Design, synthesis, molecular modelling and in vitro cytotoxicity analysis of novel carbamate derivatives as inhibitors of Monoacylglycerol lipase

Article abstract of DOI:10.1016/j.bmc.2018.04.024

Monoacylglycerol lipase (MAGL) has an essential role in the catabolic pathway of the endocannabinoid 2-arachidonoylglycerol, which makes it a potential target for highly specific inhibitors for the treatment of a number of diseases. We designed and synthesized a series of carbamate analogues of URB602. We evaluated their inhibitory activity toward human MAGL in vitro both in cell culture and lysates. The target compounds exhibited moderate to excellent inhibitory activity against MAGL. The most promising compound 2b showed good inhibitory activity with IC₅₀ value of 4.5 ± 0.70 μM reducing MAGL activity to 82% of controls at 10 μM compared to 66% for the parent compound URB602. Interestingly, compounds 2b and 2c induce cell death through the inhibition of MAGL. Molecular modelling approaches and docking studies, used to investigate inhibitory profiles, indicated that trifluoromethyl substitutions of the aryl group and the benzene ring present at the oxygen side of the carbamate molecule had a significant impact on the activity.

Full text of DOI:10.1016/j.bmc.2018.04.024

Accepted Manuscript
Design, synthesis, molecular modelling and in vitro cytotoxicity analysis of
novel carbamate derivatives as inhibitors of Monoacylglycerol lipase
Simone Lauria, Cristiana Perrotta, Silvana Casati, Ilaria Di Renzo, Roberta
Ottria, Ivano Eberini, Luca Palazzolo, Chiara Parravicini, Pierangela Ciuffreda
PII:
S0968-0896(17)32482-3
https://doi.org/10.1016/j.bmc.2018.04.024
BMC 14307
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 December 2017
27 March 2018
10 April 2018
Please cite this article as: Lauria, S., Perrotta, C., Casati, S., Renzo, I.D., Ottria, R., Eberini, I., Palazzolo, L.,
Parravicini, C., Ciuffreda, P., Design, synthesis, molecular modelling and in vitro cytotoxicity analysis of novel
carbamate derivatives as inhibitors of Monoacylglycerol lipase, Bioorganic & Medicinal Chemistry (2018), doi:
https://doi.org/10.1016/j.bmc.2018.04.024
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Design, synthesis, molecular modelling and in vitro cytotoxicity analysis of
novel carbamate derivatives as inhibitors of Monoacylglycerol lipase
Simone Lauriaa, Cristiana Perrottaa, Silvana Casatia, Ilaria Di Renzoa, Roberta Ottriaa, Ivano
Eberinib, Luca Palazzolob, Chiara Parravicinib, Pierangela Ciuffredaa*
a Dipartimento di Scienze Biomediche e Cliniche “Luigi Sacco”, Università degli Studi di
Milano, Italy
bDipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano,
Italy
*Corresponding author:
Pierangela Ciuffreda
Università degli Studi di Milano-Dipartimento di Scienze Biomediche e Cliniche “Luigi
Sacco”
Via G.B. Grassi 74, 20157 Milano, Italy
Tel: ++39-02-50319695
e-mail: pierangela.ciuffreda@unimi.it
1

Abstract
Monoacylglycerol lipase (MAGL) has an essential role in the catabolic pathway of the
endocannabinoid 2-arachidonoylglycerol, which makes it a potential target for highly specific
inhibitors for the treatment of a number of diseases. We designed and synthesized a series of
carbamate analogues of URB602. We evaluated their inhibitory activity toward human
MAGL in vitro both in cell culture and lysates. The target compounds exhibited moderate to
excellent inhibitory activity against MAGL. The most promising compound 2b showed good
inhibitory activity with IC50 value of 4.5 ± 0.70 M reducing MAGL activity to 82% of
controls at 10 M compared to 66% for the parent compound URB602. Interestingly,
compounds 2b and 2c induce cell death through the inhibition of MAGL. Molecular
modelling approaches and docking studies, used to investigate inhibitory profiles, indicated
that trifluoromethyl substitutions of the aryl group and the benzene ring present at the oxygen
side of the carbamate molecule had a significant impact on the activity.
Keywords: Monoacylglycerol lipase, carbamate, structure-activity relationships, cytotoxicity,
anti-proliferative
1. Introduction
Monoacylglycerol lipase (MAGL) is a soluble serine hydrolase that peripherally associates
with cell membranes and was originally isolated from adipose tissues where it catalyzes the
final step in lipolysis, thereby liberating free fatty acids and glycerol for fuel or lipid
synthesis.1 The enzyme is found both in the brain (cortex, hippocampus, cerebellum,
thalamus, striatum) and in peripheral tissues such as the kidney, ovary, testis, adrenal gland,
adipose tissue and heart.2 2-arachidonoylglycerol (2-AG), the main substrate of MAGL,3,4 is
an endocannabinoid that activates cannabinoid (CB) receptors CB1 and CB2. The principal
route for 2-AG inactivation is via enzymatic hydrolysis generating arachidonic acid and
glycerol. MAGL is the first hydrolase implicated in 2-AG degradation, responsible for
approximately 85% of 2-AG hydrolysis.5
The involvement of endocannabinoids (ECs) in a wide range of physiological and
pathological processes, including anxiety, pain, feeding, cognition, food intake, inflammation,
memory, and emotions,6,7 has focused on the development of MAGL inhibitors for their
therapeutic potential as new analgesics and anxiolytics.8–10 Furthermore, studies using both
non-selective and highly selective MAGL inhibitors and MAGL knockout mice have found
that MAGL is the conjunction between ECs and prostaglandin signaling networks being the
2

primary source of arachidonic acid for the production of pro-inflammatory eicosanoids in the
nervous system, in the liver, and in the lungs.11 Blocking the activity of this enzyme by
specific inhibitors12 or genetic knock-out13 increases local levels of ECs leading to lesser side
effect potential compared to global CB receptor activation.14 Moreover genetic as well as
pharmacological inactivation of MAGL in mice causes a strong increase of monoglycerides in
many tissues including brain, liver, adipose tissue, intestine, and others, demonstrating a
major role of the enzyme in monoglyceride catabolism.3,4,7
MAGL inhibitors have been shown to have anti-cancer properties not only through
modulating the endocannabinoid eicosanoid network, but also by controlling fatty acid release
for the synthesis of protumorigenic signaling lipids.15 The therapeutic potential of MAGL
inhibitors, the fact that this enzyme is well characterized and its crystal structure has been
reported,16 have pointed out MAGL as target for several studies. Several classes of MAGL
inhibitors have been identified from early reversible ones, such as URB602,17 to
carbamoylating agents that react with the catalytic serine, such as JZL184.18 Over the last few
years, MAGL inhibitors with novel structures have been developed such as JJKK-048 lead
MAGL inhibitor from piperidine triazole urea class.19
The work presented here has focused on synthesis, structure-activity relationship (SAR), and
biological evaluation of novel carbamate derivatives, potentially targeting MAGL. These
derivatives were obtained on the basis of structural modifications of the parent compound
URB602. URB602 is a suitable scaffold for designing MAGL inhibitors since it increases
levels of 2-AG without altering levels of N–arachidonoylethanolamide (anandamide, AEA) in
both in vitro and in vivo models20 and does not affect the levels of other lipid metabolizing
enzymes in the endocannabinoid system.21 Moreover a very recent paper22 shed light on
chemo-preventive and curative effects of MAGL inhibition by URB602 in murine models of
colorectal cancer in vivo.
Structural modifications included isosteric replacement, ring size and para substitution of the
biphenyl moiety with substituents promoting hydrogen bound with MAGL site active, as
suggested by molecular modeling23 (Fig. 1). The biological evaluation includes kinetic
enzyme assays and cell-based assays using B16-F10 melanoma cells lysate. Cancer cells are a
good model for these studies due to the naturally high expression of MAGL in cancer.24
Moreover, we replaced carbamate with urea function to investigate what happens by this
modification.
3

