ω-Quinazolinonylalkyl aryl ureas as reversible inhibitors of monoacylglycerol lipase

Article abstract of DOI:10.1016/j.bioorg.2019.103352

The serine hydrolase monoacylglycerol lipase (MAGL) is involved in a plethora of pathological conditions, in particular pain and inflammation, various types of cancer, metabolic, neurological and cardiovascular disorders, and is therefore a promising target for drug development. Although a large number of irreversible-acting MAGL inhibitors have been discovered over the past years, there are only few compounds known so far which inhibit the enzyme in a reversible manner. Therefore, much effort is put into the development of novel chemical entities showing reversible inhibitory behavior, which is thought to cause less undesired side effects. To explore a wide range of chemical structures as MAGL binders, we have applied a virtual screening approach by docking small molecules into the crystal structure of human MAGL (hMAGL) and envisaged a library of 45 selected compounds which were then synthesized. Biochemical investigations included the determination of the inhibitory potency on hMAGL and two related hydrolases, i.e. human fatty acid amide hydrolase (hFAAH) and murine cholesterol esterase (mCEase). The most promising candidates from theses analyses, i.e. three ω-quinazolinonylalkyl aryl ureas bearing alkyl spacers of three to five methylene groups, exhibited IC₅₀ values of 20–41 μM and reversible, detergent-insensitive behavior towards hMAGL. Among these compounds, the inhibitor 1-(3,5-bis(trifluoromethyl)phenyl)-3-(4-(4-oxo-3,4-dihydroquinazolin-2-yl)butyl)urea (96) was selected for further kinetic characterization, yielding a dissociation constant K_i = 15.4 μM and a mixed-type inhibition with a pronounced competitive component (α = 8.94). This mode of inhibition was further supported by a docking experiment, which suggested that the inhibitor occupies the substrate binding pocket of hMAGL.

Full text of DOI:10.1016/j.bioorg.2019.103352

BioorganicChemistryxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
ω-Quinazolinonylalkyl aryl ureas as reversible inhibitors of
monoacylglycerol lipase
⁎
Florian M. Datoa,b, Jörg-Martin Neudörflb, Michael Gütschowc, Bernd Goldfussb,
,
⁎
Markus Pietscha,
a Institute II of Pharmacology, Center of Pharmacology, Medical Faculty, University of Cologne, Gleueler Strasse 24, 50931 Cologne, Germany
b Institute of Organic Chemistry, Department of Chemistry, University of Cologne, Greinstrasse 4, 50939 Cologne, Germany
c Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany
A R T I C L E I N F O
A B S T R A C T
Keywords:
The serine hydrolase monoacylglycerol lipase (MAGL) is involved in a plethora of pathological conditions, in
particular pain and inflammation, various types of cancer, metabolic, neurological and cardiovascular disorders,
and is therefore a promising target for drug development. Although a large number of irreversible-acting MAGL
inhibitors have been discovered over the past years, there are only few compounds known so far which inhibit
the enzyme in a reversible manner. Therefore, much effort is put into the development of novel chemical entities
showing reversible inhibitory behavior, which is thought to cause less undesired side effects. To explore a wide
range of chemical structures as MAGL binders, we have applied a virtual screening approach by docking small
molecules into the crystal structure of human MAGL (hMAGL) and envisaged a library of 45 selected compounds
which were then synthesized. Biochemical investigations included the determination of the inhibitory potency
on hMAGL and two related hydrolases, i.e. human fatty acid amide hydrolase (hFAAH) and murine cholesterol
esterase (mCEase). The most promising candidates from theses analyses, i.e. three ω-quinazolinonylalkyl aryl
ureas bearing alkyl spacers of three to five methylene groups, exhibited IC50 values of 20–41 µM and reversible,
detergent-insensitive behavior towards hMAGL. Among these compounds, the inhibitor 1-(3,5-bis(tri-
fluoromethyl)phenyl)-3-(4-(4-oxo-3,4-dihydroquinazolin-2-yl)butyl)urea (96) was selected for further kinetic
characterization, yielding a dissociation constant Ki = 15.4 µM and a mixed-type inhibition with a pronounced
competitive component (α = 8.94). This mode of inhibition was further supported by a docking experiment,
which suggested that the inhibitor occupies the substrate binding pocket of hMAGL.
Serine hydrolases
Cholesterol esterase
Monoacylglycerol lipase
Fatty acid amide hydrolase
Inhibitors
Promiscuous inhibition
Enzyme kinetics
1. Introduction
the migration, invasion and growth of cancer cells. In case of aggressive
prostate cancer cells, MAGL also suppresses anti-tumorigenic en-
The serine hydrolase monoacylglycerol lipase (MAGL, EC 3.1.1.23)
is part of the endocannabinoid system and has been found in both the
central nervous system and peripheral tissues, including liver, kidneys,
testis, lung, prostate and small intestine [1–4]. MAGL is important for
the regulation of a vast number of different pathological processes,
particularly cancer, but also inflammation and pain, metabolic, neu-
rological, and cardiovascular disorders [5–17]. In aggressive human
cancer cells (e.g. breast, ovarian, colorectal, melanoma, liver and
prostate), for example, MAGL is highly expressed [4,5,18,19] and acts
as a key metabolic hub “regulating a fatty acid network enriched in pro-
tumorigenic lipids” [20]. These signaling lipids promote the survival,
docannabinoid signals [4,5,12,13,21]. Knocking down MAGL or
blocking its activity by the irreversible carbamate inhibitor JZL 184
(108, Fig. 1) were shown to result in a reduced tumor growth rate and a
decreased cancer cell migration accompanied by a lower conversion of
monoacylglycerols to pro-tumorigenic signal lipids [4,5,21]. However,
complete inhibition of MAGL results in a functional antagonism of the
cerebral cannabinoid receptor CB1, and, thus, might be associated with
psychiatric side effects [22]. These side effects might be prevented by
using reversibly acting MAGL inhibitors [15,22]; however, the super-
iority of such compounds over irreversible enzyme modifiers will have
to be proven by future studies [23].
⁎ Corresponding authors at: Institute II of Pharmacology, Center of Pharmacology, Medical Faculty, University of Cologne, Gleueler Str. 24, D-50931 Cologne,
Germany. (Markus Pietsch). Institute of Organic Chemistry, Department of Chemistry, University of Cologne, Greinstr. 4, D-50939 Cologne, Germany. (Bernd
Golfuss).
E-mail addresses: goldfuss@uni-koeln.de (B. Goldfuss), markus.pietsch@uk-koeln.de (M. Pietsch).
https://doi.org/10.1016/j.bioorg.2019.103352
Received 16 August 2019; Received in revised form 2 October 2019; Accepted 9 October 2019
0045-2068/©2019ElsevierInc.Allrightsreserved.
Pleasecitethisarticleas:FlorianM.Dato,etal.,BioorganicChemistry,https://doi.org/10.1016/j.bioorg.2019.103352

F.M. Dato, et al.
