Structural Optimization of 4-Chlorobenzoylpiperidine Derivatives for the Development of Potent, Reversible, and Selective Monoacylglycerol Lipase (MAGL) Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.6b01459

Monoacylglycerol lipase (MAGL) inhibitors are considered potential therapeutic agents for a variety of pathological conditions, including several types of cancer. Many MAGL inhibitors are reported in literature; however, most of them showed an irreversible mechanism of action, which caused important side effects. The use of reversible MAGL inhibitors has been only partially investigated so far, mainly because of the lack of compounds with good MAGL reversible inhibition properties. In this study, starting from the (4-(4-chlorobenzoyl)piperidin-1-yl)(4-methoxyphenyl)methanone (CL6a) lead compound that showed a reversible mechanism of MAGL inhibition (K_i = 8.6 μM), we started its structural optimization and we developed a new potent and selective MAGL inhibitor (17b, K_i = 0.65 μM). Furthermore, modeling studies suggested that the binding interactions of this compound replace a structural water molecule reproducing its H-bonds in the MAGL binding site, thus identifying a new key anchoring point for the development of new MAGL inhibitors.

Full text of DOI:10.1021/acs.jmedchem.6b01459

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Article
Structural optimization of 4-chlorobenzoylpiperidine
derivatives for the development of potent, reversible
and selective monoacylglycerol lipase (MAGL) inhibitors
Carlotta Granchi, Flavio Rizzolio, Stefano Palazzolo, Sara Carmignani, Marco Macchia, Giuseppe
Saccomanni, Clementina Manera, Adriano Martinelli, Filippo Minutolo, and Tiziano Tuccinardi
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01459 • Publication Date (Web): 03 Nov 2016
Downloaded from http://pubs.acs.org on November 7, 2016
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Structural optimization of 4ꢀchlorobenzoylpiperidine
derivatives for the development of potent, reversible and
selective monoacylglycerol lipase (MAGL) inhibitors
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†
ϕ
ϕ,§
†
†
Carlotta Granchi, Flavio Rizzolio, Stefano Palazzolo, Sara Carmignani, Marco Macchia,
†
†
†
†
Giuseppe Saccomanni, Clementina Manera, Adriano Martinelli, Filippo Minutolo, Tiziano
,†
Tuccinardi*
†
Department of Pharmacy, University of Pisa, Via Bonanno 6, 56126 Pisa, Italy
ϕ
Division of Experimental and Clinical Pharmacology, Department of Molecular Biology and
Translational Research, National Cancer Institute and Center for Molecular Biomedicine, IRCCS,
3081 Aviano (PN), Italy
3
§
Graduate School in Nanotechnology, University of Trieste, Trieste, Italy
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ABSTRACT
Monoacylglycerol lipase (MAGL) inhibitors are considered potential therapeutic agents for a
variety of pathological conditions, including several types of cancer. Many MAGL inhibitors are
reported in literature; however, most of them showed an irreversible mechanism of action, which
caused important side effects. The use of reversible MAGL inhibitors has been only partially
investigated so far, mainly because of the lack of compounds with good MAGL reversible
inhibition properties. In this study, starting from the (4ꢀ(4ꢀchlorobenzoyl)piperidinꢀ1ꢀyl)(4ꢀ
methoxyphenyl)methanone (CL6a) lead compound that showed a reversible mechanism of MAGL
0
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0
inhibition (K
i
= 8.6 µM), we started its structural optimization and we developed a new potent and
= 0.65 µM). Furthermore, modeling studies suggested that the
selective MAGL inhibitor (17b, K
i
binding interactions of this compound replace a structural water molecule reproducing its Hꢀbonds
in the MAGL binding site, thus identifying a new key anchoring point for the development of new
MAGL inhibitors.
INTRODUCTION
Among the different endogenous lipids with endocannabinoidꢀlike activity, anandamide (AEA) and
2
ꢀarachidonoylglycerol (2ꢀAG) are the two most important endogenous ligands that are able to
1
activate the G proteinꢀcoupled cannabinoid receptors, CB
1
and CB
2
. These endocannabinoids are
produced on demand through stimulusꢀdependent cleavage of phospholipid precursors and they
modulate multiple physiological processes including pain, inflammation, appetite, memory, and
2
, 3
emotion and their signaling functions terminate by enzymatic hydrolysis. In the nervous system,
2
ꢀAG is produced by phospholipase C and diacylglycerol lipase and it is deactivated through
hydrolysis mediated by monoacylglycerol lipase (MAGL). Because of the prominent role in 2ꢀAG
degradation, selective inactivation of MAGL represents a potential target for pharmacological
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agents able to treat diverse pathological conditions such as cancer, chronic pain and Alzheimer’s
4
disease. In the past ten years, massive efforts have been implemented in order to obtain potent
5ꢀ10
MAGL inhibitors;
however, almost all the compounds found showed irreversible inhibition
1
1
properties. In this context the reference inhibitors that have been used in the literature for most
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cellular
and
animal
experiments
are
4ꢀnitrophenylꢀ4ꢀ[bis(1,3ꢀbenzodioxolꢀ5ꢀ
8
yl)(hydroxy)methyl]piperidineꢀ1ꢀcarboxylate 1 (JZL184, Figure 1) and the benzyl(4ꢀ(5ꢀmethoxyꢀ2ꢀ
5
oxoꢀ1,3,4ꢀoxadiazolꢀ3(2H)ꢀyl)ꢀ2ꢀmethylphenyl)carbamate 2 (CAY10499, Figure 1). As reported
by Scholsburg and coꢀworkers, the irreversible inhibition of MAGL, induced by repeated
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administration of 1, yielded crossꢀtolerance to CB
1
agonists in mice. Chronic MAGL inhibition
also produced physical dependence, desensitized brain CB
1
receptors and damaged
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endocannabinoidꢀdependent synaptic plasticity. Genetic inactivation of MAGL or prolonged
pharmacological blockage of MAGL by an irreversible inhibitor provokes the persistence of
elevated 2ꢀAG levels in the brain. This fact leads to a tolerance of the effects induced by MAGL
inhibition. Moreover, responses to CB
1
agonists were significantly reduced and there was an
agonists were administered, resulting in a general CB
evident crossꢀtolerance when exogenous CB
1
1
desensitization. Furthermore, profound alterations to CB receptor expression and function in the
1
brain were observed. Considering all these drawbacks associated to an irreversible MAGL
inhibition, the need to discover selective and reversible MAGL inhibitors remains urgent. To our
knowledge, the only compounds described as good reversible MAGL inhibitors are the naturally
1
3
occurring terpenoids pristimerin and euphol (Figure 1). However, these two compounds act on a
large number of secondary targets and this makes their use and study as MAGL inhibitors very
1
4,15
difficult.