Fig. 1. Structural modifications of hit compound URB602.
2. Results and Discussion
2.1. Chemistry
Starting from the hit compound URB602, we aimed to explore (i) the influence of different
para substituent on the biphenyl moiety, (ii) the replacement of cyclohexyl ring by aromatic
or heteroaryl moieties, and (iii) the role of carbamate group for the inhibition of MAGL. (Fig.
1). The target compounds 1-2a-c 3a-b (Scheme 1) were prepared through Suzuki coupling
reaction via the key biphenylamine intermediates (4a-c), involving phenylboronic acid, 3-
bromoaniline and a suitable palladium catalyst. These intermediates were isolated in good
yield (58-72%) and were reacted with carbonyldiimidazole (CDI), dimethylaminopyridine
(DMAP) and an appropriate alcohol (Scheme 1).
Scheme 1. Synthesis of compounds 1-2a-c 3a-b. Reagents and conditions: (a) Pd(PPh3)4,
Na2CO3, CH3OH, reflux, 12 h; (b) CDI, DMAP, CH3CN, reflux, overnight; (c) R1-OH,
CH3CN, reflux.
4

The urea analogue 5 was synthesized with a one pot reaction of CDI, DMAP and 4a in dry
CH3CN (Scheme 2). Product 5 precipitates out from the reaction mixture and can be obtained
in high purity by filtration (89 % yield).25
Scheme 2. Synthesis of compound 5. Reagents and conditions: (a) CDI, DMAP, CH3CN,
reflux, overnight.
All the prepared compounds have been characterized by IR, 1H NMR, 13C NMR, as well as
elemental analysis. In the 13C NMR spectra of compounds 2a-c, and 3a-b carbon-fluorine
coupling constants were observed. In 13C NMR spectra of compounds 2a-c trifluorinated
methyl group comes in the aromatic region of the spectrum and fluorinated carbons were
often difficult to find due to the 1, 2 and 3 bond J coupling to 19F. Indeed the recognition of
the multiplicities in the 13C resonances with low signal-to-noise ratios is challenging as the
signal is spread over multiple lines and can be buried in the noise.
2.2. Enzyme kinetics studies
The inhibitory activities (IC50 values) of the synthesized derivatives were obtained measuring
the residual activity of the enzyme with a method based on the enzymatic hydrolysis of a red
fluorogenic substrate 7-hydroxyresorufinyl-arachidonate (7-HRA).26 For our in vitro
experiments, we chose the commercially available human recombinant enzyme (Cayman
Chemical) to have a known reference activity independent from the variability of cell extracts.
A series of dilutions of test compounds (1:10) from 1nM to 100 M (six concentrations in
triplicate) was performed using 7-HRA as substrate. The activity data for a handful of
compounds at the highest concentration of 100 M were not determined due to the emergence
of solubility issues. 10% DMSO was used in the assay to ensure solubility of the hydrophobic
substrate 7-HRA, since hMAGL retains its activity in the presence of DMSO.27 Furthermore,
7-HRA was stable in 10% DMSO for at least 36 h at room temperature and for at least 6
months at 4 °C.
5

The unrepeatability of results with the analogue 1c (Table 1) did not allow to obtain any IC50
value for this compound, possibly for severe solubility problems. The results of inhibitory test
are summarized in Table 1. In Fig. S1 (Supplementary data) the linear regression between pKi
(-LogKi) and IC50 values is plotted from column 2 data. The test results show that compounds
2a-c, with trifluoromethyl and fluoro substitutions of the aryl group were very active MAGL
inhibitors. Whereas, unexpectedly, compound 5 increases activity of MAGL. The EC50 value
for 5 was reported in Table 1.
Table 1. Chemical structures, calculated partition coefficient (log P), dissociation constant
values, and inhibitory activity of compounds 1a-c, 2a-c, 3a-b, 5.
LogPa
In silico
In vitro
Cell based assay
Compound
Kib
IC50c (µM)
IC50c (µM)
5.32
4.69
5.44
4.27
6.22
5.91
5.16
5.49
5.17
8.9e-06
7.4e-06
6.0e-06
1.3e-05
1.6e-05
3.0e-06
3.3e-06
7.7e-06
4.8e-06
Kib
9.1 ± 0.8d
11.1 ± 2.3
11.3 ± 1.4
n.a.
10.5 ± 2.0
4.5 ± 0.7
6.7 ± 0.7
13.4 ± 0.9
7.9 ± 0.8
EC50e (µM)
2.8 ± 0.6e
19.79 ± 0.079
16.48 ± 0.096
17.14 ± 0.083
n.a.
17.40 ± 0.102
9.414 ± 0.04
13.19 ± 0.038
26.69 ± 0.147
15.61 ± 0.161
EC50e (µM)
5.76 ± 0.02e
URB602
1a
1b
1c
2a
2b
2c
3a
3b
6.68
2.73e-06
5
a Calcd. using the Molinspiration Property Engine 2016.10 Program software. bThe dissociation constant
(Ki) values were computed through the binding free energy, according to the following equation: Ki =
eΔG/RT, where R represents the gas constant and T the absolute temperature, set at 300 K. cIC50 values are
the means of three separate determinations and were determined by more than five concentrations of
each inhibitor. dThe compound URB602 was used as a positive control in the fluorometric assay. EC50
e
values are the means of three separate determinations and were determined by more than five
concentrations of compound 5. Statistical calculation of IC50 and EC50 values was performed on
GraphPad Prism 5.02 (GraphPad Software, Inc.).
2.3. In vitro cell analysis
To assess the inhibitory effect of the molecules on cellular samples, an in vitro experiment
was performed using cell lysates derived from B16-F10 melanoma, a cancer cell line known
to express MAGL.24,28 B16-F10 melanoma cells lysate was pre-treated for 60 min with
DMSO (vehicle) or the compounds at different concentrations in a 96 well plate. Then, the 7-
HRA fluorogenic substrate was added to each well and fluorescence was measured every 2.5
min for 2 h. Each molecule was tested in a range of concentration from 0 to 75 μM, as
suggested from previous studies on URB602.29
The general trend of enzyme inhibition among all molecules is very similar to that of
URB602: in terms of potency, they showed activities that are appreciable when administered
6

at μM concentrations. The molecules 2b and 2c showed an increase in activity (IC50 = 9.4 μM
± 0.04 and 13.2 μM ± 0.038 respectively) that is significantly different from URB602 (IC50 =
19.8 μM ± 0.079) (Supplementary Table 1 and Fig. S2). Ureic molecule 5 showed no activity
at all as inhibitor but seemed to increase the activity of the enzymatic apparatus
(Supplementary Fig. S2).
The reported calculated partition coefficient between water and octanol (log P) for the
compound 1a-c, 2a-c, 3a-b, 5. (Table 1) shows that these molecules have comparable
lipophilicity, so the ability to enter lipid membranes by passive diffusion are good for all
compounds. For the two compounds, i.e. 2b and 2c, with the best IC50 in vitro and in cell
based assay, the ability to inhibit MAGL was tested also in intact cells. Both the compounds
were able to significantly reduce MAGL activity at the concentration of 10 µM
(Supplementary Fig. S2b).
Given the potential of inhibiting MAGL as antitumor therapeutic strategy, we tested in vitro
the cytotoxic capacity of the synthesised compounds. To this end B16-F10 cells were treated
with 10 µM of each compound for 48 h and cell proliferation was analysed by flow cytometry
using the fluorescent dye Cyto Track red.30 As shown in Fig. 2a at 10 µM only the
compounds 2b and 2c were able to inhibit cell proliferation significantly, with 2c showing the
highest activity against B16-F10; all the other compounds (1a, 1b, 2a, 3a and 3b), including
URB602 did not modified the proliferation index of B16-F10 cells with respect to the vehicle
(DMSO) treated cells. These results demonstrate the effective anti-proliferative role of
compounds 2b and 2c in melanoma cells. For these two compounds, we then analysed the
ability to induce cell death, comparing them with URB602. Flow cytometry analysis of
phosphatidylserine exposure on the outer leaflet of the plasma membrane (a typical feature of
apoptotic cell death) revealed that 10ꢀµM of 2b and 2c for 48ꢀh induced a significant increase
in the Annexin V+ cells (Fig. 2b) no apoptosis was induced by the administration of URB602.
Of notice, exposure to 10 µM of 2c for 48h resulted in a massive induction of cell death both
by apoptosis (c.a. 65% of cells) and necrosis (c.a. 15% of cells). The hydrolysis of 2-AG is
catalysed not only by MAGL, but also by two novel serine hydrolases, alpha-beta-hydrolase
domain 6 and 12 (ABHD6 and ABHD12).5 In B16-F10 the expression of MAGL and
ABHD6, evaluated by real-time PCR was very similar (CT=11.050.92 for MAGL and
CT=10.730.52 for ABHD6), while ABHD12 was almost undetectable (CT=16.842.28).
To date, little is known about the role of ABHD6 in tumors. This serine hydrolase has been
found differentially expressed among various tumor cell lines,31 but its down-regulation
seems not to be related with inhibition of tumor growth.32 Taken together these data,
7

