Discovery of Aryl Formyl Piperidine Derivatives as Potent, Reversible, and Selective Monoacylglycerol Lipase Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.9b02137

Most of the current monoacylglycerol lipase (MAGL) inhibitors function by an irreversible mechanism of action, causing a series of side effects. Herein, starting from irreversible inhibitors, 25 compounds were synthesized and evaluated in vitro for MAGL inhibition, among which, compound 36 showed the most potent inhibitory activity (IC50 = 15 nM). Crucially, docking studies demonstrated that the m-chlorine-substituted aniline fragment occupied a hydrophobic subpocket enclosed by side chains of Val191, Tyr194, Val270, and Lys273, which creatively identify a new key anchoring point for the development of new MAGL inhibitors. Furthermore, in vivo evaluation innovatively revealed that this reversible inhibitor 36 significantly ameliorated depressive-like behaviors induced by reserpine. To the best of our knowledge, this is the first time that reversible inhibitors of MAGL were developed to support MAGL as a potential therapeutic target for depression.

Full text of DOI:10.1021/acs.jmedchem.9b02137

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Article
Discovery of Aryl Formyl Piperidine Derivatives as Potent,
Reversible, and Selective Monoacylglycerol Lipase Inhibitors
Zhuoer Zhi, Wenting Zhang, Jingchun Yao, Yanguo Shang, Qingjing
Hao, Zhong Liu, Yushan Ren, Jie Li, Guimin Zhang, and Jinxin Wang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b02137 • Publication Date (Web): 20 May 2020
Downloaded from pubs.acs.org on May 20, 2020
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Discovery of Aryl Formyl Piperidine
Derivatives as Potent, Reversible, and
Selective Monoacylglycerol Lipase Inhibitors
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Zhuoer Zhi a,1, Wenting Zhang a,1, Jingchun Yao , Yanguo Shang , Qingjing
b
a
Hao , Zhong Liu , Yushan Ren , Jie Li , Guimin Zhang b, *, Jinxin Wang a, *
a
b
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a
Jiangsu Key Laboratory of Drug Design and Optimization, Department of Medicinal
Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing, 211198,
China
b
Lunan Pharmaceutical Group Corporation, Linyi, Shandong, 276006, China
Abstract
Most of the current MAGL inhibitors function by an irreversible mechanism of
action, causing a series of side effects. Herein, starting from irreversible inhibitors, 25
compounds were synthesized and evaluated in vitro for MAGL inhibition, among
which, compound 36 showed the most potent inhibitory activity (IC50 = 15
nM).Crucially, docking studies demonstrated that the m-chlorine-substituted aniline
fragment occupied a hydrophobic sub-pocket enclosed by side chains of Val191,
Tyr194, Val270, and Lys273, which creatively identify a new key anchoring point for
the development of new MAGL inhibitors. Furthermore, in vivo evaluation
innovatively revealed that this reversible inhibitor 36 significantly displayed the
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depressive-like behaviors induced by reserpine. To the best of our knowledge, this is
the first time that reversible inhibitors of MAGL were developed to support MAGL as
a potential therapeutic target for depression.
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Introduction
Clinical depression is a prevalent psychiatric disorder, and its incidence of
diagnosis is increasing. Although many antidepressants such as monoamine oxidase
inhibitors, tricyclic antidepressants, and serotonin reuptake inhibitors are in current
clinical use, the efficacy of these drugs is unsatisfactory, which may reflect the
1
complicated pathogenesis of depression. Therefore, the research for antidepressants
with new mechanisms of action is a compelling line of investigation.
