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Article
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Design, Synthesis, and Evaluation of Reversible and Irreversible
Monoacylglycerol Lipase Positron Emission Tomography (PET)
Tracers Using a “Tail Switching” Strategy on a Piperazinyl Azetidine
Skeleton
Zhen Chen,†,‡,○ Wakana Mori,§,○ Xiaoyun Deng,† Ran Cheng,† Daisuke Ogasawara,∥ Genwei Zhang,⊥
Michael A. Schafroth,∥ Kenneth Dahl,† Hualong Fu,† Akiko Hatori,§ Tuo Shao,† Yiding Zhang,§
Tomoteru Yamasaki,§ Xiaofei Zhang,† Jian Rong,† Qingzhen Yu,† Kuan Hu,§ Masayuki Fujinaga,§
Lin Xie,§ Katsushi Kumata,§ Yuancheng Gou,# Jingjin Chen,# Shuyin Gu,# Liang Bao,# Lu Wang,†
Thomas Lee Collier,† Neil Vasdev,† Yihan Shao,⊥ Jun-An Ma,‡ Benjamin F. Cravatt,∥
Christopher Fowler,∇ Lee Josephson,† Ming-Rong Zhang,*,§ and Steven H. Liang*,†
†
Division of Nuclear Medicine and Molecular Imaging, Massachusetts General Hospital & Department of Radiology, Harvard
Medical School, Boston, Massachusetts 02114, United States
‡
Department of Chemistry, School of Science, Tianjin University, 92 Weijin Road, Nankai District, Tianjin 300072, China
§
Department of Radiopharmaceuticals Development, National Institute of Radiological Sciences, National Institutes for Quantum
and Radiological Science and Technology, Chiba 263-8555, Japan
∥
The Skaggs Institute for Chemical Biology and Department of Chemical Physiology, The Scripps Research Institute, SR107 10550
North Torrey Pines Road, La Jolla, California 92037, United States
⊥
Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019, United States
#
ChemShuttle, Inc., 1699 Huishan Blvd., Wuxi, Jiangsu 214174, China
∇
Department of Pharmacology and Clinical Neuroscience, Umeå University, SE-901 87 Umeå, Sweden
S Supporting Information
*
ABSTRACT: Monoacylglycerol lipase (MAGL) is a serine
hydrolase that degrades 2-arachidonoylglycerol (2-AG) in the
endocannabinoid system (eCB). Selective inhibition of MAGL
has emerged as a potential therapeutic approach for the
treatment of diverse pathological conditions, including chronic
pain, inflammation, cancer, and neurodegeneration. Herein, we
disclose a novel array of reversible and irreversible MAGL
inhibitors by means of “tail switching” on a piperazinyl
azetidine scaffold. We developed a lead irreversible-binding
MAGL inhibitor 8 and reversible-binding compounds 17 and 37, which are amenable for radiolabeling with 11C or 18F. [11C]8
([11C]MAGL-2-11) exhibited high brain uptake and excellent binding specificity in the brain toward MAGL. Reversible
radioligands [11C]17 ([11C]PAD) and [18F]37 ([18F]MAGL-4-11) also demonstrated excellent in vivo binding specificity
toward MAGL in peripheral organs. This work may pave the way for the development of MAGL-targeted positron emission
tomography tracers with tunability in reversible and irreversible binding mechanisms.
■
INTRODUCTION
As a lipid signaling network, membrane-bound G-coupled
cannabinoid receptors, namely, CB1 and CB2, and their
endogenous ligands, 2-arachidonoylglycerol (2-AG) and Narachidonoylethanolamine (AEA) established the backbone of
endocannabinoid (eCB) system.1−5 The eCB system is
prominent in both central and peripheral nervous systems,
and its dysfunction has been implicated in a wide range of
pathological conditions, including pain, appetite, inflammation,
memory and cognition, and cancer.6−10 Since direct regulation
of CB1 receptors is often accompanied with a series of
© 2019 American Chemical Society
debilitating adverse effects, such as substance abuse and loss of
motor and cognition functions,11,12 recent drug discovery
efforts have been shifted to regulating the levels of AEA or 2AG. As the most essential endogenous ligands with
endocannabinoid-like activity, AEA and 2-AG are synthesized
“on request” in vivo and in the brain primarily degraded by
fatty acid amide hydrolase (FAAH) and monoacylglycerol
lipase (MAGL), respectively.13−16 In particular, MAGL
Received: November 14, 2018
Published: March 4, 2019
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DOI: 10.1021/acs.jmedchem.8b01778
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Journal of Medicinal Chemistry
Article
Figure 1. Representative PET tracers for imaging brain MAGL from previous work and our work.
tracers was limited to irreversible and no reversible MAGL
PET ligand has been reported to date. In fact, a reversible
MAGL tracer would enable the access to critical quantitative
kinetic analysis, including facilitated measures of binding
potential and volume of distribution, for monitoring neurological response to therapeutics.44,45 As a result, there is a
critical demand for the development of both irreversible and
reversible MAGL PET tracers with favorable lipophilicity and
brain kinetics.
As part of our continuing interest in the development and
translation of novel MAGL PET tracers,37,39,41 herein we
described a novel class of MAGL inhibitors using a “tail
switching” strategy,46,47 wherein the “tail” refers to the group
that is attached to the unique piperazinyl azetidine skeleton
(Figure 1B).48−50 In detail, our medicinal chemistry efforts
focused on the synthesis of an array of (4-(azetidin-3yl)piperazin-1-yl)(thiazol-2-yl)methanone-derived carbamates
or ureas as irreversible candidate MAGL inhibitors and (4(azetidin-3-yl)piperazin-1-yl)(thiazol-2-yl)methanone-derived
amides as reversible candidates, with amenability for radiolabeling with carbon-11 or fluorine-18. Pharmacological
studies, molecule docking, and physicochemical evaluations
were performed to identify our compound 8 as the most
promising irreversible MAGL inhibitor and compounds 17 and
37 as the most promising reversible MAGL inhibitors, worthy
of radiolabeling and in vivo PET translational studies. With
innovative and efficient 11C- and 18F-labeling strategies, we
evaluated the brain permeability, binding specificity, and
kinetics of these lead radioligands 48 ([11C]8), 49 ([11C]
17), and 50 ([11C]37) by PET experiments in rodents. While
irreversible MAGL tracer 48 demonstrated excellent in vitro
potency and selectivity, in vivo binding specificity, and stability
in the brain, our reversible MAGL tracers 49 and 50
demonstrated high-level specific binding to MAGL in a
peripherally restricted manner. As a proof of concept, we
were able to unveil the underlying cause of low brain
accumulation for 49 and 50, the most potent reversible
MAGL tracers in our design, thus paving the way for future
development of reversible MAGL PET tracers.
belongs to the serine hydrolase superfamily, which is associated
with the eCB system as well as eicosanoid and other lipidsignaling pathways.17 In rodents, MAGL is highly expressed in
the central nervous system (CNS) as well as several peripheral
organs, including liver, kidneys, adrenal glands, and brown
adipose tissue.18 In humans, there is a similar CNS MAGL
distribution to that in rodents with high levels of activity in the
cerebral cortex, hippocampus, and cerebellum, and low levels
in the hypothalamus and pons.19 Considering its prime role in
2-AG hydrolysis in the brain, selective inhibition of MAGL
may represent an alternative and potential therapeutic target
for treatment of diverse pathological conditions, including
chronic pain, inflammation, cancer, and neurodegeneration,
without apparent side effects related with direct CB 1
regulation.20−31
Positron emission tomography (PET) is a noninvasive and
highly sensitive technology in the realm of molecular imaging
and serves as an ideal tool to quantify biochemical and
pharmacological processes in vivo under normal and disease
conditions.32−34 PET studies of MAGL would allow us to
achieve in-depth knowledge of MAGL-related pathological
changes between normal and disease states and in vivo
interaction of novel MAGL inhibitors with the target.
Development of MAGL PET tracers would remarkably help
to validate promising MAGL inhibitors in clinical trials. As a
result, in the past few years, considerable efforts have been
exerted toward this goal but with limited success. The first
attempt for PET imaging of MAGL was performed by Hicks et
al. with several carbon-11-labeled MAGL inhibitors, including
[11C]KML29 and [11C]JJKK-0048. However, all of these
compounds had limited brain uptake, which impeded their
further translation.35 To date, only three potent MAGL PET
tracers,36 namely, [11C]SAR12730337−39 and [11C]MA-PB-1,40
based on a piperidyl carbamate scaffold, and [11C]MAGL051941 based on an azetidinyl oxadiazole scaffold, have been
developed to image MAGL in living brains of rats and
nonhuman primates (NHPs) (Figure 1A). However, most
reported MAGL PET tracers are highly lipophilic (c Log P ca.
3−5), which is often linked with fast metabolic clearance, poor
in vivo stability, and high propensity for off-target promiscuity.42,43 For example, the 2,5-regioisomer of LY2183240
exhibited poor selectivity between MAGL and FAAH, which
could be, to some extent, attributed to a high c Log P value of
4.03.43 Furthermore, the binding mechanism of these PET
■
RESULTS AND DISCUSSION
Chemistry. A focused library of (4-(azetidin-3-yl)piperazin1-yl)(thiazol-2-yl)methanone-derived carbamates or ureas 8−
13 as irreversible MAGL inhibitor candidates and (4-(azetidin-
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Article
Scheme 1. Synthesis of Irreversible MAGL Inhibitors 8−13a
a
Conditions: (i) DIPEA, MeCN, 80 °C for 12 h; 85% yield; (ii) TFA, CH2Cl2, rt, 12 h; 99% yield; (iii) HOBT, EDC·HCl, Et3N, DMF, rt, 12 h;
78% yield; (iv) 1-chloroethyl chloroformate, CH2Cl2, rt, 2 h; then MeOH, 35 °C, 2 h; 86% yield; (v) 1,1,1,3,3,3-hexafluoro-2-propanol, 4nitrophenyl chloroformate, DMAP, pyridine, Et3N, CH2Cl2, rt, 5 h; 20% yield for 8; (vi) 2,2,2-trifluoroethanol (for 9) or 1,2,4-triazole (for 10) or
1H-benzo[d][1,2,3]triazole (for 11) or 2-hydroxyisoindoline-1,3-dione (for 12), triphosgene, DMAP, Et3N, CH2Cl2, rt, 4 h; 43% yield for 9; 28%
yield for 10; 7% yield for 11; 29% yield for 12; (vii) N,N′-disuccinimidyl carbonate, Et3N, CH2Cl2, rt, overnight; 14% yield for 13; DIPEA = N,Ndiisopropylethylamine; TFA = trifluoroacetic acid; HOBT = 1-hydroxybenzotriazole hydrate; EDC·HCl = N-(3-dimethylaminopropyl)-N′ethylcarbodiimide hydrochloride; Et3N = triethylamine; DMF = N,N-dimethylformamide; DMAP = 4-dimethylaminopyridine.
yield over two steps. For Suzuki-type reactions with phenyl
boronic acids, Pd(PPh3) proved to be the optimal catalyst
whereas in the case of heteroaryl boronic acids as coupling
partners, PdCl2(dppf) was utilized to provide good yields.
