A Novel Radiotracer for Imaging Monoacylglycerol Lipase in the Brain Using Positron Emission Tomography

Article abstract of DOI:10.1021/acschemneuro.5b00293

Monoacylglycerol lipase (MAGL) is a serine hydrolase that hydrolyzes monoacylglycerols to glycerol and fatty acid and plays an important role in neuroinflammation. MAGL inhibitors are a class of molecules with therapeutic potential for human diseases of t

Full text of DOI:10.1021/acschemneuro.5b00293

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Letter
A Novel Radiotracer for Imaging Monoacylglycerol
Lipase in the Brain Using Positron Emission Tomography
Changning Wang, Michael S. Placzek, Genevieve C. Van
de Bittner, Frederick A Schroeder, and Jacob M. Hooker
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.5b00293 • Publication Date (Web): 22 Dec 2015
Downloaded from http://pubs.acs.org on December 26, 2015
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A Novel Radiotracer for Imaging Monoacylglycerol Lipase in the Brain Using Positron Emission
Tomography
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Changning Wang1,*, Michael S. Placzek 1, 2, Genevieve C. Van de Bittner1, Frederick A.
Schroeder1, Jacob M. Hooker1
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1Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts
General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129, United States
2 Department of Psychiatry, McLean Imaging Center, McLean Hospital, Harvard Medical School,
Belmont, Massachusetts 02478, United States
ABSTRACT
Monoacylglycerol lipase (MAGL) is a serine hydrolase that hydrolyzes monoacylglycerols to
glycerol and fatty acid and plays an important role in neuroinflammation. MAGL inhibitors are a
class of molecules with therapeutic potential for human diseases of the central nervous system
(CNS), in areas such as pain and inflammation, immunological disorders, neurological and
psychiatric conditions. Development of a non-invasive imaging probe would elucidate the
distribution and functional roles of MAGL in the brain and accelerate medical research and drug
discovery in this domain. Herein, we describe the synthesis and pilot rodent imaging of a novel
MAGL imaging agent, [11C]SAR127303. Our imaging results demonstrate the high specificity,
good selectivity, and appropriate kinetics and distribution of [11C]SAR127303, validating its utility
for imaging MAGL in the brain. Our findings support the translational potential for human CNS
MAGL imaging.
KEYWORDS: Monoacylglycerol lipase, PET, radiotracer, brain, CNS
INTRODUCTION
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Monoacylglycerol lipase (MAGL) is a serine hydrolase that hydrolyzes monoacylglycerols to
glycerol and fatty acid, with highest expression in brain, white adipose tissue, and liver. 1-3 MAGL
primarily degrades the endogenous cannabinoid, 2-arachidonoyglycerol (2-AG), one of the two
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1, 4, 5
main transmitters of the endogenous cannabinoid system.
Low levels of 2-AG have been
observed in pain and inflammation 6, 7, immunological disorders , neurological and psychiatric
conditions 9, obesity and metabolic syndromes 10, 11 and cancer. 12
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In neuroinflammatory regions, MAGL is upregulated to produce less 2-AG. 13-15 Both genetic and
pharmacological blockades of MAGL show anti-inflammatory effects in the brain and
neuroprotective effects in mouse models of Parkinson's disease and Alzheimer's disease, two
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examples of profound brain dysfunction involving neuroinflammation.
In addition, a recent
study found that MAGL releases arachidonic acid (AA), the precursor for pro-inflammatory
prostaglandin synthesis in certain tissues. Inhibiting MAGL lowered AA levels in the brain
(elevation of 2-AG), which resulted in reduced levels of pro-inflammatory prostaglandin and
thromboxane.14 The importance of MAGL in neuroinflammatory diseases has led to the
development of MAGL-selective inhibitors 1, 16, 17, such as JZL184 and KML29, which are in the
O-aryl carbamate class 13, 18. MAGL blockade by JZL184 reduced colonic pro-inflammatory
cytokines in a trinitrobenzene sulfonic acid-induced colitis model resulting in reduced brain
inflammation. 19 Currently, it is not possible to interrogate target engagement of MAGL inhibitors
in vivo. One result of this is the slowed optimization of small molecule inhibitors for clinical
application, including leads from the carbamate class.
