(4-Phenoxyphenyl)tetrazolecarboxamides and related compounds as dual inhibitors of fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL)

Article abstract of DOI:10.1016/j.ejmech.2013.01.050

Inhibitors of the enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), the principle enzymes involved in the degradation of endogenous cannabinoids like anandamide and 2- arachidonoylglycerol, have potential utility in the treatment of several disorders including pain, inflammation and anxiety. In the present study, the effectivity and selectivity of eight known FAAH and MAGL inhibitors for inhibition of the appropriate enzyme were measured applying in vitro assays, which work under comparable conditions. Because many of the known FAAH and MAGL inhibitors simply consist of a lipophilic scaffold to which a heterocyclic system is bound, furthermore, different heterocyclic structures were evaluated for their contribution to enzyme inhibition by attaching them to the same lipophilic backbone, namely 4-phenoxybenzene. One of the most active compound synthesized during this investigation was N,N-dimethyl-5-(4-phenoxyphenyl)-2H- tetrazole-2-carboxamide (16) (IC₅₀ FAAH: 0.012 μM; IC₅₀ MAGL: 0.028 μM). This inhibitor was systematically modified in the lipophilic 4-phenoxyphenyl region. Structure-activity relationship studies revealed that the inhibitory potency against FAAH and MAGL, respectively, could still be increased by replacement of the phenoxy residue of 16 by 3-chlorophenoxy (45) or pyrrol-1-yl groups (49). Finally, the tetrazolecarboxamide 16 and some related compounds were tested for metabolic stability with rat liver S9 fractions showing that these kind of FAAH/MAGL inhibitors are readily inactivated by cleavage of the bond between the tetrazole ring and its carboxamide substituent.

Full text of DOI:10.1016/j.ejmech.2013.01.050

European Journal of Medicinal Chemistry 63 (2013) 64e75
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
(4-Phenoxyphenyl)tetrazolecarboxamides and related compounds as dual
inhibitors of fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase
(MAGL)
*
Angela Holtfrerich, Walburga Hanekamp, Matthias Lehr
Institute of Pharmaceutical and Medicinal Chemistry, University of Münster, Hittorfstrasse 58-62, 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:
Inhibitors of the enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), the
principle enzymes involved in the degradation of endogenous cannabinoids like anandamide and 2-
arachidonoylglycerol, have potential utility in the treatment of several disorders including pain,
inflammation and anxiety. In the present study, the effectivity and selectivity of eight known FAAH and
MAGL inhibitors for inhibition of the appropriate enzyme were measured applying in vitro assays, which
work under comparable conditions. Because many of the known FAAH and MAGL inhibitors simply
consist of a lipophilic scaffold to which a heterocyclic system is bound, furthermore, different hetero-
cyclic structures were evaluated for their contribution to enzyme inhibition by attaching them to the
same lipophilic backbone, namely 4-phenoxybenzene. One of the most active compound synthesized
during this investigation was N,N-dimethyl-5-(4-phenoxyphenyl)-2H-tetrazole-2-carboxamide (16) (IC50
Received 21 December 2012
Received in revised form
11 January 2013
Accepted 15 January 2013
Available online 8 February 2013
Keywords:
Fatty acid amide hydrolase
Monoacylglycerol lipase
Inhibitors
FAAH: 0.012 mM; IC50 MAGL: 0.028 mM). This inhibitor was systematically modified in the lipophilic 4-
Maleimides
Thiazolidinediones
Tetrazolecarboxamides
Metabolic stability
phenoxyphenyl region. Structureeactivity relationship studies revealed that the inhibitory potency
against FAAH and MAGL, respectively, could still be increased by replacement of the phenoxy residue of
16 by 3-chlorophenoxy (45) or pyrrol-1-yl groups (49). Finally, the tetrazolecarboxamide 16 and some
related compounds were tested for metabolic stability with rat liver S9 fractions showing that these kind
of FAAH/MAGL inhibitors are readily inactivated by cleavage of the bond between the tetrazole ring and
its carboxamide substituent.
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
effects. Therefore, inhibition of FAAH and MAGL, respectively, may
be attractive therapeutic approaches in the treatment of pain,
Arachidonoyl ethanolamide (anandamide) and 2-arachidonoyl-
glycerol (2-AG) are the most important endocannabinoids. Both are
synthesized on demand from membrane phospholipids and func-
tion through the G-protein coupled cannabinoid receptors CB1 and
CB2 [1,2]. Endocannabinoids play principal physiological roles in
the nervous system as well as in peripheral tissues. Especially, they
are of major importance in the regulation of analgesia, inflamma-
tion and cognition. The duration of action of anandamide and 2-AG
is relatively short. Anandamide is mainly inactivated by fatty acid
amide hydrolase (FAAH), while monoacylglycerol lipase (MAGL) is
thought to be the major enzyme responsible for hydrolytic cleavage
of 2-AG. Inactivation of FAAH and MAGL would lead to elevated
endogenous levels of anandamide and 2-AG producing analgetic,
anxiolytic, antidepressant, sleep enhancing and anti-inflammatory
inflammation, anxiety and other disorders [3e5]. Meanwhile a va-
riety of highly active FAAH inhibitors have been identified [1,3,4,6e
10]. In contrast, the number of known potent MAGL inhibitors is
much smaller [2,5,6,8e12]. One reason for this is the fact that
bioassays to investigate MAGL activity and inhibition were set up
later than those for FAAH [7].
One aim of the present study was to compare the effectivity and
selectivity of several known FAAH and MAGL inhibitors (1e8)
(Fig. 1) for inhibition of the appropriate enzyme applying assays,
which work under similar reaction conditions. Since many of the
known FAAH and MAGL inhibitors, respectively, like 5e8 simply
consist of a lipophilic scaffold to which a heterocyclic system is
bound, furthermore we evaluated different heterocyclic structures
for their contribution to enzyme inhibition by attaching them to the
same lipophilic backbone, namely 4-phenoxybenzene, which is
present in the MAGL inhibitor 5 [12] (for structures see Table 2).
Finally, structural variation of the 4-phenoxybenzene part of one of
the most active compounds obtained during this investigation was
* Corresponding author. Tel.: þ49 251 83 33331; fax: þ49 251 83 32144.
E-mail address: lehrm@uni-muenster.de (M. Lehr).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.01.050

A. Holtfrerich et al. / European Journal of Medicinal Chemistry 63 (2013) 64e75
65
O
O
H2N
O
O
O
N
N
N
NO2
OCH3
N
N
H
N
O
O
O
N
OH
O
N
H
O
O
O
O
O
O
CH3
2 (PHOP)
4 (CAY10499)
1 (URB597)
3 (JZL184)
O
O
O
H3C
OCH3
N
N
N
N
CH3
N
N
N
N
N
N
N
C5H11
6 (NAM)
Fig. 1. Chemical structures of known FAAH and MAGL inhibitors.
CH3
O
O
O
N
O
7 (LY2183240)
8
CH3
5
performed, and structure activity relationships on inhibition of
FAAH and MAGL were examined.
converted to the acetonitrile 17 by reaction with trimethylsilyl cy-
anide (Scheme 2) [18]. Reduction of this intermediate by LiAlH4 [19]
led to the 2-aminoethanol 18. Formation of the oxazolidinone ring
was finally achieved using trichloromethyl chloroformate [20].
Treatment of 19 with cyclohexyl isocyanate in the presence of
tetrakis(triphenylphosphine)palladium [21] led to the carbamoy-
lated oxazolidinone 20, which should also be tested for enzyme
inhibition.
Reaction of 4-phenoxybenzonitrile with hydroxylamine hy-
drochloride yielded the N-hydroxybenzenecarboximidamide 21
[22], which in turn was cyclized with phenyl chloroformate [23]
to give the desired oxadiazolone 22 (Scheme 3). The synthesis
of the thiazolidinedione target compound 23 has already been
described [24].
2. Results and discussion
2.1. Chemistry
The maleimide methyl-substituted phenoxybenzene 9 [13] was
synthesized from 4-phenoxybenzyl alcohol and maleimide under
Mitsunobu conditions analogously to a published procedure [14].
The corresponding derivative with a maleimide group directly
attached to the phenoxyphenyl backbone (10) could be obtained as
described recently [15].
