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Cite This: J. Med. Chem. 2019, 62, 3336−3353
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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
J. Med. Chem. 2019, 62, 3336−3353
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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DOI: 10.1021/acs.jmedchem.8b01778
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Journal of Medicinal Chemistry
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%
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Journal of Medicinal Chemistry
Article
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
J. Med. Chem. 2019, 62, 3336−3353
Journal of Medicinal Chemistry
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 dete …
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