Biaryl tetrazolyl ureas as inhibitors of endocannabinoid metabolism: Modulation at the N-portion and distal phenyl ring

Article abstract of DOI:10.1016/j.ejmech.2013.02.005

In the present study, we have further extended the structure-activity relationships for the tetrazolyl ureas class of compounds as potential FAAH and/or MAGL inhibitors, by replacing the dimethylamino group of the parent compounds 1 and 2 with bulkier groups or by introducing on the distal phenyl ring of 1 and 2 a selected set of substituents. Some of the new compounds (16, 20, 21, 25, and 28) inhibited FAAH potently (IC₅₀ = 3.0-9.7 nM) and selectively (39- to more than 141-fold) over MAGL, while tetrazole 27 turned out to be a promising dual FAAH-MAGL inhibitor of potential therapeutic use. Covalent docking studies on FAAH indicated that the binding modes of tetrazoles 1-32 did not display a unique pattern. The ability of tetrazoles 1-32 to act as TRPV1 and TRPA1 modulators was also investigated.

Full text of DOI:10.1016/j.ejmech.2013.02.005

European Journal of Medicinal Chemistry 63 (2013) 118e132
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Biaryl tetrazolyl ureas as inhibitors of endocannabinoid metabolism:
Modulation at the N-portion and distal phenyl ring
,
a
b
c
Giorgio Ortar a , Enrico Morera , Luciano De Petrocellis , Alessia Ligresti ,
*
Aniello Schiano Moriello b, Ludovica Morera a, Marianna Nalli a, Rino Ragno a
,
,
d
Adele Pirolli a d, Vincenzo Di Marzo c
,
a Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, Piazzale Aldo Moro 5, 00185 Roma, Italy
b Endocannabinoid Research Group, Istituto di Cibernetica, Consiglio Nazionale delle Ricerche, via dei Campi Flegrei 34, 80078 Pozzuoli, Napoli, Italy
c Endocannabinoid Research Group, Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, via dei Campi Flegrei 34,
80078 Pozzuoli, Napoli, Italy
d Rome Center for Molecular Design (RCMD), Sapienza Università di Roma, Piazzale Aldo Moro 5, 00185 Roma, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present study, we have further extended the structureeactivity relationships for the tetrazolyl
ureas class of compounds as potential FAAH and/or MAGL inhibitors, by replacing the dimethylamino
group of the parent compounds 1 and 2 with bulkier groups or by introducing on the distal phenyl ring of
1 and 2 a selected set of substituents. Some of the new compounds (16, 20, 21, 25, and 28) inhibited FAAH
potently (IC50 ¼ 3.0e9.7 nM) and selectively (39- to more than 141-fold) over MAGL, while tetrazole 27
turned out to be a promising dual FAAHeMAGL inhibitor of potential therapeutic use. Covalent docking
studies on FAAH indicated that the binding modes of tetrazoles 1e32 did not display a unique pattern.
The ability of tetrazoles 1e32 to act as TRPV1 and TRPA1 modulators was also investigated.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Received 6 September 2012
Received in revised form
21 December 2012
Accepted 7 February 2013
Available online 15 February 2013
Keywords:
Endocannabinoids
Fatty acid amide hydrolase (FAAH)
Monoacylglycerol lipase (MAGL)
TRPV1 channel
TRPA1 channel
Biaryl tetrazolyl ureas
1. Introduction
accumulation, and hydrolysis of AEA has generated a pluriannual
intense debate [3]. In this respect, Moore et al. described in 2005 a
Endogenous ligands of cannabinoid receptors (endocannabi-
noids) are involved as lipid messengers in a wide array of physio-
logical and pathological conditions and the modulation of their
levels appears to be of remarkable therapeutic interest in a variety
of human disorders, ranging from neurological, affective and
neuropsychiatric disorders and various types of chronic pain to
gastrointestinal and hepatic diseases, allergic and inflammatory
disorders, cancer and osteoporosis [1].
Among different metabolic pathways that can control the extent and
duration of action of the two main endocannabinoids, N-arach-
idonoylethanolamine (anandamide, AEA) and 2-arachidonoylglycerol
(2-AG), fatty acid amide hydrolase (FAAH) and monoacylglycerol
lipase (MAGL) have definitely emerged as key players [2].
potent, competitive carbamoyl tetrazole inhibitor of AEA cellular
uptake, LY2318912 (Fig. 1) [4], but the same group subsequently
reported that LY2183240 (1), the parent compound of LY2318912,
inhibited enzyme activity in purified FAAH preparations [5], and
later Alexander and Cravatt demonstrated that 1 (actually a mixture
of 1 and its 2,5-regioisomer 2) [6] was a potent, covalent inhibitor
not only of FAAH but also of several other serine hydrolases,
including MAGL [7]. Mass spectral studies of the FAAHeLY2183240
adduct verified that FAAH inhibition was due to covalent modifi-
cation of the catalytic Ser241 [7], analogous to the mechanism of
action of carbamate-based inhibitors [8].
In order to gain further insights into the action of the carbamoyl
tetrazole class of compounds, we previously investigated a first
series of 1,5 and 2,5-disubstituted carbamoyl tetrazoles, including
LY2318912, 1, and 2, and found that five of the 18 compounds,
evaluated for their activity on AEA uptake, FAAH, MAGL, and DAGL,
were active on AEA uptake, although all potently inhibited FAAH
and some of them MAGL and DAGL as well [6]. In particular, tet-
razole 2 turned out to be a more potent MAGL inhibitor than
In contrast, the existence of a carrier-mediated transport of AEA
across the membrane preceding intracellular trafficking,
* Corresponding author. Tel.: þ39 6 49913612; fax: þ39 6 49913133.
E-mail address: giorgio.ortar@uniroma1.it (G. Ortar).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.02.005

G. Ortar et al. / European Journal of Medicinal Chemistry 63 (2013) 118e132
119
Fig. 1. Structures of compounds LY2318912, 1, and 2.
regioisomer 1, a result confirmed shortly afterwards by Makriyan-
nis et al. who demonstrated in addition that the inhibition involved
a covalent interaction with Ser129 [9]. With the aim of ‘designing
out’ cross activities of the first tetrazolyl urea series and attain a
good selectivity towards the AEA uptake process, we subsequently
investigated the replacement of the N,N-dimethylaminocarbonyl
portion with analogous groups devoid of inherent carbamylating
activity and found that this modification resulted in the identifi-
cation of some tetrazole-based selective AEA uptake inhibitors [10].
In the present study, we have further extended the structuree
activity relationships (SARs) for the tetrazolyl ureas class of com-
pounds as potential FAAH and/or MAGL inhibitors, by replacing the
dimethylamino group of the parent compounds 1 and 2 with
bulkier groups (compounds 3e10, Fig. 2) or by introducing on the
distal phenyl ring of 1 and 2 a selected set of substituents (com-
pounds 11e32, Fig. 2). The distal ring was selected in order to
minimize electronic effects of substituents upon the urea group and
to correlate therefore potencies of compounds directly with non-
covalent binding interactions.
In view of the aforementioned activity-based protein profiling
(ABPP) of the reference compound 1, revealing that it is a nonse-
lective FAAH inhibitor acting on several other serine hydrolases [7],
we have no reason to believe that the analogues described in our
study behave in a different way, i.e. they represent a ‘chemotype
with potentially excessive protein reactivity’ [7], since they were
not designed to behave otherwise. Therefore, we did not perform
on compounds 3e32 any ABPP assay and limited ourselves to
investigating whether some of them inhibited FAAH selectively
over MAGL (or vice versa) or behaved as dual FAAH/MAGL
inhibitors.
The ability of tetrazoles 1e32 to act as modulators of the tran-
sient receptor potential (TRP) cation channels of vanilloid type-1
(TRPV1) [11] and ankyrin type-1 (TRPA1) [11a,b,d,12] was also
investigated. A series of experiments carried out in the late 1990’s
has in fact established important connections between the function
of endocannabinoids and that of TRP channels [11a]. Since then, the
overlap between the ligand recognition properties of some TRP
channels and proteins of the endocannabinoid system has been
actively explored. In particular, TRPV1 was found to be activated by
AEA and can therefore be activated indirectly also following FAAH
inhibition and subsequent elevation of AEA levels [13], whereas
TRPA1 was reported to be activated by the carbamate URB597, a
well known covalent FAAH inhibitor [14].
silica gel chromatography, the less polar 2,5-isomer eluting prior
than 1,5-counterpart (Scheme 1) [15]. Carbamoyl chlorides were
obtained from the corresponding amines by treatment with tri-
phosgene. Non commercially available amines N-methyl-4-
phenylbutan-1-amine and N-methyl-1-(naphtalen-2-yl)methan-
amine were prepared via reductive alkylation of 4-phenylbutan-1-
amine via the ethyl carbamate [16] and by reduction of the corre-
sponding imine, in turn obtained by reaction of 2-naphtaldehyde
with methylamine [17], respectively.
The second group of carbamoyl tetrazoles (compounds 11e32)
was synthesized starting from the appropriate monosubstituted
4-biphenylacetonitriles 46e55 (Scheme 2). For the preparation of
these precursors, a palladium-catalyzed SuzukieMiyaura cross-
coupling reaction between 4-iodophenylacetonitrile (35) and the
appropriate phenylboronic acids 36e45 was exploited. The pro-
tection of phenolic OH group of nitriles 50e52 with benzyl bromide
furnished nitriles 56e58. Nitrile 60 was prepared from nitrile 47 via
alkaline hydrolysis and conversion HOBt/EDC mediated of the
resulting acid 59 into the primary amide 60. Tetrazoles 11e32
were eventually obtained by refluxing monosubstituted 4-
biphenylacetonitriles 46e49, 53e58, 60 with tri-n-butyltin azide
[18], followed by an acidic hydrolysis step to remove the tin group
from the tetrazole ring, acylation of the resulting tetrazoles 61e71
with N,N-dimethylcarbamoyl chloride, and, in the case of tetrazoles
15e17 and 26e28, hydrogenolysis of the mixtures of tetrazoles
72 þ 73, 74 þ 75, and 76 þ 77. All the mixtures of the 1,5 (com-
pounds 11e21) and 2,5-regioisomers (compounds 22e32) could be
separated by silica gel chromatography, the less polar 2,5-isomer
eluting, as in the case of tetrazoles 3e10, prior than 1,5-isomer.
The structure of the regioisomeric tetrazoles 3e32 was assigned on
the basis of their 13C NMR spectra. 13C NMR spectra of disubstituted
tetrazoles, in particular those of our two previous papers [6,10],
have in fact shown that the CN4 carbon atom of 1,5-disubstituted
tetrazoles is more shielded than the corresponding atom of the
2,5-disubstituted tetrazoles.
2.2. Biological evaluation
The effect of tetrazoles 3e32 on: (a) [14C]AEA hydrolysis by rat
brain membranes (which express FAAH as the only AEA hydrolyz-
ing enzyme); (b) [3H]2-AG hydrolysis by COS-7 cell cytosolic frac-
tions (which express MAGL); (c) intracellular Ca2þ elevation in
HEK293 cells stably transfected with either the human transient
receptor potential vanilloid type 1 (TRPV1) or the rat transient re-
ceptor potential ankyrin type 1 (TRPA1) cDNAs are shown in
Tables 1 and 2. Data previously recorded for tetrazoles 1 and 2 are
also included in Tables 1 and 2.
2. Results and discussion
2.1. Chemistry
In the first set of tetrazoles (compounds 3e10), one or both
methyl groups of the dimethylamino moiety of the parent com-
pounds 1 and 2 were replaced by cycloalkyl or arylalkyl groups. In
particular, groups like cyclohexyl, 4-phenylbutyl, and 2-
naphthylmethyl which were shown to produce significant po-
tency improvements in the series of biphenyl-3-yl alkyl carbamates
The tetrazoles in Fig. 2 were synthesized as summarized in
Schemes 1 and 2. Acylation of 5-[(biphenyl-4-yl)methyl]-1H(2H)-
tetrazole (33) [6] with the appropriate carbamoyl chlorides 34
afforded mixtures of the 1,5- (compounds 3e6) and 2,5-
regioisomers (compounds 7e10) which could be separated by