Very recently, the reversible MAGL inhibitor benzo[d][1,3]dioxolꢀ5ꢀylmethyl 6ꢀ
phenylhexanoate 3 (compound c21, Figure 1) was tested in vivo using the experimental allergic
encephalomyelitis (EAE) mouse model, which is broadly studied as an animal model of human
CNS demyelinating diseases, including multiple sclerosis and acute disseminated
16
encephalomyelitis. This molecule was able to ameliorate the clinical progression of the multiple
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sclerosis mouse model and, very importantly, the therapeutic effects were not accompanied by
catalepsy or other motor impairments that have been observed after the administration of
irreversible MAGL inhibitors, thus supporting the hypothesis that reversible inhibitors could be
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profitably used in in vivo models.
We have recently reported a virtual screening study for the discovery of new reversible MAGL
inhibitors. Among the tested compounds, (4ꢀ(4ꢀchlorobenzoyl)piperidinꢀ1ꢀyl)(4ꢀ
methoxyphenyl)methanone 4 (CL6a, Figure 2) proved to be an interesting reversible MAGL
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inhibitor lead due to its inhibition activity (K
i
= 8.6 µM and IC50 = 11.7 µM), the absence of
17
stereocenters in its structure and its good synthetic accessibility. With the aim of identifying
potent and selective MAGL inhibitors, chemical modifications guided by molecular modeling
predictions of the probable binding poses were made to the structure of the initial compound 4 as a
first step of our attempt to improve the inhibition potency of 4 on MAGL. The synthesis of new
analogues of 4, their biological characterization on the isolated enzyme as well as their
antiproliferative activities on a series of cancer cells are reported in this study.
Figure 1. Structures of known MAGL inhibitors.
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RESULTS AND DISCUSSION
Design and Molecular Modeling. As shown in Figure 2, by using a plane that divides the
piperidine ring we can observe that compound 4 is symmetrical, with the exception of the piperidine
nitrogen atom and the different aryl substituents at the two opposite sides of the molecule (a chloro
atom in position 4 of the phenyl ring of the ketone side and a methoxy group in the same position of
the phenyl ring of the amidic portion).
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Figure 2. 2D analysis of compound 4.
Before proceeding with the synthesis of 4 analogues, an extensive docking analysis was carried out
in order to establish the binding disposition of this ligand. Figure S1 shows the clustering analysis
of the docking results. This plot highlights the presence of two possible binding dispositions of the
ligand, as there are two highly populated clusters of docking poses with very similar energy
interaction scoring values. The most energetically favorable binding mode shows that the amidic
C=O group of 4 forms two Hꢀbonds with the nitrogen backbone of A51 and M123 and the 4ꢀ
methoxyphenyl fragment is directed towards an open cavity of the protein showing lipophilic
interactions with L148, L213 and L241. The 4ꢀchlorobenzoyl moiety is inserted into a small pocket
of the protein and shows lipophilic interactions of the phenyl ring with Y194 and V270 (binding
mode A of Figure 3). Figure 3B shows a representative docking pose of the second cluster which is
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more populated than the first one, but it shows a slightly lower energy interaction score. As
expected, this binding disposition is symmetrical to the first one: the 4ꢀchlorobenzoyl moiety of the
4
is directed towards the open cavity of the protein showing lipophilic interactions with L148, L213
and L241 and forms two Hꢀbonds with the nitrogen backbone of A51 and M123. With regards to
the 4ꢀmethoxybenzoyl fragment, it is inserted into the small pocket of the protein and the phenyl
ring shows lipophilic interactions with Y194 and V270.
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Figure 3. Docking of compound 4 into hMAGL. (A) Binding mode A; (B) binding mode B.
In order to identify which one is the preferred binding mode among the two proposed solutions by
the docking analysis (see Figure 3), we planned to synthesize two analogues of compound 4 that are
respectively able to adopt only one of these binding modes. In fact, biphenylic compounds 5 and 6
(Table 1) were designed considering: a) the small volume of the pocket delimited by E53, R57,
Y58, Y194, V270 and H272, and b) the open shape of the cavity on the opposite side of the binding
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site. These two compounds are characterized by the presence of a bulky biphenyl ring that we
hypothesized could lie only in the open cavity of the binding site surrounded by hydrophobic
residues L148, L213 and L241. As shown in Figure 4 and Figure S2, our hypothesis was confirmed
by the docking studies, since there was only one predicted binding disposition for each compound,
with the biphenyl ring that was placed in the open cavity of the binding site. Due to this disposition,
compound 5 shows the binding mode A, with the C=O amidic group forming two Hꢀbonds with the
backbone NꢀH of A51 and M123. Conversely, compound 6 shows the binding mode B, with the
ketone group that forms two Hꢀbonds with the backbone nitrogen of A51 and M123.
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Figure 4. Docking results of compound 5 (A) and 6 (B) into hMAGL.
The two compounds were thus synthesized and tested for their hMAGL inhibition activity together
with the piperidineꢀ1,4ꢀdiylbis((4ꢀchlorophenyl)methanone) derivative (7a, Table 1), which was
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6
used as a reference compound, since it possesses the paraꢀchloro substituted phenyl ring on both
sides of the molecule, without the bulky biphenyl part that is present in the amidic portion of
compound 5 or in the ketone moiety of compound 6. As shown in Table 1, in agreement with the
docking results, both binding modes are possible since both compounds show inhibition of MAGL
activity. However, the binding mode B is the preferred one, since compound 6 shows a 5ꢀfold
higher activity (IC50 = 2.1 µM) than that of compound 5 (IC50 = 9.9 µM). Subsequently, two
derivatives of compound 4 were synthesized and tested for their hMAGL inhibition activity in order
to inspect the role of the central scaffold (Table 1). Compound 8, which is characterized by the
replacement of the two C=O groups with two methylene moieties, showed a complete loss of
inhibition activity (IC50 greater than 100 µM). Similar results were also obtained for compound 9,
which possesses a central piperazine ring instead of piperidine, thus introducing a second amidic
group that further rigidifies the structure by reducing the rotational freedom of the pꢀchlorobenzoyl
portion. Both results supported the important role of the central piperidine ring bearing a carbonyl
group in the opposite position to the amidic fragment for the establishment of an efficient
interaction with the enzyme, thus confirming the key role played by the central scaffold of 4, which
justifies the development of a series of analogues of this compound.
0
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0
Table 1. Structure and hMAGL activity of compounds 5, 6, 7a, 8 and 9.