The synthesis of the tetrazolecarboxamides 12 and 13 was
performed as outlined in Scheme 1. Carbamoylation of the tetrazole
11 [16,17] with N,N-dimethylcarbamoyl chloride led to a mixture of
12 and 13, which could be separated by semi-preparative reversed
phase HPLC. Since 13C NMR studies of disubstituted tetrazoles have
revealed that the carbon atom of 2,5-substituted tetrazoles shows
resonance at higher ppm values (about 165 ppm) than the corre-
sponding carbon of the 1,5-disubstituted compounds (about
155 ppm) [17], the structure of the two regioisomers could be
assigned with the aid of their 13C NMR data. The corresponding
derivatives 15 and 16, in which the tetrazole is directly bound to the
phenoxyphenyl ring system, were obtained from 14 [16] in the
same way (Scheme 1). Separation of the two isomers here could be
achieved by simple silica gel chromatography. Structural assign-
ments were also performed by 13C NMR applying the principles
mentioned above.
The derivatives of 16, in which the phenoxy residue was replaced
by substituted phenoxy moieties (40e48) or heteroaryl systems
(49e55) were synthesized from the appropriate substituted tetra-
zoles 24e39 (Scheme 4), which were available by reaction of the
corresponding benzonitriles with trimethylsilyl azide. The
substituted benzonitrile starting compounds themselves were pre-
pared by microwave synthesis of 4-bromo- or 4-fluorobenzonitrile
and substituted phenols with K2CO3 in DMSO [25], by reaction of
4-fluorobenzonitrile or 4-bromomethylbenzonitrile and the appro-
priate nitrogen heterocycle with caesium carbonate in DMF [26], or
by reaction of 4-aminobenzonitrile or 4-aminomethylbenzonitrile
with hexane-2,5-dione with p-toluenesulfonic acid in toluene [27].
The acylated nitrile precursors 56aee necessary for the syn-
thesis of the target compounds 58aee via the tetrazoles 57aee
(Scheme 5) were obtained by Friedel Crafts reaction [28]. The N,N-
diethyl and N,N-diisopropyl-derivatives 59 and 60 could be afforded
by reaction of 15 with the appropriate carbamoyl chlorides.
The preparation of the oxazolidinone substituted target com-
pound 19 started from 4-phenoxybenzaldehyde, which was first
N
N
(CH2)nCN
a
(CH2)n
NH
b
N
O
O
11: n = 1
14: n = 0
N
N
N
N
N
N
CH3
(CH2)n
(CH2)n
N
N
N
+
CH3
O
O
O
O
CH3
N
12: n = 1
15: n = 0
13: n = 1
16: n = 0
H3C
Scheme 1. Reagents and conditions: (a) trimethylsilyl azide, tetrabutylammonium fluoride hydrate, 120 ꢀC, 3 h, 89%; (b) N,N-dimethylcarbamoyl chloride, triethylamine, aceto-
nitrile, 11: 0e70 ꢀC, 20 h, 14: 0 ꢀC, 17 h, 6e10%.

66
A. Holtfrerich et al. / European Journal of Medicinal Chemistry 63 (2013) 64e75
Si(CH3)3
O
O
b
a
H
CN
17
O
O
O
O
OH
O
NH
NH2
c
18
19
O
O
N
O
O
d
HN
20
O
Scheme 2. Reagents and conditions: (a) trimethylsilyl cyanide, triethylamine, CH2Cl2, 0 ꢀC, 2 h and rt, overnight; (b) lithium aluminium hydride in THF (2 M), THF, 0 ꢀC, 1 h and rt,
2 h, 51%; (c) trichloromethyl chloroformate, pyridine, CH2Cl2, 0 ꢀC to rt, 20 h, 76%; (d) cyclohexyl isocyanate, toluene, tetrakis(triphenylphosphine)palladium, rt, overnight, 60%.
2.2. Biological evaluation
aggregated form. The great influence of assay conditions on enzyme
inhibition can be seen on the basis of the IC50 values published in
literature for the FAAH inhibitor URB597 (1), which vary from
2.2.1. General
Inhibition of rat brain microsomal FAAH and human recombinant
MAGL by the test compounds were determined with closely related
test systems [29,30]. Derivatives of the natural substrates ananda-
mide (for FAAH) and 2-arachidonoylglycerol (for MAGL), in which
the C5eC20 carbons of the arachidonyl-chain were replaced by
a fluorogenic pyren-1-yl residue [30,31], were used as substrates. In
both assays, the micelle forming agentTritonX-100 was added tothe
reaction mixtures in a concentration of about 0.2%, allowing the
enzyme reactions taking place at an interface of water and an
aggregated lipophilic system. By this way, conditions comparable to
that prevailing in vivo at the natural phospholipid membranes were
achieved. The FAAH and MAGL activities were determined by
measuring the amount of the cleavage product 4-pyren-1-
ylbutanoic acid released by the enzymes from the two substrates
with reversed phase HPLC and fluorescence detection, applying in-
cubation times of 45 min. Some of the target compounds were also
tested for metabolic stability by incubation with S9 fractions of rat
liver in presence and absence of NADPH. The samples were inves-
tigated for parent compounds and the metabolites, respectively,
with HPLC and UV-detection.
0.0038 to 0.99 mM [34e38] (for an overview of published inhibition
data of compounds 1e8 see Supplementary material). Therefore, for
a true comparison of the inhibitory potency of FAAH and MAGL in-
hibitors it is necessary to evaluate them in analogous test systems.
The evaluation of the published compounds 1e8 for FAAH and
MAGL inhibition in our assays led to the following results (Table 1):
The N-cyclohexylcarbamate 1 (URB597) and the
2 (PHOB) [39], originally developed as FAAH inhibitors, strongly
inhibited this enzyme (IC50: 0.062 M and 0.004 M, respectively),
while they did not affect MAGL at the highest test concentration
(10 M).
The carbamates 3 (JZL184) [5] and 4 (Cay10499) [40], which
were one of the first MAGL inhibitors published, inhibited MAGL in
our assay with IC50 values of 1.7 M and 0.48 M, respectively.
While the inhibition value of 4 shows a good correlation with the
values published (IC50: 0.092 Me0.5 M [12,40]), the measured
IC50 for 3 is much higher than most of the IC50 given in literature for
this compound (IC50: 0.002 Me0.057 M [5,10,41,42]). However,
in a recent study also a lower inhibition activity of 3 on MAGL was
determined (IC50: 0.52 M) [43]. As mentioned above, one reason
a-keto heterocycle
m
m
m
m
m
m
m
m
m
m
for these discrepancies could lie in the applied times of pre-
incubation and/or incubation. Furthermore, since the hydrolysis
of the natural substrate 2-AG by MAGL in cells takes place at the
interface between cytosol and membrane bilayer, the relevant
concentration of inhibitors for this membrane bound enzyme is
their surface concentration [44]. This depends on the amount of the
substrate and, if present, surface active additives (like Triton X-100)
as well as the lipophilicity of the inhibitor [45].
2.2.2. Inhibition data of known FAAH and MAGL inhibitors
FAAH and MAGL are serine hydrolases. Most of their known in-
hibitors affect the enzymes by a covalent modification of their active
siteserine[6,7,11,32]. Becauseformationofcovalentbondstakessome
time, inhibition data strongly depend on the incubation conditions
such as pre-incubation and incubation time as well as pH value [33].
Furthermore, since FAAH and MAGL cleave their lipophilic substrates
at the lipid water interface of the cells, it is also of importance,
whetherthesubstrateispresentedtotheenzymesinmonomericorin
Compound 3 seems to be a relatively selective MAGL inhibitor,
since at the highest test concentration applied (10
mM) FAAH
O
NH
HN
O
CN
a
OH
b
N
N
H
O
O
O
21
22
Scheme 3. Reagents and conditions: (a) hydroxylamine hydrochloride, methanol, aqueous NaOH, reflux, 11 h, 81%; (b) phenyl chloroformate, triethylamine, CH2Cl2, 0 ꢀC, 2 h, 65%.

A. Holtfrerich et al. / European Journal of Medicinal Chemistry 63 (2013) 64e75
67
N
N
N
a
b
N
NH
N
N
CH3
N
R
CN
R
R
N
CH3
O
24-39
40-55
24-32 and 40-48: R = substituted phenoxy (see Table 3)
33-39 and 49-55: R = heteroaryl or heteroarylmethyl (see Table 4)
Scheme 4. Reagents and conditions: (a) trimethylsilyl azide, tetrabutylammonium fluoride hydrate, 120 ꢀC, 1.5 he9 h, 53e90%; (b) N,N-dimethylcarbamoyl chloride, triethylamine,
acetonitrile, 0 ꢀC, 0.5e2 h, rt, 1e24 h, 70 ꢀC, 1e4.5 h, 11e73%.
activity was not reduced by this compound. In contrast, 4 blocks the
activity of FAAH to nearly the same extend and, therefore, it can be
considered as dual MAGL/FAAH inhibitor. Replacement of the car-
bamate part of 4 by a phenoxyphenyl residue led to compound 5
[12], which possessed an even slightly increased inhibitory potency
0.0033
mM; IC50 MAGL: 0.02 mM) [17]. The same activity ratio could
be measured with our assays (Table 1). With an IC50 of 0.006
against MAGL, 8 was about 300fold more active against the enzyme
than 3 and about 80fold more active than 4.
mM
against MAGL (IC50: 0.31
mM) and FAAH (IC50: 0.72
mM).
