Tetrazolylpropan-2-ones as inhibitors of fatty acid amide hydrolase: Studies on structure-activity relationships and metabolic stability

Article abstract of DOI:10.1016/j.ejmech.2018.10.021

A series of derivatives of 1-(4-octylphenoxy)-3-(2H-tetrazol-2-yl)propan-2-one (3) and 1-(4-octylphenoxy)-3-(1H-tetrazol-1-yl)propan-2-one (4) was synthesized and tested for fatty acid amide hydrolase (FAAH) inhibitory potency and phase I metabolic stability. Introduction of certain substituents like 4-chlorophenyl, 4-methoxycarbonylphenyl and carboxyl in position 5 of the tetrazole ring of 3 led to a significant increase of the metabolic stability of the scissile ketone pharmacophore, while the high activity towards FAAH was not affected markedly. In contrast, substituents in position 5 of the heterocyclic system of 4 did not have a considerable impact on the undesired ketone reduction. Furthermore, the effect of shielding the ketone group of some derivatives of 3 by a methyl substituent in position 3 of the propan-2-one scaffold and the consequences of the replacement of the lipophilic octyl residue of these compounds by more drug-like substituents were examined.

Full text of DOI:10.1016/j.ejmech.2018.10.021

European Journal of Medicinal Chemistry 160 (2018) 183e192
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Tetrazolylpropan-2-ones as inhibitors of fatty acid amide hydrolase:
Studies on structure-activity relationships and metabolic stability
David Garzinsky, Stefan Zahov, Merlin Ekodo Voundi, Walburga Hanekamp,
Matthias Lehr*
Institute of Pharmaceutical and Medicinal Chemistry, University of Münster, Corrensstrasse 48, D-48149, Münster, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A
series of derivatives of 1-(4-octylphenoxy)-3-(2H-tetrazol-2-yl)propan-2-one (3) and 1-(4-
Received 20 June 2018
Received in revised form
8 October 2018
Accepted 9 October 2018
Available online 11 October 2018
octylphenoxy)-3-(1H-tetrazol-1-yl)propan-2-one (4) was synthesized and tested for fatty acid amide
hydrolase (FAAH) inhibitory potency and phase I metabolic stability. Introduction of certain substituents
like 4-chlorophenyl, 4-methoxycarbonylphenyl and carboxyl in position 5 of the tetrazole ring of 3 led to
a significant increase of the metabolic stability of the scissile ketone pharmacophore, while the high
activity towards FAAH was not affected markedly. In contrast, substituents in position 5 of the hetero-
cyclic system of 4 did not have a considerable impact on the undesired ketone reduction. Furthermore,
the effect of shielding the ketone group of some derivatives of 3 by a methyl substituent in position 3 of
the propan-2-one scaffold and the consequences of the replacement of the lipophilic octyl residue of
these compounds by more drug-like substituents were examined.
Keywords:
Fatty acid amide hydrolase
Covalent reversible inhibitors
Propan-2-one
© 2018 Elsevier Masson SAS. All rights reserved.
Metabolic stability
1. Introduction
treatment of pain, inflammation and neurological disorders [6e9].
In the recent years a large number of potent inhibitors of FAAH has
The endocannabinoid system comprises of the G-protein-coupled
cannabinoid receptors CB1 and CB2, their endogenous ligands named
endocannabinoids, and the enzymes for the synthesis and degrada-
tion of these substances [1,2]. The principal endocannabinoids are
arachidonoyl ethanolamide (anandamide) and 2-arachidonoyl glyc-
erol. They are generated as needed (“on demand”) during numerous
physiological and pathophysiological processes and mediate anal-
gesic, anti-inflammatory and neuropharmacological effects via acti-
vation of the cannabinoid receptors CB1 and CB2. The distribution of
these two receptors differs across the body. The CB1 receptor is
preferentially found in neuronal tissues, while the CB2 receptor is
mainlyexpressedincellsoftheimmunesystemsuchasmacrophages.
An important degrading enzyme in the endocannabinoid sys-
tem is the membrane-associated fatty acid amide hydrolase
(FAAH), a member of the serine hydrolase family [3]. FAAH in-
activates the lipid mediator anandamide by its cleavage into
arachidonic acid and ethanolamine [4,5]. Because the inhibition of
FAAH may result in an accumulation of anandamide at the receptor
site, inhibitors of this enzyme could be useful therapeutics for the
been developed such as the carbamates URB 597 (1) [10] and SSR
411298 [11], the urea derivatives PF-04457845 [12], V158866 [13]
and JNJ-42165279 [14], and the a-ketoheterocycle 2 [15] (Fig. 1).
During their preclinical development, promising effects of these
substances were observed in diverse animal models [11,14,16,17].
However, recent events and results have caused a setback in
FAAH inhibitor development. At first, the FAAH inhibitor PF-
04457845 from Pfizer failed to produce analgesia in a clinical trial
with patients suffering from pain due to osteoarthritis in knee
[18,19]. Similarly, the FAAH inhibitor V158866 from Vernalis did not
show the expected effects in a phase II study for treatment of
neuropathic pain resulting from spinal cord injury [20]. Moreover,
the FAAH inhibitor SSR 411298 from Sanofi was no more effective
thanplacebo in a PhaseII study for the treatmentof majordepressive
disorder in elderly patients [21]. Even worse, one volunteer in a
phase I study of the FAAH inhibitor BIA 10e2474 died and five others
werehospitalized withsevereneurologicalsymptomsaftertheyhad
received multiple doses of the compound, which possesses a reac-
tive carbonyl imidazole structural element [20,21]. On the other
hand, PF-04457845, V158866 and the Johnson & Johnsoncompound
JNJ-42165279, which all are urea derivatives, as well as the carba-
mate compound SSR411298 had been well tolerated in phase I
studies [14,21e24]. This indicates that the severe adverse effects of
* Corresponding author.
E-mail address: lehrm@uni-muenster.de (M. Lehr).
https://doi.org/10.1016/j.ejmech.2018.10.021
0223-5234/© 2018 Elsevier Masson SAS. All rights reserved.

184
D. Garzinsky et al. / European Journal of Medicinal Chemistry 160 (2018) 183e192
Fig. 1. Structures of FAAH inhibitors.
BIA 10e2474 did not result from the inhibition of FAAH, but from the
affection of other biochemical targets [25].
was reacted with 2-[(4-octylphenoxy)methyl]oxirane under
solvent-free conditions. In some cases, the bases 4-
Regardless of these negative outcomes, inhibitors of FAAH are
still considered to be new treatment options for some neurological
disorders. Thus, just recently PF-04457845 was demonstrated to
possess clinical efficacy in patients suffering from cannabis with-
drawal symptoms [26]. Furthermore, FAAH inhibitors could be
useful for the treatment of fear behaviours and Parkinson's disease
[27,28].
In previous studies, we have found that the tetrazolyl-substituted
propan-2-ones 3 and 4 (Fig.1) are potent inhibitors of FAAH with IC50
valuesof0.010 mMand0.039 mM, respectively[29]. Animportantpart
of the pharmacophore of these substances is the activated electro-
philic ketone moiety of their propan-2-one substructure. This
structural element is supposed to form reversible covalent binding
interactions with a serine residue of the active site of FAAH and is,
therefore, a so-called “serine trap”. Unfortunately, 3 and 4 are
extensively metabolized in vitro. Particularly, their activated ketone
group is reduced to an alcohol resulting in the loss of activity. Even at
dimethylaminopyridine
and
1,4-diazabicyclo[2.2.2]octane
(DABCO), respectively, were added. The reaction products consisted
of mixtures of 1H-tetrazol-1-yl- and 2H-tetrazol-2-ylpropan-2-ols,
which could be separated by silica gel chromatography. Oxidation
of these compounds with Dess-Martin reagent led to the corre-
sponding 1H-tetrazol-1-yl- and 2H-tetrazol-2-ylpropan-2-ones.
The structure assignment of the isomeric tetrazoles primarily based
on NOE/1H NMR data.
For the synthesis of the carboxylic acid derivatives 23e27 and
45e48 the appropriate ester precursors 11,12,20e22,34,35,43 and
44 (Scheme 1) were saponified with NaOH in ethanol at room
temperature.
The preparation of the two benzyltetrazolylpropan-2-ones 19
and 42 is shown in Scheme 2. Reaction of 5-benzyltetrazole with
epichlorohydrin in presence of KOH and tetrabutylammonium
bromide at room temperature resulted in the formation of a
mixture of isomeric 1- and 2-oxiranylmethyl-substituted de-
rivatives. After separation by silica gel chromatography, the
appropriate isomer was heated with 4-octylphenol and catalytic
amounts of 4-dimethylaminopyridine at 100 ꢀC to afford propan-2-
ol derivatives, which were oxidized to the target compounds with
DesseMartin reagent.