Understanding the relationship between MAGL engagement in the brain and inhibitor dosing is
essential to advancing the MAGL therapeutic discovery and dose selection process. Molecular
imaging, such as positron emission tomography (PET), is an ideal tool to study the drug-target
interaction as it is non-invasive, has exquisite sensitivity and selectivity, and can be translated to
human brain imaging. Developing novel MAGL PET imaging probes will significantly accelerate
the entire process of investigating MAGL expression and activity in the brain, quickly identifying
lead MAGL inhibitors in preclinical and translational research.
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However, to our knowledge, no PET radiotracers targeting MAGL have been reported. Although
efforts have been made to evaluate several radiolabeled MAGL inhibitors, these compounds lack
sufficient brain uptake required for PET brain imaging.20 Herein, we report a novel MAGL PET
radiotracer ([11C]SAR127303), which is based on a highly potent and selective MAGL inhibitor
(SAR127303) 4 and demonstrates suitable pharmacokinetic properties in a pilot rodent study.
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RESULTS AND DISCUSSION
Physiochemical properties of SAR127303
A number of major factors determine the success of a radiotracer candidate for imaging MAGL in
the brain. The binding affinity and selectivity for MAGL must be high enough to produce sufficient
signal for detection. SAR127303 has been recently developed and evaluated by Sanofi.4
SAR127303 is a highly selective inhibitor of mouse and human MAGL (IC50 = 3.8 and 29 nM,
respectively).4 The binding selectivity of SAR127303 was tested on more than 200 targets
(receptors, ion channels, enzymes, transporters and kinases), including cannabinoid receptors
(CB1 and CB2) and other human serine hydrolases (e.g. FAAH) at 10 mM. No significant
inhibition was observed against these targets, with the exception of α/β-hydrolase 6 (ABHD6, a
serine hydrolase for 2-AG hydrolysis). 4 However, MAGL is responsible for approximately 85%
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of CNS-centered 2-AG hydrolysis
brain 2-AG hydrolase activity 22, 23, indicating radiolabeled SAR127303 could be used as a probe
while ABHD6 only accounts for approximately 4% of
for monitoring MAGL-regulated 2-AG hydrolysis.
High brain uptake of the radiotracer is important for imaging MAGL in the brain. The molecular
weight, measured log D and total polar surface area (tPSA) of SAR127303 are within the typical
range for small molecules that demonstrate good brain penetration (Figure 1). Indeed, the brain to
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plasma ratio of SAR127303 is above 10 after p.o. administration to male mice at 10 mg/kg.
Therefore, SAR127303 is an ideal good probe candidate for radiolabeling and subsequent brain
imaging of MAGL with PET.
Synthesis of SAR127303 and the PET imaging agent, [11C]SAR127303
To date, most MAGL inhibitors, including SAR127303, are carbamate-based inhibitors that form
irreversible, covalent bonds between the carbamoyl moiety and Ser132 of the enzyme’s catalytic
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triad with the expulsion of a leaving group.24 Therefore, the carbamoyl group is an ideal
radiolabeling site. The chemical synthesis of SAR127303 (Scheme 1) and its intermediates were
performed by SAI Life Sciences Ltd. Briefly, intermediate 3 was prepared by the coupling reaction
with 1 and 2. Compound 3 was allowed to react with the intermediate formed from 4 and 5 to
afford the desired product SAR127303.
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To insert the radiolabel at the carbonyl of the carbamate, a one-pot [11C]CO2 fixation method was
employed (Scheme 2)25, which is a base-catalyzed reaction that forms carbamates from [11C]CO2.
Briefly described, [11C]SAR127303 was synthesized by bubbling cyclotron-produced [11C]CO2
into a sealed vial with 3 in acetonitrile in the presence of 2-tert-butylimino-2- diethylamino-1,3-
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dimethylperhydro-1,3,2-diazaphosphorine (BEMP). When activity peaked, POCl3 was added
and the mixture was stirred for 1 minute to form the putative mixed anhydride.