2.2.3. Evaluation of new inhibitors of FAAH and MAGL
The maleimide derivative 6 (NAM) has initially been reported to
As mentioned above, many of the known FAAH and/or MAGL
inhibitors like 5e8 simply consist of a lipophilic scaffold to which
a heterocycle is bound. A second purpose of this study was to
compare the contribution of different heterocyclic systems to
enzyme inhibition. Starting point here was the dual MAGL/FAAH
inhibitor 5. The oxadiazolone ring system of this compound bound
to a lipophilic 4-phenoxybenzene scaffold was replaced by different
heterocycles. Introduction of a maleimide methyl- or maleimide-
residue, like it is present in 6, resulted in a significant drop of
inhibitory potencyagainst both enzymes. Interestingly, the obtained
compounds 9 and 10 were still slightly more active than 6 itself
(Table 2). In contrast, N,N-dimethylcarbamoyltetrazolylmethyl
substituents had a very high impact on enzyme inhibition. The in-
hibition values obtained for 12 and 13 were comparable to those
measured for the structurally related known inhibitors 7 and 8.
Direct attachment of the tetrazole ring system to the phenoxy
benzene scaffold only slightly altered inhibition values. The result-
ing substances 15 and 16 were still nanomolar inhibitors of MAGL
and FAAH.
Since the heterocyclic system of the dual FAAH/MAGL inhibitor
5 bears a carbamate structural element, we decided to synthesize
and test the related heterocyclic compounds 19 and 22. Fur-
thermore, compound 20 bearing a N-cyclohexyl-2-oxooxazolidine-
3-carboxamide ring system was investigated, because a series of
published inhibitors of the serine hydrolase cytosolic phospholi-
pase A2a (cPLA2a) contain this structural element [52]. However,
none of the obtained derivatives 19, 20 and 22 led to inhibition of
MAGL and FAAH even at the highest test concentration.
inhibit MAGL with an IC50 of 0.14
m
M [46], while in later studies
significantly higher IC50 values (1.1
m
M and 2.8 M) were evaluated
m
[47,48]. In our assay, 6 was even inactive against this enzyme at the
highest test concentration (10 M). These findings are not very
m
surprising at all, since it is known that the maleimide ring system is
a very critical structural element in inhibitor molecules, because it
performs Michael reactions with nucleophiles, such as thiols and
amines, of many biomolecules. For example, for the putative
phospholipase C inhibitor U73122, which also bears a maleimide
moiety, it has been found that the compound is readily inactivated
when dithiothreitol or L-glutamine is present in the reaction me-
dium [49]. In our MAGL assay, 6 probably is inactivated by reaction
with nucleophilic components present in the incubation buffer. In
the FAAH assay some inhibition of the enzyme by 6 could be seen,
however this was very weak (38% inhibition at 10 mM).
The tetrazolecarboxamide 7 (LY2183240), which had been ini-
tially developed as an endocannabinoid transport blocker [50], was
later found to be a strong inhibitor of FAAH and a weak inhibitor of
MAGL [17]. Furthermore, several derivatives of 7 were described as
dual FAAH/MAGL inhibitors [51]. With the enzyme assays applied
in the published investigations [17], a more than 4000fold selec-
tivity for FAAH over MAGL was measured for 7 (IC50 FAAH:
0.0021
a very high FAAH inhibitory potency was determined (IC50
0.005 M) too, but also a considerable inhibition of MAGL (IC50
0.29
mM; IC50 MAGL: 8.1 mM). With our assays, for this compound
:
:
m
m
M) was registered. The selectivity factor here was only about
60. The regioisomer of 7, in which the carbamoyl residue was
attached at position 2 of the tetrazole ring (8), was reported to
inhibit MAGL about twice as effective than FAAH (IC50 FAAH:
In the last years many compounds with a methylidenene thia-
zolidinedione system have been reported to have biological activity
CH3
CH3
a: R = acetyl
a
b: R = benzoyl
c: R = pivaloyl
N
CN
N
CN
d: R = hexanoyl
e: R = 4-phenylbenzoyl
R
CH3
CH3
56a-e
CH3
N
CH3
N
N
N
N
b
c
N
NH
N
N
CH3
N
N
R
R
CH3
CH3
CH3
O
57a-e
58a-e
Scheme 5. Reagents and conditions: (a) acyl chloride, 1,2-dichloroethane, SnCl4, 0 ꢀC, 30 mine1 h, rt, 3.5e20 h, 50e69%; (b) trimethylsilyl azide, tetrabutylammonium fluoride
hydrate, 120 ꢀC, 1.5 he9 h, 56e92%; (c) N,N-dimethylcarbamoyl chloride, triethylamine, acetonitrile, 0 ꢀC, 0.5e2 h, rt, 1e24 h, 70 ꢀC, 1e4.5 h, 23e47%.

68
A. Holtfrerich et al. / European Journal of Medicinal Chemistry 63 (2013) 64e75
Table 1
The reference compounds 7 and 8 as well as the structurally
related new compounds 12e16, 45, 49 and 58aee were tested for
metabolic stability. After incubation with rat liver S9 fractions in
presence of NADPH for 30 min, none of these compounds could be
detected any more in the incubation solutions. In each case, the
main metabolite was the corresponding unsubstituted tetrazole.
For compound 16 it was shown, that the hydrolysis product (14)
was virtually inactive against FAAH and MAGL. This was not sur-
prising, since it had been shown, that tetrazolecarboxamides
inactivate FAAH and MAGL by carbamoylation of the catalytical
serines of the active site of the enzymes [47]. Thus, an intact car-
bamoyltetrazole structure is necessary for activity.
Inhibition of MAGL and FAAH by known inhibitors (for structures see Fig. 1).
Comp.
Inhibition of
MAGL
FAAH
IC50
IC50
(
m
M)a
(m
M)a
1 (URB597)
2 (PHOB)
3 (JZL184)
4 (CAY 10499)
5
6 (NAM)
7 (LY2183240)
8
n.a.b
n.a.b
1.7
0.48
0.31
n.a.b
0.29
0.062
0.004
n.a.b
1.3
0.72
>10c
0.005
0.012
0.006
Finally we investigated, whether it is possible to increase met-
abolic stability of the carbamoyl tetrazoles against hydrolytic en-
zymes by replacing the methyl groups of the dimethylcarbamoyl
moiety by more bulky substituents. The derivative with a dieth-
ylamino substituent (59) still was totally metabolized by the rat
liver S9 fractions under the conditions applied (Table 6). Intro-
duction of two voluminous isopropyl residues led to a measurable
increase of metabolic stability. However, the obtained compound
60 did not inhibit MAGL and only weakly inhibited FAAH at the
a
Values are the means of at least two independent determinations, errors are
within ꢁ20%.
b
c
Not active at 10
mM.
38% inhibition at 10
mM.
[53]. Concomitantly, the concern has raised that this Michael
acceptor group undergoes non-specific interactions with proteins
resulting in a promiscuous inhibition of diverse target proteins [53].
Despite of these objections, we finally also wanted to evaluate this
heterocyclic system and attached it to our basic lipophilic scaffold.
The biochemical testing of obtained compound 23 revealed that it
did not inhibit the two enzymes. Thus neither a specific nor a non-
specific interaction of 23 with the enzymes occurs.
highest test concentration (10 mM).
3. Conclusion
The evaluation of several of the most convenient known FAAH
and MAGL inhibitors for inhibition of these enzymes show that
there exist great differences between these substances in regard of
their enzyme inhibitory potencies. Furthermore, structureeactivity
relationship studies of a novel series of 5-phenyl-2H-tetrazole-2-
carboxamides are presented. The most active dual FAAH/MAGL
inhibitors synthesized were the 3-chlorophenoxy substituted de-
From the new active compounds we had found, we chose 5-(4-
phenoxyphenyl)-2H-tetrazole-2-carboxamide (16) for further
structure activity studies. The IC50 of this inhibitor against FAAH
and MAGL was 0.012 mM and 0.028 mM, respectively. First, fluoro-,
chloro-, methyl- and methoxy-substituents were introduced in
para- or meta-position of the terminal phenyl residue of 16. Fur-
thermore, the 3,4-dichlorophenyl derivative 48 was synthesized.