For the synthesis of propan-2-one derivatives with a methyl
substituent at the carbon between the ketone moiety and the
phenoxy substituent (¼ butan-2-ones), 2-(1-aryloxyethyl)oxiranes
like 54 (Scheme 3) should be used as central intermediates. Com-
pound 54 was prepared as previously described [29] from 4-
phenoxyphenol and 3-chlorobut-1-ene by Williamson ether syn-
thesis followed by conversion of the exocyclic double bond into an
epoxide with m-chloroperoxybenzoic acid. Opening of the epoxide
ring of 54 by 5-phenyltetrazole followed by Dess-Martin oxidation
of the generated secondary alcohol 55 led to the target ketone 56. A
atestconcentrationof10 mMthealcoholderivativesof3and4 [29]do
not inhibit FAAH. In this study, we wanted to investigate the effect of
substituents in position 5 of the tetrazole ring of 3 and 4 on the
metabolic stabilityandFAAHinhibitory potencyof thesecompounds.
Furthermore, it should be elucidated, whether the metabolic reduc-
tion of the ketone group of selected tetrazole-substituted propan-2-
ones can be decreased by introduction of a sterically hindering
methyl substituent at one of the neighboring methylene groups.
2. Results and discussion
2.1. Chemistry
Most of the tetrazolylpropan-2-ones investigated were synthe-
sized as outlined in Scheme 1. The appropriate substituted tetrazole

D. Garzinsky et al. / European Journal of Medicinal Chemistry 160 (2018) 183e192
185
Scheme 1. Reagents and conditions: (a) Solvent-free heating until a white vapor is given off; in case of 15e17 and 38e40 4-dimethylaminopyridine was added to the starting
compound; in case of 30 heating was carried out in the presence of DABCO at 90e100 ꢀC for 5 h; (b) Dess-Martin periodinane, CH2Cl2, room temperature, 4 h.
Scheme 2. Reagents and conditions: (a) Epichlorohydrin, KOH pulv., tetrabutylammonium bromide, room temperature, 4 h; (b) 4-octylphenol, 4-dimethylaminopyridine, 100 ꢀC,
4 h; (c) Dess-Martin periodinane, CH2Cl2, room temperature, 4 h.
Scheme 3. Reagents and conditions: (a) 3-Chlorobut-1-ene, NaOH, DMF, 45 ꢀC, 24 h; (b) m-chloroperoxybenzoic acid, CH2Cl2, room temperature, 6 d; (c) phenyltetrazole, aceto-
nitrile, reflux, 8 h; (d) Dess-Martin periodinane, CH2Cl2, room temperature, 3 h.
drawback of this synthetic route is the very long reaction time
(about 6 days) required for the formation of the epoxide. Therefore,
an alternative synthetic route was developed outlined for com-
pound 56 in Scheme 4. First, the appropriate para-substituted
phenol was reacted with 3-bromobutan-2-one in butan-2-one in
presence of 18-crown-6 and K2CO3. The obtained 3-aryloxy-
substituted butan-2-one 57 was brominated in position 1 via its
enol silyl ether 58 by a published procedure [30]. Compound 58
was generated by trapping the enolate, formed from the ketone 57
in THF in presence of lithium bis(trimethylsilyl)amide at low

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D. Garzinsky et al. / European Journal of Medicinal Chemistry 160 (2018) 183e192
Scheme 4. Reagents and conditions: (a) 3-Bromobutan-2-one, 18-crown-6, K2CO3, butan-2-one, room temperature, overnight; (b) lithium bis(trimethylsilyl)amide, tert-butyldi-
methylsilyl chloride, THF, ꢁ78 ꢀC, 30 min followed by room temperature, 4 h; (c) bromine, CH2Cl2, 0 ꢀC, 2e5 h; (d) 5-phenyltetrazole, K2CO3, DMF, room temperature, 1 h.
Table 2
temperatures with tert-butyldimethylsilyl chloride. Subsequently,
the enol silyl ether 58 was converted into the -bromoketone 59 by
FAAH inhibitory potency and metabolic stability of (1H-tetrazol-1-yl)propan-2-
ones.
a
treatment with bromine in dichloromethane. Reaction of 59 with 5-
phenyltetrazole led to a mixture of 1- and 2-substituted tetrazoles,
from which only the 2-substituted derivative 56 was isolated and
characterized. All compounds presented in Table 3 were synthe-
sized by this procedure. For the preparation of the tetrazole-5-
carboxylic acids 70e74, ethyl tetrazole-5-carboxylate was reacted
with the appropriate a-bromoketone and the ester group of the
formed intermediates was hydrolyzed with NaOH or KOH in
aqueous ethanol at room temperature. 1H/13C and 1H/15N 2D-
gHMBC NMR spectroscopy was used for the verification of the
Comp.
R
Inhibition of FAAH
IC50 (nM)a
Metabolic
stability (%)b
4
H
39
41
22
71
22
32
21
36
45
41
42
40
24
24
20
<5
27
26
38
33
6
28
29
30
31
32
33
36
37
38
39
40
41
42
45
46
47
48
Methyl
Methylsulfanyl
Phenyl
4-Chlorphenyl
3-Chlorphenyl
2-Chlorphenyl
4-(Dimethylsulfamoyl)phenyl
3-(Dimethylsulfamoyl)phenyl
Pyridin-4-yl
Pyridin-3-yl
Pyridin-2-yl
2-Thienyl
Benzyl
Table 1
FAAH inhibitory potency and metabolic stability of (2H-tetrazol-1-yl)propan-2-
ones.
130
360
4600
1400
1900
190
190
880
55
320
1000
940
3400
1800
Carboxymethyl
Carboxyethyl
4-Carboxyphenyl
3-Carboxyphenyl
Comp
R
Inhibition of FAAH
IC50 (nM)a
Metabolic
stability (%)b
3
5
6
7
8
9
H
10
27
31
24
35
24
32
29
34
590
420
24
23
18
32
66
41
88
30
90
36
60
<5
<5
<5
33
74
69
28
64
19
65
9
<5
12
30
8
<5
52
<5
<5
<5
<5
82
<5
Methyl
Methylsulfanyl
Phenyl
a
Values are the means of at least two independent determinations, errors are
within 20%.
b
Percent of parent compound remaining after metabolism in rat liver S9 fractions
4-Chlorphenyl
3-Chlorphenyl
2-Chlorphenyl
4-Methoxycarbonylphenyl
3-Methoxycarbonylphenyl
4-(Dimethylsulfamoyl)phenyl
3-(Dimethylsulfamoyl)phenyl
Pyridin-4-yl
Pyridin-3-yl
Pyridin-2-yl
2-Thienyl
Benzyl
in presence of NADPH compared with samples without NADPH; values are the
means of at least two independent determinations.
10
11
12
13
14
15
16
17
18
19
23
24
25
26
27
1
structures.
2.2. Biological evaluation
All target compounds were tested for FAAH inhibition using rat
brain microsomes as enzyme source and the fluorogenic substrate
N-(2-hydroxyethyl)-4-pyren-1-ylbutanamide. The FAAH product 4-
pyren-1-ylbutanoic acid was determined by reversed HPLC with
fluorescence-detection [29,31]. Furthermore, the stability of the
compounds towards phase I metabolic enzymes was determined
with rat liver S9 fractions [31] applying HPLC/MS for analysis and
processing.
The unsubstituted tetrazolylpropan-2-ones 3 and 4 were highly
potent inhibitors of FAAH exhibiting IC50 values of 10 and 39 nM,
respectively (Tables 1 and 2). However, their metabolic stability
against phase I reactions was low. The amount of parent compound
Carboxyl
Carboxymethyl
2-Carboxyethyl
4-Carboxyphenyl
3-Carboxyphenyl
(URB 597)
a
Values are the means of at least two independent determinations, errors are
within 20%.
b
Percent of parent compound remaining after metabolism in rat liver S9 fractions
in presence of NADPH compared with samples without NADPH; values are the
means of at least two independent determinations; compound 8: 74 5%
(mean standard deviation, n ¼ 5).

D. Garzinsky et al. / European Journal of Medicinal Chemistry 160 (2018) 183e192
187
Table 3
FAAH inhibitory potency and metabolic stability of 1-(2H-tetrazol-1-yl)butan-2-ones.
Comp.
