Synthesis and preclinical evaluation of [11C]MA-PB-1 for in?vivo imaging of brain monoacylglycerol lipase (MAGL)

Article abstract of DOI:10.1016/j.ejmech.2017.04.066

MAGL is a potential therapeutic target for oncological and psychiatric diseases. Our objective was to develop a PET tracer for in?vivo quantification of MAGL. We report [¹¹C]MA-PB-1 as an irreversible MAGL inhibitor PET tracer. The in?vitro inh

Full text of DOI:10.1016/j.ejmech.2017.04.066

European Journal of Medicinal Chemistry 136 (2017) 104e113
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
11
Synthesis and preclinical evaluation of [ C]MA-PB-1 for in vivo
imaging of brain monoacylglycerol lipase (MAGL)
a
,
1
a
,
1
a
a
a
Muneer Ahamed , Bala Attili , Daisy van Veghel , Maarten Ooms , Philippe Berben ,
a
b
a
c
c
Sofie Celen , Michel Koole , Lieven Declercq , Juha R. Savinainen , Jarmo T. Laitinen ,
Alfons Verbruggen , Guy Bormans Prof.
a
a, *
a
Laboratory for Radiopharmaceutical Research, Department of Pharmaceutical and Pharmacological Sciences, Campus Gasthuisberg O&N2, Herestraat 49
Box 821, BE-3000 Leuven, Belgium
b
Department of Nuclear Medicine & Molecular Imaging, UZ Herestraat 49, 3000 Leuven, Belgium
Institute of Biomedicine, Faculty of Health Sciences, The University of Eastern Finland, Finland
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 February 2017
Received in revised form
MAGL is a potential therapeutic target for oncological and psychiatric diseases. Our objective was to
develop a PET tracer for in vivo quantification of MAGL. We report [ C]MA-PB-1 as an irreversible MAGL
11
inhibitor PET tracer. The in vitro inhibitory activity, ex vivo distribution, brain kinetics and specificity of
21 April 2017
11
[
C]MA-PB-1 binding were studied. Ex vivo biodistribution and microPET showed good brain uptake
Accepted 24 April 2017
Available online 25 April 2017
which could be blocked by pretreatment with both MA-PB-1 and a structurally non-related MAGL in-
hibitor MJN110. These initial results suggest that [11C]MA-PB-1 is a suitable tracer for in vivo imaging of
MAGL.
Keywords:
©
2017 Elsevier Masson SAS. All rights reserved.
MAGL
[
11C]MA-PB-1
MJN110
Biodistribution
PET imaging
1
. Introduction
Abbreviations: AA, arachidonic acid; AEA, N-arachidonoyl ethanolamine; 2-AG,
-arachidonoyl glycerol; BBB, blood brain barrier; Bmax, enzyme expression; BBr
boron tribromide; CB1, cannabinoid 1 receptors; CDCl , deuterated chloroform;
CH CN, acetonitrile; clog D, calculated distribution coefficient; cPSA, calculated
polar surface area; Cs CO , cesium carbonate; DCM, dichloromethane; DIPEA, dii-
sopropylethylamine; DMF, dimethylformamide; DMSO, dimethylsulfoxide; EtOAc,
ethyl acetate; EtOH, ethanol; ESI, electron spray ionization; FAAH, fatty acid amide
hydrolase; GBq/mmol, Gigabecquerel per micromole; hMAGL, human mono-
acylglycerol lipase; HRMS, high resolution mass spectrometry; IC50, minimum
2
3
,
The endocannabinoid system consists of cannabinoid receptors,
3
endogenous ligands and other proteins for their synthesis, degra-
dation and transport [1,2]. In the central nervous system, the
endocannabinoid system plays an important role in the regulation
of several physiological functions such as cognition, pain, move-
ment and memory [3,4]. Two of the most important and widely
studied endogenous lipid transmitters are N-arachidonoyl etha-
nolamine or anandamide (AEA) and 2-arachidonoylglycerol (2-AG)
3
2
3
inhibitory concentration (50% enzyme inhibition);
EDTA, ethylenediaminetetraacetic acid dipotassium salt; LPA, lysophosphatidic
acid; MAGL, monoacylglycerol lipase; MBq, megabecquerel; MgSO , magnesium
sulfate; NaBH(OAc) , sodium triacetoxyborohydride; NaHCO , sodium bicarbonate;
D
K , dissociation constant;
K
2
4
[5]. These two endocannabinoids are synthesized on demand in
3
3
response to neuronal activity. The signal is then rapidly terminated
due to the breakdown of the endocannabinoids by the enzymatic
activity of two serine hydrolases: fatty acid amide hydrolase (FAAH)
and monoacylglycerol lipase (MAGL), respectively [6].
MAGL is a 33-kDa cytosolic serine hydrolase enzyme that pref-
erentially cleaves monoacylglycerols into fatty acids and glycerol
and plays a crucial role in coordinating multiple lipid signalling
pathways. In the brain, MAGL was found to be co-localized with
CB1 receptors in axon terminals where it is responsible for
approximately 85% of 2-AG hydrolysis [7,8]. Therefore, MAGL is an
NaOAc, sodium acetate; NMR, nuclear magnetic resonance spectroscopy; NMRI,
naval medical research institute; NaI(Tl), thallium-doped sodium iodide; PET,
positron emission tomography; PGE , prostaglandin E ; p.i., post injection; RP-
2 2
HPLC, reverse phase-high performance liquid chromatography; SUV, standardized
uptake value; TAC, time activity curve; TFA, trifluoroacetic acid; TMS, tetrame-
thylsilane; UHPLC, ultra-high performance liquid chromatography; UHR-TOF, ultra-
high resolution time of flight; VOI, volume of interest; %ID, percentage injected
dose; [18F]FDG, 2-[18F]fluoro-2-deoxy-
D
-glucose; 3D-MAP, 3D maximum a poste-
riori iterative reconstruction.
*
Corresponding author. O&N2, Herestraat 49, box 821, BE-3000 Leuven, Belgium.
E-mail address: guy.bormans@kuleuven.be (G. Bormans).
These authors contributed equally.
1
http://dx.doi.org/10.1016/j.ejmech.2017.04.066
223-5234/© 2017 Elsevier Masson SAS. All rights reserved.
0

M. Ahamed et al. / European Journal of Medicinal Chemistry 136 (2017) 104e113
105
important terminating factor of the endocannabinoid signalling
activity. Additionally, MAGL also plays an important role in the
release of arachidonic acid (AA) and other free fatty acids like
prostaglandin E2 (PGE2) and lysophosphatidic acid (LPA). It is well
established that AA is metabolized to pro-inflammatory prosta-
glandins and the free fatty acids generate pro-tumorigenic signal-
ling lipids [9,10].
Because of its important role in several lipid signalling path-
ways, MAGL is known to be involved in many pathological condi-
tions, such as inflammation, cancer, neurodegenerative disorders
and several psychological disorders such as anxiety and depression
Hooker et al. published [11C]-SAR127303 as a specific MAGL tracer
in rats [23] and Liang et al. published comparative study between
1
1
11
11
[
C]-SAR127303 and [ C]-TZPU [24]. In their results [ C]-TZPU
11
does not show adequate brain penetration whereas
[ C]-
SAR127303 does display good brain penetration and awaits further
evaluation in non-human primates.