The endocannabinoid system (ECS), which characterized by a series of
neuromodulatory lipids and their receptors (Figure 1), modulates various physiological
and pathological processes such as pain, inflammation, anxiety, depression, memory,
2
and emotion. Anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are the two
major endocannabinoids that are capable of activating the G protein-coupled
cannabinoid receptors, cannabinoid receptor 1 (CB ), and cannabinoid receptor 2
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(
CB2).3-4 Some studies have shown that monoacylglycerol lipase (MAGL) is
responsible for approximately 85% of 2-AG hydrolysis in brain, which is a potential
therapeutic target for many physiological processes, including the neurodegenerative
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diseases, depression, etc, and the remaining 15% of 2-AG is degraded by α/β-
hydrolase domain-containing 6 (ABHD6) and α/β-hydrolase domain-containing 12
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(ABHD12) , 6-7 while Fatty acid amide hydrolase (FAAH) hydrolyzes AEA into
arachidonic acid (AA) and ethanolamine. 8-9
Even though numerous potent and selective agonists for cannabinoid receptors
have been discovered over the last two decades, this class of compounds suffers from
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clinical limitations, mainly because of the direct CB -mediated side effects. Hence,
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the strategy to indirectly activate ECS by increasing 2-AG levels in brain through
inhibition of MAGL has emerged.11-12 Furthermore, pharmacological inhibition of
MAGL has been strongly manifested physiologically by decreasing pain, inflammation,
anxiety, and depression in rodent models without the undesired side effects observed
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with direct CB agonists. Therefore, greater attention has been focused on the
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development of MAGL inhibitors.
Great efforts have been applied to obtain potent MAGL inhibitors in the past 10
years. Most of these compounds are irreversible (see Figure 2), forming covalent
carbamylated enzyme adducts with Ser122. Although these compounds inhibit MAGL
with high activity and some of them have been considered effective in animal models,
side effects caused by sustained activation of CB may occur because of the permanent
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blockade of the MAGL. Taking into account all the negative effects, developing
inhibitors that could temporarily inhibit the enzyme in a reversible manner has become
a significative challenge in this field. It has been encouraging to observe the number of
reversible MAGL inhibitors reported in the past few years (Figure 3).15-21 In 2014,
Martinelli et al. reported a reversible inhibitor 12 (IC = 11.7 μM), and subsequently
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activity in 2018. Even though these inhibitors exhibit good properties of reversibility
and selectivity, at present the studies of the detail docking binding mode and structure-
activity relationships of reversible MAGL inhibitors were not deep enough. And the
need for further exploration still remains.
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ethanolamine arachidonic acid
glycerol
Figure 1 Endogenous cannabinoid pathway.
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Figure 2 Structures of representative irreversible MAGL inhibitors.
Figure 3 Structures of representative reversible MAGL inhibitors.
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Results and discussion
Design and Molecular Modeling.
These reported irreversible MAGL inhibitors share the common feature of the
presence of two H-bonds between the carbonyl oxygen and the nitrogen backbone of
Ala51 and Met123 in the oxyanion hole, immediately adjacent to the catalytic Ser122
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residue of the MAGL enzyme. They usually contain a carbamate or urea moiety in
combination with a high potency leaving group. The hydroxyl group of Ser122
nucleophilic attacks the urea or carbamate moiety of the inhibitor to produce a
carbamylated enzyme adduct, accompanied by the release of the leaving group,
producing a sustained, irreversible inhibitory effect. Analysis of the data related to
available reversible MAGL inhibitors indicates several common features: (a) a carbonyl
group forming at least one hydrogen bond with the nitrogen backbone of Ala51 and
Met123 in the oxyanion hole; (b) an aromatic portion entering into a large hydrophobic
cavity of the enzyme; and (c) a second aromatic portion, which inserts into a narrow
closed hydrophobic pocket, forming a π-π stacking interaction with Tyr194.
Compounds with these properties usually have good inhibitory activity and thus, these
features can guide the design of novel inhibitors.
Following this binding analysis, we used compounds 2 (MJN110) and 4 (SAR629)
(Figure 2) as starting hits. The first round of structural investigation held the simple
scaffold fixed and replaced (1) one carbon atom of the central piperazine moiety with
a nitrogen atom, and (2) substituted the leaving group with either aniline or a substituted
aniline (Figure 4). We expected to convert irreversible inhibitors into reversible
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inhibitors by reducing the reactivity of the compound through these modifications
(Table 1). However, we were disappointed to note that this series of compounds did not
show effective inhibitory activity against human MAGL and only compound 19 had
moderate inhibitory activity. As shown in Figure 5A, the most energetically favorable
binding mode shows that the amidic carbonyl group of 19 formed two H-bonds with
the nitrogen backbone of Ala51 and Met123. The piperidyl and the two 4-fluorophenyl
fragments were directed toward an open cavity of the protein and showed lipophilic
interactions with Leu148, Leu213, and Leu241. Moreover, the two fluorine atoms
formed two halogen bonds with Pro178 and Gly210, respectively. However, the 3-
methoxyaniline moiety could not be inserted into a narrow hydrophobic pocket of the
protein. After analyzing the binding mode of 19 with MAGL, we found that the whole
conformation is more inclined toward the large hydrophobic cavity and away from the
narrow hydrophobic pocket. This configuration may explain why the activity of this
series was unsatisfactory.