R1
R2
R3
Inhibition of FAAH
IC50 (nM)a
Metabolic
log P
(HPLC)
stability (%)b
7
C8H17
C8H17
4-F-Phenoxy
4-Cl-Phenoxy
4-CF3-Phenoxy
Phenyl
H
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
24
24
24
51
160
15
33
40
<5
<5
6
8.0
>8.0
5.3
6.0
6.2
5.3
49
50
51
52
56
CH3
CH3
CH3
CH3
CH3
<5
8
C8H17
C8H17
4-F-Phenoxy
4-Cl-Phenoxy
4-CF3-Phenoxy
Phenyl
H
4-Cl-Phenyl
4-Cl-Phenyl
4-Cl-Phenyl
4-Cl-Phenyl
4-Cl-Phenyl
4-Cl-Phenyl
35
35
42
76
300
25
74
72
<5
<5
9
>8.0
>8.0
6.2
6.8
7.0
60
61
62
63
64
CH3
CH3
CH3
CH3
CH3
18
6.1
11
65
66
67
68
69
C8H17
C8H17
4-F-Phenoxy
4-Cl-Phenoxy
4-CF3-Phenoxy
Phenyl
H
4-COOCH3-Phenyl
4-COOCH3-Phenyl
4-COOCH3-Phenyl
4-COOCH3-Phenyl
4-COOCH3-Phenyl
4-COOCH3-Phenyl
29
25
36
47
130
23
64
62
<5
<5
15
21
8.0
>8.0
5.4
6.1
6.2
5.4
CH3
CH3
CH3
CH3
CH3
23
70
71
72
73
74
C8H17
C8H17
4-F-Phenoxy
4-Cl-Phenoxy
4-CF3-Phenoxy
Phenyl
H
COOH
COOH
COOH
COOH
COOH
COOH
41
50
100
90
160
83
52
55
33
32
36
30
5.3
5.9
2.7
3.3
3.5
2.6
CH3
CH3
CH3
CH3
CH3
a
Values are the means of at least two independent determinations, errors are within 20%.
Percent of parent compound remaining after metabolism in rat liver S9 fractions in presence of NADPH compared with samples without NADPH; values are the means of at
b
least two independent determinations.
remaining after incubation with liver S9 fractions in presence of the
co-substrate NADPH was less than 5% in case of the 2H-tetrazole
derivative 3 and about 20% in case of the corresponding 1H-tetra-
zole 4. The main metabolites detected were the alcohol derivatives
formed by the reduction of the ketone moiety by carbonyl
reductases.
compound 11, which is substituted with a voluminous 4-
methoxycarbonylphenyl residue. However, the volume of the
substituent obviously must not exceed a certain value, since the
potency dropped significantly, when the bulkier 3- and 4-(dime-
thylsulfamoyl)phenyl substituents were introduced. An increase of
metabolic stability was generated in particular by 4-substituted
phenyl residues or an electron-withdrawing carboxyl moiety.
Several of the 2H-tetrazolyl-substituted propan-2-ones exhibited a
slightly better FAAH inhibitory potency than the known reference
inhibitor URB 597. However, none of these compounds possessed a
higher Phase I in vitro stability than this reference (Table 1).
Substituents in position 5 of the tetrazole ring of the 1H-tetra-
zole 4 affected FAAH inhibition to a higher degree than seen in the
case of the 2H-tetrazole derivatives (Table 2). While the small
methyl residue did not change activity, a methylsulfanyl group (29)
even increased FAAH inhibitory potency about twofold. Introduc-
tion of a phenyl (30) or a bioisosteric 2-thienyl substituent (41)
resulted in slightly less potent inhibitors. Substitution of the phenyl
residue of 30 with a chloro atom in 4- or 3-position (31 and 32) or
its replacement by pyridine-4-yl (38), pyridine 3-yl (39) or benzyl
(42) led to a further small drop of activity. In contrast, a chloro atom
in position 2 of the phenyl ring as well as dimethylsulfamoyl and
carboxyl moieties in position 3- and 4 were not well tolerated. The
IC50 values of these compounds (33, 36, 37, 47 and 48) were about
40e120-fold higher than that of the lead 4. A significant decrease of
inhibitory potency (about 25-fold) also occurred, when the tetra-
zole hydrogen of 4 was replaced by carboxymethyl (45) or
Introduction of differing substituents in position 5 of the het-
erocyclic ring of the 2H-tetrazole derivative 3 did not result in
drastic changes in FAAH inhibition in general. For most of the
compounds a moderate (2e9 fold) decrease of activity was
observed (Table 1). A marked drop of potency (40e50 fold) only
took place after introduction of bulky (dimethylsulfamoyl)phenyl
residues (13 and 14). Fortunately, several of the introduced sub-
stituents in 5-position had a pronounced impact on the metabolic
stability of the ketone moiety of the compounds. The most prom-
inent effect could be achieved with 4-chlororphenyl (8), 3-
chlorophenyl (9), 4-methoxycarbonylphenyl (11), 4-(dime-
thylsulfamoyl)phenyl (13) and carboxyl groups (23). The metabolic
stability of the obtained compounds exceeded a value of 50% that
means that still more than 50% of their initial amounts were pre-
sent in the incubation mixture at the end of the metabolic re-
actions. In general, no clear correlation between the steric and
electronic properties of the substituents in position 5 of the tetra-
zole ring of 3 and the inhibitory activity could be seen. Thus,
compound 5 substituted with an electron-donating methyl group
inhibited FAAH to about the same extent as compound 23, which
bears an electron withdrawing carboxyl group, as well as

188
D. Garzinsky et al. / European Journal of Medicinal Chemistry 160 (2018) 183e192
carboxyethyl (46). Unlike in the case of the 2H-tetrazole series, the
metabolic stability of the 1H-tetrazoles could not be increased
markedly by substituents in position 5 of the tetrazole. While about
20% of compound 4 remained after the incubation with liver S9
fractions, the metabolic stability of the most stable 5-substituted
derivatives of 4 was about 40%. Taken together, in case of the 1H-
tetrazole derivatives there was a certain correlation between the
steric property of the substituents in position 5 and the FAAH
inhibitory potency. Thus, all compounds with phenyl residues were
significantly less active than the derivatives with the smaller
methyl and methylsulfanyl groups (28 and 29). Furthermore, sub-
stituents with ionizable carboxyl moieties were not well tolerated
by the enzyme. In contrast, no correlation between the electronic
and steric properties and the metabolic stability of the 1H-tetrazole
derivatives could be seen.
During structure activity relationship studies with analogous
benzotriazole-substituted propan-2 ones it was found that a
methyl substituent at the methylene group between the ketone
moiety and the heterocycle decreased activity, while a methyl
residue at the methylene group linking the carbonyl functionality
with the aryloxy substituent did not change inhibitory potency
[29]. Simultaneously, the metabolic stability was increased by the
latter structural variation probably due to the steric shielding of the
inhibition can be registered already after 5 min incubation and the
discrepancy between the 5 min and the 60 min inhibition values is
not too big, the covalent reaction seems to proceed rather fast. The
reversibility of the covalent reaction was studied with rat liver
homogenate (S9 fraction). In these experiments, we took advantage
of the fact that in liver tissue high levels of FAAH are expressed
[36e38] besides the reductases, which are responsible for the
metabolism of the propan-2-one derivatives [39]. We initially
measured the IC50 value of 50 against the liver enzyme using the
same conditions as in the screening experiments with rat brain
enzyme (incubation time 60 min without pre-incubation). As
shown in Table 4, the IC50 values obtained with the different
enzyme sources corresponded well (24 nM vs. 31 nM). Next, we
pre-incubated compound 50 with the liver homogenate for 15 min,
to enable the formation of covalent binding interactions between
enzyme and inhibitor. Subsequently, NADPH was added to this
mixture (final concentration of 50: 10 mM), to start inactivation of
the unbound propan-2-one by liver carbonyl reductases. After
30 min, 50 could not be detected any more by LC/MS, while the two
diastereomeric alcohol metabolites of 50 emerged. In the following,
the IC50 value of 50 was determined under application of this pre-
incubation procedure. The enzyme reaction was started after the
NADPH treatment by addition of a DMSO solution of the substrate,
the incubation time was 60 min. For comparative purposes, the IC50
of 50 was measured in the same way without addition of NADPH.
While the IC50 obtained in absence of NADPH was still about 30 nM,
pre-incubation with NADPH resulted in a more than 30-fold drop of
activity (Table 4). This indicates that the proposed covalent reaction
between FAAH and inhibitor must be reversible. The unbound in-
hibitor is removed from the equilibrium between bound and un-
bound form by the metabolic reaction, finally resulting in a
decrease of the amount of 50 bound to FAAH. In case of an irre-
versible covalent reaction between enzyme and inhibitor pre-
incubation with NADPH would not have an effect on the IC50 value.