120
G. Ortar et al. / European Journal of Medicinal Chemistry 63 (2013) 118e132
Fig. 2. Structures of carbamoyl tetrazoles synthesized and tested in this study.
[19] were selected. Unlike this series, the modifications of the
dimethylamino moiety proved however to be generally detrimental
to the inhibitory potency on both FAAH and MAGL, except for
compounds 9 and 10 which showed IC50 values on FAAH of the
same magnitude as their prototype 2. The most significant decrease
was observed by substituting one of the two methyl groups with a
cyclohexyl group (compare compounds 1,2 and 4,8). Furthermore,
1,5-regioisomers were negatively affected to a greater extent than
2,5-analogues. Anyway, the first group of tetrazoles, compounds 4
excluded, were endowed with some selectivity for FAAH over
MAGL. Interestingly, most of tetrazoles 3e10 exhibited significant
TRPV1 and/or TRPA1 agonist/desensitizing activities (Table 2). A
special mention needs to be made for compound 8 which turned
out to be a very potent TRPA1 activator, displaying a submicromolar
horn of the spinal cord was shown to counteract pain by leading to
inhibition of voltage-activated Naþ channels [20]. Therefore, given
the analgesic actions of FAAH inhibitors [1c,d,2bef,i], compounds
like 8 might represent useful leads for the development of intra-
thecal analgesics.
With the second group of tetrazoles (compounds 11e32), the
effect of phenyl ring substitution on FAAH and MAGL inhibition was
explored, introducing on the distal phenyl ring of 1 and 2 a set of
substituents. Some of the substituents we selected, in particular
small polar groups like COCH3, CH2OH, OH, and CONH2, have pre-
viously yielded FAAH inhibitors with potencies in the low nano-
molar concentration range in the series of biphenyl-3-yl alkyl
carbamates [21]. As far the FAAH inhibitory activity is concerned, in
some cases 1,5-isomers proved to be more potent than 2,5-isomers
(compare compounds 15, 16, 20, 21 and 26, 27, 31, 32), while in
other cases the opposite trend was observed (compare compounds
14, 17, 18 and 25, 28, 29). Ortho-substituted compounds were
potency (EC50 ¼ 0.15
(EC50 ¼ 24.5 M) [14]. Although this type of activity may also cause
pain in peripheral sensory afferents, TRPA1 activation in the dorsal
mM), w150-fold greater than that of URB597
m
Scheme 1. Synthesis of carbamoyl tetrazoles 3e10.

G. Ortar et al. / European Journal of Medicinal Chemistry 63 (2013) 118e132
121
Scheme 2. Synthesis of carbamoyl tetrazoles 11e32.
generally less active than the corresponding meta- and para-
this compound displayed
a
kinact/Ki value for FAAH of
isomers (compare compounds 15, 19, 26 and 16, 20, 21, 27, 28).
Both hydrophilic and lipophilic substituents were compatible with
a good inhibitory potency. Although none of compounds 11e32
proved to be more potent than 1, some of them (compounds 16,
20, 21, 25, and 28) inhibited FAAH potently (IC50 ¼ 3.0e9.7 nM) and
selectively (39- to more than 141-fold) over MAGL.
Since kinact/Ki values are, unlike IC50 values, independent of
preincubation times and substrate concentration and are consid-
ered the best measure of potency for irreversible inhibitors [22], we
have also derived for one of the two most FAAH-selective inhibitors,
i.e. compound 20, the kinact/Ki value from time-dependent IC50
values [23], using a standard software tool (XLfitÒ) and found that
33,485 ꢀ 9200 Mꢁ1 sꢁ1 which is comparable to that of the highly
efficacious FAAH inhibitor PF-04457845 (32,400 ꢀ 8600) [22]. This
observation may suggest that also the other potent FAAH inhibitors
in this series may have similar kinact/Ki values.
With the only partial exception of compounds 13 and 16, none of
the 1,5-regioisomers inhibited appreciably MAGL, while 2,5-
isomers proved to be invariably more potent, with IC50 values
<1 mM. Tetrazole 27, which exhibited an excellent inhibitory ac-
tivity for both FAAH and MAGL, has the potential to serve as a useful
pharmacological probe for the evaluation of behavioural conse-
quences of simultaneous elevation of the tissue levels of the two
main endocannabinoids [24]. Although the issue of selectivity of

122
G. Ortar et al. / European Journal of Medicinal Chemistry 63 (2013) 118e132
Table 1
Table 3
Effect of tetrazoles 1e32 on [14C]AEA hydrolysis by rat brain membranes and [3H]2-
Effect of tetrazole 27 on endocannabinoid levels in intact COS-7 cells.
AG hydrolysis by COS-7 cell cytosolic fractions.a
AEA
OEA
2-AG
Compd IC50 (nM)
FAAH
Compd IC50 (nM)
FAAH
Vehicle
Ionomycin 3
0.16 ꢀ 004
0.37 ꢀ 0.06
0.41 ꢀ 0.18
1.08 ꢀ 0.17
3.99 ꢀ 0.04
7.25 ꢀ 0.97a
2.82 ꢀ 0.75
mM
15.66 ꢀ 2.46
MAGL
>1000b
>1000
MAGL
20.0b
27 (1
(3
m
mM)
M) þ Ionomycin
412.84 ꢀ 182.78b
1
3
4
5
2.1b
38.2 ꢀ 2.4
2
33.0b
7
8
9
10
22
23
68.1 ꢀ 13.2 346.5 ꢀ 2.0
a
725.0 ꢀ 82.0 >1000
21.3 ꢀ 2.6 >1000
96.3 ꢀ 10.4 601.5 ꢀ 49.8
Data are expressed as pmol/mg of lipid extracted and are means ꢀ SD of n ¼ 4
11.6 ꢀ 1.5
16.9 ꢀ 2.5
34.2 ꢀ 7.4
12.3 ꢀ 2.2
10.7 ꢀ 1.7
7.5 ꢀ 0.9
250.0 ꢀ 33.5
84.5 ꢀ 12.1
807.4 ꢀ 35.6
498.5 ꢀ 11.7
27.0 ꢀ 3.1
separate determinations.
b
6
86.7 ꢀ 13.8 >1000
P < 0.05 as assessed by the ANOVA followed by the Bonferroni’s test.
11
12
13
14
15
16
17
18
19
20
21
14.7 ꢀ 2.2
12.1 ꢀ 0.4
10.6 ꢀ 1.0
37.4 ꢀ 2.3
15.6 ꢀ 3.0
3.0 ꢀ 0.2
>1000
>1000
520.2 ꢀ 113.9 24
endocannabinoid were higher due to the use of a cell line over-
expressing the biosynthetic enzyme for 2-AG.
>1000
>1000
25
26
27
28
29
30
31
32
372.2 ꢀ 24.4
98.1 ꢀ 13.0
10.0 ꢀ 1.5
40.2 ꢀ 13.0
12.0 ꢀ 2.1
5.4 ꢀ 0.6
In contrast with the first set of new tetrazoles described here,
the compounds of the second group, with the exception of 32,
displayed weak or no activity at TRPV1 and TRPA1 channels. An
analogous result was essentially observed for parent compounds 1
and 2, therefore suggesting that the presence of groups bulkier than
methyl on the nitrogen of the carbamoyl moiety is required for an
efficient TRPV1 and/or TRPA1 modulation.
170.9 ꢀ 39.5
16.7 ꢀ 1.6
11.0 ꢀ 3.4
35.5 ꢀ 4.8
9.7 ꢀ 0.7
>1000
209.4 ꢀ 33.3
137.2 ꢀ 18.2
>1000
>1000
>1000
>1000
7.1 ꢀ 1.9
51.7 ꢀ 13.3 121.4 ꢀ 51.5
48.5 ꢀ 8.0
46.2 ꢀ 8.2
139.2 ꢀ 13.0
334.9 ꢀ 25.1
7.1 ꢀ 0.4
a
Data are means ꢀ SD of n ¼ 4 separate determinations.
b
Data from Ref. [6].
In Figs. 3e5 the concentrationeresponse curves for inhibition of
FAAH by the FAAH-selective compounds 16, 20, 21, 25, and 28 and
for inhibition of FAAH and MAGL by the dual FAAHeMAGL inhibitor
27, respectively, are presented.
In Fig. 6 the SARs for the tetrazolyl ureas FAAH and/or MAGL
inhibitors described in the present study are summarized.
inhibition for FAAH versus MAGL (or vice versa) is certainly the
most important one [25], the identification of inhibitors with
nanomolar potency on both targets appears to be of interest in view
of their possible therapeutic exploitation in those pathological
conditions in which the levels of both endocannabinoids need to be
elevated. In this respect, Makriyannis et al. has very recently re-
ported that the dual FAAH/MAGL inhibitor 2 is more protective
against seizure pathology than regioisomer 1 which is more se-
lective for FAAH than MAGL [26]. The effect of tetrazole 27 on AEA,
N-oleoylethanolamine (OEA, an alternate FAAH substrate) [2ceg]
and 2-AG levels in COS-7 cells overexpressing the human recom-
2.3. Molecular modelling
Although mass spectrometry analysis has unequivocally
demonstrated that tetrazole 1 covalently inhibits FAAH by carba-
mylation of a nucleophilic Ser241 residue with releasing the
biphenylmethyl tetrazole moiety [7], no 3D-structure or molecular
modelling studies have been so far reported on its possible binding
mode. Molecular docking studies were therefore conducted on
tetrazoles 1e32 with AutoDock Vina [27] docking program. Auto-
Dock Vina usage was assessed through an extensive re-docking and
cross-docking previously reported protocol [28] and using the
available experimental three-dimensional structures of FAAH co-
crystallized with different inhibitors (PDB IDs: 1MT5, 2VYA,
2WAP, 2WJ1, 2WJ2, 3K7F, 3K83, 3K84, 3LJ6, 3LJ7, 3OJ8, 3QJ9, 3QK5,
binant diacylglycerol lipase-a (DAGL-a) was also measured, by
incubating cells with vehicle, or ionomycin with/or without pre-
incubation with compound 27 (Table 3). As predicted from its ac-
tivity, 27 elevated the stimulated levels of the MAGL substrate, 2-
AG, and of the FAAH substrate OEA. It also slightly elevated the
levels of the other FAAH substrate, AEA, but in a non-statistically
significant manner. Again unsurprisingly, the effect on 2-AG was
stronger, as expected from the fact that the stimulated levels of this
Table 2
Effect of tetrazoles 1e32 on intracellular Ca2þ elevation in HEK293 cells stably transfected with either the human TRPV1 or the rat TRPA1 cDNAs.a
Compd TRPV1
(efficacy)b (EC50
<10 ND
67.4 5.9
TRPV1
TRPV1
TRPA1
TRPA1
TRPA1
Compd TRPV1
TRPV1
TRPV1
TRPA1
TRPA1
TRPA1
M) (IC50, m
M)e
,
m
M) (IC50
,
m
M)c (efficacy)d (EC50
,
m
M) (IC50
,
m
M)e
(efficacy)b (EC50
,
m
M) (IC50
,
m
M)c (efficacy)d (EC50
,
m
1
3
>100
22.6
18.3
7.1
7.6
101.1
106.5
84.7
89.9
53.9
<10
<10
<10
58.7
26.9
6.5
4.9
20.1
13.5
ND
49.4
12.5
14.9
83.7
15.0
>100
99.8
>100
52.6
>100
>100
>100
>100
45.2
41.0
15.9
2
7
8
9
14.5
66.7
33.6
15.9
36.1
5.0
1.8
0.25
1.7
31.3
7.4
9.2
47.6
4.5
>100
>100
>100
>100
>100
>100
>100
91.8
>100
78.9
20.6
186.5
130.2
45.2
<10
<10
<10
<10
<10
67.3
445.0
<10
109.4
<10
201.6
187.0
275.0
20.1
3.7
0.15
ND
ND
ND
ND
ND
24.4
53.3
ND
14.6
ND
15.9
15.0
10.3
11.2
21.5
15.4
92.1
24.3
97.2
91.2
>100
70.6
28.0
98.9
30.2
83.1
18.9
17.8
10.5
4
5
6
45.6
45.7
34.1
13.4
1.5
2.5
10
22
23
24
25
26
27
28
29
30
31
32
11.9
11
12
13
14
15
16
17
18
19
20
21
<10
ND
ND
ND
ND
ND
25.0
ND
32.3
ND
ND
22.5
>100
<10
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
<10
<10
<10
<10
20.0
<10
21.3
<10
<10
28.0
>100
>100
>100
>100
ND
ND
<10
<10
<10
<10
<10
<10
<10
<10
<10
24.2
59.7
26.8
21.7
50.1
48.7
32.8
19.7
228.5
35.7
40.5
67.4
>100
>100
>100
>100
>100
30.7
348.8
233.4
388.0
33.3
4.8
a
Data are means of n ¼ 4 separate determinations. Standard errors are not shown for the sake of clarity and were never higher that 10% of the means.
b
c
As percent of ionomycin (4
mM).
Determined against the effect of capsaicin (0.1
m
M).
d
e
As percent of allyl isothiocyanate (100
mM).
Determined against the effect of allyl isothiocyanate (100
m
M). ND, not determined when efficacy is lower than 10%.