Hexafluoroisopropanol (HFIP) was then introduced and the mixture was stirred for 1 minute before
quenching with water. All radiochemistry was carried out with no heating or cooling, as previously
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described, and purified by semi-preparative HPLC. Radiosynthesis of [11C]SAR127303 was
prepared 30–34 min after end of bombardment (EOB) with adequate radiochemical yields (3–6%,
non-decay corrected, calculated from initial trapped [11C]CO2) and final formulation ready for
injection with high radiochemical purity (>95%) and good specific activity (1.6-2.3 mCi/nmol at
time of injection).
In vivo PET-CT imaging with [11C]SAR127303 in rodents
Initial evaluation of [11C]SAR127303 in vivo was conducted in rodents with dynamic brain PET.
[11C]SAR127303 exhibited high BBB penetration and sustained binding over the 60 min scan
when administered by intravenous bolus injection (0.2-0.5 mCi (80-310 ng) per animal), as shown
in Figure 2. [11C]SAR127303 displayed highest uptake in the cortex, thalamus and cerebellum.
Based on a whole-brain analysis, the concentration of [11C]SAR127303 in the brain reached a
maximum at ∼2 min post-injection.
To investigate the specificity of [11C]SAR127303, we performed PET imaging studies in rats with
5 min i.v. pretreatment of unlabeled SAR127303 at 0.05, 0.1 , 1.0 and 2.0 mg/kg. At 0.05 mg/kg,
we found a 48 % reduction in binding, estimated as the percent change in whole-brain radioactivity
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between peak uptake at 2 min and the lowest uptake at 60 min (Figure 2). Increasing the dose of
unlabeled SAR127303 to 0.1 mg/kg resulted in a ~67% reduction in [11C]SAR127303 brain
uptake, and we observed a similar blockade with 1.0 and 2.0 mg/kg unlabeled SAR127303,
indicating saturation at 0.1 mg. This finding demonstrates a high saturability of [11C]SAR127303
for MAGL, with a dose-dependent response to self-blockade and ~30% of uptake attributed to
non-specific binding.
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To support the notion that [11C]SAR127303 was not effluxed from the brain by the P-glycoprotein
(P-gp), we measured radioligand uptake in brain after treatment with the P-gp inhibitor,
cyclosporin A (CsA) and found no difference compared to control treatment (Figure 3A).
Additionally, [11C]SAR127303 binding in rat brain was not disrupted (koff = 0) by i.v. challenge
with unlabeled SAR127303 20 min after tracer administration.26 This provided confirmation of the
irreversibility of [11C]SAR127303 binding to MAGL (Figure 3B).
To test the ability of [11C]SAR127303 as a tool for evaluation of MAGL inhibitors in the brain,
we performed a study with 5 min pretreatment of two MAGL selective inhibitors, JZL184 and JW
642. From our imaging results, JW642 showed good blockade in the brain, however, JZL184 did
not, suggesting that JW642 has good brain permeability and MAGL inhibition, while JZL184 has
limited brain uptake or poor MAGL engagement at this dose (Figure 4).
Our data in rodents support [11C]SAR127303 as a new tool to visualize MAGL density in the living
brain. The robust brain uptake of [11C]SAR127303 potentially offers a method for quantification
of MAGL density in inflammation-associated brain disorders, such as neurodegenerative diseases
and mood disorders. We also anticipate utility of [11C]SAR127303 for in vivo evaluation of target
engagement of novel therapeutics early in compound development pipeline.
CONCLUSION
In summary, [11C]SAR127303 binds to MAGL with high selectivity and specificity and provides
a potential tool for quantitative imaging of MAGL density in the brain of living subjects. We are
progressing [11C]SAR127303 forward for evaluation as a novel MAGL PET radiotracer in non-
human primates and humans.
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METHODS
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General methods and materials
Compound 3 and SAR127303 were prepared form SAI Life Sciences Ltd, Telangana, India.