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Fig. 1. A selection of reported reversible inhibitors of rat (1, 2, 5) and human (3, 4, 6–15) MAGL. In this study, quinazolinone 96 was investigated on human MAGL,
with ω-phthalimidoalkyl 3,5-bis(trifluoromethyl)phenyl ureas 106 and 107 as well as irreversibly acting carbamate JZL 184 (1 0 8) being used as reference inhibitors
[43].
In recent years, a plethora of MAGL inhibitors have been published
often containing reactive carbamate or urea moieties, which lead to an
irreversible inactivation of the enzyme by covalent modification of the
active serine residue [12,23–26]. In contrast, only few reversibly acting
MAGL inhibitors are known so far [23,24] and, thus, “novel chemical
classes displaying a reversible mechanism of action” towards this
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enzyme [23] are much-needed. Examples of reversible MAGL inhibitors
(Fig. 1) include the steroids pristimerin (1) and euphenol (2), which
have been identified by screening a commercially available compound
library [27]. Furthermore, the piperazinyl azetidinyl amide ZYH (3)
developed by Janssen Pharmaceutica [28] was the first reversible in-
hibitor co-crystallized with MAGL (PDB: 3PE6) [29]. Many efforts in
investigating reversibly acting compounds have also been made by the
groups of Lopez-Rodríguez and Tuccinardi, who identified, for example,
the 4-benzylphenylacetyl esters 4 [30] and 5 [31], the N-acyl piper-
idines 6 and 7–9, [32–35], and the biphenyl derivatives 10–13
[36–38]. In addition, Aida et al. [39] discovered the piperazinyl pyr-
rolidin-2-one 14 and Patel et al. [40] described the loratadine analogue
15 as novel MAGL inhibitors exhibiting reversible behavior.
(trifluoromethyl)benzylamine were obtained from commercial sources,
the 3′,5′-bis(trifluoromethyl)-substituted biphenylamines 102–104 and
1-(3,5-bis(trifluoromethyl)phenyl)piperazine (105) (Scheme S7) were
prepared as previously described by Dato et al. [43]
Synthesis of nine oxalamides with various substitution patterns
(Scheme 2) started from ethyl chlorooxoacetate by reaction with var-
ious commercially available amines or previously synthesized 1-(3,5-bis
(trifluoromethyl)phenyl)piperazine (105)[43] to obtain the respective
ethyl-2-amino-2-oxoacetates 23–26 in 71–92% yield. After saponifica-
tion, the free carboxylic acids 27–30 formed in 70–90% yield were then
coupled with various amines, amongst them compounds 103–105,
yielding the target compounds 31–39 (23–85%). The molecular struc-
ture of oxalamide 37 was proven by X-ray crystallography (Fig. S17).
To obtain the three squaramides 42–44 (38–72%, Scheme 3), 3,4-
diethoxy-3-cyclobutene-1,2-dione was initially treated with 3,5-bis
(trifluoromethyl)aniline and 3′,5′-bis(trifluoromethyl)-[1,1′-biphenyl]-
4-amine (104) to obtain the anilides 40 (72%) and 41 (93%), respec-
tively, which were then reacted with the aliphatic amines 3-phenyl-
propylamine and 2-(4-phenylpiperazin-1-yl)ethanamine (100, Scheme
S6). Squareamides have recently drawn our attention, since derivatives
of the compounds investigated herein were found to act as efficient
hydrogen-bonding catalysts mediating the “enantioselective Michael
addition of 4-hydroxycoumarin to β-nitrostyrenes” [44].
The objective of our study was the identification of novel chemical
entities reversibly acting on human MAGL (hMAGL) as potent and se-
lective inhibitors. For this purpose, we initially applied an in silico ap-
proach by virtual screening of low molecular weight compounds
(among others, structures from the ZINC database) [41,42] using mo-
lecular docking on the crystal structure of hMAGL (PDB: 3PE6). A se-
lection of identified in silico hits was then synthesized and characterized
in terms of its inhibitory activity on hMAGL. To investigate the se-
lectivity of inhibitors for MAGL over other serine hydrolases, all com-
pounds were also assayed on human fatty acid amide hydrolase
(hFAAH, EC 3.5.1.99) and murine cholesterol esterase (mCEase, EC
3.1.1.13), both of which accept fatty acid derivatives as physiological
substrates similar to MAGL. Those inhibitors most active on the three
serine hydrolases were re-tested in the presence of Triton X-100 (0.01%
(v/v)) to exclude promiscuous inhibition, leaving three quinazolinones
(85, 96 and 97) which inhibited hMAGL with IC50 values in the mid-
micromolar range. Finally, the binding of compound 96 (Fig. 1) to
hMAGL was analyzed by a detailed kinetic investigation, yielding a
mixed-type mode of inhibition with a pronounced competitive com-
ponent (α = 8.94) and a Ki value of 15.4 µM.
Thiazoles 48–51, 55 and 56 were prepared via two different
synthesis routes (Schemes 4 and S1). The synthetic route to obtain
thiazoles 55 and 56 (Scheme 4) started from benzoyl chloride by re-
action with thiourea and ammonium hydroxide to obtain N-carba-
mothioylbenzamide 52 (52%), which was reacted with ethyl bromo-
pyruvate yielding thiazole 53 (94%) and subsequently saponified to the
free carboxylic acid 54 (99%). Final amide coupling using 1-phe-
nylpiperazine and 1-(3,5-bis(trifluoromethyl)phenyl)piperazine (1 0 5)
gave the target thiazoles 55 (68%) and 56 (68%), respectively. The
structures of 55 and 56 were confirmed by X-ray crystallography (Figs.
S19 and S20).
Our series was further completed by imidazolones 59 and 60
(Scheme S2), triazoles 62, 65, 67, 72, 74 (Schemes S3 and S4) and
(oxa)naphthalenes 75, 77–81 (Scheme S5).
2. Results and discussion
2.1. Chemistry
Synthesis of eight quinazolinone derivatives (Schemes 5 and 6)
containing either a urea (84–86, 96 and 97) or an amide moiety
(87–89) started by preparing the quinazolinone heterocycle bearing 2-
alkyl spacers of 2–5 methylene units with terminal carboxylic acid (82,
54%; 83, 81%) or primary amino groups (94, 95; overall yields based
on 90 and 91 [43] were 53% and 62%, respectively). Then, amide
coupling of the carboxylic acids with commercially available amines
and 1-(3,5-bis(trifluoromethyl)phenyl)piperazine (1 0 5) using standard
conditions (EDC × HCl, HOBt) gave the target compounds 87–89
(76–79%), whereas the urea derivatives 84–86 (8–21%) were acces-
sible by Curtius rearrangement of the carboxylic acid azides of 82 and
83 followed by trapping of the formed isocyanates with anilines
(Scheme 5). The final quinazolinones 96 (51%) and 97 (53%) resulted
from the amines 94 and 95, respectively, by reaction with 3,5-bis(tri-
fluoromethyl)phenyl isocyanate (Scheme 6). Successful synthesis of 96
We prepared a library of 45 compounds from eight substance
classes, including sulfonacetamides, oxalamides, squaramides, thia-
zoles, quinazolinones, imidazolones, triazoles, and (oxa)naphthalenes
(Schemes 1–6 and S1–S5). Representative synthetic routes to bioactive
compounds are outlined below; for others, see supplementary material.