complemented by the knowledge that MAGL accounts for approximately 85% of hydrolysis
of 2-AG and ABHD6 and ABHD12 are responsible only for almost the remaining 15% of
hydrolysis,5 suggested that the effect of compounds 2b and 2c is dependent on MAGL
inhibition. This was supported by the effect of decreasing doses of the compound 2c (75 nM
and 35 nM). Indeed, the compounds were synthetized based on a specific MAGL inhibitor
and reducing the dose should guarantee the interaction with the highly affine receptor. The
two low doses of 2c were still able to inhibit cell proliferation and trigger cell death via
apoptosis in a dose-dependent manner (Fig. 2c and 2d).
Fig. 2. Effect of synthetic compounds on cell proliferation and apoptosis. (a) B16-F10 cells
were cultured in the presence of 10 µM of each compound for 48 h. Cell proliferation was
analysed by flow cytometry. Data are expressed as the fold change of proliferation index over
control (DMSO) (n = 3). *pꢀ<ꢀ0.05 and ***pꢀ<ꢀ0.0001 vs the respective control. (b) B16-F10
cells were treated with 10 µM of compounds 2b and 2c for 48 h. Cell death was analysed by
8

flow cytometry. Images on the left are representative contour plots of Annexin V-FITC/PI
staining. The graph on the right show the mean ± S.E.M. percentage of apoptotic cells
(Annexin V+/PI− + Annexin V+/PI+ fraction; the Annexin V−/PI+ fraction, regarded as necrotic
stage, was excluded) for 3 independent experiments. ***pꢀ<ꢀ0.0001 vs the respective control.
(c, d) Evaluation of cell proliferation (c) and apoptosis (d) after treatment of cells with 35 nM
and 75 nM of the compound 2c for 48 h. Images on the left are representative proliferation (c)
and contour plots (d). The vertical black line in the proliferation plots represents the
undivided cell peak used as point of reference. Data in the graphs on the right are expressed as
the fold change of proliferation index over control (DMSO treated cells) (c) and % of
apoptotic cells (d) (n = 3). **pꢀ<ꢀ0.001 and ***pꢀ<ꢀ0.0001 vs the respective control.
To further investigate the dependence of the cytotoxic effect of compound 2c by MAGL
inhibition we evaluated two cell lines expressing different levels of the three enzymes (Fig.
3a), the human breast adenocarcinoma MCF7 and embryonic kidney HEK293T. As shown in
Fig. 3a, MCF7 expressed MAGL at higher levels with respect to HEK293T, while ABHD6
and ABHD12 were expressed at significantly lower levels. As result, MCF7 were more
sensible to apoptosis induced by compounds 2c with respect to HEK293T (Fig. 3b), thus
indicating a predominant role for MAGL inhibition in the induction of apoptosis. The
cytotoxic effect on MCF7 was further confirmed by the evaluation of MAGL activity. In the
presence of both compounds 2b and 2c enzyme activity was indeed inhibited in a significant
manner (Fig. 3c). The lower sensitivity of MCF7 to compound 2c with respect to B16-F10
may be due to different mechanisms of chemoresistance, as for instance the lack of expression
of caspase 3 in this cell line.33 To investigate whether the inhibition of MAGL activity by 2c
accounted for its protective effect, we treated MCF7 cells with the compound alone and
together with JZL184, a known specific inhibitor of MAGL (Supplementary Fig. S3a). The
two molecules induced apoptosis at the same levels when administered alone or in
combination, thus corroborating the idea that they act through the same target i.e. MAGL
(Supplementary Fig. S1a). These data were also confirmed by the fact that both oleoyl ethyl
amide (OEtA), an inhibitor of fatty acid amide hydrolase enzyme (FAAH) and KT195, an
inhibitor of ABHD6, were not able to induce MCF7 apoptosis (Supplementary Fig. S3b).
9

Fig. 3. (a) evaluation of MAGL, ABHD6 and ABHD12 expression in MCF7 and HEK293T
human cell lines. Data are expressed as fold change vs HEK293T (n=4). **pꢀ<ꢀ0.001;
***pꢀ<ꢀ0.0001 vs HEK293T. (b) Cell death analysis by flow cytometry. Right panel:
representative contour plots of Annexin V-FITC/PI staining of HEK293T and MCF7 cells
treated with 10 µM of compound 2c for 48 h (n=3). The data in the graph are expressed as
means ± S.E.M.. *pꢀ<ꢀ0.05 vs DMSO treated cells. (c) MCF7 cells were cultured for 6 h in
the presence of the compounds 2b and 2c (10 µM). MAGL activity was measured on total cell
lysate. *pꢀ<ꢀ0.05; **pꢀ<ꢀ0.001 vs DMSO treated cells.
All these data combined with the results obtained in the in vitro experiments (Table 1) and
with the knowledge that URB602 selectively block MAGL29 seem to indicate that compounds
2b and 2c induce cell death through the inhibition of MAGL.
10

2.4. Study of inhibitor selectivity
To assess the selectivity of more active compounds (2b, 2c) as MAGL inhibitors, we
evaluated also their activity toward fatty acid amide hydrolase enzyme (FAAH), the other
major endocannabinoids hydrolytic enzyme. First the fluorogenic probe for FAAH,
arachidonoyl-aminomethylcoumarine amide (AAMCA), was synthesized starting from
arachidonic acid and 7-amino-4-methyl coumarine, by a coupling reaction.34 NMR confirmed
the structure and purity grade of the product (Supplementary data).
Inhibitor activity of 2b and 2c has been assessed by a well-known fluorescence based method
on hFAAH35 and oleoyl ethyl amide36 has been selected as positive control. In our
experimental conditions compounds 2b and 2c, at cells bioactive concentration of 10 μM,
showed an inhibitory activity of 15% and 10% respectively while OEtA displayed 99%
inhibition of hFAAH hydrolytic activity (Supplementary fig.S4).
2.5. Molecular mechanism of MAGL inhibition
Fig. 4 shows the MAGL binding site, according to the MOE Site Finder, a region located in
the near proximity of the catalytic active site that has already been described by Szabo and
colleagues21 as the most likely binding site for the interaction with potential inhibitors.
Fig. 4. MAGL binding site according to the MOE Site Finder. 3JW8 is represented as ribbon
and the binding site as a gray shaded molecular surface.
Table 1 reports the dissociation constant value (Ki) for the top-scoring pose of each tested
inhibitor, showing the ability of all the tested compounds to bind and block the MAGL active
site. Globally, our docking simulations provided overlapping poses for all the tested
compounds and a good superposition of their common functional groups, confirming the
accuracy of this approach.
11

At an atomistic level, the top-scoring pose of the parent compound URB602 well fits the
enzyme binding site, as also suggested by its good Ki value, in agreement with the
experimental IC50 (Fig. 5a,b). The carbamate group lies close to the catalytic Ser132,
confirming that the actual mechanism of these compounds is the inhibition of MAGL activity.
Due to its nonpolar nature, URB602 establishes interactions with the hydrophobic residues
that characterize the MAGL pocket. In detail, the mono-substituted aromatic ring of URB602
engages Leu215 and Ile189 sidechains via C-H-π interactions. Moreover, the carbonyl oxygen
of the ligand interacts with Ala61 backbone via H-bond.
Compound 2b, associated to the best Ki value, also showed the best inhibitory activity using
the fluorescent method previously described.
Fig. 5a,c reports the MAGL::2b interactions: the molecule accommodates in proximity of
hydrophobic residues such as Leu and Ile; in particular, as for URB602, an arene-H
interaction occurs between the side chain of Ile189 and the biphenyl group, so that the
trifluoromethyl substituent is stretched toward Ile189 and Leu21. The benzyl ring occupies
the same region of the URB602 cyclohexyl ring, and the two carbamate groups are
superposed. Moreover, the ligand carbonyl oxygen is placed in proximity of Met133 and
Ala61, establishing two H-bond interactions with their backbones.
Fig. 5. Molecular recognition mechanism of MAGL inhibitors. (a) URB602 and compound
2b docked to MAGL are represented in cyan and yellow stick, respectively. MAGL is
represented as ribbon. (b) Detailed molecular interactions between MAGL residues and
URB602 (b) and compound 2b (c) in the catalytic pocket. Catalytic Ser132 is highlighted in
magenta.
Overall, our in silico results suggest that the introduction of classical small lipophilic
substituent on the biphenyl group, like fluorine or trifluoromethyl, improves the affinity for
the enzyme, likely due to the hydrophobic environment of the pocket. Similarly, the
replacement of the aliphatic cyclohexyl group with an aromatic one produces a more efficient
12