Subsequent coupling reactions of 15 with piperazinyl azetidine
7 in the presence of HOBT and EDC·HCl proceeded
smoothly to give amide-type MAGL inhibitors 16−22 in
21−40% yields. For the synthesis of 36−38, ethyl 4fluorobenzoate was utilized as the starting material (Scheme
2B). Amination reactions with hydroxyl azetidine, hydroxyl
pyrazole, or hydroxyl piperidine occurred smoothly under basic
conditions, thus delivering 24−26 in moderate-to-high
efficiencies (38−82% yields). Activation of the hydroxyl
group by treatment with methane sulfonyl chloride afforded
the mesylate compounds 27−29 in excellent yields (80−84%),
which were readily fluorinated by tetrabutylammonium
fluoride (TBAF) to generate 30−32 in 23−38% yields. The
poor yields observed in the fluorination reactions can likely be
attributed to the propensity of β-elimination of methanesulfonate derivatives 27−29 under basic conditions. Ultimately,
candidates 36−38 were obtained in 18−28% yield over two
steps, namely, LiOH-promoted hydrolysis of 30−32 followed
by condensation with piperazinyl azetidine 7.
Pharmacology. Compounds 8−13, 16−22, and 36−38
were investigated for their potency and selectivity toward
MAGL in vitro. For irreversible candidates 8−13, we
determined their in vitro ability to inhibit MAGL hydrolysis
of [3 H]2-oleoylglycerol ([ 3H]2-OG), a tritiated 2-AG
analogue, according to our previously reported protocol.51 As
outlined in Table 1, Figure 2A, and Figure S1, candidate 8
containing a hexafluoroisopropanol leaving group demonstrated the most promising potency toward inhibition of MAGL
3-yl)piperazin-1-yl)(thiazol-2-yl)methanone-derived amides
16−22 and 36−38 as reversible MAGL inhibitor candidates
were synthesized, the scaffolds of which are amenable for 11Cor 18F-labeling. As summarized in Scheme 1, the SN2
displacement reaction between tert-butyloxycarbonyl (Boc)protected piperazine 1 and 1-benzhydrylazetidin-3-yl methanesulfonate 2 readily proceeded to give 3 in 85% yield.
Trifluoroacetic acid (TFA)-induced deprotection of the Boc
group from 3 led to isolation of 4 in nearly quantitative yield,
which subsequently coupled with thiazole-2-carboxylic acid 5
to produce 6 in high efficiency in the presence of 1hydroxybenzotriazole hydrate (HOBT) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCl).
1-Chloroethyl chloroformate-triggered deprotection of the
diphenylmethyl group from 6 released azetidine 7 in 86%
yield, which served as a crucial precursor for the following 11Clabeling. To synthesize irreversible MAGL inhibitor candidates
8−13, we deployed several parallel approaches for the
introduction of different carbonyl-R groups. The combination
of 1,1,1,3,3,3-hexafluoro-2-propanol, 4-nitrophenyl chloroformate, 4-dimethylaminopyridine (DMAP), pyridine, and Et3N
in CH2Cl2 proved optimal to generate 8 in 20% yield. For
candidates 9−12, triphosgene was found to be a superior
activating reagent, thus producing the corresponding carbamates 9 and 12, as well as triazolyl carbonyls 10 and 11 in 7−
43% yields. Candidate 13 was isolated in 13% yield by the
treatment of azetidine 7 with N,N′-disuccinimidyl carbonate.
To synthesize reversible candidate MAGL inhibitors 16−22
and 36−38, methyl 4-bromo-3-methoxybenzoate was used as
the starting material (Scheme 2A). Cross-coupling reactions
with aryl boronic acid followed by LiOH-mediated hydrolysis
readily provided [1,1′-biaryl]-4-carboxylic acid 15 in 32−89%
3338
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Scheme 2. Synthesis of Reversible MAGL Inhibitors 16−22 and 36−38a
Conditions: (i) Pd(PPh3)4, K2CO3, toluene/H2O, 100 °C, overnight; (ii) PdCl2(dppf), K2CO3, 1,4-dioxane/H2O, 105 °C, overnight; (iii) LiOH,
THF/H2O, rt, overnight; (iv) HOBT, EDC·HCl, Et3N, DMF, rt, 12 h; 22% yield for 16; 25% yield for 17; 21% yield for 18; 23% yield for 19; 24%
yield for 20; 39% yield for 21; 40% yield for 22; 32% yield for 36; 30% yield for 37; 19% yield for 38; (v) azetidin-3-ol hydrochloride (for 24) or
pyrrolidin-3-ol hydrochloride (for 25) or piperidin-4-ol (for 26), K2CO3, DMSO, 180 °C for 2 h (for 24) or 120 °C for 24 h (for 25 and 26); 38%
yield for 24; 79% yield for 25; 82% yield for 26; (vi) MsCl, Et3N, CH2Cl2, rt, overnight; 81% yield for 27; 84% yield for 28; 80% yield for 29; (vii)
TBAF, THF, 70 °C, 2 h; 23% yield for 30; 38% yield for 31; 26% yield for 32; (viii) LiOH, THF/MeOH/H2O, 40 °C, 16 h; 73% yield for 33; 92%
yield for 34; 95% yield for 35; dppf = 1,1′-bis(diphenylphosphino)ferrocene; HOBT = 1-hydroxybenzotriazole hydrate; EDC·HCl = N-(3dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride; DMF = N,N-dimethylformamide; DMSO = methyl sulfoxide; MsCl =
methanesulfonyl chloride; TBAF = tetrabutylammonium fluoride.
a
Table 1. IC50 Values of Compounds 8−13 for Inhibition of MAGL Activity with MAGL-0519, a Known Irreversible MAGL
Inhibitor, as Reference
entry
8
9
10
11
12
13
MAGL-0519
IC50 (nM)
0.88 ± 0.05
>1000
10.0 ± 4.2
87.1 ± 12.4
>1000
27.5 ± 8.1
8.4 ± 0.2
tration of 1 μM utilizing two different assays: (1) activity-based
protein profiling (ABPP) studies and (2) hydrolysis of [3H]2OG. As depicted in Figures 3A and S2, both methods
demonstrated compounds 17, 37, and 38 as the most potent
MAGL inhibitors. The IC50 values of candidates 17, 37, and 38
were further determined as 2.7, 11.7, and 15.0 nM,
respectively, via ABPP assays (Figure 3B). As a proof of
concept, reversibility of the binding mechanism for candidates
17 and 37 was further investigated. In the case of 17, we
utilized an ABPP assay (Figure 3C). In this case, enzyme
activity was measured by treatment with FP-rhodamine. For a
reversible inhibitor, the compound would compete with FPrhodamine and each dissociation from the enzyme would
present a new target for FP-rhodamine. Therefore, for
reversible inhibitors, the levels of MAGL labeled with FPrhodamine would increase over time. This was shown to be the
case for 17 but not for the irreversible inhibitor MJN110. In
activity with an IC50 value of 0.88 nM, whereas all of the other
candidates 9−13 indicated inferior potency (Figure S1). The
residual activity seen in Figure 2B reflects the fact that whereas
MAGL is the predominant hydrolytic enzyme in the brain, it is
not the only one: Blankman et al.15 reported that MAGL was
responsible for ∼85% of the hydrolysis of 2-AG in the brain,
and the present data is consistent with this report. Inhibitor 8
exhibited preincubation-time-dependent inhibition at three
tested concentrations (0.3, 1, and 3 nM, Figure 2B), which is
in line with the irreversible binding mechanism. Although
compound 10 possessing a triazole leaving group also exhibited
good potency (IC50 = 10 nM), no further evaluation was
conducted considering the poor blood-brain barrier (BBB)
penetration ability for an irreversible MAGL PET tracer with a
triazole leaving group.39
For reversible candidates 16−22 and 36−38, we initially
evaluated their potency toward MAGL at a single concen3339
DOI: 10.1021/acs.jmedchem.8b01778
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Article
inhibition of the 2-OG hydrolytic activity of the samples,
which would have been expected for an irreversible inhibitor
(Figure 3D). Jump dilution experiments, whereby samples are
preincubated with inhibitor and then diluted prior to addition
of a substrate suggested no recovery of inhibition following a
20-fold dilution (Figure 3E). However, this behavior can be
found under certain conditions (Kiapp/[E] ≥ 10, short
incubation times with the substrate) for tight-binding
reversible inhibitors when the high potency is due to longer
residence times, i.e., slower rates of dissociation.52 On the basis
of these preliminary results, we selected 17 for 11C-labeling and
37 for 18F-labeling, as lead reversible inhibitors for further in
vitro and in vivo evaluation.
As shown in Table 2, the selectivity of these three lead
compounds 8, 17, and 37 toward MAGL over FAAH was
further investigated in ABPP assays and none of them showed
significant inhibition toward FAAH at a concentration up to 10
μM. In addition, for all three lead compounds, no significant
interactions were observed with CB1 and CB2 receptors (up to
a concentration of 30 μM, Figure 4) as well as ABHD6 and
ABHD12 (up to a concentration of 10 μM), both of which also
belong to the serine hydrolase superfamily and play a vital role
in the metabolism of 2-AG in the brain.
Lipophilicity of candidate compounds is an essential factor
for the prediction of BBB permeability with 1.0−3.5 as the
favorable range.53−55 By means of the “shake flask method”,
namely, liquid−liquid partition between PBS and n-octanol,56
the LogD values of 8, 17, and 37 were 1.90 ± 0.38, 3.35 ±
0.50, and 2.70 ± 0.04, respectively (n = 3) (Table 1). The
topological polar surface areas (tPSA) for these compounds
were determined by in silico prediction with ChemBioDraw
Figure 2. (A) Concentration−response curves of 8 for inhibition of
[3H]2-OG hydrolysis by rat brain MAGL (1 h preincubation between
the enzyme source and inhibitor prior to addition of a substrate); (B)
Time-dependent inhibition of [3H]2-OG hydrolysis at three different
concentrations (0.3, 1, and 3 nM).
the case of 37, we investigated the mode of inhibition using
[3H]2-OG as substrate (Figure 3D,E). There was no
preincubation time-dependent increase in the observed
Figure 3. Inhibition properties of MAGL activity by compounds 18−22 and 36−38. (A) Mouse brain MAGL activity in the presence of
compounds 16−22 and 36−38 at 1 μM concentration determined by ABPP assay; (B) concentration−response curves of 17 and 37−38 for
inhibition of mouse brain MAGL activity determined by ABPP assay with pristimerin, a known reversible MAGL inhibitor, as reference (Figure S3
in the Supporting Information); (C) MAGL activity determined at different incubation time points with 17 and the irreversible inhibitor MJN110
as control. Recovered MAGL activity over time indicates the binding of compound 17 is reversible, whereas the irreversible inhibitor MJN110
shows no significant time-dependent changes. (D) Time-dependent inhibition of [3H]2-OG hydrolysis at three different concentrations of
compound 37 (30, 100, and 300 nM). (E) Inhibition of 0.5 μM [3H]2-OG hydrolysis by compound 37 without preincubation (red and black bars)
or with 60 min of preincubation followed by dilution to reduce the free concentration by 20-fold (gray bar). Given that this compound showed no
time dependence toward inhibition of [3H]2-OG hydrolysis (data not shown), the data are consistent with a tight-binding but reversible inhibitor.