G. Ortar et al. / European Journal of Medicinal Chemistry 63 (2013) 118e132
123
Fig. 3. Concentration-dependent inhibition of FAAH by compounds 16, 20, 21, 25, 28.
Fig. 5. Concentration-dependent inhibition of MAGL by compound 27.
3QKV). Twelve out of 14 experimental inhibitors were covalently
bound to the catalytic Ser241 [29]. Therefore, a covalent docking
protocol was performed with AutoDock Vina. The inhibitors were
modelled and bound to Ser241 in their transition state conforma-
tions (TS, Fig. 7).
leaving groups were binding in either the CA cavity (1,5-
regioisomers 3, 5, 12, 17, and 20 and 2,5-regioisomers 8, 22, 24, and
27) or the ABP pocket (1,5-regioisomers 4, 11, 13, and 16 and 2,5-
regioisomers 7, 9, 15, 28, and 31).
We speculate that, the orientation of the leaving groups seems
to play an important role. Five out of the six single digit nanomolar
active derivatives (1, 28, 25, 16, and 21 in decreasing order of po-
tency) were predicted to bind with their leaving moieties in the ABP
pocket, with their small dimethylaminocarbonyl carbamylating
portions residing in the CA channel (Supplementary material,
Fig. SM 2).
As AutoDock Vina proved to correctly reproduce the experi-
mental binding mode of the covalent FAAH inhibitors (see
Experimental section and Supplementary material, Table SM 1), the
same covalent cross-docking protocol was applied to compounds
1e32. Although it would be reasonable to expect that in their TS
conformation the compounds would bind by locating the biphe-
nylmethyl tetrazole leaving groups in the cytosolic port (or cyto-
plasmatic access channel, CA) [2c,e,f], due to the lack of any
structural insights, it was decided to investigate all the possible
binding TSs. Therefore, each inhibitor/FAAH complex was built by
modelling the tetrazoles 1e32 either in the CA cavity or in the acyl-
chain binding pocket (ABP), leading to either (S)- or (R)-TS config-
urations. Upon cross-docking into the 12 experimental FAAH con-
formations on the basis of non-docking interaction energies, the
(S)-TS configuration was found to be the preferred one (Supple-
mentary material, Table SM 2).
Surprisingly, AutoDock Vina proposed TS binding modes of
tetrazoles 1e32 endowed with the lowest docking energy did not
display a unique pattern, and were determined by the regioisomery
of compounds, the nature of nitrogen substituents of the carba-
mylating moiety, and the nature and position of the distal phenyl
ring substituents. In seven out of 16 regioisomeric couples (Sup-
plementary material, Table SM 3), both 1,5 and 2,5-regioisomers (1
and 2, 6 and 10, 14 and 25, 15 and 26, 18 and 29, 19 and 30, and 21
and 32) displayed the biphenylmethyl tetrazole moieties accomo-
dated in the ABP pocket, while for the other nine couples, the
Regarding the influence of the regioisomery on the binding
mode of tetrazoles, the major difference among the non-bonding
interactions that could account for the decreased activity of the
2,5-regioisomer 2 compared to 1,5-regioisomer 1, was a missing
pe
p
interaction that could be observed in 1 between the proximal
phenyl ring and the Phe192 side chain (Fig. 8; phenyl centroid
ꢀ
distance is about 3.4 A). When examining, among the new com-
pounds 3e32, the regioisomeric couple with the highest difference
in their inhibitory potencies (compounds 14 and 25), the same pep
interaction was also observed in the compound 14/FAAH complex.
However, the presence of the 4-hydroxymethyl group on the distal
phenyl group induced the adoption of a different rotamer, con-
straining the bonding Ser241 to a disfavoured eclipsed conforma-
tion, thus attenuating the positive
pep interaction. On the other
hand, the 4-hydroxymethyl group of compound 25, partially
overlapping the membrane access channel (MAC) (Supplementary
material, Fig. SM 3), points towards outside the enzyme (Fig. 9),
thus possibly making some hydrophilic intermolecular stabilizing
interactions that could account for its enhanced activity compared
to both its analogue 2 and its regioisomer 14.
3. Conclusions
The replacement of the dimethylamino group of the parent
tetrazoles 1 and 2 with bulkier groups resulted invariably in sig-
nificant decreases in the inhibitory potency against both FAAH and
MAGL, while the impact of the introduction of substituents at the
ortho, meta or para positions of the distal phenyl ring of 1 and 2
appeared more variable. However, some of the new tetrazoles, that
is 16, 20, 21, 25, and 28, still inhibited FAAH potently and selectively
versus MAGL. On the other hand, tetrazole 27, whilst not selective
versus FAAH, demonstrated also an excellent MAGL inhibitory ac-
tivity in the low nanomolar range.
Molecular modelling and covalent docking studies suggested
that binding modes with FAAH of tetrazoles 1e32 do not display a
unique pattern. In twenty-three out of 32 cases, the biphenylmethyl
tetrazole moiety might bind in the hydrophobic acyl chain channel
Fig. 4. Concentration-dependent inhibition of FAAH by compound 27.

124
G. Ortar et al. / European Journal of Medicinal Chemistry 63 (2013) 118e132
4.1.3. General procedure for the preparation of carbamoyl tetrazoles
3e10
To a stirred solution of 33 [6] and Et3N (2 equiv) in acetonitrile
(1.5 mL/mmol) the appropriate carbamoyl chloride 34 (1.5 equiv)
was added at 0 ꢂC, and the mixture was stirred at 0 ꢂC overnight.
The mixture was diluted with brine and extracted with AcOEt. The
organic phase was washed twice with brine, dried (Na2SO4), and
evaporated under vacuum. The residue was chromatographed on
silica gel using CH2Cl2 or CH2Cl2/AcOEt mixtures as eluents, the less
polar 2,5-disubstituted tetrazole eluting prior to the 1,5-
disubstituted regioisomer.
Fig. 6. Summary of SARs for tetrazolyl ureas inhibitors of FAAH and/or MAGL.
of FAAH, with the disubstituted amino group extending into the
cytosolic port. The investigation of the ability of tetrazoles 1e32 to
act as TRPV1 and TRPA1 modulators led to the identification of a
potent TRPA1 activator (compound 8) which should be tested in
future studies as a spinal analgesic.
4.1.3.1. (5-(Biphenyl-4-ylmethyl)-1H-tetrazol-1-yl)(piperidin-1-yl)
methanone (3) and (5-(biphenyl-4-ylmethyl)-2H-tetrazol-2-
yl)(piperidin-1-yl)methanone (7). Piperidine-1-carbonyl chloride
and CH2Cl2/AcOEt ¼ 98/2 were used as acylating agent and as
eluent for the chromatographic separation of regioisomers,
respectively. Compound 7 (less polar, 33%): mp 104e105 ꢂC; IR
2948, 2862, 1735, 1488, 1434, 1238, 988 cmꢁ1; 1H NMR
(6H, m), 3.38 (2H, t, J ¼ 5.6 Hz), 3.75 (2H, t, J ¼ 5.5 Hz), 4.36 (2H, s),
7.32e7.57 (9H, m); 13C NMR
23.99, 25.30, 26.00, 31.41, 46.71, 48.76,
d 1.60e1.74
4. Experimental protocols
d
4.1. Chemistry
127.04, 127.29, 127.49, 128.76, 129.35, 134.98, 140.09, 140.68, 146.57,
165.16. Anal. Calcd for C20H21N5O: C, 69.14; H, 6.09; N, 20.16. Found:
C, 69.18; H, 6.07; N, 20.19. Compound 3 (more polar, 16%): mp 102e
103 ꢂC; IR 2948, 2861, 1723, 1504, 1434, 1258, 1008 cmꢁ1; 1H NMR
4.1.1. General
All chemical reagents were commercially available unless
otherwise indicated. Melting points were determined on a Kofler
hot-stage apparatus and are uncorrected. IR spectra were recorded
on a PerkineElmer 1000 FT-IR spectrophotometer as CHCl3 solu-
tions unless otherwise indicated. 1H and 13C NMR spectra were
obtained on a Varian Gemini 200 or a Bruker Avance 400 spec-
trometer using CDCl3 as solvent unless otherwise indicated and
TMS as internal standard. Chromatographic separations were per-
formed on Merck 60 silica gel (230e400 mesh). The preparations of
N-methyl-4-phenylbutan-1-amine [16] and of N-methyl-1-(naph-
talen-2-yl)methanamine [17] have been described in the literature.
d
1.14 (2H, m), 1.49 (2H, m), 1.57 (2H, m), 3.01 (2H, t, J ¼ 5.6 Hz), 3.58
(2H, t, J ¼ 5.5 Hz), 7.31e7.54 (9H, m); 13C NMR
d 23.73, 25.09, 25.45,
29.57, 46.01, 48.51, 126.96, 127.58, 127.63, 128.90, 129.69, 133.43,
140.37, 140.75, 146.40, 155.95. Anal. Calcd for C20H21N5O: C, 69.14;
H, 6.09; N, 20.16. Found: C, 69.19; H, 6.05; N, 20.18.
4.1.3.2. 5-(Biphenyl-4-ylmethyl)-N-cyclohexyl-N-methyl-1H-tetra-
zole-1-carboxamide (4) and 5-(biphenyl-4-ylmethyl)-N-cyclohexyl-
N-methyl-2H-tetrazole-2-carboxamide (8).
Cyclohexyl(methyl)carbamoyl chloride and CH2Cl2/AcOEt ¼ 98/2
were used as acylating agent and as eluent for the chromatographic
separation of regioisomers, respectively. Compound 8 (less polar,
4.1.2. General procedure for the preparation of carbamoyl chlorides
34
30%): oil; IR 2938, 2860, 1731, 1488, 1204, 1064 cmꢁ1 1H NMR
;
To a stirred solution of bis(trichloromethyl)carbonate and dry
pyridine (6 equiv) in dry CH2Cl2 (2 mL/mmol) a solution of the
appropriate amine (3 equiv) in dry CH2Cl2 (0.5 mL/mmol) was
added. The mixture was stirred at room temperature for 2 h,
acidified with 2 N HCl and extracted with CH2Cl2. The organic phase
was washed with saturated NaHCO3 and brine, dried (Na2SO4), and
evaporated under vacuum to give the title compounds which were
used in the next step without further purification.
d
1.07e1.90 (10H, m), 2.87 and 3.09 (3H, 2s), 3.29 and 4.27 (1H, 2m),
4.37 (2H, s), 7.31e7.57 (9H, m); 13C NMR
d
25.43, 29.41, 29.72, 29.95,
30.23, 31.51, 57.12, 59.26, 127.10, 127.32, 127.54, 128.79, 129.36,
135.16, 140.23, 140.84, 147.54, 165.09. Anal. Calcd for C22H25N5O: C,
70.38; H, 6.71; N, 18.65. Found: C, 70.45; H, 6.66; N, 18.59. Com-
pound 4 (more polar, 25%): oil; IR 2938, 2860, 1720, 1411, 1224,
1203, 1072 cmꢁ1; 1H NMR
d 0.92e1.79 (10H, m), 2.47 and 2.90 (3H,
A
B
Fig. 7. Examples of the transition state of a co-crystallized inhibitor (A, PDB entry code 3K83) and of that proposed for compound 1 (B) as used in the covalent dockings. The circled
arrows indicate the rotatable bonds during the covalent docking.

