Carbamoyl tetrazoles as inhibitors of endocannabinoid inactivation: A critical revisitation

Article abstract of DOI:10.1016/j.ejmech.2007.02.023

We have synthesized a series of 18 1,5- and 2,5-disubstituted carbamoyl tetrazoles, including LY2183240 (1) and LY2318912 (7), two compounds previously described as potent inhibitors of the cellular uptake of the endocannabinoid anandamide, and their regioisomers 2 and 8. We confirm that compound 1 is a potent inhibitor of both the cellular uptake and, like the other new compounds synthesized here, the enzymatic hydrolysis of anandamide. With the exception of 9, 12, 15, and the 2,5-regioisomer of LY2183240 2, the other compounds were all found to be weakly active or inactive on anandamide uptake. Several compounds also inhibited the enzymatic hydrolysis of the other main endocannabinoid, 2-arachidonoylglycerol, as well as its enzymatic release from sn-1-oleoyl-2-arachidonoyl-glycerol, at submicromolar concentrations. Four of the novel compounds, i.e. 3, 4, 17, and 18, inhibited anandamide hydrolysis potently (IC₅₀ = 2.1-5.4 nM) and selectively over all the other targets tested (IC₅₀ ≥ 10 μM), thus representing new potentially useful tools for the inhibition of fatty acid amide hydrolase.

Full text of DOI:10.1016/j.ejmech.2007.02.023

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 62e72
http://www.elsevier.com/locate/ejmech
Original article
Carbamoyl tetrazoles as inhibitors of endocannabinoid
inactivation: A critical revisitation
Giorgio Ortar a, Maria Grazia Cascio b,c, Aniello Schiano Moriello b, Mercedes Camalli d,
Enrico Morera a, Marianna Nalli a, Vincenzo Di Marzo b,
*
a Dipartimento di Studi Farmaceutici, Universita` di Roma ‘La Sapienza’, piazzale Aldo Moro 5, 00185 Roma, Italy
b Endocannabinoid Research Group, Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche,
via dei Campi Flegrei 34, 80078 Pozzuoli (Napoli), Italy
c Dipartimento di Scienze Farmaceutiche, Universita` degli Studi di Salerno, via Ponte Don Melillo, 84084 Fisciano (SA), Italy
d Istituto di Cristallografia, Consiglio Nazionale delle Ricerche, Area della Ricerca Roma 1, 00016 Monterotondo Stazione, Roma, Italy
Received 24 November 2006; accepted 28 February 2007
Available online 19 March 2007
Abstract
We have synthesized a series of 18 1,5- and 2,5-disubstituted carbamoyl tetrazoles, including LY2183240 (1) and LY2318912 (7), two
compounds previously described as potent inhibitors of the cellular uptake of the endocannabinoid anandamide, and their regioisomers 2 and 8.
We confirm that compound 1 is a potent inhibitor of both the cellular uptake and, like the other new compounds synthesized here, the enzymatic
hydrolysis of anandamide. With the exception of 9, 12, 15, and the 2,5-regioisomer of LY2183240 2, the other compounds were all found to be
weakly active or inactive on anandamide uptake. Several compounds also inhibited the enzymatic hydrolysis of the other main endocannabinoid,
2-arachidonoylglycerol, as well as its enzymatic release from sn-1-oleoyl-2-arachidonoyl-glycerol, at submicromolar concentrations. Four of the
novel compounds, i.e. 3, 4, 17, and 18, inhibited anandamide hydrolysis potently (IC50 ¼ 2.1e5.4 nM) and selectively over all the other targets
tested (IC50 ꢀ 10 mM), thus representing new potentially useful tools for the inhibition of fatty acid amide hydrolase.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Endocannabinoids; Anandamide cellular uptake inhibitors; Fatty acid amide hydrolase inhibitors; Carbamoyl tetrazoles
1. Introduction
are regulated by specific biosynthetic and degradative path-
ways, most of which have been now fully characterized and
the relevant enzymes cloned [9]. The termination of the action
of AEA is regulated by its cellular uptake, followed by intra-
cellular hydrolysis by fatty acid amide hydrolase (FAAH). The
mechanism of AEA transport, first characterized in 1994 by Di
Marzo et al. [10], has been proposed to be an ATP-indepen-
dent process of facilitated diffusion. However, a protein able
to specifically bind AEA and facilitate its migration across
the cell membrane has not yet been identified, its existence
is still controversial, and alternative hypotheses have been pro-
posed, including passive diffusion, endocytosis, and intracellu-
lar sequestration [11e17]. Much of the discussion in recent
years has been centred on the issue of whether or not AEA
uptake is driven uniquely by the FAAH-catalyzed hydrolysis
of the compound.
Endocannabinoids, i.e. endogenous agonists of CB1 and
CB2 cannabinoid receptors [1,2], are involved in an extraordi-
narily large number of physiological and pathological condi-
tions in mammals [3,4]. Increasing evidence suggests that
the modulation of endocannabinoid levels might be of valu-
able therapeutic relevance in the treatment of a variety of path-
ological conditions including anorexia, anxiety, inflammation,
nausea and emesis, neuropsychiatric disorders, pain and spas-
ticity [5]. The levels of the two main endocannabinoids, anan-
damide (AEA) [6] and 2-arachidonoylglycerol (2-AG) [7,8],
* Corresponding author. Tel.: þ39 081 8675093; fax: þ39 081 8041770.
E-mail address: vdimarzo@icmib.na.cnr.it (V. Di Marzo).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.02.023

G. Ortar et al. / European Journal of Medicinal Chemistry 43 (2008) 62e72
63
While the nature of the mechanism responsible for AEA
uptake is still a matter of debate, a large number of arachi-
donic or oleic acid-based compounds that effectively inhibit
this process have been described over the last decade.
Although the ability of many of them to inhibit also FAAH
has hampered the accurate characterization of their selectivity
for either target, the recent individuation of novel selective
AEA transport inhibitors that do not appear to inhibit FAAH
such as UCM707 [18], AM1172 [13] and OMDM1,2 [19]
has stimulated further studies to distinguish between the rela-
tive contributions of the putative AEA transporter and FAAH
in AEA removal process.
regioisomers, we have synthesized a series of nine regioisomeric
couples of 1,5- and 2,5-disubstituted carbamoyl tetrazoles,
including LY2183240, LY2318912 and their regioisomers
(Fig. 1), by varying the substituent in the 5-position, and
have investigated the effects of the regiochemistry and of
the 5-substitution on the activity of these compounds on
both AEA uptake and FAAH. The capability of these com-
pounds to also inhibit the MAGL as well as the diacylglycerol
lipase catalysing the biosynthesis of 2-AG [3] has also been
assessed.
2. Chemistry
From a medicinal chemistry perspective, the identification
of potent and selective inhibitors of AEA transport lacking
a long acyl side chain would be advantageous in terms of
more precise target identification and of their potential of be-
ing made into therapeutic drugs. In this respect, Hopkins and
Wang reported in 2004 in abstract form a non-arachidonoyl
compound, SEP-0200228 that inhibited AEA uptake in human
monocytes with an IC50 of 90 nM [20]. However, a full publi-
cation of this study has not yet appeared. More recently,
Moore et al. described a potent, competitive small molecule
inhibitor of AEA uptake, LY2318912, which has allowed the
identification of a high-affinity AEA binding site distinct
from FAAH in RBL-2H3 cell plasma membranes [21]. They
also tested the effect of the parent compound of LY2318912,
LY2183240, in a pharmacological test of persistent pain.
As underlined by Mechoulam and Deutsch in the commen-
tary accompanying Moore et al.’s paper [22], ‘the identifica-
tion of a high-affinity binding site involved in the transport
of AEA by Eli Lilly group represents a major step in under-
standing endocannabinoid metabolism and will present drug
researchers with a very valuable tool’. However, these authors
also raised a number of questions regarding the possibility that
the novel compounds developed at Eli Lilly might act via
additional mechanisms other than inhibition of AEA cellular
uptake. Indeed, the Eli Lilly group subsequently reported that
LY2183240 does inhibit enzyme activity in purified FAAH
preparations [23], whereas very recently Alexander and Cravatt
have shown that LY2183240 is a potent, covalent inhibitor not
only of FAAH but also of several other serine proteases, includ-
ing the monoacylglycerol lipase (MAGL) that catalyses the
hydrolysis of 2-arachidonoylglycerol [24].