All data are mean ± SD, n = 3−5. *p < 0.05, **p ≤ 0.01, and ***p ≤ 0.001. 3340 DOI: 10.1021/acs.jmedchem.8b01778 J. Med. Chem. 2019, 62, 3336−3353 Journal of Medicinal Chemistry Article Table 2. Binding Potency and Physicochemical Properties of Compounds 8, 17, and 37a,b,c,d a Determined by ABPP assay on a rat brain membrane (MJN110 as positive control). bDetermined by CB1/CB2 agonist and antagonist assays (CP55940, rimonabant, and SR144528 as positive control in CB1/CB2 agonist assays, CB1 antagonist assay, and CB2 antagonist assay, respectively). c Measured by “shake-flask” method quantified by LC-MS/MS. dTopological polar surface area (tPSA) was calculated by ChemBioDraw Ultra 14.0. Figure 4. Pharmacological interactions of compounds 8, 17, and 37 with CB1/CB2 receptors. (A) Agonism of 8, 17, and 37 in the CB1 assay with CP55940 as control; (B) antagonism of 8, 17, and 37 in the CB1 assay with rimonabant as control; (C) agonism of 8, 17, and 37 in the CB2 assay with CP55940 as control and (D) antagonism of 8, 17, and 37 in the CB2 assay with SR144528 as control. All data are mean ± SD, n = 3−5. Figure 5. Molecular docking structures of compounds 8 (yellow in A; precovalent docking state), 17 (blue in B), and 37 (magenta in C) onto MAGL. The catalytic triad was labeled as Asp239, His269, and Ser122. Hydrogen bonds between compounds and the MAGL protein were connected with solid lines. The bottom-right insets at each panel showed the docking pose of each compound into (or at the opening end of) the 2-AG binding pocket. The PDB ID of the protein structure is 3PE6. Ultra 14.0, and all of them exhibited reasonable tPSA (Table 2), which, together with the lipophilicity results, indicated a high possibility of sufficient brain permeability. Molecular Docking Studies. On the basis of the fact of excellent binding affinities for lead compounds 8, 17, and 37 (0.88, 2.7, 11.7 nM, respectively), we then performed molecular docking studies with the purpose of identifying their possible interactions with MAGL and the corresponding binding domain (Figure 5). A 1.35 Å resolution crystal structure of a soluble form of human monoglyceride lipase (MGLL) was downloaded (PDB ID: 3PE6), and its original ligand57 was removed before performing the molecular docking studies. First, we reproduced the binding pose of the original ligand within 3PE6 using Autodock Vina, which yielded an excellent overlapping between the docked and original poses (Figure S4, Supporting Information). Subsequently, selected MAGL inhibitors 8, 17, and 37 were docked onto the 3PE6 structure (Figure 5 and Figure S5, Supporting Information). As shown in Figure 5A,B, both compounds 8 and 17 entered the 2-AG binding pocket and were located near the catalytic triad site, formed by Ser122, His269, and Asp239. Since the catalytic triad is essential for catalyzing 2-AG hydrolysis, compounds 8 and 17 reduced the accessibility of the active site to 2-AG, therefore inhibiting the 2-AG hydrolysis. Meanwhile, compound 37 was only partially docked into the 2-AG binding pocket, which blocked the “lid” of the pocket and prevented the 2-AG substrate from accessing the catalytic site (Figure 5C). It is also worth mentioning that although the Vina docking studies suggested that the docked pose of 37 did not bind to the oxyanionic hole or 37 was situated in a different binding pocket, more in-depth computational studies, including molecular dynamic simulation, are necessary to verify this hypothesis. Inside the binding pocket, we found π−π stacking interactions and hydrogen bonds. The π−π stackings were observed between the phenyl group of compounds 17, 37, and nearby Phe159 side chains (Figure 5B,C), and the hydrogen bonds were found between compound 8 and Ala51 (Figure 5A) and also between compound 17 and nearby Ala51 and Met123 (Figure 5B). Overall, these docking results provided supportive evidence for the inhibitory mechanism of aforementioned compounds. Radiochemistry. The encouraging results of pharmacology and physiochemical property and docking studies prompted us to perform radiolabeling of 8, 17, and 37 with carbon-11 or fluorine-18. In the case of 37, we used its racemic form for 3341 DOI: 10.1021/acs.jmedchem.8b01778 J. Med. Chem. 2019, 62, 3336−3353 Journal of Medicinal Chemistry Article Scheme 3. Synthesis of Labeling Precursors 44 and 47 for MAGL PET Tracersa a Conditions: (i) MOMCl, K2CO3, acetone, rt, overnight; 95% yield; (ii) Pd (PPh3)4, K2CO3, toluene/H2O, reflux, overnight; then (iii) LiOH, THF/H2O, rt, overnight; 90% yield over two steps; (iv) HOBT, EDC·HCl, Et3N, DMF, rt, 12 h; 24% yield for 43; 22% yield for 46; (v) 6 N HCl, MeOH, 66 °C, 2 h; 80% yield; (vi) pyrrolidin-3-ol hydrochloride, K2CO3, DMSO, 120 °C, 24 h; 79% yield; (vii) LiOH, THF/MeOH/H2O, 40 °C, overnight; 97% yield; (viii) MsCl, Et3N, CH2Cl2, rt, overnight; 70% yield; HOBT = 1-hydroxybenzotriazole hydrate; EDC·HCl = N-(3Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride; DMF = N,N-dimethylformamide; DMSO = methyl sulfoxide; MsCl = methanesulfonyl chloride. radiochemical purity was greater than 99% (n = 2), and the molar activity was as high as 288.6 GBq/μmol (7.8 Ci/μmol). For lead compounds 17 and 37 with a reversible binding mechanism, we deployed two distinct labeling methods, namely, 11C-methylation of phenolic precursor 44 for lead compound 17 and 18F-fluorination of mesylate precursor 47 for lead compound 37, respectively (Scheme 4B,C). The general procedure for preparation of precursors 44 and 47 is outlined in Scheme 3A,B, respectively. Specifically, synthesis of phenolic precursor 44 commenced with the protection of phenol derivative 39 with chloromethyl methyl ether (MOMCl) to give bromobenzene derivative 40 in 90% yield. Subsequently, Pd(PPh3)4-catalyzed cross-coupling reaction of 40 with (4-chloro-3-(trifluoromethyl)phenyl)boronic acid 41, followed by LiOH-triggered hydrolysis of the ester group, readily proceeded to give [1,1′-biphenyl]-4-carboxylic acid 42 in 89% yield over two steps. Coupling of 42 with piperazinyl azetidine 7 in the presence of HOBT and EDC occurred to deliver azetidine amide 43 in 24% yield. The following deprotection of the methoxylmethyl ether (MOM) group from 43 was achieved with 6 N HCl, thus affording the desired phenolic precursor 44 in 80% yield. On the other hand, the synthetic route to mesylate precursor 47 involved a K2CO3 promoted displacement of ethyl 4-fluorobenzoate 23 by pyrrolidin-3-ol hydrochloride, followed by LiOH-mediated hydrolysis of the ester group, thus producing 4-(3-hydrox- preliminary proof-of-concept studies for brain permeability and binding specificity. The significance of labeling site selection in irreversible serine hydrolase PET tracers has been demonstrated by Wilson58,59 and our group.39,41 Therefore, for irreversible lead compound 8, we utilized two 11C-carbonylation approaches with [11C]COCl2 and [11C]CO2, respectively (Scheme 4A). The piperazinyl azetidine precursor 7 for 11 C-carbonylation was synthesized as per the method depicted in Scheme 1. Initial radiosynthesis of 48 ([11C]8) was performed with [11C]COCl2 generated from our previously reported procedure. 39 Reaction of [ 11 C]COCl 2 with 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) facilitated by 1,2,2,6,6-pentamethylpiperidine (PMP) followed by addition of azetidine 7 smoothly occurred to deliver 11C-labeled carbamate 48 in an average 18% (n = 6) radiochemical yield (RCY) relative to starting [11C]CO2 (decay corrected). The radiochemical purity was ≥99%, and the molar activity was 30−52 GBq/μmol (0.81−1.40 Ci/μmol). Alternative radiosynthesis of 48 was carried out on the basis of our previously reported “in-loop” [11C]CO2 fixation method.60 Treatment of [11C]CO2 with 2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphorine (BEMP) and piperazinyl azetidine 7, followed by successive addition of HFIP and phosphorus(V) oxychloride (POCl3) in acetonitrile (MeCN), readily produced 48 in an average 2.5% decay-corrected RCY based on starting [11C]CO2 at the end-of-synthesis. The 3342 DOI: 10.1021/acs.jmedchem.8b01778 J. Med. Chem. 2019, 62, 3336−3353 Journal of Medicinal Chemistry Article Scheme 4. Radiosynthesis of Tracers 48−50a Conditions: (i) [11C]COCl2, HFIP, PMP, THF; 30 °C, 1 min. 18% decay-corrected RCY (ii) [11C]CO2, BEMP, HFIP, POCl3, rt, 1 min. 2.5% decay-corrected RCY; (iii) [11C]MeI, NaOH, DMF, 70 °C, 5 min, 25% decay-corrected RCY; (iv) [18F]KF, DMSO, 120 °C, 10 min, 39% decaycorrected RCY. HFIP = 1,1,1,3,3,3-hexafluoro-2-propanol; PMP = 1,2,2,6,6-pentamethylpiperidine; BEMP = 2-tert-butylimino-2-diethylamino-1,3dimethylperhydro-1,3,2-diazaphosphorine. a ypyrrolidin-1-yl)benzoic acid 45 in 77% overall yield. Coupling of 45 with piperazinyl azetidine 7, EDC, and HOBT led to the isolation of hydroxyl-containing azetidine amide 46, which was subsequently protected with methanesulfonyl chloride (MsCl) to generate the desired mesylate precursor 47 in 16% yield over two steps. As shown in Scheme 4B, the radiotracer 49 ([11C]17) with a reversible binding mechanism was prepared by reacting the phenolic precursor 44 with [11C]CH3I in 25% decay-corrected RCY based on starting [11C]CO2 at the end of synthesis with molar activity greater than 97.5 GBq/μmol. The 18 F-labeled radiotracer 50 ([18F]37) was obtained by reacting the mesylate precursor 47 with [18F]fluoride in the presence of K2CO3 and 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8,8,8]hexacosane (Kryptofix222) in an average RCY of 39% (decay corrected) with high radiochemical purity (>99%) and
excellent molar activity (>110 GBq/μmol). All of these
radiotracers exhibited excellent in vitro stability without
radiolysis up to 90 min after formulation. The efficient
radiosynthesis, as well as excellent radiochemical purity and
molar activity of 48−50 paved the way for subsequent in vivo
investigation.
Preliminary PET Imaging Studies of Irreversible MAGL
Tracer 48 ([11C]8). Dynamic PET acquisitions of radioligand
48 were carried out in Sprague−Dawley rats for 90 min.
Representative PET images in the brain (sagittal and coronal,
summed images 0−90 min) and time−activity curves are
shown in Figure 6 and Figure S6 in the Supporting
Information. Compound 48 demonstrated excellent BBB
penetration with a maximum standard uptake value (SUV)
of 2.26 at 3.5 min in the whole brain and heterogeneous
distribution with high uptake in cerebellum, striatum, and
hippocampus and low uptake in pons (Figure 6A and Figure
S6, Supporting Information). No significant washout (ratio of
SUV3.5min/SUV90min = 1.2) was observed during PET scans,
Figure 6. Representative PET images and time−activity curves of 48
in rat brain: (A) baseline; (B) blocking studies with KML29 (3 mg/
kg); (C) whole body ex vivo biodistribution studies. The brain uptake
was measured and quantified by the standardized uptake value (SUV).