Another important enzyme involved in the process of endo-
cannabinoid inactivation is the serine hydrolase monoacylglycerol
lipase (MAGL) [40]. To get some information about the selectivity of
the investigated substance class, we measured the effect of all
compounds depicted in Tables 1 and 2 on this enzyme [41]. At a
carbonyl group by the methyl substituent in
a-position, which
hinders the attack of carbonyl reductases. Therefore, we speculated
that the metabolic stability of the relatively stable 2H-tetrazoles 7,
8, 11 and 23 could be further increased by an analogous modifica-
tion. Since the terminal octyl residue of the planned 2H-tetrazoles
causes a high lipophilicity, which could impair bioavailability, the
octyl chain should be replaced by a phenyl residue or substituted
phenoxy groups additionally to convert the molecules into more
drug-like compounds. As to be expected, the introduction of the
additional methyl group in
a-position did not change inhibitory
potency of the compounds (49, 60, 65 and 70) (Table 3). Unex-
pectedly, the metabolic stability also was not influenced by this
modification. In the case of the 5-phenyl-2H-tetrazolyl derivatives
49, 60 and 65, the replacement of the octyl residue by 4-
fluorophenoxy (50) or phenyl (56) did not change the high inhib-
itory potency. The substitution of the fluoro atom of the 4-
fluorophenoxy residue by chloro decreased activity about
twofold, a trifluoromethyl moiety at this position even dropped
activity about five-to eightfold. In case of the carboxyl-substituted
compound 70, the exchange of the octyl residue by phenyl or
substituted phenoxy groups resulted in two-to threefold less active
compounds. Reducing the lipophilicity of the 5-aryl-substituted
2H-tetrazoles 49, 60 and 65 by replacement of the octyl chain
decreased metabolic stability remarkably. With stability values of
about 20%, the two derivatives with rigid phenylphenoxy residues
64 and 69 still were the most stable compounds of the investigated
derivatives. The 2H-tetrazole-5-carboxylic acids 71e74 possessed
about the same metabolic stability. With values of 30e36% the loss
of metabolic stability of these compounds was nearly constant and
less pronounced when compared with the stability of the starting
compound 70 (55% stability).
concentration of 10 mM none of these tetrazolyl-substituted
propan-2-one derivatives showed a significant inhibition of MAGL
indicating their high selectivity for FAAH over MAGL.
In conclusion, we have seen that the metabolic stability of the
lead compound 3 can be increased by introduction of substituents
at position 5 of the tetrazole ring under preservation of their FAAH
inhibitory potency. In contrast, with substituents in position 5 of
the heterocyclic system of 4 the undesired ketone reduction could
not be diminished significantly. Furthermore, introduction of a
shielding methyl substituent in position 3 of the propan-2-one
scaffold of some derivatives of 3 and replacement of the lipo-
philic octyl residue of these compounds by more drug-like sub-
stituents also did not improve metabolic stability. For further
inhibitor development, the carboxylic acids 71e74 are the most
interesting of the investigated compounds, since they have an
adequate lipophilicity, a fairly good metabolic stability and still a
submicromolar activity towards FAAH.
There is some evidence that the investigated propan-2-ones
bind covalently to FAAH via the formation of a reversible hemi-
ketal with the active site serine [32], like it is described for
a-
ketoheterocyle based inhibitors [33]. To support this assumption,
we performed some additional experiments with compound 50.
First, we reduced the incubation time of the inhibition experiment
with rat brain FAAH from 60 min to 5 min. By doing this, the IC50 of
50 increased about fourfold from 24 nM to 89 nM (Table 4). This
time-dependency of the inhibition value indicates a covalent in-
hibition mode of the inhibitor [34,35] due to the fact that the co-
valent binding process takes some time. Since a considerable
3. Experimental section
3.1. Chemistry
3.1.1. General
Column chromatography was performed on silica gel 60, parti-
cle size 0.040e0.063 mm, from Macherey & Nagel (Düren, Ger-
many). Melting points were determined on
a Büchi B-540

D. Garzinsky et al. / European Journal of Medicinal Chemistry 160 (2018) 183e192
189
Table 4
Inhibition experiments with FAAH of rat brain cytosol and rat liver homogenate with compound 50 under different conditions for proving the covalent reversible inhibition
mode of the inhibitors.
Enzyme source
Incubation
time (min)
Pre-incubation
Inhibition of FAAH
IC50 (nM)a
Rat brain
Rat brain
60
5
none
none
24
89
Rat liver
Rat liver
Rat liver
60
60
60
none
31
29
1060
45 min without NADPH
15 min without þ 30 min with NADPH
a
Values are the means of two independent determinations, errors are within 20%.
apparatus (Essen, Germany) and are uncorrected. 1H NMR and 13C
NMR spectra were recorded on an Agilent DD2 400 spectrometer
(400 MHz), an Agilent DD2 600 spectrometer (600 MHz) (Agilent
Technologies, Richardson, USA), a Bruker AV 300 spectrometer
(300 MHz) or a Bruker AV 400 spectrometer (400 MHz). The high
resolution mass spectra (HRMS) were recorded on a Bruker
micrOTOF-Q II spectrometer (Bremen, Germany) applying atmo-
spheric pressure chemical ionization (APCI). For preparative
reversed phase HPLC a RP18 Eurospher II 5 mm column (20 mm
inside diameter ꢂ 250 mm) (Knauer, Berlin, Germany) protected
with a RP18 Eurospher II 5 mm guard column (20 mm inside
diameter ꢂ 30 mm) was used. Elution was performed in isocratic
mode using acetonitrile/water as mobile phase. The compounds
were dissolved in DMSO and the injected sample volume was
0.5e1 mL. The flow rate was 25 mL/min and detection was carried
out at 254 nm. Purity of the target compounds was determined by
normal phase HPLC. The test compounds (1e2 mg) were dissolved
3.79 mmol), 5-phenyltetrazole (550 mg, 3.76 mmol), and 1,4-
diazabicyclo[2.2.2]octane (DABCO) (60 mg, 0.53 mmol) was heat-
ed at 90e100 ꢀC for 5 h. The reaction mixture was dissolved in a
small amount of toluene and purified by silica gel chromatography
(hexane/ethyl acetate, 8:2 to 7:3) to give 1-(4-octylphenoxy)-3-(5-
phenyl-1H-tetrazol-1-yl)propan-2-ol (176 mg, 11%) and 1-(4-
octylphenoxy)-3-(5-phenyl-2H-tetrazol-2-yl)propan-2-ol (431 mg,
28%) as oils. Under a nitrogen atmosphere, a solution of the 1H-
tetrazol-1-yl-substututed isomer (170 mg, 0.42 mmol) in dry
CH2Cl2 (5 mL) was treated with Dess-Martin periodinane (286 mg,
0.67 mmol). After stirring at room temperature overnight,
a
mixture of equal volumes of aqueous sodium thiosulfate solution
(5%) and saturated aqueous sodium bicarbonate solution was
added and the reaction mixture exhaustively extracted with ethyl
acetate. The combined organic layers were dried (Na2SO4),
concentrated and purified by chromatography on silica gel (hexane/
ethyl acetate, 9:1 to 8:2 to 7:3) to give 30 (138 mg, 82%) as an oil.
in THF (0.5 mL). The injection volume was 5
performed using a LiChrospher 100 CN cyano phase (3.0 mm inside
m) (Merck, Darmstadt, Ger-
mL. Separation was
C d (ppm) 0.88 (t,
24H30N4O2(406.5); 1H NMR (600 MHz, CDCl3):
J ¼ 7.0 Hz, 3H), 1.17e1.37 (m, 10H), 1.58 (m, 2H), 2.50e2.62 (m, 2H),
4.69 (s, 2H), 5.62 (s, 2H), 6.69e6.84 (m, 2H), 7.06e7.19 (m, 2H),
7.48e7.52 (m, 2H), 7.53e7.58 (m, 3H); 13C NMR (151 MHz, CDCl3):
diameter ꢂ 250 mm, particle size 5
m
many) with a hexane/THF gradient at a flow rate of 0.5 mL/min and
a detection wavelength of 254 nm. Alternatively, purity of the
target compounds was determined by reversed phase HPLC on a
Nucleosil 100 RP18 column (3 mm inside diameter ꢂ 125 mm,
d
(ppm) 14.2, 22.8, 29.4, 29.4, 29.6, 31.8, 32.0, 35.2, 54.5, 72.3, 114.3,
123.6, 128.7, 129.5, 130.0, 131.6, 137.5, 155.1, 155.7, 198.8; HRMS
(APCI, direct probe) m/z [MþH]þ calculated: 407.2442, found:
407.2415.
particle size 3 mm) (Macherey & Nagel, Düren, Germany) at a flow
rate of 0.4 mL/min with a gradient consisting of acetonitrile/water/
trifluoroacetic acid (42:58:0.1 to 86:14:0.1, v/v/v). In this case, the
3.1.3. 3-(4-Phenylphenoxy)-1-(5-phenyl-2H-tetrazol-2-yl)butan-2-
ol (55)
samples were prepared by mixing 20
mL of a 5 mM solution of the
compound in DMSO with 180 L of acetonitrile. 5
m
mL of this solution
Under a nitrogen atmosphere and anhydrous conditions, 2-[1-
(4-phenylphenoxy)ethyl]oxirane (54) (237 mg, 0.99 mmol), 5-
phenyltetrazole (145 mg, 0.99 mmol), anhydrous K2CO3 (278 mg,
2.01 mmol) and 18-crown-6 ether (63 mg, 0.24 mmol) were dis-
solved in dry DMF (20 mL). The solution was stirred at room tem-
perature for 12 h and then heated under reflux for 15 h. After
addition of saturated aqueous NaCl solution, the reaction mixture
was exhaustively extracted with ethyl acetate. The combined
organic layers were dried over anhydrous Na2SO4, filtered and
concentrated. The resulting residue was purified by chromatog-
raphy on silica gel (cyclohexane/ethyl acetate, 49:1 to cyclohexane/
was injected into the HPLC-system. UV-absorbance was also
measured at 254 nm. Under these conditions all compounds
showed purities greater than 95%.