#
5
Structure
IC50 (µM)
9.9 ± 0.6
6
2.1 ± 0.4
5.4 ± 0.1
7
a
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> 100
> 100
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Hence, once we identified the preferred binding mode of this class of compounds into hMAGL, we
proceeded with the lead optimization of 4. As a first step in the development of this scaffold, we
decided to improve the interaction of the compound inside the small pocket delimited by E53, R57,
Y58, Y194, V270 and H272 while maintaining the rest of the molecule fixed. On this basis, we
replaced the pꢀmethoxyphenyl ring present in the amidic portion of the initial compound 4 with
differently substituted aryl groups and kept the pꢀchlorobenzoyl unit fixed. Hence, we explored the
ortho/meta/para effects generated by the presence in the amidic phenyl ring of halogen atoms (F,
Cl, Br, I), methyl, trifluoromethyl, methoxy, trifluoromethoxy, hydroxy, amino and nitro
substituents, by testing the inhibitory activities of the series of novel compounds reported in Table
2
.
Enzymatic experiments. The inhibitory effects of the newly synthesized compounds on human
isoforms of MAGL by using 4ꢀnitrophenylacetate substrate are reported in Table 2, together with
those of the reference irreversible inhibitor 1 and of the recently reported reversible inhibitor 3.
Considering the activity of compound 20 (IC50 = 6.5 µM), which possesses an unsubstituted phenyl
ring, the introduction in all the three o/m/p positions of the aromatic ring of bromine and iodine
(compounds 11aꢀc and 12aꢀc) determined an increase of the activity (IC50 values in the range 2.4ꢀ
4.8 µM). The presence of the other substituents generally led to a certain reduction of inhibition
activity, which resulted to be either lower than that of reference compound 20 (IC50 values in the
range 9.9ꢀ57.3 µM) or comparable (IC50 = 5.4 µM for 7a, IC50 = 7.1 µM for 13c). It is worth noting
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
that almost all the reported compounds show an activity that is similar or even better than that of the
reference reversible inhibitor 3.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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0
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0
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9
0
Table 2. Structures and hMAGL inhibitory activities of the synthesized compounds.
1
2
3
Compounds
R
R
R
IC50 (µM)
5.4 ± 0.7
7
a
H
H
Cl
H
H
F
H
H
Br
H
H
I
H
H
CH
H
H
H
Cl
H
H
F
H
H
Br
H
H
I
H
H
CH
H
H
Cl
H
H
F
H
H
Br
H
H
I
H
H
CH
H
H
7
b
17.1 ± 0.1
10.4 ± 0.5
17.8 ± 0.9
20.4 ± 0.1
13.4 ± 0.6
2.4 ± 0.1
4.8 ± 0.4
4.0 ± 0.4
4.4 ± 0.2
4.3 ± 0.4
7
c
10a
10b
1
0c
1
1
1a
1b
1
1c
1
1
2a
2b
12c
13a
2.4 ± 0.2
3
11.8 ± 0.4
10.9 ± 0.8
7.1 ± 0.1
29.8 ± 1.3
11.9 ± 0.4
26.3 ± 2.4
11.7 ± 2.2
9.9 ± 1.7
11.5 ± 0.3
26.6 ± 2.6
16.3 ± 1.0
17.3 ± 1.2
11.7 ± 1.7
0.84 ± 0.04
32.8 ± 4.3
16.7 ± 1.7
57.3 ± 2.2
15.5 ± 1.9
14.2 ± 0.3
24.9 ± 3.5
24.0 ± 0.4
6.5 ± 0.1
1
1
3b
3c
3
3
1
1
4a
4b
CF
H
3
CF
H
3
1
4c
CF
3
H
OCH
H
H
OCF
H
H
OH
H
H
NH
H
H
NO
H
H
H
4
15b
15c
H
H
OCH
H
H
OCF
H
H
OH
H
H
NH
H
H
NO
H
H
OCH
H
H
OCF
H
H
OH
H
H
NH
H
3
3
3
1
1
6a
6b
3
3
1
6c
3
1
1
7a
7b
1
7c
18a
18b
2
2
1
8c
2
1
1
9a
9b
H
NO
H
2
2
1
9c
2
2
0
H
1
0.048 ± 0.005
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0.134 ± 0.015
9.0 ± 1.5
Among the 34 reported derivatives, the mꢀhydroxyꢀsubstituted derivative 17b showed a high
inhibition activity (IC50 = 0.84 µM), about 14 fold more potent than the starting 4 lead compound.
In order to better rationalize the activity of this compound, 17b was subjected to docking
calculations, together with its analogues 17a and 17c. Binding mode B of compound 17b resulted to
be more energetically favorable than the other one (see Figure S3). The overall disposition of the
molecule is comparable to that observed for compound 4, with the 4ꢀchlorobenzoyl moiety that is
directed towards the open cavity of the protein showing lipophilic interactions with L148, L213 and
L241 and the formation of two Hꢀbonds with the backbone nitrogen of A51 and M123 (see Figure
10
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17
18
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45
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48
49
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51
52
53
54
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58
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5
). The mꢀhydroxybenzoyl fragment is inserted into the small pocket of the protein and shows
lipophilic interactions between its aryl ring and residues Y194 and V270. Furthermore, the mꢀ
hydroxyl participates to a highly energetic Hꢀbond network with residues E53 and H272, where it
behaves as a Hꢀbond acceptor with H272 and as a Hꢀbond donor with E53. It is interesting to note
1
8ꢀ21
that, analyzing the main crystal structures of hMAGL
and superimposing them with the
hMAGLꢀ17b complex, the hydroxyl group of the ligand replaces a structural water molecule that
acts as a Hꢀbond bridge between E53 and H272, thus supporting the high activity of this compound
(
see Figure S4). The replacement of the mꢀhydroxyl with the oꢀ and pꢀhydroxyl group determined a
marked decrease of the MAGL inhibition activity (see compounds 17c and 17a, respectively). As
shown in Figure S5, the docking results suggested that for both compounds the shift of the hydroxyl
group from the meta to para and ortho position determines the loss of the Hꢀbonds with E53 and
H272, without establishing any further interaction.
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9
0
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2
3
4
5
6
7
8
9
0
Figure 5. Docking results of compound 17b into hMAGL.
The replacement of the mꢀhydroxy with the mꢀamine group led to a marked decrease of activity
(
compound 18b, IC50 = 57.3 µM). Due to the planar geometry of the aniline nitrogen atom and to
the delocalization of its electron lone pair in the adjacent πꢀsystem, it is unlikely that this group is
able to act as a Hꢀbond acceptor, thus supporting the low activity of this compound.