interaction between the potential inhibitor and the enzyme, especially when the carbamate and
the ring are separated by a single carbon atom chain, while elongating the alkyl chain does not
result in an improved affinity.
Common features of the tested inhibitors in the binding MAGL pocket are recapitulated by
the proposed pharmacophoric model. In detail, an acceptor, a donor, an aromatic and a
hydrophilic feature are highlighted in Fig. 6 as spheres. This model has been built keeping
into account not only the common feature of the functional groups but also the activity for
each inhibitor. No differences exist between two different models based on activities on
purified MAGL and cultured cells. All the pharmacophoric features are present in the 100%
of the inhibitors compounds, with an alignment score of 7.5, and seem to be crucial for the
activity of this series of compounds. By definition, only the inactive compound does not
match the pharmacophoric query.
Fig. 6. Pharmacophoric model and structure activity relationship for MAGL inhibitors. The
pharmacophore features are represented as colored spheres: orange for aromatic or Pi ring
center, green for hydrophobic, cyan for H-bond acceptor and magenta for H-bond donor.
2.6. Molecular mechanism of MAGL activation
During this synthetic project a symmetric compound 5 (Scheme 2) was designed, synthesized
and tested, and it was demonstrated to be an interesting MAGL activator. In order to better
understand its activating mechanism at a molecular level, we checked the 3D MAGL structure
(RCSB PDB: 3JV823) and we looked for all the other crystallographic structures of MAGLs,
identifying a human form co-crystallized with an irreversible inhibitor (RCSB PDB: 3JWE23).
A careful inspection of both the structures revealed a molar ratio of 4 molecules of 2-methyl-
pentane-2,4-diol (MPD) per 1 enzyme in 3JV8 and a molar ratio of 2 molecules of SAR per 1
enzyme in 3JVE.23 In detail, in 3JV8 crystal, approx. two MPD molecules occupy the
13

catalytic site, while two other molecules are located in a second pocket (external binding site),
close to the α-helix 4 of the lid domain,23 suggesting that the MAGL secondary pocket is not a
crystallographic artifact. In 3JVE, one SAR molecule covalently binds Ser132 of the catalytic
triad, after the elimination of triazole as a leaving group, while the second one is non-
covalently bound in the second pocket in contact with the α-helix 4. Fig. 7a shows the
superposition between 3JV8 and 3JVE binding sites in which MPD and SAR molecules
occupy both active and secondary pockets with very similar distribution in space.
These observations suggested us that our activator could preferentially bind to this second
pocket, modifying the orientation of the α-helix 4, and consequently increasing the
accessibility of substrates to, and their affinity for the catalytic site.
A first run of molecular docking was carried out on this crystallographic structure, after
removing both the two inhibitor molecules. Our activator was docked on the 3JWE 3D
structure, setting the whole protein as binding site. We obtained two different clusters of
accepted solutions. The most energetically favored pose belongs to the cluster located in the
external binding site, and has a binding free energy of -6.94 kcal/mol with an associated
affinity value of -7.56 kcal/mol; whereas the top-scoring pose belonging to the cluster located
into the catalytic site is characterized by less favorable energetic values: -6.88 kcal/mol and -
6.97 kcal/mol, respectively (Fig. 7b).
14

b
a
Fig. 7. MAGL binding site. (a) Focus on two different crystals of MAGL binding site: in
magenta, crystal structure of 3JW8, with 4 co-crystallized MPD molecules; in light blue,
crystal structure of 3JWE, with 2 co-crystallized SAR molecules. (b) Superposition of two
activator::MAGL (3JWE) complexes: in orange, the activator in placed in the enzyme
catalytic site; in green, the activator in the external binding site.
Furthermore, a reference substrate, 2-AG, was docked both on an apo- and on a holo-MAGL
form bound to the studied activator. The latter form was generated by molecular docking of
the activator on the MAGL external binding site. The solutions were characterized by the
following affinity values: -8.49 kcal/mol, and -9.09 kcal/mol, for the apo- and holo-MAGL
form (Fig. 8), respectively. This result suggests a higher affinity for this reference substrate
when our activator is bound to the MAGL external binding site.
15

Fig. 8. MAGL can contemporarily bind out activator and a model substrate. Top scoring
docking pose of 2-AG (dark green) with respect to the activator::MAGL complex (PDB:
3JWE) in the catalytic site. Ser132 of the catalytic triad is depicted in blue.
3. Conclusions
In this study, we have designed and synthesized a new series of carbamates as MAGL
inhibitors. The best activity values were obtained with compounds 2b, 2c, and 3b (IC50 4.5,
6.7, and 7.9 M, respectively) bearing the 4'-trifluoromethyl substitution of the biphenyl
moiety and aromatic functionality at the oxygen side of the carbamate molecule. Compounds
containing fluoro moieties on the 4' position of biphenyl ring resulted in a decreased potency
for hMAGL enzyme. Molecular modelling studies further demonstrated that compound 2b
establishes favorable interactions, due to its improved lipophilicity, within the active site of
hMAGL, rich of hydrophobic residues that participate in the molecular recognition
mechanism.
Taken together, docking scores confirm that an increase of the hydrophobic and aromatic
functional groups of the inhibitors improves their affinity for the enzyme. Our molecular
modelling strategy allowed us to develop a first pharmacophoric model, connecting the
structure and activity relationships of the investigated series of inhibitors. The
characterization of further MAGL active compounds is mandatory for refining our structure
and activity relationship model in order to identify novel hit compounds.
In vitro experiments on cancer cells demonstrated that compounds 2b and 2c, were able to
block cell proliferation as well as to induce cell death via apoptosis thus indicating this
molecule as very promising anti-cancer drug. Moreover in present studies, three series of data,
determined separately, with hMAGL, with cell lysate and modeling studies are in agreement
16

and gave precise indications that compound 5 is an activator, allowed us to identify, to our
knowledge, first MAGL activator posing the bases for the development of novel enzymatic
MAGL modulators with promising applications. Further studies are needed to better
understand this activity.
4. Experimental section
4.1. Chemistry
All of the reagents and solvents, analytically pure, were obtained from commercial sources
and used without further purification. Reaction progress was monitored by TLC on pre-coated
silica gel plates (Kieselgel 60 F254, Merck) and visualized by UV254 light. Flash column
chromatography was performed on silica gel (particle size 40-63 m, 60 Å). Anhydrous
solvents were obtained by passing solvents through activated alumina columns. When stated,
reactions were carried out under an inert atmosphere. Purity of all products ( 98 %) was
verified by thin-layer chromatography and NMR measurements. Elemental analyses were
obtained for all intermediates and are within  0.4% of theoretical values. Human
recombinant C-terminal hexahistidine-tagged protein expressed in E. coli MAGL (hMAGL),
human recombinant C-terminal His-tagged protein expressed in Sf21 cells (hFAAH) and
oleoylethylamide (OEtA) were purchased from Cayman Chemical.
Melting points were determined with a Stuart Scientific SMP3 melting point apparatus. 1H
NMR spectra were recorded in CDCl3 (isotopic enrichment 99.95%) or DMSO-d6 (isotopic
enrichment 99.98%) or CD3OD (isotopic enrichment 99.98%), solutions at 300 K using a
Bruker AVANCE 500 instrument (500.13 MHz for 1H, 125.76 MHz for 13C) using 5 mm
inverse detection broadband probes and deuterium lock. Chemical shifts () are given in parts
per million (ppm) and were referenced using residual signals of the solvent as internal
standard (1H: CHCl3, 7.26 ppm; CD3OD, 3.30 ppm; DMSO, 2.49 ppm. 13C: CDCl3, 77.0 ppm;
CD3OD, 47.6 ppm, central line; DMSO-d6, 39.5 ppm). Coupling constants (J) are in hertz
(Hz) and the experimental error in the measured 1H-1H coupling constants is  0.5 Hz. The
results are presented as the chemical shift in ppm, number of protons, multiplicity, J values in
Hertz, proton position, or carbon position. The splitting pattern abbreviations are as follows:
s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet, and bs, broad signal. For two-
dimensional experiments, Bruker microprograms using gradient selection (gs) were applied.
IR spectra were determined on a Jasco FTIR 4100 spectrometer as a thin film on NaCl plates
and were expressed in cm-1 scale.
17