The tetrazole compounds in Fig. 1 were prepared as de-
tailed below and as summarized in Schemes 1e3, following
some of the newer methods for 5-substituted tetrazole
O
N
N
N
N
O
N
N
N
N
N
N
2
1 (LY2183240)
O
N
N
N
O
R
N
N
R
N
N
N
N
N
3: R = PhCH2
5: R = PhCH2
6: R = Ph(CH2)2
4: R = Ph(CH2)2
O
O
N
O
N
N
I
N
N
I
O
N
N
H
N
N
H
N
N3
N
N
N3
7 (LY2318912)
8
The compound described by Alexander and Cravatt is,
however, in all evidence a mixture of regioisomeric 1,5- and
2,5-disubstituted tetrazoles (see Section 2), while neither de-
tails of the syntheses of LY2183240 and LY2318912 nor any
information on the activity of their 2,5-regioisomers has
been presented in the Eli Lilly papers. This despite the fact
that the main procedure for the preparation of disubstituted
tetrazoles, i.e. the alkylation (arylation, acylation) of 5-
substituted tetrazoles, affords generally mixtures of regio-
isomers and difficulties are often encountered in their separation
and/or when assigning their regiochemistry [25].
O
O
O
N
N
N
O
R
N
H
N
N
R
N
H
N
N
N
N
N
14: R = 3-biphenyl
15: R = 4-biphenyl
9: R = 3-biphenyl
10: R = 4-biphenyl
16: R = 4-biphenylmethyl
17: R = Ph(CH2)6
11: R = 4-biphenylmethyl
12: R = Ph(CH2)6
In light of these uncertainties of both synthetic and bio-
logical nature and in an effort to gain further insight into
the actions of tetrazolic ureas, in particular the individual
18: R = Ph(CH2)7
13: R = Ph(CH2)7
Fig. 1. Structures of carbamoyl tetrazoles synthesized and tested in this study.

64
G. Ortar et al. / European Journal of Medicinal Chemistry 43 (2008) 62e72
N
a
b
R
1 + 2 (or 3 + 5 or 4 + 6)
N
H
R
CN
N
N
19: R = 4-biphenyl
20: R = CH2Ph
22: R = 4-biphenyl
23: R = CH2Ph
21: R = (CH2)2Ph
24: R = (CH2)2Ph
Scheme 1. Synthesis of tetrazoles 1e6. Reagents and conditions: (a) n-Bu3SnN3, dioxane, reflux, 20 h, then dry HCl, rt, 15 min; (b) Me2NCOCl, Et3N, MeCN,
rt, 15 h.
formation from organic nitriles [26]. Refluxing the commer-
cially available 4-biphenylacetonitrile 19 with tri-n-butyltin
azide [27], followed by an acidic hydrolysis step to remove
the tin group from the tetrazole ring and acylation of the
resulting tetrazole 22 [28] with N,N-dimethylcarbamoyl chlo-
ride gave a 0.58:1 mixture of 1,5- and 2,5-regioisomers 1
(LY2183240) and 2 which could be separated by silica gel
chromatography, the less polar 2,5-disubstituted tetrazole 2,
eluting prior to the 1,5-disubstituted tetrazole 1 (Scheme 1).
A mixture of regioisomers was also obtained using the same
conditions employed by Alexander and Cravatt [24] for the
carbamoylation of 22 and, indeed, the 1H NMR spectrum
reported by them for the compound assumed to be pure 1
was the sum of those of 1 and 2.
The structures of the regioisomeric tetrazoles were first ten-
tatively assigned on the basis of 13C NMR spectra. 13C NMR
studies of disubstituted tetrazoles have shown that the CN4
carbon atom of 1,5-disubstituted tetrazoles is more shielded
than the corresponding carbon atom of the 2,5-disubstituted
tetrazoles [29e31], but exceptions to this general rule can
be found [32]. Based on these reported observations, the tetra-
zole which was first eluted was designated the 2,5-disubsti-
tuted tetrazole 2, since it showed a CN4 13C resonance at
d 164.99 ppm, while the more polar tetrazole 1 had a CN4
13C resonance at d 155.53 ppm. However, more spectroscopic
evidence for the regiochemical assignment of the two
regioisomers could not be obtained by a heteronuclear multi-
ple bond correlation (HMBC) experiments, since both the
compounds showed similar HMBC correlations. The 2,5-di-
substituted structure of 2 was thus unambiguously determined
by X-ray crystal structural analysis. Perspective view of 2 with
atom numbering is shown in Fig. 2, while crystal data col-
lection procedures, structure determination methods and re-
finement results are summarized in Table 1.
Repetition of the above procedure on nitriles 20 and 21 fur-
nished similar and separable mixtures of 1,5- and 2,5-re-
gioisomers 3 þ 5 and 4 þ 6, respectively.
As concerns the preparation of regioisomers
7
(LY2318912) and 8, methyl 4-aminobenzoate (25) was con-
verted into methyl 4-amino-3-iodobenzoate (26) according to
Spivey et al. [33]. Reaction of 26 with sodium nitrite and
sodium azide in acidic medium followed by alkaline hydrolysis
of the ester 27 as described previously [34] afforded 4-azido-
3-iodobenzoic acid (28). Reaction of the acyl chloride derived
from 28 by the action of SOCl2 with aminoacetonitrile bisul-
fate furnished the amide 29. Refluxing of 29 with tri-n-butyltin
azide followed by acidic hydrolysis and acylation of tetrazole
30 with N,N-dimethylcarbamoyl chloride afforded a 0.52:1
mixture of 1,5- and 2,5-regioisomers 7 and 8 which could be
separated by silica gel chromatography, again the less polar
2,5-disubstituted tetrazole 8 eluting prior to the 1,5-disubsti-
tuted tetrazole 7 (Scheme 2).
Repetition of the three-step procedure on acids 31e35
furnished mixtures of 1,5- and 2,5-regioisomers which could
be separated in the case of tetrazoles 9 þ 14, 12 þ 17 and
13 þ 18, while from the mixture of regioisomers 10 þ 15
CO2Me
I
CO2Me
I
CO2Me
a
b
H2N
H2N
N3
25
26
27
O
I
I
CO2H
N
H
CN
c
d,e
f
N3
N3
28
29
O
I
g
N
N
N
H
7 + 8
N
H
N
N3
30
Scheme 2. Synthesis of tetrazoles 7 and 8. Reagents and conditions: (a) ICl, AcOH, rt, 1 h; (b) 4 N HCl, NaNO2, AcOH/H2O, 0e5 ꢁC, 15 min, then NaN3, H2O, rt,
15 min; (c) 1 M LiOH, THF/H2O, rt, 15 h; (d) SOCl2, 100 ꢁC, 1 h; (e) H2NCH2CN$H2SO4, Et3N, CH2Cl2, rt, 15 h; (f) n-Bu3SnN3, dioxane, reflux, 20 h, then dry
HCl, rt, 15 min; (h) Me2NCOCl, Et3N, MeCN, rt, 15 h.

G. Ortar et al. / European Journal of Medicinal Chemistry 43 (2008) 62e72
65
O
O
N
N
R
N
H
a
b
N
H
R CO2H
R
N
H
CN
N
31: R = 3-biphenyl
32: R = 4-biphenyl
36: R = 3-biphenyl
37: R = 4-biphenyl
38: R = 4-biphenylmethyl
39: R = Ph(CH2)6
41: R = 3-biphenyl
42: R = 4-biphenyl
43: R = 4-biphenylmethyl
44: R = Ph(CH2)6
33: R = 4-biphenylmethyl
34: R = Ph(CH2)6
35: R = Ph(CH2)7
40: R = Ph(CH2)7
45: R = Ph(CH2)7
c
9 + 14 (or 10 + 15 or 11 + 16 or 12 + 17 or 13 + 18)
Scheme 3. Synthesis of tetrazoles 9e18. Reagents and conditions: (a) HOBt/EDC, rt, 1 h, then H2NCH2CN$H2SO4, Et3N, CH2Cl2, rt, 15 h; (b) n-Bu3SnN3,
dioxane, reflux, 20 h, then dry HCl, rt, 15 min; (c) Me2NCOCl, Et3N, MeCN, rt, 15 h.
only 2,5-regioisomer 15 could be isolated in a pure form,
owing to the propensity of 1,5-regioisomer 10 to isomerise
during chromatographic separation, and attempts to separate
regioisomers 11 þ 16 were unsuccessful (Scheme 3).