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Figure 4 First round of structural investigation from 2 and 4.
Table 1. Inhibitory Activities of 14-19 against human MAGL
R1
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N
H
compound
R1
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H
IC (μM)
50
14
H
H
>>100
>>100
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3,5-di-Me
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H
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H
>>100
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3,5-di-Me
3-OMe
>>100
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2.465 ± 0.230
0.2045 ± 0.011
JZL195
Data are expressed as means ± SD of 3 experiments.
To address this deficiency, we designed a new series of compounds based on 19.
Another carbonyl was introduced at the central piperidinyl moiety to form an amide
group, and the two aromatic rings forming a Y configuration were replaced by one
single aromatic ring. Once the scaffold was confirmed, different substituents (OH, OMe,
F, Cl) were introduced into two aromatic rings (20-35).
We were delighted to observe that most of these compounds exhibited a significant
increase in their inhibitory activity as compared to 19. Notably, the inhibitory activity
decreased as long as there was a chlorine substituent at the para-position of the benzoyl
moiety (20, 21 and 31), with IC values ranging from 1.9 to 4.0 μM. On the other hand,
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in compounds 23, 24 and 25, an ortho-hydroxyl group led to a sharp decrease of activity
IC value of 10.81 μM for 24). This outcome may be due to the intramolecular H-
(
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bond formed between the hydroxyl and the amide, which limits the conformation of the
compounds in MAGL. Compound 26 confirmed this hypothesis with no inhibitory
activity detected.
Among the 16 reported derivatives, compound 28 with the 3-methoxybenzoyl moiety
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and m-chloroaniline moiety showed a high inhibition activity (IC = 0.33 μM), about
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-fold more potent than the starting compound 19. To better rationalize the activity of
this compound, 28 was subjected to docking calculations. The carbonyl group of the
benzamide, rather than another amide group, forms two H-bonds with the nitrogen
backbone of Ala51 and Met123 (Figure 5B). Accordingly, the m-chlorine-substituted
aniline moiety of 28 is inserted into the closed hydrophobic region of the binding site
and shows lipophilic interactions with Tyr194, Val270, Lys273 and a π-π interaction
with Tyr194 due to its sufficient molecular length. Meanwhile, the 3-methoxybenzoyl
moiety that is directed toward the open cavity of the protein shows lipophilic
interactions with Leu148, Leu213, and Leu241. It is worth noting that the chlorine
atom shows hydrophobic interaction with Val191 and Tyr194 as well, and this might
be one of the critical factors for the high activity. Comparing compounds 28 and 30,
we observed that the replacement of the hydroxyl with the more hydrophobic methoxy
group, which is inserted deeper into the hydrophobic cavity, led to very positive effects.
Similar results were also obtained for compounds 22 and 23. Both results supported
the important roles of a larger hydrophobic group on the benzoyl moiety and the m-
chlorine atom on the aniline moiety. In particular, the activities were reduced in
different degrees when replacing a chlorine atom with fluorine (IC = 12.17 μM for
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32) or introducing another halogen (IC values in the range 1.774-4.026 μM for 33,
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4 and 35).
Table 2. Inhibitory Activities of 20-35 against human MAGL.