With the aim of evaluating the reversible or irreversible mechanism of inhibition, the effects of
dilution and preincubation on the inhibitory ability of compound 17b and of reference compounds 2
and 3 were evaluated. In the dilution experiments, if the compound is an irreversible inhibitor, then
its inhibition potency should not drop upon dilution, whereas inhibition levels should be
substantially reduced upon dilution in the presence of a reversible inhibitor. As shown in Figure 6,
1
7b showed reversible inhibition, since the inhibition produced by 20 µM of the compound was
significantly higher compared with that observed with a 40X dilution, which appears similar to that
produced by a 0.5 µM concentration of the compound. The same results were also obtained for
compound 3, whereas compound 2 showed an irreversible inhibition since the inhibition produced
by 4 µM of the compound was substantially unchanged upon a 40X dilution, which appears
significantly higher to that produced by a 0.1 µM concentration of this compound.
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Figure 6. Dilution assay for compounds 17b, 3 and 2: the first two columns indicate the inhibition
percentage of compounds at a concentration of 20 µM and 0.5 µM (17b), 40 µM and 1.0 µM (3)
and 4 µM and 0.1 µM (2). The third column indicates the inhibition percentage of compounds after
a 40X dilution (final concentration = 0.5 µM, 1.0 µM and 0.1 µM for compounds 17b, 3 and 2,
respectively).
In order to further support these results, the activity of 17b, 2 and 3 were tested at different
preincubation times of the inhibitors with the enzyme. An irreversible inhibitor will increase its
ability to block MAGL over gradually longer incubation times. On the contrary, unchanged IC50
22
values in these experiments support a reversible mechanism of action. As expected, compound
17b and 3 showed a constant MAGL inhibition activity even after 60 min of preincubation, thus
supporting a reversible mode of action (see Figure 7). As expected, instead, a timeꢀdependent
increase of potency was observed for reference compound 2, which further supports its already
5
known irreversible inhibitor character.
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0
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2
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9
0
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2
3
4
5
6
7
8
9
0
Figure 7. IC50 (µM) values of 17b, 3 and 2 at different preincubation times with hMAGL (0 min
and 60 min).
Once we confirmed the reversibility of the inhibitory action of compound 17b, we then evaluated its
inhibition mode by measuring Michaelis–Menten kinetics. The dataset was plotted as substrate
concentration versus enzyme activity and analyzed by applying the mixedꢀmodel inhibition fit of
m
GraphPad Prism 5.0. This model, beyond the Vmax and K values, also calculates the α parameter,
which can be useful for establishing the inhibition mechanism. Its value is greater than zero and
determines the extent to which the binding process of the inhibitor changes the affinity of the
enzyme for the substrate. If the inhibitor does not modify the binding of the substrate to the enzyme,
α
is equal to one and the mixedꢀmodel corresponds to a noncompetitive inhibition. When
large value, the binding of the inhibitor prevents that of the substrate and the mixedꢀmodel
corresponds to a competitive inhibition. Finally, when is a very small value, the binding of the
inhibitor increases the binding of the substrate to the enzyme, and the mixedꢀmodel corresponds to
α is a very
α
an uncompetitive model. Kinetic studies indicate for 17b a K
i
value of 0.65 ± 0.05 µM and an
α
value greater than 10000, thus suggesting a competitive binding for this compound (Figure 8).
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50
51
52
53
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57
58
59
60
Figure 8. Michaelis–Menten plot for the determination of the K
i i
of compound 17b for hMAGL (K
=
0.65 ± 0.05 µM, >10000).
α
As a further analysis, in order to explore the potential activity of these compounds for other
hydrolases, the selectivity versus the analogue enzyme fatty acid amide hydrolase (FAAH) of some
of the most promising inhibitors was evaluated. As shown in Table 4, all the tested compounds
showed MAGL selectivity. In particular, compound 17b displayed a MAGL selectivity greater than
119ꢀfold.
Table 4. MAGL selectivity versus FAAH for the best compounds.
Compd MAGL IC50 (µM) FAAH IC50 (µM) Selectivity Index
1
1b
12c
3c
4.8 ± 0.4
2.4 ± 0.2
7.1 ± 0.2
0.84 ± 0.04
9.0 ± 1.5
> 100
86.7 ± 5.1
> 100
> 21
36
1
> 14
> 119
3
1
7b
3
> 100
27.7 ± 1.1
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6
Molecular dynamic simulation. In order to evaluate the reliability of the docking results and to
carry out an analysis of the ligand–protein interaction, the hMAGLꢀ17b complex was used as a
starting structure for 51 ns of molecular dynamic (MD) simulation. As shown in Figure S6, the
complex is stable during the simulations and the analysis of the rootꢀmeanꢀsquare deviation (rmsd)
of all the heavy atoms from the Xꢀray structures highlights a stabilization of the rmsd value around
0.8 Å. With regard to the geometry of the compounds, analyzing the rmsd of the position of the
ligand during the simulations with respect to the starting structure, it maintains its starting
disposition with an rmsd value between 0.3 and 0.7 Å. With regard to the Hꢀbond analysis (Table
S1 in the Supporting Information), the interaction of the hydroxyl group with E53 and H272
appears to be very stable, similarly to the interaction with the nitrogen backbone of A51. Differently
from what reported in the docking studies, the interaction with the backbone nitrogen of M123 is
not highly conserved; however, this is due to a small shift of the ligand with the formation of a very
stable Hꢀbond between the hydroxyl group of S181 and the amidic oxygen of the compound (see
Figure S7).
0
1
2
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8
9
0
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9
0
1
2
3
4
5
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8
9
0
Antiproliferative assays. Compound 17b was also further tested in in vitro experiments for
evaluating its antiproliferative activity against cancer cells. Compound 2 and 3 were used as
2
3
reference compounds. Due to the potential role of MAGL as a therapeutic target in ovarian cancer,
four human ovarian cancer cell lines (OVSAHO, OVCAR3, COV318 and CAOV3) were chosen
and a western blot analysis was carried out in order to measure the overexpression of MAGL in
these cells. As shown in Figure 9A, western blot analysis highlighted an overexpression of MAGL
in OVCAR3 and CAOV3 compared to OVSAHO and COV318 cell lines. Compound 17b caused a
considerable inhibition of cell viability, with IC50 values ranging from 31.5 to 43.9 ꢁM in the
OVCAR3 and CAOV3 cell lines (Table S2), whereas it proved to be remarkably less potent against
ovarian cancer cells that do not overexpress MAGL, such as OVSAHO and COV318 cells.