by Alexander and Cravatt, showing that carbamoyl tetrazoles
act as inhibitors for several serine hydrolases, including
FAAH and MAGL [24], we have used membranes from rat
brain to assess the FAAH inhibitory activity. This method is
now to be considered even more suitable than in the past since
recent data indicate that in some mammals, but not in rats or
mice, a second FAAH isoform (FAAH-2) is also abundantly
expressed [36]. In order to determine whether the compounds
would also act as inhibitors of 2-AG hydrolysis via the
MAGL, and of 2-AG biosynthesis via the sn-1-selective diac-
ylglycerol lipases (DAGLs), we have used the cytosolic and
membrane fractions, respectively, of COS cells overexpressing
DAGLa, since COS cells exhibit high levels of a cytosolic
3. Biological evaluation
Apart from testing for the first time 13 new compounds
(two of which as a mixture of regioisomers) and the 2,5-
isomers 2, 8 of the Eli Lilly compounds, whose biological
activity was not reported in Moore et al.’s paper [21], we have
also re-examined in this study the activity of the previously
described 1,5-isomers 1, 7, using different conditions for the
assay of [14C]anandamide cellular uptake. These conditions,
by using higher concentrations of the [14C]anandamide substrate,
have proved in the past to be more restrictive than other assays
regarding potency and/or efficacy of competitive uptake inhib-
itors [35] and, by using incubation times with intact rat baso-
philic leukemia RBL-2H3 cell that are shorter (90 s) than
those used by Moore et al., minimize the interference of
FAAH inhibition by the compounds on their net effect on
[14C]anandamide uptake [11]. We have also used a 10 min
pre-incubation of cells with the compound to maximize their
potential competition with the specific site(s) involved in
anandamide cellular uptake. In view of the results published
Table 1
Crystal data of compound 2
Empirical formula
Formula weight
Crystal size (mm)
Z
C17H17N5O
307.36
0.4 ꢂ 0.3 ꢂ 0.3
4
Density (calculated) (g/cm3)
Crystal system
Space group
1.294
Monoclinic
P21/c
˚
a (A)
6.112 (4)
8.907 (4)
29.07 (2)
94.27 (9)
1578.2 (17)
1.54178
293
˚
b (A)
˚
c (A)
b (ꢁ)
Volume (A )
3
˚
˚
Wavelength (A)
Temperature (K)
Reflections number (total)
Reflections number (observed)
Reflections threshold expression
Diffraction measurement method
qmin, qmax
2483
1946
F > 3.0sjFj
we2w
1.50e31.04
0.698
208
m (mmꢃ1
)
Refine number parameters
R factor
0.065
0.083
wR factor
Goodness of fit
0.88
0.000
Shift/error-max
Shift/su_mean
0.000
0.64
SIR200a
SIR2004a
Final e.s.d. of r
Computing structure solution
Computing structure refinement
a
Fig. 2. ORTEP projection of the tetrazole 2.
Ref. [39].

66
G. Ortar et al. / European Journal of Medicinal Chemistry 43 (2008) 62e72
constitutive MAGL activity whereas their overexpressed DAGL
activity is concentrated in the membranes [37]. Only 15 of
the 18 compounds could be tested in a regioisomerically pure
form. Compound 10 was unstable and easily got converted to
15, and therefore could not be tested. The two regioisomers
11 and 16 were tested in mixture, as they could not be separated
chromatographically.
Although we have confirmed here that the most potent
anandamide uptake inhibitor identified by Moore et al., i.e.
compound 1, is also the most potent compound in this assay
under our conditions, we could not confirm here the same
potency (IC50 ¼ 0.27 nM, i.e. w55-fold higher) published pre-
viously for this compound. The discrepancy is even more
evident with compound 7, which was previously reported to
inhibit AEA uptake in the same cells with an IC50 of 7.2 nM
and was found here to exhibit w140-fold lower potency.
The exact reasons for these differences are not known, but
are very likely due to the fact that we used experimental con-
ditions different from those used in the previous study (namely
much shorter incubation times and higher concentrations of
substrate [14C]anandamide).
4. Results and discussion
The results of the pharmacological assays shown in Table 2
can be summarized as follows: (1) we could confirm that com-
pound 1 is a potent inhibitor of both the cellular uptake and the
enzymatic hydrolysis of anandamide [21,24]; (2) with the
exception of 9, 12, 15, and the 2,5-regioisomer of LY2183240
(2), which exhibited lower potency than 1, but were still capa-
ble of efficaciously inhibiting anandamide cellular uptake with
IC50 values between 0.1 and 1 mM, the other compounds were
all weakly active or inactive in this assay; (3) all compounds
inhibited anandamide hydrolysis by rat brain membranes
with IC50 values ranging from 2.1 to 33 nM; (4) several com-
pounds also inhibited the enzymatic hydrolysis of the other
endocannabinoid, 2-AG, as well as its enzymatic release
from sn-1-oleoyl-2-arachidonoyl-glycerol, at submicromolar
concentrations; (5) four of the novel compounds, i.e. 3, 4,
Furthermore, as predicted by Mechoulam and Deutsch [22],
and later shown by both the Eli Lilly group [23] and Alexander
and Cravatt [24], we found that all compounds also potently
inhibit rat brain FAAH. However, additional data also indicate
that the effect of compound 1 on AEA uptake is only in part
due to its capability of inhibiting AEA hydrolysis. First,
we observed that under the incubation conditions used here,
the extent of temperature-sensitive [14C]anandamide uptake
(16.2 ꢄ 3.2% of total incubated anandamide) was significantly
higher than that of [14C]anandamide hydrolysis (1.9 ꢄ 0.1% of
total incubated anandamide, means ꢄ SD of N ¼ 10). Further-
more, when this was tested in the same experiment in intact
RBL-2H3 cells, compound 1 inhibited the uptake of
[14C]anandamide more efficaciously than it inhibited the for-
mation of [14C]ethanolamine from [14C]anandamide (respec-
tively, 66.5 ꢄ 11.4 and 74.2 ꢄ 8.1% inhibition of uptake and
41.1 ꢄ 6.1 and 36.7 ꢄ 5.0% inhibition of hydrolysis at 0.1
and 1 mM, respectively, p < 0.01 in both cases, N ¼ 4). These
findings suggest that, in intact cells, compound 1 inhibits
anandamide hydrolysis, which is an intracellular process,
because it inhibits anandamide uptake and not vice versa.
Thus, although part of the inhibitory effect on anandamide
uptake by compound 1 is clearly due to its inhibition of
FAAH, which contributes to driving cellular uptake, this com-
pound seems capable of influencing the former process also in
a way independent from anandamide hydrolysis. This con-
clusion is also supported by the data recently published by
Dickason-Chesterfield et al., who also found a clear dissocia-
tion between the FAAH- and anandamide uptake-inhibitory
activities of several compounds, including compound 1 [23].
As could be predicted again by the study by Alexander and
Cravatt [24], several of the carbamoyl tetrazoles synthesized in
this study also potently inhibited the hydrolysis of [14C]2-AG
by a MAGL-like activity present in the cytosol of COS cells.
Furthermore, we showed here for the first time that these com-
pounds can also be good inhibitors of 2-AG biosynthesis from
sn-1-[14C]oleoyl-2-arachidonoyl-glycerol. However, we agree
only in part with the conclusions drawn by these authors
that ‘the promiscuity of LY2183240 designates the heterocy-
clic urea as a chemotype with potentially excessive protein
reactivity for drug design’. In fact, we did find that out of the
17 tetrazoles screened, four compounds exhibited potent activ-
ity for FAAH selectively over all other targets tested here.
17, and 18, inhibited anandamide hydrolysis potently (IC50
2.1e5.4 nM) and with high (500e2000-fold) selectivity over
¼
all the other targets tested.
Table 2
Effect of the compounds synthesized in this study on [14C]anandamide hydro-
lysis by rat brain membranes (which express FAAH as the only anandamide
hydrolysing enzyme), [14C]anandamide uptake by intact RBL-2H3 cells
(where a putative anandamide transporter has been characterized pharma-
cologically), [14C]2-AG hydrolysis by COS cell cytosolic fractions (which
express MAGL), and sn-1-[14C]oleoyl-2-arachidonoyl-glycerol (DAG) conver-
sion into 2-AG and [14C]oleic acid by COS cells membranes overexpressing
human recombinant DAGLaa
Compound
AEA
hydrolysis
AEA
uptake
2-AG
hydrolysis
DAG hydrolysis
to 2-AG
1
0.0021
0.0330
0.0054
0.0027
0.0035
0.0032
0.0230
0.0250
0.0320
0.0290
0.0037
0.0100
0.0230
0.0060
0.0021
0.0032
0.015
0.998
8.10
0.02
10
>10
0.51
10
2
3
>10 (15.8%)
>10 (18.0%)
>10 (10.9%)
>10 (16.0%)
>1 (32.4%)
>1 (9.0%)
0.090
4
10
10
5
0.48
0.11
0.35
>10
0.85
0.10
0.02
0.04
10
0.60
0.50
0.13
4.40
0.34
1.00
0.20
0.57
>10
>10
10
6
7
8
9
11 þ 16
>10 (35.2%)
0.851
12
13
14
15
17
18
a
>10 (37.5%)
>10 (23.0%)
0.985
>10
>10
10
>10 (30.0%)
>10 (18.8%)
10
Data are expressed as IC50 (mM) and in the case of weak inhibitors as the
maximal percent inhibition observed (between parentheses), and are means of
n ¼ 3e4 separate determinations. Standard errors are not shown for the sake of
clarity and were never higher than 10% of the means.