Asterisks indicate statistical significance. *p < 0.05, **p ≤ 0.01, and ***p ≤ 0.001. AGs = adrenal glands; SI = small intestine; WAT = white adipose tissue; BAT = brown adipose tissue. which was aligned with characteristic plateaued time−activity curves of typically irreversible binding PET tracers.40,58,61 3343 DOI: 10.1021/acs.jmedchem.8b01778 J. Med. Chem. 2019, 62, 3336−3353 Journal of Medicinal Chemistry Article Figure 7. Representative PET images and time−activity curves of 49: (A) baseline PET images in SD rat (summed 0−90 min); (B) baseline PET images (summed 0−90 min) and time−activity curve of 49 in rat brain; (C) whole body ex vivo biodistribution in mice at five different time points (1, 5, 15, 30, and 60 min) post injection of 49. All data have mean ± SD, n = 4. (D) ex vivo specificity measured by the uptake of 49 in MAGL-rich tissues of adrenal glands and brown adipose tissue (baseline and blocking with KML29 (3 mg/kg)); (E) representative PET images of wild-type and Pgp/Bcrp knockout mouse (summed 0−90 min); (F) representative PET images and time−activity curve of 49 of wild-type and Pgp/Bcrp knockout mouse (summed 0−90 min). Asterisks indicate statistical significance. *p < 0.05, **p ≤ 0.01, and ***p ≤ 0.001. AGs = adrenal glands; SI = small intestine; WAT = white adipose tissue; BAT = brown adipose tissue. SUV, respectively, Figures 7A−B and 8A−B). High levels of radioactivity were observed in brown adipose tissue (BAT), kidneys, and liver (Figures 7A and 8A), which is consistent with the expression of MAGL in the periphery and confirmed by subsequent ex vivo biodistribution studies (Figures 7C, 8C and Tables S2, S3, Supporting Information).62 In contrast to the irreversible radioligand 48, both 49 and 50 exhibited an initially high uptake (>15% ID/g at 1 min) in heart and lungs
followed by fast clearance (ratio of % ID/g 1min/60min in heart:
3.49 for 49, 6.34 for 50; ratio of % ID/g1min/60min in lungs: 6.80
for 49, 10.38 for 50). Besides, the radioactivity of 49 and 50
was also rapidly washed out from blood with the SUV1min/
SUV60min ratios as 5.1 and 3.5, respectively. These results
together with high bound activity in the kidneys, liver, and
small intestine suggested fast urinary and hepatobiliary
elimination of 49 and 50. To test the in vivo specificity of
49 and 50 toward MAGL, we carried out blocking experiments
by pretreatment of KML29 (3 mg/kg). While 50 exhibited a
significant blocking effect in two MAGL-rich organs, including
adrenal glands (80%) and BAT (88%), 49 demonstrated a
comparable result in BAT (63% decrease), and no statistical
significance was observed for the uptake in adrenal glands
(13% decrease) between the baseline and blocking conditions
(Figures 7D and 8D). The increased level of nonspecific
binding of compound 49 in adrenal glands and BAT was likely
attributed to its higher lipophilicity. In tissues with low MAGL
expression, such as lungs and muscle, no obvious contrast was
Pretreatment with KML29 (3 mg/kg), a widely used MAGL
inhibitor, remarkably decreased the uptake of 48 in various
brain regions and the whole brain uptake (∼50% reduction
based on area under curve, AUC), which demonstrated
excellent in vivo specificity of 48 toward MAGL (Figures 6B
and S6). We then investigated the uptake, biodistribution, and
clearance of 48 by whole body distribution in mice at five time
points (1, 5, 15, 30, and 60 min) after tracer injection (Figure
6C and Table S1, Supporting Information). Several organs,
including liver, adrenal glands, kidney, and brown adipose
tissue (BAT), exhibited high radioactivity levels at 30 min
(>5% ID/g, injected dose per gram of wet tissue), which is
consistent with the expression of MAGL in rodents.62 The
radioactivity levels in heart and lungs reached a plateau at 5
min, followed by a slow washout. These results together with
high activity remaining in the kidneys, liver, and small intestine
suggested urinary and hepatobiliary elimination of 48. To
investigate the in vivo stability of 48, we carried out
radiometabolic analysis at 30 min post injection of the
radiotracers in mouse brain homogenate for 48. The fraction
of the remaining parent 48 was determined to be 96% by
radioHPLC, indicating excellent in vivo stability in the brain.
Preliminary PET Imaging Studies of Reversible MAGL
Tracers 49 ([11C]17) and 50 ([18F]37). Dynamic PET
acquisitions of reversible radioligands 49 and 50 were also
performed in Sprague−Dawley rats for 90 min, and both
exhibited insufficient BBB permeability in rats (ca. 0.4 and 0.3
3344
DOI: 10.1021/acs.jmedchem.8b01778
J. Med. Chem. 2019, 62, 3336−3353
Journal of Medicinal Chemistry
Article
Figure 8. Representative PET images and time−activity curves of 50: (A) baseline PET images in SD rat (summed 0−90 min); (B) baseline PET
images (summed 0−90 min) and time−activity curve of 50 in rat brain; (C) whole body ex vivo biodistribution in mice at five different time points
(1, 5, 15, 30, and 60 min) post injection of 50. All data have mean ± SD, n = 4; (D) ex vivo specificity measured by the uptake of 50 in MAGL-rich
tissues, including adrenal glands (AGs) and brown adipose tissue (BAT) (baseline and blocking with KML29 (3 mg/kg)); (E) representative PET
images of wild-type and Pgp/Bcrp knockout mouse (summed 0−90 min); (F) representative PET images and time−activity curve of 50 of wildtype and Pgp/Bcrp knockout mouse (summed 0−90 min). Asterisks indicate statistical significance. *p < 0.05, **p ≤ 0.01, and ***p ≤ 0.001. AGs = adrenal glands; SI = small intestine; WAT = white adipose tissue; BAT = brown adipose tissue. homogenate. The fractions of parent 49 and 50 were determined by radioHPLC to be 62 and 45%, respectively, indicating reasonable metabolic stability. observed for both 49 and 50 between baseline and blocking conditions, indicating a limited window to show specific binding in MAGL-deficient organs. Furthermore, in MAGLdeficient organs, such as lungs and muscle, no significant changes of uptake were observed for both 49 and 50. The peripherally restricted distributions of 49 and 50 may be attributed to their interaction with P-glycoprotein (Pgp) and breast cancer resistance protein (Bcrp), both of which are crucial components of the ATP-binding cassette (ABC) efflux transporter family colocalizing at the BBB. To verify this hypothesis, we conducted PET imaging studies of 49 and 50 in wild-type and Pgp/Bcrp knockout (ABCB1a/ 1b−/−ABCG2−/−) mice. As outlined in Figures 7E,F and 8E,F, the brain uptake was significantly increased in Pgp/Bcrp knockout mice compared with that of wide-type mice, and reached a plateau of 1.4 SUV at 2.5 min for 49 and 1.0 SUV at 11 min for 50, respectively. These results demonstrated that 49 and 50 had intensive interactions with ABC efflux transporters at the BBB, including Pgp and Bcrp, which may explain their limited brain accumulation at tracer dose. The extensive interactions of 49 and 50 with ABC efflux transporters are probably attributed to their nature of being nitrogenous compounds and characterized by one or more positive charges in the physiological conditions. To investigate the in vivo stability of 49 and 50, we performed radiometabolic analysis 30 min post injection of the radiotracers in mouse plasma ■ CONCLUSIONS We have established a focused library of MAGL inhibitors with irreversible or reversible binding mechanisms by the utilization of a tail switching strategy on a piperazinyl azetidine scaffold. Potency, selectivity, as well as irreversible or reversible binding profiles were determined in vitro by two different tissue-based assays, namely, [3H]2-OG hydrolysis and ABPP. Lipophilicity (LogD) of candidate compounds was also measured by the “shake-flask” method to estimate the brain permeability. As a result, compound 8 was identified as the most promising irreversible MAGL inhibitor and compounds 17 and 37 were identified as the most promising reversible inhibitors. Possible molecular interactions between these lead compounds and MAGL, as well as the corresponding binding domain were also identified through molecular docking studies. The corresponding 11C- and 18F-isotopologues 48 ([11C]8, also known as [11C]MAGL-2-11); 49 ([11C]17, also known as [11C]PAD); and 50 ([11C]37, also known as [18F]MAGL-4-11) were synthesized in excellent radiochemical yield, high molar activity, and high radiochemical purity, with diverse radiolabeling strategies, including [11C]COCl2 cyclization, in-loop 3345 DOI: 10.1021/acs.jmedchem.8b01778 J. Med. Chem. 2019, 62, 3336−3353 Journal of Medicinal Chemistry Article [11C]CO2 fixation, 11C-methylation, and 18F-fluorination. The pharmacokinetic profiles of these radioligands, including brain uptake, clearance, and binding specificity were further investigated by PET imaging and ex vivo whole-body distribution studies in rodents. The irreversible tracer 48 demonstrated excellent BBB penetration and heterogenous regional brain uptake and high binding specificity, which was in line with MAGL distribution. The reversible tracers 49 and 50 showed limited brain uptake but exhibited excellent binding specificity toward MAGL in the periphery, as evidenced by ex vivo blocking biodistribution studies. We also performed PET experiments in Pgp/Bcrp knockout mice, in which 49 and 50 exhibited substantially increased activity accumulation in the brain, indicating that the underlying cause of their limited brain uptake was probably attributed to the significant interaction with ABC efflux