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R1
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compound
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3-OH
4-Cl
IC (μM)
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20
4-Cl
4-Cl
4.073 ± 0.728
1.963 ± 0.084
0.5757 ± 0.069
1.335 ± 0.150
10.81 ± 0.801
0.6621 ± 0.074
>>100
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1
22
3-OMe
3-OH
2-OH
4-OH
2-OH
3-OH
3-OMe
3-OMe
4-OH
4-Cl
2
2
3
4
4-Cl
4-Cl
25
4-Cl
2
6
3-Cl
27
2-OMe
3-Cl
0.9263 ± 0.140
0.3331 ± 0.043
>>100
2
2
8
9
3-OH
3-Cl
30
9.923 ± 1.759
>>100
3
1
3-Cl
32
3-OH
3-OH
3-OH
3-OH
3-F
12.17 ± 0.455
1.774 ± 0.012
4.026 ± 0.713
2.922 ± 0.324
3
3
3
4
3,4-di-Cl
3,5-di-Cl
3-F,4-Cl
35
Data are expressed as means ± SD of 3 experiments.
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5
5
5
5
5
5
5
5
6
In order to verify the hypothesis above, we designed another series of
compounds based on 28. A remarkable increase of inhibitory activity was
observed when benzene was replaced with naphthalene (36) (IC50 values of
0.01475 μM) which permitted occupancy of the larger hydrophobic cavity. To
further investigate our conjecture, a molecular docking study was conducted.
As shown in Figure 5C, the carbonyl group of the diphenyl ether formyl of
compound 36 forms two H-bonds with the nitrogen backbones of Ala51 and
Met123. Similar to compound 28, the m-chlorine-substituted aniline moiety of
36 is inserted inside the closed hydrophobic region of the binding site and
shows lipophilic interactions with Val191, Tyr194, Val270, Lys273 and the π-π
interaction with Tyr194. Meanwhile, the chlorine atom shows hydrophobic
interactions with Val191 and Tyr194. With respect to the naphthalene group,
the larger hydrophobic cavity is occupied and lipophilic interactions are formed
with Leu148, Leu213, and Leu241. These results support the importance of the
larger hydrophobic group on the benzoyl moiety and the m-chlorine atom on
the aniline moiety. The ADME properties of all designed compounds were predicted
the drugability as CNS drugs by DS 3.0.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. Inhibitory Activities of 36-38 against human MAGL
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R1
O
N
R2
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O
N
H
compound
R1
R2
IC (μM)
50
3
6
3-Cl
0.01475 ± 0.003
3
3
7
8
3-Cl
3-Cl
2.819 ± 0.163
7.328 ± 1.625
O
Data are expressed as means ± SD of 3 experiments.
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8
9
0
Figure 5 Docking results of compound 19 (A), 28 (B) and 36 (C) into hMAGL (PDB
code 3PE6).
Reversibility Analysis.
With the aim of establishing whether the mechanism of inhibition was reversible or
irreversible, the effect of dilution and preincubation time on the inhibitory activity of
23
compound 36 were evaluated. In the dilution assay, the inhibition produced by
preincubation with a 1 μM concentration of compound 36 was measured after a 10X
dilution and compared with the potency of a 1 μM and a 0.1 μM of compound 36. A
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reversible inhibitor will gradually reduce the potency level after dilution. In contrast, in
the case of an irreversible inhibition, the potency should not decrease after dilution. As
expected in Figure 6A, the inhibition efficiency of 0.1 μM was similar to that after a
10X dilution with 1 μM and was different to that produced by a 1 μM concentration of
compound 36, thus this assay confirmed the reversible property of compound 36.
In preincubation experiment, compound 36 was preincubation with the enzyme for
1
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0
, 30 and 60 min before adding the substrate to start the enzymatic reaction. An
irreversible inhibitor should show a higher potency after longer incubation times,
whereas a reversible inhibitor should show a constant potency and not change with
incubation time. As shown in Figure 6B, the results were consistent with a reversible
inhibition mechanism, as it did not show any significant increase in inhibitory potency
at longer incubation times.
Figure 6 (A) Dilution assay: the first two columns indicate the inhibition percentage of
compound 36 at a concentration of 1 μM and 0.1 μM, and the third column indicates
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the inhibition percentage of compound 36 after dilution (final concentration = 0.1 μM).
(
B) Preincubation assay: IC (nM) values of compound 36 at different preincubation
50
times with MAGL (0 min, 30 min and 60 min). Data are obtained from 3 independent
experiments.