G. Ortar et al. / European Journal of Medicinal Chemistry 43 (2008) 62e72
67
These compounds, i.e. 3, 4, 17, and 18, might represent new
useful FAAH inhibitors exploitable for therapeutic use in
pathologies as diverse as those listed in Section 1.
6. Experimental protocols
6.1. Chemistry
Our results in 12 carbamoyl tetrazoles, which represent six
1,5- and 2,5-regioisomeric couples, together with those
obtained with 1 and 7 and their 2,5-regioisomers 2 and 8 allow
us to delineate for the first time some structureeactivity rela-
tionships for the interactions of this class of compounds with
the putative anandamide membrane transporter, FAAH,
MAGL and DAGLa, and hence provide new information for
the future design of new pharmacological tools interacting
with these targets. In particular, the following conclusions
can be drawn from the data in Table 2: (1) regarding the inhi-
bition of anandamide cellular uptake, most 1,5-isomers are
significantly more potent than the corresponding 2,5-isomers.
Furthermore, conformationally less flexible substituents in the
5-position seem to yield better inhibitors than more flexible
ones. In any case, the features of 5-substitution necessary for
potent uptake inhibition appear to be rather stringent, as seem-
ingly minor structural differences produce remarkable varia-
tions of the activity (e.g. compounds 14, 15 and 12, 13); (2)
by contrast, 2,5-isomers can also be more potent FAAH inhib-
itors than their 1,5-counterparts; the requirements of the 5-sub-
stitution are less critical and conformationally more flexible
substituents appear to afford more potent inhibitors. These
observations represent yet another indirect piece of evidence
in favour of the existence of a FAAH-independent mechanism
for anandamide uptake; (3) regarding the inhibition of MAGL
and DAGL, 1,5-isomers were significantly more potent than
the corresponding 2,5-isomers in the case of compounds
with an amide function in the 5-side chain, while the opposite
seems to apply for compounds with non-functionalized side
chains; (4) with a few exceptions, carbamoyl tetrazoles seem
to be significantly more potent at inhibiting MAGL than
DAGL. Nevertheless, this property is not likely to be exploited
for the development of selective MAGL inhibitors since the
most potent inhibitors of 2-AG hydrolysis synthesized in the
present study were as potent as FAAH inhibitors.
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 re-
corded on a Perkin-Elmer 1000 FT-IR spectrophotometer as
KBr disks unless otherwise indicated. 1H and 13C NMR spectra
were obtained on a Varian Mercury 300 spectrometer using
CDCl3 as solventunless otherwiseindicatedand TMS as internal
standard. Analyses indicated by the symbols of the elements or
functions were within ꢄ0.4% of the theoretical values (Table 3).
Compounds 26 [33], 27 [34], 28 [34], and 31 [38] have
been described in the literature.
6.1.1. 5-((Biphenyl-4-yl)methyl)-1H-tetrazole (22)
A solution of 4-biphenylacetonitrile (19) (1.449 g, 7.5 mmol)
and n-Bu3SnN3 [27] (3.736 g, 11.25 mmol) in 1,4-dioxane
(15 mL) was refluxed for 20 h, cooled at room temperature,
and gaseous HCl was added over 15 min. The mixture was
diluted with water, neutralized with 2 N NaOH, and extracted
with AcOEt. The organic phase was washed with water, dried
(Na2SO4), and evaporated under vacuum to leave a residue
(5.20 g) which was triturated with hot hexane. The resulting
solid was filtered off, washed with hexane, and dried under
vacuum to give 1.460 g (82%) of 22. Mp 215e217 ꢁC; IR
3117, 3029, 2853, 2727, 2623, 1575, 1487, 1424, 1256,
1
1106, 1053 cmꢃ1; H NMR (300 MHz) d 4.37 (2H, s), 7.36e
7.67 (9H, m); 13C NMR (75 MHz) d 28.53, 126.53, 126.94,
127.37, 128.85, 129.21, 135.05, 138.93, 139.69, 155.17. Anal.
(C14H12N4) C, H, N.
6.1.2. 5-((Biphenyl-4-yl)methyl)-N,N-dimethyl-1H-
tetrazole-1-carboxamide (1) and 5-((biphenyl-4-yl)methyl)-
N,N-dimethyl-2H-tetrazole-2-carboxamide (2)
To a stirred solution of 22 (1.460 g, 6.18 mmol) and Et3N
(1.73 mL, 12.36 mmol) in acetonitrile (10 mL) Me2NCOCl
(0.85 mL, 9.27 mmol) was added at 0 ꢁC, and the mixture
was stirred at 0 ꢁC for 15 h. The mixture was diluted with
water and extracted with AcOEt. The organic phase was
washed twice with brine, dried (Na2SO4), and evaporated
under vacuum. The residue (1.91 g) was chromatographed
on silica gel (200 g) with CH2Cl2/AcOEt ¼ 99/1 as eluent to
give 2 (1.041 g, 55%) followed by 1 (609 mg, 32%). Com-
pound 2: mp 80e81 ꢁC; IR (CHCl3) 3010, 1732, 1488,
5. Conclusions
We have provided here new methods for the syntheses of
carbamoyl tetrazoles with activity as inhibitors of endocanna-
binoid inactivation, as well as new data on the structuree
activity relationships for the interactions of these compounds
with the putative anandamide membrane transporter, FAAH,
MAGL and DAGLa. Our data provide further indirect evi-
dence that a specific and FAAH-independent process underlies
part of the cellular uptake of anandamide by RBL-2H3 cells,
but argue against the possibility of developing selective inhib-
itors of such process from carbamoyl tetrazoles. On the other
hand, we have shown here that it might be possible to develop
selective as well as potent FAAH inhibitors from the chemical
modification of the compounds previously reported by Eli
Lilly. Four such new inhibitors i.e. 3, 4, 17, and 18, have
been described here.
1
1396, 1257, 1231, 1088 cmꢃ1; H NMR (300 MHz) d 3.11
(3H, s), 3.25 (3H, s), 4.37 (2H, s), 7.33e7.58 (9H, m); 13C
NMR (75 MHz) d 31.26, 37.98, 39.00, 126.89, 127.14,
127.35, 128.60, 129.16, 134.78, 140.00, 140.55, 147.66,
164.99. Anal. (C17H17N5O) C, H, N. Compound 1: mp 87e
88 ꢁC; IR (CHCl3) 3011, 1742, 1489, 1393, 1235, 1166,
1
1130, 1068 cmꢃ1; H NMR (300 MHz) d 2.69 (3H, s), 3.07
(3H, s), 4.48 (2H, s), 7.32e7.57 (9H, m); 13C NMR
(75 MHz) d 29.43, 37.22, 38.57, 126.75, 127.26, 127.37,
128.66, 129.26, 133.07, 140.07, 140.46, 147.71, 155.53.
Anal. (C17H17N5O) C, H, N.