transporters. Therefore, further optimization is necessary to increase brain permeability by minimizing the interaction with Pgp/Bcrp efflux transporters and further validation in comprehensive in vivo MAGL inhibitory experiments is also desirable to evaluate the therapeutic potential of these novel molecules. Multiparameter optimization (MPO), namely, the approach to simultaneously optimize and balance basicity (pKa), lipophilicity (c log P and c log D), number of hydrogen bond donors (HBD), polar surface area (tPSA), and molecular weight (MW), has demonstrated its usefulness in the improvement of molecular properties and ADME profile, including brain penetration.42,63−66 We will utilize this strategy to design next-generation brain penetrant radioligands for reversible binding mechanism. In conclusion, this work not only provides the first synergetic medicinal chemistry approach to both irreversible and reversible MAGL inhibitors based on a unique piperazinyl azetidine scaffold but also paves the way toward the development of next-generation reversible PET tracers for translational PET imaging studies. ■ (male; 7 weeks, 34−36 g), Sprague−Dawley rats (male; 7 weeks; 210−230 g) were kept on a 12 h light/12 h dark cycle and were allowed food and water ad libitum. Male Pgp/Bcrp knockout (Mdr1a/1b(−/−) and Abcg(−/−)) mice and male wild-type mice (FVB) were purchased from Taconic (Hudson, NY). Chemistry. General Procedure for Synthesis of 1,1,1,3,3,3Hexafluoropropan-2-yl 3-(4-(Thiazole-2-carbonyl)piperazin-1-yl)azetidine-1-carboxylate (8). Under a nitrogen atmosphere, a solution of 4-nitrophenyl chloroformate (201.6 mg, 1.0 mmol) in CH2Cl2 (2.5 mL) was added dropwise to a solution of 1,1,1,3,3,3-hexafluoro-2propanol (420.2 mg, 2.5 mmol), pyridine (197.8 mg, 2.5 mmol), and 4-dimethylaminopyridine (DMAP, 12.3 mg, 0.1 mmol) in CH2Cl2 (2.5 mL) at 0 °C. The resulting mixture was stirred at room temperature overnight, followed by addition of 7 (303.2 mg, 1.05 mmol) and Et3N (404.8 mg, 4.0 mmol) in CH2Cl2 (4.0 mL). The reaction mixture was stirred at room temperature for 5 h, then evaporated under reduced pressure to dryness and redissolved in ethyl acetate (25 mL). After successively washing with H2O, 1 M aqueous potassium carbonate solution, and brine, the organic phase was dried over MgSO4 and evaporated to dryness. The residue was purified by silica column chromatography (PE/EA = 3/1) to give product 8 as a white solid in 20% yield. Melting point: 102−103 °C. 1H NMR (300 MHz, CDCl3) δ 7.87 (d, J = 3.2 Hz, 1H), 7.54 (d, J = 3.2 Hz, 1H), 5.75−5.54 (m, 1H), 4.48 (s, 2H), 4.20−3.97 (m, 4H), 3.86−3.84 (m, 2H), 3.35−3.16 (m, 1H), 2.48−2.46 (m, 4H); 19F NMR (282 MHz, CDCl3) δ −69.74 (d); 13C NMR (75 MHz, CDCl3) δ 165.06, 159.34, 151.89, 143.35, 124.42, 120.79 (d, J = 281.9 Hz), 68.76−66.88 (m), 54.23, 54.09, 53.35, 50.18, 49.60, 46.02, 43.08. HRMS (ESI): m/z calculated for C15H16F6N4O3SNa+ [M + Na]+: 469.0740, found: 469.0739. General Procedure for Synthesis of 2,2,2-Trifluoroethyl 3-(4(Thiazole-2-carbonyl)piperazin-1-yl) Azetidine-1-carboxylate (9). A solution of triphosgene (0.2 mmol) in dichloromethane (DCM, 0.5 mL) was added dropwise to a solution of CF3CH2OH (0.6 mmol) and Et3N (0.6 mmol) in DCM (1 mL) at 0 °C. The mixture was stirred at room temperature for 4 h, followed by the addition of 7 (0.5 mmol) and a solution of Et3N (2 mmol) in DCM (2 mL). After stirring at room temperature for 4 h, the mixture was evaporated to dryness and redissolved in ethyl acetate (10 mL). The organic phase was washed with H2O (10 mL), and the aqueous phase was extracted with ethyl acetate (10 mL × 2). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by silica column chromatography (eluent: ethyl acetate) to give product 9 as a white solid in 43% yield. Melting point: 95−97 °C. 1H NMR (300 MHz, CDCl3) δ 7.85 (s, 1H), 7.52 (s, 1H), 4.54−4.32 (m, 4H), 4.14−3.76 (m, 6H), 3.25−3.13 (m, 1H), 2.44 (s, 4H); 19F NMR (282 MHz, CDCl3) δ −70.36 (t, J = 8.4 Hz); 13C NMR (75 MHz, CDCl3) δ 164.90, 159.11, 153.99, 143.11, 124.15, 123.02 (q, J = 277.4 Hz), 60.91 (q, J = 36.4 Hz), 53.99, 53.65, 52.93, 49.96, 49.36, 45.89, 42.94. HRMS (ESI): m/z calculated for C14H17F3N4O3SNa+ [M + Na]+: 401.0866, found: 401.0867. Preparation of (4-(1-(1H-1,2,4-Triazole-1-carbonyl)azetidin-3-yl) Piperazin-1-yl) (Thiazol-2-yl) Methanone (10). Compound 10 was prepared in a manner similar to that described for 9 in 28% yield as a white solid. Melting point: 164−165 °C. 1H NMR (300 MHz, CDCl3) δ 8.88 (s, 1H), 7.97 (s, 1H), 7.87 (d, J = 3.2 Hz, 1H), 7.54 (d, J = 3.2 Hz, 1H), 4.82−4.65 (m, 1H), 4.62−4.39 (m, 3H), 4.33− 4.20 (m, 1H), 4.16−4.05 (m, 1H), 3.85 (s, 2H), 3.34−3.19 (m, 1H), 2.50 (t, J = 4.9 Hz, 4H); 13C NMR (75 MHz, CDCl3) δ 165.09, 159.33, 153.01, 147.62, 144.92, 143.32, 124.38, 58.86, 54.62, 53.23, 50.23, 49.64, 46.10, 43.17. HRMS (ESI): m/z calculated for C14H17N7O2SNa+ [M + Na]+: 370.1057, found: 370.1059. Preparation of (4-(1-(1H-Benzo[d][1,2,3]triazole-1-carbonyl) azetidin-3-yl)piperazin-1-yl) (thiazol-2-yl)methanone (11). Compound 11 was prepared in a manner similar to that described for 9 in 7% yield as a white solid. Melting point: 218−219 °C. 1H NMR (300 MHz, CDCl3) δ 8.26 (dt, J = 8.4, 0.9 Hz, 1H), 8.08 (dt, J = 8.3, 0.9 Hz, 1H), 7.88 (d, J = 3.2 Hz, 1H), 7.60 (ddd, J = 8.3, 7.1, 1.1 Hz, 1H), 7.54 (d, J = 3.2 Hz, 1H), 7.46 (ddd, J = 8.2, 7.1, 1.1 Hz, 1H), EXPERIMENTAL SECTION General Consideration. The general procedure for the Experimental section was described previously41 with minor modification in this work. All chemicals employed in the synthesis were purchased from commercial vendors and used without further purification. Thin-layer chromatography (TLC) was conducted with 0.25 mm silica gel plates (60F254) and visualized by exposure to UV light (254 nm) or stained with potassium permanganate. Flash column chromatography was performed using silica gel (particle size 0.040−0.063 mm). Nuclear magnetic resonance (NMR) spectra were obtained on a Bruker spectrometer at 300 MHz. Chemical shifts (δ) are reported in ppm, and coupling constants are reported in Hertz. The multiplicities are abbreviated as follows: s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, sext = sextet, sept = setpet, m = multiplet, br = broad signal, and dd = doublet of doublets. For mass spectrometer measurement, the ionization method is ESI using Agilent 6430 Triple Quad LC/MS. Lipophilicity was calculated by ChemBioDraw Ultra 14.0 (CambridgeSoft Corporation, PerkinElmer). All tested MAGL inhibitors showed high purity (≥95%), as determined by a reverse-phase HPLC (Gemini 5 μm, NX-C18 110 Å column (3 mm ID × 150 mm)). The lead compounds 8, 17, and 37 did not show any promiscuous moieties in the Pan Assay Interference Compounds Assay (PAINS) using two different in silico filters (http://www.swissadme.ch/index.php and http://zinc15.docking. org/patterns/home/).67 Molar activity determinations are reported at the end of synthesis, unless otherwise stated. The animal experiments were approved by the Institutional Animal Care and Use Committee of Massachusetts General Hospital or the Animal Ethics Committee at the National Institutes for Quantum and Radiological Science and Technology, National Institute of Radiological Sciences. CD-1 mice (female; 7 weeks, 22−24 g), DdY mice 3346 DOI: 10.1021/acs.jmedchem.8b01778 J. Med. Chem. 2019, 62, 3336−3353 Journal of Medicinal Chemistry Article Preparation of (4-(1-(4′-Chloro-2-methoxy-3′-(trifluoromethyl)[1,1′-biphenyl]-4-carbonyl)azetidin-3-yl)piperazin-1-yl)(thiazol-2yl)methanone (17). Compound 17 was prepared in a manner similar to that described for 16 in 25% yield as a white solid. Melting point: 82−83 °C. 1H NMR (300 MHz, CDCl3) δ 7.86 (d, J = 3.2 Hz, 1H), 7.82 (d, J = 1.6 Hz, 1H), 7.66−7.60 (m, 1H), 7.57−7.49 (m, 2H), 7.35−7.33 (m, 1H), 7.28 (t, J = 7.3 Hz, 1H), 7.23−7.18 (m, 1H), 4.54−4.44 (m, 2H), 4.39−4.20 (m, 3H), 4.17−4.07 (m, 1H), 3.91− 3.83 (m, 5H), 3.32−3.22 (m, 1H), 2.51−2.49 (m, 4H); 19F NMR (282 MHz, CDCl3) δ −58.52 (s); 13C NMR (75 MHz, CDCl3) δ 169.97, 165.03, 159.33, 156.66, 143.32, 136.59, 134.44, 133.91, 131.40 (q, J = 1.8 Hz), 131.26, 130.88, 130.35, 128.63 (q, J = 5.4 Hz), 128.24 (q, J = 31.3 Hz), 124.40, 123.03 (q, J = 273.3 Hz), 120.27, 111.32, 57.28, 55.96, 54.44, 52.79, 50.27, 49.68, 46.03, 43.09. HRMS (ESI): m/z calculated for C26H24F3N4O3SClNa+ [M + Na]+: 587.1102, found: 587.1101. Preparation of (4-(1-(3-Methoxy-4-(pyridin-3-yl)benzoyl)azetidin-3-yl)piperazin-1-yl)(thiazol-2-yl)methanone (18). Compound 18 was prepared in a manner similar to that described for 16 in 28% yield as a white solid. Melting point: 90−91 °C. 1H NMR (300 MHz, CDCl3) δ 8.71 (s, 1H), 8.52 (s, 1H), 7.83−7.81 (m, 2H), 7.51−7.49 (m, 1H), 7.32−7.16 (m, 4H), 4.48−4.20 (m, 5H), 4.10− 4.06 (m, 1H), 3.81 (s, 5H), 3.24−3.20 (m, 1H), 2.45 (s, 4H); 13C NMR (75 MHz, CDCl3) δ 169.88, 164.84, 159.17, 156.75, 149.70, 148.05, 143.17, 137.07, 134.22, 133.49, 130.29, 129.65, 124.26, 123.10, 120.15, 111.08, 57.20, 55.78, 54.26, 52.68, 50.11, 49.51, 45.93, 42.98. HRMS (ESI): m/z calculated for C24H25N5O3SNa+ [M + Na]+: 486.1570, found: 486.1568. Preparation of (4-(1-(3-Methoxy-4-(pyridin-4-yl)benzoyl)azetidin-3-yl)piperazin-1-yl)(thiazol-2-yl)methanone (19). Compound 19 was prepared in a manner similar to that described for 16 in 23% yield as a white solid. Melting point: 85−86 °C. 1H NMR (300 MHz, CDCl3) δ 8.64−8.62 (m, 2H), 7.85 (d, J = 3.1 Hz, 1H), 7.54−7.46 (m, 3H), 7.41−7.30 (m, 2H), 7.27−7.19 (m, 1H), 4.52− 4.22 (m, 5H), 