Six 2-sulfonacetamide derivatives containing either aromatic or
aliphatic amide residues (Scheme 1) were synthesized starting from 1-
bromo-3-phenylpropane and thioglycolic acid followed by oxidation of
the resulting thioether to the corresponding sulfonyl derivative with
hydrogen peroxide in glacial acetic acid to yield sulfonylacetic acid 16
in 62% yield. The carboxylic acid was then converted to the amides
17–22 (51–87%) by means of 1-ethyl-3-(3-dimethylaminopropyl)car-
bodiimide hydrochloride (EDC × HCl) and 1-hydroxybenzotriazole
hydrate (HOBt). While 3,5-bis(trifluoromethyl)aniline and 3,5-bis
Scheme 1. Synthesis of 2-sulfonacetamides 17–22. Reagents and conditions: (a) NaOH, MeOH, 14 h, RT; (b) H2O2, AcOH, 14 h, RT; (c) amine, EDC × HCl, HOBt,
CH2Cl2, 14 h, 0 °C → RT. Pip, 1,4-disubstituted piperazine.
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Scheme 2. Synthesis of oxalamides 31–39. Reagents and conditions: (a) amine, TEA, THF, 14 h, 0 °C → RT; (b) KOH, H2O, EtOH, 2 h, 0 °C → RT; (c) LiOH, H2O, THF,
2 h, 0 °C → RT; (d) amine, EDC × HCl, HOBt, CH2Cl2, 14 h, 0 °C → RT. Pip, 1,4-disubstituted piperazine.
was proven by X-ray crystallography (Fig. S22).
and 4-MUB, respectively. Screening for inhibition of the three enzymes
by our library of 45 compounds was initially done at a concentration of
25 µM, with the residual enzymatic activities being given in Figs.
S1–S7. Eight of these compounds (amongst them one 2-sulfonacetamide
(21), two oxalamides (32 and 34), one squareamide (42), one thiazole
(56) and three quinazolinones (85, 96 and 97)) caused residual enzy-
matic activities ≤65% towards at least one of the hydrolases (which
corresponds to a calculated IC50 value ≤46 µM) and, thus, were se-
lected for further investigations (Table 1).
All intermediates and final products were structurally analyzed by
1H and 13C NMR spectroscopy, with the respective spectra being shown
in the supplementary material (Figs. S23–S106).
2.2. Screening for inhibitors of hMAGL, hFAAH and mCEase
The hydrolytic activity of hMAGL, hFAAH and mCEase was de-
termined as previously described by Dato et al. [43,45] using the
chromogenic compound 4-NPB and the fluorogenic substrates D-MAP
Promiscuous behavior of the identified hits was excluded by
Scheme 3. Synthesis of squaramides 42–44. Reagents and conditions: (a) aniline, MeOH, 48 h, RT; (b) amine, CH2Cl2, 24 h, RT. Pip, 1,4-disubstituted piperazine.
4

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Scheme 4. Synthesis of thiazoles 55 and 56. Reagents and conditions: (a) KSCN, acetone, 30 min, RT; (b) NH4OH, 30 min, RT; (c) ethyl bromopyruvate, EtOH, 2 h,
reflux; (d) NaOH, H2O, EtOH, 4 h, 60 °C; (e) amine, EDC × HCl, HOBt, CH2Cl2, 14 h, 0 °C → RT.
repeating the inhibition experiments in the presence of Triton X-100
(0.01% (v/v)) [46]. All compounds investigated on mCEase showed a
drop in inhibitory activity under these conditions, with thiazole 56
having been exemplarily characterized as promiscuous inhibitor by
determination of the inhibitor concentration resulting in 50% inhibition
(IC50) in the absence and presence of detergent (Fig. S12). In contrast,
promiscuous inhibition of FAAH by 56 (IC50 = 52.4 µM, Fig. S13) could
be excluded due to the applied assay conditions, [47] since the re-
spective assay buffer already contained Triton X-100 (0.1% (w/v)) in
the initial screening and the complete solubility of 56 at the chosen
concentrations had been shown by UV–Vis spectroscopy (Fig. S8) ac-
cording to Huang et al. [48].
concentration of 25 µM) showed a detergent-sensitive behavior,
whereas inhibition by quinazolinones 85, 96 and 97 was not much
influenced by Triton X-100 (Fig. 2, Figs. S14A and S14B). The latter
three inhibitors exhibited values of IC50 between 19.6 µM and 41.4 μM,
which were only slightly increased by factors of 1.2–1.8 when detergent
was added. The comparable inhibitory behavior of 85, 96 and 97 can be
attributed to the structural similarities of the three compounds
(Schemes 5 and 6), which only differ in the alkyl chain spacer between
the quinazolinone and the urea motifs, containing three, four and five
methylene groups, respectively. The insensitivity of the MAGL inhibi-
tion towards the presence of Triton X-100, which had been observed for
compounds 85, 96 and 97, might be explained by the inhibitor con-
centrations chosen for the IC50 determinations. At these concentrations,
In case of hMAGL, compounds 21 and 42 (analyzed at a single
Scheme 5. Synthesis of quinazolinones 84–89. Reagents and conditions: (a) benzene/diethyl ether/1,4-dioxane (2:2:1), 2 h, RT; (b) NaOH, EtOH, 40 min, 0 °C → RT;
(c) diphenylphosporyl azide, TEA, acetone, 3 h, RT; (d) xylene, 20 min, 135 °C; (e) aniline, 14 h, reflux; (f) amine, EDC × HCl, HOBt, DMF, 14 h, 0 °C → RT. Pip, 1,4-
disubstituted piperazine.
5

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Scheme 6. Synthesis of quinazolinones 96 and 97. Reagents and conditions: (a) neat, 3 h, 170 °C; (b) SOCl2, toluene, 14 h, 0 °C → RT; (c) anthranilamide, TEA, THF,
5 h, 0 °C → RT; (d) diphenyl ether, 1 h, 220 °C; (e) hydrazine hydrate, MeOH, 4 h, reflux; (f) 3,5-bis(trifluoromethyl)phenyl isocyanate, CHCl3, 14 h, RT.
Table 1
determined the dependency of IC50 on the pre-incubation time of in-
hibitor and enzyme before starting the enzymatic reaction. As pre-
viously shown [43,49], irreversibly acting compounds, such as the
potent MAGL inhibitor JZL 184 (108, Figs. 1 and 2) [50], show a strong
increase in inhibitory potency after pre-incubation with hMAGL
(IC50 = 0.013 µM after 30 min pre-incuabation [43]). In contrast, va-
lues of IC50 for the inhibition of hMAGL by 96 showed no time-de-
pendency within 60 min (Fig. 3), which strongly points to a reversible
mode of interaction. Similar results have been obtained with other re-
versible inhibitors of hMAGL [51], including compounds 6 [32], 8 [34],
9 [35], and 11 [37] (Fig. 1).
Residual enzymatic activities of hMAGL, hFAAH and mCEase in the presence of
selected compounds.
