Radiosynthesis and in vivo evaluation of [11C]MOV as a PET imaging agent for COX-2

Article abstract of DOI:10.1016/j.bmcl.2018.06.015

Radiosynthesis and in vivo evaluation of [¹¹C]4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide (methoxy analogue of valdecoxib, [¹¹C]MOV), a COX-2 inhibitor, was conducted in rat and baboon. Synthesis of t

Full text of DOI:10.1016/j.bmcl.2018.06.015

Accepted Manuscript
Radiosynthesis and in vivo evaluation of [11C]MOV as a PET imaging agent for
COX-2
Jaya Prabhakaran, Mark Underwood, Francesca Zanderigo, Norman R.
Simpson, Anna R. Cooper, Jeffrey Matthew, Harry Rubin-Falcone, Ramin V.
Parsey, J. John. Mann, J.S. Dileep Kumar
PII:
S0960-894X(18)30503-1
https://doi.org/10.1016/j.bmcl.2018.06.015
BMCL 25896
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 March 2018
8 June 2018
9 June 2018
Please cite this article as: Prabhakaran, J., Underwood, M., Zanderigo, F., Simpson, N.R., Cooper, A.R., Matthew,
J., Rubin-Falcone, H., Parsey, R.V., Mann, J.J., Dileep Kumar, J.S., Radiosynthesis and in vivo evaluation of
[11C]MOV as a PET imaging agent for COX-2, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://
doi.org/10.1016/j.bmcl.2018.06.015
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Radiosynthesis and in vivo evaluation of [11C]MOV as a PET
imaging agent for COX-2
Jaya Prabhakarana,b, Mark Underwooda,b, Francesca Zanderigoa,b, Norman R. Simpsonb, Anna R.
Coopera, Jeffrey Matthewa, Harry Rubin-Falconeb, Ramin V. Parseyc, J. John. Manna,b, and J. S. Dileep
Kumarb*
aDepartment of Psychiatry, Columbia University Medical Center, New York, USA; bMolecular Imaging and
Neuropathology Division, New York State Psychiatric Institute, New York, cDepartment of Psychiatry, Stony Brook Medical
Center, Stony Brook, New York, USA
Abstract— Radiosynthesis and in vivo evaluation of [11C]4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-
yl]benzenesulfonamide (methoxy analogue of valdecoxib, [11C]MOV), a COX-2 inhibitor, was conducted in rat and
baboon. Synthesis of the reference standard MOV (3), and its desmethyl precursor 2 for radiolabeling were performed using
1,2-diphenylethan-1-one as the starting material in five steps with 15% overall yield. Radiosynthesis of [11C]MOV was
accomplished in 40 ± 10% yield and >99% radiochemical purity by reacting the precursor 2 in dimethyl formamide (DMF)
with [11C]CH3I followed by removal of the dimethoxytrityl (DMT) protective group using trifluroacetic acid. PET studies in
anesthetized baboon showed very low uptake and homogeneous distribution of [11C]MOV in brain. The radioligand
underwent rapid metabolism in baboon plasma. MicroPET studies in male Sprague Dawley rats revealed [11C]MOV binding
in lower thorax. The tracer binding in rats was partially blocked in heart and duodenum by the administration of 1mg/kg
oral dose of COX-2 inhibitor valdecoxib.
Cyclooxygenase (COX) is the key enzyme required for
the conversion of arachidonic acid to prostaglandin.1 COX- are observed in myocardial infarction and cardiac allograft
1, one of the three isoforms of COX, is constitutively rejection.9, 11 Since, COX-2 is also expressed in heart, the
expressed and it is responsible for the production of psychiatric illnesses.15-18 Similarly, elevation in COX-2
prostanoids associated with the normal homeostatic levels cardiac side effects of COX-2 inhibitors are a major
functions, whereas, COX-2 is mainly induced by concern due the possibility of the inhibition of platelet
inflammatory stimuli. Several studies suggest that COX-2 is aggregation in the blood.
involved in the development of certain types of cancer,
COX-2 selective inhibitors (COXIBs) are non-steroidal
arthritis, stroke, pain, transplant rejection, and anti-inflammatory drugs (NSAIDs).5-8,19-22 COXIBs are used
neurodegenerative disorders such as Alzheimer’s and to treat pain and inflammation and they are less likely to
Parkinson’s diseases.2-11 Quantification of the expression of cause significant gastrointestinal bleeding and other side
COX-1 and COX-2 proteins and mRNA in postmortem effects seen with the commonly used non-selective NSAIDs.
human subjects is reported using western blot, Because of cardiac side effects, COXIBs such as rofecoxib
immunocytochemistry and RT-PCR techniques.12-14 The (Vioxx) and valedecoxib (Bextra) were withdrawn from the
above experiments show that COX-2 levels for normal market and celecoxib (Celebrex) is the only COXIB
tissues are highest in kidney followed in decreasing order by currently approved by the FDA.23-26 Positron Emission
brain > spleen = liver = heart = intestine. Upregulation of Tomography (PET) imaging may allow in vivo monitoring
COX-2 plays a significant role in many malignances, of COX-2 induction in disorders with COX-2 pathology,
neurologic and
________
Key words: PET, Inflammation, COX-2, Radiotracer
*Corresponding author: Fax: 617-774-7521;
Email: kumardi@nyspi.columbia.edu
monitor organ rejection and evaluate target engagement of
new NSAIDs and COXIB medications. Although significant
progress has been reported in the development of high
affinity COXIBs, a target-specific validated PET tracer is
not available currently for COX-2 imaging in vivo. Progress
in the development of COX-2 PET tracers in the past two