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Vitro Kinetic Parameters
Once the reversible mechanism of compound 36 was confirmed, its mode of
inhibition was then evaluated by measuring Michaelis−Menten kinetics with various
inhibitor concentrations. The data set was drawn as substrate concentration versus
enzyme activity and analyzed by using the mixed-model inhibition fit of GraphPad
Prism software, which includes the terms of competitive, uncompetitive, and
noncompetitive inhibition. The model evaluates the Vmax, K , and the α parameter,
m
which indicate the inhibition mechanism. When α corresponds to one, the inhibitor does
not alter the binding of the substrate to the enzyme, and the mixed-model can be
considered as a noncompetitive inhibition. When α is very large, the interaction of
inhibitor prevents the binding of substrates and the mixed-model corresponds to a
competitive inhibition. Finally, when α is very small, the binding of the inhibitor
increases the binding of the substrate and the mixed-model corresponds to an
uncompetitive model. Kinetic studies indicate for 36 a Ki value of 13.4 ± 0.2 nM and
an α value greater than 10000, thus supporting a competitive behavior for compound
36 (Figure 7).
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0
Figure 7 Inhibition of the activity of MAGL and competitive nature of compound 36.
4
-NPA = 4-nitrophenylacetate.
Selectivity Analysis.
Once the reversible mechanism of compound 36 was confirmed, the selectivity of
its activities against FAAH (another major endocannabinoid degrading enzyme) was
tested. As shown in Table 4, compound 36 did not display any inhibition on FAAH at
the concentration of 100 μM (41.34%). On the other hand, the biological activity of
compound 36 for the cannabinoid receptors CB and CB were also evaluated. As
1
2
reported in Table 4, this compound did not show a significant binding to CB and CB
1
2
receptors. Thus, the tested compound 36 displayed excellent MAGL selectivity (6780
times more selective than FAAH).
Table 4. Biological activity of compound 36 on the key enzymes in the
endocannabinoid systema
compound
MAGL
FAAH
CB receptor
CB receptor
2
1
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36
0.01475 ±0.0027
>100 (41.34%)
>10 (29.71%)
>10 (20.35%)
a
Effects on FAAH is expressed as IC values (mean ± SD, μM), effects on CB and
50
1
CB receptors are expressed as EC values (mean ± SD, μM). Data are expressed as
2
50
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means ± SD of 3 experiments.
Liver microsome stability Analysis.
Three species of liver microsomes, including human, mouse, and rat, were used to
study the stability of compound 36 in vitro. The remaining substrates in reaction
systems at different time points were illustrated in Figure 8A. Stability of inhibitors
exhibited species difference in different liver microsomes. Content of compound 36 in
mouse and rat microsome were 57.87% and 17.31%, respectively, after 60 min of
incubation. Despite the unsatisfactory stability in mouse and rat liver microsomes,
content of compound 36 in human microsome maintained a high level (88.46%) after
60 min of incubation, which suggested the good stability in human liver microsomes.
Cell Viability Assay
To investigate the function of MAGL inhibitor on cell activity, mouse fibroblasts
(
L929) were used. The results showed that compound 36 did not inhibit L929 cells
growth obviously at 24, 48 and 72 hours, which suggested that 36 displayed a very low
toxicity in vitro. At 20 μM, 36 could only inhibit L929 at 8.6%. (Figure 8B)
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Figure 8 (A) Content of compound 36 in human, mouse, rat liver microsomes. (B)
Cytotoxicity of Compound 36 in Mouse Fibroblasts. Data are obtained from 3
independent experiments.
Pharmacokinetic Analysis
The experiments were determined in SD rats, and relevant pharmacokinetic
parameters are reported in Table 5. Upon intragastric administration of a single dose of
compound 36 at 10 mg/kg in rats, the values of Cmax and Tmax achieved in plasma were
83.122 μg/L, 34 min, respectively, which indicate compound 36 had a fast onset time.
Compared with intravenous injection, the plasma half-life (t ) of intragastric
1
/2
administration was longer (about 5 h), which showed the effect of compound 36 lasted
long in vivo (Figure 9A). In addition, oral bioavailability was observed dosing
compound 36 at 10 mg/kg (F = 21.6%).