Residual enzymatic activity (%) at [I] = 25 µMa
Compd
hMAGL
hFAAH
mCEase
21
59.8
83.8
94.6
59.6
88.6
56.4
64.5
58.1
64.1
68.5
1.9b
2.5
65.7
89.0
88.7
80.0
48.0
93.4
82.1
88.4
78.2
85.0
6.1
1.3
2.8
2.4
8.7c
2.2
2.0
2.5
3.8
2.0
102
3
32
44.2
55.5
66.5
29.8
55.8
63.7
71.2
5.25
15.6
0.8b
34
1.9
0.7b
3.3
42
3.4b
3.5
56
1.0d
2.6b
2.4b
2.4
85
2.6d
9.1d
3.7d
10.4
5.3
96
Binding of 96 to hMAGL was quantified by determining the dis-
sociation constant, Ki, and the mode of inhibition. Enzymatic rates
obtained with various concentrations of the substrate 4-NPB and in-
hibitor 96 were analyzed by nonlinear regression (Michaelis-Menten
plot, Fig. 4) using an equation of mixed-type inhibition [52]. This de-
termination yielded values of Ki = 15.4 µM and α = 8.94, with the
latter parameter indicating a mixed-type inhibition with a pronounced
competitive component. The mode of inhibition was confirmed by
subjecting the original data to several linear transformation methods as
shown in Fig. S15. Literature-known MAGL inhibitors, such as com-
pounds 4 [30] and 5 [31] (Fig. 1) have been shown to act in a non-
competitive manner, whereas 6–9 [32–35] exhibit an extraordinarily
clear competitive behavior with parameter α > 10000.
97
106e
107e
1.63
3.1
a
Mean
SEM value (n = 3–4). Residual activity ≤ 65% results in
IC50 ≤ 46 µM. Values in italics and bold represent enzyme-inhibitor interac-
tions proven to be promiscuous and shown to be not affected by promiscuous
inhibition, respectively.
b
Residual activities in the presence of Triton X-100 (0.01% (v/v)): 21 and
42 on hMAGL: 82.8
96 on mCEase, 73.6
2.3%, and 87.7
1.4%, 101
3.9%, respectively; 32, 34, 85 and
5%, 95.1
1.0% and 93.1
2.3%
respectively.
c
Determination of IC50 on hFAAH in the presence of Triton X-100 (0.1% (w/
v)) was done as shown in Fig. S13.
d
Determination of IC50 and checking for promiscuous inhibition with Triton
The ω-quinazolinonylalkyl aryl urea 96 represents a novel chemical
entity that acts as a reversible inhibitor of the serine hydrolase hMAGL.
So far, there is no quinazolinone known to affect the activity of MAGL.
In contrast, the urea motif is quite common in potent irreversible MAGL
inhibitors [23–25]. Those ureas which most probably act in a reversible
manner often show lower inhibitory activities [53,54] or behave
as activator of MAGL [55]. Recently, we have discovered that
ω-phthalimidoalkyl 3,5-bis(trifluoromethyl)phenyl ureas 106 and 107
(Fig. 1, Table 1), which are structurally related to quinazolinones 85
and 96, respectively, exhibit inhibitory potencies on hMAGL and
hFAAH comparable to those of the two quinazolinones. However, in
contrast to 85 and 96, their phthalimide analogs affected both mCEase
and the respective human isoenzyme (hCEase) to greater extent
(Table 1) [43]. In addition to 96, only few chemical entities have been
identified so far that inhibit MAGL in a reversible manner. Examples for
X-100 (0.01% (v/v)) were done as shown in Fig. S14A (85 on hMAGL), Fig. 2
(96 on hMAGL), Fig. S14B (97 on hMAGL) and Fig. S12 (56 on mCEase).
e
ω-Phthalimidoalkyl 3,5-bis(trifluoromethyl)phenyl ureas investigated in a
previous study [43].
the compounds were shown to be completely soluble under MAGL assay
conditions by means of UV/Vis spectroscopy (Figs. S9–S11) [48].
2.3. Determination of Ki and mode of inhibition of quinazolinone 96 on
MAGL
To obtain further insights into the inhibition of hMAGL by com-
pounds of the class of quinazolinones, we first analyzed the reversibility
of the enzyme-inhibitor interaction in case of 96. For this purpose, we
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Fig. 2. (A) Values of IC50 for the inhibition
of hMAGL by reference compound JZL 184
(1 0 8) (taken from [43]) and the quina-
zolinones 85, 96 and 97, which were de-
termined without pre-incubation of en-
zyme and inhibitor. (B) Exemplarily
shown are residual enzymatic activities
(mean
SEM values of three in-
dependent triplicate experiments) in the
presence of 96 relative to the control in
the absence of compound 96 (vE = 100%)
without (open circles) and with (full cir-
cles) Triton X-100 (0.01% (v/v)). IC50
(mean
SEM value, n = 3) of 85, 96 and
97 were determined according to the
equation vE = 100%/(1 + ([I]/IC50)),
with vE representing the rate of enzymatic
product formation in the presence of in-
hibitor, I. IC50 values of 85, 96 and 97
obtained in the absence and presence of
Triton X-100 were not significantly dif-
ferent from each other (P > 0.05, un-
paired Student’s t test).
such compounds are summarized in Fig. 1, with the first generation
inhibitor 7 [33] (competitive) and the second generation inhibitor 4
[30] (non-competitive) exhibiting Ki values in a similar range to that of
96.
to decrease the activity of hMAGL. The quinazolinone 96 possessing
suitable ADME properties (Table S1) was identified as most promising
candidate for further investigation.
To have an idea of a possible interaction of 96 with hMAGL, the
computed positioning of this predominantly competitive acting com-
pound in the enzyme’s active site (Figs. 5 and S16) was analyzed in
comparison to the orientation of the re-docked ZYH (3) (Fig. S16).
