Synthesis of [O-methyl-11C]1-(2-chlorophenyl)-5-(4- methoxyphenyl)-4-methyl-1H-pyrazole-3-carboxylic acid piperidin-1-ylamide: A potential PET ligand for CB1 receptors

Article abstract of DOI:10.1016/j.bmcl.2004.03.034

Synthesis and in vitro evaluation of [O-methyl-¹¹C]1-(2- chlorophenyl)-5-(4-methoxyphenyl)-4-methyl-1H-pyrazole-3-carboxylic acid piperidin-1-ylamide ([¹¹C]-1), a potential imaging agent for CB ₁ receptors using PET is described. 1-(2-Chlorophenyl)-5-(4- hydroxyphenyl)-4-methyl-1H-pyrazole-3-carboxylic acid piperidin-1-ylamide (5), the precursor for radiolabeling, was synthesized from 4-OTBDPS-propiophenone (2) in five steps with 30% overall yield. The reaction of alcohol 5 with [ ¹¹C]MeOTf at 60°C afforded [¹¹C]-1 with an average radiochemical yield of 14.5% (EOS) and >2000Ci/mmol specific activity. The radiotracer was found to selectively label CB₁ receptors in slide-mounted sections of postmortem human brain containing prefrontal cortex as demonstrated by in vitro autoradiography using phosphor imaging.

Full text of DOI:10.1016/j.bmcl.2004.03.034

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2393–2396
Synthesis of [O-methyl-11C]1-(2-chlorophenyl)-5-(4-
methoxyphenyl)-4-methyl-1H-pyrazole-3-carboxylic acid
piperidin-1-ylamide: a potential PET ligand for CB1 receptors
a
a,c
J. S. Dileep Kumar,a,c, Jaya Prabhakaran, Victoria Arango, Ramin V. Parsey,a,c
Mark D. Underwood,a,c Norman R. Simpson,b,c Suham A. Kassir,c Vattoly J. Majo,a
Ronald L. Van Heertumb,c and J. John Manna,b,c
*
aDepartment of Psychiatry, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA
bDepartment of Radiology, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA
cDepartment of Neuroscience, New York State Psychiatric Institute, New York, NY 10032, USA
Received 9 February 2004; revised 10 March 2004; accepted 12 March 2004
Abstract—Synthesis and in vitro evaluation of [O-methyl-11C]1-(2-chlorophenyl)-5-(4-methoxyphenyl)-4-methyl-1H-pyrazole-3-
carboxylic acid piperidin-1-ylamide ([11C]-1), a potential imaging agent for CB1 receptors using PET is described. 1-(2-Chloro-
phenyl)-5-(4-hydroxyphenyl)-4-methyl-1H-pyrazole-3-carboxylic acid piperidin-1-ylamide (5), the precursor for radiolabeling, was
synthesized from 4-OTBDPS-propiophenone (2) in five steps with 30% overall yield. The reaction of alcohol 5 with [11C]MeOTf at
60 ꢀC afforded [11C]-1 with an average radiochemical yield of 14.5% (EOS) and >2000 Ci/mmol specific activity. The radiotracer was
found to selectively label CB1 receptors in slide-mounted sections of postmortem human brain containing prefrontal cortex as
demonstrated by in vitro autoradiography using phosphor imaging.
ꢁ 2004 Elsevier Ltd. All rights reserved.
Cannabis has been used for recreational and medicinal
purposes for centuries and is the third most commonly
used drug after tobacco and alcohol.1 The potential
therapeutic effects of cannabinoids are broad and
include antiemetic, bronchodilation, antiinflammatory,
antiglaucoma, analgesic, anticonvulsant, as well as
treatment of obesity and alcoholism.2 Also therapeutic
benefit for pain is reported. However, the pharmaco-
logical mechanisms of these actions are still unclear.
Cannabinoid actions are thought to be mediated
through two types of cannabinoid receptors designated
as CB1 and CB2.3 The CB1 receptor is the most abun-
dant CB receptor subtype found in the CNS and is also
expressed in peripheral neurons and tissues.3;4 CB2
receptors are not present in CNS, but are found in
immune system cells, especially in B-cells, monocytes,
and T-cells.3 Extensive preclinical as well as clinical
studies of CB1 agonists and antagonists suggest that
altered levels of CB1 receptor binding may underlie the
pathogenesis of a diverse range of neuropsychiatric
disorders, neurodegenerative diseases, stroke, pain dis-
orders some cancers, diabetes, glaucoma, emesis, asth-
ma, diarrhea, and cardiovascular diseases.5 We recently
demonstrated an upregulation of CB1 receptor density
in the dorsolateral prefrontal cortex (DLPFC) of
depressed suicides (DS) and alcoholic suicides (AS)
compared with age-matched controls.6;7 To better
understand the role of CB1 receptors in disease and
therapeutics, it would be of great benefit to be able to
measure CB1 receptor binding in vivo, noninvasively
and repeatedly over time. Highly selective and high
affinity CB1 antagonists capable of penetrating the
blood brain barrier are candidates for PET imaging
probes for CB1 receptors. To date, most efforts at in vivo
imaging of human brain CB1 receptors have been dis-
appointing.8 We chose 1-(2-chlorophenyl)-5-(4-meth-
oxyphenyl)-4-methyl-1H-pyrazole-3-carboxylic
acid
piperidin-1-ylamide (1) as our candidate PET ligand for
CB1 receptors due to its high affinity for CB1 receptors
(8 nM) and log P value 3.45, which allows sufficient
lipophilicity for 1 to enter into brain.9
Keywords: CB1 receptors; Autoradiography; Positron emission tomo-
graphy; Biological imaging; Human postmortem.
* Corresponding author. Tel.: +1-212-543-5909; fax: +1-212-543-6017;
e-mail: dk2038@columbia.edu
0960-894X/$ - see front matter ꢁ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.03.034