4.1.1. General procedure for the Suzuki coupling reaction to obtain compounds 4a-c. To a
solution of phenylboronic acid (4.0 mmol) in 10 mL of MeOH, Na2CO3 (8.0 mmol) and 3-
bromoaniline (4.0 mmol) were added sequentially. To this suspension, Pd tetrakis (0.14
mmol) was added and the reaction was heated to reflux for 12 h; the suspension was cooled at
room temperature, diluted with MeOH and the black precipitate was removed by filtration.
The filtrate was diluted with water and extracted with CH2Cl2 (3 x 10 ml). The organic phase
was dried on Na2SO4 and concentrated under reduced pressure.
4.1.2. [1,1'-biphenyl]-3-amine (4a). The product, prepared starting from phenylboronic acid
and 3-bromoaniline and purified by chromatography on silica gel (petroleum ether:CH3OH,
80:20 v/v) was obtained as a white solid in 72% yield. mp: 29-30.5 °C (lit.37 28-30 °C); 1H
NMR (CDCl3) δ: 7.58 (2H, d, J=7.4 Hz, 2', 6'-H), 7.43 (2H, dd, J=7.4, 7.4 Hz, 3', 5'-H), 7.35
(1H, t, J=7.4 Hz, 4'-H), 7.24 (1H, dd, J=7.6, 7.6 Hz, 5-H), 7.01 (1H, ddd, J=7.6, 1.0, <1.0 Hz,
6-H), 6.92 (1H, dd, J=1.3, <1.0 Hz, 2-H), 6.69 (1H, ddd, J= 7.8, 1.3, 1.0 Hz, 4-H), 3.75 (2H, s,
7-NH2). 13C NMR δ: 146.7 (3), 142.5 (1), 141.4 (1'), 129.7 (5), 128.6 (3', 5'), 127.2 (6', 2'),
127.1 (4'), 117.7 (6), 114.1 (4), 114.0 (2).
4.1.3. 4'-(trifluoromethyl)-[1,1'-biphenyl]-3-amine (4b). The product, prepared starting from
4-(trifluoromethyl)phenylboronic acid and 3-bromoaniline, purified by chromatography on
silica gel (petroleum ether:CH3OH, 70:30 v/v), was obtained as a white solid in 58% yield.
mp: 90-91 °C (lit.38 91-92 °C); 1H NMR (CDCl3) δ:7.41-7.68 (4H, m, 2, 3, 5and 6), 5.29
(1H, dd, J=7.9 Hz, 5-H), 5.60 (1H, dd, J=1.3, <1.0 Hz, 2-H), 5.59 (1H, ddd, J=7.5, 1.3, 1.3
Hz, 6-H), 5.37 (1H, ddd, J= 7.5, 1.3, <1.0 Hz, 4-H); 13C NMR δ: 146.9 (3), 144.9 (1'), 140.9
(1), 129.9 (5), 129.2 (q, 2JC-F = 32.6 Hz, 4'), 127.3 (2', 6'), 126.2 (q, 1JC-F = 271.6 CF3), 125.6
(q, 3JC-F = 4.1 Hz, 3', 5'), 117.7 (6), 114.6 (4), 113.8 (2).
4.1.4. 4'-fluoro-[1,1'-biphenyl]-3-amine (4c). The product, prepared starting from 4'-
fluorophenylboronic acid and 3-bromoaniline and purified by chromatography on silica gel
(petroleum ether: CH3OH, 70:30 v/v), was obtained as a white solid in 68% yield. mp: 29-
30.5 °C (lit.39 28-30 °C); 1H NMR (CD3OD)  7.59-7.55 (2H, AA part of AABB system,
2'- and 6'-H), 7.17 (1H, dd, J=7.8, 7.8 Hz, 5-H), 7.16-7.11 (2H, BB part of AABB system,,
3'- and 5'-H), 6.96 (1H, dd, J=1.3, 1,3 Hz, 2-H), 6.91 (1H, ddd, J=7.8, 1.3, 1.3 Hz, 6-H), 6.72
18

(1H, ddd, J=7.8, 1.3, 1.3 Hz, 4-H); 13C NMR δ: 162.3 (d, 1JC-F = 244.5 Hz, 4'), 147.8 (3),
140.9 (1), 137.9 (d, 4JC-F = 2.9 Hz, 1'), 129.1 (5), 128.2 (d, 3JC-F = 8.0 Hz, 2', 6'), 116.5 (6),
114.8 (d, 2JC-F = 21.5 Hz, 3', 5'), 114.2 (4), 113.6 (2).
4.1.5. General procedure for the preparation of compounds 1-2a-c, 3a-b. A stirred solution of
the previously obtained amine (1 mmol), CDI (4 mmol, 0.648 g), and DMAP (0.2 mmol,
0.024 g) in CH3CN (8 mL) was heated at 100 °C for 15 h under N2 atmosphere. Then a
solution of the suitable alcohol (1.2 mmol) in CH3CN (1 mL) was added. The mixture was
stirred at 100 °C for 8 h and concentrated.
4.1.6. Benzyl [1,1'-biphenyl]-3-ylcarbamate (1a). The product, prepared starting from 4a and
phenyl methanol and purified by chromatography on silica gel (petroleum ether:EtOAc, 90:10
v/v), was obtained as a white solid in 62% yield. mp: 73.2-73.9 °C. TLC (petroleum ether:
EtOAc, 95:5 v/v): Rf=0.33; IR (Nujol): 3324, 1941, 1700, 1550, 1376, 1274, 1076, 754, 696
cm-1; 1H NMR (CDCl3) 7.68 (1H, bs, 2-H), 7.61 (2H, bd, J=7.3 Hz, 2', 6'-H), 7.47-7.37
(10H, m, 3', 4', 5', 5, 6, 5 x Ar-H), 7.33 (1H, dd, J=8.7, 1.8 Hz, 4-H), 6.79 (1H, bs, NH), 5.26
(2H, s, -CH2); 13C NMR δ: 153.4 (CO), 142.3 (1), 140.7 (1'), 138.2 (3), 136.1 (1), 129.5,
128.8, 128.7, 128.4, 128.4, 127.5, 127.2 and 122.4 (C-Ar), 67.1 (CH2). Anal. Calcd. for
C20H17NO2: C, 79.19; H, 5.65; N, 4.62. Found: C, 79.30; H, 5.47; N, 4.60.
4.1.7. Phenethyl [1,1'-biphenyl]-3-ylcarbamate (1b). The product, prepared starting from 4a
and phenylethanol and purified by chromatography on silica gel (petroleum ether:EtOAc,
80:20 v/v), was obtained as a white solid in 74% yield. mp 98.2-99.4 °C. TLC (petroleum
ether: EtOAc, 80:20 v/v): Rf=0.41; IR (Nujol): 3414, 1955, 1729, 1573, 1376, 1275, 1078,
754, 699 cm-1; 1H NMR (CDCl3)  7.66 (1H, bs, 2-H), 7.61 (2H, d, J=7.6 Hz, 2', 6'-H), 7.46
(2H, t, J=7.6 Hz, 3', 5'-H), 7.42-7.27 (10H, m, 3', 4', 5', 5, 6, 5 x Ar-H), 6.69 (1H, bs, NH),
4.45 (2H, t, J=7.0 Hz, -CH2), 3.04 (2H, t, J=7.0 Hz, -CH2). 13C NMR δ: 153.5 (CO), 142.3 (1),
140.7 (1'), 138.3 (1), 137.8 (3), 129.5, 128.9, 128.8, 128.6, 127.5, 127.2, 126.7 (C-Ar), 65.7
(CH2), 35.5 (CH2). Anal. Calcd. for C21H19NO2: C, 79.47; H, 6.03; N, 4.41. Found: C, 79.57;
H, 6.12; N, 4.36.
4.1.8. Furan-2-ylmethyl [1,1'-biphenyl]-3-ylcarbamate (1c). The product, prepared from 4a
and 2-furfuryl alcohol and purified by chromatography on silica gel (petroleum ether:EtOAc,
19