G. Ortar et al. / European Journal of Medicinal Chemistry 63 (2013) 118e132
125
Fig. 8. Binding modes of 1 (magenta) and 2 (blue) regioisomers. In yellow are highlighted the phenyl rings centroids that account for the
proximal phenyl ring. In wire are also reported the residues in a 5 A distance from the covalent inhibitors. For sake of clarity hydrogen atoms were undisplayed. Gly240 and Ile242
p
e
p
interaction between Phe192 and the 1
ꢀ
are depicted in black wire. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
2s), 2.73 and 4.09 (1H, 2m), 4.48 (2H, s), 7.26e7.53 (9H, m); 13C
NMR 25.24, 29.17, 29.46, 29.69, 30.20, 31.50, 57.12, 59.26, 127.00,
127.60, 128.90, 129.48, 133.61, 140.43, 140.87, 147.38, 155.74. Anal.
Calcd for C22H25N5O: C, 70.38; H, 6.71; N, 18.65. Found: C, 70.46; H,
6.65; N, 18.58.
127.31, 127.52, 128.44, 128.77, 129.33, 135.06, 140.19, 140.76, 147.79,
165.13. Anal. Calcd for C26H27N5O: C, 73.39; H, 6.40; N,16.46. Found:
C, 73.32; H, 6.46; N, 16.51. Compound 5 (more polar, 23%): oil; IR
d
2940, 2862, 1727, 1488, 1405, 1077 cmꢁ1; 1H NMR
d 1.32e1.66 (4H,
m), 2.41 and 2.62 (2H, 2t, J ¼ 7.4 Hz), 2.65 and 3.01 (3H, 2s), 2.97 and
3.40 (2H, t, J ¼ 7.4 Hz), 4.46 and 4.48 (2H, 2s), 7.02e7.53 (14H, m);
4.1.3.3. 5-(Biphenyl-4-ylmethyl)-N-methyl-N-(4-phenylbutyl)-1H-
tetrazole-1-carboxamide (5) and 5-(biphenyl-4-ylmethyl)-N-methyl-
N-(4-phenylbutyl)-2H-tetrazole-2-carboxamide (9).
13C NMR
d 25.96, 29.70, 35.33, 36.81, 50.17, 51.28, 126.04, 127.00,
127.51, 127.61, 128.25, 128.46, 128.90, 129.46, 129.61, 140.21, 140.70,
147.86. Calcd for C26H27N5O: C, 73.39; H, 6.40; N, 16.46. Found: C,
73.31; H, 6.45; N, 16.50.
Methyl(4-phenylbutyl)carbamoyl chloride and CH2Cl2 were used as
acylating agent and as eluent for the chromatographic separation of
regioisomers, respectively. Compound 9 (less polar, 49%): oil; IR
4.1.3.4. 5-(Biphenyl-4-ylmethyl)-N-methyl-N-(naphthalen-2-
ylmethyl)-1H-tetrazole-1-carboxamide (6) and 5-(biphenyl-4-
ylmethyl)-N-methyl-N-(naphthalen-2-ylmethyl)-2H-tetrazole-2-
2940, 2862, 1736, 1488, 1406, 1230, 1067 cmꢁ1; 1H NMR
d 1.48e1.74
(4H, m), 2.52 and 2.68 (2H, 2t, J ¼ 7.5 Hz), 3.01 and 3.15 (3H, 2s),
3.26 and 3.58 (2H, 2t, J ¼ 7.5 Hz), 4.34 (2H, s), 7.07e7.54 (14H, m);
carboxamide
(10). Methyl(naphthalen-2-ylmethyl)carbamoyl
13C NMR
d
26.18, 31.46, 35.39, 37.14, 50.83, 51.42, 126.00, 127.06,
chloride and CH2Cl2 were used as acylating agent and as eluent for
ꢀ
Fig. 9. Binding modes of 14 (yellow) and 25 (cyan) regioisomers. In wire are also reported the residues in a 5 A distance from the covalent inhibitors. For sake of clarity hydrogen
atoms were undisplayed. Gly240 and Ile242 are depicted in black wire. For comparison purposes the docked pose for 1 (magenta) is also reported. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)

126
G. Ortar et al. / European Journal of Medicinal Chemistry 63 (2013) 118e132
the chromatographic separation of regioisomers, respectively.
4.1.4.5. 2-(20-Hydroxybiphenyl-4-yl)acetonitrile (50).
Compound 10 (less polar, 42%): oil; IR 3017, 2932, 1736, 1488, 1402,
2-Hydroxyphenylboronic acid (40) and hexane/AcOEt ¼ 75/25
were used as arylboronic acid and as eluent for the chromato-
graphic purification of 50 (66%), respectively: mp 141e143 ꢂC; IR
1066 cmꢁ1; 1H NMR
d
3.03 and 3.16 (3H, 2s), 4.33 and 4.37 (2H, 2s),
4.69 and 4.92 (2H, 2s), 7.30e7.85 (16H, m); 13C NMR
d
31.48, 36.67,
54.40, 126.46, 126.61, 127.07, 127.31, 127.52, 127.80, 127.87, 128.76,
129.07, 129.33, 133.23, 133.37, 134.94, 140.19, 140.76, 165.31. Calcd
for C27H23N5O: C, 74.81; H, 5.35; N, 16.16. Found: C, 74.75; H, 5.39;
N, 16.21. Compound 6 (more polar, 33%): mp 153e154 ꢂC; IR 2933,
3353, 2260, 1591, 1498, 1451, 1361, 1196 cmꢁ1
;
1H NMR (CD3OD)
d
3.90 (2H, s), 6.87e7.58 (8H, m); 13C NMR (CD3OD)
d 23.29, 117.04,
119.76, 121.03, 128.70, 129.15, 129.78, 130.47, 131.09, 131.60, 140.08,
155.49. Calcd for C14H11NO: C, 80.36; H, 5.30; N, 6.69. Found: C,
80.42; H, 5.28; N, 6.65.
2855, 1726, 1488, 1403, 1266, 1077 cmꢁ1
;
1H NMR
d
2.67 and 3.01
(3H, 2s), 4.43 and 4.76 (2H, 2s), 4.50 and 4.55 (2H, 2s), 7.23e7.73
(16H, m); 13C NMR
29.74, 36.33, 53.77, 124.67, 125.78, 126.50,
d
4.1.4.6. 2-(30-Hydroxybiphenyl-4-yl)acetonitrile (51). 3-Hydroxy-
phenylboronic acid (41) and hexane/AcOEt ¼ 7/3 were used as
arylboronic acid and as eluent for the chromatographic purification
of 51 (75%), respectively: mp 123e125 ꢂC; IR (KBr) 3317, 2918, 2255,
126.63, 126.96, 127.34, 127.51, 127.62, 127.82, 128.85, 129.11, 129.27,
129.60, 133.16, 135.18, 140.81, 155.84. Calcd for C27H23N5O: C, 74.81;
H, 5.35; N, 16.16. Found: C, 74.77; H, 5.37; N, 16.19.
1586, 1568, 1452, 1303, 1209 cmꢁ1; 1H NMR (CD3OD)
d
3.88 (2H, s),
4.1.4. General procedure for the preparation of 20- or 30- or 40-
monosubstituted 2-(biphenyl-4-yl)acetonitriles 46e55
6.77e7.59 (8H, m); 13C NMR (CD3OD)
d 23.18, 114.72, 115.49, 119.22,
119.52, 128.49, 129.37, 130.87, 131.04, 142.03, 142.94, 158.80. Calcd
for C14H11NO: C, 80.36; H, 5.30; N, 6.69. Found: C, 80.41; H, 5.27; N,
6.66.
A stirred mixture of 4-iodophenylacetonitrile (35), arylboronic
acid (36e45) (1.1 equiv), Pd(PPh3)4 (0.03 equiv), KBr (1.1 equiv), and
K3PO4 (2.5 equiv) in dioxane (5 mL/mmol) was purged with N2 for
10 min at room temperature and then stirred overnight at 85 ꢂC
under N2. The mixture was diluted with water and extracted with
AcOEt. The organic phase was washed three times with water, dried
(Na2SO4), and evaporated under vacuum. The residue was chro-
matographed on silica gel.
4.1.4.7. 2-(40-Hydroxybiphenyl-4-yl)acetonitrile (52). 4-Hydroxy-
phenylboronic acid (42) and hexane/AcOEt ¼ 7/3 were used as
arylboronic acid and as eluent for the chromatographic purification
of 52 (70%), respectively: mp 188e190 ꢂC; IR (KBr) 3398, 2260, 1611,
1591, 1501, 1213 cmꢁ1; 1H NMR (CD3OD)
6.84e7.56 (8H, m); 13C NMR (CD3OD)
d
3.88 (2H, s), 6.62 (1H, s),
d
23.15, 116.72, 116.85, 128.01,
4.1.4.1. 2-(30-Acetylbiphenyl-4-yl)acetonitrile (46). 3-Acetylphenyl-
boronic acid (36) and hexane/AcOEt ¼ 75/25 were used as aryl-
boronic acid and as eluent for the chromatographic purification of
46 (69%), respectively: mp 71e72 ꢂC; IR (KBr) 3057, 2916, 2247,
129.03,129.45,130.23,132.94,142.09,158.45. Calcd for C14H11NO: C,
80.36; H, 5.30; N, 6.69. Found: C, 80.42; H, 5.33; N, 6.67.
4.1.4.8. 2-(20-Methylbiphenyl-4-yl)acetonitrile
(53).
2-Methyl-
1676, 1584, 1412, 1359,1300, 1240 cmꢁ1; 1H NMR
(2H, s), 7.42e8.17 (8H, m); 13C NMR
d
2.66 (3H, s), 3.81
phenylboronic acid (43) and hexane/AcOEt ¼ 9/1 were used as
arylboronic acid and as eluent for the chromatographic purification
of 53 (87%), respectively: oil; IR 3024, 2927, 2254, 1602, 1514, 1484,
d
23.34, 26.72, 117.66, 126.79,
127.60, 127.92, 128.55, 129.19, 129.57, 131.60, 137.80, 140.12, 140.76.
Calcd for C16H13NO: C, 81.68; H, 5.57; N, 5.95. Found: C, 81.71; H,
5.55; N, 5.93.
1417, 1110, 1008 cmꢁ1; 1H NMR
(8H, m); 13C NMR
d
2.26 (3H, s), 3.79 (2H, s), 7.19e7.38
d
20.40, 23.39,117.87,125.88,127.57,127.72,128.39,
129.68, 129.94, 130.42, 135.27, 140.94, 141.90. Calcd for C15H13N: C,
86.92; H, 6.36; N, 6.76. Found: C, 86.96; H, 6.34; N, 6.77.
4.1.4.2. Methyl
40-(cyanomethyl)biphenyl-3-carboxylate
(47).
3-Methoxycarbonylphenylboronic acid (37) and hexane/
CH2Cl2 ¼ 3/7 were used as arylboronic acid and as eluent for the
chromatographic purification of 47 (63%), respectively: mp 155e
156 ꢂC; IR (KBr) 2921, 2247, 1715, 1443, 1309, 1298, 1250,
4.1.4.9. 2-(30-Methylbiphenyl-4-yl)acetonitrile
(54).
3-Methyl-
phenylboronic acid (44) and hexane/AcOEt ¼ 9/1 were used as
arylboronic acid and as eluent for the chromatographic purification
of 54 (48%), respectively: oil; IR 2924, 2860, 2255, 1604, 1486, 1460,
1113 cmꢁ1
13C NMR
;
1H NMR
d
3.80 (2H, s), 3.95 (3H, s), 7.41e8.26 (8H, m);
d
23.34, 52.23, 117.69, 127.87, 128.18, 128.51, 128.71, 129.00,
1378, 1115 cmꢁ1; 1H NMR
m); 13C NMR
d
2.42 (3H, s), 3.77 (2H, s), 7.11e7.60 (8H,
129.46, 130.88, 131.41, 140.04, 140.49, 166.91. Calcd for C16H13NO2:
C, 76.48; H, 5.21; N, 5.57. Found: C, 76.50; H, 5.20; N, 5.58.
d 21.51, 23.29, 112.29, 116.03, 121.45, 124.18, 127.83,
128.29, 128.39, 128.78, 129.36, 138.49, 140.20, 141.26. Calcd for
C15H13N: C, 86.92; H, 6.36; N, 6.76. Found: C, 86.97; H, 6.33; N, 6.80.
4.1.4.3. 2-(30(Hydroxymethyl)biphenyl-4-yl)acetonitrile
(48).
3-Hydroxymethylphenylboronic acid (38) and hexane/AcOEt ¼ 6/4
were used as arylboronic acid and as eluent for the chromato-
graphic purification of 48 (65%), respectively: mp 60e62 ꢂC; IR
4.1.4.10. 2-(40-Methylbiphenyl-4-yl)acetonitrile
(55)
[30].
4-Methylphenylboronic acid (45) and hexane/AcOEt ¼ 88/12 were
used as arylboronic acid and as eluent for the chromatographic
purification of 55 (35%), respectively: mp 121e122 ꢂC; IR 3009,
3605, 3424, 3009, 2932, 2255, 1484, 1415, 1020 cmꢁ1
d
;
1H NMR
23.29,
3.77 (2H, s), 4.74 (2H, s), 7.34e7.60 (8H, m); 13C NMR
d
2924, 2254,1602,1502,1417,1113,1007 cmꢁ1; 1H NMR
d
2.40 (3H, s),
65.18, 117.82, 125.33, 126.20, 126.31, 127.84, 128.37, 128.96, 129.10,
140.51, 140.90, 141.60. Calcd for C15H13NO: C, 80.69; H, 5.87; N, 6.27.
Found: C, 80.73; H, 5.84; N, 6.30.
3.78 (2H, s), 7.23e7.54 (8H, m); 13C NMR
d 21.09, 23.28, 115.08,
117.84,126.89,127.60,128.31,129.59,137.32,137.48,141.04. Calcd for
C15H13N: C, 86.92; H, 6.36; N, 6.76. Found: C, 86.95; H, 6.38; N, 6.78.
4.1.4.4. 2-(40(Hydroxymethyl)biphenyl-4-yl)acetonitrile
(49).
4.1.4.11. 2-(20-Benzyloxybiphenyl-4-yl)acetonitrile (56). A mixture
of 50 (376 mg, 1.8 mmol), K2CO3 (298 mg, 2.16 mmol), benzyl
4-Hydroxymethylphenylboronic acid (39) and hexane/AcOEt ¼ 55/
45 were used as arylboronic acid and as eluent for the chromato-
graphic purification of 49 (38%), respectively: mp 160e162 ꢂC; IR
(KBr) 3212, 2922, 2250, 1497, 1403, 1206, 1041, 998 cmꢁ1; 1H NMR
bromide (246
mL, 2.07 mmol) in acetone (15 mL) was refluxed
overnight, filtered, and the filtrate was evaporated under vacuum.
The residue (580 mg) was chromatographed on silica gel (23 g)
using hexane/AcOEt ¼ 85/15 as eluent to give 56 (388 mg, 72%): mp
71e75 ꢂC; IR (KBr) 3029, 2924, 2853, 2251, 1597, 1583, 1513, 1487,
d
1.91 (1H, br s), 3.79 (2H, s), 4.75 (2H, s), 7.38e7.65 (8H, m); 13C
NMR
d 23.33, 65.00, 117.78, 127.24, 127.53, 127.77, 128.40, 128.91,
139.59,140.36,140.76. Calcd for C15H13NO: C, 80.69; H, 5.87; N, 6.27.
Found: C, 80.72; H, 5.89; N, 6.29.
1447, 1264, 1226, 1125, 1022 cmꢁ1; 1H NMR
s), 6.97e7.56 (13H, m); 13C NMR
d
3.62 (2H, s), 5.02 (2H,
d
23.00, 71.15, 116.58, 117.90,