The synthesis of the target compounds 5e19, 23e29, 31e33,
36e42 and 45e48 has been described previously [42]. For the
synthesis of 49e52 and 60e74 see Supplementary material.
3.1.2. 1-(4-Octylphenoxy)-3-(5-phenyl-1H-tetrazol-1-yl)propan-2-
one (30)
A
mixture of 2-[(4-octylphenoxy)methyl]oxirane (994 mg,

190
D. Garzinsky et al. / European Journal of Medicinal Chemistry 160 (2018) 183e192
ethyl acetate, 8:2) to yield 55 (72 mg, 19%) as a mixture of two di-
astereomers.
23H22N4O2 (386.5); mp 150e151 ꢀC; 1H NMR
(400 MHz, CDCl3): (ppm) 1.46e1.51 (m, 3H), 4.35e4.44 (m, 1H),
slowly added to a solution of 57 (101 mg, 0.42 mmol) in dry THF
(20 mL). The mixture was cooled down to - 78 ꢀC and stirred at this
temperature for 30 min. Then tert-butyldimethylsilyl chloride
(196 mg, 1.30 mmol) was added rapidly. After 20 min, the mixture
was allowed to warm up to room temperature and further stirred at
this temperature overnight. The reaction mixture was diluted with
equal volumes of saturated aqueous NaCl solution and aqueous
NaHCO3 solution (5%) and extracted exhaustively with ethyl ace-
tate. The combined organic layers were dried over anhydrous
Na2SO4 and concentrated. The residue was purified by chroma-
tography on silica gel (cyclohexane to cyclohexane/ethyl acetate,
97:3) to yield 58 as an oil (131 mg, 88%). C22H30O2Si (354.6); 1H
C
d
4.45e4.51 (m, 0.7H), 4.51e4.61 (m, 0.3H), 4.86e4.94 (m, 1.4H), 5.01
(dd, J ¼ 14.6 Hz and 3.2 Hz, 0.6H), 6.86e7.08 (m, 2H), 7.29e7.36 (m,
1H), 7.37e7.46 (m, 2H), 7.46e7.51 (m, 3H), 7.51e7.59 (m, 4H),
8.03e8.26 (m, 2H); 13C NMR (101 MHz, CDCl3):
d (ppm) 15.5, 15.9,
55.4, 55.7, 72.7, 73.0, 74.1, 74.8, 116.4, 116.6, 126.9, 126.93, 127.0,
127.0, 127.0, 128.4, 128.5, 128.5, 128.9, 129.1, 129.1, 130.6, 134.9,
134.9, 140.7, 140.7, 156.6, 156.7, 165.4, 165.5; HRMS (APCI, direct
probe) m/z [MþH]þ calculated: 387.1821, found: 387.1823.
3.1.4. 3-(4-Phenylphenoxy)-1-(5-phenyl-2H-tetrazol-2-yl)butan-2-
one (56)
NMR (400 MHz, CDCl3): d (ppm) 0.14 (s, 3H), 0.19 (s, 3H), 0.95 (s,
9H), 1.51 (d, J ¼ 6.4 Hz, 3H), 4.18 (d, J ¼ 1.6 Hz, 1H), 4.37 (d, J ¼ 1.5 Hz,
1H), 4.58 (q, J ¼ 6.4 Hz, 1H), 6.95e6.99 (m, 2H), 7.28e7.32 (m, 1H),
7.37e7.43 (m, 2H), 7.47e7.51 (m, 2H), 7.53e7.57 (m, 2H); 13C NMR
Synthesis via compound 55: Under a nitrogen atmosphere, a
solution of 55 (60 mg, 0.16 mmol) in dry CH2Cl2 (20 mL) was treated
with Dess-Martin periodinane (79 mg, 0.19 mmol). The mixture
was stirred at room temperature for 3 h. After addition of equal
volumes of aqueous sodium thiosulfate solution (5%) and saturated
aqueous NaHCO3 solution, the resulting mixture was exhaustively
extracted with ethyl acetate. The combined organic layers were
dried over anhydrous Na2SO4 and concentrated. The residue was
purified by chromatography on silica gel (cyclohexane/ethyl ace-
tate, 19:1 to 7:3) to yield 56 as a solid (21 mg, 35%).
(101 MHz, CDCl3):
d
(ppm) ꢁ4.8, ꢁ4.4, 18.1, 20.0, 24.8, 75.2, 79.5,
115.4, 126.9, 127.0, 128.6, 128.9, 133.9, 141.0, 157.7, 158.2; HRMS
(APCI, direct probe) m/z [MþH]þ calculated: 355.2093, found:
355.2088.
3.1.7. 1-Bromo-3-(4-phenylphenoxy)butan-2-one (59)
Underanitrogenatmosphere, asolution of 58(2.01 g, 5.67 mmol)
in dry CH2Cl2 (15 mL) was stirred at ꢁ20 ꢀC for 10 min. Then bromine
Synthesis via compound 59:
A
solution of 59 (103 mg,
(901 mg, 5.64 mmol, 289 mL) was added dropwise and the mixture
0.32 mmol) in dry DMF (3 mL) was added dropwise to a stirred
mixture of 5-phenyltetrazole (46 mg, 0.31 mmol), K2CO3 (88 mg,
0.64 mmol) and dry DMF (10 mL). After stirring at room tempera-
ture for 1 h, the reaction mixture was diluted with saturated
aqueous NaCl solution and exhaustively extracted with ethyl ace-
tate. The combined organic layers were dried over anhydrous
Na2SO4 and concentrated. The resulting residue was purified by
chromatography on silica gel (cyclohexane to cyclohexane/ethyl
acetate, 49:1) to give 56 as a solid (41 mg, 34%). C23H20N4O2
was allowed to warm to 0 ꢀC and further stirred at this temperature
for additional 4 h. After the addition of saturated aqueous NaCl so-
lution, the mixture was extracted twice with ethyl acetate. The
combined organic layers were dried over anhydrous Na2SO4 and
concentrated. The residue was purified by chromatography on silica
gel (cyclohexane to cyclohexane/ethyl acetate, 49:1) and subse-
quently by preparative RP-HPLC (acetonitrile/H2O/HCOOH, 9:1:0.1).
The organic solvent was distilled off and the remaining aqueous
solution lyophilized to yield 59 as an oil (1.06 g, 59%). C16H15BrO2
(384.4); mp 173e174 ꢀC; 1H NMR (400 MHz, CDCl3):
d
(ppm) 1.68
(319.2); 1H NMR (400 MHz, CDCl3):
d
(ppm) 1.61 (d, J ¼ 6.8 Hz, 3H),
(d, J ¼ 6.7 Hz, 3H), 4.99 (q, J ¼ 6.8 Hz, 1H), 5.70 (d, J ¼ 18.2 Hz, 1H),
5.96 (d, J ¼ 18.2 Hz, 1H), 7.01e7.07 (m, 2H), 7.31e7.38 (m, 1H),
7.41e7.47 (m, 2H), 7.47e7.51 (m, 3H), 7.54e7.64 (m, 4H), 8.09e8.19
4.17 (d, J ¼ 14.2 Hz, 1H), 4.33 (d, J ¼ 14.2 Hz, 1H), 4.97 (q, J ¼ 6.8 Hz,
1H), 6.88e7.04 (m, 2H), 7.29e7.39 (m, 1H), 7.39e7.49 (m, 2H),
7.52e7.54 (m, 2H), 7.55e7.60 (m, 2H); HRMS (APCI, direct probe) m/z
[MþH]þ calculated: 319.0334, found: 319.0329.
(m, 2H); 13C NMR (101 MHz, CDCl3):
d (ppm) 17.6, 57.8, 78.7, 115.6,
127.0, 127.1, 127.3, 127.3, 128.9, 129.0, 129.0, 130.6, 136.0, 140.4,
156.2, 165.8, 200.9; HRMS (APCI, direct probe) m/z [MþH]þ calcu-
lated: 385.1665, found: 385.1683.