90:10 v/v) was obtained as an oil 68% yield. TLC (petroleum ether: EtOAc, 75:25 v/v):
Rf=0.61; IR (Nujol): 3330, 1934, 1737, 1598, 1265, 1156, 744, 704 cm-1; 1H NMR
(CDCl3)7.67 (1H, bs, 2-H), 7.60 (2H, d, J=7.8, 2, 6-H), 7.48 (1H, dd, J=1.9, <1 Hz, 5-
H), 7.46 (2H, dd, J=7.8, 7.8 Hz, 3, 5-H), 7.41-7.36 (3H, m, 4-, 5-, 4-H), 7.33 (1H, ddd,
J=7.1, 1.7, 1.7 Hz, 6-H), 6.78 (1H, ds, NH), 6.50 (1H, dd, J=3.3, <1.0 Hz, 3-H), 6.41 (1H,
dd, J=3.3, 1.9 Hz, 4-H), 5.21 (2H, s, CH2).13C NMR δ: 153.0 (CO), 149.6 (2), 143.4 (5),
142.3 (1), 140.7 (1), 138.1 (3), 129.4, 128.8, 127.5, 127.2, (5, 2, 3, 4, 5, 6), 122.5 (6),
117.6 (2, 4), 110.8 (4), 110.6 (3), 58.8 (CH2). Anal. Calcd. for C18H15NO3: C, 73.71; H,
5.15; N, 4.78. Found: C, 73.86; H, 5.26; N, 4.60.
4.1.9. Cyclohexyl (4'-(trifluoromethyl)-[1,1'-biphenyl]-3-yl)carbamate (2a). The product,
prepared starting from 4b and cyclohexanol and purified by chromatography on silica gel
(petroleum ether:EtOAc, 90:10 v/v), was obtained as a white solid in 74% yield. mp 124.8-
126.2 °C. TLC (Rf=0.62, petroleum ether: EtOAc, 80:20 v/v). IR (Nujol): 3342, 1912, 1697,
1548, 1329, 1235, 1118, 1070, 781 cm-1; 1H NMR (CDCl3) 7.78 (1H, bs, 2-H), 7.73-7.69
(4H, m, 2', 3', 5' and 6'-H), 7.42 (1H, dd, J=7.8, 7.8 Hz, 5-H), 7.36 (1H, ddd, J=7.8, 1.4, 1.4
Hz, 4-H), 7.31 (1H, ddd, J=7.8, 1.4, 1.4 Hz, 6-H), 6.69 (1H, s, NH), 4.83-4.77 (1H, m, 1-H),
2.00-1.98 (2H, m, 2-Heq and 6-Heq), 1.80-1.76 (2H, m, 3-Heq and 5-Heq), 1.62-1.59 (1H,
m, 4-Heq), 1.54-1.39 (4H, m, 2-Hax , 3-Hax, 5-Hax and 6-Hax), 1.34-1.26 (1H, m, 4-Hax);
13C NMR δ: 153.2 (CO), 144.3 (1'), 140.7 (1), 138.8 (3), 129.6 (5), 129.4 (q, 2JC-F = 32.2 Hz,
4'), 127.4 (2, 6), 126.4 (q, 1JC-F = 270.2 CF3), 125.7 (q, 3JC-F = 4.2 Hz, 3', 5'), 122.1 (6),
118.2 (4), 117.4 (2), 73.9 (1), 31.9 (2, 6), 25.4 (4), 23.8 (3, 5). Anal. Calcd. for
C21H22F3NO2: C, 66.83; H, 5.88; N, 3.71. Found: C, 66.96; H, 5.90; N, 3.84.
4.1.10. Benzyl (4'-(trifluoromethyl)-[1,1'-biphenyl]-3-yl)carbamate (2b). The product,
prepared starting from 4b and phenyl methanol and purified by chromatography on silica gel
(petroleum ether:EtOAc, 80:20 v/v), was obtained as a white solid in 84% yield. mp 114.6-
116.0 °C. TLC (petroleum ether: EtOAc, 80:20 v/v): Rf=0.59; IR (Nujol): 3292, 1935, 1687,
1574, 1542, 1330, 1273, 1114, 1072, 782 cm-1; 1H NMR (CDCl3) 7.75 (1H, bs, 2-H), 7.70
(4H, m, 2', 3', 5' and 6'-H), 7.46-7.37 (8H, m, 4, 5, 6, 2, 3, 4, 5and 6), 7.32 (1H, ddd,
J=7.9, 1.2, 1.2 Hz, 6-H), 6.80 (1H, s, NH), 5.27 (2H, s, CH2); 13C NMR δ: 153.3 (CO), 144.3
(1'), 140.8 (1), 138.5 (3), 135.9 (1''), 129.7 (5), 129.5 (q, 2JC-F = 32.0 Hz, 4'), 128.7, 128.5,
128.4 (2, 3, 4, 5, 6), 127.5 (2, 6), 126.4 (q, 1JC-F = 270.2 CF3), 125.7 (q, 3JC-F = 4.2 Hz,
20

3', 5'), 122.5 (6), 118.4 (4), 117.6 (2), 67.2 (CH2). Anal. Calcd. for C21H16F3NO2: C, 67.92; H,
4.34; N, 3.77. Found: C, 68.02; H, 4.21; N, 3.74.
4.1.11. Furan-2-ylmethyl 4'-(trifluoromethyl)biphenyl-3-ylcarbamate (2c). The product,
prepared starting from 4b and furfuryl alcohol and purified by chromatography on silica gel
(petroleum ether:EtOAc, 80:20 v/v), was obtained as a white solid in 88% yield. mp 119.2-
120.0 °C. TLC (petroleum ether: EtOAc, 80:20 v/v): Rf=0.63; IR (Nujol): 3378, 1921, 1696,
1546, 1339, 1275, 1111, 1075, 786 cm-1; 1H NMR (CDCl3)7.74 (1H, bs, 2-H), 7.72-7.68
(4H, m, 2'- 3'- 5' and 6'-H), 7.48 (1H, dd, J=1.7, <1 Hz, 5''-H), 7.42 (1H, dd, J=7.8, 7.8 Hz, 5-
H), 7.36 (1H, bd, J=7.5 Hz, 4-H), 7.33 (1H, ddd, J=7.5, 1.4, 1.4 Hz, 4-H), 6.78 (1H, bs, NH),
6.51 (1H, dd, 3.3, <1 Hz, 4''-H), 6.42 (1H, dd, J=3.3, 1.7 Hz, 3''-H), 5.21 (2H, s, CH2); 13C
NMR δ: 153.0 (CO), 149.4 (2''), 144.2 (1'), 143.4 (5), 140.8 (1), 138.3 (3), 129.7 (5), 129.4
(q, 2JC-F = 32.2 Hz, 4), 127.5 (2, 6), 125.7 (q, 1JC-F = 270.6 CF3), 125.3 (q, 3JC-F = 4.0 Hz, 3',
5'), 122.5 (6), 118.4 (4), 117.5 (2), 110.9 (4), 110.7 (3), 58.8 (CH2). Anal. Calcd. for
C19H14F3NO3: C, 63.16; H, 3.91; N, 3.88. Found: C, 63.32; H, 3.79; N, 3.84.
4.1.12. Cyclohexyl (4'-fluoro-[1,1'-biphenyl]-3-yl)carbamate (3a). The product, prepared
starting from 4c and cyclohexanol and purified by chromatography on silica gel (petroleum
ether:EtOAc, 80:20 v/v), was obtained as a white solid in 84% yield. mp 124.6-125.0 °C.
TLC (Rf=0.59, petroleum ether: EtOAc, 80:20 v/v). IR (Nujol): 3338, 1693, 1586, 1548,
1232, 1213, 1174, 1101, 1063, 790 cm-1; 1H NMR (CDCl3) 7.70 (1H, bs, 2-H), 7.59-7.55
(2H, AA part of AABB system, 2'- and 6'-H), 7.38 (1H, dd, J=7.8, 7.8 Hz, 5-H), 7.32 (1H,
ddd, J=7.8, 1.4, 1.4 Hz, 4-H), 7.25 (1H, ddd, J=7.8, 1.4, 1.4 Hz, 6-H), 7.15-7.11 (2H, BB part
of AABB system, 3'- and 5'-H), 6.68 (1H, s, NH), 4.83-4.75 (1H, m, 1-H), 2.01-1.96 (2H,
m, 2-Heq and 6-Heq), 1.81-1.76 (2H, m, 3-Heq and 5-Heq), 1.62-1.57 (1H, m, 4-Heq),
1.54-1.38 (4H,m, 2-Hax, 3-Hax, 6-Hax, and 5-Hax), 1.30 (1H, ddd, J=12.6, 4.4 Hz, 4-Hax);
13C NMR δ: 162.6 (d, 1JC-F = 246.4 Hz, 4'), 153.2 (CO), 141.2 (1), 138.6 (3), 136.9 (d, 4JC-F
=
3.1 Hz, 1'), 129.5 (5), 128.7 (d, 3JC-F = 8.0 Hz, 2', 6'), 121.9 (6), 117.4 (d, 2JC-F = 21.5 Hz, 3',
5'), 115.7 (4), 115. 5 (2), 73.9 (1''), 32.0 (2,6), 25.4 (4), 23.8 (3, 5). Anal. Calcd. for
C20H22FNO2: C, 73.37; H, 6.77; N, 4.28. Found: C, 73.47; H, 6.72; N, 4.21.
4.1.13.Benzyl (4'-fluoro-[1,1'-biphenyl]-3-yl)carbamate (3b). The product, prepared starting
from 4c and phenyl methanol and purified by chromatography on silica gel (petroleum
21