JZL184 and JW642 were purchased from Tocris Bioscience. Minneapolis, MN. All other reagents
and solvents were of ACS-grade purity or higher and used without further purification. NMR data
were recorded on a Varian 400 MHz magnet and were reported in ppm units downfield from
trimethylsilane. Analytical separation was conducted on an Agilent 1100 series HPLC fit with a
diode-array detector, quaternary pump, vacuum degasser, and autosampler. Mass spectrometry
data were recorded on an Agilent 6310 ion trap mass spectrometer (ESI source) connected to an
Agilent 1200 series HPLC equipped with a quaternary pump, vacuum degasser, diode-array
detector, and an autosampler. [11C]CO2 (1.2 Ci) was obtained via the 14N (p, α) 11C reaction with
nitrogen in the presence of 2.5% oxygen, with 11 MeV protons (Siemens Eclipse cyclotron), and
trapped on molecular sieves in a TRACERlab FX-MeI synthesizer (General Electric).
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All animal studies were carried out at Massachusetts General Hospital (PHS Assurance of
Compliance No. A3596-01). The Subcommittee on Research Animal Care (SRAC) serves as the
Institutional Animal Care and Use Committee (IACUC) for the Massachusetts General Hospital
(MGH). SRAC reviewed and approved all procedures detailed in this paper.
PET/CT imaging was performed in anesthetized (isoflurane) Sprague-Dawley rats. Highly-trained
animal technicians monitored animal safety throughout all procedures and veterinary staff were
responsible for daily care. All animals were socially housed in cages appropriate for the physical
and behavioral health of the animals. Animals were given unlimited access to food and water.
Chemical synthesis
4-chloro-N-(piperidin-4-ylmethyl)benzenesulfonamide (3)
4-chlorobenzenesulfonyl chloride (1) (0.84 g, 4 mmol) and tert-butyl 4-(aminomethyl)piperidine-
1-carboxylate (2) (0.85 g, 4 mmol) were added to a chilled solution (0 °C, ice water bath) in the
presence of diisopropylethyl amine (DIPEA) (0.78 g, 6 mmol) and 4-dimethylaminopyridine
(DMAP) (0.049 g, 0.4 mmol) in dichloromethane (DCM) (15 mL). After 16 h, the solvent was
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removed under vacuum and the residue partitioned between DCM and water (20 mL each). The
organic layer was washed with brine (20 mL), dried with Na2SO4, filtered, and the solvent was
removed under vacuum. Crude products were purified by flash chromatography. Pure fractions
were combined, the solvent removed and solids were collected (1.12 g, yield 72%). 1H-NMR (400
MHz, CDCl3): δ 7.80 (d, J = 8.4 Hz, 2H), 7.51 (d, J = 8.4 Hz, 2H), 4.59-4.62 (m, 1H), 4.11 (s, 2H),
2.83-2.86 (m, 2H), 2.62-2.65 (m, 2H), 1.58-1.67 (m, 2H), 1.45 (s, 9H), 1.02-1.27 (m, 2H). The
product from the previous step (1.1 g, 2.8 mmol) was dissolved in DCM chilled over an ice water
bath (0 °C). TFA (3.2 g, 28 mmol) was added and stirred for 16 h. The solvent was removed under
vacuum and the residue partitioned between DCM and sodium bicarbonate solution (20 mL each).
The organic layer was washed with brine (20 mL), dried (Na2SO4), filtered, and the solvent was
removed under vacuum. Crude products were purified by flash chromatography. Pure fractions
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were combined, the solvent removed and the product (3) was collected (0.7 g, yield 85%). H-
NMR (400 MHz, CDCl3): δ 7.80 (d, J = 8.4 Hz, 2H), 7.50 (d, J = 8.4 Hz, 2H), 4.62 (s, 1H), 3.05
(d, J = 12 Hz, 2H), 2.83 (d, J = 9.6 Hz, 2H), 2.51-2.57 (m, 2H), 1.57-1.66 (m, 2H), 1.02-1.10 (m,
2H). LC-MS calculated for C12H17ClN2O2S [M]: 288.1; found [M+H]+: 289.1.