68
G. Ortar et al. / European Journal of Medicinal Chemistry 43 (2008) 62e72
Table 3
Elemental analysis data
Compound
Molecular formula
Calculated C, H, N
Found C, H, N
1
C
17H17N5O
17H17N5O
C, 66.43; H, 5.58; N, 22.79
C, 66.43; H, 5.58; N, 22.79
C, 58.76; H, 6.16; N, 28.55
C, 60.21; H, 6.61; N, 27.01
C, 58.76; H, 6.16; N, 28.55
C, 60.21; H, 6.61; N, 27.01
C, 32.67; H, 2.74; N, 28.57
C, 32.67; H, 2.74; N, 28.57
C, 61.70; H, 5.18; N, 23.99
C, 60.32; H, 7.31; N, 23.45
C, 61.27; H, 7.58; N, 22.56
C, 61.70; H, 5.18; N, 23.99
C, 61.70; H, 5.18; N, 23.99
C, 60.32; H, 7.31; N, 23.45
C, 61.27; H, 7.58; N, 22.56
C, 71.17; H, 5.12; N, 23.71
C, 62.05; H, 5.79; N, 32.16
C, 63.81; H, 6.43; N, 29.77
C, 33.05; H, 1.85; N, 21.41
C, 29.21; H, 1.91; N, 30.28
C, 76.25; H, 5.12; N, 11.86
C, 76.25; H, 5.12; N, 11.86
C, 76.78; H, 5.64; N, 11.19
C, 73.74; H, 8.25; N, 11.47
C, 70.04; H, 8.08; N, 10.21
C, 64.51; H, 4.69; N, 25.07
C, 64.51; H, 4.69; N, 25.07
C, 65.52; H, 5.15; N, 23.88
C, 62.70; H, 7.37; N, 24.37
C, 63.76; H, 7.69; N, 23.24
C, 66.49; H, 5.49; N, 22.65
C, 66.37; H, 5.62; N, 22.87
C, 58.59; H, 6.31; N, 28.67
C, 60.37; H, 6.44; N, 26.83
C, 59.02; H, 6.03; N, 28.18
C, 60.40; H, 6.47; N, 27.33
C, 32.72; H, 2.72; N, 28.57
C, 32.59; H, 2.70; N, 28.49
C, 61.48; H, 5.09; N, 24.08
C, 60.14; H, 7.28; N, 23.47
C, 61.03; H, 7.49; N, 22.48
C, 61.54; H, 5.07; N, 24.16
C, 61.67; H, 5.19; N, 23.90
C, 59.97; H, 7.44; N, 23.22
C, 61.30; H, 7.67; N, 22.67
C, 71.39; H, 5.18; N, 23.64
C, 62.16; H, 5.65; N, 32.47
C, 63.58; H, 6.19; N, 29.75
C, 32.89; H, 1.97; N, 21.37
C, 29.36; H, 1.87; N, 30.35
C, 76.47; H, 5.07; N, 11.68
C, 76.32; H, 5.02; N, 12.01
C, 76.68; H, 5.60; N, 11.13
C, 73.56; H, 8.32; N, 11.53
C, 69.78; H, 7.99; N, 10.34
C, 64.47; H, 4.39; N, 25.40
C, 64.39; H, 4.51; N, 24.93
C, 65.72; H, 5.07; N, 23.80
C, 62.76; H, 7.45; N, 24.12
C, 63.46; H, 7.58; N, 23.24
2
C
3
C12H15N5O
C13H17N5O
4
5
C
12H15N5O
13H17N5O
6
C
7
C12H12IN9O2
C12H12IN9O2
8
9
C
18H18N6O2
18H26N6O2
12
13
14
15
17
18
22
23
24
29
30
36
37
38
39
40
41
42
43
44
45
C
C19H28N6O2
C18H18N6O2
C
18H18N6O2
18H26N6O2
C
C19H28N6O2
C14H12N4
C9H10N4
C10H12N4
C9H6IN5O
C9H7IN8O
C15H12N2O
C
15H12N2O
C16H14N2O
15H20N2O
C
C16H22N2O
C15H13N5O
C15H13N5O
C16H15N5O
C15H21N5O
C16H23N5O
6.1.3. 5-Phenethyl-1H-tetrazole (23)
13C NMR (75 MHz) d 25.65, 33.61, 37.58, 39.12, 126.68,
128.58, 139.24, 147.84, 156.37. Anal. (C12H15N5O) C, H, N.
The title compound was prepared from 3-phenylpropaneni-
trile (20) following the same procedure that was used for the
synthesis of 22. Yield 70%; mp 80e85 ꢁC; IR 3134, 3029,
2859, 2730, 2626, 1560, 1497, 1454, 1258, 1108,
6.1.5. 5-(3-Phenylpropyl)-1H-tetrazole (24)
The title compound was prepared from 4-phenylbutyroni-
trile (21) following the same procedure that was used for the
synthesis of 22. Yield 55%; mp 80e85 ꢁC; IR 3130, 3024,
2864, 2730, 2622, 1568, 1495, 1450, 1421, 1256, 1107,
1
1050 cmꢃ1; H NMR (300 MHz) d 3.15 (2H, t, J ¼ 7.6 Hz),
3.38 (2H, t, J ¼ 7.6 Hz), 7.13e7.25 (5H, m); 13C NMR
(75 MHz) d 25.29, 33.67, 126.72, 128.30, 128.67, 139.10,
156.11. Anal. (C9H10N4) C, H, N.
1
1047 cmꢃ1; H NMR (300 MHz) d 2.19 (2H, m), 2.71 (2H,
t, J ¼ 7.5 Hz), 3.08 (2H, t, J ¼ 7.7 Hz), 7.11e7.27 (5H, m);
13C NMR (75 MHz) d 22.91, 29.03, 34.99, 126.25, 128.42,
128.52, 140.54, 156.77. Anal. (C10H12N4) C, H, N.
6.1.4. N,N-Dimethyl-5-phenethyl-1H-tetrazole-1-
carboxamide (3) and N,N-dimethyl-5-phenethyl-2H-
tetrazole-2-carboxamide (5)
The title compounds were prepared from 23 following the
same procedure that was used for the synthesis of 1, 2 using
hexane/AcOEt ¼ 1/1 as eluent for the chromatographic separa-
tion of regioisomers. Compound 5 (less polar, 23%): oil; IR
(CHCl3) 3009, 2941, 1740, 1498, 1393, 1311, 1252, 1168,
6.1.6. N,N-Dimethyl-5-(3-phenylpropyl)-1H-tetrazole-1-
carboxamide (4) and N,N-dimethyl-5-(3-phenylpropyl)-2H-
tetrazole-2-carboxamide (6)
The title compounds were prepared from 24 following the
same procedure that was used for the synthesis of 1, 2 using
preparative layer chromatography (silica gel, 0.5 cm thick)
and hexane/acetone ¼ 7/3 as eluent for the separation of
regioisomers. Compound 6 (less polar, 38%): oil; IR (CHCl3)
3024, 2942, 2864, 1740, 1603, 1497, 1453, 1393, 1310,
1
1130, 1070 cmꢃ1; H NMR (300 MHz) d 3.01 (3H, s), 3.22
(3H, s), 3.11e3.34 (4H, m), 7.14e7.32 (5H, m); 13C NMR
(75 MHz) d 27.12, 33.79, 38.05, 39.00, 126.39, 128.37,
128.52, 140.09, 147.92, 165.53. Anal. (C12H15N5O) C, H, N.
Compound 3 (more polar, 17%): oil; IR (CHCl3) 3014,
1
1167, 1071 cmꢃ1; H NMR (300 MHz) d 2.16 (2H, m), 2.70
1
2941, 1728, 1497, 1396, 1257, 1236, 1088, 1076 cmꢃ1; H
(2H, t, J ¼ 7.5 Hz), 2.99 (2H, t, J ¼ 7.6 Hz) 3.09 (3H, s),
3.24 (3H, s), 7.14e7.33 (5H, m); 13C NMR (75 MHz)
d 24.76, 29.26, 35.11, 38.06, 39.10, 126.03, 128.42, 128.49,
NMR (300 MHz) d 2.76 (3H, s), 3.09 (3H, s), 3.13 (2H, t,
J ¼ 7.6 Hz), 3.48 (2H, t, J ¼ 7.6 Hz), 7.13e7.31 (5H, m);

G. Ortar et al. / European Journal of Medicinal Chemistry 43 (2008) 62e72
69
141.19, 147.96, 166.14. Anal. (C13H17N5O) C, H, N. Com-
pound 4 (more polar, 23%): oil; IR (CHCl3) 3024, 2942,
d 3.13 (3H, s), 3.27 (3H, s), 4.97 (2H, d, J ¼ 6.0 Hz), 7.13
(1H, d, J ¼ 8.4 Hz), 7.48 (1H, m), 7.86 (1H, dd, J ¼ 8.4,
2.1 Hz), 8.25 (1H, d, J ¼ 2.1 Hz); 13C NMR (75 MHz)
d 35.25, 38.11, 39.13, 87.33, 117.87, 128.44, 131.02, 138.93,
145.06, 147.39, 163.12, 165.06. Anal. (C12H12IN9O2) C, H, N.
1
2863, 1728, 1603, 1497, 1453, 1396, 1258, 1088 cmꢃ1; H
NMR (300 MHz) d 2.17 (2H, m), 2.74 (2H, t, J ¼ 7.5 Hz),
3.06 (5H, m), 3.20 (3H, s), 7.15e7.32 (5H, m); 13C NMR
(75 MHz) d 23.59, 28.48, 35.02, 37.76, 39.33, 126.17,
128.48, 140.80, 148.00, 156.89. Anal. (C13H17N5O) C, H, N.