decades is reported in several reviews.27-30 In general, failure Bases such as sodium hydride and sodium hydroxide
of the previously developed radioligands can be partially resulted in dimethylation as the major reaction via
attributed to their partially attributed to their poor methylation of the NH group of the sulfonamide compound
pharmacology (low affinity and COX-2/COX-1 selectivity), 2. Radiosynthesis of [11C]MOV was achieved by reacting
de[18F]fluorination and inability to detect low basal level of the precursor molecule 2 with [11C]CH3I in DMF using
COX-2 under normal conditions. Our pilot PET imaging KOtBu, followed by deprotection with 20% TFA in
experiments
with
[18F]fluorovaldecoxib
indicated dichloromethane. [11C]MOV was obtained in 40+10% yield
de[18F]fluorination in rodents and nonhuman primates.31 Our at the end of synthesis (EOS), with 100% chemical purity
attempts to evaluate [18F]Fluoroethylvaldecoxib in rodents and a molar activity of 2+0.5 Ci/μmol (n=15). The total
also resulted de[18F]fluorination. Similarly, incorporation of synthesis time was 40 minutes at EOS.36
[11C]methyl group to the 5-position of valdecoxib methyl
Subsequently, in vivo evaluation of [11C]MOV was
position was not successful. Hydroxymetyl valdecoxib (1) is performed in anesthetized baboons (n=2) using PET
the major metabolite of valdecoxib in vivo and is also a imaging. PET images showed that [11C]MOV penetrated the
COX-2 inhibitor.32-34 Several ether analogues of the blood brain barrier (BBB), however, a very low level of
valdecoxib derivative 1 are COX-2 inhibitors indicating radioactivity was retained in the brain (Figure 1).37 Time-
derivatization of the methoxy group may not significantly activity curves (TACs) based on standard uptake values
alter the affinity and in vivo biological property of (SUV) of [11C]MOV also indicate a very low binding with
compound 1 (Table 1).31-34 Although some of these homogeneous distribution across the brain regions (Figure
analogues (eg. methylbenzoate) have potential [C-11] 2). [11C]MOV underwent rapid metabolism in baboon
labeling sites, their radiolabeled versions may lead to polar plasma with 89+0.8%, 64.8+1.1%, 45.7+0.3%, 27+5%,
radioactive metabolites in vivo. Therefore, we sought to 13+6.6% and 14+0.6% retention of parent radiotracer at 2, 4,
synthesize a methoxy-valdecoxib (MOV) derivative, the 12, 30, 60 and 90 minutes respectively (Figure 3). The
closest analogue of valdecoxib with a suitable site for the radioactive metabolites of [11C]MOV were found to be
introduction of 11CH3 group. A large number of Carbon-11 polar, suggesting an inability to penetrate the BBB. Figure 1
(t1/2 = 20.38 min.; positron emission; 99.8%; Emax; 0.96 shows that the signals due to the binding of [11C]MOV in
MeV) labeled radiotracers is widely used for PET imaging brain is lesser than that of the background activity. The
owing to their feasibility of radiosynthesis, high specific preliminary PET imaging results are therefore not
activity, and possibility to perform test-retest and test-block encouraging for further evaluating [11C]MOV in brain.
PET scans within few hours of the same day. We anticipate
that [11C]MOV also produce nonradioactive 1 as the major
1.6
metabolite by O-11C-demethylation, which do not compete
with the parent radiotracer making facile tracer
quantification. Herein, we describe the radiosynthesis and in
vivo evaluation of [11C]MOV in baboon and rodents.
SUV
(g/mL)
0
Figure 1. Sum of 0-120 minute PET images of [11C]MOV in
a representative anesthetized baboon (left: sagittal, middle:
coronal, right: transaxial).
0.16
0.12
0.08
THA
Scheme 1. Synthesis and radiosynthesis of [11C]MOV.
PUT
CER
PFC
HIP
0.04
0
The key intermediate, hydroxymethyl valdecoxib (1),
for MOV (3) synthesis is prepared using a previously
described procedure.33 The sulfonamide group of compound
1 was protected with dimethoxytrityl (DMT) by reacting it
with DMT-chloride in presence of triethylamine in 70%
yield.31 The reference standard was 3 was then prepared by
reaction of the DMT-protected compound 2 with methyl
iodide, followed by removal of DMT group with 20%
trifluoroacetic acid (TFA) in dichloromethane. An optimal
yield (85%) of MOV (3) was obtained by performing the
reaction with potassium t-butoxide (KOtBu) in DMF.35
0
20
40
60
80
100
Time (min.)
Figure 2. TACs of [11C]MOV in baboon brain (CER:
cerebellum; HIP: hippocampus; PFC: prefrontal cortex;
PUT: putamen; THA: thalamus).
We then examined the in vivo distribution of
[11C]MOV in periphery organs in male Sprague Dawley rats
with microPET (Figure 4). The radioligand showed binding