1,1,1,3,3,3-hexafluoropropan-2-yl
carboxylate (SAR127303)
4-(((4-chlorophenyl)sulfonamido)methyl)piperidine-1-
HFIP (4) (0.17 g, 1 mmol) was added to a chilled solution (0 °C, ice water bath) of 4-nitrophenyl
carbonochloridate (5) (0.4 g, 2 mmol), pyridine (0.16 g, 2 mmol) and DMAP (0.006 g, 0.05 mmol)
in DCM (3 mL). After 16 h, a solution of 3 (0.3g, 1 mmol) and DIPEA (0.33 g, 2.5 mmol) in DCM
(15 mL) was added dropwise and after 30 min the solvent was removed under vacuum and the
residue partitioned between DCM and water (20 mL each). The organic layer was washed with
brine (20 mL), dried (Na2SO4), filtered, and the solvent was removed under vacuum. Crude
products were purified by flash chromatography. Pure fractions were combined, the solvent
removed and solids were precipitated from pentane. The final product (SAR127303) was collected
(0.06 g, yield 8%). Purity of SAR127303 was >99% as measured by HPLC. 1H-NMR (400 MHz,
CDCl3): δ 7.80 (d, J = 8.0 Hz, 2H), 7.52 (d, J = 8.4 Hz, 2H), 5.71-5.76 (m, 1H), 4.51-4.55 (m, 1H),
4.14-4.21 (m, 2H), 2.81-2.93 (m, 4H), 1.71-1.80 (m, 2H), 1.12-1.18 (m, 2H). LC-MS calculated
for C16H17ClF6N2O4S [M]: 482.0; found [M-H]+: 480.7.
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Radiosynthesis of [11C]SAR127303
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A conical vial (1 mL) sealed with a Teflon-lined silicone septum was purged with N2 (15 mL/min
for 5 min) 10 min prior to the end-of-bombardment and then charged with a solution of 3 (1 mg)
and BEMP (7 μL) in anhydrous CH3CN (100 μL). Carbon-11 labeled CO2 was dispensed in a
stream of nitrogen (15 mL/min) into the conical vial until radioactivity peaked. One min later, a
solution of POCl3 (0.3 μL) in CH3CN (100 μL) was added followed 1 min later by a CH3CN
solution (100 μL) of HFIP (1.5 μL). After 1 min, the reaction was quenched with water (700 μL)
and purified on a reverse phase semi-preparative HPLC (Agilent Eclipse XDB-18 column, 5µm,
9.4×250 mm, 5.0 mL/min, 35% H2O + 0.1% TFA / 65% CH3CN + 0.1% TFA, 254 nm) and the
desired fraction was collected. The final product was diluted with 20 mL water and reformulated
by loading onto a solid-phase exchange (SPE) C-18 cartridge, rinsing with water (5 mL), eluting
with EtOH (1 mL), and diluting with saline (9 mL). The chemical and radiochemical purity of the
final product was tested by analytical HPLC (Agilent Eclipse XDB-C18, 150 mm x 4.6 mm). The
identity of the product was confirmed by analytical HPLC via co-injection with a SAR127303
reference standard. Specific activity of [11C]SAR127303 (at time of injection): 1.6-2.3 mCi/nmol.
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Log D Determination
An aliquot (∼50 μL) of the formulated radiotracer was added to a test tube containing 2.5 mL of
octanol and 2.5 mL of phosphate buffer solution (pH 7.4).The test tube was mixed by vortex for 2
min and then centrifuged for 2 min to fully separate the aqueous and organic phase. A sample
taken from the octanol layer (0.1 mL) and the aqueous layer (1.0 mL) was saved for radioactivity
measurement. An additional aliquot of the octanol layer (2.0 mL) was carefully transferred to a
new test tube containing 0.5 mL of octanol and 2.5 mL of phosphate buffer solution (pH 7.4). The
previous procedure (vortex mixing, centrifugation, sampling, and transfer to the next test tube)
was repeated until six sets of aliquot samples had been prepared. The radioactivity of each sample
was measured in a well counter (Perkin-Elmer, Waltham, MA).The log D of each set of samples
was derived by the following equation: log D = log (decay-corrected radioactivity in octanol
sample x 10/decay-corrected radioactivity in phosphate buffer sample).