4.15−4.06 (m, 1H), 3.87−3.80 (m, 5H), 3.30−3.19 (m, 1H), 2.50−2.46 (m, 4H); 13C NMR (75 MHz, CDCl3) δ 169.84, 164.99, 159.29, 156.87, 149.05, 146.09, 143.28, 134.99, 130.31, 130.23, 124.46, 124.36, 120.22, 111.37, 57.27, 55.93, 54.39, 52.80, 50.23, 49.64, 46.04, 43.09. HRMS (ESI): m/z calculated for C24H25N5O3SNa+ [M + Na]+: 486.1570, found: 486.1571. Preparation of (4-(1-(3-Methoxy-4-(pyrimidin-5-yl)benzoyl)azetidin-3-yl)piperazin-1-yl)(thiazol-2-yl)methanone (20). Compound 20 was prepared in a manner similar to that described for 16 in 23% yield as a white solid. Melting point: 89−90 °C. 1H NMR (300 MHz, CDCl3) δ 9.16 (s, 1H), 8.90 (s, 2H), 7.86 (d, J = 3.2 Hz, 1H), 7.53 (d, J = 3.2 Hz, 1H), 7.40−7.30 (m, 2H), 7.27−7.22 (m, 1H), 4.54−4.22 (m, 5H), 4.16−4.06 (m, 1H), 3.87 (s, 5H), 3.32− 3.18 (m, 1H), 2.52−2.48 (m, 4H); 13C NMR (75 MHz, CDCl3) δ 169.73, 164.98, 159.31, 157.38, 156.95, 156.91, 143.31, 135.28, 131.51, 130.09, 126.22, 124.41, 120.42, 111.34, 57.23, 55.98, 54.42, 52.78, 50.25, 49.66, 45.94, 43.04. HRMS (ESI): m/z calculated for C23H24N6O3SNa+ [M + Na]+: 487.1523, found: 487.1526. Preparation of (4-(1-(4-(Furan-3-yl)-3-methoxybenzoyl)azetidin3-yl)piperazin-1-yl)(thiazol-2-yl)methanone (21). Compound 21 was prepared in a manner similar to that described for 16 in 39% yield as a white solid. Melting point: 91−92 °C. 1H NMR (300 MHz, CDCl3) δ 8.04 (d, J = 0.5 Hz, 1H), 7.85 (d, J = 3.2 Hz, 1H), 7.52 (d, J = 3.2 Hz, 1H), 7.51−7.43 (m, 2H), 7.31 (d, J = 1.0 Hz, 1H), 7.17 (dd, J = 7.9, 1.4 Hz, 1H), 6.80−6.76 (m, 1H), 4.47 (d, J = 19.2 Hz, 2H), 4.37−4.20 (m, 3H), 4.14−4.10 (m, J = 2.7 Hz, 1H), 3.94 (s, 3H), 3.90−3.81 (m, 2H), 3.29−3.20 (m, 1H), 2.50−2.48 (m, 4H); 13 C NMR (75 MHz, CDCl3) δ 170.27, 164.99, 159.29, 156.61, 143.29, 142.65, 132.08, 127.43, 124.36, 121.14, 120.19, 111.01, 109.33, 57.30, 55.73, 54.43, 52.72, 50.23, 49.65, 46.00, 43.05. HRMS (ESI): m/z calculated for C23H24N4O4SNa+ [M + Na]+: 475.1410, found: 475.1411. Preparation of (4-(1-(3-Methoxy-4-(thiophen-3-yl)benzoyl)azetidin-3-yl)piperazin-1-yl)(thiazol-2-yl)methanone (22). Compound 22 was prepared in a manner similar to that described for 16 in 40% yield as a white solid. Melting point: 103−104 °C. 1H 4.86 (dd, J = 9.8, 7.5 Hz, 1H), 4.69 (dd, J = 10.2, 5.1 Hz, 1H), 4.51 (s, 2H), 4.43−4.31 (m, 1H), 4.23 (dd, J = 9.4, 4.6 Hz, 1H), 3.89 (s, 2H), 3.45−3.26 (m, 1H), 2.55 (d, J = 4.5 Hz, 4H); 13C NMR (75 MHz, CDCl3) δ 165.06, 159.40, 149.50, 145.24, 143.37, 132.48, 129.86, 125.68, 124.39, 119.91, 114.44, 59.30, 54.82, 53.33, 50.31, 49.72, 46.17, 43.22. HRMS (ESI): m/z calculated for C18H19N7O2SNa+ [M + Na]+: 420.1213, found: 420.1215. General Procedure for Synthesis of 1,3-Dioxoisoindolin-2-yl 3(4-(Thiazole-2-carbonyl)piperazin-1-yl) Azetidine-1-carboxylate (12). A solution of Et3N (0.84 mmol) in DCM (0.5 mL) was added dropwise to a solution of 2-hydroxyisoindoline-1,3-dione (0.7 mmol) and triphosgene (0.14 mmol) in DCM (1.5 mL) at 0 °C. The mixture was stirred at room temperature for 16 h, followed by addition of 7 (0.5 mmol) and a solution of Et3N (1 mmol) in DCM (1 mL). After stirring at room temperature for another 4 h, the mixture was evaporated to dryness and redissolved in ethyl acetate (10 mL). The organic phase was washed with H2O (10 mL), and the aqueous phase was extracted with ethyl acetate (10 mL × 2). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by silica column chromatography (eluent: ethyl acetate) to give product 12 in 29% yield as a light yellow solid. Melting point: 180−182 °C. 1H NMR (300 MHz, CDCl3) δ 7.90−7.84 (m, 3H), 7.77 (dd, J = 5.5, 3.1 Hz, 2H), 7.55 (d, J = 3.2 Hz, 1H), 4.58−4.43 (m, 2H), 4.37−4.02 (m, 4H), 3.87 (s, 2H), 3.39−3.26 (m, 1H), 2.56−2.43 (m, 4H); 13C NMR (75 MHz, CDCl3) δ 165.00, 162.50, 159.28, 151.35, 143.28, 134.81, 129.00, 124.33, 124.00, 54.59, 54.43, 53.84, 50.01, 49.47, 46.06, 43.12. HRMS (ESI): m/z calculated for C20H19N5O5SNa+ [M + Na]+: 464.0999, found: 464.0998. General Procedure for Synthesis of 2,5-Dioxopyrrolidin-1-yl 3-(4(Thiazole-2-carbonyl) Piperazin-1-yl) Azetidine-1-carboxylate (13). Bis(2,5-dioxopyrrolidin-1-yl) carbonate (0.6 mmol) was added to a solution of (4-(azetidin-3-yl)piperazin-1-yl)(thiazol-2-yl)methanone hydrochloride 7 (0.5 mmol) and Et3N (1 mmol) in DCM (2 mL). After stirring at ambient temperature for 5 h, the mixture was evaporated to dryness and redissolved in ethyl acetate (10 mL). The organic phase was washed with H2O (10 mL) and then extracted with ethyl acetate (10 mL × 2). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by silica column chromatography (eluent: ethyl acetate) to give product 13 in 14% yield as a white solid. Melting point: 209−211 °C. 1H NMR (300 MHz, CDCl3) δ 7.87 (s, 1H), 7.54 (s, 1H), 4.47 (s, 2H), 4.15 (dd, J = 38.7, 31.8 Hz, 4H), 3.84 (s, 2H), 3.30 (s, 1H), 2.81 (s, 4H), 2.46 (s, 4H); 13C NMR (75 MHz, CDCl3) δ 169.83, 165.03, 159.27, 150.52, 143.27, 124.33, 54.53, 53.77, 49.99, 49.45, 46.06, 43.11, 25.59. HRMS (ESI): m/z calculated for C16H19N5O5SNa+ [M + Na]+: 416.0999, found: 416.0997. General Procedure for Synthesis of (4-(1-(2-Methoxy-[1,1′biphenyl]-4-carbonyl)azetidin-3-yl)piperazin-1-yl)(thiazol-2-yl)methanone (16). Under a nitrogen atmosphere, a mixture of 2methoxy-[1,1′-biphenyl]-4-carboxylic acid (182.6 mg, 0.8 mmol), 7 (277.3 mg, 0.96 mmol), 1-hydroxybenzotriazole (HOBT, 14% wet with H2O, 326.8 mg, 2.08 mmol), N-(3-dimethylaminopropyl)-Nethylcarbodiimide hydrochloride (EDC·HCl, 398.8 mg, 2.08 mmol), and Et3N (647.6 mg, 6.4 mmol) in DMF (8 mL) was stirred at room temperature for 16 h. Then the mixture was poured into saturated aqueous NaHCO3 solution and extracted with ethyl acetate three times. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by silica column chromatography (PE/EA = 1/3) to give 16 as a white solid in 22% yield. Melting point: 92−93 °C. 1H NMR (300 MHz, CDCl3) δ 7.86 (d, J = 3.2 Hz, 1H), 7.54−7.48 (m, 3H), 7.45−7.37 (m, 2H), 7.37−7.29 (m, 3H), 7.22−7.17 (m, 1H), 4.53− 4.10 (m, 6H), 3.96−3.76 (m, 5H), 3.32−3.17 (m, 1H), 2.50 (s, 4H); 13 C NMR (75 MHz, CDCl3) δ 170.31, 165.01, 159.28, 156.74, 143.29, 137.67, 133.66, 133.30, 130.60, 129.54, 128.20, 127.58, 124.35, 120.12, 111.14, 57.29, 55.87, 54.42, 52.70, 50.24, 49.65, 46.00, 43.06. HRMS (ESI): m/z calculated for C25H26N4O3SNa+ [M + Na]+: 485.1618, found: 485.1619. 3347 DOI: 10.1021/acs.jmedchem.8b01778 J. Med. Chem. 2019, 62, 3336−3353 Journal of Medicinal Chemistry Article NMR (300 MHz, CDCl3) δ 7.86 (d, J = 3.2 Hz, 1H), 7.67 (dd, J = 2.9, 1.2 Hz, 1H), 7.53 (d, J = 3.2 Hz, 1H), 7.49 (d, J = 7.9 Hz, 1H), 7.44 (dd, J = 5.0, 1.2 Hz, 1H), 7.38−7.31 (m, 2H), 7.17 (dd, J = 7.9, 1.5 Hz, 1H), 4.52−4.21 (m, 5H), 4.15−4.06 (m, 1H), 3.91 (s, 3H), 3.90−3.81 (m, 2H), 3.30−3.19 (m, 1H), 2.50−2.46 (m, 4H); 13C NMR (75 MHz, CDCl3) δ 170.26, 165.04, 159.32, 156.66, 143.30, 137.44, 132.77, 129.33, 128.41, 127.95, 124.89, 124.37, 120.18, 111.36, 57.36, 55.86, 54.43, 52.78, 50.25, 49.67, 46.08, 43.13. HRMS (ESI): m/z calculated for C23H24N4O3S2Na+ [M + Na]+: 491.1182, found: 491.1180. Preparation of (4-(1-(4-(3-Fluoroazetidin-1-yl)benzoyl)azetidin3-yl)piperazin-1-yl)(thiazol-2-yl)methanone (36). Compound 36 was prepared in a manner similar to that described for 16 in 32% yield as a white solid. Melting point: 171−172 °C. 1H NMR (300 MHz, CDCl3) δ 7.85 (d, J = 2.8 Hz, 1H), 7.57−7.50 (m, 3H), 6.39 (d, J = 8.3 Hz, 2H), 5.64−5.23 (m, 1H), 4.56−3.93 (m, 10H), 3.82 (s, 2H), 3.27−3.13 (m, 1H), 2.48−2.43 (m, 4H); 19F NMR (282 MHz, CDCl3) δ −175.66−−178.14 (m); 13C NMR (75 MHz, CDCl3) δ 170.69, 165.02, 159.27, 152.59, 143.25, 129.66, 124.28, 122.01, 110.78, 82.66 (d, J = 204.9 Hz), 59.30 (d, J = 24.2 Hz), 57.42, 54.41, 52.66, 50.18, 49.61, 46.07, 43.12. HRMS (ESI): m/z calculated for C21H24FN5O2SNa+ [M + Na]+: 452.1527, found: 452.1529. Preparation of (4-(1-(4-(3-Fluoropyrrolidin-1-yl)benzoyl)azetidin-3-yl)piperazin-1-yl)(thiazol-2-yl)methanone (37). Compound 37 was prepared in a manner similar to that described for 16 in 30% yield as a white solid. Melting point: 165−166 °C. 1H NMR (300 MHz, CDCl3) δ 7.85 (d, J = 3.2 Hz, 1H), 7.63−7.48 (m, 3H), 6.50 (d, J = 8.8 Hz, 2H), 5.48−5.23 (m, 1H), 4.51 (s, 2H), 4.38−4.04 (m, 4H), 3.87 (s, 2H), 3.64−3.59 (m, 1H), 3.56−3.41 (m, 3H), 3.32−3.18 (m, 1H), 2.54 (s, 4H), 2.44−2.29 (m, 1H), 2.28− 2.01 (m, 1H); 19F NMR (282 MHz, CDCl3) δ −170.11−−171.72 (m); 13C NMR (75 MHz, CDCl3) δ 170.93, 165.07, 159.30, 149.29, 143.27, 129.99, 124.28, 120.18, 111.05, 92.86 (d, J = 176.1 Hz), 57.51, 54.49, 54.40 (d, J = 23.2 Hz), 52.59, 50.21, 49.64, 46.11, 45.36, 43.16, 32.38 (d, J = 21.8 Hz). HRMS (ESI): m/z calculated for C22H26FN5O2SNa+ [M + Na]+: 466.1683, found: 466.1685. Preparation of (4-(1-(4-(4-Fluoropiperidin-1-yl)benzoyl)azetidin3-yl)piperazin-1-yl)(thiazol-2-yl)methanone (38). Compound 38 was prepared in a manner similar to that described for 16 in 19% yield as a white solid. Melting point: 137−138 °C. 1H NMR (300 MHz, CDCl3) δ 7.86 (d, J = 3.2 Hz, 1H), 7.59−7.50 (m, 3H), 6.90− 6.83 (m, 2H), 4.84 (ddt, J = 14.6, 9.8, 4.2 Hz, 1H), 4.54−4.04 (m, 6H), 3.84 (s, 2H), 3.52−3.14 (m, 5H), 2.47 (s, 4H), 2.10−1.83 (m, 4H); 19F NMR (282 MHz, CDCl3) δ −177.11−−177.63 (m); 13C NMR (75 MHz, CDCl3) δ 170.49, 165.07, 159.30, 152.66, 143.28, 129.78, 124.30, 122.72, 114.49, 88.19 (d, J = 171.3 Hz), 57.40, 54.47, 52.65, 50.22, 49.65, 46.11, 88.19 (d, J = 171.3 Hz), 43.15, 30.82 (d, J = 19.8 Hz). HRMS (ESI): m/z calculated for C23H28FN5O2SNa+ [M + Na]+: 480.1840, found: 480.1841. Pharmacology. The[3H]2-OG hydrolysis assay was