G. Ortar et al. / European Journal of Medicinal Chemistry 63 (2013) 118e132
127
121.56, 127.11, 127.73, 127.92, 128.31, 128.62, 128.98, 129.32, 130.00,
131.91, 136.72, 137.88. Calcd for C21H17NO: C, 84.25; H, 5.72; N, 4.68.
Found: C, 84.32; H, 5.67; N, 4.72.
127.39, 128.34, 128.47, 130.25, 130.36, 132.48, 136.38, 138.84, 140.23,
142.00, 156.85. Calcd for C16H14N4O: C, 69.05; H, 5.07; N, 20.13.
Found: C, 69.11; H, 5.03; N, 20.09.
4.1.4.12. 2-(30-Benzyloxybiphenyl-4-yl)acetonitrile
(57).
4.1.5.2. Methyl 40-((1H(2H)-tetrazol-5-yl)methyl)biphenyl-3-
Prepared from 51 following the same procedure that was used for
the preparation of 56 using hexane/AcOEt ¼ 85/15 as eluent for the
chromatographic purification of 57 (86%): mp 96e97 ꢂC; IR (KBr)
2932, 2868, 2255, 1605, 1581, 1563, 1480, 1294, 1198, 1008 cmꢁ1; 1H
carboxylate (62). Yield 89%; mp 169e171 ꢂC; IR (KBr) 2956, 2852,
2691, 2613, 1719, 1446, 1409, 1297, 1251, 1043 cmꢁ1 1H NMR
;
(CD3OD)
(CD3OD)
d
3.92 (3H, s), 4.38 (2H, s), 7.40e8.23 (8H, s); 13C NMR
29.67, 52.60,128.23, 128.41, 129.12, 130.12,130.30,131.72,
d
NMR
d
3.76 (2H, s), 5.11 (2H, s), 6.97e7.59 (13H, m); 13C NMR
d
23.28,
132.27, 136.43, 139.72, 141.68, 167.69. Calcd for C16H14N4O2: C,
65.30; H, 4.79; N, 19.04. Found: C, 65.36; H, 4.75; N, 18.99.
70.15, 113.85, 113.96, 117.76, 119.81, 127.51, 127.83, 128.02, 128.33,
128.62, 129.02, 129.92, 136.92, 140.89, 141.73, 159.26. Calcd for
C21H17NO: C, 84.25; H, 5.72; N, 4.68. Found: C, 84.29; H, 5.69; N, 4.65.
4.1.5.3. (40-((1H(2H)-Tetrazol-5-yl)methyl)biphenyl-3-yl)methanol
(63). Yield 86%; mp 116e118 ꢂC; IR (KBr) 3158, 2852, 1563, 1493,
4.1.4.13. 2-(40-Benzyloxybiphenyl-4-yl)acetonitrile
(58).
1251, 1040 cmꢁ1; 1H NMR (CD3OD)
7.61 (8H, m); 13C NMR (CD3OD)
d
4.34 (2H, s), 4.66 (2H, s), 7.30e
30.02, 65.18, 126.48, 126.84,
Prepared from 52 following the same procedure that was used for
the preparation of 56 using hexane/CH2Cl2 ¼ 65/35 as eluent for the
chromatographic purification of 58 (70%): mp 165e167 ꢂC; IR (KBr)
d
127.09, 128.57, 129.92, 130.23, 135.65, 141.63, 141.85, 143.37, 157.19.
Calcd for C15H14N4O: C, 67.65; H, 5.30; N, 21.04. Found: C, 67.71; H,
5.27; N, 21.00.
2923, 2863, 2251, 1607, 1499, 1249 cmꢁ1; 1H NMR
d
3.76 (2H, s), 5.11
(2H, s), 7.03e7.56 (13H, m); 13C NMR
d 23.29, 70.14, 115.29, 127.37,
127.46, 128.03, 128.12, 128.19, 128.33, 128.63, 132.98, 136.90, 140.69,
158.65. Calcd for C21H17NO: C, 84.25; H, 5.72; N, 4.68. Found: C,
84.31; H, 5.68; N, 4.71.
4.1.5.4. (40-((1H(2H)-Tetrazol-5-yl)methyl)biphenyl-4-yl)methanol
(64). Yield 66%; mp 154e156 ꢂC; IR (KBr) 3160, 2856, 1567, 1498,
1249, 1031 cmꢁ1
;
1H NMR (DMSO-d6)
d
4.34 (2H, s), 4.54 (2H, s),
7.34e7.65 (8H, m); 13C NMR (DMSO-d6)
d
28.45, 62.51, 115.34,
4.1.4.14. 40-(Cyanomethyl)biphenyl-3-carboxylic acid (59). A solu-
tion of 47 (338 mg, 1.35 mmol) and 1 N LiOH (2.06 mL) in THF/
H2O ¼ 5/1 (23 mL) was stirred at room temperature overnight.
After acidification to pH 4 with 2 N HCl, the mixture was concen-
trated under vacuum and extracted with AcOEt. The organic phase
was washed with brine until neutral, dried (Na2SO4), and evapo-
rated under vacuum to give the acid 59 (310 mg, 97%), which was
used in the next step without further purification.
126.17,126.74,126.93,129.13,134.81,137.98,138.76,141.74. Calcd for
C15H14N4O: C, 67.65; H, 5.30; N, 21.04. Found: C, 67.70; H, 5.28; N,
21.08.
4.1.5.5. 5-((20-(Benzyloxy)biphenyl-4-yl)methyl)-1H(2H)-tetrazole
(65). Yield 88%; mp 118e119 ꢂC; IR (KBr) 3029, 2865, 2615, 1581,
1485, 1448, 1221, 1106, 1020, 752 cmꢁ1
;
1H NMR (CD3OD)
d
4.35
(2H, s), 5.05 (2H, s), 7.00e7.54 (13H, m); 13C NMR (CD3OD)
d 30.12,
71.64, 114.72, 122.44, 128.29, 128.71, 129.28, 129.40, 129.92, 131.32,
131.74, 132.06, 135.12, 138.68, 139.38, 157.02. Calcd for C21H18N4O:
C, 73.67; H, 5.30; N, 16.36. Found: C, 73.70; H, 5.28; N, 16.33.
4.1.4.15. 40-(Cyanomethyl)biphenyl-3-carboxamide (60). To a stirred
solution of 59 (300 mg, 1.26 mmol) in DMF (5 mL) 1-
hydroxybenzotriazole (HOBt, 204 mg, 1.33 mmol) and N-ethyl-N0-
(3-dimethylaminopropyl)carbodiimide
hydrochloride
(EDC,
4.1.5.6. 5-((30-(Benzyloxy)biphenyl-4-yl)methyl)-1H(2H)-tetrazole
255 mg, 1.33 mmol) were added at 0 ꢂC. The mixture was stirred for
(66). Yield 90%; mp 144e145 ꢂC; IR (KBr) 2867, 2608, 1581, 1565,
15 min at 0 ꢂC and for 1 h at room temperature. Then, a 25% NH3
1479, 1407, 1294, 1197, 1049 cmꢁ1; 1H NMR (CD3OD)
d
4.32 (2H, s),
aqueous solution (153
mL, 1.89 mmol) wad added and the mixture
5.09 (2H, s), 6.93e7.56 (13H, m); 13C NMR (CD3OD)
d 30.03, 71.09,
was stirred overnight at room temperature. The mixture was
diluted with brine and extracted with AcOEt. The organic phase was
washed with 2 N HCl, brine, saturated NaHCO3, and brine, dried
(Na2SO4), and evaporated under vacuum to give 60 (298 mg, 100%):
mp 193e195 ꢂC; IR (KBr) 3328, 3147, 2254, 1668, 1628, 1580, 1447,
114.72, 114.93, 120.64, 128.61, 128.88, 129.51, 130.20, 130.94, 135.75,
138.71, 141.49, 143.26, 160.66. Calcd for C21H18N4O: C, 73.67; H,
5.30; N, 16.36. Found: C, 73.73; H, 5.27; N, 16.31.
4.1.5.7. 5-((40-(Benzyloxy)biphenyl-4-yl)methyl)-1H(2H)-tetrazole
1389, 1121 cmꢁ1; 1H NMR (DMSO-d6)
m); 13C NMR (DMSO-d6)
d
4.11 (2H, s), 7.47e8.19 (8H,
(67). Yield 83%; mp 216e218 ꢂC; IR (KBr) 3034, 2864, 1607, 1500,
d
22.01, 119.08, 125.56, 126.67, 127.27,
1252 cmꢁ1
;
1H NMR (DMSO-d6)
d
4.32 (2H, s), 5.15 (2H, s), 7.08e
128.65, 128.89, 129.27, 130.72, 134.92, 138.84, 139.38, 167.71. Calcd
for C15H12N2O: C, 76.25; H, 5.12; N, 11.86. Found: C, 76.31; H, 5.09;
N, 11.82.
7.64 (13H, m); 13C NMR (DMSO-d6)
d
28.40, 69.15, 115.16, 126.38,
126.57, 127.54, 127.57, 127.72, 128.34, 128.50, 129.07, 132.19, 136.96,
138.45, 157.89. Calcd for C21H18N4O: C, 73.67; H, 5.30; N, 16.36.
Found: C, 73.75; H, 5.26; N, 16.30.
4.1.5. General procedure for the preparation of tetrazoles 61e71
A solution of the appropriate 20-, 30- or 40-monosubstituted 4-
biphenylacetonitrile (46e49, 53e58, 60) and n-Bu3SnN3 [18]
(1.5 equiv) in 1,4-dioxane (2 mL/mmol) was refluxed overnight,
cooled at room temperature, and gaseous HCl was added over
15 min. The mixture was evaporated under vacuum and the residue
was triturated with hot hexane. The resulting solid was filtered off,
washed with hexane, and dried under vacuum.
4.1.5.8. 40-((1H(2H)-Tetrazol-5-yl)methyl)biphenyl-3-carboxamide
(68). Yield 75%; mp 222e224 ꢂC; IR (KBr) 3304, 3132, 2253, 1670,
1627, 1580, 1447, 1415, 1390, 1122 cmꢁ1; 1H NMR (DMSO-d6)
d
4.36
(2H, s), 7.38e8.15 (8H, m); 13C NMR (DMSO-d6)
d 28.46, 125.48,
126.49, 127.03, 128.81, 129.16, 129.21, 134.89, 135.35, 138.24, 139.57,
167.66. Calcd for C15H13N5O: C, 64.51; H, 4.69; N, 25.07. Found: C,
64.59; H, 4.65; N, 25.01.
4.1.5.1. 1-(40-((1H(2H)-Tetrazol-5-yl)methyl)biphenyl-3-yl)ethanone
4.1.5.9. 5-((20-Methylbiphenyl-4-yl)methyl)-1H(2H)-tetrazole (69).
Yield 82%; mp 188e189 ꢂC; IR (KBr) 2978, 2719, 2615, 1575, 1483,
(61). Yield 76%; mp 159e161 ꢂC; IR (KBr) 2855, 2783, 2617, 1684,
1437, 1359, 1236, 1051, 790 cmꢁ1
;
1H NMR (CD3OD)
d
2.64 (3H, s),
1430, 1256, 1106,1049, 759 cmꢁ1; 1H NMR (DMSO-d6)
d
2.22 (3H, s),
4.37 (2H, s), 7.39e8.19 (8H, m); 13C NMR (CD3OD)
d
26.85, 29.85,
4.36 (2H, s), 7.17e7.35 (8H, m); 13C NMR (DMSO-d6)
d 20.07, 28.49,