Rodent PET/CT Acquisition and Post Processing
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Male Sprague-Dawley rats were utilized in pairs, anesthetized with inhalational isoflurane at 3%
in a carrier of 1.5 L/min medical oxygen and maintained at 2% isoflurane for the duration of the
scan. The rats were arranged side-by-side in a Triumph Trimodality PET/CT/SPECT scanner
(Gamma Medica, Northridge, CA). Rats were injected with unlabeled SAR127303 or vehicle via
a lateral tail vein catheterized at the start of PET acquisition. Dynamic PET acquisition lasted for
60 min and was followed by computed tomography (CT) for anatomic co-registration and PET
attenuation correction. PET data were reconstructed using a 3D-MLEM method resulting in a full
width at half-maximum resolution of 1 mm. Reconstructed images were exported from the scanner
in DICOM format along with an anatomic CT for rodent studies. These files were imported to
PMOD (PMOD Technologies, Ltd.) and manually co-registered using six degrees of freedom.
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Rodent PET/CT Image Analysis
Volumes of interest (VOIs) were drawn manually as spheres in brain regions guided by high
resolution CT structural images and summed PET data, with a radius no less than 1mm to minimize
partial volume effects. Time-activity curves (TACs) were exported as decay-corrected activity per
unit volume. The TACs were expressed as percent injected dose per unit volume for analysis.
AUTHOR INFORMATION
Corresponding Author:
*Mailing address: Athinoula A. Martinos Center for Biomedical Imaging Massachusetts General
Hospital, CNY 149, Room 5.022A 149 13th Street, Charlestown, MA 02179. Phone: 617.724.9390.
E-mail: cwang15@mgh.harvard.edu.
Author Contributions:
C.W. discovered, synthesized and purified [11C]SAR127303. C.W. and J.M.H. planned
experiments, performed data analysis. C.W., M.S.P., G.V..d.B. and F.A.S. performed the
experiments. The manuscript was written through contributions of all the authors. All authors have
given approval to the final version of the manuscript.
Funding:
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This research was supported by the Harvard/MGH Nuclear Medicine Training Program from the
Department of Energy under Grant DE-SC0008430 (C.W.) and NIH-NIDA T32 postdoctoral
fellowship T32DA015036 (M.S.P.). This research was carried out at the Athinoula A. Martinos
Center for Biomedical Imaging at the Massachusetts General Hospital, using resources provided
by the Center for Functional Neuroimaging Technologies, P41EB015896, a P41 Regional
Resource supported by the National Institute of Biomedical Imaging and Bioengineering (NIBIB),
National Institutes of Health. This work also involved the use of instrumentation supported by the
NIH Shared Instrumentation Grant Program and/or High-End Instrumentation Grant Program;
specifically, grant numbers: S10RR015728.
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Notes:
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors are grateful to the Martinos Center radiopharmacy staff (Judit Sore, Kari Phan, Garima
Gautam and Samantha To) for help with radiotracer production. The authors are grateful to Dr.
Aijun Zhu for assisting the rodent PET-CT studies.
ABBREVIATIONS
MAGL, monoacylglycerol lipase; PET, positron emission tomography; CT, computed tomography;
EOS, end of synthesis; RCY, radiochemical yield; VOI, volume of interest; TAC, time-activity
curves;
BEMP,
(2-tert-butylimino-2-
diethylamino-1,3-dimethylperhydro-1,3,2-
diazaphosphorine); HFIP, hexafluoroisopropanol; tPSA, total polar surface area; FAAH, fatty acid
amide hydrolase; ABHD6, α/β-hydrolase 6; TFA, Trifluoroacetic acid; SPECT, single-photon
emission computed tomography; SPE, solid-phase exchange; DIPEA, diisopropylethyl amine;
DCM, dichloromethane; DMAP, 4-Dimethylaminopyridine; LPS, lipopoly-saccharide; AA,
arachidonic acid; AEA, N-arachidonoylethanolamine; CNS, central nervous system; PNS,
peripheral nervous system; EOB, end of bombardment.