Inhibition of FAAH was not affected substantially by these modifi-
cations (Table 3). The IC50s of these derivatives of 16 against this
rivative 45 (IC50 FAAH: 0.009
pyrrol-1-yl substituted derivative 49 (IC50 FAAH: 0.005
MAGL 0.015 M). The potency of these two compounds is compa-
rable to that of the potent 2H-tetrazole-2-carboxamide reference
inhibitor 8 (IC50 FAAH: 0.012 M; IC50 MAGL: 0.006 M) (Fig. 1).
mM; IC50 MAGL 0.006
m
M) and the
m
M; IC50
m
enzyme only ranged from 0.009 mM to 0.024 mM. In contrast, the
effects on MAGL inhibition were slightly more pronounced. While
m
m
a chlorine atom in meta position increased activity about fivefold
The investigation of the metabolism of several of these new and
structurally related previously published compounds (7 and 8)
show that the tertrazoledimethylcarboxamide pharmacophore is
readily inactivated by cleavage of the bond between heterocycle
and carboxamide moiety. Despite this metabolic liability, it seems
to be possible to achieve in vivo effects with such inhibitors,
because 7 has been reported to have activity in several animal
models [50,54,55]. A more serious problem in the development of
tetrazolecarboxamides as drug candidates could be their pro-
miscuity. For 7 it has been found that beside FAAH and MAGL some
more serine hydrolases are inhibited [56]. However, this inhibition
is not merely nonspecific, because we have found that the serine
hydrolase cytosolic phospholipase A2a is not inhibited by the ref-
erences 7 and 8 as well as the related new compounds 12, 13,15 and
(IC50 of 45: 0.006
inhibitory potency sevenfold (IC50 of 43: 0.19
against MAGL and FAAH of 0.006 M and 0.009
showed the same high potency as the tetrazolecarboxamide ref-
erence inhibitor 8 (IC50: 0.006 M and 0.012 M, respectively).
m
M), a methoxy group in para position reduced
M). With IC50 values
M, compound 45
m
m
m
m
m
Next, the phenoxy residue of 16 was replaced by pyrrol-1-yl,
2,5-dimethypyrrol-1-yl, pyrazol-1-yl or indol-1-yl residues (49e
52). These structural modifications did not exert greater impact
on the inhibition of both enzymes (Table 4). Likewise, in case of the
pyrrole 49 introduction of a methylene spacer inserted between
phenyl ring and heterocycle (53) did not lead to a drastic change in
activity. In contrast, the (2,5-dimethylpyrrol-1-yl)methyl and
(indol-1-yl)methyl homologues 54 and 55, which possess a more
voluminous heterocyclic system than 53, possessed a significantly
lower inhibitory potency against FAAH and/or MAGL than the
parent compounds 50 and 52.
In the further investigations different acyl residues were intro-
duced in the pyrrole ring of the 2,5-dimethylpyrrole substituted
derivative 50. From MAGL only an acetyl residue was well tolerated
(Table 5). The other acyl groups decreased inhibition of this enzyme
up to 14fold. Inhibition of FAAH was not altered by the small acetyl
and the linear hexanoyl residue (58a, 58d). Like in the case of
MAGL, the greatest loss of activity occurred after introduction of the
bulky 4-phenylbenzoyl substituent (58e). Inhibitory activity on
FAAH was lowered fourfold in this case. Nevertheless, with an IC50
16 even at the highest test concentration of 10 mM. Since intro-
duction of substituents into the terminal aryl residue of the new
2H-tetrazole-2-carboxamides is tolerated by both FAAH and MAGL
to some extent, structural modification in this region of the mole-
cules could offer the opportunity to control selectivity of the in-
hibitors against other serine hydrolase targets.
4. Experimental section
4.1. Chemistry
4.1.1. General
value against FAAH and MAGL of 0.65
tively, 58e still has considerable activity.
mM and 0.040
m
M, respec-
Column chromatography was performed on silica gel 60, parti-
cle size 0.040e0.063 mm, from Merck or Macherey & Nagel. For

A. Holtfrerich et al. / European Journal of Medicinal Chemistry 63 (2013) 64e75
69
Table 2
Inhibition of MAGL and FAAH by a series of heteroaryl-substituted 4-phenoxybenzene derivatives.
O
O
N
O
O
OCH3
N
N
N
O
O
O
O
O
9
10
5
CH3
O
N
N
O
CH3
N
N
N
CH3
N
N
H3C
N
N
O
N
O
13
12
H3C
N
N
N
CH3
N
O
N
N
N
N
CH3
N
N
O
O
16
CH3
15
O
O
O
O
O
O
O
HN
NH
N
O
N
HN
O
O
O
20
22
19
O
NH
O
S
O
23
Comp.
Inhibition of
MAGL
FAAH
IC50
(m
M)a
IC50 (m
M)a
5
9
0.31
0.72
4.9
5.3
>10b
9.7
10
12
13
15
16
19
20
22
23
0.31
0.006
0.009
0.007
0.012
n.a.c
0.016
0.060
0.028
n.a.c
n.a.c
n.a.c
n.a.c
n.a.c
n.a.c
n.a.c
a
Values are the means of at least two independent determinations, errors are within ꢁ20%.
b
c
44% inhibition at 10
Not active at 10 M.
mM.
m
preparative HPLC a RP18 column was applied (Kromasil 100, 5
m
m,
applying gradient elution from acetonitrile/water/trifluoroacetic
acid 10/90/0.1 (v/v/v) to acetonitrile/water/trifluoroacetic acid 90/
10/0.1 (v/v/v) at a flow rate of 0.4 mL/min. The samples were pre-
10 mm (I.D.) ꢂ 250 mm protected with an analogously filled guard
column 10 mm (I.D.) ꢂ 50 mm). Melting points were determined on
a Büchi B-540 apparatus and are uncorrected. 1H NMR spectra
(400 MHz) and 13C NMR spectra (100 MHz) were recorded on
a Varian Mercury Plus 400 spectrometer. Electron ionization (EI)
mass spectra were obtained on a Finnigan GCQ apparatus. The high
resolution mass spectra were recorded on a Bruker micrOTOF-Q II
spectrometer applying electro spray chemical ionization (ESI) or
atmospheric pressure chemical ionization (APCI). Purity of the
target compound was determined by reversed phase HPLC on
pared by dilution of 20
compounds in DMSO with 180
was 5 L. UV-absorbance was measured at 254 nm. The purity of all
mL of 5 mM stock solutions of the test
mL acetonitrile. The injection volume
m
target compounds was greater or equal than 95%, with exception of
compounds 9, 10 and 19, whose purity was about 90%.
4.1.2. 5-(4-Phenoxybenzyl)tetrazole (11)
A mixture of 4-phenoxyphenylacetonitrile (200
trimethylsilyl azide (214 L, 1.59 mmol) and tetrabutylammonium
fluoride hydrate (139 mg, 0.53 mmol) was heated at 120 ꢀC for 3 h.
mL, 1.06 mmol),
a Nucleosil 100 RP 18 column (125 ꢂ 3 mm, 3
m
) protected with
m
a Phenomenex C18 guard column (3 mm inside diameter ꢂ 4 mm)

70
A. Holtfrerich et al. / European Journal of Medicinal Chemistry 63 (2013) 64e75
Table 3
Table 5
Inhibitory activity of different (4-phenoxyphenyl)tetrazolecarboxamides against
MAGL and FAAH.
Inhibitory activity of acylated (4-heteroarylphenyl)tetrazolecarboxamides against
MAGL and FAAH.
H3C
N
N
N
N
N
CH3
CH3
N
R1
R2
N
N
H3C
O
N
O
R
CH3
Comp.
R1
R2
Inhibition of
MAGL
FAAH
IC50
Comp.
R
Inhibition of
MAGL
IC50
(
m
M)a
(
m
M)a
FAAH
IC50
16
40
41
42
43
44
45
46
47
48
H
F
Cl
CH3
OCH3
H
H
H
H
H
H
H
H
F
Cl
CH3
OCH3
Cl
0.028
0.049
0.037
0.067
0.19
0.015
0.006
0.015
0.028
0.075
0.012
0.014
0.016
0.024
0.013
0.010
0.009
0.013
0.015
0.011
IC50
(
m
M)a
(
m
M)a
50
H
0.048
0.036
0.18
0.11
0.37
0.65
0.009
0.009
0.016
0.029
0.011
0.040
58a
58b
58c
58d
58e
Acetyl
Benzoyl
Pivaloyl
Hexanoyl
4-Phenylbenzoyl
H
Cl
a
Values are the means of at least two independent determinations, errors are
within ꢁ20%.
a
Values are the means of at least two independent determinations, errors are
within ꢁ20%.
