. The yield of [11C]1 obtained was 30–
40 mCi (10% yield at EOS) with specific activity in the range of 2–
ligible protein binding in human and baboon plasma. The radioac-
tivity distribution is almost homogeneous except for thalamus,
amygdala, insular cortex, and hippocampus.
The target to cerebellum ratios for thalamus, amygdala, cau-
date, putamen, hippocampus, cingulate cortex, temporal cortex,
and occipital cortex are 1.66, 166, 1.4, 1.52, 1.34, 1.28, and 0.9,
respectively.14 This distribution pattern is not in agreement with
the reported 5-HTT distributions and this rules out the possibility
of [11C]1 binding to 5-HTT for which it has 16 nM affinity.31–33
The bindings in occipital cortex of four selective SERT tracers in
non-human primates are higher than in cerebellum unlike
2.4 Ci/lmol (n = 4). However, reaction of precursor 2 with aqueous
KOH and [11C]CH3I in DMSO (Scheme 2) resulted in better radio-
chemical yield (25% at EOS, n = 6). Therefore, we adopted the later
experimental condition for the production of [11C]1 for further stud-
ies. Chemical and radiochemical purities obtained were >99% and
theimprovedspecific activitywas 3–4 Ci/l
mol.28 Thepartition coef-
ficient for [11C]1 obtained by standard shake flask method is 2.5.29
To determine the BBB permeability and distribution of [11C]1,
we conducted baboon PET scanning experiments adopting our
established procedures.30 Anesthesia (isoflurane 1.5–2.0%) was in-
duced in a fasted baboon (male, 5 year) immobilized with keta-
mine (10 mg/kg, im) via endotracheal tube. After
a
10 min
mol)
transmission scan, 4.43 mCi of [11C ]1 (specific activity 3 Ci/
l
was injected as an iv bolus and emission data were collected for
10 min in 3–D mode in a Siemens ECAT EXACT HR+ camera (CPS/
Knoxville, TN). Figure 2, the sum of last 60 min images, shows that
[
11C ]1 enter the BBB and the radioactivity is retained in brain with
homogeneous distribution. The time activity curves (TACs) demon-
strate the uptake is consistent with the known distribution of al-
pha2A-AR, the predominant subtype of alpha2-AR (Fig. 3).14
Metabolite analyses show 35 5% and 21 3% of unmetabolized
Figure 3. Time activity curves of [11C]1 in baboon. AMY = Amygdala; CAU = Cau-
date; CER = Cerebellum; HIP = Hippocampus; INS = Insula; FRT = Frontal cortex;
OCC = Occipital cortex; PAR = Parietal cortex; PFC = Prefrontal cortex; THA = Thal-
amus, TEM = temporal cortex; CIN = Cingulate cortex; PUT = Putamen.
[
11C]1 at 30 and 90 min, respectively. The radioligand exhibits neg-
N
N
O
N
O
N
N
N
i, ii
N
N
11CH3
11C]1
H
2
[
Scheme 2. Radiosynthesis of [11C]1. Reagent and conditions: (i) KOH, DMSO, 11CH3I, 5 min; (ii) HPLC purification, 25% (EOS).
Figure 2. PET and MRI images of [11C]1 in baboon. First row: MRI; second row: Baseline scan First column: axial, middle column: coronal, last column: sagittal views.

J. Prabhakaran et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3654–3657
3657
20. Van der Mey, M.; Windhorst, A. D.; Klok, R. P.; Herscheid, J. D. M.; Kennis, L. E.;
Bischoff, F.; Bakker, M.; Langlois, X.; Heylen, L.; Jurzak, M.; Leysen, J. E. Bioorg.
Med. Chem. 2006, 14, 4526.
[
[
11C]1.31 Also another discrepancy of SERT ligands compared to
11C]1 is their higher binding in hippocampus, compared to stria-
tum (caudate and putamen) and higher binding in temporal cortex
than cingular cortex.31 These findings further rule out the 5-HTT
binding of [11C]1 in baboon brain.30
21. Enas, J. D.; Clark, R. D.; VanBrocklin, H. F.; Budinger, T. F. Abstracts of papers,
213th ACS National Meeting, San Francisco, 1997, April 13–17.
22. Data collected from PDSP database http://pdsp.med.unc.edu/indexR.html.
23. Smith, D. F.; Dyve, S.; Minuzzi, L.; Jakobsen, S.; Munk, O. L.; Marthi, K.;
Cumming, P. Synapse (Hoboken, NJ, United States) 2006, 59, 463.
24. Marthi, K.; Jakobsen, S.; Bender, D.; Hansen, S. B.; Smith, S. B.; Hermansen, F.;
Rosenberg, R.; Smith, D. F. Psychopharmacology 2004, 174, 260.
25. Andres, J.; Alcazar, J.; Alonso, J. M.; Alvarez, R. M.; Bakker, M. H.; Biesmans, I.;
Cid, J. M.; De Lucas, A. I.; Fernandez, J.; Font, L. M.; Hens, K. A.; Iturrino, L.;
Lenaerts, I.; Martinez, S.; Megens, A. A.; Pastor, J.; Vermote, P. C. M.; Steckler, T.
J. Med. Chem. 2005, 48, 2054.
In summary, we successfully synthesized [11C]1, a PET tracer
agent for alpha2A-AR. The total time required for the radiosynthe-
sis was 30 min from EOB using [11C]methyl iodide in DMSO. [11C]1
was obtained in 25% yield (EOS) with >99% chemical and radio-
chemical purities and the specific activity ranged from 3–4 Ci/
l
[
mol (n = 6). PET studies in anesthetized baboon show that
26. Andres, J. I.; Alcazar, J.; Alonso, J. M.; De Lucas, A. I.; Iturrino, L.; Biesmans, I.;
Megens, A. A. Bioorg. Med. Chem. 2006, 14, 4361.
11C]1 penetrates BBB and accumulates in brain areas enriched
with alpha2A-AR. Experiments are under progress to further quan-
27. Zeng, Z.; Chen, T.-B.; Miller, P. J.; Dean, D.; Tang, Y. S.; Sur, C.; Williams, D. L., Jr.
Nucl. Med. Biol. 2006, 33, 555.
tify alpha2A-AR with [11C]1.
28. Radiosynthesis of [11C]1: The precursor desmethyl-1 (0.5–1.0 mg) was dissolved
in 500 lL of DMSO in a capped 5 mL V-vial containing potassium hydroxide
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