Furthermore, 17b proved to be completely inactive also against noncancerous human fibroblast
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lung cells (MRC5, Figure 9B). Differently, the covalent reference inhibitor 2 showed inhibition of
cell viability in all the four ovarian cancer cell lines, with IC values ranging from 17.2 to 83.1 ꢁM
50
(Table S2). This behavior may be due to the lack of targetꢀselectivity of this compound, thus we
cannot exclude that the involvement of different targets could contribute to its antiproliferative
potential. Surprisingly, the known MAGL inhibitor 3 displayed a substantial inactivity against all
the tested cell lines. Overall, these data suggest that the reversible inhibition of MAGL operated by
the herein reported class of compounds may be a possible profitable alternative to the irreversible
inhibition treatment.
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51
52
53
54
55
56
57
58
59
60
Figure 9. A) Western blot analysis of MAGL expression in ovarian cancer cell lines. Vinculin was
utilized to normalize the amount of total loaded proteins. B) Cell growth inhibitory activities (IC50
)
of compounds 2, 17b and 3.
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6
Chemistry. Benzoyl piperidine derivatives 4, 7aꢀc, 10aꢀc, 11aꢀc, 12aꢀc, 13aꢀc, 14aꢀc, 15bꢀc, 16aꢀc,
1
9aꢀc and 20 were synthesized by an amide coupling between properly substituted benzoic acids
and 4ꢀ(4ꢀchlorobenzoyl)piperidine 21 in the presence of the condensing agent HATU and DIPEA as
17
the base in dry N,Nꢀdimethylformamide as the solvent, as previously reported for compound 4.
0
1
2
3
4
5
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8
9
0
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0
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2
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8
9
0
1
2
3
4
5
6
7
8
9
0
Hydroxyꢀsubstituted derivatives 17aꢀc were obtained after BBr ꢀpromoted deprotection of the
3
corresponding methoxylated precursors 4, 15bꢀc. A particular case was the orthoꢀamino substituted
compound 18c which, unlike its analogues paraꢀ and metaꢀNH ꢀbearing compounds 18aꢀb (see
2
Scheme 2), was directly obtained from condensation of 4ꢀ(4ꢀchlorobenzoyl)piperidine with
anthranilic acid (Scheme 1), since the protection of the amino moiety with tertꢀbutyloxycarbonyl
group, similarly adopted for the paraꢀ and metaꢀanalogues, needed very long reaction times with the
simultaneous formation of many sideꢀproducts, therefore the direct condensation was preferred,
although this caused a decrease in the reaction yield.
a
Scheme 1. Synthesis of benzoylꢀpiperidine derivatives.
a
Reagents and conditions: (a) properly substituted benzoic acid (1 eq), HATU (1.05 eq), DIPEA (4
3 2 2
eq), dry DMF, RT, 6ꢀ8 h; (b) 1M BBr , dry CH Cl
, −78 to 0 °C, then RT, 1ꢀ2 h.
As anticipated in the previous discussion, the synthesis of aminoꢀsubstituted compounds 18aꢀb
started with the formation of the methyl esters 24 and 25 from the corresponding aminoꢀbenzoic
acids 22 and 23 by refluxing them in MeOH in the presence of SOCl
2
(Scheme 2). Bocꢀprotection
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of the amino groups with diꢀtertꢀbutyl dicarbonate in the presence of triethylamine in THF gave
compounds 26 and 27, which were then hydrolyzed to give Bocꢀprotected benzoic acids 28 and 29
ready to be condensed with piperidine 21. The amino groups of the intermediate amides 30 and 31
were finally deprotected by reaction with trifluoroacetic acid in DCM to obtain compounds 18a and
10
11
12
13
14
15
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17
18
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44
45
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48
49
50
51
52
53
54
55
56
57
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1
8b in good yields.
Scheme 2. Synthesis of meta and paraꢀaminoꢀsubstituted benzoylꢀpiperidine derivatives 18aꢀ
a
b.
a
2 2 3
Reagents and conditions: a) MeOH, SOCl (2.5 eq), reflux, 3 h; (b) (Boc) O (2 eq), Et N (2 eq),
dry THF, RT, 24 h; (c) aq. 2N LiOH (6 eq), THF/MeOH 1:1 v/v, RT, overnight; (d) (4ꢀ
chlorophenyl)(piperidinꢀ4ꢀyl)methanone 21 (1 eq), HATU (1.05 eq), DIPEA (4 eq), dry DMF, RT,
3
3ꢀ4 h; (e) CF COOH, dry DCM, 0 °C to RT, 3ꢀ5 h.
The synthetic approaches for the synthesis of the biphenyl derivatives 5 and 6 are reported in
Schemes 3 and 4. For the synthesis of compound 5, [1,1'ꢀbiphenyl]ꢀ4ꢀcarboxylic acid 35 was
obtained after a sequence of reactions starting from 4ꢀbromobenzoic acid 32, consisting in a Fischer
esterification to obtain the methyl ester 33, followed by a palladiumꢀcatalyzed crossꢀcoupling
reaction to replace the bromine atom with a phenyl ring. This step required anhydrous conditions to
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2
2
2
2
2
2
2
2
3
3
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5
5
5
5
5
6
prevent the hydrolysis of the methyl ester group under prolonged heating in the presence of alkaline
solutions, since the isolation and purification of the biphenyl methyl ester was preferred. Finally,
hydrolysis of the methyl ester gave compound 35 which was condensed with 4ꢀ(4ꢀ
chlorobenzoyl)piperidine 21 in the presence of HATU and DIPEA to yield the desired compound.
The synthesis of compound 6 started with the protection of piperidine 21, then a crossꢀcoupling
reaction adopting the Fuꢀtype conditions, which are suitable for the coupling of aryl chlorides with
the appropriate phenylboronic acid, using the catalytic system containing Pd (dba) and
0
1
2
3
4
5
6
7
8
9
0
1
2
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8
9
0
1
2
3
4
5
6
7
8
9
0
2
3
tricyclohexylphosphine, and cesium carbonate as the base, gave compound 37. TFAꢀpromoted
deprotection of the piperdine Nꢀatom gave compound 38, which was reacted with 4ꢀchlorobenzoic
acid in the same conditions adopted for all the amidic condensation of this class of compounds to
produce compound 6 in high yields.
a
Scheme 3. Synthesis of biphenyl derivative 5.
a
Reagents and conditions: (a) MeOH, H
0.03 eq), PPh (0.15 eq), K CO (1.5 eq), toluene, 100 °C, 18 h; (c) aq. 2N LiOH (6 eq),
THF/MeOH 1:1 v/v, RT, 20 h; (d) (4ꢀchlorophenyl)(piperidinꢀ4ꢀyl)methanone 21 (1 eq), HATU
1.05 eq), DIPEA (4 eq), dry DMF, RT, 3 h.