Whereas the quinazolinone moiety of 96 interacts via π-stacking with
Tyr194 similar to ZYH’s pyrimidine ring, the bulky 3,5-bis(tri-
fluoromethyl)phenyl substituent of 96 “projects into a more spacious
void”[29] that is occupied by the hydrophobic cyclohexane portion of
ZYH. The urea motif in 96 confers planarity to the portion of the in-
hibitor adjacent to the terminal bulky moiety as found for the ben-
zoxazole ring within ZYH. A comparison of the structure of co-crys-
tallized ZYH with that of the docked substrate 2-arachidonyl glycerol
revealed that the cyclohexane and benzoxazole portions of the inhibitor
occupy the same space as the arachidonyl moiety of the natural sub-
strate (Figs. 4B and 4C in Schalk-Hihi et al. [29]). The increased in-
hibitory activity of ZYH (IC50 = 10 nM)[28] in comparison to 96 might
be explained by the two hydrogen bonds formed between the carbonyl
oxygen atom of ZYH and the backbone NH groups of Ala51 and Met123
(oxyanion hole of hMAGL) which are absent in the docking structure of
2.4. Molecular docking
To identify new reversible inhibitors of MAGL, we initially performed
a virtual screening of compounds originated from the ZINC database and
structures derived thereof using the docking program Glide (version 6.1,
Schrödinger, LLC, New York, NY, 2013). As a target for the docking
experiments, we chose the crystal structure of hMAGL co-crystallized
with the non-covalent binding inhibitor ZYH (3, Fig. 1). Validation of the
docking procedure was done by removing the co-crystallized ZYH and re-
docking of the structure resulting in an excellent reproduction of the
inhibitor orientation (rmsd of 0.6 Å). The initial docking was performed
in the standard precision mode of glide and afterwards, a selection of the
best matching compounds was re-docked in the extra precise mode. The
resulting library is depicted in Table S1. Our initial docking results in-
dicated that representatives of various structural classes might be able to
interact with the active site of hMAGL in a productive manner. Hence all
compounds of Table S1 were synthesized and analyzed for their potential
Fig. 3. Reversible inhibition of hMAGL by
96. Values of IC50 for the inhibition of
hMAGL by quinazolinone 96 were de-
termined after pre-incubation of enzyme
and inhibitor for 0, 30, and 60 min. Shown
are
residual
enzymatic
activities
(mean
SEM values of three independent
triplicate experiments) relative to the con-
trol without inhibitor (vE = 100%). *Data of
a
previous IC50 determination shown in
Fig. 2 (performed in the absence of Triton X-
100 without pre-incubation of MAGL with
96) are depicted again for reasons of com-
parison. IC50 (mean
SEM value, n = 3)
was calculated according to the equation
vE = 100%/(1+([I]/IC50)). Values of IC50
obtained without pre-incubation within a
previous analysis (see also Fig. 2) and this
series of experiments were not significantly
different from each other (P > 0.05, un-
paired Student’s t test). Furthermore, a one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test of the experiments of this series without pre-
incubation and both with 30 min and 60 min pre-incubation yielded no significant differences (P > 0.05) between the respective values of IC50
.
7

F.M. Dato, et al.
BioorganicChemistryxxx(xxxx)xxxx
reversible manner, since no time-dependency of the inhibitory potency
has been observed. Both a kinetic analysis and a molecular docking
study with 96 indicated that this compound occupies the substrate
binding pocket of MAGL and, thus, exhibits a mixed-type inhibition
with a predominant competitive behavior (α = 8.94). The activities of
hFAAH and mCEase were either inhibited to lesser extent or affected in
a detergent-sensitive manner by the ω-quinazolinonylalkyl aryl ureas.
In conclusion, our findings indicate that ω-quinazolinonylalkyl aryl
ureas, in particular 96, may be leads worth to be pursued to yield po-
tent reversible MAGL inhibitors for studying the effects of inhibiting
this enzyme in various diseases.
4. Experimental section
General information about material and methods can be found in
the Supplement.
Fig. 4. Determination of the dissociation constant, Ki, and the mode of in-
hibition for the interaction of hMAGL (20 ng mL−1) with compounds 96.
4.1. Chemistry
Depicted is
a Michaelis-Menten plot with enzymatic rates from four in-
dependent triplicate experiments (mean
SEM values). Concentrations of 96
4.1.1. General procedures A/B/C for the 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide mediated amide coupling reactions
were as follows: 0 µM (●), 2.5 µM (○), 5 µM (■), 10 µM (□), 20 µM (▾), and
30 µM (∇). Nonlinear regression analysis was performed using an equation of
mixed-type inhibition [52] in dependence on the concentration of both the
To a stirred solution of 1 eq amine, 1.2 eq (A) or 1 eq (B/C) carboxylic
acid and 1.15 eq 1-hydroxybenzotriazole hydrate dissolved in DCM or DMF
was added 1.25 eq 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hy-
drochloride at 0 °C in one portion. The reaction was warmed to room
temperature and stirred for further 14 h. After completion of the reaction in
DMF, the solvent was evaporated and the crude product was purified by
flash chromatography or recrystallized if mentioned. When DCM was used
as solvent, the mixture was washed three times with a saturated sodium
carbonate solution (A/B/C) followed by 10% (w/w) hydrochloric acid (C),
the organic layer was separated, the solvent was evaporated under reduced
pressure and the crude product was purified by flash chromatography or
recrystallized if mentioned.
substrate 4-NPB and inhibitor 96, resulting in values Ki = 15.4
1.8 µM and
α = 8.94
5.04 (mean
SEM values, n = 4).
96. Although interaction of quinazolinone 96 with hMAGL lacks hy-
drogen bonds and is predominantly mediated by both π-π and lipophilic
interactions, we observed a strong similarity to the binding mode of
ZYH (Fig. S16).
3. Conclusions
In the search of reversible inhibitors of MAGL we have initially
chosen an in silico approach to explore a variety of compound classes for
their ability to effectively bind to hMAGL. Synthesis of primary hits and
their biochemical investigation on hMAGL as well as on the related
enzymes hFAAH and mCEase by spectrophotometric and fluorometric
assays led to the identification of ω-quinazolinonylalkyl aryl ureas as a
new class of inhibitors of the former enzyme which exhibit IC50 values
in the range of 20–41 µM. As exemplarily shown for 96, i.e. the most
potent derivative (Ki = 15.4 µM) of the investigated ω-quinazolinony-
lalkyl aryl ureas, this class of compounds interacts with MAGL in a
4.1.2. General procedure D for the Suzuki coupling
Under an argon atmosphere 1 eq bromoaniline, 1 eq 3,5-bis(tri-
fluoromethyl)phenylboronic acid, 0.05 eq tri(o-tolyl)phosphine and
2 eq potassium carbonate were added to a solution containing 8 mL
DMF, 4 mL toluene and 2 mL water. The mixture was degassed under
reduced pressure and purged with argon for 30 min. Then, 0.0125 eq
palladium(II) acetate was added and the mixture was heated to 80 °C
for 1.5 h. The solvent was evaporated in vacuo and the crude product
was purified by flash chromatography.
Fig. 5. Result of docking quinazolinone 96 (orange carbon atoms) into the active site of hMAGL (PDB: 3PE6). Non-polar hydrogen atoms are omitted. Residues of the
catalytic triad (Ser122-His269-Asp239) and those of the oxyanion hole (Ala51, Met123) are shown.
8

F.M. Dato, et al.
BioorganicChemistryxxx(xxxx)xxxx
4.1.3. Synthetic procedures for selected compounds listed in Table 1
4.1.3.1. N-(3′,5′-Bis(trifluoromethyl)-[1,1′-biphenyl]-4-yl)-2-((3-
7.23 (m, 5H), 3.65 (d, J = 4.7 Hz, 2H), 2.66 (t, J = 7.6 Hz, 2H), 1.90 (m,
2H); 13C NMR (75 MHz, DMSO-d6) δ = 180.45, 169.85, 162.37, 141.12,
131.31 (q, J = 31.9 Hz), 128.33, 128.23, 125.85, 123.20 (q, J = 273.1 Hz),
117.97, 114.62, 43.60, 32.19, 31.98; IR ṽ [cm−1] = 3674 (m), 3221 (w),
2987–2901 (s), 2341 (w), 2186 (w), 2126 (w), 1929 (w), 1787 (w), 1657
(m), 1565 (m), 1380 (s), 1277 (m), 1132 (s), 1066 (s), 894 (m), 697 (m);
HR-MS (ESI) m/z calcd for C21H16F6N2O2: 443.11887 (M+H)+, found:
443.11886; elemental analysis calcd for C21H16F6N2O2: C 57.02, H 3.65, N
6.33, found: C 57.22, H 3.89, N 6.53.