6.1.10. N-(Cyanomethyl)biphenyl-3-carboxamide (36)
To a stirred solution of biphenyl-3-carboxylic acid (31) [35]
(223 mg, 1.12 mmol) in DMF (4.5 mL) were added at 0 ꢁC
HOBt (181 mg, 1.18 mmol) and EDC (226 mg, 1.18 mmol).
The mixture was stirred for 15 min at 0 ꢁC and for 1 h at
room temperature, and H2NCH2CN$H2SO4 (207 mg,
1.35 mmol) and Et3N (0.78 mL, 5.61 mmol) were added and
the mixture was stirred overnight at room temperature. The
mixture was diluted with brine and extracted with AcOEt.
The organic phase was washed with 2 N HCl solution, brine,
saturated NaHCO3 and brine, dried (Na2SO4), and evaporated
under vacuum. The residue (349 mg) was chromatographed on
silica gel (11 g) using CH2Cl2/AcOEt ¼ 95/5 as eluent to give
224 mg (85%) of 36 as an oil; IR (CHCl3) 3453, 3339, 3065,
6.1.7. 4-Azido-N-(cyanomethyl)-3-iodobenzamide (29)
A solution of 28 [34] (7.250 g, 25.08 mmol) in SOCl2
(30 mL) was stirred at 100 ꢁC for 2 h. Excess SOCl2 was
evaporated under vacuum and the residue of the acid chlo-
ride (7.711 g, 100%) was dissolved in dry CH2Cl2 (50 mL),
and H2NCH2CN$H2SO4 (4.253 g, 27.59 mmol) and Et3N
(17.6 mL, 125.71 mmol) were added under stirring at 0 ꢁC.
The mixture was stirred at room temperature for 15 h, diluted
with water, and extracted with AcOEt. The organic phase was
washed with 2 N HCl, water until neutral, 2 N NaOH, and wa-
ter until neutral, dried (Na2SO4), and evaporated under vac-
uum to leave a residue (5.239 g, 64%) of 29 as a white
solid. Mp 65e68 ꢁC; IR 3337, 3230, 3059, 3000, 2116,
2943, 1676, 1509, 1476, 1304, 1257, 1202, 1145, 1091 cmꢃ1
;
1643, 1588, 1538, 1474, 1298 cmꢃ1 1H NMR (300 MHz)
;
1H NMR (300 MHz) d 4.31 (2H, d, J ¼ 5.6 Hz), 7.30e7.99
(10H, m); 13C NMR (75 MHz) d 28.10, 116.36, 125.93,
126.06, 127.09, 127.91, 128.94, 129.23, 131.01, 133.06,
139.79, 141.83, 167.82. Anal. (C15H12N2O) C, H, N.
d 4.38 (2H, d, J ¼ 5.8 Hz), 6.81 (1H, m), 7.18 (1H, d,
J ¼ 8.4 Hz), 7.83 (1H, dd, J ¼ 8.4, 2.0 Hz), 8.21 (1H, d,
J ¼ 2.0 Hz); 13C NMR (75 MHz) d 28.15, 87.68, 115.87,
118.28, 128.66, 130.18, 139.11, 146.07, 165.06. Anal.
(C9H6IN5O) C, H, N.
6.1.11. N-((1H-Tetrazol-5-yl)methyl)biphenyl-3-
carboxamide (41)
The title compound was prepared from 36 following the
same procedure that was used for the synthesis of 22. Yield
86%; oil; IR (CHCl3) 3410, 3338, 3031, 2960, 2925, 1656,
1518, 1476, 1306, 1225, 1088 cmꢃ1 1H NMR (300 MHz,
;
CDCl3/CD3OD ¼ 9/1) d 4.86 (2H, s), 7.32e8.05 (9H, m);
13C NMR (75 MHz, CDCl3/CD3OD ¼ 9/1) d 33.62, 126.01,
126.12, 127.13, 127.17, 127.85, 128.95, 129.10, 130.66,
133.84, 140.07, 141.70, 168.77. Anal. (C15H13N5O) C, H, N.
6.1.8. N-((1H-Tetrazol-5-yl)methyl)-4-azido-3-
iodobenzamide (30)
The title compound was prepared from 29 following the
same procedure that was used for the synthesis of 22. Yield
(39%); mp 188e190 ꢁC; IR 3287, 2855, 2125, 1636, 1447,
1301, 1035 cmꢃ1 1H NMR (300 MHz, DMSO-d6) d 3.38
;
(1H, br s), 4.76 (2H, s), 7.47 (1H, d, J ¼ 8.1 Hz), 7.98 (1H,
dd, J ¼ 8.1, 1.8 Hz), 8.36 (1H, d, J ¼ 1.8 Hz), 9.35 (1H, m);
13C NMR (75 MHz, DMSO-d6) d 33.14, 33.24, 87.71,
118.99, 129.00, 131.11, 138.42, 144.30, 154.48, 164.37,
164.45. Anal. (C9H7IN8O) C, H, N.
6.1.12. 5-(Biphenyl-3-ylcarboxamidomethyl)-N,N-dimethyl-
1H-tetrazole-1-carboxamide (9) and 5-(biphenyl-3-
ylcarboxamidomethyl)-N,N-dimethyl-2H-tetrazole-2-
carboxamide (14)
6.1.9. 5-((4-Azido-3-iodobenzamido)methyl)-N,N-dimethyl-
1H-tetrazole-1-carboxamide (7) and 5-((4-azido-3-
iodobenzamido)methyl)-N,N-dimethyl-2H-tetrazole-2-
carboxamide (8)
The title compounds were prepared from 41 following the
same procedure that was used for the synthesis of 1, 2 using
hexane/acetone ¼ 65/35 as eluent for the chromatographic
separation of regioisomers. Compound 14 (less polar, 14%):
oil; IR (CHCl3) 3451, 3033, 2944, 1731, 1667, 1510, 1396,
The title compounds were prepared from 30 following the
same procedure that was used for the synthesis of 1, 2 using
hexane/AcOEt ¼ 45/55 as eluent for the chromatographic sep-
aration of regioisomers. Compound 8 (less polar, 29%): mp
125 ꢁC; IR 3274, 2928, 2121, 1739, 1651, 1534, 1477, 1291,
1
1252, 1091 cmꢃ1; H NMR (300 MHz) d 3.10 (3H, s), 3.20
(3H, s), 5.03 (2H, d, J ¼ 5.8 Hz), 7.27e8.02 (10H, m); 13C
NMR (75 MHz) d 34.34, 38.61, 39.47, 125.82, 126.02,
127.16, 127.83, 128.91, 129.11, 130.71, 133.65, 140.02,
141.82, 148.06, 154.55, 167.70. Anal. (C18H18N6O2) C, H,
N. Compound 9 (more polar, 10%): oil; IR (CHCl3) 3452,
3414, 3022, 2941, 1746, 1675, 1509, 1392, 1251, 1072,
1
1085 cmꢃ1; H NMR (300 MHz) d 3.17 (3H, s), 3.26 (3H,
s), 4.99 (2H, d, J ¼ 5.8 Hz), 7.10 (1H, d, J ¼ 8.4 Hz), 7.81
(1H, dd, J ¼ 8.4, 2.2 Hz), 8.00 (1H, m), 8.20 (1H, d,
J ¼ 2.2 Hz); 13C NMR (75 MHz) d 34.22, 37.85, 39.38,
87.33, 117.87, 128.32, 130.53, 138.99, 145.26, 147.73,
154.49, 165.16. Anal. (C12H12IN9O2) C, H, N. Compound 7
(more polar, 15%): mp 119 ꢁC; IR 3279, 2933, 2123, 1735,
1
1000 cmꢃ1; H NMR (300 MHz) d 3.09 (3H, s), 3.23 (3H,
s), 5.03 (2H, d, J ¼ 5.6 Hz), 7.26e8.02 (10H, m); 13C NMR
1638, 1536, 1390, 1314, 1131 cmꢃ1 1H NMR (300 MHz)
;
(75 MHz) d 35.42, 38.11, 39.20, 125.97, 126.28, 127.18,

70
G. Ortar et al. / European Journal of Medicinal Chemistry 43 (2008) 62e72
127.77, 128.88, 129.05, 130.54, 134.28, 140.14, 141.78,
163.42, 167.66. Anal. (C18H18N6O2) C, H, N.
(300 MHz, DMSO-d6) d 3.60 (2H, s), 4.09 (2H, m), 7.30e
7.59 (9H, m); 13C NMR (75 MHz, DMSO-d6) d 27.53,
42.42, 116.36, 127.13, 127.56, 127.66, 128.97, 129.81,
133.36, 140.51, 140.73, 172.26. Anal. (C16H15N5O) C, H, N.