Crossing the blood-brain-barrier was critical to whether the central nervous system
drugs could produce efficacy. Compound 36 was evaluated for its BBB penetration by
intragastric administration in rats. Concentrations of 36 in brain were quantified by LC-
MS/MS. From Figure 9B, after intragastric administration the Tmax of compound 36
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reached the brain was short (about 40 min). And the brain half-life (t ) of intragastric
1
/2
administration was long (about 2.5 h). All these results strongly proved that 36
penetrated the BBB very well. Moreover, it was found that the relative bioavailability
of 36 in brain (F = 36.13%) was higher than in plasma (F = 21.6%).
0
1
2
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9
0
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2
3
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9
0
Table 5. Pharmacokinetic Studies of compound 36 in Rats
plasma
brain
I.G
Parameters
Units
I.V
I.G
3
3
582.71±1116.1
7
AUC(0-t) a μg/L*min 80893.0±5100.67
9913.54±2693.94
720.04±1173.0
6
AUC(0-∞) μg/L*min 82362.19±3754.91 17791.86±4103.97
t1/2z b
Tmax c
min
min
μg/L
%
65.76±6.06
---
297.74±179.93
34±6.93
146.68±34.08
40±8.66
16．65±2.9
36.13
Cmax d
5476.14±2445.01
83.122±11.91
F(0-t) e
F(0-∞)
12.25
21.6
%
---
aAUC, Area Under the Cure. b t1/2z, drug half-life. cTmax, peak time. Cmax, peak
d
e
concentration. F, bioavailability.
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Figure 9 (A) Drug concentration-time curve of plasma by intragastric injection. (B)
Drug concentration-time curve of brain by intragastric injection. Data are obtained
from two independent experiments.
In order to evaluate the plasma protein binding rate of compound 36,
ultracentrifugation was used to investigate the plasma protein binding rate of compound
3
6 in the concentration range of 20-500 ng/mL, which was 77.1% in rats (Table S2).
Compound 36 was existed in the form of protein binding compounds. There was no
concentration dependence of plasma protein binding in the range of 20-500 ng/mL.
In addition, the preliminary physicochemical properties of compound 36 was also
determined (Table S3). Lipid−water distribution coefficient (log D) and intrinsic
aqueous solubility of compound 36 was determined by the LC-MS based shake-flask
method.29
Antidepressant Effect of 36 in an Animal Model induced by Reserpine
The effect of compound 36 was evaluated in a mouse model of depressive behavior
caused by reserpine. Reserpine-induced depression model is a classic depression model
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with rapid modeling and stable effect. Thus, we decided to use this model to evaluate
the preliminary antidepressant effect of compound 36. Mice were administered
compounds 36 (4, 8 and 16 mg/kg) by subcutaneous injection (s.c.), and reserpine (4
mg/kg) was used to induce depression of mice. Control mice were administrated with
phosphate buffer (4 mg/kg) alone. The results showed that reserpine could increase
awakening, finger contact, fear, head touch, autonomous movement, straight and eyelid
closure of mice, while compound 36 can improve these indexes (Figure 10).
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Meanwhile, in forced swim test and tail suspension test, fluoxetine hydrochloride
capsules (FHC) was chosen as positive control. Mice were intragastric administered
(i.g.) with compound 36 (5 mg/kg) and FHC (5 mg/kg). Results from forced swim test
and tail suspension test proved that compound 36 could significantly shorten the
cumulative immobility time in depressive mice induced by reserpine (Table 6-7). All
these results indicated that compound 36 could improve reserpine-induced depression
in mice and that compound 36 could be effective in the treatment of depression.
Figure 10 Effect of compound 36 on awakening (A), finger contact (B), fear (C), head
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touch (D), autonomous movement (E), straight (F) and eyelid closure (G). Control,
injected with phosphate buffer (4 mg/kg) alone. Model, injected with reserpine (4
mg/kg) alone. Values are expressed as mean ± SD from 3 independent experiments. *,
P value compared to control group (*p<0.05, **p<0.01, ***p<0.001). #, P value
compared the model group (# p<0.05, # # p<0.01, # # # p<0.001).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
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0
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0
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3
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9
0
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2
3
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9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 6. Effect of compound 36 on forced swim test in depression Mice.
group
Dose (mg/kg)
Animal number
immobile time (s)
136.84±46.69
MODEL a
FHC b
0
5
5
10
10
10
92.64±29.17**
89.38±41.67**
Compound 36
a
b
Model, normal saline was injected. FHC, fluoxetine hydrochloride capsules. Values
are expressed as mean ± SD from 3 independent experiments. *, P value compared to
model group (*p<0.05, **p<0.01).