ether:EtOAc, 85:15 v/v), was obtained as a white solid in 84% yield. mp 82.2-82.8 °C. TLC
(petroleum ether: EtOAc, 80:20 v/v): Rf=0.55; IR (Nujol): 3278, 1696, 1612, 1554, 1512,
1494, 1241, 1218, 1102, 1077, 790 cm-1; 1H NMR (CDCl3, 500 MHz)  7.74 (1H, bs, 2-H),
7.58-7.53 (2H, AA part of AABB system, 2'- and 6'-H), 7.46-7.36 (6H, m, 5, 2, 3, 4, 5,
6-H), 7.33 (1H, ddd, J=7.8, <1.0, <1.0 Hz, 4-H), 7.27 (1H, ddd, J=7.8, <1.0, <1.0 Hz, 6-H),
6.79 (1H, bs, NH), 5.25 (2H, s, CH2); 13C NMR δ: 162.6 (d, 1JC-F = 246.5 Hz, 4'), 153.4 (CO),
141.3 (1), 138.3 (3), 136.9 (d, 4JC-F = 3.1 Hz, 1'), 136.0 (1''), 129.5 (5), 128.8 (d, 3JC-F = 8.0
Hz, 2', 6'), 128.7, 128.6, 128.5, 128.4 (1'', 2, 3, 4, 5, 6), 122.3 (6), 117.4 (d, 2JC-F = 22.7
Hz, 3', 5'), 115.7 (4), 115.6 (2), 67.2 (CH2). Anal. Calcd. for C20H16FNO2: C, 74.75; H, 5.02;
N, 4.36. Found: C, 74.77; H, 5.14; N, 4.56.
4.1.14. Synthesis of 1,3-di([1,1'-biphenyl]-3-yl)urea (5). CDI (115 mg, 7.08 mmol, 4 eq.), and
4a (300 mg, 1.77 mmol, 1 eq.) were mixed in dry toluene (8 ml) and heated at reflux for 12 h
under nitrogen atmosphere. After cooling to room temperature white precipitates were
collected by filtration, washed with diethyl ether and cold CH2Cl2/ CH3CN to obtain 255 mg
(0.70 mmol, 89%) of 2 as a white crystalline solid. mp 258.2-259.0 °C; TLC (petroleum ether:
EtOAc, 90:10 v/v): Rf=0.24; IR (Nujol): 3300, 1635, 1601, 1555, 1497, 1398, 1271, 1075,
785, 698 cm-1; 1H NMR (d6-DMSO) 8.85 (2H, s, 2 x NH), 7.83 (2H, dd, J=1.9, 1.9 Hz, 2 x
2-H), 7.63 (4H, dd, J=7.7, 1.0 Hz, 2 x 2'- and 6'-H), 7.48 (4H, dd, J=7.7, 7.7 Hz, 2 x 3'- and 5'-
H), 7.42 (2H, ddd, J=8.1, 1.9, 1.2 Hz, 2 x 4-H), 7.40-7.35 (4H, m, 2 x 5- and 4'-H), 7.27 (2H,
ddd, J=8.1, 1.9, 1.2 Hz, 2 x 6-H). 13C NMR δ: 153.2 (CO), 141.3 (1'), 140.8 (1), 140.7 (3),
129.8 (5), 129.4 (3', 5'), 128.0 (4'), 127.1 (2', 6'), 120.8 (6), 117.9 (4), 117.0 (2). Anal. Calcd.
for C25H20N2O: C, 82.39; H, 5.53; N, 7.69. Found: C, 82.42; H, 5.68; N, 7.52.
4.2. Biological Section.
4.2.1. Enzyme kinetics study on human recombinant MAGL (hMAGL). For kinetic experiments,
the assay was performed in a 96-well black plate in a total volume of 100 μl per well. The
reaction mixture consisted of 25 ng/well of hMAGL and 5 μM (final concentration) of 7-HRA
substrate (prepared in DMSO) in assay buffer solution consisting of 50 mM Tris-HCl (pH 7.4,
1 mM EDTA) and 10% DMSO (final concentration) at room temperature.
For hMAGL inhibitors testing, 25 ng/well of hMAGL dissolved in assay buffer (50mM Tris-
HCl pH 7.4, 1 mM EDTA) are added to 80 μl of buffer solution; 5 μl of inhibitor solution in
DMSO (between 10 to 75 M) or DMSO alone were added and then incubated at room
22

temperature for 60 minutes. After incubation, the reaction was started by the rapid addition of
5 μl of 7-HRA intermediate solution to obtain the final concentration of 5 μM in the final
volume of 100 μl. The fluorescence was measured at different time by Promega-GloMax
multidetection system and the fluorescence unit converted to amount of resorufin produced
(ex = 571 nm, em = 588 nm) basing on a calibration curve by using standard solutions in
DMSO (10 μl of standard in 90 μl of buffer).
In the kinetic study, different concentrations of hMAGL and 7-HRA were tested and no
inhibitor solutions were added.
The reaction rate was determined by linear regression analyses (r2 0.98) of the fluorescence
emission increase over time. The background reaction rate with no enzyme present was
subtracted and the reaction rates were normalized to the reaction rate with no inhibitor present
(100%).
The IC50 values were calculated after 30 minutes and derived from two independent
experiments performed in triplicate, calculated as nonlinear regressions using sigmoid dose–
response setting with variable Hill slope by GraphPad Prism 5.0 for Windows.
4.2.2. Inhibitor selectivity of compounds 2b and 2c with hFAAH. For selectivity tests, in a 96
well black plate to each well, containing 184.5 µl of reaction buffer (50 mM TRIS-HCl, 1mM
EDTA, pH 9), were added 1U of hFAAH and 2 µl of 2b or 2c or OEtA (1mM in DMSO) to
reach a final concentration of 10 μM. After 1h of incubation at room temperature was added 1
µl of AAMCA (2mM in DMSO) to reach final concentration of 20 μM. The reaction was run
for 30 minute in a JASCO fluorimeter with fluorescence readings taken every 3 minutes at a
wavelength of 460 nm applying an excitation wavelength of 360 nm. As done for MAGL the
reaction rate was determined by linear regression analyses (r2 0.98) of the fluorescence
emission increase over time. The background reaction rate with no enzyme present was
subtracted and the reaction rates were normalized to the reaction rate with no inhibitor present
(100%) (Supplementary fig. S4).
4.2.3. Cell lines and culture techniques. B16-F10, B16-F1 melanoma cell lines, MCF7
adenocarcinoma and HEK293T embryonic kidney cell lines were obtained from American
Type Culture Collection (Rockville, Md., USA). Melanoma cells were cultured in Iscoves’s
medium supplemented with 10% heat-inactivated (56 °C) fetal bovine serum (FBS), 2 mM L-
glutamine, 100 IU/ml penicillin G sodium, 100 lg/ml streptomycin sulfate (Euroclone, Milan,
23