in heart, liver, blood vessels, kidney and duodenum (Figures
4 and 5) of rats. TACs of thoracic regions showed peak
uptake less than a minute and retention of radioactivity in
heart and blood vessels.
Liver
Liver-B
Duodenum-B
Duodenum
Kidney
1.2
B
100
0.8
0.4
0
75
50
25
0
0
20
40
Time (min.)
60
80
Figure 5. TACs of [11C]MOV in rats (A: Upper thorax; B:
Lower thorax, B: blocking with 1 mg/kg valdecoxib) (IVC:
inferior vena cava; RCA: Right coronary artery; LCA: left
coronary artery).
0
25
50
Time (min)
75
100
Figure 3. Unmetabolized [11C]MOV in baboon plasma (n=2).
The radioligand binding in heart was 1.4 times higher than
that found in aorta, 1.3 times at inferior vena cava (IVC) and
twice as high as the left and right carotid arteries at 16
minutes (Figure 5A). This indicates that part of the binding
of the radiotracer in heart is not due to blood flow effect.
PET images of the lower thorax in rats showed high binding
of the tracer in duodenum (Figure 5B), however, the binding
did not reach equilibrium even at 80 minutes. In order to
determine COX-2 specific binding of [11C]MOV, PET
imaging studies were also performed in rats pretreated with
the COX-2 inhibitor valdecoxib (1 mg/kg, oral). We noticed
a partial blocking of the binding in heart (22% at 16 minute)
and duodenum (30% at 80 minute) respectively (Figures 5A
and 5B). However, we did not find any significant blocking
in blood vessels and liver. The unexpected higher binding of
[11C]MOV in duodenum is intriguing. One possibility is the
leakage of radioactivity from liver or radioactive metabolite
deposition in duodenum. Although the binding is partially
blocked with valdecoxib, further ex vivo analysis is required
to verify the [11C]MOV binding in duodenum.
In summary, we synthesized COX-2 radioligand
[11C]MOV by a simple radiomethylation with excellent
purity and molar activity. As evident from SUV uptake,
radiotracer did not show significant accumulation in baboon
brain. The radiotracer show uptake in heart and duodenum
and was partially blocked with COX-2 inhibitor valdecoxib.
Further in vivo studies in animal models of inflammation
may establish the utility of [11C]MOV as a PET imaging
agent for peripheral COX-2 imaging. The above results also
indicate that structure activity relationship (SAR) studies of
valdecoxib with PET approach may lead to a successful PET
tracer for in vivo imaging of COX-2.
Figure 4. MicroPET images of [11C]MOV in rats (left:
thorax; middle: lower thorax; right: thorax, blocking with 1
mg/kg valdecoxib).
1.5
Heart
Aorta
IVC
RCA
LCA
A
1.2
0.9
0.6
0.3
0
Heart-B
0
4
8
12
16
Time (min)
Acknowledgement
Pfizer Inc. provided funding for this investigator initiated
grant request (JSDK and JJM).
Supporting Information
Table 1. COX-2 affinities of valdecoxib, hydroxymethyl
valdecoxib and ether analogues.
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35. 4-(5-(methoxymethyl)-3-phenylisoxazol-4-yl)benzenesulfona-
mide (3): KOtBu (33 mg, 0.3 mmol) was added to a solution of
2 (0.15 mmol, 95 mg) in 3 mL of DMF at 0 °C. The resulting
solution was stirred for 20 minutes and added 20 μL of CH3I
(0.3 mmol) and the mixture was heated at 40 oC for 2 h.
Subsequently, the reaction mixture was treated with freshly
prepared 0.1 mL of 20% trifluoroacetic acid in dichloromethane
o
and allowed to heat at 70 C for 5 minutes, The reaction was