Table 7. Effect of compound 36 on tail suspension test in depression Mice.
group
Dose (mg/kg)
Animal number
immobile time (s)
132.38±44.51
MODEL a
FHC b
0
5
5
10
10
10
92.19±23.63**
89.87±29.68**
Compound 36
a
b
Model, normal saline was injected. FHC, fluoxetine hydrochloride capsules. Values
are expressed as mean ± SD from 3 independent experiments. *, P value compared to
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model group (*p<0.05, **p<0.01).
Chemistry.
The synthesis of the key intermediates 4-piperidine formylaniline hydrochlorides
8a-k started with the formation of 47a-k from the starting material N-Boc-piperidine-
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4
4-carboxylic acid by an amide coupling between partially substituted anilines in the
presence of the condensing agent HATU and DIPEA as the base in CH Cl as the
2
2
solvent. Boc-protection of the amino groups were finally deprotected by reaction with
HCl in EtOAc to obtain compounds 48a-k in good yields.
For diphenylmethyl piperidine derivatives 14-19, the starting compounds
benzophenone and bis(4-fluorophenyl)-methanone were commercially available and,
therefore, the carbonyl groups were directly reduced by sodium borohydride in absolute
ethanol as the solvent to obtain compounds 44a-b. Hydroxyl compounds 44a-b were
reacted with commercially available phosphorus tribromide in dichloromethane as the
solvent to give bromo-compounds 45a-b. Then, the key intermediates 48a-c were
substituted in the presence of hydroxyl compounds 44a-b, sodium hydride as the base,
and N,N-dimethylformamide as the solvent to give compounds 14-19.
Aryl formyl piperidine derivatives 20-42 were synthesized by an amide coupling
between properly substituted benzoic acids and properly substituted 4-piperidine
formylaniline hydrochlorides 48c-k in the presence of the condensing agent HATU and
TEA as the base in N,N-dimethylformamide as the solvent.
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Scheme 1. Synthesis of compounds 14-19α
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Reagents and conditions: (a)NaBH , EtOH, RT, 1 h; (b) PBr , CH Cl , RT, 10 min;
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(c) substituted anilines, HATU, DIPEA, CH Cl , RT, 6 h; (d) 4 M HCl in EtOAc,
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EtOAc, RT, 12 h; (e) NaH, DMF, RT, 2 h.
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Scheme 2. Synthesis of compounds 20-42α
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αReagents and conditions: (a) aromatic acids, HATU, TEA, DMF, RT, 2h.
Conclusions
Overall, starting from compounds 2 (MJN110) and 4 (SAR629), we have designed
and synthesized a new class of aryl formyl piperidine compounds, exemplified by 36,
which displayed high MAGL inhibition activity with an IC value of 15 nM as well as
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a high selectivity for MAGL vs CB , CB and FAAH (EC /IC value > 10 μM).
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Moreover, the biochemical experiments confirmed the reversible properties of
compound 36. Molecular docking and detailed SAR studies obviously gain insight into
the extended binding mode that the lipophilic interactions between the m-chlorine-
substituted aniline fragment and Val191, Tyr194, Val270, and Lys273 play a crucial
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role for inhibitory activity.
This reversible inhibitor 36 also reflected the excellent stability in human liver
microsomes and low toxicity in mouse fibroblasts. Besides, pharmacokinetic analysis
revealed that compound 36 could smoothly cross the blood-brain barrier.
Antidepressant effcet of 36 in an animal model by intragastric administration explained
that it could significantly shorten the cumulative immobility time in the forced swim
test and tail suspension test induced by reserpine, indicating its efficacy in the treatment
of depression. Furthermore, these innovative studies positively and firstly validated that
inhibiting MAGL with a reversible inhibitor can be a promising antidepressant strategy
in the future.
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Experimental Section
Synthesis: General Procedures and Materials.