128
G. Ortar et al. / European Journal of Medicinal Chemistry 63 (2013) 118e132
125.85, 127.23, 128.42, 129.19,129.38, 130.25, 134.46, 134.59, 139.90,
140.73, 155.16. Calcd for C15H14N4: C, 71.98; H, 5.64; N, 22.38.
Found: C, 72.03; H, 5.60; N, 22.34.
for C19H19N5O3: C, 62.46; H, 5.24; N, 19.17. Found: C, 62.41; H, 5.27;
N, 19.20.
4.1.6.3. 5-((30-(Hydroxymethyl)biphenyl-4-yl)methyl)-N,N-dimethyl-
1H-tetrazole-1-carboxamide (13) and 5-((30-(hydroxymethyl)
biphenyl-4-yl)methyl)-N,N-dimethyl-2H-tetrazole-2-carboxamide
(24). CH2Cl2/acetone ¼ 96/4 was used as eluent for the chro-
matographic separation of regioisomers. Compound 24 (less polar,
4.1.5.10. 5-((30-Methylbiphenyl-4-yl)methyl)-1H(2H)-tetrazole (70).
Yield 71%; mp 149e152 ꢂC; IR (KBr) 2856, 2717, 1572, 1485, 1409,
1258, 1050, 766 cmꢁ1; 1H NMR (DMSO-d6)
d
2.37 (3H, s), 4.34 (2H,
d 21.01, 28.43, 123.62,
s), 7.16e7.64 (8H, m); 13C NMR (DMSO-d6)
126.87, 127.13, 127.20, 127.98, 128.71, 129.10, 134.91, 134.93, 137.95,
138.95, 139.59. Calcd for C15H14N4: C, 71.98; H, 5.64; N, 22.38.
Found: C, 72.06; H, 5.59; N, 22.31.
31%): oil; IR 3603, 3435, 3009, 2932, 1742, 1484, 1393, 1068 cmꢁ1
;
1H NMR
7.60 (8H, m); 13C NMR
d
3.09 (3H, s), 3.23 (3H, s), 4.35 (2H, s), 4.73 (2H, s), 7.26e
31.40, 38.14, 39.19, 65.26, 125.63, 125.90,
d
126.28, 127.52, 128.99, 129.34, 135.05, 139.90, 140.97, 141.53, 147.81,
165.12. Calcd for C18H19N5O2: C, 64.08; H, 5.68; N, 20.76. Found: C,
64.00; H, 5.72; N, 20.81. Compound 13 (more polar, 8%): oil; IR
4.1.5.11. 5-((40-Methylbiphenyl-4-yl)methyl)-1H(2H)-tetrazole (71).
Yield (63%); mp 214e215 ꢂC; IR (KBr) 2856, 2659, 2613, 1575, 1499,
1408, 1259, 1194, 1049 cmꢁ1; 1H NMR (DMSO-d6)
d
2.34 (3H, s), 4.33
3603, 3435, 3007, 2938,1732,1483,1396,1068 cmꢁ1; 1H NMR
d
2.69
(3H, s), 3.06 (3H, s), 4.45 (2H, s), 4.75 (2H, s), 7.27e7.56 (8H, m); 13C
NMR 29.59, 37.45, 38.80, 65.11, 125.51, 126.16, 127.48, 129.07,
(2H, s), 7.25e7.62 (8H, m); 13C NMR (DMSO-d6)
d
20.55, 28.45,
126.30, 126.62, 126.71, 129.11, 129.41, 134.69, 136.64, 136.73, 138.76.
Calcd for C15H14N4: C, 71.98; H, 5.64; N, 22.38. Found: C, 72.04; H,
5.61; N, 22.33.
d
129.46, 129.63, 133.34, 140.44, 140.52, 141.69, 147.88, 155.72. Calcd
for C18H19N5O2: C, 64.08; H, 5.68; N, 20.76. Found: C, 63.99; H, 5.73;
N, 20.82.
4.1.6. General procedure for the preparation of carbamoyl tetrazoles
11e14, 18e25, 29e32, 72e77
4.1.6.4. 5-((40-(Hydroxymethyl)biphenyl-4-yl)methyl)-N,N-dimethyl-
1H-tetrazole-1-carboxamide (14) and 5-((40-(hydroxymethyl)
biphenyl-4-yl)methyl)-N,N-dimethyl-2H-tetrazole-2-carboxamide
(25). CH2Cl2/acetone ¼ 95/5 was used as eluent for the chro-
matographic separation of regioisomers. Compound 25 (less polar,
16%): mp 91e93 ꢂC; IR (KBr) 3299, 2922, 1734, 1499, 1394, 1166,
To a stirred solution of the appropriate tetrazole (61e71) and
Et3N (2 equiv) in acetonitrile (1.5 mL/mmol) Me2NCOCl (1.5 equiv)
was added at 0 ꢂC, and the mixture was stirred at 0 ꢂC overnight.
The mixture was diluted with brine and extracted with AcOEt. The
organic phase was washed twice with brine, dried (Na2SO4), and
evaporated under vacuum. The residue was chromatographed on
silica gel using CH2Cl2/acetone or CH2Cl2/MeOH mixtures as elu-
ents, the less polar 2,5-disubstituted tetrazole eluting prior to the
1,5-disubstituted regioisomer.
1002 cmꢁ1; 1H NMR
(2H, s), 4.71 (2H, s), 7.39e7.55 (8H, m); 13C NMR
d
2.08 (1H, br s), 3.09 (3H, s), 3.23 (3H, s), 4.35
31.39, 38.15,
d
39.20, 64.96, 127.15, 127.44, 129.35, 134.98, 139.74, 139.98, 140.09,
165.12. Calcd for C18H19N5O2: C, 64.08; H, 5.68; N, 20.76. Found: C,
64.02; H, 5.71; N, 20.79. Compound 14 (more polar, 9%): mp 134e
135 ꢂC; IR (KBr) 3279, 2926, 1731, 1495, 1392, 1252, 1087 cmꢁ1; 1H
4.1.6.1. 5-((30-Acetylbiphenyl-4-yl)methyl)-N,N-dimethyl-1H-tetra-
zole-1-carboxamide (11) and 5-((30-acetylbiphenyl-4-yl)methyl)-
N,N-dimethyl-2H-tetrazole-2-carboxamide (22).
NMR
d
1.94 (1H, br s), 2.71 (3H, s), 3.07 (3H, s), 4.48 (2H, s), 4.74 (2H,
29.64, 37.46, 38.83, 64.94, 127.12,
s), 7.27e7.56 (8H, m); 13C NMR
d
CH2Cl2/acetone ¼ 98/2 was used as eluent for the chromatographic
127.40, 127.52, 129.51, 133.33, 139.61, 140.28, 140.38, 155.75. Calcd
for C18H19N5O2: C, 64.08; H, 5.68; N, 20.76. Found: C, 64.04; H, 5.69;
N, 20.81.
separation of regioisomers. Compound 22 (less polar, 38%): oil; IR
3007, 2930, 1743, 1684, 1496, 1393, 1238, 1068 cmꢁ1; 1H NMR
d
2.64
(3H, s), 3.10 (3H, s), 3.24 (3H, s), 4.37 (2H, s), 7.43e8.15 (8H, m); 13C
NMR 26.70, 31.44, 38.13, 39.18, 126.85, 127.25, 127.59, 129.08,
d
4.1.6.5. 5-((30-Carbamoylbiphenyl-4-yl)methyl)-N,N-dimethyl-1H-
tetrazole-1-carboxamide (18) and 5-((30-carbamoylbiphenyl-4-yl)
129.54, 131.60, 135.63, 137.72, 139.13, 141.23, 147.81, 165.05, 197.89.
Calcd for C19H19N5O2: C, 65.32; H, 5.48; N, 20.04. Found: C, 65.25; H,
5.52; N, 20.10. Compound 11 (more polar, 16%): mp 153e154 ꢂC; IR
methyl)-N,N-dimethyl-2H-tetrazole-2-carboxamide
(29).
CH2Cl2/MeOH ¼ 97/3 was used as eluent for the chromatographic
separation of regioisomers. Compound 29 (less polar, 28%): mp
161e163 ꢂC; IR (KBr) 3310, 3133, 1739, 1673, 1629, 1604, 1417, 1389,
3005, 2940, 1731, 1684, 1497, 1397, 1238, 1088 cmꢁ1; 1H NMR
d
2.65
(3H, s), 2.78 (3H, s), 3.10 (3H, s), 4.50 (2H, s), 7.36e8.15 (8H, m); 13C
NMR 26.72, 29.72, 37.55, 38.95, 126.69, 127.55, 127.59, 129.18,
d
1126, 1069 cmꢁ1; 1H NMR (CDCl3/CD3OD ¼ 9/1)
d 3.12 (3H, s), 3.26
129.65, 131.54, 134.00, 137.80, 139.65, 140.90, 147.93, 155.73, 197.88.
Calcd for C19H19N5O2: C, 65.32; H, 5.48; N, 20.04. Found: C, 65.28; H,
5.51; N, 20.07.
(3H, s), 4.37 (2H, s), 7.35e8.06 (8H, m); 13C NMR (CDCl3/CD3OD ¼ 9/
1) d 31.45, 38.23, 39.28,126.32,126.37,127.65,129.15,129.57,130.59,
134.02, 135.57, 139.25, 141.22, 148.02, 165.23, 170.44. 170.49. Calcd
for C18H18N6O2: C, 61.70; H, 5.18; N, 23.99. Found: C, 61.64; H, 5.21;
N, 24.04. Compound 18 (more polar, 13%): oil; IR 3530, 3413, 2929,
2855, 1731, 1677, 1586, 1441, 1396, 1369, 1230, 1088 cmꢁ1; 1H NMR
4.1.6.2. Methyl 40-((1-(dimethylcarbamoyl)-1H-tetrazol-5-yl)methyl)
biphenyl-3-carboxylate (12) and methyl 40-((2-(dimethylcarbamoyl)-
2H-tetrazol-5-yl)methyl)biphenyl-3-carboxylate
(23).
d
2.77 (3H, s), 3.10 (3H, s), 4.48 (2H, s), 6.12 (1H, br s), 6.40 (1H, br s),
CH2Cl2/acetone ¼ 98/2 was used as eluent for the chromatographic
7.27e8.05 (8H, m); 13C NMR
d
29.69, 37.55, 38.93, 126.16, 126.29,
separation of regioisomers. Compound 23 (less polar, 28%): oil; IR
127.56, 129.14, 129.60, 130.43, 133.92, 134.10, 139.57, 140.91, 147.89,
155.71, 169.27. Calcd for C18H18N6O2: C, 61.70; H, 5.18; N, 23.99.
Found: C, 61.62; H, 5.23; N, 24.05.
3007, 2953, 1742, 1495, 1443, 1394, 1311, 1252 cmꢁ1; 1H NMR
d
3.09
(3H, s), 3.23 (3H, s), 3.92 (3H, s), 4.37 (2H, s), 7.42e8.25 (8H, m); 13C
NMR 31.38, 38.10, 39.14, 52.14, 127.47, 128.07, 128.36, 128.88,
d
129.46, 130.73, 131.35, 135.54, 138.93, 140.88, 147.77, 164.99, 166.92.
Calcd for C19H19N5O3: C, 62.46; H, 5.24; N, 19.17. Found: C, 62.38; H,
5.28; N, 19.22. Compound 12 (more polar, 16%): mp 153e154 ꢂC; IR
4.1.6.6. N,N-Dimethyl-5-((20-methylbiphenyl-4-yl)methyl)-1H-tetra-
zole-1-carboxamide (19) and N,N-dimethyl-5-((20-methylbiphenyl-4-
yl)methyl)-2H-tetrazole-2-carboxamide (30). CHCl3/acetone ¼ 99/1
was used as eluent for the chromatographic separation of
regioisomers. Compound 30 (less polar, 38%): oil; IR 3034, 2943,
3007, 2953, 1727, 1444, 1397, 1310, 1251 cmꢁ1; 1H NMR
d
2.76 (3H,
s), 3.10 (3H, s), 3.95 (3H, s), 4.50 (2H, s), 7.35e8.24 (8H, m); 13C NMR
29.68, 37.52, 38.90, 52.23, 127.52, 128.09, 128.62, 128.99, 129.61,
130.87, 131.33, 133.88, 139.55, 140.57, 147.91, 155.69, 166.93. Calcd
d
1743, 1601, 1484, 1393, 1166, 1068 cmꢁ1; 1H NMR
(3H, s), 3.22 (3H, s), 4.36 (2H, s), 7.19e7.39 (8H, m); 13C NMR
d
2.24 (3H, s), 3.09
20.42,
d