cooled to room temperature, added 5 mL water and extracted
with ethyl acetate (2x 25 mL). Combined ethyl acetate extracts
were washed with water, brine, and evaporated under high
vacuum to afford the crude product which upon silica gel
chromatography with 60% ethyl acetate in hexane afforded 40
mg (85%) of compound 3 as a white solid.
3: H NMR (300 MHz, CDCl3) δ: 3.47 (s, 3H, CH3), 4.2 (s,
2H, CH2), 4.9 (bs, NH2), 7.4 (7H, m), 8 (d, 2H). HRMS (EI+)
calculated for C17H17N2O4S: 345.0812; Found: 345.0802.
previously. Followed by transmission scan, [11C]MOV (4.5±
0.5 mCi) was injected as an i.v. bolus and the emission data
were collected for 120 min in 3D mode. Plasma samples were
taken every 10 seconds for the first 2 minutes, using an
automated system, and thereafter manually for a total of 34
samples over 2 hours.38 PET data were reconstructed with
transmission-based attenuation correction and model-based
scatter correction.38 A T1-weighted magnetic resonance image
(MRI) of the head was acquired on a GE 1.5-T Signa
Advantage system. Regions of interests were drawn on the MRI
using MEDX software (Sensor Systems, Inc., Sterling, VA,
USA). PET data were co-registered to the MRI using AIR
software and TACs generated for each regions of interest
(ROI).38,39
1
36.Radiosynthesis of [11C]4-(5-(methoxymethyl)-3-phenylisoxazol-
4-yl)benzenesulfonamide ([11C]MOV): Precursor alcohol (2)
(~0.5-1 mg) was dissolved in 400 μL of DMF in a capped 1
mL reaction vial. KOtBu (1mg) was added and the solution was
allowed to stand for 5 minutes at room temperature. High
specific active [11C]CO2 produced from RDS112 Cyclotron ( 37
GBq) was subsequently converted into [11C]CH3I via wet
chemistry by sequential reaction with lithium aluminum
hydride and hydrogen iodide in a semi-automated system.
[11C]CH3I was transported into the vial by a stream of argon
(20-30 mL/min) over a period of 5 min at room temperature
and the mixture was heated at 40°C for 5 min. Freshly prepared
TFA/dichloromethane (0.5 mL, 20%) was added to the above
solution and heated under argon flow at 50 oC for 5 minutes to
reduce the reaction volume to 0.5-0.6 mL. The solution was
then diluted with 0.5 mL of the mobile phase and was injected
onto a semi-preparative RP-HPLC column (Phenomenex,
progidy ODS(3) 10 x 250 mm, 10 μ column) and eluted with
acetonitrile : 0.1 M ammonium formate solution: acetic acid
(34:65:1) at a flow rate of 10 mL/min. The deprotected
compound 2 appeared around 6 min. The radioproduct fraction
with a retention time of 12-13 min based on a γ-detector was
collected, diluted with 100 mL of deionized water, and passed
through a classic C-18 Sep-Pak cartridge. Reconstitution of the
product in 1 mL of absolute ethanol afforded [11C]3 in 40%
yield based on [11C]CH3I at EOB. A portion of the ethanol
solution was analyzed by analytical HPLC (Phenomenex