6.1.13. N-(Cyanomethyl)biphenyl-4-carboxamide (37)
The title compound was prepared from acid 32 following
the same procedure that was used for the synthesis of 36. Yield
76%; mp 197e198 ꢁC; IR 3307, 2923, 1645, 1608, 1531,
6.1.18. 5-((2-(Biphenyl-4-yl)acetamido)methyl)-N,N-
dimethyl-1H-tetrazole-1-carboxamide (11) and 5-((2-
(biphenyl-4-yl)acetamido)methyl)-N,N-dimethyl-2H-
tetrazole-2-carboxamide (16)
1484, 1413, 1309 cmꢃ1
;
1H NMR (300 MHz, CDCl3/
CD3OD ¼ 1/1) d 4.32 (2H, d, J ¼ 8.2 Hz), 7.38e7.95 (9H,
m); 13C NMR (75 MHz, CDCl3/CD3OD ¼ 1/1) d 28.02,
116.69, 127.38, 127.45, 128.20, 128.39, 129.17, 131.58,
140.03, 145.34, 168.50. Anal. (C15H12N2O) C, H, N.
The title compounds were obtained as an inseparable 0.82:1
mixture from 43 following the same procedure that was used
for the synthesis of 1, 2. Yield 31%; IR (CHCl3) 3433,
3009, 2942, 1740, 1676, 1504, 1489, 1395, 1257, 1165,
1
6.1.14. N-((1H-Tetrazol-5-yl)methyl)biphenyl-4-
carboxamide (42)
The title compound was prepared from 37 following the
same procedure that was used for the synthesis of 22. Yield
67%; mp 275e276 ꢁC; IR 3288, 2856, 1638, 1532, 1484,
1433, 1310, 1246, 1222, 1054 cmꢃ1 1H NMR (300 MHz,
;
DMSO-d6) d 3.28 (1H, br s), 4.49 (2H, d, J ¼ 5.6 Hz),
7.07e7.73 (9H, m), 9.02 (1H, t, J ¼ 5.6 Hz); 13C NMR
(75 MHz, DMSO-d6) d 33.23, 126.44, 126.78, 128.02,
128.93, 131.45, 132.31, 139.00, 143.02, 154.72, 166.26.
Anal. (C15H13N5O) C, H, N.
1064 cmꢃ1; H NMR (300 MHz) d 3.01 (3H, s), 3.14 and
3.19 (3H, 2s), 3.60 and 3.66 (2H, 2s), 4.76 and 4.77 (2H,
2d, J ¼ 5.8 Hz), 6.72 and 7.03 (1H, 2m), 7.26e7.58 (9H,
m); 13C NMR (75 MHz) d 33.96, 35.00, 37.83, 38.11, 39.10,
39.32, 42.73, 42.97, 126.98, 127.38, 127.41, 127.57, 128.81,
129.79, 129.88, 133.37, 133.57, 140.23, 140.26, 140.47,
140.55, 147.56, 147.81, 154.46, 163.25, 171.27, 171.60.
6.1.19. N-(Cyanomethyl)-7-phenylheptanamide (39)
The title compound was prepared from 7-phenylheptanoic
acid (34) following the same procedure that was used for the
synthesis of 36 using hexane/AcOEt ¼ 7/3 as eluent for the
chromatographic purification. Yield 61%; oil; IR (CHCl3)
3452, 3318, 3034, 2934, 2858, 1686, 1602, 1501, 1414,
6.1.15. 5-(Biphenyl-4-ylcarboxamidomethyl)-N,N-dimethyl-
2H-tetrazole-2-carboxamide (15)
The title compound was prepared from 42 following the
same procedure that was used for the synthesis of 1, 2 using
hexane/AcOEt ¼ 45/55 as eluent for the chromatographic sep-
aration of regioisomers. Attempts to isolate 1,5-regioisomer 10
in a pure form failed owing to its propensity to isomerise.
Compound 15 (49%): mp 186e188 ꢁC; IR 3289, 2857,
1234, 1204 cmꢃ1 1H NMR (300 MHz) d 1.31e1.36 (4H,
;
m), 1.61 (4H, m) 2.21 (2H, t, J ¼ 7.4 Hz), 2.59 (2H, t,
J ¼ 7.6 Hz), 4.10 (2H, d, J ¼ 5.8 Hz), 6.60 (1H, m), 7.14e
7.30 (5H, m); 13C NMR (75 MHz) d 25.22, 27.41, 28.86,
28.98, 31.20, 35.81, 35.88, 116.33, 125.64, 128.25, 128.38,
142.59, 173.57. Anal. (C15H20N2O) C, H, N.
1
1725, 1627, 1552, 1487, 1397, 1307, 1249, 1084 cmꢃ1; H
NMR (300 MHz, DMSO-d6) d 2.91 (3H, s), 3.08 (3H, s),
4.90 (2H, d, J ¼ 5.4 Hz), 6.88 (1H, br s), 7.41e8.00 (9H,
m); 13C NMR (75 MHz, DMSO-d6) d 30.24, 33.36, 36.91,
124.69, 126.44, 126.69, 127.81, 128.82, 131.77, 138.86,
143.09, 147.31, 151.28, 154.17. Anal. (C18H18N6O2) C, H, N.
6.1.20. N-((1H-Tetrazol-5-yl)methyl)-7-phenylheptanamide
(44)
The title compound was prepared from 39 following the
same procedure that was used for the synthesis of 22. Yield
56%; mp 124e132 ꢁC; IR 3297, 3025, 2934, 2854, 1659,
1
1521, 1434, 1333, 1250, 1235, 1080, 1053 cmꢃ1; H NMR
6.1.16. 2-(Biphenyl-4-yl)-N-(cyanomethyl)acetamide (38)
The title compound was prepared from 2-(biphenyl-4-yl)-
acetic acid (33) following the same procedure that was used
for the synthesis of 36. Yield 69%; mp 146e147 ꢁC; IR
(KBr) 3249, 3046, 1683, 1644, 1541, 1486, 1407, 1256,
(300 MHz, CDCl3/CD3OD ¼ 9/1) d 1.27e1.38 (4H, m),
1.52e1.66 (4H, m) 2.21 (2H, t, J ¼ 7.6 Hz), 2.58 (2H, t,
J ¼ 7.5 Hz), 4.66 (2H, s), 7.11e7.31 (5H, m); 13C NMR
(75 MHz, CDCl3/CD3OD ¼ 9/1) d 25.46, 28.95, 29.11,
31.27, 32.71, 35.89, 36.06, 125.69, 128.31, 128.43, 142.69,
154.76, 175.12. Anal. (C15H21N5O) C, H, N.
1
1160, 1029 cmꢃ1; H NMR (300 MHz, CD3OD) d 3.61 (2H,
s), 4.43 (2H, s), 7.34e7.56 (9H, m); 13C NMR (75 MHz,
CD3OD) d 27.62, 42.43, 116.53, 127.24, 127.65, 127.71,
129.09, 129.88, 133.65, 140.56, 140.95, 172.80. Anal.
(C16H14N2O) C, H, N.