G. Ortar et al. / European Journal of Medicinal Chemistry 63 (2013) 118e132
129
31.41, 38.08, 39.13, 125.76, 127.27, 128.58, 129.54, 129.71, 130.31,
134.41,135.26, 140.77,141.36,147.80, 165.90. Calcd for C18H19N5O: C,
67.27; H, 5.96; N, 21.79. Found: C, 67.21; H, 6.01; N, 21.74. Com-
pound 19 (more polar, 12%): oil; IR 3034, 2937, 1729, 1602, 1483,
solution of the crude mixture of 72 and 73 (524 mg, w1.27 mmol) in
AcOEt (8 mL) and MeOH (2 mL) was hydrogenated in the presence
of 10% Pd/C (175 mg) at room temperature and atmospheric pres-
sure for 15 h. The suspension was filtered through a short pad of
silica gel. The filtrate was evaporated under vacuum and the residue
(347 mg) was chromatographed on silica gel (35 g) using CH2Cl2/
acetone ¼ 95/5 as eluent for the chromatographic separation of
regioisomers. Compound 26 (less polar, 150 mg, 37%): oil; IR 3556,
1396, 1258, 1088 cmꢁ1; 1H NMR
s), 4.49 (2H, s), 7.15e7.32 (8H, m); 13C NMR
d
2.24 (3H, s), 2.70 (3H, s), 3.08 (3H,
20.43, 29.69, 37.43,
d
38.73, 125.91, 127.55, 128.82, 129.66, 130.44, 132.83, 135.12, 141.06,
141.51, 147.98, 155.78. Calcd for C18H19N5O: C, 67.27; H, 5.96; N,
21.79. Found: C, 67.20; H, 6.00; N, 21.75.
3011, 1744, 1483, 1394, 1180 cmꢁ1; 1H NMR
s), 4.33 (2H, s), 6.52 (1H, br s), 6.93e7.45 (8H, m); 13C NMR
d
3.08 (3H, s), 3.21 (3H,
31.40,
d
4.1.6.7. N,N-Dimethyl-5-((30-methylbiphenyl-4-yl)methyl)-1H-tetra-
zole-1-carboxamide (20) and N,N-dimethyl-5-((30-methylbiphenyl-4-
yl)methyl)-2H-tetrazole-2-carboxamide (31). CH2Cl2/acetone ¼ 98/2
was used as eluent for the chromatographic separation of
regioisomers. Compound 31 (less polar, 11%): oil; IR 3023, 2939,
38.14, 39.18, 116.02, 120.74, 127.78, 129.09, 129.48, 129.55, 130.31,
135.21, 136.38, 147.79, 152.73, 164.98. Calcd for C17H17N5O2: C,
63.15; H, 5.30; N, 21.66. Found: C, 63.08; H, 5.25; N, 21.72. Com-
pound 15 (91 mg, more polar, 22%): mp 142e144 ꢂC; IR 3560, 3009,
1730, 1483, 1397, 1257, 1088 cmꢁ1; 1H NMR
s), 4.44 (2H, s), 6.93e7.48 (8H, s); 13C NMR
d
d
2.69 (3H, s), 3.05 (3H,
29.66, 37.43, 38.79,
1742, 1604, 1490, 1394, 1216 cmꢁ1; 1H NMR
s), 3.24 (3H, s), 4.36 (2H, s), 7.14e7.55 (8H, m); 13C NMR
d
2.40 (3H, s), 3.10 (3H,
21.51,
d
116.17, 120.81, 127.59, 129.14, 129.36, 129.69, 130.33, 133.09, 137.40,
147.81, 152.85, 155.75. Calcd for C17H17N5O2: C, 63.15; H, 5.30; N,
21.66. Found: C, 63.09; H, 5.27; N, 21.70.
31.40, 38.13, 39.19, 124.14, 127.50, 127.82, 128.05, 128.66, 129.25,
134.81, 138.32, 140.23, 140.65, 165.15. Calcd for C18H19N5O: C, 67.27;
H, 5.96; N, 21.79. Found: C, 67.19; H, 6.01; N, 21.73. Compound 20
(more polar, 10%): oil; IR 3011, 2930, 1732, 1604, 1485, 1397, 1256,
4.1.6.13. 5-((30-(Hydroxy)biphenyl-4-yl)methyl)-N,N-dimethyl-1H-
tetrazole-1-carboxamide (16) and 5-((30-(hydroxy)biphenyl-4-yl)
methyl)-N,N-dimethyl-2H-tetrazole-2-carboxamide (27). The title
compounds were obtained from the mixture of 74 and 75 following
the same procedure that was employed for the preparation of 15
and 26 using CH2Cl2/acetone ¼ 96/4 as eluent for the chromato-
graphic separation of regioisomers. Compound 27 (less polar,
81 mg, 20%): oil; IR 3595, 3316, 3006, 2940, 1740, 1598, 1494, 1393,
1089 cmꢁ1
;
1H NMR
d
2.41 (3H, s), 2.67 (3H, s), 3.06 (3H, s), 4.48
21.52, 29.61, 37.39, 38.75,
(2H, s), 7.17e7.56 (8H, m); 13C NMR
d
124.07, 127.47, 127.73, 128.34, 128.79, 129.41, 133.14, 138.50, 140.22,
140.76, 147.91, 155.73. Calcd for C18H19N5O: C, 67.27; H, 5.96;
N, 21.79. Found: C, 67.18; H, 6.02; N, 21.72.
4.1.6.8. N,N-Dimethyl-5-((40-methylbiphenyl-4-yl)methyl)-1H-tetra-
zole-1-carboxamide (21) and N,N-dimethyl-5-((40-methylbiphenyl-4-
yl)methyl)-2H-tetrazole-2-carboxamide (32). CH2Cl2/acetone ¼ 98/2
was used as eluent for the chromatographic separation of
regioisomers. Compound 32 (less polar, 31%): oil; IR 3013, 2941,
1307, 1174 cmꢁ1
;
1H NMR
d
3.09 (3H, s), 3.23 (3H, s), 4.32 (2H, s),
31.35, 38.21, 39.23,
6.52 (1H, br s), 6.80e7.48 (8H, m); 13C NMR
d
114.10, 114.46, 119.24, 127.45, 129.25, 129.91, 134.92, 139.78, 142.23,
147.86, 156.36, 165.19. Calcd for C17H17N5O2: C, 63.15; H, 5.30; N,
21.66. Found: C, 63.07; H, 5.35; N, 21.72. Compound 16 (62 mg,
more polar, 15%): mp 158e160 ꢂC; IR 3595, 3312, 3015, 2939, 1732,
1741, 1602, 1500, 1394, 1223, 1068 cmꢁ1; 1H NMR
(3H, s), 3.23 (3H, s), 4.35 (2H, s), 7.22e7.54 (8H, m); 13C NMR
d
2.38 (3H, s), 3.09
21.07,
d
31.40, 38.12, 39.17, 126.86, 127.30, 129.28, 134.62, 137.07, 137.79,
140.05, 147.80, 165.18. Calcd for C18H19N5O: C, 67.27; H, 5.96; N,
21.79. Found: C, 67.19; H, 5.92; N, 21.82. Compound 21 (more polar,
18%): oil; IR 3014, 2928, 1731, 1602, 1500, 1398, 1257, 1088 cmꢁ1; 1H
1599, 1480, 1397, 1307, 1088 cmꢁ1; 1H NMR
s), 4.46 (2H, s), 6.82e7.55 (8H, s); 13C NMR
d
2.66 (3H, s), 3.06 (3H,
29.63, 37.45, 38.79,
d
114.06, 114.75, 118.54, 127.56, 129.48, 130.04, 133.13, 140.83, 141.81,
147.98, 155.96, 157.38. Calcd for C17H17N5O2: C, 63.15; H, 5.30; N,
21.66. Found: C, 63.09; H, 5.26; N, 21.71.
NMR
d
2.39 (3H, s), 2.67 (3H, s), 3.05 (3H, s), 4.47 (2H, s), 7.23e7.55
21.08, 29.61, 37.39, 38.74, 126.78, 127.24, 129.43,
(8H, m); 13C NMR
d
129.60, 132.93, 137.35, 137.45, 140.56, 147.91, 155.74. Calcd for
C18H19N5O: C, 67.27; H, 5.96; N, 21.79. Found: C, 67.20; H, 5.91;
N, 21.85.
4.1.6.14. 5-((40-(Hydroxy)biphenyl-4-yl)methyl)-N,N-dimethyl-1H-
tetrazole-1-carboxamide (17) and 5-((40-(hydroxy)biphenyl-4-yl)
methyl)-N,N-dimethyl-2H-tetrazole-2-carboxamide (28). The title
compounds were obtained from the mixture of 76 and 77 following
the same procedure that was employed for the preparation of 15
and 26 using CH2Cl2/acetone ¼ 95/5 as eluent for the chromato-
graphic separation of regioisomers. Compound 28 (less polar, 27%):
mp 222 ꢂC; IR (KBr) 3260, 2926, 1741, 1611, 1499, 1386, 1271,
4.1.6.9. 5-((20-(Benzyloxy)biphenyl-4-yl)methyl)-N,N-dimethyl-1H-
tetrazole-1-carboxamide (72) and 5-((20-(benzyloxy)biphenyl-4-yl)
methyl)-N,N-dimethyl-2H-tetrazole-2-carboxamide
(73).
Prepared according to the general procedure and used in the next
step as a mixture without further purification.
1172 cmꢁ1; 1H NMR (CD3OD)
6.82e7.50 (8H, m); 13C NMR (CD3OD)
d
3.03 (3H, s), 3.21 (3H, s), 4.32 (2H, s),
31.93, 38.20, 39.39, 116.66,
d
4.1.6.10. 5-((30-(Benzyloxy)biphenyl-4-yl)methyl)-N,N-dimethyl-1H-
tetrazole-1-carboxamide (74) and 5-((30-(benzyloxy)biphenyl-4-yl)
127.75, 128.97, 130.25, 133.32, 135.73, 141.27, 158.25, 166.72. Calcd
for C17H17N5O2: C, 63.15; H, 5.30; N, 21.66. Found: C, 63.11; H, 5.27;
N, 21.70. Compound 17 (more polar, 9%): mp 205 ꢂC; IR (KBr) 3143,
methyl)-N,N-dimethyl-2H-tetrazole-2-carboxamide
(75).
Prepared according to the general procedure and used in the next
step as a mixture without further purification.
1736, 1608, 1497, 1383, 1251, 1084 cmꢁ1
;
1H NMR (CD3OD)
d
2.67
(3H, s), 3.08 (3H, s), 4.46 (2H, s), 6.87e7.61 (8H, m); 13C NMR
(CD3OD) 29.84, 37.54, 38.91, 116.25, 127.41, 128.50, 129.90, 132.24,
d
4.1.6.11. 5-((40-(Benzyloxy)biphenyl-4-yl)methyl)-N,N-dimethyl-1H-
tetrazole-1-carboxamide (76) and 5-((40-(benzyloxy)biphenyl-4-yl)
132.64, 141.35, 156.54, 157.56. Calcd for C17H17N5O2: C, 63.15; H,
5.30; N, 21.66. Found: C, 63.10; H, 5.28; N, 21.69.
methyl)-N,N-dimethyl-2H-tetrazole-2-carboxamide
(77).
Prepared according to the general procedure and used in the next
4.2. Biological evaluation
step as a mixture without further purification.
4.2.1. Assay of fatty amide hydrolase (FAAH)
4.1.6.12. 5-((20-(Hydroxy)biphenyl-4-yl)methyl)-N,N-dimethyl-1H-
tetrazole-1-carboxamide (15) and 5-((20-(hydroxy)biphenyl-4-yl)
methyl)-N,N-dimethyl-2H-tetrazole-2-carboxamide (26). A stirred
The effect of compounds on the enzymatic hydrolysis of anan-
damide was obtained using membranes prepared from rat brain,
incubated with the test compounds and [14C]AEA (2.4
mM) in