2 4 2 2
SO , reflux, overnight; (b) PhB(OH) (2 equiv), Pd(OAc)
(
3
2
3
(
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a
Scheme 4. Synthesis of biphenyl derivative 6.
a
Reagents and conditions: (a) (Boc)
2 3 2
O (1.2 eq), Et N (2 eq), dry THF, RT, 2 h; (b) PhB(OH) (1.6
equiv), Pd (dba) (0.032 eq), Cy P 20% toluene (0.08 eq), Cs CO (1.7 eq), dioxane, 100 °C,
2
3
3
2
3
3
overnight; (c) CF COOH, dry DCM, 0 °C to RT, 2 h; (d) 4ꢀchlorobenzoic acid (1 eq), HATU (1.05
eq), DIPEA (4 eq), dry DMF, RT, 6 h.
The synthesis of compounds 8 and 9 is reported in Scheme 5 and for both derivatives a oneꢀpot
procedure gave the desired products. Derivative 8 was obtained by alkylation of 4ꢀ(4ꢀ
chlorobenzyl)piperidine hydrochloride 39 with 4ꢀmethoxybenzyl chloride 40 using potassium
carbonate as the base. Differently, the amidic condensation between 1ꢀ(4ꢀchlorobenzoyl)piperazine
4
1 and 4ꢀmethoxybenzoic acid 42 under the same conditions previously described for the synthesis
of other compounds (Schemes 1ꢀ4) gave derivative 9.
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2
2
2
2
2
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4
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
6
a
Scheme 5. Synthesis of compounds 8 and 9.
a
Reagents and conditions: (a) an. K CO (3 eq.), dry DMF, RT, overnight; (b) HATU (1.05 eq),
2
3
DIPEA (4 eq), dry DMF, RT, 3 h.
0
1
2
3
4
5
6
7
8
9
0
1
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0
1
2
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9
0
For the synthesis of compound 43 (see Conclusions section), the synthetic scheme described for the
preparation of biphenylic piperidine 38 (Scheme 4) was adopted, then piperidine 38 was submitted
to a condensation with 3ꢀmethoxybenzoic acid to get amide 44, followed by deprotection of the
methoxy group with boron tribromide, to obtain the final compound 43 (Scheme 6).
a
Scheme 6. Synthesis of compound 43.
a
Reagents and conditions: (a) (Boc)
(dba) (0.032 eq), Cy P 20% toluene (0.08 eq), Cs
overnight; (c) CF COOH, dry DCM, 0 °C to RT, 2 h; (d) 3ꢀmethoxybenzoic acid (1 eq), HATU
1.05 eq), DIPEA (4 eq), dry DMF, RT, 4 h; (e) 1M BBr , dry CH Cl , −78 to 0 °C, then RT, 1 h.
2
O (1.2 eq), Et
3
N (2 eq), dry THF, RT, 2 h; (b) PhB(OH)
2
(1.6
equiv), Pd
2
3
3
2
CO (1.7 eq), dioxane, 100 °C,
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A detailed analysis of H and
CꢀNMR spectra of some (1ꢀbenzoylpiperidinꢀ4ꢀyl)(4ꢀ
chlorophenyl)methanone derivatives revealed the presence of two rotational conformers generated
by the rotation of the bond between the variously substituted phenyl ring and the amidic carbonyl
1
13
group. The splitting of H and CꢀNMR signals was observed for orthoꢀsubstituted compounds,
since the presence of bulky groups in this position such as Cl (7c), Br (11c), I (12c), CF (14c), CH
(13c), OCH (15c), and OCF (16c) led to the presence of two rotational conformers. Conversely
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when the substituents are either absent or are present in meta or para positions the rotation is free at
room temperature and a unique conformer is observed by NMR analysis. In the case of compounds
1
2
7c and 18c, the presence of orthoꢀOH (17c) or orthoꢀNH (18c) groups allows the formation of an
intramolecular Hꢀbond between the hydrogen atom of hydroxyl or amino group and the amide C=O
oxygen atom, thus inducing a stabilization of only one conformer. Surprisingly, for orthoꢀfluoro
derivative 10c no splitting of signals was observed, despite the nonꢀnegligible steric hindrance of
the halogen atom. This observation may be explained considering the partial negative charge of
both the fluorine atom and the carbonyl oxygen atom, which cause a charge repulsion leading to the
stabilization of the conformer where these two atoms maximize their distance. Similarly, the single
conformer observed for the orthoꢀnitro derivative 19c may have the nitro group and the carbonyl
oxygen atom distant, due to the repulsion between the partially negatively charged oxygen of the
2
amide C=O and the negatively charged oxygens of the NO group, causing the block of the rotation
which leads to the presence of a single conformer. The presence of conformer was verified for a
representative compound, (4ꢀ(4ꢀchlorobenzoyl)piperidinꢀ1ꢀyl)(2ꢀiodophenyl)methanone 12c, by
1
acquiring HꢀNMR spectrum at high temperature (80 °C) in a suitable solvent (DMSOꢀd ), in order
6
to overcome the energetic rotational barrier for the different conformers and thus collapse the
splitting of signals. As shown in Figures S8ꢀS10, the high temperature increased the interconversion
rate between the two rotamers and the spectrum revealed a collapse of the signals of the two
conformers, which is particularly evident in the aromatic region of the spectrum.
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CONCLUSIONS
In summary, molecular modeling predictions of the probable binding pose guided chemical
modifications of the structure of the initial compound 4 as a first attempt to improve the inhibition
potency of this compound on hMAGL. The first part of the structural optimization of 4 led to the
identification of compound 17b, which displayed remarkably high MAGL inhibition activity,
selectivity for MAGL over FAAH, reversible interaction properties and antiproliferative activity in
cancer cells. Therefore, to the best of our knowledge 17b can now be considered as one of the most
active reversible MAGL inhibitors ever reported so far in literature. Finally, the mꢀhydroxyl
substituent of this compound replaces a structural water molecule present in the main crystal
0
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8ꢀ21
structures of hMAGL.