Italy). MCF7 were cultured in 50% Dulbecco’s modified Eagle’s medium (DMEM) high
glucose and 50% Ham’s F-12 nutrient mixture, supplemented 10% heat-inactivated (56 °C)
fetal bovine serum (FBS), 2 mM L-glutamine, 100 IU/ml penicillin G sodium, 100 lg/ml
streptomycin sulfate. HEK293T were cultured in 50% Dulbecco’s modified Eagle’s medium
(DMEM) high glucose supplemented 10% heat-inactivated (56 °C) fetal bovine serum (FBS),
2 mM L-glutamine, 100 IU/ml penicillin G sodium, 100 lg/ml streptomycin sulfate. Cells
were maintained in a humidified atmosphere of 5% CO2 in air at 37 °C.
4.2.4. Enzyme kinetic study on cancer cell lysates and intact cells. B16-F10 melanoma
cells,19,28 after 36 h of culture, were homogenised for 30 min at 4° C in lysis buffer (10 mM
Tris, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100 with protease inhibitor
mixture, pH 7.4) and lysates were then centrifuged for 5 min at 1,500 × g.
The assay was carried out in a 96-well format using 1 g of cell lysate and 10 M of
substrate. To test the inhibitors, B16-F10 melanoma cells lysate was pre-treated for 60 min
with DMSO or the compounds at different concentrations. Then, the substrate was added to
each well and fluorescence was measured every 2.5 min for 2 h at 588 nm (λex.571
nm/λem.588 nm) by a Glomax-Microplate Multimode Reader (Promega). The graphs show
the results after 5 min of incubation. Values are expressed as mean ± SEM (n = 3).
For the evaluation of MAGL activity in intact cells, B16-F10 melanoma cells were cultured in
presence of compounds 2b-c or DMSO for 6 h and then homogenised for 30 min at 4° C in
lysis buffer. Lysates were then centrifuged for 5 min at 1,500 × g and the assay was carried
out as described above using 1 g of cell lysate and 10 M of substrate. The graphs show the
results after 5 min of incubation. Values are expressed as mean ± SEM (n = 3).
4.2.5. Proliferation assay. B16-F10 cell proliferation was assessed by measuring the cell
divisions by flow cytometry.30 Cells were stained by the CytoTrack Cell Proliferation Assays
(CytoTrack Red 628/643; Bio-Rad, Hercules, CA, USA), according to the manufacturer’s
protocol and analysed by Gallios Flow Cytometer (Beckman-Coulter, Brea, CA, USA). The
proliferation index, i.e. the average number of cells that an initial cell became, was calculated
using the FCS Express 4 software (De Novo System, Portland, OR, USA).
4.2.6. Apoptosis assay. Cell death was evaluated by annexin V/propidium iodide (PI) staining
via flow cytometry.30,40 Briefly, cells were incubated with Alexa Fluor® 488 annexin V
(ThermoFisher Scientific, Waltham, MA, USA) (5 μl x 106 cells) to assess the
24

phosphatidylserine exposure on the cell surface, and 5 μg/ml PI to exclude necrotic cells in
binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2). Annexin V (FL-1) and PI
(FL-3) fluorescences were measured in 10,000 cells per sample by Gallios Flow Cytometer.
4.2.7. Real-time PCR. The analysis of mRNA expression was performed as previously
described.30,40 Briefly, total RNA from B16-F10, B16-F10, MCF7 and HEK293T cells was
extracted with the Purezol RNA Isolation Kit (Bio-rad), according to the manufacturer’s
protocol. First-strand cDNA was generated from 1ꢀμg of total RNA using iScript Reverse
Transcription Supermix (Bio-Rad). A set of primer pairs was designed to hybridize to unique
regions of the appropriate gene sequence (Supplementary Table S1). PCR was performed
using SsoAdvanced Universal SYBR Green Supermix and the CFX96 Touch Real-Time PCR
Detection System (Bio-Rad). The fold change was determined relative to the control after
normalizing to β-actin (internal standard) through the use of the formula 2-ΔΔCT.
4.3. Molecular Modelling.
4.3.1. In silico molecular docking simulations. Molecular docking of all the investigated
compounds has been carried out on a crystallographic hMAGL 3D structure obtained from
RCSB PDB, code 3JW8,23 while hMAGL 3D structure obtained from RCSB PDB, code
3JWE23 was used for the binding studies of the activator compound 5. The protein was
prepared through the Structure Preparation program of the Molecular Operating Environment
(MOE) Prepare module [https://www.chemcomp.com/MOE-
Mocular_Operating_Environment. htm]. This tool is useful for checking and correcting
structures and preparing macromolecular data for further computational analysis.
A database, containing target compounds, was manually built by the MOE Builder program.
All the compounds were energy minimized by the Energy Minimize program with default
parameters of the MOE Compute module, to produce a single low-energy conformation for
each putative ligand.
The enzyme binding site was identified by the use of the MOE Site Finder program, which
uses a geometric approach to calculate possible binding sites in a receptor starting from its 3D
atomic coordinates. This method is based not on energy models but on alpha spheres, which
are a generalization of convex hulls.
The in silico screening was carried out with the MOE Dock program contained in the
Compute module. The full MAGL structure was set as receptor and the binding site was
delimited by a set of dummy atoms previously defined by the Site Finder analysis.
25

Before starting with the placement procedure, conformations were generated for each ligand
by sampling their rotatable bonds. The selected placement methodology was Triangle
Matcher, in which the poses are generated by superposing triplets of ligand atoms and triplets
of receptor site points. The receptor site points are alpha spheres centers that represent
locations of tight packing. Before scoring all the generated poses, duplicate complexes were
removed. Poses are considered as duplicates if the same set of ligand-receptor atom pairs are
involved in hydrogen bond interactions and the same set of ligand atom receptor residue pairs
are involved in hydrophobic interactions. The accepted poses were scored according to the
London dG scoring, which estimates the free energy of binding of the ligand from a given
pose. The top-scoring 30 poses were passed to a refinement step based on molecular
mechanics and then rescored according to the GBVI/WSA dG, a forcefield-based scoring
function that estimates the free energy of binding of the ligand from a given pose. It has been
trained using the MMFF94x and Amber99 force field on the 99 protein–ligand complexes of
the SIE training set.41 In order to speed up the calculation, residues over a 6 Å cut-off distance
away from the pre-refined pose were ignored, both during the refinement and in the final
energy evaluation. All receptor atoms were held fixed during the refinement. By default,
during the refinement, solvation effects are calculated using the reaction field functional form
for the electrostatic energy term and a dielectric constant of 4 and the final energy is evaluated
using the Generalized Born solvation model (GB/VI).
The estimated binding free energy (affinity) of the top-scoring solution (selected according to
the docking score) for all the investigated compounds, the complexes were further refined
through the use of a set of specific MOE procedures, named QuickPrep, aimed at the
minimization of ligands in the receptor binding site. During molecular docking procedures,
the MMFF94x force field was used both for protein and ligands. Dissociation constant (Ki)
was computed through the following equation: Ki = eΔG/RT, where R represents the gas
constant and T the absolute temperature, set at 300 K.
4.3.2. Pharmacophoric model.
The pharmacophoric model was built by the MOE Pharmacophore Elucidator using default
settings. The IC50 value of the inactive compound was defined as the cut-off for satisfying the
pharmacophoric queries. The unified scheme was selected as set of rules for defining the
available features. Only the top scoring poses from molecular docking were selected for
building the model, after a refinement flexible alignment of the chemicals.
26

Statistical analysis. Data are expressed as mean ± S.E.M. of single determinations performed
in three or four independent experiments at different days. IC50 values were calculated by
nonlinear regression using sigmoid dose–response setting with variable Hill slope by
GraphPad Prism 5.0 for Windows. A p value < 0.05 (*) was considered significant. For
proliferation and apoptosis assays statistical significance of raw data between the groups in
each experiment was evaluated using one-way ANOVA followed by the Tukey’s post-test
(multiple comparisons).
Acknowledgements
This work was supported by: “Università di Milano, Linea 2 – Azione A 2016-2017” funding
to P.C., C.P. and I.E.; MIUR Progetto Eccellenza.
A. Supplementary data
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