phenylpropyl)sulfonyl)acetamide (21). The reaction was performed
according to the general procedure B with 0.24 g 2-((3-phenylpropyl)
sulfonyl)acetic acid (16, 1.0 mmol), 0.31 g 3′,5′-bis(trifluoromethyl)-
[1,1′-biphenyl]-4-amine (104, 1.0 mmol), 0.16 g HOBt (1.15 mmol) and
0.24 g EDC × HCl (1.25 mmol) in 15 mL DCM. The crude product was
purified via column chromatography (35% EtOAc/n-hexane), yielding a
white solid (0.27 g, 0.51 mmol, 51%); Rf = 0.30 (35% EtOAc/n-
hexane); mp: 211 °C; 1H NMR (300 MHz, DMSO-d6) δ = 10.63 (s,
1H), 8.31 (s, 2H), 8.05 (s, 1H), 7.89 (d, J = 8.5 Hz, 2H), 7.74 (d,
J = 8.6 Hz, 2H), 7.26 (m, 5H), 4.33 (s, 2H), 3.33 (m, 2H), 2.76 (t,
J = 7.6 Hz, 2H), 2.08 (m, 2H); 13C NMR (75 MHz, DMSO-d6)
δ = 160.77, 142.03, 140.60, 139.12, 132.33, 130.95 (q, J = 32.8 Hz),
128.42, 128.37, 127.98, 126.86, 126.12, 123.36 (q, J = 273.0 Hz),
120.52, 119.69, 59.28, 52.42, 33.44, 23.03; IR ṽ [cm−1] = 3675 (m),
2987–2901 (s), 2360 (w), 2173 (w), 2119 (w), 1926 (w), 1707 (w),
1394 (m), 1250 (m), 1066 (s), 891 (m); HR-MS (ESI) m/z calcd for
4.1.3.5. N-(4-(4-(3,5-Bis(trifluoromethyl)phenyl)piperazine-1-carbonyl)
thiazol-2-yl)benzamide (56). The reaction was performed according to
the general procedure A with 0.60 g 2-benzamidothiazole-4-carboxylic
acid (54, 2.4 mmol), 0.60 g 1-(3,5-bis(trifluoromethyl)phenyl)
piperazine (105, 2.0 mmol), 0.31 g HOBt (2.3 mmol) and 0.48 g
EDC × HCl (2.5 mmol) in 30 mL DMF. The crude product was
purified via column chromatography (90% CHCl3/acetone), yielding a
yellow solid (0.72 g, 1.37 mmol, 68%); Rf = 0.33 (90% CHCl3/
acetone); mp: 219 °C; 1H NMR (300 MHz, DMSO-d6) δ = 12.80 (s,
1H), 8.12 (d, J = 7.1 Hz, 2H), 7.71 (s, 1H), 7.65 (t, J = 7.3 Hz, 1H),
7.56 (t, J = 7.4 Hz, 2H), 7.51 (s, 2H), 7.33 (s, 1H), 3.86 (s, 4H), 3.46 (s,
4H); 13C NMR (75 MHz, DMSO-d6) δ = 165.52, 162.85, 158.16, 151.43,
144.39, 132.70, 131.91, 131.15 (q, J = 32.3 Hz), 128.59, 128.21,
C
25H21F6NO3S: 530.12191 (M+H)+, found: 530.12209.
4.1.3.2. 2-(4-(3,5-Bis(trifluoromethyl)phenyl)piperazin-1-yl)-2-oxo-N-
phenylacetamide (32). The reaction was performed according to the
general procedure A with 0.40 g 2-oxo-2-(phenylamino)acetic acid (27,
2.4 mmol), 0.60 g 1-(3,5-bis(trifluoromethyl)phenyl)piperazine (105,
2.0 mmol), 0.31 g HOBt (2.3 mmol) and 0.48 g EDC × HCl (2.5 mmol)
in 30 mL DCM. The crude product was purified via column
123.58 (q, J = 273.0 Hz), 117.80, 114.55, 110.42, 47.27; IR
ṽ
[cm−1] = 3670 (w), 2988–2901 (s), 1360 (w), 1610 (m), 1537 (m),
1394 (m), 1273–1232 (m), 1066 (s), 865 (w), 698 (w); HR-MS (ESI) m/
z calcd for C23H18F6N4O2S: 529.11274 (M+H)+, found: 529.11272.
chromatography (100% DCM), yielding
a white solid (0.60 g,
1.35 mmol, 67%); Rf = 0.29 (100% DCM); mp: 122 °C; 1H NMR
(300 MHz, DMSO-d6) δ = 10.80 (s, 1H), 7.67 (d, J = 7.9 Hz, 2H),
7.52 (s, 2H), 7.36 (m, 3H), 7.13 (t, J = 7.3 Hz, 1H), 3.69 (m, 4H),
3.48 (m, 4H); 13C NMR (75 MHz, DMSO-d6) δ = 162.49, 161.61,
151.25, 137.78, 131.14 (q, J = 32.3 Hz), 128.87, 124.33, 123.54 (q,
J = 272.9 Hz), 119.83, 114.68, 110.59, 47.38, 46.75, 45.04, 40.55; IR ṽ
[cm−1] = 3307 (w), 2363 (w), 1687 (m), 1633 (m), 1395 (m), 1280
(s), 1178 (m), 1128 (s), 963 (m), 761 (m), 696 (m); HR-MS (ESI) m/z
calcd for C20H17F6N3O2: 446.12977 (M+H)+, found: 446.13016.