Considering that almost all the known MAGL inhibitors show Hꢀbond
interactions only in the proximity of the oxyanion hole, constituted by the backbone NH groups of
A51 and M123, the possibility of replacing a water molecule by forming profitable Hꢀbonds also
with E53 and H272 residues could provide a new key anchoring point for the development of new
MAGL inhibitors. As a further attempt to improve the MAGL inhibition activity of the reported
compounds, the replacement of the pꢀchlorophenyl group of compound 17b with fragments that are
characterized by different size and interaction points is currently underway. In the present paper we
have modified only the amidic phenyl portion of the scaffold with the insertion of simple and small
groups as a firstꢀstage optimization study. It is our purpose now to carry out further modifications of
the structure of the best compound 17b, with the aim of obtaining derivatives that are characterized
by an improved MAGL inhibition activity. As a preview of our future optimization work, we have
observed that the replacement of the pꢀchlorophenyl ring with a biphenyl ring (compound 6) leads
to about a twoꢀfold increase of activity (compared to compound 7a). Therefore, as a preliminary
attempt to improve the activity of compound 17b, we synthesized and tested the (4ꢀ([1,1'ꢀbiphenyl]ꢀ
4
ꢀcarbonyl)piperidinꢀ1ꢀyl)(3ꢀhydroxyphenyl)methanone derivative (compound 43, Scheme 6). As
shown in Figure S13, in an agreement with the herein reported data, adding a second phenyl ring in
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compound 43 led to a twoꢀfold increase of activity with respect to 17b (IC = 0.46 ± 0.02 µM),
5
0
thus confirming the promising possibilities of further improving the inhibition activity of the best
derivative of this series of compounds by modifying the pꢀchlorophenyl region of the molecule.
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EXPERIMENTAL SECTION
Pan Assay Interference Compounds (PAINS) analysis.
The herein reported class of compounds could act as artifacts and promiscuous bioactive molecules.
Therefore, a PAINS analysis for the most active compound (17b) was carried out in order to
analyze and exclude this possibility.
PAINS substructural features screening. Baell and Holloway published an interesting paper on the
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analysis of frequent hitters from screening assays. This report described a number of substructural
features which could help to identify compounds that appear as PAINS in many biochemical high
throughput screens. In the supplementary information they provided the corresponding filters that
TM
have been included in the Filterꢀit software. Compound 17b was thus filtered by using this
program and as a result it was recognized as no PAIN molecule because it did not possess any of
the substructural features shared by the most common PAINS.
Interference analysis. Some PAINS are associated with color that could interfere photometrically
with the assay. As reported in the part of MAGL inhibition assay of the Experimental Section, in
order to avoid this possibility for each compound concentration a blank analysis was carried out,
and the final absorbance values resulted from the subtraction of the absorbance determined by the
presence of all the components except the enzyme in the same conditions. Furthermore, a specific
analysis for compound 17b was carried out. The 4ꢀnitrophenylacetate (4ꢀNPA) to 4ꢀnitrophenol (4ꢀ
NP) conversion curve was prepared at 100 µM total concentration of 4ꢀNPA+4ꢀNP in the presence
and absence of 17b at a concentration of 200 µM. As shown in the Figure S11, the two resulting
curves were superimposable. As a positive control, we analyzed in the same conditions the effects
of quercetin at 200 µM, that resulted in a shifted curve. Finally, we analyzed the results in the first
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part of the plot, in the 100ꢀ80 µM concentration range of 4ꢀ NPA (corresponding to 0ꢀ20%
conversion of 4ꢀNAP to 4ꢀNP). As shown in Figure S11 the two straight lines measured in the
presence and absence of 17b were almost identical, differently from what observed for quercetin,
that produced a shifted line, thus suggesting that 17b did not interfere with the reading at 405 nm in
the enzymatic assay.
0
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Aggregation analysis. All the enzymatic assays were carried out in the presence of bovine serum
albumin (BSA) 0.1 mg/ml. However, in order to further verify the possible formation of aggregates,
the MAGL inhibition activity of compound 17b was also tested in the presence of 0.01% (v/v)
Triton Xꢀ100 as detergent. With respect to the IC50 value obtained in the presence of BSA (IC50
=
0
.84 ± 0.04 µM), a similar result was obtained (IC50 = 0.76 ± 0.02 µM) in the presence of Triton Xꢀ
25
1
00, supporting the notion that the ligand is operating as a monomer rather than an aggregate.
2
6
Thiol reactive analysis. As reported by Walters and coꢀworkers, promiscuous enzymatic inhibition
could be due in some cases to covalent reaction with cysteines on multiple proteins. MAGL
inhibition activity of compound 17b was also tested in the presence of the thiolꢀcontaining agent
7
1
,4ꢀdithioꢀDLꢀthreitol (DTT). As shown in Figure S12, the MAGLꢀIC50 value of compound 17b
was not influenced by the presence of DTT, as it showed a IC = 0.80 ± 0.05 µM when assayed
50
with 100
µM DTT, thus excluding the interaction of this compound with the cysteine residues of the
MAGL enzyme.
2
7
Selectivity testing and orthogonal assay. As reported by Dahlin and Walters the selective activity
of a compound and the measurement of the activity against the target by using a different readout
method are two other steps that contribute to elucidate the PAINS properties. As reported in Table 4
we measured the activity of compound 17b against FAAH and the results highlighted a MAGL
selectivity greater than 119ꢀfold. Furthermore, very recently Saccomanni and coꢀworkers reported
28
the development of an HPLC/UV assay for the evaluation of MAGL inhibitors. Therefore, the
activity of compound 17b against MAGL was also tested by using this method. An aliquot of stock
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solutions of hMAGL was diluted 1:125 with Tris buffer (pH = 7.2; 10 mM, containing 1.0 mM
EDTA) to obtain the working solutions of hMAGL at 1.6 ng/µl. The stock and working solutions
were stored at
containing 1.0 mM EDTA), 5 µl of working solution hMAGL (8 ng), and 5 µl inhibitor (or solvent
as control) were added. Samples were preꢀincubated for 30 min at 37 C and then 5 µl of a
solution of 4ꢀNPA in absolute ethanol (4.25 mM) were added. Samples were incubated for 10 min
at 37 C. The enzymatic reaction was stopped by cooling in an ice bath for 10 min and then 20 µl
−20 °C. In a 0.5 ml spin tube containing 75 µl of Tris buffer (pH = 7.2; 10 mM
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°
°
of the reaction mixture were taken and analyzed by HPLC. Thermo Finnigan HPLC system was
used to quantify 4ꢀNP formed after enzymatic hydrolysis of 4ꢀNPA by using a UV detector at
operation wavelength of 315 nm. Separation of compounds was carried out on to reverseꢀphase
column (150 mm
× 4.6 mm; 5 µm). The mobile phase, delivered at a flow rate of 1.0 ml/min and
consisted of methanol and ammonium acetate buffer (pH 4.0; 10 mM) (53:47, v/v). The sample
injection volume was 20 µl. The HPLC system consists of a Thermo Finnigan SpectraSystem
SN4000 system controller, coupled with P2000 pump, a SCM1000 degasser and a UV2000 UV
detector at operation wavelength of 315 nm. Data were monitored and analyzed using ChromQuest
software (Thermo Finnigan, Waltham, MA, USA). Separation of compounds was carried out at r.t.
on to reverseꢀphase Chrompack HPLC column SS 150 mm × 4.6 mm; 5 m, with ChromSep guard
column intersil 5 ODSꢀ3 (Varian, Palo Alto California).