3.1.8. 2-[3-(4-Phenylphenoxy)-2-oxobutyl]-2H-tetrazole-5-
carboxylic acid (74)
A solution of 59 (101 mg, 0.32 mmol) in dry DMF (5 mL) was
3.1.5. 3-(4-Phenylphenoxy)butan-2-one (57)
added dropwise to a stirred mixture of ethyl tetrazole-5-
Under a nitrogen atmosphere, a mixture of 4-phenylphenol
(1.35 g, 7.93 mmol), 18-crown-6 ether (50 mg, 0.19 mmol), K2CO3
(1.10 g, 7.96 mmol) and dry butan-2-one (20 mL) was stirred at
carboxylate (38 mg, 0.27 mmol), K2CO3 (100 mg, 0.72 mmol) and
dry DMF (5 mL). After stirring at room temperature for 2 h, the
reaction mixture was diluted with saturated aqueous NaCl solution
and exhaustively extracted with ethyl acetate. The combined
organic layers were dried over anhydrous Na2SO4 and concen-
trated. The resulting residue was purified by chromatography on
silica gel (cyclohexane to cyclohexane/ethyl acetate, 8:2) to give a
mixture of ethyl 2-[3-(4-phenylphenoxy)-2-oxobutyl]-2H-tetra-
zole-5-carboxylate and 1-[3-(4-phenylphenoxy)-2-oxobutyl]-1H-
tetrazole-5-carboxylate as a solid (76 mg, 74%). To an aliquot of this
mixture (40 mg, 0.11 mmol) dissolved in EtOH (10 mL) was added
dropwise with stirring an aqueous NaOH solution (1 M, 1.0 mL,
1.0 mmol). Stirring was continued at room temperature for 30 min.
Then the organic solvent was distilled off. The residue was acidified
with aqueous 1 M HCl solution (15 mL, 15 mmol) and exhaustively
extracted with ethyl acetate. The combined organic layers were
dried over anhydrous Na2SO4 and concentrated. The resulting res-
idue was purified by chromatography on silica gel (cyclohexane/
ethyl acetate/HCOOH, 8:2:0.1 to ethyl acetate/HCOOH, 99:1) and
subsequently by preparative RP-HPLC (acetonitrile/H2O/HCOOH,
70:30:0.1). The organic solvent was distilled off and the remaining
room temperature for 30 min. Then 3-chlorobutan-2-one (530 mL,
502 mg, 4.71 mmol) was added dropwise and stirring was
continued at room temperature for 23 h. The reaction mixture was
diluted with saturated aqueous NaCl solution and exhaustively
extracted with ethyl acetate. The combined organic layers were
dried over anhydrous Na2SO4 and concentrated. The residue was
purified by chromatography on silica gel (cyclohexane to cyclo-
hexane/ethyl acetate, 9:1) to yield 57 as a solid (1.13 g, 100%).
C
d
16H16O2 (240.3); mp 38e39 ꢀC; 1H NMR (400 MHz, DMSO‑d6):
(ppm) 1.45 (d, J ¼ 6.8 Hz, 3H), 2.19 (s, 3H), 4.95 (q, J ¼ 6.8 Hz, 1H),
6.92e7.00 (m, 2H), 7.26e7.36 (m, 1H), 7.38e7.49 (m, 2H), 7.55e7.65
(m, 4H); HRMS (APCI, direct probe) m/z [MþH]þ calculated:
241.1229, found: 241.1243.
3.1.6. {[3-(4-Phenylphenoxy)but-1-en-2-yl]oxy)}(tert-butyl)
dimethylsilane (58)
Under a nitrogen atmosphere, a solution of lithium bis(-
trimethylsilyl)amide in dry THF (1.0 M, 1.30 mL, 1.30 mmol) was

D. Garzinsky et al. / European Journal of Medicinal Chemistry 160 (2018) 183e192
191
aqueous solution lyophilized to give 74 (18 mg, 49%) as a solid.
C
d
flow rate was 0.2 mL/min. Detection was performed in ESIþ (pyr-
idine compounds) or ESIe mode (all other compounds). The main
metabolic reaction was the reduction of the ketone group of the
propan-2-one scaffold leading to propan-2-ol derivatives. In case of
the methoxycarbonylphenyl derivatives 11, 12 and 65e69
concomitantly ester hydrolysis took place. In the negative ion mode
(ESIe) often formate adduct ions were detected as base peaks.
18H16N4O4 (352.4); mp 145e146 ꢀC; 1H NMR (300 MHz, CDCl3):
(ppm) 1.67 (d, J ¼ 6.8 Hz, 3H), 5.00 (q, J ¼ 6.8 Hz, 1H), 5.78 (d,
J ¼ 18.2 Hz, 1H), 6.06 (d, J ¼ 18.2 Hz, 1H), 6.98e7.06 (m, 2H),
7.30e7.39 (m, 1H), 7.40e7.49 (m, 2H), 7.52e7.57 (m, 2H), 7.58e7.62
(m, 2H); 13C NMR (101 MHz, CDCl3):
d (ppm) 17.6, 53.3, 78.6, 115.5,
126.8, 127.3, 128.8, 129.0, 136.1, 140.0, 149.8, 155.7, 169.5, 200.9,;
HRMS (APCI, direct probe) m/z [MþH]þ calculated: 353.1250,
found: 353.1296.
3.2.4. Partition coefficient (log P)
Partition coefficients (log P) were determined by reversed phase
HPLC applying a published OECD-method [43]. The following
reference compounds were used: anisole, acetophenone, ethyl
benzoate, benzophenone, naphthalene, diphenyl, phenanthrene,
3.2. Evaluation of biological and physicochemical properties
3.2.1. Inhibition of fatty acid amide hydrolase (FAAH) from rat brain
Inhibition of FAAH was measured as described previously [31].
Briefly, the substrate N-(2-hydroxyethyl)-4-pyren-1-ylbutanamide
dibenzyl, fluoranthene, triphenylamine, DDT. To 2
solution of a reference and a test compound, respectively, in DMSO
(5 mM) was added 2 L of a solution of thiourea in DMSO (5 mM)
mL of a stock
(100
mM) solubilized with Triton X-100 (0.2%) was incubated with
m
rat brain microsomes in absence and in presence of a test com-
pound. Enzyme reaction was terminated after 60 min by addition of
a mixture of acetonitrile/methanol including the internal standard
6-pyren-1-ylhexanoic acid. FAAH inhibition was determined by
measuring the amount of 4-pyren-1-ylbutanoic acid released by
the enzyme in absence and presence of a test compound by
reversed phase HPLC with fluorescence detection. Values are listed
with two significant figures.
applied for dead time determination and 1 mL of the mobile phase,
which consisted of acetonitrile/water/phosphoric acid (85%)
(55:45:0.1, v/v/v). From each sample 20
HPLC system. Analysis was performed on a Kromasil 100-5C18
m)
mL were injected onto the
column (3 mm inside diameter ꢂ 60 mm, particle size
5 m
(AzkoNobel, Bohus, Sweden). The temperature of the column oven
was maintained at 25 ꢀC. The flow rate was 0.6 mL/min and the
effluents were monitored at 254 nm.
The linear fit of the log k values (k: capacity factor) and the log P
3.2.2. Inhibition of human recombinant monoacylglycerol lipase
(MAGL)
The effect of selected compounds on the activity of the
endocannabinoid-degrading MAGL was evaluated as previously
described using human recombinant MAGL (Cayman Chemical via
Biomol, Hamburg, Germany) [41]. Briefly, the substrate 1,3-
values of the 11 references showed a correlation factor of
R ¼ 0.9926. With this method, for indomethacin and ibuprofen log
P values of 2.9 and 3.2, respectively, were obtained. For measuring
the log P values of the target compounds, the retention times of the
peak of their ketone forms were used [44].
dihydroxypropan-2-yl 4-pyren-1-ylbutanoate (100
m
M) solubi-
3.2.5. Inhibition of FAAH from rat liver homogenate by compound
50
lized with Triton X-100 (0.2%) was incubated with the enzyme
solution in absence and in presence of a test compound. The
enzyme reaction was terminated after 45 min by addition of a
mixture of acetonitrile⁄methanol including the internal standard 6-
pyren-1-ylhexanoic acid. MAGL inhibition was determined by
measuring the amount of 4-pyren-1-ylbutanoic acid released by
the enzyme in absence and presence of a test compound by
reversed phase HPLC with fluorescence detection.
3.2.5.1. Experiments without pre-incubation. The experiments were
carried out as described above for inhibitor evaluation (see 3.2.1)
with the following modification: Instead of rat brain microsomal
suspension an equal volume of an S9 fraction of rat liver homoge-
nate [31] was used (protein content of the S9 fraction determined
with the Bradford method: 8 mg/mL). The incubation time was
60 min and 5 min, respectively.