6.1.21. N,N-Dimethyl-5-((7-phenylheptanamido)methyl)-
1H-tetrazole-1-carboxamide (12) and N,N-dimethyl-5-((7-
phenylheptanamido)methyl)-2H-tetrazole-2-carboxamide
(17)
6.1.17. N-((1H-Tetrazol-5-yl)methyl)-2-(biphenyl-4-
yl)acetamide (43)
The title compounds were prepared from 44 following the
same procedure that was used for the synthesis of 1, 2 using
hexane/acetone ¼ 65/35 as eluent for the chromatographic
separation of regioisomers. Compound 17 (less polar, 38%):
mp 74e76 ꢁC; IR 3310, 3062, 3027, 2926, 2853, 1727,
The title compound was prepared from 38 following the
same procedure that was used for the synthesis of 22. Yield
67%; mp 187e189 ꢁC; IR 3334, 3034, 1646, 1548, 1517,
1487, 1423, 1261, 1231, 1160, 1076 cmꢃ1
;
1H NMR
1653, 1542, 1420, 1397, 1251, 1087 cmꢃ1 1H NMR
;

G. Ortar et al. / European Journal of Medicinal Chemistry 43 (2008) 62e72
71
(300 MHz) d 1.27e1.35 (4H, m), 1.52e1.62 (4H, m) 2.21
(2H, t, J ¼ 7.5 Hz), 2.58 (2H, t, J ¼ 7.6 Hz), 3.10 (3H, s),
3.22 (3H, s), 4.80 (2H, d, J ¼ 5.8 Hz), 6.94 (1H, m), 7.13e
7.30 (5H, m); 13C NMR (75 MHz) d 25.38, 28.88, 29.03,
31.18, 33.69, 35.82, 36.09, 37.91, 39.42, 125.62, 128.24,
128.38, 142.63, 147.91, 154.65, 173.72. Anal. (C18H26N6O2)
C, H, N. Compound 12 (more polar, 26%): mp 55e57 ꢁC;
IR 3312, 3066, 3029, 2937, 2846, 1732, 1651, 1548, 1410,
m); 13C NMR (75 MHz) d 25.45, 29.07, 29.13, 31.36, 33.69
35.90, 36.12, 37.90, 39.42, 125.58, 128.22, 128.37, 142.75,
147.91, 154.64, 173.75. Anal. (C19H28N6O2) C, H, N. Com-
pound 13 (more polar, 26%): mp 55e57 ꢁC; IR 3294, 3068,
2927, 2849, 1732, 1655, 1549, 1490, 1384, 1300, 1248,
1133, 1075 cmꢃ1 1H NMR (300 MHz) d 1.32 (6H, m),
;
1.61e1.68 (4H, m) 2.26 (2H, t, J ¼ 7.6 Hz), 2.59 (2H, t,
J ¼ 7.6 Hz), 3.09 (3H, s), 3.25 (3H, s), 4.81 (2H, d,
J ¼ 5.8 Hz), 6.37 (1H, m), 7.12e7.30 (5H, m); 13C NMR
(75 MHz) d 25.51, 29.11, 29.18, 31.38, 34.86, 35.93, 36.41,
38.17, 39.18, 125.58, 128.23, 128.39, 142.79, 147.59,
163.42, 173.30. Anal. (C19H28N6O2) C, H, N.
1383, 1277, 1215, 1134, 1073 cmꢃ1 1H NMR (300 MHz)
;
d 1.26e1.39 (4H, m), 1.54e1.69 (4H, m) 2.26 (2H, t,
J ¼ 7.5 Hz), 2.60 (2H, t, J ¼ 7.7 Hz), 3.09 (3H, s), 3.24 (3H,
s), 4.80 (2H, d, J ¼ 5.6 Hz), 6.40 (1H, m), 7.13e7.31 (5H,
m); 13C NMR (75 MHz) d 25.47, 28.95, 29.09, 31.25, 34.87,
35.87, 36.38, 38.17, 39.17, 125.63, 128.26, 128.41, 142.70,
147.61, 163.46, 173.31. Anal. (C18H26N6O2) C, H, N.
6.2. X-ray structure determination of tetrazole 2
X-ray diffraction data for compound 2 (slow crystallisation
from CH2Cl2/hexane) were collected on a Rigaku AFC5R dif-
fractometer at 298 K, using graphite-monochromated Cu Ka
6.1.22. N-(Cyanomethyl)-8-phenyloctanamide (40)
The title compound was prepared from 8-phenyloctanoic
acid (35) following the same procedure that was used for the
synthesis of 36 using hexane/AcOEt ¼ 7/3 as eluent for the
chromatographic purification. Yield 47%; mp 39e40 ꢁC; IR
(CHCl3) 3452, 3323, 3034, 2932, 2857, 2337, 1686, 1602,
˚
radiation (l ¼ 1.54178 A) and a rotating anode generator.
The structure was solved by direct methods and refined by
full-matrix least-squares with anisotropic thermal parameters,
using the SIR2004 structure determination package [39]. The
1498, 1453, 1348, 1263, 1263, 1206, 1031 cmꢃ1; H NMR
hydrogen atoms were idealised (CeH ¼ 0.96 A). Each H atom
1
˚
(300 MHz) d 1.30 (6H, m), 1.59 (4H, m), 2.20 (2H, t,
J ¼ 7.5 Hz), 2.57 (2H, t, J ¼ 7.6 Hz), 4.10 (2H, d,
J ¼ 5.8 Hz), 6.52 (1H, m), 7.11e7.30 (5H, m); 13C NMR
(75 MHz) d 25.30, 27.42, 29.08, 29.13, 31.34, 35.90, 35.95,
116.30, 125.62, 128.26, 128.39, 142.76, 173.54. Anal.
(C16H22N2O) C, H, N.
was assigned the equivalent isotropic temperature factor of
the parent atom and allowed to ride it. The final difference
Fourier map withꢃa3 root-mean-square deviation of electron
˚
density of 0.64 eA showed no significant features.
6.3. Biological evaluation
6.1.23. N-((1H-Tetrazol-5-yl)methyl)-8-phenyloctanamide
(45)
6.3.1. Assay of AEA cellular reuptake
The effect of compounds on the uptake of [14C]anandamide
by rat basophilic leukemia (RBL-2H3) cells was studied by us-
ing 2.4 mM (10,000 cpm) of [14C]anandamide [40]. The
method consisted of incubating the cells with [14C]ananda-
mide for 90 s at 37 ꢁC, in the presence or absence of varying
concentrations of the inhibitors, which were administered to
cells 10 min prior to incubation with [14C]anandamide. In all
cases, residual [14C]anandamide in the incubation medium af-
ter extraction with CHCl3/CH3OH ¼ 2/1 (by volume), deter-
mined by scintillation counting of the lyophilized organic
phase, was used as a measure of the amount of anandamide
that was taken up by cells. In some cases, the formation of
[14C]ethanolamine from [14C]anandamide was measured by
counting the water phase of the incubation medium extract.
Data are expressed as the concentration exerting 50% inhibi-
tion of anandamide uptake (IC50) calculated by GraphPadÒ.
Non-specific binding of [14C]anandamide to cells and plastic
dishes was determined in the presence of 100 mM anandamide
and was never higher than 40% of total uptake.
The title compound was prepared from 40 following the
same procedure that was used for the synthesis of 22. Yield
79%; mp 118e120 ꢁC; IR 3291, 3024, 2930, 2852, 1659,
1
1526, 1433, 1333, 1251, 1231, 1083, 1053 cmꢃ1; H NMR
(300 MHz, CDCl3/CD3OD ¼ 9/1) d 1.29 (6H, m), 1.57e1.61
(4H, m) 2.26 (2H, t, J ¼ 7.6 Hz), 2.58 (2H, t, J ¼ 7.7 Hz),
4.70 (2H, m), 7.13e7.31 (5H, m), 7.67 (1H, m); 13C NMR
(75 MHz, CDCl3/CD3OD ¼ 9/1) d 25.48, 29.11, 31.35,
32.46, 32.57, 35.92, 36.12, 36.16, 125.63, 128.27, 128.40,
142.76, 153.20, 175.43. Anal. (C16H23N5O) C, H, N.
6.1.24. N,N-Dimethyl-5-((7-phenyloctanamido)methyl)-1H-
tetrazole-1-carboxamide (13) and N,N-dimethyl-5-((7-
phenyloctanamido)methyl)-2H-tetrazole-2-carboxamide
(18)
The title compounds were prepared from 45 following the
same procedure that was used for the synthesis of 1, 2 using
preparative layer chromatography (silica gel, 0.5 cm thick)
and hexane/acetone ¼ 60/40 as eluent for the chromatographic
separation of regioisomers. Compound 18 (less polar, 38%):
mp 74e76 ꢁC; IR 3302, 3074, 2927, 2852, 1728, 1655,
6.3.2. Assay of fatty acid amide hydrolase (FAAH)
The effect of compounds on the enzymatic hydrolysis of
anandamide was studied as described previously [40] using
membranes prepared from either rat brain, incubated with the
test compounds and [14C]anandamide (2.4 mM) in 50 mM
triseHCl, pH 9, for 30 min at 37 ꢁC. [14C]ethanolamine
1
1548, 1413, 1397, 1334, 1251, 1123, 1087 cmꢃ1; H NMR
(300 MHz) d 1.29 (6H, m), 1.55e1.61 (4H, m) 2.21 (2H, t,
J ¼ 7.6 Hz), 2.58 (2H, t, J ¼ 7.7 Hz), 3.09 (3H, s), 3.22 (3H,
s), 4.81 (2H, d, J ¼ 6.0 Hz), 6.95 (1H, m), 7.14e7.30 (5H,

72
G. Ortar et al. / European Journal of Medicinal Chemistry 43 (2008) 62e72
produced from [14C]anandamide 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). With active compounds, data are expressed as
the concentration exerting 50% inhibition of AEA hydrolysis
(IC50), calculated by GraphPad.
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