By using this assay method, compound 17b showed an IC50 against MAGL of 1.1 ± 0.3 µM, thus
confirming the inhibition activity measured by the colorimetric assay.
General Procedures and Materials.
All solvents and chemicals were used as purchased without further purification. Chromatographic
separations were performed on silica gel columns by flash chromatography (Kieselgel 40,
0
.040−0.063 mm; Merck). Reactions were followed by thin layer chromatography (TLC) on Merck
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aluminum silica gel (60 F254) sheets that were visualized under a UV lamp. Evaporation was
performed in vacuo (rotating evaporator). Sodium sulfate was always used as the drying agent.
1
13
Proton ( H) and carbon ( C) NMR spectra were obtained with a Bruker Avance III 400 MHz
spectrometer using the indicated deuterated solvents. Chemical shifts are given in parts per million
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(
ppm) (δ relative to residual solvent peak for H and C). HꢀNMR spectra are reported in this
order: multiplicity and number of protons. Standard abbreviation indicating the multiplicity were
used as follows: s = singlet, d = doublet, dd = doublet of doublets, t = triplet, tt = triplet of triplets,
dt = doublet of triplets, td = triplet of doublets, q = quartet, m = multiplet, bm = broad multiplet and
bs = broad signal. HPLC analysis: all target compounds (i.e., assessed in biological assays) were ≥
95% pure by HPLC, confirmed via UV detection (λ = 254 nm). Analytical reversedꢀphase HPLC
was conducted using a Kinetex EVO C18 column (5 ꢁm, 150 × 4.6 mm, Phenomenex, Inc.); eluent
A, water; eluent B, CH CN; after 5 min. at 25% B, a gradient was formed from 25% to 75% of B in
3
5
2
min and held at 75% of B for 10 min; flow rate was 1 mL/min. HPLC analyses were performed at
54 nm, with the exception of compound 8 which was analyzed at 226 nm (UVꢀvisible spectrum
and superimposition of HPLC chromatograms at 254 e 226 nm of compound 8 are reported in the
Supporting Information section). Yields refer to isolated and purified products derived from nonꢀ
optimized procedures. Compounds 1 and 2 were purchased from Cayman Chemical and 3 was
16
17
synthesized as previously reported. Compound 4 was characterized as previously reported.
General procedure for the synthesis of benzoylpiperidines 5, 6, 7aꢀc, 10aꢀc, 11aꢀc, 12aꢀc, 13aꢀc,
4aꢀc, 15bꢀc, 16aꢀc, 18c, 19aꢀc, 20, 30, 31, 44 and benzoylpiperazine 9. HATU (1.05 equiv) was
1
added to a solution of the appropriate benzoic acid (0.447 mmol, 1 equiv) in dry DMF (2.1 mL),
then DIPEA (0.31 mL, 4 equiv) was added dropwise. The resulting mixture was stirred at room
temperature for 30 min and then 4ꢀ(4ꢀchlorobenzoyl)piperidine 21 (100 mg, 1 equiv) or [1,1'ꢀ
biphenyl]ꢀ4ꢀyl(piperidinꢀ4ꢀyl)methanone 38 (for compounds
6
and 44), or 1ꢀ(4ꢀ
chlorobenzoyl)piperazine 41 (for compound 9) was added and left under stirring at room
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temperature until consumption of starting material (TLC). After this time, DMF was evaporated
under reduced pressure and the residue was diluted with water and extracted with EtOAc. The
2 4
organic layer was washed sequentially with water, saturated brine, dried over Na SO and the
solvent was removed under reduced pressure. The residue was purified with a flash column
chromatography (silica gel, appropriate mixture of nꢀhexane/ethyl acetate) and pure fractions
containing the desired compound were evaporated to dryness affording the amides.
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Synthesis of 4ꢀ(4ꢀchlorobenzyl)ꢀ1ꢀ(4ꢀmethoxybenzyl)piperidine (8). A solution of 4ꢀ(4ꢀ
2 3
chlorobenzyl)piperidine 39 (0.812 mmol, 1 eq) in 6.8 mL of DMF was treated with K CO (3 eq)
and 4ꢀmethoxybenzyl chloride 40 (1.1 eq) and the reaction mixture was stirred at room temperature
overnight. The mixture was diluted with water and extracted into ethyl acetate. The organic extracts
were washed successively with water and brine, and the organic solvent was removed under
vacuum on a rotary evaporator. Crude product was purified by flash chromatography over silica gel.
1
Elution with nꢀhexane/EtOAc (6:4) afforded the desired compound as a white solid (yield 53%). Hꢀ
NMR (CDCl
3
) δ (ppm): 1.27ꢀ1.37 (m, 2H), 1.41ꢀ1.52 (m, 1H), 1.54ꢀ1.62 (m, 2H), 1.83ꢀ1.94 (m,
2H), 2.49 (d, 2H, J = 7.0 Hz), 2.83ꢀ2.89 (m, 2H), 3.44 (s, 2H), 3.79 (s, 3H), 6.84 (AA’XX’, 2H, JAX
=
8.7 Hz, JAA’/XX’ = 2.5 Hz), 7.04 (AA’XX’, 2H, JAX = 8.4 Hz, JAA’/XX’ = 2.2 Hz), 7.19ꢀ7.24 (m, 4H).
13
CꢀNMR (CDCl ) δ (ppm): 32.18 (2C), 38.01, 42.64, 53.71 (2C), 55.38, 62.87, 113.66 (4C),
3
1
28.38, 130.55 (4C), 131.64, 139.33, 158.81. HPLC analysis: retention time = 11.718 min; peak
area, 99% (226 nm).
Procedure for the synthesis of
Oꢀdeprotected benzoylpiperidines 17aꢀc and 43. A solution of
pure amides 4, 15bꢀc or 44 (0.23 mmol) in anhydrous CH Cl (2.7 mL) was cooled to −78 °C and
2
2
treated dropwise with a 1.0 M solution of BBr in CH Cl (0.73 mL) under argon. The mixture was
3
2
2
left under stirring at the same temperature for 5 min and then at 0 °C for 1 h and finally at RT until
starting material was consumed (TLC). The mixture was then diluted with water and extracted with
ethyl acetate. The organic phase was washed with brine, dried and concentrated. The crude product
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