2 h. After cooling to room temperature, silica gel was added and the
solvent was evaporated under reduced pressure. The residue was
transferred to the top of a silica gel column and eluted with hexane/
ethyl acetate (8:2) to afford a mixture of the two isomers 12 and 13.
Separation of this mixture was achieved on a semi-preparative RP-
HPLC column (eluent: methanol/water 7:3) to afford 12 as an oily
substance after distilling off most of the methanol and freeze-
drying the remaining aqueous phase (16 mg, 10%). 1H NMR
The obtained yellowish, high-viscous liquid was cooled to room
temperature and purified by silica gel chromatography (hexane/
ethyl acetate, 8:2; ethyl acetate þ 0.2% formic acid) to give 11 [57] as
a solid (237 mg, 89%); mp 147 ꢀC, 1H NMR (DMSO-d6):
d 4.27 (s, 2H),
6.95e7.01 (m, 4H), 7.13 (t, J ¼ 7.4 Hz, 1H), 7.29 (d, J ¼ 8.5 Hz, 2H),
7.35e7.41 ppm (m, 2H); MS (EI, 70 eV) m/z (%): 252 (19) Mþ, 183
(100).
(CDCl3):
d 2.76 (s, 3H), 3.10 (s, 3H), 4.42 (s, 2H), 6.92e7.00 (m, 4H),
7.11 (t, J ¼ 7.4 Hz, 1H), 7.22 (d, J ¼ 8.4 Hz, 2H), 7.30e7.37 ppm (m,
4.1.3. N,N-Dimethyl-5-(4-phenoxybenzyl)-1H-tetrazole-1-
carboxamide (12)
Compound 11 (120 mg, 0.48 mmol) was suspended in dry ace-
tonitrile (2 mL) and dissolved by addition of triethylamine (133
2H); 13C NMR (CDCl3):
d 29.1, 37.3, 38.7, 118.8, 123.4, 128.7, 129.7,
130.3, 147.7, 155.7, 156.6, 156.7 ppm; MS (ESI) m/z [M þ Na]þ: 346.
mL,
4.1.4. N,N-Dimethyl-5-(4-phenoxybenzyl)-2H-tetrazole-2-
carboxamide (13)
Compound 13 was obtained during semi-preparative RP-HPLC
of 12. After distilling off most of the methanol and freeze-drying the
0.95 mmol). N,N-Dimethylcarbamoyl chloride (66
mL, 0.71 mmol)
was added dropwise at 0 ꢀC and the mixture was stirred at the same
temperature for 16 h, at room temperature for 2 h, and at 70 ꢀC for
Table 4
Table 6
Inhibitory activity of different (4-heteroarylphenyl)tetrazolecarboxamides against
MAGL and FAAH.
Effect of the substitution pattern at the carboxamide moiety on enzyme inhibition
and metabolic stability.
H3C
R
N
N
N
N
CH3
N
N
N
R
N
N
O
N
O
R
O
Comp.
R
Inhibition of
MAGL
Comp.
R
Inhibition of
MAGL
IC50
Metabolic
FAAH
IC50
stability (%)d
FAAH
IC50
IC50
(
m
M)a
(m
M)a
(
m
M)a
(m
M)a
16
49
50
51
52
53
54
55
Phenoxy
Pyrrol-1-yl
2,5-Dimethylpyrrol-1-yl
Pyrazol-1-yl
Indol-1-yl
Pyrrol-1-ylmethyl
(2,5-Dimethylpyrrol-1-yl)methyl
Indol-1-ylmethyl
0.028
0.015
0.048
0.033
0.023
0.012
0.14
0.012
0.005
0.009
0.008
0.012
0.011
0.14
16
59
60
Methyl
Ethyl
Isopropyl
0.028
0.87
0.012
0.037
0
0
n.a.b
>10c
27 ꢁ 7
a
Values are the means of at least two independent determinations, errors are
within ꢁ20%.
b
c
Not active at 10
17% inhibition at 10
Percent of parent compound remaining after metabolism in rat liver S9 frac-
mM.
0.22
0.088
m
M.
a
d
Values are the means of at least two independent determinations, errors are
within ꢁ20%.
tions; in case of 16 and 59: n ¼ 2; in case of 60: mean ꢁ standard deviation, n ¼ 3.

A. Holtfrerich et al. / European Journal of Medicinal Chemistry 63 (2013) 64e75
71
remaining aqueous phase 13 was obtained as an oily substance
(16 mg, 10%). 1H NMR (CDCl3):
3.11 (s, 3H), 3.26 (s, 3H), 4.30 (s,
2H), 6.93e6.97 (m, 2H), 6.99 (d, J ¼ 7.7 Hz, 2H), 7.09 (t, J ¼ 7.4 Hz,
1H), 7.28e7.36 ppm (m, 4H); 13C NMR (CDCl3):
31.2, 38.3, 39.4,
methanol/conc. NH3, 9:1:0.01) yielded 18 [59] as a solid (238 mg,
51%); mp 96e97 ꢀC; 1H NMR (CDCl3):
2.36 (s broad, 3H), 2.83 (dd,
J ¼ 12.7 Hz and 8.1 Hz, 1H), 3.03 (dd, J ¼ 12.7 Hz and 3.8 Hz, 1H),
4.65 (dd, J ¼ 8.0 Hz and 3.8 Hz, 1H), 6.91e7.05 (m, 4H), 7.10 (t,
J ¼ 7.4 Hz, 1H), 7.28e7.39 ppm (m, 4H); HRMS (APCI) m/z [M þ H]þ
calculated: 230.1176, found: 230.1235.
d
d
d
119.0, 119.2, 123.5, 129.9, 130.4, 130.8, 147.9, 156.5, 157.2, 165.4 ppm;
MS (EI, 70 eV) m/z (%): 323 (8) Mþ, 295 (100).
4.1.5. 5-(4-Phenoxyphenyl)tetrazole (14)
4.1.10. 5-(4-Phenoxyphenyl)oxazolidin-2-one (19)
A mixture of 4-phenoxybenzonitrile (231 mg, 1.18 mmol), tri-
A solution of 18 (222 mg, 0.97 mmol) in dry CH2Cl2 (5 mL) was
methylsilyl azide (238
mL, 1.77 mmol) and tetrabutylammonium
treated at 0 ꢀC with dry pyridine (1 mL) followed bycareful dropwise
fluoride hydrate (156 mg, 0.60 mmol) was heated at 120 ꢀC for 9 h.
The obtained yellowish, high-viscous liquid was cooled to room
temperature and purified by silica gel chromatography (hexane/
ethyl acetate, 8:2; ethyl acetate þ 0.1% formic acid) to give 14 [58] as
addition of trichloromethyl chloroformate (117 mL, 0.97 mmol). After
the reaction had slowed down, the ice bath was removed and the
mixture was allowed to stir at room temperature for 20 h. The re-
action mixture was diluted with CH2Cl2 (10 mL), poured into water,
and extracted three times with CH2Cl2. The combined organic layers
were washed with water, dried (Na2SO4) and evaporated under
reduced pressure. Purification on silica gel (CH2Cl2/methanol, 98:2)
gave 19 as a solid (189 mg, 76%); mp 117e118 ꢀC; 1H NMR (CDCl3):
crystals (250 mg, 89%); mp 220 ꢀC; 1H NMR (DMSO-d6):
d 7.10e7.16
(m, 2H), 7.16e7.21 (m, 2H), 7.23 (t, J ¼ 7.4 Hz,1H), 7.41e7.50 (m, 2H),
8.01e8.07 ppm (m, 2H); MS (EI, 70 eV) m/z (%): 238 (9) Mþ, 210
(100); HRMS (ESI) m/z [M þ H]þ calculated: 239.0927, found:
239.0922.
d
3.56 (t, J ¼ 8.2 Hz, 1H), 3.97 (t, J ¼ 8.6 Hz, 1H), 5.54 (s, 1H), 5.61 (t,
J ¼ 8.2 Hz,1H), 6.96e7.08 (m, 4H), 7.14 (td, J ¼ 7.6 Hz and 1.0 Hz,1H),
7.30e7.40 ppm (m, 4H); 13C NMR (CDCl3):
48.4, 77.8, 119.0, 119.4,
4.1.6. N,N-Dimethyl-5-(4-phenoxyphenyl)-1H-tetrazole-1-
carboxamide (15)
d
123.9, 127.6, 130.0, 132.9, 156.7, 158.2, 159.6 ppm; HRMS (APCI) m/z
[M þ H]þ calculated: 256.0968, found: 256.1015.