130
G. Ortar et al. / European Journal of Medicinal Chemistry 63 (2013) 118e132
50 mM TriseHCl, pH 9, for 30 min at 37 ꢂC. [14C]Ethanolamine
produced from [14C]AEA hydrolysis was measured by scintillation
counting of the aqueous phase after extraction of the incubation
mixture with 2 volumes of CHCl3/CH3OH ¼ 2/1 (by volume). Data
are expressed as the concentration exerting 50% inhibition of AEA
hydrolysis (IC50), calculated by GraphPad.
transfected clones were selected by G-418 (Invitrogen; 600 mg/ml).
The effect of the substances on intracellular Ca2þ concentration
([Ca2þ]i) was determined by using Fluo-4-AM, a selective intracel-
lular fluorescent probe for Ca2þ. On the day of the experiment, cells
were loaded for 1 h at room temperature with the methyl ester
Fluo-4-AM (4
Tyrode’s buffer (145 mM NaCl, 2.5 mM KCl, 1.5 mM CaCl2, 1.2 mM
MgCl2, 10 mM -glucose, and 10 mM HEPES, pH 7.4), resuspended
mM; Invitrogen) in EMEM, then were washed twice in
4.2.2. Assay of 2-AG hydrolysis
D
The 10,000 g cytosolic fraction from COS-7 cells, which contains
high levels of MAGL, was incubated in TriseHCl 50 mM, at pH 7.0 at
in Tyrode’s buffer, and transferred to the quartz cuvette of the
spectrofluorimeter (PerkineElmer LS50B; PerkinElmer Life and
Analytical Sciences, Waltham, MA) under continuous stirring.
[Ca2þ]i was determined before and after the addition of various
concentrations of test compounds by measuring cell fluorescence
37 ꢂC for 20 min, with synthetic [3H]2-AG (1.0 mCi/mmol, 25
mM).
After the incubation, lipids were extracted with 2 volumes of CHCl3/
CH3OH ¼ 2/1 (by volume), and the extracts were lyophilized under
vacuum. Extracts were fractionated by TLC on silica on plastic plates
using CHCl3/MeOH/NH4OH (85/15/1 by volume) as the eluting
system. Bands corresponding to [3H]arachidonic acid were cut, and
(
lEX ¼ 488 nm, lEM ¼ 516 nm). Curve fitting (sigmoidal dosee
response variable slope) and parameter estimation were per-
formed with GraphPad PrismÒ (GraphPad Software Inc., San Diego,
CA). Potency was expressed as the concentration of test substances
exerting a half-maximal agonist effect (i.e. half-maximal increases
in [Ca2þ]i) (EC50). The efficacy of TRPV1 agonists was first deter-
mined by normalizing their effect to the maximum Ca2þ influx
their radioactivity was counted with a b-counter.
4.2.3. Z0-Factor evaluation
The Z0-factor was used to evaluate the quality of the assays on
FAAH and MAGL and was calculated from the following equation:
effect on [Ca2þ]i observed with application of 4
(Sigma). The effects of TRPA1 agonists are expressed as a percent-
age of the effect obtained with 100 M allyl isothiocyanate (AITC).
mM ionomycin
Z0 ¼ 1 ꢁ (3scþ þ 3scꢁ)/jmcꢁ
ꢁ
mcþj where scþ is the standard devi-
M for FAAH test and
M for MAGL test) and mcþ is the means of
ation of the positive control (URB597 0.1
m
m
OMDM169 [31] 1.0
m
For both TRPA1 and TRPV1 agonism, the values of the effect on
[Ca2þ]i in wild type HEK293 (i.e. not transfected with any TRP
construct) were taken as baselines and subtracted from the values
obtained from transfected cells. Antagonist/desensitizing behav-
sample signals, while scꢁ and mcꢁ are values of the negative control,
i.e. the test without inhibitor. Z0-Factor was found to be 0.77 ꢀ 0.05
for FAAH and 0.78 ꢀ 0.10 for MAGL denoting an excellent assay [32].
iour was evaluated against capsaicin (0.1
mM) for TRPV1 and against
4.2.4. Assay of endocannabinoid levels in intact COS-7 cells
AITC (100 M) for TRPA1, by adding the test compounds in the
m
For each data point, about 106 COS-7 cells overexpressing the
quartz cuvette 5 min before stimulation of cells with agonists. Data
are expressed as the concentration exerting a half-maximal inhi-
human recombinant DAGL-
a
[33] were incubated for 20 min at
M with or without pre-
37 ꢂC with vehicle, or ionomycin 3
m
bition of agonist-induced [Ca2þ
] elevation (IC50), which was
i
incubation (10 min) with compound 27. Cells were then homoge-
nized in 5 volumes of CHCl3/CH3OH/TriseHCl 50 mM pH 7.4 (2/1/1)
containing 5 pmol of d8-AEA, d5-2-AG and d4-OEA as internal
standards. Homogenates were centrifuged at 13,000ꢃ g for 15 min
(4 ꢂC), the aqueous phase plus debris was collected and extracted
again twice with 1 volume of CHCl3. Lipid-containing organic
phases from the three extractions were pooled and the organic
solvents evaporated under vacuum. Lyophilized extracts were re-
suspended in chloroform/methanol ¼ 99/1 (by volume). The solu-
tions were then purified by open bed chromatography on silica gel
as described in Bisogno et al. [34] and analyzed by isotope dilution
liquid chromatography (LC)-atmospheric pressure chemical ioni-
zation (APCI)-mass spectrometry (MS) (LC-APCI-MS) using a Shi-
madzu HPLC apparatus (LC-10ADVP) coupled to a Shimadzu
(LCMS-2010) quadrupole MS via a Shimadzu APCI interface [35].
MS analyzes were carried out in the selected ion-monitoring mode.
AEA, 2-AG, and OEA levels were quantified on the basis of their area
ratio with deuterated internal standards signal area. For 2-AG, the
areas of the peaks corresponding to 1(3)-and 2-isomers were added
together. Amounts in pmols were normalized per mg of lipid
extract. All determinations are at least in triplicate, data were sta-
tistically evaluated by one-way ANOVA followed by the Bonferro-
ni’s test.
calculated again using GraphPad PrismÒ software. The effect on
[Ca2þ]i exerted by agonist alone was taken as 100%. Dose response
curves were fitted by a sigmoidal regression with variable slope. All
determinations were performed at least in triplicate. Statistical
analysis of the data was performed by analysis of variance at each
point using ANOVA followed by the Bonferroni’s test.
4.3. Molecular modelling
4.3.1. FAAH experimental structure preparation
Fourteen FAAH experimental structures (Supplementary mate-
rial, Table SM 1) co-crystallized with 12 covalent and 2 reversible
inhibitors were retrieved from Brookheaven Data Bank (PDB) [36].
In 50% of the covalent complexes (PDB IDs: 1MT5, 2BYA, 2WAP,
3LJ6, 3LJ7, and 3QKV) the found experimental PDB structures dis-
played only a portion of them bound to the catalytic Ser214 oxygen
and all in the acyl-chain binding pocket (ABP), while the other 50%
(PDB IDs: 2WJ1, 2WJ2, 3K7F, 3K83, 3K84, 3OJ8) clearly all displayed
the more hydrophilic molecule ends to be in the cytosolic port
region (CA) and their hydrophobic ones all overlapped in the ABP
region. Therefore for the covalent docking studies the TS for 1MT5,
2BYA, 2WAP, 3LJ6, 3LJ7, and 3QKV were modelled by building the
leaving groups in the CA channel. A third pocked is also visible for
the non-covalent inhibitors 3QJ9, 3QK5 (membrane access channel,
MAC) partially overlapped with ABP.
4.2.5. TRPV1 and TRPA1 channel assays
HEK293 (human embryonic kidney) cells were grown as
monolayers in EMEM supplemented with nonessential amino
acids, 10% fetal bovine serum, and 2 mM glutamine, maintained
under 5% CO2 at 37 ꢂC plated on 100-mm diameter Petri dishes.
Cells were transfected at approximately 80% confluence with Lip-
ofectamine 2000 (Invitrogen, Carlsbad, CA) by using a plasmid
pcDNA3 (Invitrogen) containing the human TRPV1-cDNA, or the rat
TRPA1-cDNA, according to the manufacturer’s protocol. Stably
The structures of the 14 complexes were arbitrary super-
imposed using as template the better resolved complex (PDB
ꢀ
ID ¼ 2WJ1, R ¼ 1.84 A). The superimpositions of the FAAH com-
plexes were carried out by means of the program UCSF Chimera
[37] using the implementation of MatchMaker [38]. All crystal
waters were discarded, and hydrogen atoms were added using the
Leap module of AMBER suite [39]. All ligands were individually
inspected, and the correct protonation states at pH 7.4 were

G. Ortar et al. / European Journal of Medicinal Chemistry 63 (2013) 118e132
131
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assumed to be in the ionized form and the parameter were calcu-
lated by means of the Antechamber module of AMBER suite. The
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complexes were solvated (SOLVATEOCT command) in
a box
ꢀ
extending 10 A with water molecules (TIP3 model) and neutralized
with Naþ and Clꢁ ions. The solvated complexes were then refined
by a single point minimization using the Sander module of AMBER
suite. The minimized complexes were realigned with MatchMaker
using the same reference complex.
4.3.2. Covalent docking protocol
For the docking, the AutoDock Vina program [27] was used in a
similar fashion as described by Ahn [40]. A full docking protocol
similarly as described [28] was performed for docking program
assessment. In Table SM 1 (Supplementary material) are reported
the root mean square deviation errors (RMSDs) supporting that
AutoDock Vina could reproduce the experimental binding modes.
In the herein applied protocol, covalent docking was applied by
simply docking a water molecule while setting as flexible residue
the covalent inhibitors bound to the nucleophile Ser241 residue.
AutoDock 4 [41] was also tested as possible alternative docking
method. RMSDs were however much higher than those obtained
with AutoDock Vina. To select the binding, all the AutoDock Vina
poses were collected in a single bin and clustered using the clus-
tering feature of the AutoDock program. To inspect the binding
modes, the program UCSF Chimera 1.5.3 was used.
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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.2013.02.005.
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