In this study, we modified the structure of JW642 in which the
phenoxy group is substituted with a methoxy group resulting in
MA-PB-1. We herein report the synthesis, radiosynthesis and pre-
11
liminary biological evaluation of [ C]MA-PB-1 for in vivo imaging
of MAGL with PET.
[10]. Blockage of MAGL leads to increased 2-AG levels and
enhanced endocannabinoid signalling [11]. Additionally, blockage
of MAGL also lowers AA levels, which in turn leads to decreased
production of pro-inflammatory prostaglandins. Therefore, MAGL
inhibition could play an important role in several diseases. Indeed,
previous studies have shown that MAGL blockade has several
beneficial outcomes in inflammation, pain, anxiety, neuro-
degeneration and cancer [10,12e15]. In the recent past, a number of
selective and potent MAGL inhibitors have been developed to
modulate MAGL activity [16e20]. Cravatt et al. identified some
interesting carbamate containing small molecule inhibitors of
MAGL, particularly JW-642 was reported to have an IC50 value of
2. Results and discussion
Non-invasive imaging of MAGL using PET will allow quantifi-
cation of regional enzyme expression in vivo in healthy and path-
ological conditions. In general, a successful PET ligand should
provide a clear specific signal (signal/noise ratio ꢀ 1.5) to allow
quantitative mapping of the target of interest. Ideally, a brain PET
tracer should have a high binding affinity with B /K ꢀ 10 and a
max
d
2
log D of 2e3.5 with PSA value of less than 90 Å for adequate brain
penetration and optimum specific to nonspecific activity ratio
[30e32].
3
.7 nM for hMAGL [16].
Non-invasive imaging of MAGL using positron emission to-
2.1. Chemistry
mography (PET) can assist in the translational evaluation of
promising drug candidates targeting MAGL. Furthermore, PET im-
aging of MAGL also allows quantifying MAGL expression in vivo
thus allowing to identifying modulation of MAGL in relevant pa-
thologies. Therefore, the development of a suitable PET radioligand
to visualize MAGL is important.
Synthesis of the target compounds MA-PB-1 and MA-PB-2 were
carried out based on literature procedures [16] (Scheme 1). Com-
pound 4 was synthesized by NaBH(OAc) mediated reductive
amination followed by boc deprotection using trifluoroacetic acid
TFA). After complete conversion (monitored by LC-MS) the reac-
3
(
Structures of recently reported MAGL PET tracers and of MA-PB-1
2
tion mixture was neutralized, extracted (DCM/H O), dried and used
(
radiotracer developed and studied in this work), MJN 110 MAGL in-
in the next step without any further purification. The free amine 4
was converted into MA-PB-1 by adding 4 to a mixture of hexa-
fluoroisopropanol and triphosgene with DIPEA as a base. Finally,
hibitor used for blocking studies.
Hicks and co-workers reported PET radioligands for imaging of
MAGL [26]. Four ligands labeled with carbon-11 at a carbonyl car-
MA-PB-1 was demethylated with BBr
sor MA-PB-2.
3
to yield the phenol precur-
bon, namely [1 C]-KML29, [ C]-JW642, [ C]-ML-30 and [ C]-JJKK-
1
11
11
11
0
048 and a [11C]CH
3
-labeled 1,2,4-triazole urea compound were
studied. These compounds were reported to have moderate BBB
penetration (0.2e0.8 SUV at 2 and 40 min in mice), but brain
retention could not be self-blocked. The lack of MAGL-specific brain
uptake led to the conclusion that none of the tracers was suitable
for in vivo imaging of brain MAGL. While preparing this manuscript,
2
.2. In vitro affinity studies
Table 1 compares the hMAGL IC50 values and the clog D and
cPSA values of various reported MAGL inhibitors. IC50 values should
be interpreted with caution as values were compiled from

106
M. Ahamed et al. / European Journal of Medicinal Chemistry 136 (2017) 104e113
Scheme 1. Synthesis of reference and precursor.
Table 1
compound resulting in hepatobiliary clearance from the blood.
Apart from the hepatobiliary clearance, [ C]MA-PB-1 showed a
significant clearance through the renal pathway (7.9 %ID in bladder
MAGL inhibitors along with their corresponding IC50 values, and calculated log D
and calculated polar surface area (PSA) values (using Marvin sketch 14.11.10.0),
11
*
results from our assays.
IC50 (hMAGL)
5.916 (3.620) nM
plus urine at 60 min p.i.). Despite the excretion through both the
clog D (pH 7.4)
cPSA (Å2)
11
renal and hepatobiliary system, clearance of [ C]MA-PB-1 from
KML29
JJKK-048
JW642
4.3
2.4
86.7
87.9
42.0
75.7
54.3
97.2
42.0
66.9
blood was rather slow (3.8 ± 0.2% ID at 60 min p.i.). This retention
2
0
0.4 nM
11
does not seem to be caused by MAGL binding of [ C]MA-PB-1 in
37.0*(3.716) nM
5.0
2
3
3.7 (1.923)
blood cells or plasma [33] as we did not observe any blocking effect
in the blood (Fig. 3). However, plasma radiometabolite analysis
SAR127303
ML30
TZPU
MA-PB-1
MJN110
29.0 nM
0.5 (1.520) nM
17
3.9
35.924 nM
0.9 (0.724)
3.4
11
indicated fast metabolism with only 6% of the intact [ C]MA-PB-1
26.0* nM
present in blood at 30 min p.i. Retention of radiometabolites in the
blood may thus explain the slow blood clearance.
11.0* (9.125) nM
3.1
11
Importantly, [ C]MA-PB-1 showed a high brain uptake (average
SUVwhole brain ¼ 2.6 at 2 min p.i.) which can be explained by its
favourable clog D and cPSA values (Table 1). Investigation of the
regional brain distribution of the tracer showed a uniform distri-
literature reports and were thus not obtained using the same assay
except for JW642, MA-PB-1 and MJN110 which were assayed in this
study.
11
bution of [ C]MA-PB-1 amongst the studied brain regions (Fig. 3)
which are in accordance to the reported uniform MAGL distribution
11
in the brain [34]. After the high initial uptake, [ C]MA-PB-1
JW642 was reported to have a hMAGL IC50 of 3.7 nM [16] and
calculated log D and PSA values of 5.0 and 42.0, respectively. In our
experiments assessing hMAGL-catalyzed hydrolysis of 2-AG [21,22]
we observed an IC50 of 37 nM for JW642. In the parallel assay, we
found an IC50 (hMAGL) value of 26 nM for MA-PB-1. Calculated log
D and PSA values of MA-PB-1 were 3.4 and 42.0, respectively. The
substitution of the phenoxy with a methoxy group although not
affecting inhibitor potency in a statistically significant manner
decreased the clog D value and is still within the good range for
efficient BBB passage.
partially washed out followed by accumulation in the brain be-
tween 10 and 60 min to reach a steady state concentration (SUVtotal
brain ¼ 1.6 ± 0.3). These observations are in accordance with the
kinetics of an irreversible tracer. Because of the retention of tracer
in the brain, high brain-to-blood ratios (calculated as SUV brain/
SUV blood) were achieved (4.1 and 3.7 at 30 and 60 min p.i.,
respectively) despite the slow clearance of radioactivity from the
blood.