3.2.3. Metabolic stability in rat liver S9 fractions
3.2.5.2. Experiments with pre-incubation. To 2
mL of a DMSO solu-
The metabolic stability was tested using S9 fractions of rat liver
homogenate [31]. Briefly, the test compounds were incubated un-
der aerobic conditions in presence of the co-factor NADPH. The
metabolic reactions were terminated after 30 min by addition of
acetonitrile. Controls without NADPH were prepared by addition of
the test compounds to a mixture of S9 fractions and acetonitrile. In
deviation to the published procedure, the extent of metabolism was
evaluated by reversed phase HPLC with MS-detection. The HPLC/
MS system from Shimadzu (Kyoto, Japan) consisted of two LC-
20ADXR HPLC-pumps, a SIL-30AC autosampler, and a LCMS-2020
tion of 50 (concentration variable) was added a mixture of 125 mL
S9 fraction (protein content determined with the Bradford method:
8 mg/mL). The mixture was pre-incubated at 37 ꢀC for 15 min. Then
363 mL of a NADPH solution (11.2 mg NADPH-Na4 in a mixture of
9.5 mL of potassium phosphate buffer (0.1 M, pH 7.4), which con-
tained 0.5 mM EDTA-Na2, and 0.5 mL of a 0.1 M magnesium chlo-
ride solution) was added and pre-incubation at 37 ꢀC was
continued for 30 min. After addition of 50 mL of a 2% solution of
Triton X-100 in phosphate buffered saline (0.01 M, pH 7.4) con-
taining 1 mM EDTA-Na2, the FAAH reaction was started by addition
single quad detector. Aliquots of 2
mL were injected onto a ACE 3
of 10 mL of a 5 mM solution of the substrate N-(2-hydroxyethyl)-4-
C18 column (2.1 mm inside diameter ꢂ 100 mm, particle size 3
m
m)
pyren-1-ylbutanamide in DMSO. The mixture was incubated at
(HICHROM, Berkshire, UK) protected with a C18 guard column
(3 mm inside diameter ꢂ 4 mm) (Phenomenex, Aschaffenburg,
Germany). The autosampler temperature was 10 ꢀC, column oven
temperature was set to 30 ꢀC. The mobile phase consisted of
acetonitrile/10 mM ammonium acetate 10:90 (v/v), adjusted to pH
5 with formic acid (A) and acetonitrile/10 mM ammonium acetate
90:10 (v/v), adjusted to pH 5 with formic acid. For most of the
compounds the following gradient was applied (A%): 0 min: 50;
2 min: 50; 6 min: 5; 27 min: 5; 29 min: 50; 35 min: 50. For com-
pounds 71, 72 and 74 an alternative gradient was used (A%): 0 min:
90; 1 min: 90; 9 min: 0; 11 min: 0; 11.5 min: 90; 18 min: 90. The
37 ꢀC for 60 min. The enzyme reaction was terminated by the
addition of 1000
mL acetonitrile/methanol (1:1, v/v), which con-
tained the internal standard 6-pyren-1-ylhexanoic acid (0.025
mg/
200 mL). After cooling in an ice bath for 15 min, the samples were
centrifuged at 12000 g at 4 ꢀC for 5 min. In the supernatants, the
amount of the enzyme product 4-pyren-1-ylbutanoic acid was
determined by reversed phase HPLC with fluorescence detection
[31]. Control incubations (n ¼ 3) with 2
mL of DMSO instead of 2 mL
of a DMSO solution of 50 as well as a blank incubation (with DMSO
instead of the DMSO solution of the test compound and a potas-
sium phosphate buffer (0.1 M, pH 7.4) containing 0.5 mM EDTA-Na2

192
D. Garzinsky et al. / European Journal of Medicinal Chemistry 160 (2018) 183e192
of the pharmacology and tolerability of PF-04457845, an irreversible inhibitor
of fatty acid amide hydrolase-1, in healthy subjects, Br. J. Clin. Pharmacol. 73
(2012) 706e716.
instead of the S9 fraction) were carried out and analyzed in the
same way in parallel and used for the calculation of the inhibition
data. The IC50 values were determined via Probit analysis.
In the same way, an experiment was carried out, in which the
NADPH solution was replaced by a phosphate buffer consisting of a
mixture of 9.5 mL of potassium phosphate buffer (0.1 M, pH 7.4),
which contained 0.5 mM EDTA-Na2, and 0.5 mL of a 0.1 M magne-
sium chloride solution.
[23] S. Pawsey, M. Wood, H. Browne, K. Donaldson, M. Christie, S. Warrington,
Safety, tolerability and pharmacokinetics of FAAH inhibitor V158866:
a
double-blind, randomised, placebo-controlled phase I study in healthy vol-
unteers, Drugs R. D. 16 (2016) 181e191.
[24] A. Postnov, M.E. Schmidt, D.J. Pemberton, J. de Hoon, A. van Hecken, M. van
den Boer, P. Zannikos, P. van der Ark, J.A. Palmer, S. Rassnick, S. Celen,
G. Bormans, K. van Laere, Fatty acid amide hydrolase inhibition by JNJ-
42165279: a multiple-ascending dose and a positron emission tomography
study in healthy volunteers, Clin. Transl. Sci. 11 (2018) 397e404.
[25] A.C.M. van Esbroeck, A.P.A. Janssen, A.B. Cognetta, D. Ogasawara, G. Shpak,
Appendix A. Supplementary data
ꢁ
M. van der Kroeg, V. Kantae, M.P. Baggelaar, F.M.S. de Vrij, H. Deng, M. Allara,
F. Fezza, Z. Lin, T. van der Wel, M. Soethoudt, E.D. Mock, H. den Dulk, I.L. Baak,
B.I. Florea, G. Hendriks, L. De Petrocellis, H.S. Overkleeft, T. Hankemeier, C.I. De
Zeeuw, V. Di Marzo, M. Maccarrone, B.F. Cravatt, S.A. Kushner, M. van der
Stelt, Activity-based protein profiling reveals off-target proteins of the FAAH
inhibitor BIA 10-2474, Science 356 (2017) 1084e1087.
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2018.10.021.
References
[26] D. D'Souza, FAAH Inhibitor PF-04457845 in the treatment of cannabis
dependence, Neuropsychopharmacology 42 (2017) S72.
ꢀ
ꢀ
[27] M. Celorrio, D. Fernandez-Suarez, E. Rojo-Bustamante, V. Echeverry-Alzate,
[1] C.J. Fowler, P. Doherty, S.P.H. Alexander, Endocannabinoid turnover, Adv.
Pharmacol. 80 (2017) 31e66.
[2] F.A. Iannotti, V. Di Marzo, S. Petrosino, Endocannabinoids and
endocannabinoid-related mediators: targets, metabolism and role in neuro-
logical disorders, Prog. Lipid Res. 62 (2016) 107e128.
ꢀ
ꢀ
M.J. Ramírez, C.J. Hillard, J.A. Lopez-Moreno, R. Maldonado, J. Oyarzabal,
R. Franco, M.S. Aymerich, Fatty acid amide hydrolase inhibition for the
symptomatic relief of Parkinson's disease, Brain Behav. Immun. 57 (2016)
94e105.
[28] E.M. Marco, C. Rapino, A. Caprioli, F. Borsini, G. Laviola, M. Maccarrone, Po-
tential therapeutic value of a novel FAAH inhibitor for the treatment of anx-
iety, PloS One 10 (2015), e0137034.
[29] S. Zahov, D. Garzinsky, W. Hanekamp, M. Lehr, 1-Heteroarylpropan-2-ones as
inhibitors of fatty acid amide hydrolase: studies on structure-activity re-
lationships and metabolic stability, Bioorg. Med. Chem. 25 (2017) 825e837.
[30] L. Blanco, P. Amice, J.M. Conia, Halogenation of enol silyl ethers. Synthesis of
[3] D.A. Bachovchin, B.F. Cravatt, The pharmacological landscape and therapeutic
potential of serine hydrolases, Nat. Rev. Drug Discov. 11 (2012) 52e68.
[4] D.G. Deutsch, N. Ueda, S. Yamamoto, The fatty acid amide hydrolase (FAAH),
Prostaglandins Leukot. Essent. Fatty Acids 66 (2002) 201e210.
[5] G. Labar, C. Michaux, Fatty acid amide hydrolase: from characterization to
therapeutics, Chem. Biodivers. 4 (2007) 1882e1902.
ꢀ
[6] P. Pacher, S. Batkai S, G. Kunos, The endocannabinoid system as an emerging
various types of a-bromocarbonyl compounds, Synthesis (1976) 194e196.
target of pharmacotherapy, Pharmacol. Rev. 58 (2006) 389e462.
[7] A. Lodola, R. Castelli, M. Mor, S. Rivara, Fatty acid amide hydrolase inhibitors: a
patent review (2009-2014), Expert Opin. Ther. Pat. 25 (2015) 1247e1266.