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J. S. D. Kumar et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2393–2396
The synthesis of 1-(2-chlorophenyl)-5-(4-hydroxyphen-
yl)-4-methyl-1H-pyrazole-3-carboxylic acid piperidin-1-
ylamide (5), the precursor for radiolabeling, has been
achieved in five steps from the OTBDPS protected
propiophenone (2) (Scheme 1). Compound 2 was ini-
tially treated with LiHMDS and diethyloxalate followed
by coupling the enolate with 2-chlorophenylhydrazine
hydrochloride and cyclization of the imine derivative
obtained with acetic acid to afford the pyrazole ester (3).
The ester 3 upon hydrolysis followed by condensation
with 1-aminopiperidine in presence of HOBT, TPTU,
and diisopropylethylamine in acetonitrile provided the
precursor 5 in 30% overall yield.10 Alternatively, syn-
thesis of the standard 1 was achieved from 40-meth-
oxyacetophenone using a reported procedure.9
Even though, under optimized reaction conditions, the
methylation of 5 was achieved using methyl iodide in
presence of tetrabutylammonium hydroxide (TBAOH)
or NaOH as base, our labeling experiments under these
conditions with [11C]CH3I did not provide [11C]-1 in
sufficient yield and purity. However, using [11C]MeOTf
in the presence of 5 N NaOH in acetone at room tem-
perature provided [11C]-1 with a 14.5% radiochemical
yield (EOS, Scheme 2, Fig. 1).11 The chemical identity of
[11C]-1 was confirmed by co-injection with an authentic
CO Et
2
1. LiHDMS, (Et) O, (CO Et) , -78°C
2
2
2
N
O
N
2. 2-Chlorophenylhydrazine, HCl, EtOH, rt
3. AcOH, reflux
Cl
TBDPSO
TBDPSO
2
3
O
CO H
2
N
N
H
N
LiOH, THF-H O
2
reflux
TPTU, HOBT, DIEA
Aminopiperidine, DMF, 48 h
N
N
N
Cl
Cl
HO
HO
4
5
Scheme 1. Synthesis of radiolabeling precursor 5.
O
O
N
N
N
N
H
H
1. [11C]MeOTf, NaOH, acetone, 5'
2. HPLC purification
N
N
N
N
Cl
Cl
H311CO
HO
[
11C]-1
5
Scheme 2. Radiosynthesis of [11C]-1.
Figure 1. (A) Radiosynthesis of [11C]-1; (B) co-injection with cold 1.

J. S. D. Kumar et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2393–2396
2395
Figure 2. Phosphor image of PFC brain sections with [11C]-1: (A) total; (B) nonspecific VLPFC (ventral lateral prefrontal cortex); OPFC (orbital
prefrontal cortex); MPFC (medial prefrontal cortex).
sample of 1 on analytical reverse-phase HPLC (Fig. 1).
The chemical and radiochemical purity of [11C]-1 was
found to be >99% with a specific activity >2000 Ci/mmol
(EOB). The average time required for the [11C] labeling
was 30 min (EOB).
The authors also thank Ms. Julie Arcement for her
assistance in the radiolabeling studies.
References and notes
After successful synthesis and specific activity determi-
nation of [11C]-1, phosphor image autoradiographic
studies were undertaken to determine the distribution of
CB1 binding sites in the prefrontal cortex (PFC) post-
mortem.12 Figure 2 illustrates the total and nonspecific
binding of 10 nM [11C]-1 incubated for 600. Nonspecific
binding was determined by co-incubating with 1 lM
SR141716A, a known CB1 antagonist.13 As is evident
from Figure 2, specific binding was observed in the gray
matter of DLPFC, ventral lateral prefrontal cortex
(VLPFC), orbital prefrontal cortex (OLPFC), medial
prefrontal cortex (MPFC), and anterior cingulate cortex
with [11C]-1. The distribution of CB1 binding is consis-
tent with what is reported in other in vitro studies.4;6;7
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In summary, we successfully synthesized [11C]-1, a
potential imaging agent for CB1 receptors. The total
time required for the synthesis of [11C]-1 is 30 min from
EOB using [11C]methyl triflate in acetone, with a 14.5%
yield at EOS based on [11C]MeOTf. The chemical and
radiochemical purities are >99% with a specific activity
>2000 Ci/mmol. The radiotracer binds to PFC post-
mortem human brain as visualized by phosphor imaging
suggesting specific affinity for quantifying CB1 recep-
tors. The phosphor image studies indicate that the newly
developed [11C] labeled CB1 ligand has the potential for
the quantification of CB1 receptors in brain, and further
detailed in vitro and in vivo studies are under progress.
Acknowledgements
This work was supported by research grants from the
National Institutes of Health MH40210 and MH62185.