carried out using cytosolic preparation from rat brain according to our previous procedure.41,68 Briefly, the hydrolysis was measured as per the method described by Boldrup et al.,69 whereby test compounds, brain samples, and assay buffer (10 mM Tris-HCl and 1 mM EDTA, pH 7.4) are preincubated for 0−60 min prior to addition of the substrate ([3H]2OG for MAGL, obtained from American Radiolabeled Chemicals Inc. St. Louis, MO) and diluted with nonradioactive 2-OG, as appropriate (Cayman Chemical Co., Ann Arbor, MI) to give the final assay concentration (0.5 μM in assay buffer containing 0.125% w/v assay concentration of fatty acid-free bovine serum albumin) in an assay volume of 200 μL. Reactions were stopped by addition of 400 μL of a solution containing active charcoal in 0.5 M HCl. Phases were separated by centrifugation, and the aqueous phase, containing the reaction products, was taken and measured for tritium content using liquid scintillation spectroscopy with quench correction. pIC50 and hence IC50 values were determined from the data expressed as a percentage of control using the log(inhibitor) vs response−variable slope algorithm of GraphPad Prism. Using this assay, the prototypical MAGL inhibitor JZL184 inhibits rat cerebellar 2-OG hydrolysis with an IC50 value, following a 60 min preincubation phase, of 5.8 nM and a residual activity of ∼10%.68 Statistical Analysis. Statistical analysis was performed by a student’s two-tailed t-test, and we used asterisks to indicate the statistical significance: *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ***p ≤ 0.0001. Activity-Based Protein Profiling (ABPP). The general procedure for ABPP assays was described previously.41,70 Briefly, mouse brain membrane proteomes (1 mg/mL) were preincubated with either DMSO or inhibitors (1 and 10 μM) at 37 °C. After 30 min, FPrhodamine (1 μM final concentration) was added and the mixture was incubated for another 1−180 min at room temperature. Reactions were quenched with 4× SDS loading buffer and run on SDS−PAGE. Samples were visualized by in-gel fluorescence scanning using a ChemiDoc MP system. For time course experiment, proteomes are treated with 1 μM compounds 16−22 and 36−38 for 30 min at 37 °C followed by labeling with FP-Rh (1 μM final concentration) for varying times at room temperature. DMSO is used as negative control, and MJN110 [2,5-dioxopyrrolidin-1-yl-4-(bis(4chlorophenyl)methyl) piperazine-1-carboxylate],71 a validated irreversible MAGL inhibitor, is used as positive control. The relative intensity was compared to that of the DMSO-treated proteomes, which was set to 100%. All data were acquired in average of 3 runs. The percentage of enzyme activity remaining was determined by measuring the integrated optical intensity of the fluorescent protein bands using image lab 5.2.1. Binding Affinities to CB1 and CB2 Receptors. CB1 and CB2 binding profiles of 8, 17, and 37 were determined according to published literatures72,73 and supported by the National Institute of Mental Health’s Psychoactive Drug Screening Program. The detailed procedures of “assay protocol book” are listed on the website (https://pdspdb.unc.edu/pdspWeb/). Compound CP55940 was used as positive control in CB1/CB2 agonist assays. Rimonabant was used as positive control in CB1 antagonist assay, and SR144528 was used as positive control in CB2 antagonist assay. All data were acquired in an average of 3−5 runs. The results are shown in Table 2, and the corresponding dose−response curves are shown in Figure 4. Molecular Docking Studies. Candidate compounds 8, 17, and 37 were docked onto the selected protein structure (PDB ID: 3PE6) using the Autodock Vina module in the UCSF Chimera software. Briefly, the mol2 file of protein was prepared by deleting the solvent and adding hydrogen atoms and charges using the default settings. Standard residues were determined according to the AMBER 14 force field, whereas nonstandard residues were ignored. Note that the predicted free energy of binding was calculated in AutoDock Vina using a hybrid scoring function (combined knowledge-based and empirical approaches).74 Measurement of the Distribution Coefficient (Log D) (“Shake Flask Method”). The general procedure for lipophilicity measurement was described previously39,41 with minor modification in this work. Briefly, the measurement of Log D values was carried out by mixing the test compound (20 μM in DMSO, 50 μL) with noctanol (475 μL) and PBS (475 μL) in a test tube. Both n-octanol and PBS were presaturated with each other prior to use. After vortexing for 1 min, the tube was shaken at 37 °C overnight. PBS phase and n-octanol phase were aliquoted (200 μL each). The amount of the test compound in each phase was determined by Agilent 6430 Triple Quad LC/MS. LogD was calculated by Log [the ratio between the amount of test compound in n-octanol and PBS]. Radiochemistry: Radiosynthesis of [11C]8 (48) Using [11C]COCl2 (1,1,1,3,3,3-Hexafluoropropan-2-yl 3-(4-(Thiazole-2-carbonyl)piperazin-1-yl)azetidine-1-carboxylate-11C). The general procedure for [11C]COCl2 formation was described previously39,41 with minor modification in this work. Briefly, [11C]CO2 was produced by 14N(p, α)11C nuclear reactions in a cyclotron and transferred into a preheated methanizer packed with nickel catalyst at 400 °C to generate [11C]CH4, which was subsequently reacted with chlorine gas at 560 °C to produce [11C]CCl4. [11C]COCl2 was generated by reacting [11C]CCl4 with iodine oxide and sulfuric acid39 and trapped in a solution of hexafluoroisopropanol (5.00 mg) and 1,2,2,6,63348 DOI: 10.1021/acs.jmedchem.8b01778 J. Med. Chem. 2019, 62, 3336−3353 Journal of Medicinal Chemistry Article pentamethylpiperidine (PMP; 5.4 μL) in THF (200 μL) at 0 °C. A solution of piperazinyl azetidine 7 (1.00 mg) and PMP (1.0 μL) in THF (200 μL) was added into the mixture and heated at 30 °C for 3 min before cooling to ambient temperature. The reaction mixtures were concentrated to remove THF, then diluted with HPLC mobile phase (500 μL), followed by injection into an HPLC column. HPLC purification was performed on a Capcell Pak C18 column (10 × 250 mm, 5 μm) using a mobile phase of CH3CN/H2O + 0.1% Et3N (55/ 45, v/v) at a flowrate of 5.0 mL/min. The retention time of 48 was 8.5 min. The product solution was concentrated by evaporation and reformulated in a saline solution (3 mL) containing 100 μL of 25% ascorbic acid in sterile water and 100 μL of 20% Tween 80 in ethanol. (Note: We added ascorbic acid to prevent potential radiolysis and Tween 80 to improve aqueous solubility.) The radiochemical and chemical purity were measured by an analytical HPLC (Capcell Pak C18, 4.6 × 250 mm, 5 μm). The identity of 48 was confirmed by coinjection with unlabeled 8. The radiochemical yield was 17.8 ± 10.7% (n = 6) decay-corrected based on starting [11C]CO2 with >
99% radiochemical purity and the molar activity was 29.7−52.4 GBq/
μmol (0.83−1.41 Ci/μmol).
Radiosynthesis of [11C]8 (48) Using [11C]CO2 (1,1,1,3,3,3Hexafluoropropan-2-yl 3-(4-(thiazole-2-carbonyl)piperazin-1-yl)azetidine-1-carboxylate-11C). The general experimental section was
described previously75 with minor modification as follows: (a) Setup:
Prior to the start of synthesis, a solution of a dehydrating (0.2 μL,
POCl3) agent in MeCN (100 μL) was loaded into the reagent loop
using a 1 mL syringe. Furthermore, to the same loop, an additional
amount of MeCN (800 μL) was added. A solution of the piperazinyl
azetidine precursor 7, BEMP (2.5 μL) in MeCN (40−50 μL), was
loaded onto the reactor loop (section A). Furthermore, a solution of
HFIP (50 μL) in MeCN (50 μL) was loaded onto another part of the
reactor loop (section B). To enable reagent injection to the different
sections of the reactor loop, the inlet nuts on union connectors were
replaced by a needle injection port. It is important to note that prior
to use, all solutions were degassed with a helium flow for 15 min and
kept over 4 Å molecular sieves to minimize the introduction of
atmospheric CO2. (b) Trapping and reaction (synthetic procedure).
[11C]CO2 was produced by 14N(p, α)11C nuclear reactions in a
cyclotron, then transferred into a liquid nitrogen trap of [11C]CO2fixation apparatus. The stainless-steel tube was removed from the
liquid nitrogen by an air-pressure-driven lift, whereby [11C]CO2 was
passed through the coated reaction loop in a controlled stream of N2
(20 mL/min). When radioactivity peaked in the reaction loop, as
measured by the proximal radiation detector (typically within 2 min),
the flow of N2 was stopped and the reaction was allowed to proceed
for an additional 1 min. Following the [11C]carbamate-ion formation,
the position of the injection valve was switched and the content of the
reagent loop was eluted into the reactor loop by a stream of nitrogen
(80 mL/min), where the dehydration reagent (POCl3) reacts with
[11C]carbamate ion to form an [11C]isocyanate. Finally, the
[11C]anhydride reacts with the HFIP in section B of the reactor
loop, to form the final 11C-labeled product 48. The crude reaction
mixture is then eluted to an empty receiving vial preloaded with 3.5
mL of water and injected into an HPLC column. HPLC purification
was performed on a ACE 5 C-18-HL column (10 × 250 mm, 5 μm,
Waters) using a mobile phase of CH3CN/0.1 M CH3CO2NH4 (55/
45, v/v) at a flowrate of 5.0 mL/min. The retention time of 48 was 12
min. The radiochemical and chemical purity were measured by an
analytical HPLC (XSelect HSS T3, 3.5 μm, 4.6 × 150 mm, Waters).