In order to verify the binding specificity, biodistribution studies
were performed at 30 and 60 min post tracer injection in mice that
were pretreated with cold MA-PB-1 (10 mg/kg, i.p.) 60 min before
injection of the tracer. Additionally, a blocking experiment was
conducted in which the biodistribution was studied at 30 min post
tracer injection in mice that were injected with the non-
structurally related MAGL inhibitor MJN110 (10 mg/kg, i.p.)
30 min before tracer injection. Results of both blocking experi-
ments are shown in Fig. 3. The pretreated mice showed a signifi-
2.3. Radiochemistry
The radiolabeled compound [11C]MA-PB-1 was obtained by
11
alkylation of the phenolic precursor (MA-PB-2) with [ C]methyl
ꢁ
iodide at 70 C in DMF in the presence of a base (Fig. 1).
11
[
C]MA-PB-1 was synthesized with a radiochemical yield of
11
cantly lower [ C]MA-PB-1 retention in the brain compared to
untreated animals (p ¼ 0.0007). Whole brain SUV values were
about 70% lower in mice pretreated with cold MA-PB-1 compared
to vehicle-treated animals at both the 30 and 60 min post tracer
injection sacrification time points. As a result, brain-to-blood ratios
were significantly lower in the MA-PB-1 pretreated mice (1.2 and
4
0% (n ¼ 11, based on HPLC recovered activity). The radiolabeled
reaction product could be efficiently purified from unreacted pre-
cursor and other chemical and radiochemical impurities using the
11
described preparative HPLC system, resulting in [ C]MA-PB-1 with
a high radiochemical purity (ꢀ99%) and high specific activity
(
58e400 GBq/
mmol, n ¼ 11), in a total synthesis time of 45 min.
1
.1 at 30 and 60 min p.i., respectively). Pretreatment of animals with
11
MJN110 also resulted in a significant reduction of total [ C]MA-PB-
2.4. Ex vivo biodistribution and regional brain distribution in mice
1
retention (SUV ¼ 0.7 ± 0.1, 30 min p.i.) in the brain compared to
vehicle treated animals. It is noteworthy that blocking studies
performed at 1 mg/kg, 5 mg/kg and 10 mg/kg MJN110 demon-
strated a significant difference in brain SUV with respect to the
control. Although blocking generally follows a dose-dependent
The data of the baseline biodistribution studies are summarized
11
in Fig. 2 and show a transient accumulation of [ C]MA-PB-1 in liver
14.4 %ID at 60 min p.i.) with transfer to the intestines (12.8 %ID at
0 min p.i.). This can be explained by the lipophilic nature of the
(
6

M. Ahamed et al. / European Journal of Medicinal Chemistry 136 (2017) 104e113
107
Fig. 1. MA-PB-2 was methylated with [11C]MeI yielding [11C]-MA-PB-1 with ꢀ99% radiochemical purity after preparative HPLC purification. Retention times of reference and
radiolabeled MA-PB-1 were identical (co-injection of 10 mg/mL MA-PB-1 onto analytical HPLC system). Chromatographic conditions of the quality control: XBridge C18 RP column
(
3.5
m
m, 3.0 mm ꢂ 100 mm), Mobile phase: CH
3
CN/NaOAc 0.05 M pH 5.5 (55:45 v/v), Flow rate: 0.8 mL/min, detection: UV 210 nm (upper chromatogram) and radioactivity (lower
chromatogram).
trend, the observed difference was not significant except between
mg/kg and 10 mg/kg (Fig. 4). We expected that at 1 mg/kg we
2.6. Perfused brain radiometabolite quantification in rats
1
would observe maximum blocking in line with previously reported
results [25] but we observed stronger blocking effect after pre-
treatment with 10 mg/kg. Likewise, the blocking effect was also
observed in peripheral organs such as spleen, pancreas, heart and
lungs, major organs where MAGL expression has been reported in
various organs [34].
In order to quantify radiometabolites formed in or entering the
brain, a perfused brain radiometabolite analysis in rats was per-
formed. The extraction efficiency from brain was low (15e30%)
suggesting irreversible binding of the tracer to brain MAGL which is
associated to the pellet of denatured proteins that precipitate upon
3
addition of CH CN to the brain homogenate. A relatively low
amount of radiometabolites more polar than the intact tracer was
found in brain (<10% of total brain activity; 90% of non-protein
bound tracer at 30 min p.i.), indicating that the radiometabolites
found in plasma at the same time point show limited brain uptake.
2.5. Plasma radiometabolite quantification in rats
Radio-HPLC of rat plasma (at 30 min post tracer injection)
2.7. In vivo microPET studies in rats
showed three peaks. From co-injection of plasma with authentic
reference compound, we could identify the peak with retention
In vivo brain distribution and kinetics of [11C]MA-PB-1 were
studied using microPET imaging in rats (n ¼ 3). A group of female
Wistar rats (n ¼ 3) was scanned at baseline conditions. Addition-
ally, a blocking and displacement study was set up using MJN110 to
check the specificity and reversibility of the tracer binding. Since
the same animals were used for baseline, blocking and chase ex-
periments, each baseline scan was used as an internal reference for
the other scans. An overview of the microPET results can be found
in Fig. 5. Averaged microPET images in baseline conditions showed
a high-intensity signal in the brain (Fig. 5A). Analysis of the
11
time of approximately 10 min as intact [ C]MA-PB-1. The major
radiometabolite was a polar radiometabolite eluting around 4 min.
A second apolar radiometabolite eluted around 15 min. Our data
suggest that [11C]MA-PB-1 is rapidly metabolized in vivo, since
11
3
0 min after injection of [ C]MA-PB-1, only 6% of the radioactivity
in plasma was assigned to intact parent tracer. 28% (n ¼ 3) of
plasma activity could not be extracted from the protein denatured
fraction which may indicate covalent binding of the tracer to
plasma.

108
M. Ahamed et al. / European Journal of Medicinal Chemistry 136 (2017) 104e113
Fig. 2. Biodistribution and regional brain distribution of [11C]MA-PB-1 in mice at 2, 10, 30 and 60 min post tracer injection in baseline conditions. The top figure shows the general
distribution (% ID) of the tracer in adult female NMRI mice at different time points post-injection (n ¼ 4/time point). The bottom figure shows the relative regional brain and blood
concentrations (SUV) at different time points post-injection (n ¼ 4/time point).
Fig. 3. Biodistribution and regional brain distribution of [11C]MA-PB-1 in mice at 30 min and 60 min p.i. in baseline conditions and 30 or 60 min after pretreatment with cold MA-
PB-1 (10 mg/kg i.p.) and 30 min after pretreatment with MJN110 (10 mg/kg, i.p.). The concentration in the different organs, brain regions and fluids is expressed as SUV values.

M. Ahamed et al. / European Journal of Medicinal Chemistry 136 (2017) 104e113
109
comparable with the recently reported [11C]-SAR127303 tracer [23]
1
1
(
SUV value of 1.7 at 2 min in rats) and 4 times higher than [ C]-
11
TZPU [24] (SUV value 0.4 at 2 min in rats). In vivo studies with [ C]-
11
SAR127303 and [ C]-TZPU showed that the radioactivity in brain
remained stable with no significant clearance even after 60 min.