[8] R. Ranieri, C. Laezza, M. Bifulco, D. Marasco, A.M. Malfitano, Endocannabinoid
system in neurological disorders, Recent Pat. CNS Drug Discov. 10 (2015)
90e112.
[31] A. Holtfrerich, W. Hanekamp, M. Lehr, (4-Phenoxyphenyl)tetrazolecarbox-
amides and related compounds as dual inhibitors of fatty acid amide hydro-
lase (FAAH) and monoacylglycerol lipase (MAGL), Eur. J. Med. Chem. 63 (2013)
64e75.
[32] S. Connolly, C. Bennion, S. Botterell, P.J. Croshaw, C. Hallam, K. Hardy,
P. Hartopp, C.G. Jackson, S.J. King, L. Lawrence, A. Mete, D. Murray,
D.H. Robinson, G.M. Smith, L. Stein, I. Walters, E. Wells, W.J. Withnall, Design
and synthesis of a novel and potent series of inhibitors of cytosolic phos-
pholipase A2 based on a 1,3-disubstituted propan-2-one skeleton, J. Med.
Chem. 45 (2002) 1348e1362.
[9] R. Kaur, S.R. Ambwani, S. Singh, Endocannabinoid system:
a multi-facet
therapeutic target, Curr. Clin. Pharmacol. 11 (2016) 110e117.
~
[10] S. Kathuria, S. Gaetani, D. Fegley, F. Valino, A. Duranti, A. Tontini, M. Mor,
G. Tarzia, G. La Rana, A. Calignano, A. Giustino, M. Tattoli, M. Palmery,
V. Cuomo, D. Piomelli, Modulation of anxiety through blockade of ananda-
mide hydrolysis, Nat. Med. 9 (2003) 76e81.
[33] M. Mileni, J. Garfunkle, C. Ezzili, F.S. Kimball, B.F. Cravatt, R.C. Stevens,
D.L. Boger, X-ray crystallographic analysis of
a-ketoheterocycle inhibitors
[11] G. Griebel, J. Stemmelin, M. Lopez-Grancha, V. Fauchey, F. Slowinski, P. Pichat,
G. Dargazanli, A. Abouabdellah, C. Cohen, O.E. Bergis, The selective reversible
FAAH inhibitor, SSR411298, restores the development of maladaptive be-
haviors to acute and chronic stress in rodents, Sci. Rep. 8 (2018) 2416.
[12] D.S. Johnson, C. Stiff, S.E. Lazerwith, S.R. Kesten, L.K. Fay, M. Morris, D. Beidler,
M.B. Liimatta, S.E. Smith, D.T. Dudley, N. Sadagopan, S.N. Bhattachar,
S.J. Kesten, T.K. Nomanbhoy, B.F. Cravatt, K. Ahn, Discovery of PF-04457845: a
highly potent, orally bioavailable, and selective urea FAAH inhibitor, ACS Med.
Chem. Lett. 2 (2011) 91e96.
bound to a humanized variant of fatty acid amide hydrolase, J. Med. Chem. 53
(2010) 230e240.
€
€
[34] J. Schroder, A. Klinger, F. Oellien, R.J. Marhofer, M. Duszenko M, P.M. Selzer,
Docking-based virtual screening of covalently binding ligands: an orthogonal
lead discovery approach, J. Med. Chem. 56 (2013) 1478e1490.
[35] B.F. Krippendorff, R. Neuhaus, P. Lienau, A. Reichel, W. Huisinga, Mechanism-
based inhibition: deriving Ki and kinact directly from time-dependent IC50
values, J. Biomol. Screen 14 (2009) 913e923.
[36] B.F. Cravatt, D.K. Giang, S.P. Mayfield, D.L. Boger, R.A. Lerner, N.B. Gilula,
Molecular characterization of an enzyme that degrades neuromodulatory
fatty-acid amides, Nature 384 (1996) 83e87.
[37] M.P. Patricelli, H.A. Lashuel, D.K. Giang, J.W. Kelly, B.F. Cravatt, Comparative
characterization of a wild type and transmembrane domain-deleted fatty acid
amide hydrolase: identification of the transmembrane domain as a site for
oligomerization, Biochemistry 37 (1998) 15177e15187.
[38] B.F. Cravatt, K. Demarest, M.P. Patricelli, M.H. Bracey, D.K. Giang, B.R. Martin,
A.H. Lichtman, Supersensitivity to anandamide and enhanced endogenous
cannabinoid signaling in mice lacking fatty acid amide hydrolase, Proc. Natl.
Acad. Sci. U.S.A. 98 (2001) 9371e9376.
[39] M. Lehr, J. Fabian, W. Hanekamp, Involvement of microsomal NADPH-
cytochrome P450 reductase in metabolic reduction of drug ketones, Bio-
pharm. Drug Dispos. 36 (2015) 398e404.
[13] S. Roughley, S. Walls, T. Hart, R. Parsons, P. Brough, C. Graham, A. Macias,
Azetidine Derivatives as Inhibitors of Fatty Acid Amide Hydrolase Useful in
the Treatment of Diseases and Preparation and Pharmaceutical Compositions
Thereof, WO 2009109743, 2009.
[14] J.M. Keith, W.M. Jones, M. Tichenor, J. Liu, M. Seierstad, J.A. Palmer, M. Webb,
M. Karbarz, B.P. Scott, S.J. Wilson, L. Luo, M.L. Wennerholm, L. Chang,
M. Rizzolio, R. Rynberg, S.R. Chaplan, J.G. Breitenbucher, Preclinical charac-
terization of the FAAH inhibitor JNJ-42165279, ACS Med. Chem. Lett. 6 (2015)
1204e1208.
[15] D.L. Boger, H. Sato, A.E. Lerner, M.P. Hedrick, R.A. Fecik, H. Miyauchi,
G.D. Wilkie, B.J. Austin, M.P. Patricelli, B.F. Cravatt, Exceptionally potent in-
hibitors of fatty acid amide hydrolase: the enzyme responsible for degrada-
tion of endogenous oleamide and anandamide, Proc. Natl. Acad. Sci. U.S.A. 97
(2000) 5044e5049.
[40] S. Petrosino, V. Di Marzo, FAAH and MAGL inhibitors: therapeutic opportu-
nities from regulating endocannabinoid levels, Curr. Opin. Invest. Drugs 11
(2010) 51e62.
ꢀ
[16] B.P. Roques, M.C. Fournie-Zaluski, M. Wurm, Inhibiting the breakdown of
endogenous opioids and cannabinoids to alleviate pain, Nat. Rev. Drug Discov.
11 (2012) 292e310.
[41] A. Holtfrerich, T. Makharadz, M. Lehr, High-performance liquid chromatog-
raphy assay with fluorescence detection for the evaluation of inhibitors
against human recombinant monoacylglycerol lipase, Anal. Biochem. 399
(2010) 218e224.
[42] M. Lehr, S. Zahov, M. Ekodo Voundi, Preparation of Tetrazolylpropan-2-ones
as Inhibitors of Cytosolic Phospholipase A2 and Fatty Acid Amide Hydrolase
Useful for Topical Applications, DE102013016573, 2015.
[17] D.L. Boger, Preparation of
a-keto Heterocycles as Inhibitors of Fatty Acid
Amide Hydrolase (FAAH), WO2004033652, 2004.
[18] J.P. Huggins, T.S. Smart, S. Langman, L. Taylor, T. Young, An efficient rando-
mised, placebo-controlled clinical trial with the irreversible fatty acid amide
hydrolase-1 inhibitor PF-04457845, which modulates endocannabinoids but
fails to induce effective analgesia in patients with pain due to osteoarthritis of
the knee, Pain 153 (2012) 1837e1846.
[43] OECD Guidelines for the Testing of Chemicals No. 117 e Partition Coefficient
(n-octanol/water, High Performance Liquid Chromatography (HPLC) Method,
2004.
[19] V. Di Marzo, Inhibitors of endocannabinoid breakdown for pain: not so FA(AH)
cile, after all, Pain 153 (2012) 1785.
[20] E. von Schaper, Bial incident raises FAAH suspicions, Nat. Biotechnol. 34
(2016) 223.
[44] A. Fritsche, A. Schulze Elfringhoff, J. Fabian, M. Lehr, 1-(2-Carboxyindol-5-
yloxy)propan-2-ones as inhibitors of human cytosolic phospholipase A2a
:
ꢀ
[21] C. Mallet, C. Dubray, C. Duale, FAAH inhibitors in the limelight, but regrettably,
synthesis, biological activity, metabolic stability, and solubility, Bioorg. Med.
Chem. 16 (2008) 3489e3500.
Int. J. Clin. Pharm. Ther. 54 (2016) 498e501.
[22] G.L. Li, H. Winter, R. Arends, G.W. Jay, V. Le, T. Young, J.P. Huggins, Assessment