Compound 14 (120 mg, 0.50 mmol) was suspended in dry ace-
tonitrile (2 mL) and dissolved by addition of triethylamine (138
mL,
1.00 mmol). N,N-Dimethylcarbamoyl chloride (69 L, 0.75 mmol)
m
4.1.11. N-Cyclohexyl-2-oxo-5-(4-phenoxyphenyl)oxazolidine-3-
carboxamide (20)
was added dropwise at 0 ꢀC and the mixture was stirred at that
temperature for 17 h. Then silica gel was added and the solvent was
evaporated under reduced pressure. The residue was transferred to
the top of a silica gel column and eluted with hexane/ethyl acetate
(8:2) to afford 15 as an oily substance (9 mg, 6%); 1H NMR (CDCl3):
To a solution of 19 (102 mg, 0.40 mmol) in dry toluene (10 mL)
was added dropwise cyclohexyl isocyanate (51
followed by tetrakis(triphenylphosphine)palladium (Pd(PPh3)4),
(5 mg, 0.004 mmol). After h, further Pd(PPh3)4 (25 mg,
mL, 0.42 mmol)
1
d
2.93 (s, 3H), 3.27 (s, 3H), 7.06e7.12 (m, 4H), 7.21 (t, J ¼ 7.4 Hz, 1H),
0.02 mmol) was added and the reaction was continued overnight.
Then the solvent was evaporated and the residue purified by col-
umn chromatography on silica gel (hexane/ethyl acetate, 19:1, 9:1)
to give 20 as an oily substance (92 mg, 60%). 1H NMR (CDCl3):
7.37e7.44 (m, 2H), 7.83e7.89 ppm (m, 2H); 13C NMR (CDCl3):
d
37.8,
39.0, 117.0, 118.3, 120.4, 124.9, 130.3, 130.5, 148.7, 153.7, 155.4,
161.3 ppm; MS (EI, 70 eV) m/z (%): 281 (100), 237 (51).
d
1.19e1.45 (m, 4H), 1.59e1.65 (m, 2H), 1.69e1.77 (m, 2H), 1.94 (d,
4.1.7. N,N-Dimethyl-5-(4-phenoxyphenyl)-2H-tetrazole-2-
carboxamide (16)
J ¼ 12.3 Hz, 2H), 3.66e3.78 (m, 1H), 3.96 (dd, J ¼ 10.6 Hz and 7.9 Hz,
1H), 4.41 (dd, J ¼ 10.6 Hz and 8.9 Hz, 1H), 5.54 (t, J ¼ 8.3 Hz, 1H),
7.00e7.06 (m, 4H), 7.15 (tt, J ¼ 7.8 Hz and 1.1 Hz, 1H), 7.30e7.40 (m,
Compound 16 was afforded as second product during silica gel
chromatography of 15 as an oily substance (10 mg, 7%). 1H NMR
4H), 7.74 ppm (d, J ¼ 7.9 Hz, 1H); 13C NMR (CDCl3):
d 24.8, 25.6, 33.1,
(CDCl3):
d
3.18 (s, 3H), 3.30 (s, 3H), 7.06e7.13 (m, 4H), 7.18 (t,
49.2, 49.8, 75.2, 119.0, 119.5, 124.1, 127.8, 130.1, 131.4, 150.7, 155.3,
156.6, 158.6 ppm; HRMS (APCI) m/z [M þ H]þ calculated: 381.1809,
found: 381.1844.
J ¼ 7.4 Hz, 1H), 7.36e7.42 (m, 2H), 8.16e8.22 ppm (m, 2H); 13C NMR
(CDCl3):
d 38.4, 39.4, 118.6, 119.9, 121.1, 124.4, 129.2, 130.1, 148.1,
156.2, 160.2, 164.3 ppm; MS (EI, 70 eV) m/z (%): 281 (100), 197 (42);
HRMS (ESI) m/z [M þ H]þ calculated: 310.1299, found: 310.1299, m/
z [M ꢃ N2 þ H]þ calculated: 282.1237, found: 282.1244.
4.1.12. N-Hydroxy-4-phenoxybenzenecarboximidamide (21)
A solution of hydroxylamine hydrochloride (97 mg, 1.39 mmol)
in methanol (5 mL) was added to an aqueous solution of sodium
hydroxide (53 mg, 1.33 mmol in 5 mL). This mixture was added to
a solution of 4-phenoxybenzonitrile (262 mg, 1.34 mmol) in
methanol (5 mL) and the resulting mixture was heated under
reflux. After 6 h, the same amount of hydroxylamine hydrochloride/
sodium hydroxide solution was added again and heating was
continued for further 5 h. While cooling to room temperature, silica
gel was added. Then the solvent was removed under reduced
pressure and the residue was transferred to the top of a silica gel
column. Elution with hexane/ethyl acetate (7:3) gave 21 [60] as
4.1.8. 2-(4-Phenoxyphenyl)-2-[(trimethylsilyl)oxy]acetonitrile (17)
A solution of 4-phenoxybenzaldehyde (347
CH2Cl2 (8 mL) was treated with trimethylsilyl cyanide (256
2.02 mmol) and triethylamine (28
L, 0.2 mmol) at 0 ꢀC. The mix-
mL, 2.02 mmol) in
mL,
m
ture was stirred at this temperature for 2 h and subsequently
allowed to warm to room temperature overnight. Then the solvent
was removed under reduced pressure without heating to yield 17
[58] as an oil. This was employed without further purification.
4.1.9. 2-Amino-1-(4-phenoxyphenyl)ethanol (18)
a solid (248 mg, 81%); mp 103e104 ꢀC; 1H NMR (CDCl3):
d 3.19 (s
A solution of the residue obtained in the synthesis of 17 in dry
THF (2.5 mL) was added dropwise under a nitrogen atmosphere at
0 ꢀC to a solution of lithium aluminium hydride in THF (2 M, 4 mL).
After stirring at the same temperature for 1 h and at room tem-
perature for 2 h, the mixture was carefully poured into ice water,
alkalized with aqueous sodium hydroxide solution (10%) and
extracted three times with diethyl ether. The combined organic
layers were dried (Na2SO4) and evaporated under reduced pres-
sure. Chromatography on silica gel (ethyl acetate; ethyl acetate/
broad, 1H), 4.99 (s broad, 2H), 6.97e7.06 (m, 4H), 7.15 (t, J ¼ 7.4 Hz,
1H), 7.36 (dd, J ¼ 8.5 Hz and 7.5 Hz, 2H), 7.59 ppm (d, J ¼ 8.8 Hz, 2H);
MS (EI, 70 eV) m/z (%): 228 (100) Mþ, 211 (91).
4.1.13. 3-(4-Phenoxyphenyl)-4,5-dihydro-1,2,4-oxadiazol-5-one (22)
A solution of 21 (118 mg, 0.52 mmol) in dry CH2Cl2 (5 mL) was
treated with triethylamine (72
1 h. Then phenyl chloroformate (65
stirring was continued at 0 ꢀC for 1 h. The reaction mixture was
m
L, 0.52 mmol) and stirred at 0 ꢀC for
mL, 0.52 mmol) was added and

72
A. Holtfrerich et al. / European Journal of Medicinal Chemistry 63 (2013) 64e75
washed once with a saturated sodium carbonate solution and then
three times with water. The combined organic layers were dried
(Na2SO4) and concentrated under reduced pressure. Toluene was
added to the residue and the mixture was stirred under reflux for
4 h. After nearly complete evaporation of the solvent, the residue
was purified by silica gel chromatography (hexane/ethyl acetate/
formic acid 8:2:0.01) to give 22 as a solid (86 mg, 65%); mp 218 ꢀC,
J ¼ 7.0 Hz, 2H), 7.06e7.14 (m, 4H), 7.15e7.22 (m, 1H), 7.35e7.44 (m,
2H), 8.16e8.22 ppm (m, 2H); 13C NMR (CDCl3):
12.5, 14.2, 43.6,
44.4, 118.6, 119.9, 121.1, 124.4, 129.1, 130.1, 147.6, 156.2, 160.2,
164.2 ppm; HRMS (APCI) m/z [M ꢃ N2 þ H]þ calculated: 310.1556,
found: 310.1558.
d
4.1.18. N,N-Diisopropyl-5-(4-phenoxyphenyl)-2H-tetrazole-2-
carboxamide (60)
1H NMR (CDCl3):
d
4.87 (s broad, 1H), 6.97e7.07 (m, 4H), 7.15 (t,
J ¼ 7.4 Hz, 1H), 7.32e7.40 (m, 2H), 7.57e7.62 ppm (m, 2H); MS (EI,
A
mixture of 5-(4-phenoxyphenyl)tetrazole (14) (88 mg,
70 eV) m/z (%): 254 (100) Mþ, 211 (32).