11
However with [ C]MA-PB-1 after the high initial brain uptake, a
significant washout from rat brain was observed, with SUV values
decreasing from ~1.6 at 2 min p.i. to ~0.6 at 60 min p.i. (Fig. 5D). This
is in contrast with the biodistribution results obtained in mice,
where the radioactivity in brain remained stable even at 60 min p.i.
and likely this could be attributed to species difference (Fig. 2).
11
Specificity of [ C]MA-PB-1 binding to MAGL in vivo was tested
in a blocking study in which the same rats from the baseline scan
(
(
n ¼ 3) were pre-treated with the structurally unrelated MJN110
10 mg/kg, i.p., 30 min before tracer injection). After blocking, the
11
retention of [ C]MA-PB-1 in the brain was significantly reduced
resulting in only background signal in the brain (Fig. 5B). The sig-
nificant blocking effect could also be observed via the TAC analysis.
Equilibrium SUV values in the different brain regions (average SUV
total brain 30mine90min ¼ 0.26) were about 60% lower compared to
SUV values in baseline conditions acquired in the same rats. Pre-
treatment with cold MA-PB-1 also showed a significant blocking
effect both in ex vivo biodistribution and in vivo micro-PET studies
Fig. 4. Blocking study with 1 mg/kg, 5 mg/kg and 10 mg/kg doses of MJN110 compared
with control conditions. (ns ¼ P ꢀ 0.05, * ¼ P ꢃ 0.05, ** ¼ P ꢃ 0.01, *** ¼ P ꢃ 0.001).
Results expressed as SUV in mouse brain at 30 min post tracer injection.
corresponding time-activity curves (TACs) showed retention of
11
11
[
(
C]MA-PB-1 in the brain. After high initial uptake of [ C]MA-PB-1
SUV of ~1.6 at 2 min), activity in the brain rapidly decreased to
reach stable values starting from 20 min p.i. (average SUV whole
(
data not shown). These microPET data confirm that a high pro-
11
brain 30e90 min ¼ 0.6). The initial brain uptake of [ C]MA-PB-1
11
portion of the [ C]MA-PB-1 specifically binds to brain MAGL in vivo
(
1.6 and 2.6 SUV in rats and mice, respectively at 2 min p.i.) is
in rats as was also observed in biodistribution studies in mice.
Fig. 5. MicroPET images (horizontal section) showing brain distribution and corresponding TACs describing brain kinetics after i.v. injection of 55 MBq [11C]MA-PB-1 in rats under
gaseous anesthesia (isoflurane 2.5% in O , 1 L/min). The top figure shows averaged summed images (0e90 min) from three animals: baseline (A), after blocking with MJN110 (10 mg/
2
kg i.p. 30 min before tracer injection, B) and after a chase with MJN110 (10 mg/kg i.v. 20 min after tracer injection, C). The graph (D) shows the TACs, the average whole brain kinetics
in baseline conditions, after blocking and in chase conditions as a function of time.

110
M. Ahamed et al. / European Journal of Medicinal Chemistry 136 (2017) 104e113
Finally, in a microPET displacement study, the same rats (n ¼ 3)
radiotracer. For comparison, the brain SUV of [11C]-SAR127303
gradually increased as a function of time to reach about 0.8 at
steady state. A homogeneous distribution of [ C]MA-PB-1 was
recorded in all observed brain regions, with lower initial uptake in
the caudate and highest in prefrontal cortex. The SUV signal of the
pre-treatment scan was decreased (SUV of 1 after 20 min and 0.3
after 40 min in the whole brain).
were injected with [11C]MA-PB-1 followed by an i.v. challenge with
MJN110 (10 mg/kg) at 20 min post tracer injection. An averaged
microPET image of all three rats in the chase experiment is shown
in Fig. 5C. TACs of the brain after chase are similar to those acquired
in baseline conditions. This lack of displacement of radioactivity
after injection of a high dose of MJN110 was expected due to the
11
11
irreversible nature of the [ C]MA-PB-1 binding. Similarly, Hooker
et al. performed a chase study by injection of cold SAR127303
(
20 min p.i. of [11C]-SAR127303) and observed that brain uptake
3. Conclusion
was not affected.
We successfully synthesized MA-PB-1, and radiolabeled [11C]
MA-PB-1 in good yields, and high radiochemical purity and specific
radioactivity. Substitution of the phenoxy group with a methoxy
group in JW642 although not affecting inhibitor potency in a sta-
tistically significant manner, decreased the clog D which may
contribute to increased brain exposure of MA-PB-1. Significant
2.8. Non-human primate rhesus monkey microPET
The results of the 90-min baseline and pre-treatment scan of
[11
C]MA-PB-1 in monkey are shown in Fig. 6. TACs of the baseline
11
11
scan of [ C]MA-PB-1 in the brain show a fast and high initial brain
uptake (SUV of ~4.2 in the whole brain, time to peak uptake:
brain uptake of [ C]MA-PB-1 was observed in biodistribution and
microPET imaging experiments in mice, rats and monkey. Self-
blocking experiments confirmed the specificity of binding. Block-
ing and chasing studies with the structurally non-related MAGL
2
.5 min). After 20 min the brain activity reaches a steady state (SUV
value of about 2.5), suggesting irreversible binding of the
Fig. 6.
m
PET TACs for [11C]MA-PB-1 of the whole monkey brain and of various brain regions: Baseline (A) and after pre-treatment with MJN110 (1 mg/kg i.v. 10 min before tracer
11
injection, C). Transversal, sagittal and coronal
to 90 min after tracer injection.
mPET images of the baseline (B) and pre-treatment (D) experiment with [ C]MA-PB-1: averaged images from 1 to 10 min and from 45

M. Ahamed et al. / European Journal of Medicinal Chemistry 136 (2017) 104e113
111
inhibitor MJN110 in mice, rats and monkey established [11C]MA-PB-
2
under N . Compound 3 was boc deprotected using TFA in DCM, the
1
4
4
as a MAGL specific irreversible tracer meriting further evaluation.
free amine 4 (0.390 g, 1.892 mmol, 1 eq) in DCM (2 mL) was added
and the reaction mixture was further stirred at RT for 3 h. The
mixture was diluted with DCM (100 mL) and washed with H O
2
. Materials and methods
(
2 ꢂ 10 mL) and with brine (20 mL). The organic layers were dried
.1. General materials and methods
4
over anhydrous MgSO and evaporated under reduced pressure.
The crude residue was purified by flash column chromatography
All the chemicals employed in the synthesis were purchased
using EtOAc/heptane mixtures to give MA-PB-1 as pale yellow oil
from commercially reliable suppliers (Aldrich, TCI Europe or
(0.455 g, 60%).