Progidy ODS(3) 4.6 x 250 mm, 5 μ column; mobile phase:
acetonitrile : 0.1 M ammonium formate: acetic acid 34 : 65:1;
flow rate: 2 mL/min, retention time: 10 min) to determine
radiochemical purity and molar activity.
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2006, 49, 125-134.
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Tomogr. 1998, 22, 139-152.
40. Micro PET studies of [11C]MOV in rats: Adult Sprague Dawley
rats (male, n=3) weighing 300-400g (Hilltop Lab Animals, NY)
were anesthetized with isoflurane (1-5% in O2) and positioned
the head and upper thorax at the center of the field of view of a
microPET R4 camera (Concorde Siemens Microsystems, Inc.,
Model R4). A polyethylene catheter (PE90, 1.27mm o.d.,
Intramedic) filled with heparinized saline was inserted into the
left femoral vein for radioligand injection and a terminal KCl
(4M) injection at the conclusion of the experiment. The femoral
arteries were cannulated (PE50, 0.965mm o.d., Intramedic) for
measurement of arterial pressure, blood gases, and hematocrit.
A tracheotomy was performed (PE360, Intramedic) for artificial
ventilation. Transmission scans were acquired with a rotating
germanium-68 point source and used attenuation correction.
[11C]MOV (200+50 μCi) was injected into the left femoral
vein and initiated camera acquisition. List-mode data were
collected for 100 minutes and reconstructed using attenuation
correction and Fourier rebinning. The dynamic images were
reconstructed using a filtered back-projection algorithm and
interpolated to give nCi/cc (microPET Manager, Concorde
Microsystems Inc.). ROIs were drawn on the PET images and
TACs generated for each of the ROIs.
37. PET studies in baboons: All animal experiments were carried
out with the approval of the Institutional Animal Care and Use
Committees of Columbia University Medical Center and the
New York State Psychiatric Institute. PET scans were
performed in male baboon (Papio anubis, n=2) with an ECAT
EXACT HR+ scanner (Siemens, Knoxville, TN) as described

Highlights
 Induced COX-2 expression is one of the pathophysiology of inflammation.
 Upregulation of COX-2 expression is a biomarker for diseases.
 [11C]MOV, a COX-2 PET tracer did not exhibit binding in baboon brain in vivo.
 Radiotracer exhibit partial specific binding in heart and duodenum in rats.

Radiosynthesis and in vivo evaluation of [11C]MOV as a PET imaging agent for COX-2
Jaya Prabhakarana,b, Mark Underwooda,b, Francesca Zanderigoa,b, Norman R. Simpsonb, Anna R. Coopera, Jeffrey Matthewa,
Harry Rubin-Falconeb, Ramin V. Parseyc, J. John. Manna,b, and J. S. Dileep Kumara*; aDepartment of Psychiatry, Columbia
University, NY; bMolecular Imaging and Neuropathology Division, New York State Psychiatric Institute, NY 10032, USA;
cNow at Department of Psychiatry, Stony Brook University Medical Center, Stony Brook, New York
N
O
O
O
11CH3
S
H2N
[
11C]MOV
O