0.37 mmol), triethylamine (104 L, 0.74 mmol) and N,N-diisopro-
m
pylcarbamoyl chloride (91 mg, 0.56 mmol) in dry acetonitrile
(8 mL) was stirred at room temperature for 39 h. After silica gel
chromatography (hexane/ethyl acetate, 19:1) 60 was obtained as an
4.1.14. General procedure for the synthesis of the substituted
phenyltetrazoles 24e39 and 57aee
A mixture of the corresponding 4-substituted benzonitrile
(1 equiv, 0.43e1.34 mmol), trimethylsilyl azide (1.5 equiv) and
tetrabutylammonium fluoride hydrate (0.5 equiv) was heated at
120 ꢀC for 1.5 he9 h. The reaction mixture was dissolved in a small
amount of warm toluene and chromatographed on silica gel eluting
with hexane/ethyl acetate (9:1e1:1), ethyl acetate and ethyl
acetate/formic acid (100:0.2) to obtain the desired product as
a solid. Characterization of the compounds see Supplementary
material.
oil (98 mg, 72%). 1H NMR (CDCl3):
d
1.14e1.72 (m,12H), 3.73 (m, 2H),
7.05e7.15 (m, 4H), 7.15e7.21 (m, 1H), 7.36e7.45 (m, 2H), 8.14e
8.24 ppm (m, 2H); 13C NMR (CDCl3):
20.04, 47.99, 52.07, 118.63,
d
119.90, 121.35, 124.30, 129.08, 130.11, 145.96, 156.23, 160.04,
164.16 ppm; HRMS (APCI) m/z [M ꢃ N2 þ H]þ calculated: 338.1869,
found: 338.1900.
4.2. Biochemical evaluation of the target compounds
4.2.1. Reagents and materials
4.1.15. General procedure for the synthesis of the phenyltetrazole-
carboxamides 40e55 and 58aee
Brains of female Sprague Dawley rats (HarlaneWinkelmann);
livers of female Wistar rats (Pharmacological section of the Institute
of Pharmaceutical and Medicinal Chemistry, University of Münster);
dimethyl sulfoxide (DMSO) (Fluka); potassium dihydrogenphos-
phate, potassium hydrogenphosphate, EDTA (VWR); acetonitrile
HPLC-grade (Sigma); monoacylglycerol lipase (human recombinant)
The corresponding phenyltetrazole (1 equiv, 0.32e0.93 mmol)
was suspended in dry acetonitrile (9 mL/mmol) and dissolved by
addition of triethylamine (2 equiv). Then N,N-dimethylcarbamoyl
chloride (1.5 equiv) was added dropwise at 0 ꢀC and the solution
was stirred at the same temperature for 0.5e2 h, at room tem-
perature for 1e24 h, and at 70 ꢀC for 1e4.5 h to afford a mixture of
the corresponding 2H-tetrazole-2-carboxamide and 1H-tetrazole-
1-carboxamide. After cooling the reaction mixture, silica gel was
added and the solvent was evaporated under reduced pressure. The
residue was transferred to the top of a silica gel column and eluted
with hexane/ethyl acetate (9:1e1:1). In all cases only the 2H-tet-
razole-2-carboxamide was separated and characterized.
(10
mg/50 m
L), cyclohexylcarbamic acid 30-carbamoylbiphenyl-3-
ylester (URB597) (1), 1-oxazolo[4,5-b]pyridin-2-yl-6-phenylhexan-
1-one (PHOP) (2), 4-nitrophenyl 4-[bis(1,3-benzodioxol-5-yl)
hydroxymethyl]piperidine-1-carboxylate (JZL184) (3), benzyl (4-(5-
methoxy-2-oxo-1,3,4-oxadiazol-3(2H)-yl)-2-methylphenyl)carbam-
ate (CAY 10499) (4), 1-arachidonylpyrrole-2,5-dione (NAM) (5)
(Cayman Chemical); the reference compounds 5, 7 and 8 were
synthesized according to published procedures [17,20].
Data of the compounds see Supplementary material.
4.2.2. Inhibition of fatty acid amide hydrolase (FAAH)
Inhibition of FAAH by test compounds was measured with FAAH
from rat brain microsomes as described [29,31] with some
modifications.
4.1.16. General procedure for the preparation of 4-(3-acyl-2,5-
dimethylpyrrol-1-yl)benzonitriles 56aee
A solution of acyl chloride (1.2 equiv) in dry 1,2-dichloroethane
(10 mL) was treated carefully with SnCl4 (1 equiv) at 0 ꢀC, and the
mixture was stirred for 10 min at this temperature. Then a solution
of 4-(2,5-dimethylpyrrol-1-yl)benzonitrile (1 equiv, 1.54 mmole
2.24 mmol) in dry 1,2-dichloroethane (10 mL) was added, and
stirring was continued at this temperature for 30 mine1 h and at
room temperature for 3.5e20 h. The reaction was quenched by
addition of water, and the aqueous phase was extracted exhaus-
tively with diethyl ether. The combined organic layers were washed
with water, dried (Na2SO4) and evaporated under reduced pressure.
The residue was purified by column chromatography on silica gel
(hexane/ethyl acetate,19:1e8:2) to give the appropriate acylpyrrole
derivative as a solid. Characterization of the compounds see
Supplementary material.
4.2.2.1. Isolation of rat brain microsomes. Two rat brains (female
Sprague Dawley rats, Harlan-Winkelmann) were homogenized for
3 min under ice-cooling with a fivefold volume potassium phos-
phate buffer (0.1 M, pH 7.4 at 20 ꢀC) containing EDTA (1 mM) using
a Potter-Elvehjem homogenizer at 1000e1200 rpm. The homoge-
nate was centrifuged at 1000 g at 4 ꢀC for 10 min and the resulting
supernatant was centrifuged again at 10,000 g at 4 ꢀC for 30 min.
Finally, the supernatant was centrifuged at 40,000 g at 4 ꢀC for
60 min. The obtained supernatant was discharged. The pellet was
resuspended in 2 mL of ice-cold potassium phosphate buffer (0.1 M,
pH 7.4) containing EDTA (1 mM) and stored in 50
mL aliquots
at ꢃ80 ꢀC. Immediately before each incubation in dependence of
the activity of FAAH in the preparation, an aliquot of about 50 mL
4.1.17. N,N-Diethyl-5-(4-phenoxyphenyl)-2H-tetrazole-2-
was diluted with about 200e400 mL potassium phosphate buffer
carboxamide (59)
(0.1 M, pH 7.4) containing EDTA (1 mM) and homogenized
shortly (2 ꢂ 5 s) at 0 ꢀC with a Branson sonifier B15. Under the
conditions applied, in the controls the peak area of the enzyme
product 4-pyren-1-ylbutanoic acid amounted to about 70% of the
peak area of the internal standard 6-pyren-1-ylhexanoic acid.
A
mixture of 5-(4-phenoxyphenyl)tetrazole (14) (62 mg,
0.26 mmol), triethylamine (73 L, 0.52 mmol) and N,N-dieth-
m
ylcarbamoyl chloride (50 mL, 0.39 mmol) in dry acetonitrile (5 mL)
was stirred at room temperature for 18 h and heated at 70 ꢀC for
1.5 h. After silica gel chromatography (hexane/ethyl acetate, 9:1) 59
[61] was obtained as an oil (24 mg, 27%). 1H NMR (CDCl3):
d
1.31 (t,
4.2.2.2. Incubation procedure. The substrate N-(2-hydroxyethyl)-4-
J ¼ 6.9 Hz, 3H),1.37 (t, J ¼ 6.9 Hz, 3H), 3.40 (q, J ¼ 7.0 Hz, 2H), 3.64 (q,
pyren-1-ylbutanamide [31] was dissolved in methanol (2.5 mg/

A. Holtfrerich et al. / European Journal of Medicinal Chemistry 63 (2013) 64e75
73
mL). An aliquot of this solution was thoroughly dried under
a stream of nitrogen. The residue was resuspended by intense
vortexing and sonication in a sonication bath in such an amount of
a solution of 0.2% Triton X-100 in phosphate buffered saline (pre-
pared from tablets from SigmaeAldrich, P4417: one tablet dissolved
in 200 mL of deionized water yields 0.01 M phosphate buffer,
0.0027 M potassium chloride and 0.137 M sodium chloride, pH 7.4,
at 25 ꢀC) containing EDTA (1 mM), so that the concentration of the
1-ylbutanoate [30] in DMSO (5 mM), the mixture was pre-
incubated at 37 ꢀC for exactly 15 min. Then the enzymatic reac-
tion was started by adding of 5
for 45 min at 37 ꢀC. The final incubation volume of 100
100 M of the substrate and 10 ng MAGL. The enzyme reaction was
terminated by the addition of 200 L acetonitrile/methanol (1:1, v/
v) spiked with the internal standard 6-pyren-1-ylhexanoic acid
(100 ng/200 L). After cooling in an ice bath for 10 min, the samples
mL enzyme solution and continued
mL contained
m
m
m
substrate was 114
L of a DMSO solution of inhibitor or to 2
the controls. The mixture was pre-incubated for 10 min at 37 ꢀC.