1
1
ACROS) and used without further purification unless stated. H and
H NMR (CDCl
), 3.54e3.58 (m, 4H, 2CH
1H, CH), 6.80 (d, 1H, J 8.0, Ar), 6.78 (s, 1H, Ar), 6.89 (d, 1H, J 7.2, Ar),
3
, 400 MHz):
d
2.42e2.47 (m, 4H, 2CH
), 3.80 (s, 3H, OMe), 5.73e5.78 (m,
2
), 3.50 (s,
1
3
C Nuclear magnetic resonance (NMR) spectra were obtained on a
00 MHz and 101 MHz Bruker NMR spectrometer in the deuterated
2H, CH
2
2
4
13
solvents as indicated and with tetramethylsilane, TMS (
d
¼ 0), as an
3
7.23 (t,1H, J 7.9, Ar). C NMR (CDCl ,101 MHz): d 41.12, 41.52, 49.06,
49.29, 51.93, 59.48, 64.60, 109.38, 111.34, 118.09, 126.08, 135.93,
148.15, 156.48. HRMS (ESI) Calcd. for C16
401.1294. Found: 401.1296. HPLC purity: >98%.
internal standard. Chemical shifts ( ) are reported in ppm and
d
þ
coupling constants are reported in Hertz. Multiplicity is defined by
s (singlet), d (doublet), t (triplet), and m (multiplet). The mass
spectra were recorded on a Bruker maXis impact UHR-TOF mass
spectrometer coupled to a Dionex 3000RS UHPLC system (Bruker-
Daltonik, Bremen, Germany).
H
19
F
6
N
2
O
3
(MþH) :
4.2.3. 1,1,1,3,3,3-Hexafluoropropan-2-yl 4-(3-hydroxybenzyl)
piperazine-1-carboxylate, MA-PB-2
High performance liquid chromatography (HPLC) was per-
formed using a Shimadzu LC-2010A HT system connected to a UV
spectrometer. Radioactivity in the column eluent was monitored
using a 3-in NaI(Tl) scintillation detector connected to a single
channel analyzer (Gabi box, Raytest, Straubenhardt, Germany).
Radioactivity in samples of biodistribution studies and radio-
metabolite analysis was quantified with an automated gamma
counter, equipped with a 3-in NaI(Tl) well crystal coupled to a
multichannel analyzer (Wallac 1480 Wizard, Wallac, Turku,
Finland). The results were corrected for background radiation, de-
tector dead-time and physical decay during counting.
To a solution of MA-PB-1 (100 mg, 0.25 mmol) in DCM (5 mL) at
C, BBr (~0.3 mL) was added and stirred. After 30 min the re-
3
action mixture was allowed to warm to RT and stirred for about
16 h. The mixture was neutralized to pH 7, diluted with DCM
ꢁ
0
(50 mL) and washed with H
NaHCO solution (20 mL). The organic layers were dried over
anhydrous MgSO and evaporated under reduced pressure. The
2
O (2 ꢂ 10 mL) and with saturated
3
4
crude residue was purified by flash column chromatography using
EtOAc/heptane mixtures to give MA-PB-2 as a colourless powder
(97 mg, 41%).
1
H NMR (CDCl
), 3.52e3.56 (m, 4H, 2CH
1H, J 8.0, Ar), 6.78 (d, 1H, Ar), 6.87e6.90 (m, 2H, Ar), 7.16 (t, 1H, J 7.4,
3
, 400 MHz):
d
2.44e2.47 (m, 4H, 2CH
2
), 3.47 (s,
All animals were housed in individually ventilated cages in a
2H, CH
2
2
), 5.70e5.75 (m, 1H, CH), 6.71 (d,
ꢁ
thermoregulated (22 C) and humidity-controlled environment
13
under a 12 h/12 h day/night cycle with free access to food and
water. All animal experiments were approved by the local Animal
Ethics Committee of the University of Leuven (P104/2016) and was
in accordance with European Ethics Committee guidelines (decree
3
Ar). C NMR (CDCl , 101 MHz): d 44.30, 44.68, 52.48, 52.70, 53.62,
62.82, 68.32, 114.92, 116.32, 121.69, 129.84, 138.87, 151.64, 156.18.
þ
HRMS (ESI) Calcd. for C15
387.1140.
H
17
F
6
N
2
O
3
(MþH ): 387.1137. Found:
8
4
4
6/609/EEC).
4
.3. Radiosynthesis
.2. Chemistry
[11
C]MeI was produced according to the procedure described by
.2.1. Tert-butyl 4-(3-methoxybenzyl)piperazine-1-carboxylate, 3
To a solution of 3-methoxy benzaldehyde, 1 (1.0 g, 7.34 mmol, 1
Andr eꢀ s et al. [27]. and bubbled with a stream of helium through a
solution of the radiolabeling precursor (200 g) and Cs CO
L). The mixture was then heated
m
2
3
eq) and N-Boc piperazine, 2 (1.05 g, 8.08 mmol, 1.1 eq) in
dichloromethane (DCM, 10 mL) at 0 C was added portionwise
(1e3 mg) in anhydrous DMF (200
m
ꢁ
ꢁ
for 3 min at 70 C. After dilution with 1 mL water, the crude reaction
NaHB(OAc)
at room temperature (RT) for 16 h. The reaction mixture was
diluted with DCM (150 mL) and washed with H
O (2 ꢂ 30 mL) and
with brine (30 mL). The organic layers were dried over anhydrous
MgSO and evaporated under reduced pressure. The crude residue
3
(1.87 g, 8.81 mmol, 1.2 eq) and the mixture was stirred
mixture was purified by HPLC using an XBridge C18 column (5 mm,
4.6 mm ꢂ 150 mm; Waters, Milford, Connecticut) eluted with a
mixture of 0.05 M sodium acetate buffer pH 5.5 and EtOH (43/57 v/
v) at a flow rate of 1 mL/min. UV monitoring of the eluate was
2
11
4
performed at 254 nm. The peak corresponding to [ C]MA-PB-1
was purified by flash column chromatography using 0e50% EtOAc/
heptane mixtures to give compound 3 as a colorless oil (1.30 g,
was collected, diluted with saline to obtain a final ethanol con-
centration of <10% and sterile filtered through a 0.22-mm mem-
5
8%).
1H NMR (CDCl
CH ), 3.40e3.44 (m, 4H, 2CH
.79 (d, 1H, J 8.0, Ar), 6.87e6.90 (m, 2H, Ar), 7.22 (t, 1H, J 7.9, Ar).
, 101 MHz):
brane filter (Millex GV 13 mm; Millipore, Billerica, MA). The
chemical and radiochemical purity of the tracer was assessed using
3
, 400 MHz):
d
1.45 (s, 9H, Boc), 2.37e2.40 (m, 4H,
2
6
2
2
), 3.48 (s, 3H, OMe), 3.80 (s, 1H, CH
2
),
C
HPLC on an analytical XBridge
C
18 column (3.5
mm,
1
3
3.0 mm ꢂ 100 mm, Waters) eluted with a mixture of a 0.05 M
sodium acetate buffer pH 5.5 and acetonitrile (45/55 v/v) at a flow
rate of 0.8 mL/min. UV detection was done at 210 nm. Identity of the
tracer was confirmed by co-elution with authentic cold reference
on the same HPLC.
NMR (CDCl
3
d
28.61, 53.05, 55.38, 63.15, 79.72, 112.70,
114.78, 121.61, 129.40, 139.75, 154.98, 159.83. HRMS (ESI) Calcd. for
þ
C
17
27
H N
2
O
3
(MþH) : 307.2016. Found: 307.2071.