4.1.3.6. 1-(3,5-Bis(trifluoromethyl)phenyl)-3-(3-(4-oxo-3,4-
dihydroquinazolin-2-yl)propyl)urea (85). 4-(4-Oxo-3,4-dihydroquinazolin-2-
yl)butanoic acid (0.70 g, 3 mmol), 1.0 mL triethylamine (83, 0.73 g,
7.2 mmol) and 0.78 mL diphenylphosphoryl azide (0.99 g, 3.6 mmol) in
100 mL acetone were stirred for 3 h at room temperature. The solvent was
removed in vacuo, the resulting residue was dissolved in 40 mL xylene and
heated to 135 °C for 20 min. 3,5-Bis(trifluoromethyl)aniline (0.56 mL, 0.83,
3.6 mmol) was added and the mixture was refluxed for further 14 h. Then,
the reaction mixture was cooled to room temperature, the solvent was
evaporated in vacuo, and the crude product was purified by flash
4.1.3.3. N-(3,5-Bis(trifluoromethyl)phenyl)-2-(4-(3,5-bis(trifluoromethyl)
phenyl)piperazin-1-yl)-2-oxoacetamide
(34). The
reaction
was
chromatography (30% acetone/n-hexane), yielding a white solid
performed according to the general procedure A with 0.72 g 2-((3,5-
bis(trifluoromethyl)phenyl)amino)-2-oxoacetic acid (28, 2.4 mmol),
0.60 g 1-(3,5-bis(trifluoromethyl)phenyl)piperazine (105, 2.0 mmol),
0.31 g HOBt (2.3 mmol) and 0.48 g EDC × HCl (2.5 mmol) in 30 mL
DCM. The crude product was purified via column chromatography
(65% DCM/n-hexane), yielding a yellow solid (0.53 g, 0.91 mmol,
45%); Rf = 0.25 (65% DCM/n-hexane); mp: 166 °C; 1H NMR
(300 MHz, DMSO-d6) δ = 8.71 (s, 1H), 7.86 (m, 4H), 7.36 (s, 4H),
6.23 (t, J = 5.6 Hz, 1H), 3.61 (t, J = 6.9 Hz, 2H), 3.11 (dd, J = 12.6 Hz,
6.4 Hz, 2H), 1.75 (p, J = 6.7 Hz, 2H); 13C NMR (75 MHz, DMSO-d6)
δ = 161.92, 161.36, 151.23, 139.76, 130.86 (q, J = 32.8 Hz), 123.10
(q, J = 272.4 Hz), 119.79, 117.26, 114.67, 110.61, 47.45, 46.75, 45.08,
40.94; IR ṽ [cm−1] = 3671 (m), 3294 (w), 2987–2901 (s), 2357 (w),
1643 (m), 1394 (s), 1274 (s), 1066 (s), 893 (m), 683 (m); HR-MS (ESI)
m/z calcd for C22H15F12N3O2: 582.10454 (M+H)+, found: 582.10470;
elemental analyis calcd for C22H15F12N3O2: C 45.45, H 2.60, N 7.23,
found: C 45.45, H 2.64, N 7.26.
(105 mg, 0.23 mmol, 8%); Rf = 0.20 (30% acetone/n-hexane); mp:
235 °C; 1H NMR (300 MHz, DMSO-d6) δ = 12.19 (s, 1H), 9.23 (s, 1H),
8.07 (m, 3H), 7.76 (t, J = 6.9 Hz, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.53 (s,
1H), 7.45 (t, J = 7.5 Hz, 1H), 6.56 (t, J = 5.6 Hz, 1H), 3.20 (dd,
J = 12.7 Hz, 6.6 Hz, 2H), 2.65 (t, J = 7.5 Hz, 2H), 1.93 (m, 2H); 13C
NMR (101 MHz, DMSO-d6) δ = 161.65, 156.94, 154.75, 148.81, 142.55,
134.08, 130.49 (q, J = 32.5 Hz), 126.68, 125.79, 125.56, 123.29 (q,
J = 272.7 Hz), 120.78, 117.19, 113.26, 38.65, 31.83, 27.02; IR
ṽ
[cm−1] = 3674 (w), 3329 (w), 2988–2901 (m), 2357 (w), 1686 (m),
1652 (m), 1392 (m), 1285 (m), 1173 (m), 1126 (s), 1066 (s), 887 (m); HR-
MS (ESI) m/z calcd for C20H16F6N4O2: 459.12502 (M+H)+, found:
459.12531.
4.1.3.7. 1-(3,5-Bis(trifluoromethyl)phenyl)-3-(4-(4-oxo-3,4-
dihydroquinazolin-2-yl)butyl)urea (96). 3,5-Bis(trifluoromethyl)phenyl
isocyanate (0.35 mL, 0.51 g, 2 mmol) was added to solution of 0.44 g
2-(4-aminobutyl)quinazolin-4(3H)-one (94, 2 mmol) in 20 mL CHCl3
and stirred for 14 h at room temperature. The solvent was removed in
vacuo and the resulting residue was purified via column
chromatography (30% acetone/DCM), yielding a white solid (0.49 g,
1.03 mmol, 51%); Rf = 0.29 (30% acetone/DCM); mp: 215 °C; 1H NMR
(300 MHz, DMSO-d6) δ = 12.19 (s, 1H), 9.21 (s, 1H), 8.08 (s, 3H), 7.75
(t, J = 7.0 Hz, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.51 (s, 1H), 7.44 (t,
J = 7.4 Hz, 1H), 6.53 (t, J = 5.3 Hz, 1H), 3.15 (dd, J = 12.4 Hz, 6.3 Hz,
2H), 2.63 (t, J = 7.5 Hz, 2H), 1.75 (dd, J = 14.8 Hz, 7.7 Hz, 2H), 1.53
(dd, J = 14.2 Hz, 6.8 Hz, 2H); 13C NMR (75 MHz, DMSO-d6)
δ = 161.83, 157.35, 154.81, 148.95, 134.26, 130.58 (q, J = 32.4 Hz),
4.1.3.4. 3-((3,5-Bis(trifluoromethyl)phenyl)amino)-4-((3-phenylpropyl)
amino)cyclobut-3-ene-1,2-dione (42). 3-Phenyl-1-propylamine (0.14 mL,
0.135 g, 1.0 mmol) were added to
a
solution of 0.353 g
3-((3,5-bis(trifluoromethyl)phenyl)amino)-4-ethoxycyclobut-3-ene-
1,2-dione (40, 1.0 mmol) dissolved in 10 mL DCM at room temperature.
After 24 h, a white precipitate was formed. The solid was filtered off and
washed with ice-cold DCM. The product was obtained as a white solid
(0.15 g, 0.38 mmol, 38%); mp: 202 °C; 1H NMR (300 MHz, DMSO-d6)
δ = 10.15 (s, 1H), 8.02 (s, 2H), 7.71 (d, J = 27.2 Hz, 1H), 7.63 (s, 1H),
9

F.M. Dato, et al.
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126.80, 125.94, 125.70, 123.40 (q, J = 272.9 Hz), 120.83, 117.21,
4.2.3. FAAH assay
113.33, 38.95, 34.18, 29.11, 24.21; IR
ṽ
[cm−1] = 3675 (m),
Activity of FAAH was investigated as recently reported by Dato et al.
[45]. Screening for inhibition of FAAH (1 µg mL−1) was performed with
10 µM D-MAP (1.2 × Km) and 25 µM of compound to be analyzed
without pre-incubation. IC50 of 56 was determined under the same
conditions but with seven inhibitor concentrations (2.5–50 µM). The
2987–2901 (s), 1653 (m), 1558 (w), 1385 (m), 1250 (m), 1066 (s),
892 (m), 771 (w); HR-MS (ESI) m/z calcd for C21H18F6N4O2: 473.14067
(M+H)+, found: 473.14103.