Then the enzymatic reaction was started by adding 10 L rat brain
microsome preparation and continued for 45 min at 37 ꢀC. The final
incubation volume of 100 L contained a pyrenylbutanamide sub-
strate concentration of 100 M. The enzyme reaction was termi-
nated by the addition of 200 L acetonitrile/methanol (1:1, v/v),
which contained the internal standard 6-pyren-1-ylhexanoic acid
mM. 88
mL of the obtained mixture were added to
were centrifuged at 2000 g and 4 ꢀC for 5 min and stored at ꢃ20 ꢀC
until HPLC analysis of the enzyme product 4-pyren-1-ylbutanoic
acid, which was carried out as described in 4.2.2.3. The injection
volume was 5 mL. Blank incubations in the absence of the enzyme
were carried out in parallel.
2
m
mL of DMSO in case of
m
m
m
4.2.4. Inhibition of cytosolic phospholipase A2a (cPLA2a)
Inhibition of cPLA2a was measured according to a recently
published procedure [63].
m
(0.025
mg/200 mL). After cooling in an ice bath for 10 min, the
samples were centrifuged at 2000 g at 4 ꢀC for 5 min. Blank in-
cubations in the absence of the enzyme were carried out in parallel.
4.2.5. Metabolic stability in rat liver S9 fractions
4.2.5.1. Preparation of rat liver S9 fractions. Immediately after the
death of the animals (female Wistar rats) the liver was withdrawn
and washed several times with ice cold potassium chloride solution
(1.15%, m/v) to remove remaining blood. Then the cold liver was
reduced to small pieces with a scalpel. After addition of ice cold
0.1 M potassium phosphate buffer (83 mM K2HPO4 and 17 mM
KH2PO4 containing 0.5 mM EDTA and adjusted to pH 7.4 with conc.
H3PO4 at 20 ꢀC; amount: 2 mL/g liver) the liver was homogenized in
an ice-cooled Potter-Elvehjem homogenizer. After addition of fur-
ther 0.1 M potassium phosphate buffer (4 mL/g liver), the homog-
enate was centrifuged at 9000 g and 4 ꢀC for 20 min. The
supernatant was stored at ꢃ80 ꢀC until used. For these preparations
a protein concentration of 8.0 mg/mL was determined applying the
method of Bradford with bovine serum albumin (BSA) as standard.
4.2.2.3. HPLC analysis. The HPLC system consisted of a Bischoff
HPLC-compact pump model 2250, a Midas Cool Autosampler,
a Bischoff Chromatography column oven and a Waters fluorescence
detector model 2475. Data analysis was carried out using
a McDacq32 control chromatography software from Bischoff. Sep-
aration was achieved on a Nucleosil 100 C18 analytical column
(3 mm inside diameter ꢂ 125 mm, particle size 3
Nagel) protected with a Phenomenex C18 guard column (3 mm
inside diameter ꢂ 4 mm). 50 L of each sample were injected into
mm) (Macherey &
m
the HPLC system. The mobile phase consisted of methanol/water/
trifluoroacetic acid (80:20:0.1, v/v/v). Injector temperature was
maintained at 10 ꢀC, oven temperature at 20 ꢀC. The flow rate was
0.4 mL/min. The fluorescence detector was set at an excitation
wavelength of 340 nm and emission was monitored at 380 nm. To
eliminate the large substrate peak in the chromatogram occurring
between 3.0 min and 6.3 min and to protect the photomultiplier of
the detector from damage by intense light, from 3.0 to 6.3 min
excitation wavelength was set to 400 nm and emission wavelength
to 600 nm. After 7.0 min an auto zero was carried out.
4.2.5.2. Incubation procedure. To a mixture of 125
mL S9-fraction
and 373 L of a NADPH-solution (11.2 mg NADPHeNa4 in a mix-
m
ture of 9.5 mL of potassium phosphate buffer (0.1 M, pH 7.4), which
contained 0.5 mM EDTA, and 0.5 mL of a 0.1 M magnesium chloride
solution) was added 2 mL of a DMSO solution (5 mM) of the test
compound (¼parent compound). The final protein concentration in
For calculation of enzyme inhibition the peak ratio of enzyme
product and internal standard obtained in presence of a test com-
pound was compared with the mean level of this peak ratio deter-
the sample was 2 mg/mL, the concentration of NADPH was 1 mM
and the concentration of the parent compound was 20 mM. After
incubation at 37 ꢀC for 30 min, 1.0 mL acetonitrile was added. The
mixture was vortexed and allowed to stand in an ice bath for
15 min. After vigorous vortexing, the mixture was centrifuged at
2000 g and 4 ꢀC for 5 min. The supernatant of the metabolism
sample was separated and subjected to HPLC.
mined in absence of test compounds (¼control tests, n ¼ 4). The IC50
-
values were calculated with the aid of Probit transformation.
The IC50 values of the FAAH reference inhibitors 1 (URB597) and
2 (PHOB) vary from the data published recently by us [29,31,62] due
to the slightly different reaction conditions. While in the first
publication the enzyme reaction was performed in Tris buffer for
60 min [31] in a second publication an incubation time in Tris buffer
of only 45 min was used [62]. In a following publication a PBS-
buffer was used applying an incubation time of 60 min [29]. In
this publication the reaction was carried out also in PBS-buffer,
however, the incubation time was again reduced to 45 min.
A test substance blank was prepared by adding 2
solution (5 mM) of the test compound to a mixture of 125
fraction, 373 L of a 19:1 (v/v) mixture of potassium phosphate
mL of a DMSO
mL S9-
m
buffer (0.1 M, pH 7.4), which contained 0.5 mM EDTA, and a 0.1 M
magnesium chloride solution, and 1.0 mL of acetonitrile. This mix-
ture was allowed to stand at room temperature for 30 min and in an
ice bath for 15 min before centrifugation at 2000 g and 4 ꢀC for 5 min.
The supernatant was separated and subjected to HPLC.
4.2.3. Inhibition of monoacylglycerol lipase (MAGL)
Inhibition of MAGL by test compounds was measured with
human recombinant enzyme [30]. Thus, an aliquot of a solution of
To ensure that the recovery of the parent compound is almost
quantitatively in presence of S9 fractions, control samples con-
sisting of 2
125 L potassium phosphate buffer (0.1 M, pH 7.4), which con-
tained 0.5 mM EDTA, 373 L of a 19:1 (v/v) mixture of the latter
mL of a DMSO solution (5 mM) of the test compound,
human recombinant MAGL (10
HEPES buffer (50 mM, pH 7.4 pH at 20 ꢀC) containing 100 mM NaCl,
5 mM MgCl2, 0.1% Triton X-100 and 25% glycerol. To 2 L of a DMSO
solution of inhibitor, or to 2 L of DMSO in case of the controls, was
added 91
L of HEPES buffer (50 mM, pH 7.0 at 20 ꢀC) containing
1 mM EDTA and 0.2% (m/v) Triton X-100. After addition of 2 L of
a solution of the substrate 1,3-dihydroxypropan-1-yl 4-pyren-
mg/50
mL) was diluted 1:100 with
m
m
m
solution and a 0.1 M magnesium chloride solution, and 1.0 mL
acetonitrile were prepared and also subjected to HPLC.
m
m
m
4.2.5.3. HPLC analysis. The HPLC system consisted of a Waters
HPLC-pump model 515, a Waters autosampler model 717 plus,

74
A. Holtfrerich et al. / European Journal of Medicinal Chemistry 63 (2013) 64e75
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.01.050.
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