4.2.2. 1,1,1,3,3,3-Hexafluoropropan-2-yl 4-(3-methoxybenzyl)
piperazine-1-carboxylate, MA-PB-1
4.4. Biological evaluation
To a solution of triphosgene (0.185 g, 0.624 mmol, 0.33 eq) in dry
DCM (5 mL) were added hexafluoroisopropanol (0.197 mL,
4.4.1. MAGL activity assay
1
3
.892 mmol, 1 eq) and diisopropylethyl amine (DIPEA, 0.346 mL,
.784 mmol, 2 eq) and the reaction mixture was stirred for 2 h at RT
A sensitive fluorescence-based hydrolase assay detecting for-
mation of glycerol was used to determine the rate of hMAGL-

112
M. Ahamed et al. / European Journal of Medicinal Chemistry 136 (2017) 104e113
catalyzed hydrolysis of the natural monoacylglycerol substrate 2-
AG as well as to determine the potency of MAGL inhibitors (IC50
values) in a 96-well format, essentially as previously described
3
and CH CN (45:55 v/v) at a flow rate of 0.7 mL/min. The HPLC eluate
was collected as 1-mL fractions after passing through a UV detector
(210 nm), and radioactivity in the fractions was measured using an
automated gamma counter.
[21,22]. The amount of hMAGL-lysate protein per well was 0.3 mg.
This methodology has been previously used allowing comparison
of potencies between various MAGL inhibitors, such as JJKK-048,
ML30, KML29 and IDFP, under identical experimental conditions
4
.4.5. In vivo microPET studies in rats
MicroPET imaging experiments were performed on a Focus™
20 microPET scanner (Concorde Microsystems, Knoxville, TN)
[20].
2
4.4.2. Biodistribution
using female Wistar rats. During the whole procedure, animals
were kept under gas anesthesia (2.5% isoflurane in O at 1 L/min
Biodistribution studies were carried out in healthy adult female
2
NMRI mice (body mass 29e50 g) at 2, 10, 30 and 60 min post in-
jection (p.i.) (n ¼ 4 per time point). Additional biodistribution
studies (30 and 60 min post tracer injection, n ¼ 4 per time point)
were performed 60 min after pretreatment with cold MA-PB-1
flow rate). The animal's head was fixed and positioned central in
the field of view of the microPET scanner. Upon tracer injection, a
dynamic microPET scan was started for 90 min. All scans were ac-
quired in list mode. Acquisition data were Fourier rebinned in 24
time frames (4 ꢂ 15 s, 4 ꢂ 60 s, 5 ꢂ 180 s, 8 ꢂ 5 min, 3 ꢂ 10 min) and
reconstructed using maximum a posteriori iterative reconstruction.
A summed image of the reconstructed data was spatially normal-
ized to an in-house created F-FDG template of the rat brain in
Paxinos coordinates [29] using an affine transformation. The latter
was then used to normalize all time frames of the dynamic
microPET data set to allow automated and bilateral volumes of
interest (VOIs) analyses. Time activity curves (TACs) were generated
for whole brains, using PMOD software (v 3.2, PMOD Technologies,
Zürich, Switzerland).
(
10 mg/kg; subcutaneous (s.c.) administration) or 30 min after
pretreatment with MJN110 (10 mg/kg; s.c. administration). MA-PB1
and MJN110 were dissolved in a vehicle containing 10% DMSO, 10%
2
-hydroxypropyl
concentration of 2 mg/mL. During the procedure, mice were
anesthetized with 2.5% isoflurane in O at a flow rate of 1 L/min and
b-cyclodextrin and 5% tween 80 in saline at a
18
2
injected with about 9 MBq of tracer via a tail vein. The animals were
then sacrificed by decapitation at above specified time points post
tracer injection. Blood and major organs were collected in tared
tubes and weighed. The radioactivity in blood, organs and other
body parts was counted using an automated gamma counter. For
calculation of the total radioactivity in blood, blood mass was
assumed to be 7% of the total body mass [28].
In the blocking microPET studies, rats (n ¼ 3) were anesthetized
2
with 2.5% isoflurane in O at 1 L/min flow rate and i.p. injected with
MJN110 (10 mg/kg) 30 min prior to tracer injection. For the chase
study (n ¼ 3), MJN110 (10 mg/kg) was i.v. administered 20 min after
tracer injection. MA-PB1 and MJN110 were dissolved in a vehicle
containing 10% DMSO, 10% 2-hydroxypropyl b-cyclodextrin and 5%
tween 80 in saline at a concentration of 2 mg/mL.
4.4.3. Plasma radiometabolite quantification in rats
Healthy Wistar rats (n ¼ 3) were anesthetized with isoflurane
(
2
2.5% in O at 1 L/min) and injected intravenously (i.v.) with about
5
5 MBq of the tracer via a tail vein. At 30 min p.i., the rats were
sacrificed by decapitation and blood was collected in tubes con-
taining 7.2 mg K EDTA (BD vacutainer, BD, Franklin Lakes, NJ) and
2
stored on ice. The blood was centrifuged for 10 min (2330 ꢂ g) to
separate plasma. To about 0.5 mL of plasma an equal amount of
acetonitrile was added and the solution was vortexed and centri-
fuged for 5 min (2330 ꢂ g) to separate the precipitated proteins
from the supernatant. Further, 0.5 mL of supernatant was taken and
diluted with 0.1 mL of water. It was filtered through a syringe filter
4.4.6. Non-human primate rhesus monkey microPET
Dynamic 90 min
with a Focus 220 PET scanner on a female rhesus monkey (Macaca
m
PET scans with [11C]MA-PB-1 were acquired
m
mulatta, 5.2 kg). Sedation was performed by i.m. injection of a
combination of 0.3 mL Rompun (xylazine 2% solution) and 0.35 mL
Nimatek (ketamine 100 mg/mL). About 60 min after the first in-
jection, the monkey received an additional dose of 0.15 mL Rompun
and 0.175 mL Nimatek via i.v. injection. During the last part of the
scan, dosing was done less frequently, based on the heartbeat fre-
(
0.22
with 20
injected onto an HPLC system consisting of an analytical XBridge
column (C18, 5
M, 3 mm ꢂ 100 mm, Waters) eluted with a mixture
of 0.05 M sodium acetate (pH 5.5) and CH CN (45:55 v/v) at a flow
m
m nylon filter, Acrodisc 13, PALL Life Sciences) and spiked
mg of authentic MA-PB-1. A volume of 0.5 mL of extract was
m
2 2
quency. The O and CO saturation in the blood and the heartbeat
3
were constantly monitored. Temperature was regulated via a
heating pad. The breathing frequency and the eye response were
checked visually. The head of the animal was placed central in the
rate of 0.7 mL/min. The HPLC eluate was collected as 1-mL fractions
after passing through a UV detector (210 nm), and radioactivity in
the fractions was measured using an automated gamma counter.