All solvents and chemicals were obtained from commercially available sources
with high-grade purity and without further purification. Chromatographic separations
were carried out on silica gel columns by flash chromatography (Kieselgel 60, 200-300
mesh; Merck). Reactions were followed by thin layer chromatography (TLC) on Merck
silica gel (GF254; Germany) sheets that were visualized under a UV lamp (254 nm).
Evaporation was performed in vacuo (rotating evaporator). Anhydrous sodium sulfate
was always used as the drying agent. Melting points were determined on a MEL-TEMP
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Ⅱ apparatus (Laboratory Devices, MA, USA). Proton ( H) NMR and C NMR spectra
were obtained at a Bruker AV-300 MHz instrument (Bruker Biospin AG, Switzerland)
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spectrometer using the CDCl or DMSO-d with TMS as the internal standard.
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Higheresolution mass spectra (HRMS) were recorded on Q-Tof Premier hybrid mass
spectrometer with electron spray ionization (ESI). The purity of target compounds was
determined by HPLC (Shimadzu LC-20AT, Hanbon Sci.&Tech. Phecda C18, 5 μm,
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50 mm × 4.6 mm, 30℃, UV 245 nm, flow rate = 1.0 mL/min); Compounds were
dissolved in methanol; eluent A, methanol; eluent B, water; eluting with mixtures of
methanol/water (80:20) for 15 min; all target compounds were ≥95% pure by HPLC.
General Procedure for the Synthesis of Bromo-compounds 45a-b.
To a solution of the diphenyl ketones 43a-b (0.5 mmol) in absolute ethanol (10
mL) was added sodium borohydride (2 mmol). The resulting mixture was stirred at
room temperature for 2 h. 10% hydrochloric acid was added to the solvent and the
ethanol was evaporated under reduced pressure. The residue was diluted with water and
extracted with CH Cl . The organic layer was washed sequentially with water and
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saturated brine and dried over Na SO and the solvent was removed under reduced
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pressure to obtain compounds 44a-b without further purification. 44a-b were then
dissolved in anhydrous CH Cl (10 mL) and treated dropwise with PBr (2mmol). The
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mixture was left under stirring at room temperature for 10 min. The reaction was
quenched by saturated sodium bicarbonate and extracted with CH Cl . The organic
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layer was washed with water and dried over Na SO and the solvent was removed under
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reduced pressure to obtain compounds 45a-b without further purification.
General Procedure for the Synthesis of 4-piperidine Formylaniline
Hydrochlorides 48a-k.
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A mixture of N-Boc-piperidine-4-carboxylic acid (1 mmol), DIPEA (2 mmol) and
HATU (1.2 mmol) in CH Cl (10 mL) was stirred at room temperature for 10 min.
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Properly substituted aniline (2mmol) was added to the solvent and the resulting mixture
was stirred at room temperature for 6 h. The mixture was washed sequentially with
water and 10% hydrochloric acid and dried over Na SO and the solvent was removed
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under reduced pressure to obtain compounds 47a-k without further purification. Then
mL 4 M HCl in EtOAc was added to a solution of 47a-k in EtOAc (10 mL) and the
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mixture was stirred at room temperature for 12 h to precipitate white solid. The solid
was then filtrated by suction and washed with EtOAc to afford the pure 48a-k.
General Procedure for the Synthesis of Diphenylmethyl Piperidine Derivatives 14-
19.
To a solution of 4-piperidine formylaniline hydrochlorides 48a-c (1 mmol) in N,
N-dimethlformamide (10 mL) was added sodium hydrogen (3 mmol). The resulting
mixture was stirred at room temperature for 1 h. Bromo-compounds 45a-b (1 mmol)
were added to the solvent and the resulting mixture was stirred at room temperature for
2 h. The reaction was quenched by water and extracted with EtOAc. The organic layer
was washed sequentially with water and saturated brine and dried over Na SO . The
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mixture was concentrated and purified by silica gel column chromatography using as
eluent with petroleum ether /EtOAc (4:1) to give the desired products.
General Procedure for the Synthesis of Aryl Formyl Piperidine Derivatives 20-38
To a solution of 4-piperidine formylaniline hydrochlorides 48a-k (0.5 mmol) in N,
N-dimethlformamide (10 mL) was added TEA (1.67 mmol). The resulting mixture was
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