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was loaded into a semipreparative HPLC (Phenomenex
C18) column and the product fraction, between 7 and
8 min was collected based on a c-detector using 50:49:1
(acetonitrile–water–acetic acid), 12 mL/min. The collected
fraction was then diluted with deionized water (100 mL),
passed through a C-18 Sep-Pak and washed with water
(2 · 20 mL). Following a water rinse, the product [11C]-1
was eluted from the Sep-Pak with 1 mL of ethanol. A
small portion of [11C]-1 was analyzed with analytical
HPLC for chemical identity, chemical and radiochemical
purities, and specific activity. The average yield was found
to be 14.5% at EOS with a specific activity >2000 Ci/mmol.
12. In vitro phosphor imaging: Slide-mounted prefrontal
cortical sections from the right hemisphere (20 lm) were
brought to 22 ꢀC and pre-incubated in 50 mM Tris
hydrochloride buffer (pH 7.4) containing 1% BSA for
45 min (22 ꢀC). This was followed by incubation in 10 nM
of [11C]-1 for 60 min at room temperature. Adjacent
sections were incubated in parallel in with [11C]-1 and
1 lM SR141716A to determine nonspecific binding. Sec-
tions were washed in 50 mM Tris buffer (pH 7.4) at 4 ꢀC
and briefly dipped in ice-cold water to remove salts. Slides
were quickly dried under a stream of cold air and exposed
to ST-phosphor-imaging screen (Packard, wrapped in
Mylar film) with high- and low-activity [14C] standards
(American Radiolabeled Chemicals) for 90 min. Screens
were scanned with a Packard Cyclone phosphor-imaging
system and analyzed with OptiQuant Acquisition and
Analysis software (Packard).
10. 1-(2-Chlorophenyl)-5-(4-hydroxyphenyl)-4-methyl-1H-pyr-
azole-3-carboxylic acid piperidin-1-ylamide (5): (2-Chloro-
phenyl)-5-(4-hydroxyphenyl)-4-methyl-1H-pyrazol-3-yl]-
oxo-acetic acid (657.50 mg, 2.0 mmol) was dissolved in
15 mL acetonitrile. To the above solution 1-aminopiper-
idine (0.30 mL, 2.6 mmol), HOBT (540.50 mg, 4 mmol),
TPTU (713.0 mg, 2.4 mmol), and diisopropylethylamine
(1.1 mL, 6.0 mmol) were successively added and stirred for
2 days. The reaction mixture was diluted with water and
the products were extracted into ethyl acetate. The ethyl
acetate layer was washed with water (2 · 30 mL), saturated
NaHCO3 solution (2 · 30 mL), and then with brine. The
organic layer was dried over anhydrous MgSO4 and the
solvent was evaporated under reduced pressure. The crude
product was purified by silica gel column chromatography
using 60–70% ethyl acetate in hexane as the eluent. The
product was obtained as colorless crystals (600 mg, 73%),
5: mp > 270 ꢀC. 1H NMR (400 MHz, CDCl3) d 1.58 (m,
6H), 2.49 (s, 3H), 2.69 (m, 4H), 6.67 (d, J ¼ 8 Hz, 2H),
6.95 (d, J ¼ 7:8 Hz, 2H), 7.45–7.52 (m, 3H), 7.59 (d,
J ¼ 7:5 Hz, 1H), 8.95 and 9.80 (2s, 1H), HRMS calcd for
C22H24O2N4Cl 411.1588; found 411.1601.
11. Radiosynthesis of [O-Methyl-11C]1-(2-chlorophenyl)-5-(4-
methoxyphenyl)-4-methyl-1H-pyrazole-3-carboxylic acid
piperidin-1-ylamide ([11C]-1): [11C]MeOTf was trapped
into an acetone (400 lL) solution containing 0.5 mg of 5
and 10 lL of 5 N NaOH at rt for 50. The resulting mixture
13. Rinaldi, C. M.; Barth, F.; Heaulme, M.; Shire, D.;
Calandra, B.; Congy, C.; Martinez, S.; Maruani, J.;
Neliat, G.; Caput, D. FEBS Lett. 1994, 350, 240.