The identity of 48 was confirmed by the coinjection with unlabeled 8.
The radiochemical yield was 2.5 ± 0.4% (n = 2) decay-corrected
based on starting [11C]CO2 with >99% radiochemical purity, and the
molar activity was 288.6 ± 7.4 GBq/μmol (7.8 ± 0.2 Ci/μmol).
Radiosynthesis of [11C]17 (49) (11C-Carbonyl-Labeled (4-(1-(4′Chloro-2-(methoxy- 11 C)-3′-(trifluoromethyl)-[1,1′-biphenyl]-4carbonyl)azetidin-3-yl)piperazin-1-yl)(thiazol-2-yl)methano-ne).
The general procedure for [11C]CH3I formation was described
previously76 with minor modification in this work. Briefly, [11C]CH3I
was synthesized from cyclotron-produced [11C]CO2, which was
produced by 14N(p, α)11C nuclear reaction. [11C]CO2 was bubbled
into a solution of LiAlH4 (0.4 M in THF, 300 μL). After evaporation,
the remaining reaction mixture was treated with hydroiodic acid (57%
aqueous solution, 300 μL). The resulting [11C]CH3I was transferred
under helium gas with heating into a precooled (−15 to −20 °C)
reaction vessel containing the phenolic precursor 47 (1.0 mg), NaOH
(3.7 μL, 0.5 M), and anhydrous DMF (300 μL). After the
radioactivity reached a plateau during transfer, the reaction vessel
was warmed to 70 °C and maintained for 5 min. CH3CN/H2O +
0.1% Et3N (v/v, 60/40, 0.5 mL) was added to the reaction mixture,
which was then injected to a semipreparative HPLC system. HPLC
purification was completed on a Capcell Pak UG80 C18 column (10
mm i.d. × 250 mm) using a mobile phase of CH3CN/H2O + 0.1%
Et3N (v/v, 60/40) at a flow rate of 5.0 mL/min. The retention time
for 49 was 9.8 min. The radioactive fraction corresponding to the
desired product was collected in a sterile flask, evaporated to dryness
in vacuo, and reformulated in a saline solution (3 mL) containing 100
μL of 25% ascorbic acid in sterile water and 100 μL of 20% Tween 80
in ethanol. (Note: we added ascorbic acid to prevent potential
radiolysis and Tween 80 to improve aqueous solubility.) The
synthesis time was ∼30 min from the end of bombardment.
Radiochemical and chemical purity were measured by analytical
HPLC (Capcell Pak UG80 C18, 4.6 mm i.d. × 250 mm, UV at 254
nm; CH3CN/H2O + 0.1% Et3N (v/v, 60/40) at a flow rate of 1.0
mL/min, retention time: 9.5 min). The identity of 49 was confirmed
by the coinjection with unlabeled 17. Radiochemical yield was 25 ±
8% (n = 3) decay-corrected on the basis of [11C]CO2 with > 99%
radiochemical purity, and the molar activity was greater than 92.5
GBq/μmol (2.5 Ci/μmol).
Radiosynthesis of [18F]37 (50) ((4-(1-(4-(3-(Fluoro-18F)pyrrolidin1-yl)benzoyl)azetidin-3-yl)piperazin-1-yl)(thiazol-2-yl)methanone).
The cyclotron-produced [18F]F− was separated from H218O using the
Sep-Pak Accell Plus QMA Plus Light cartridge (Waters; Milford,
MA). The produced [18F]F− was eluted from the cartridge with a
mixture of aqueous K2CO3 (4 mg in 200 μL) and a solution of
4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8,8,8]hexacosane (Kryptofix222; 7.5 mg) in CH3CN (200 μL) and transferred to a reaction
vessel in the hot cell. After drying [18F]KF solution at 120 °C for 30
min to remove water and CH3CN, a solution of mesylate precursor 50
(2.0 mg) in anhydrous DMSO(300 μL) was then added. The vessel
was heated at 120 °C for 10 min, then diluted with HPCL mobile
phase (500 μL), followed by injection into an HPLC column. HPLC
purification was performed on a X Bridge Prep C18 column (10 ×
250 mm, 5 μm) using a mobile phase of CH3CN/H2O/Et3N (30/70/
0.1) at a flow rate of 5.0 mL/min. The reaction time of 50 was 13.5
min. The radioactive fraction corresponding to the desired product
was collected in a sterile flask, evaporated to dryness in vacuo, and
reformulated in a saline solution (3 mL) containing 100 μL of 25%
ascorbic acid in sterile water and 100 μL of 20% Tween 80 in ethanol.
(Note: we added ascorbic acid to prevent potential radiolysis and
Tween 80 to improve aqueous solubility.) The synthesis time was 71
min from the end of bombardment. Radiochemical and chemical
purity were measured by analytical HPLC (X Bridge Prep C18
column (4.6 × 250 mm, 5 μm) using a mobile phase of CH3CN/H2O
+ 0.1% Et3N (30/70) at a flow rate of 1.0 mL/min. The identity of 50
was confirmed by the coinjection with unlabeled 37. Radiochemical
yield was 39.3 ± 13.7% (n = 5) decay-corrected on the basis of
[18F]F− with > 99% radiochemical purity, and the molar activity was
111.8−328.7 GBq/μmol (3.03−8.87 Ci/μmol).
Small-Animal PET Imaging Studies. The general procedure for
PET studies was described previously39,41 with minor modification in
this work. Briefly, PET scans were carried out by an Inveon PET
scanner (Siemens Medical Solutions, Knoxville, TN). Sprague−
Dawley rats were kept under anesthesia using 1−2% (v/v) isoflurane
during the scan. The radiotracer (ca. 1 mCi/150 μL) was injected into
the tail vein via a preinstalled catheter. A dynamic scan in 3D list
mode was acquired for 90 min. For pretreatment studies, a solution of
KML29 (3 mg/kg) in 300 μL of saline containing 10% ethanol and
5% Tween 80 was injected via the pre-embedded tail vein catheter at
30 min prior to tracer injection. As we previously reported,39,77,78 the
PET dynamic images were reconstructed using ASIPro VW software
3349
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J. Med. Chem. 2019, 62, 3336−3353
Journal of Medicinal Chemistry
Article
(Analysis Tools and System Setup/Diagnostics Tool, Siemens
Medical Solutions). Volumes of interest, including the whole brain,
hippocampus, cerebral cortex, cerebellum, striatum, thalamus, and
pons were placed using ASIPro software. The radioactivity was decaycorrected and expressed as the standardized uptake value. SUV =
(radioactivity per mL tissue/injected radioactivity) × body weight.
Ex Vivo Whole Body Biodistribution of 48−50 in Mice. The
general procedure for ex vivo biodistribution studies was described
previously39,41 with minor modification in this work. Briefly, a
solution of 48−50 (50 μCi/150−200 μL) was injected into DdY mice
via tail vein. These mice (at each time point n = 4) were sacrificed at
1, 5, 15, 30, and 60 min post tracer injection. Major organs, including
whole brain, heart, liver, lung, spleen, kidneys, small intestine
(including contents), muscle, testes, and blood samples, were quickly
harvested and weighted. The radioactivity present in these tissues was
measured using 1480 Wizard γ counter (PerkinElmer), and all
radioactivity measurements were automatically decay-corrected on the
basis of the half-life of carbon-11 or fluorine-18. The results are
expressed as a percentage of the injected dose per gram of wet tissue
(% ID/g).
Radiometabolite Analysis. The general procedure for radiometabolite analysis was described previously39,41 with minor
modification in this work. Briefly, following the intravenous injection
of tracers 48−50, CD-1 mice were sacrificed at 30 min (n = 2). Either
whole brain (for 48) or blood (for 49 and 50) samples were quickly
removed and the blood samples were centrifuged at 15 000g for 2 min
at 4 °C to separate the plasma. The supernatant (0.5 mL) was then
collected in a test tube containing CH3CN (0.5 mL), and the
resulting mixture was vortexed for 15 s and centrifuged at 15 000g for
2 min for deproteinization. The rat brain was homogenized in an icecooled CH3CN/H2O (1 mL, 1/1, v/v) solution. The homogenate
was centrifuged at 150 000 rpm for 2 min at 4 °C, and the supernatant
was collected. The recovery of radioactivity into the supernatant for
all three tracers 48−50 was > 90% based on the total radioactivity in
the brain or blood homogenate. An aliquot of the supernatant (100
μL) obtained from the plasma or brain homogenate was injected into
the HPLC system together with unlabeled 8, 17, or 37 and analyzed
using a Phenomenex C18 column (10.0 mm ID × 250 mm). The
radioactivity collected was measured by a 1480 Wizard γ counter
(PerkinElmer). The percentage of 48, 49, or 50 to the total
radioactivity was calculated as (counts for 48, 49, or 50/total counts)
× 100.
■
Benjamin F. Cravatt: 0000-0001-5330-3492
Ming-Rong Zhang: 0000-0002-3001-9605
Steven H. Liang: 0000-0003-1413-6315
Author Contributions
○
Z.C. and W.M. contribute equally to this work. The
manuscript was written through contributions of all authors.
All authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
We thank Dr. Lei Zhang (Medicine Design, Pfizer, Inc.) and
Professor Thomas J. Brady (Nuclear Medicine and Molecular
Imaging, Radiology, MGH and Harvard Medical School) for
helpful discussion. We also acknowledge the National Institute
of Mental Health’s Psychoactive Drug Screening Program
(NIMH PDSP; directed by Bryan L. Roth at the University of
North Carolina at Chapel Hill and Jamie Driscoll at NIMH)
for in vitro screening. We thank Mona Svensson for expert
technical assistance in the [3H]2-OG hydrolysis studies.
Financial support from the NIH grants (DA038000 and
DA043507 to S.L., AG054473 to N.V., and DA033760 to
B.F.C.), CSC scholarship to Z. C. (Grant No. 201606250107)
and the Swedish Science Research Council (Grant 12158,
Medicine to C.F.) is gratefully acknowledged.
■
ABBREVIATIONS
MAGL, monoacylglycerol lipase; eCB, endocannabinoid
system; CNS, central nervous system; 2-AG, 2-arachidonylglycerol; AEA, anandamide; FAAH, fatty acid amide hydrolase; 2OG, 2-oloeylglycerol; PET, positron emission tomography;
SUV, standardized uptake value; TAC, time−activity curve; %
ID/g, the percentage of injected dose per gram of wet tissue;
PgP, P-glycoprotein; Bcrp, breast cancer resistance protein
■
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S Supporting Information
*
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmedchem.8b01778.
Experimental procedures, characterization and NMR
spectra of compounds 3, 4, 6, 7, 15b−g, 24−35, 40,
42−47, 51b−g, and 52, and supporting figures and
tables (PDF)
Molecular formula strings and some data (CSV)
■
REFERENCES
AUTHOR INFORMATION
Corresponding Authors
*E-mail: zhang.ming-rong@qst.go.jp. Tel: +81 433 823 709.
Fax: +81-43-206-3261 (M.R.Z.).
*E-mail: liang.steven@mgh.harvard.edu. Tel: +1 617 726
6107. Fax: +1-617-726-6165 (S.H.L.).
ORCID
Zhen Chen: 0000-0002-6289-4332
Michael A. Schafroth: 0000-0002-5864-6766
Neil Vasdev: 0000-0002-2087-5125
Yihan Shao: 0000-0001-9337-341X
Jun-An Ma: 0000-0002-3902-6799
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