field of view of the
m
PET scanner. The monkey was injected with
11
163 MBq of [ C]MA-PB-1 via the vena saphena. Scans were ac-
4
.4.4. Perfused brain radiometabolite quantification in rats
Healthy Wistar rats (n ¼ 3) were anesthetized with isoflurane
quired in list mode and Fourier rebinned in 24 time frames
(4 ꢂ 15 s, 4 ꢂ 60 s, 5 ꢂ 180 s, 8 ꢂ 300 s, 3 ꢂ 600 s). Data were
reconstructed using a 3D maximum a posteriori (3D-MAP) iterative
reconstruction. TACs of the whole brain were generated using VOIs
with PMOD software and radioactivity concentration in the brain is
expressed as SUV as a function of time after tracer injection. For the
pre-treatment study, MJN110 was dissolved in a vehicle containing
11
(
2
2.5% in O at 1 L/min) and i.v. injected with about 37 MBq of [ C]
MA-PB-1. At 30 min p.i., they were sacrificed by an overdose of
pentobarbital (Nembutal, CEVA Sant eꢀ Animale, Libourne, France;
2
00 mg/kg intraperitoneal) followed by cardiac perfusion with sa-
line. Brain was isolated and homogenized/denatured in 3 mL of
CH
CN, for about 2 min. After centrifugation (1710 ꢂ g, 5 min),
about 1 mL supernatant was collected, diluted with 1 mL H O, and
filtered through a 0.22- m filter (Millipore, Bedford, MA). About
.5 mL of the filtrate was diluted with 0.1 mL of water and spiked
with 20 g of authentic MA-PB-1. A volume of 0.5 mL of this brain
homogenate extract was injected onto an HPLC system consisting
of an analytical XBridge column (C18, 5
M, 3 mm ꢂ 100 mm,
Waters) eluted with a mixture of 0.05 M sodium acetate (pH 5.5)
3
20% DMSO and 40% (2-hydroxypropyl)-
treatment was done by i.v. injection of MJN110 at a dose of 1 mg/
kg 10 min before tracer injection. PET images were compared to
b-cyclodextrin. Pre-
2
m
m
0
the baseline scan, acquired in the vehicle treated monkey. Blood
samples were collected during both baseline and pre-treatment
scans at 10, 30 and 60 min p.i. via the other vena saphena and
plasma was analysed for radiometabolites according to the same
procedure as described for rats (see above).
m
m

M. Ahamed et al. / European Journal of Medicinal Chemistry 136 (2017) 104e113
113
4
.5. Data and statistical analysis
[14] C. Costola-de-Souza, A. Ribeiro, V. Ferraz-de-Paula, A.S. Calefi, T.P. Aloia,
J.A. Gimenes-Junior, V.I. de Almeida, M.L. Pinheiro, J. Palermo-Neto, Mono-
acylglycerol lipase (MAGL) inhibition attenuates acute lung injury in mice,
PLoS One 8 (2013) e77706.
Quantitative data expressed as mean ± SD. Means were
compared using an unpaired two-tailed student t-test. Values were
considered statistically significant for P ꢃ 0.05.
[15] J.Z. Long, W. Li, L. Booker, J.J. Burston, S.G. Kinsey, J.E. Schlosburg, F.J. Pavon,
A.M. Serrano, D.E. Selley, L.H. Parsons, A.H. Lichtman, B.F. Cravatt, Selective
blockade of 2-arachidonoylglycerol hydrolysis produces cannabinoid behav-
ioral effects, Nat. Chem. Biol. 5 (2009) 37e44.
Funding
[16] J.W. Chang, M.J. Niphakis, K.M. Lum, A.B. Cognetta III, C. Wang, M.L. Matthews,
S. Niessen, M.W. Buczynski, L.H. Parsons, B.F. Cravatt, Remarkably selective
inhibitors of monoacylglycerol lipase bearing a reactive group that is bio-
isosteric with endocannabinoid substrates, Chem. Biol. 19 (2012) 579e588.
[17] L. Morera, G. Labar, G. Ortar, D.M. Lambert, Development and characterization
This work was supported by IMIR (In Vivo Molecular Imaging
Research group) at KU Leuven, Belgium also funded by the Euro-
pean Union's Seventh Framework Programme (FP7/2007-2013)
under grant agreement no. HEALTH-F2-2011-278850 (INMiND)
of endocannabinoid hydrolases FAAH and MAGL inhibitors bearing
benzotriazol-1-yl carboxamide scaffold, Bioorg. Med. Chem. 20 (2012)
260e6275.
a
6
[
18] G. Hernandez-Torres, M. Cipriano, E. Hed eꢀ n, E. Bj o€ rklund, A. Canales, D. Zian,
A. Feliu, M. Mecha, C. Guaza, C.J. Fowler, S. Ortega-Gutierrez, M.L. Lopez-
Rodriguez, A reversible and selective inhibitor of monoacylglycerol lipase
ameliorates multiple Sclerosis, Angew. Chem. Int. Ed. Engl. 53 (2014)
13765e13770.
[19] J.Z. Patel, S. Ahenkorah, M. Vaara, M. Staszewski, Y. Adams, T. Laitinen,
D. Navia-Paldanius, T. Parkkari, J.R. Savinainen, K. Walczynski, J.T. Laitinen,
T.J. Nevalainen, Loratadine analogues as MAGL inhibitors, Bioorg Med. Chem.
Lett. 25 (2015) 1436e1442.
[20] N. Aaltonen, J.R. Savinainen, C.R. Ribas, J. Ronkko, A. Kuusisto, J. Korhonen,
D. Navia-Paldanius, J. Hayrinen, P. Takabe, H. Kasnanen, T. Pantsar, T. Laitinen,
M. Lehtonen, S. Pasonen-Seppanen, A. Poso, T. Nevalainen, J.T. Laitinen,
Piperazine and piperidine triazole ureas as Ultrapotent and highly selective
inhibitors of monoacylglycerol lipase, Chem. Biol. 20 (2013) 379e590.
21] J.R. Savinainen, E. Kansanen, T. Pantsar, D. Navia-Paldanius, T. Parkkari,
M. Lehtonen, T. Laitinen, T. Nevalainen, A. Poso, A.L. Levonen, J.T. Laitinen,
Robust hydrolysis of prostaglandin glycerol esters by human mono-
acylglycerol lipase (MAGL), Mol. Pharmacol. 86 (2014) 522e535.
Acknowledgements
We thank Dr. Jacqueline Blankman (Abide Therapeutics, USA)
for the useful discussions. We also thank Julie Cornelis (Laboratory
for Radiopharmacy; KU Leuven) and Ann Van Santvoort (Depart-
ment of Nuclear Medicine; KU leuven) for their help with the mice
and rat animal studies, Professor Wim Vanduffel and Christophe
Ulens form the Laboratory of Neuro-and Psychophysiology for
providing the Macaque monkey and assisting in the monkey
studies.
[
Appendix A. Supplementary data
[
[
[
22] D. Navia-Paldanius, J.R. Savinainen, J.T. Laitinen, Biochemical and pharmaco-
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2017.04.066.
logical characterization of human a/b hydrolase domain containing 6 (ABHD6)
and 12 (ABHD12), J. Lipid Res. 53 (2012) 2413e2424.
23] C. Wang, M.S. Placzek, G.C. Van de Bittner, F.A. Schroeder, J.M. Hooker, A novel
radiotracer for imaging Monoacylglycerol lipase in the brain using positron
emission tomography, ACS Chem. Neurosci. 7 (2016) 484e489.