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Figure 1. SAR127303 is a highly selective inhibitor of mouse and human MAGL. Griebel et al.
reported the pharmacokinetics of SAR127303 recetly.4 Single oral administration (P.O.) of
SAR127303 at 10 mg/kg showed high concentration in mouse brain. Plasma and brain Cmax were
observed at 4 hours after SAR127303 administration.4
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O
S
DIPEA, DMAP
TFA
NH
a).
b).
Cl
H
O
S
BocN
N
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+
O
NH2
O
Cl
2
Cl
1
3
O
N
O2
CF3
O
O
S
CF3
CF3
N
4 5
, ,
DMAP
c).
Py,
O
NH
HO
CF3
Cl
O
Cl
O
4
5
SAR127303
Scheme 1. Reagents and conditions: (a) 1 (1.0 eq), 2 (1.0 eq), DIPEA (1.5 eq), DMAP (0.1 eq),
DCM (15 mL), 0 °C to rt, 16 h, 72%; (b) ), TFA (10 eq), DCM (2 mL), 0 °C - rt, 16 h, 85%; (c) 4
(1.0 eq), 5 (2.0 eq), Pyridine (2.0 eq), DMAP (0.05 eq), DCM (3 mL), 0 °C - rt, 16 h; then 3 (1.0
eq), DIPEA (2.5 eq), DCM (15 mL), 0 °C - rt, 16 h, 8%.
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Scheme 2. [11C]CO2 fixation reaction forming [11C]SAR127303. a = POCl3, 1 min, rt; b = HFIP,
1 min, rt, then quenched with water and purified by HPLC. Radiochemical yield (RCY): 3-6%
based on the trapped [11C]CO2 (decay-uncorrected). Specific activity at time of injection:1.6-2.3
mCi/nmol.
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Figure 2. Rodent in vivo PET imaging with [11C]SAR127303 reveals blockable uptake in brain.
(A) Summed PET-CT images (30-60 min) following injection with [11C]SAR127303 at baseline
or after 5-min pretreatment with 1.0 mg/kg unlabeled SAR127303 (self-blocking); (B)
[11C]SAR127303 has high uptake in the whole brain with significant blockade after pre-treatment
with SAR127303 at 1.0 mg/kg; (C) [11C]SAR127303 shows blockade in brain regions such as
cortex (CTX), thalamus (THA) and cerebellum (CB) after pre-treatment with SAR127303 at 1.0
mg/kg; (D) SAR127303 pre-treatment at 0.05, 0.1, 1.0 and 2.0 mg/kg shows activity blockade in
the brain.
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Figure 3. (A) Brain uptake of [11C]SAR127303 in rats is equivalent 30 min after pretreatment with
vehicle (control) or the P-glycoprotein (Pgp) inhibitor cyclosporin A (CsA, 25 mg/kg, i.v.); (B)
Equilibrium binding of [11C]SAR127303 is undisrupted by administration of unlabeled
SAR127303 (1.0 mg/kg, i.v.) 20 min after tracer injection, demonstrating irreversible MAGL
binding in the brain.
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Figure 4. (A)Structures of MAGL selective inhibitor, JZL 184 (IC50 = 8 nM) and JW 642 (IC50 =
3.7 nM); (B) Brain uptake of [11C]SAR127303 can be blocked by JW 642 (5 min pretreatment,
i.v., 1 mg/kg), demonstrating specific MAGL binding of [11C]SAR127303 in the brain. The
unchanged uptake of [11C]SAR127303 following pretreatment with JZL 184 suggests limited brain
uptake of JZL 184.
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