tivity was calculated as percentage vaDlu-Me ArePl.atRiveesidtouaclonetnrzoylsmwatiitchoauct-
enzymatic reaction was started with
4.1.3.8. 1-(3,5-Bis(trifluoromethyl)phenyl)-3-(5-(4-oxo-3,4-
dihydroquinazolin-2-yl)pentyl)urea (97). 3,5-Bis(trifluoromethyl)phenyl
isocyanate (0.35 mL, 0.51 g, 2 mmol) was added to solution of 0.46 g 2-
(5-aminopentyl)quinazolin-4(3H)-one (95, 2 mmol) in 20 mL CHCl3
and stirred for 14 h at room temperature. The solvent was removed in
vacuo and the resulting residue was purified via column
chromatography (30% acetone/DCM), yielding a white solid (0.53 g,
1.09 mmol, 55%); Rf = 0.43 (30% acetone/DCM); mp: 207 °C; 1H NMR
(300 MHz, DMSO-d6) δ = 12.16 (s, 1H), 9.17 (s, 1H), 8.07 (m, 3H),
7.74 (t, J = 7.1 Hz, 1H), 7.58 (d, J = 8.1 Hz, 1H), 7.52 (s, 1H), 7.44 (t,
J = 7.4 Hz, 1H), 6.47 (t, J = 5.4 Hz, 1H), 3.11 (dd, J = 12.5 Hz, 6.4 Hz,
2H), 2.61 (t, J = 7.5 Hz, 2H), 1.76 (dt, J = 14.8 Hz, 7.4 Hz, 2H), 1.50
(m, 2H), 1.37 (dd, J = 14.3 Hz, 7.5, 2H); 13C NMR (75 MHz, DMSO-d6)
δ = 161.85, 157.41, 154.79, 148.97, 142.69, 134.22, 130.58 (q,
J = 32.5 Hz), 126.80, 125.89, 125.68, 123.41 (q, J = 272.7 Hz),
120.82, 117.17, 113.31, 39.09, 34.47, 29.34, 26.51, 25.91; IR ṽ
[cm−1] = 3675 (m), 2987–2901 (s), 2360 (w), 2176 (w), 2119 (w),
1932 (w), 1682 (w), 1650 (w), 1612 (w), 1394 (m), 1250 (m), 1066 (s),
894 (m); HR-MS (ESI) m/z calcd for C22H20F6N4O2: 487.15632 (M
+H)+, found: 487.15653.
inhibitor after correction for non-enzymatic hydrolysis of D-MAP.
4.3. Solubility of selected compounds
The solubility of the compounds 56, 85, 96 and 97 was investigated
in 96-well microtiter plates (transparent BRANDplates® with F-bottom)
in a volume of 200 μL at four to nine compound concentrations (56, 85
and 97: 25–100 μM; 96: 2.5–100 μM). For each experiment, 20 μL of
compound solution (ten-fold concentrated) prepared in assay buffer
with 25% (v/v) DMSO was added to 170 μL assay buffer (56: FAAH
assay buffer; 85, 96 and 97: MAGL assay buffer), 5 μL DMSO and 5 μL
enzyme buffer (56: FAAH enzyme buffer; 85, 96, 97: MAGL enzyme
buffer). UV/Vis spectra were measured in the range of 300–650 nm.
4.4. Molecular docking
Docking studies on hMAGL were performed using the Schrödinger
Suite 2013–3 (Schrödinger, LLC, New York, NY, 2013), including the
programs Glide (v.6.1) [56–58], LigPrep (v.2.8), Maestro (v.9.6) and
the Protein Preparation Wizard containing the programs Epik (v.2.6)
[59,60], Impact (v.6.1) and Prime (v.3.4). ADME property analysis,
including parameters of Lipinski’s rule of five (molecular weight,
number of hydrogen bond donors, number of hydrogen bond acceptors,
and predicted octanol-water partition coefficient) was done using
QikProp (v.3.4) of the Schrödinger Suite. hMAGL crystal structure in
complex with the co-crystallized non-covalent Inhibitor ZYH (3, PDB:
3PE6) was taken from the Protein Data Bank and pre-processed ap-
plying the Protein Preparation Wizard optimizing the hydrogen
bonding network, pre-defining a pH value of 7.4 and removing possible
crystallographic artifacts. Before the docking process, ligands were
prepared with LigPrep adjusting a pH value of 7.4. For virtual
screening, the unconstrained glide docking protocol was used, the grid
box was centered on the co-crystallized inhibitor ZYH (3) and the initial
docking simulation was performed using the default SP settings. Based
on these results, promising candidates were re-docked with the stan-
dard XP settings, with additional settings “rewarding intramolecular
hydrogen bonds” and “enhancing the planarity of conjugated pi-
groups” being activated.
4.2. Biological activity
4.2.1. mCEase assay
Activity of murine CEase was investigated as recently described by
Dato et al. [43]. Inhibition of mCEase (40 ng mL−1) was determined
with 25 µM of compound without pre-incubation by starting the reac-
tion with 200 µM 4-MUB (2.7 × Km). The IC50 value of compound 56
was determined under the same conditions with six inhibitor con-
centrations (2.5–40 µM). Residual enzymatic activity was calculated as
percentage value relative to controls without inhibitor after correction
for non-enzymatic hydrolysis of 4-MUB. Analysis of compounds 32, 34,
56, 85 and 96 for promiscuous inhibition was done under the same
conditions as described above in the presence of Triton X-100 (0.01%
(v/v)).
4.2.2. hMAGL assay
Activity of human MAGL was investigated as recently reported by
Dato et al. [43]. Inhibition of hMAGL (20 ng mL−1) was determined
with 25 µM of compound without pre-incubation by starting the reac-
tion with 200 µM 4-NPB (1.1 × Km). Residual enzymatic activity was
calculated as percentage value relative to controls without inhibitor
after correction for non-enzymatic substrate hydrolysis. Values of IC50
for hMAGL inhibition by 85 (5–60 µM), 96 (2.5–30 µM) and 97
(2.5–40 µM), respectively, were analyzed in the presence of five to
seven inhibitor concentrations. Promiscuous behavior of the com-
pounds was investigated by repeating the experiments with Triton X-
100 (0.01% (v/v)) being present in the reaction mixture. The reversible
character of the interaction of hMAGL with 96 was analyzed by de-
termining the values of IC50 without pre-incubation of enzyme and
inhibitor and after 30 min and 60 min pre-incubation at 30 °C. Calcu-
lation of percentage enzymatic activities was done as described above.
The dissociation constant Ki and the mode of inhibition of compound
96 were determined with four different concentrations of 4-NPB
(50–200 µM) and six different inhibitor concentrations (0–30 µM).
Rates from experiments with hMAGL were corrected by the mean value
of the corresponding rate obtained in the absence of enzyme and sub-
jected to non-linear regression (equation of linear mixed-type inhibi-
tion) [52].
Declaration of Competing Interest
The authors declare that they have no conflict of interests.
Acknowledgements
The authors thank A. W. Schüller and R. Z. Uhl for assistance in
screening for inhibitors of the three serine hydrolases. F. M. Dato and B.
Goldfuss are grateful to the Fonds der Chemischen Industrie, the
Deutsche Forschungsgemeinschaft (DFG GO-930-13), Bayer AG, BASF
AG, Wacker Chemie AG, Evonik Industries AG, Raschig GmbH, Symrise
AG, Solvay GmbH, and INEOS Manufacturing Deutschland GmbH for
support as well as to the RRZK and the Department of Chemistry
(University of Cologne) for the deployment of computer hardware
(CHEOPS) and software. M. Pietsch thanks the Graduate Program in
Pharmacology and Experimental Therapeutics of the University of
Cologne and the Bayer Health Care AG.
10

F.M. Dato, et al.
BioorganicChemistryxxx(xxxx)xxxx
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