References
24] L. Wang, W. Mori, R. Cheng, J. Yui, A. Hatori, L. Ma, Y. Zhang, B.H. Rotstein,
M. Fujinaga, Y. Shimoda, T. Yamasaki, L. Xie, Y. Nagai, T. Minamimoto,
M. Higuchi, N. Vasdev, M.R. Zhang, S.H. Liang, Synthesis and preclinical
[1] L.A. Matsuda, S.J. Lolait, M.J. Brownstein, A.C. Young, T.I. Bonner, Structure of a
cannabinoid receptor and function expression of the cloned cDNA, Nature 346
(
1990) 561e564.
11
evaluation of Sulfonamido-based [ C-Carbonyl]-Carbamates and ureas for
[
2] N. Aaltonen, C.S. Ribas, M. Lehtonen, J.R. Savinainen, J.T. Laitinen, Brain
regional cannabinoid CB1 receptor signalling and alternative enzymatic
pathways for 2-arachidonoylglycerol generation in brain sections of diac-
ylglycerol lipase deficient mice, Eur. J. Pharm. Sci. 51 (2014) 87e95.
3] V. Di Marzo, D. Melck, T. Bisogno, L. De Petrocellis, Endocannabinoids:
endogenous cannabinoid receptor ligands with neuromodulatory action,
Trends Neurosci. 21 (1999) 521e528.
imaging monoacylglycerol lipase, Theranostics 6 (8) (2016) 1145e1159.
25] M.J. Niphakis, A.B. Cognetta, J.W. Chang, M.W. Buczynski, L.H. Parsons,
F. Byrne, J.J. Burston, V. Chapman, B.F. Cravatt, Evaluation of NHS carbamates
as a potent and selective class of endocannabinoid hydrolase inhibitors, ACS
Chem. Neurosci. 4 (2013) 1322e1332.
[
[
[
26] J.W. Hicks, J. Parkesa, J. Tonga, S. Houlea, N. Vasdev, A.A. Wilson, Radiosyn-
11
thesis and ex vivo evaluation of [ C-carbonyl]carbamate- and urea-based
[
[
[
4] P. Pacher, S. Batkai, G. Kunos, The endocannabinoid system as an emerging
monoacylglycerol lipase inhibitors, Nucl. Med. Biol. 41 (2014) 688e694.
target of pharmacotherapy, Pharmacol. Rev. 58 (2006) 389e462.
5] B.F. Cravatt, A.H. Lichtman, The endogenous cannabinoid system and its role
in nociceptive behavior, J. Neurobio 61 (2004) 149e160.
6] K. Ahn, M.K. McKinney, B.F. Cravatt, Enzymatic pathways that regulate
endocannabinoid signaling in the nervous system, Chem. Rev. 108 (2008)
[
27] J.I. Andr eꢀ s, M. De Angelis, J. Alc aꢀ zar, L. Iturrino, X. Langlois, S. Dedeurwaerdere,
I. Lenaerts, G. Vanhoff, S. Celen, G. Bormans, Synthesis, in vivo occupancy, and
radiolabeling of potent Phosphodiesterase subtype-10 inhibitors as candi-
dates for positron emission tomography imaging, J. Med. Chem. 54 (2011)
5820e5835.
1687e1707.
[
[
[
[
28] A.R. Fritzberg, W.P. Whitney, C.C. Kuni, W. Klingensmith, Biodistribution and
renal excretion of 99mTc-N, N’-bis-(Mercaptoacetamido) ethylenediamine.
Effect of renal tubular transport inhibitors, Int. J. Nucl. Med. Biol. 9 (1982)
[
[
[
7] J.R. Savinainen, S.M. Saario, J.T. Laitinen, The serine hydrolases MAGL, ABHD6
and ABHD12 as guardians of 2-arachidonoylglycerol signaling through
cannabinoid receptors, Acta Physiol. (Oxf.) 204 (2012) 267e276.
8] J.L. Blankman, G.M. Simon, B.F. Cravatt, A comprehensive profile of brain en-
zymes that hydrolyze the endocannabinoid 2-arachidonoylglycerol, Chem.
Biol. 14 (2007) 1347e1356.
9] R.A. Kohnz, D.K. Nomura, Chemical approaches to therapeutically target the
metabolism and signaling of the endocannabinoid 2-AG and eicosanoids,
Chem. Soc. Rev. 43 (2014) 6859e6869.
79e82.
29] C. Casteels, P. Vermaelen, J. Nuyts, A. Van Der Linden, V. Baekelandt,
L. Mortelmans, G. Bormans, K. Van Laere, Construction and evaluation of
multitracer small-animal PET probabilistic atlases for voxel-based functional
mapping of the rat brain, J. Nucl. Med. 47 (2006) 1858e1866.
30] W.C. Eckelman, R.E. Gibson, W.J. Rzeszotarski, F. Vieras, J.K. Mazaitis,
B. Francis, B.,W.C. Reba, The design of receptor binding radiotracers, in:
L. Colombetti (Ed.), Principles of Radiopharmacology vol. 1, CRC Press, New
York, 1979, pp. 251e274.
[
[
10] M.M. Mulvihill, D.K. Nomura, Therapeutic potential of monoacylglycerol
lipase inhibitors, Life Sci. 92 (2013) 492e497.
11] J.K. Makara, M. Mor, D. Fegley, S.I. Szabo, S. Kathuria, G. Astarita, A. Duranti,
A. Tontini, G. Tarzia, S. Rivara, T.F. Freund, D. Piomelli, Selective inhibition of
31] R.N. Waterhouse, Determination of lipophilicity and its use as a predictor of
blood-brain barrier penetration of molecular imaging agents, Mol. Imaging
Biol. 5 (2003) 376e389.
2-AG hydrolysis enhances endocannabinoid signaling in hippocampus, Nat.
Neurosci. 8 (2005) 1139e1141.
[
[
[
32] V.W. Pike, PET Radiotracers: crossing the blood-brain barrier and surviving
metabolism, Trends Pharmacol. Sci. 30 (2009) 431e440.
33] C.J. Fielding, Monoglyceride hydrolase activities of rat plasma and platelets,
J. Biol. Chem. 256 (1981) 876e881.
34] M. Karlsson, J.A. Contreras, U. Hellman, H. Tornqvist, C. Holm, cDNA cloning,
tissue distribution, and identification of the catalytic triad of monoglyceride
lipase, J. Biol. Chem. 272 (1997) 27218e27223.
[
12] D.K. Nomura, B.E. Morrison, J.L. Blankman, J.Z. Long, S.G. Kinsey,
M.C.G. Marcondes, A.M. Ward, Y.K. Hahn, A.H. Lichtman, B. Conti, B.F. Cravatt,
Endocannabinoid hydrolysis generates brain prostaglandins that promote
neuroinflammation, Science 334 (2011) 809e813.
[
13] Z. Cao, M.M. Mulvihill, P. Mukhopadhyay, H. Xu, K. Erdelyi, E. Hao, E. Holovac,
G. Hasko, B.F. Cravatt, D.K. Nomura, P. Pacher, Monoacylglycerol lipase con-
trols endocannabinoid and eicosanoid signaling and hepatic injury in mice,
Gastroenterology 144 (2013) 808e817.