New N-arachidonoylserotonin analogues with potential "dual" mechanism of action against pain

Article abstract of DOI:10.1021/jm070678q

N-Arachidonoylserotonin (AA-5-HT, 1a) is an inhibitor of fatty acid amide hydrolase (FAAH) that acts also as an antagonist of transient receptor potential vanilloid-type 1 (TRPV1) channels and is analgesic in rodents. We modified the chemical structure of 1a with the aim of developing "hybrid" FAAH/TRPV1 blockers more potent than the parent compound or obtaining analogues with single activity at either of the two targets to study the mechanism of the analgesic action of 1a. Thirty-eight AA-5-HT analogues, containing a serotonin "head" bound to a variety of lipophilic moieties via amide, urea, or carbamate functionalities, were synthesized. Unlike 1a, most of the new compounds possessed activity at only one of the two considered targets. The amides 1b and 1c of α- and γ-linolenic acid, however, showed "hybrid" activity similar to 1a. The carbamate 3f (OMDM106), although unable to antagonize TRPV1 receptors, was the most potent FAAH inhibitor in this study (IC₅₀ = 0.5 μM). Compounds 3f and 1m (OMDM129), which exhibited activity at only FAAH or TRPV1, respectively, were 10-fold less potent than 1a at preventing formalin-induced hyperalgesia in mice.

Full text of DOI:10.1021/jm070678q

6554
J. Med. Chem. 2007, 50, 6554–6569
New N-Arachidonoylserotonin Analogues with Potential “Dual” Mechanism of Action
against Pain
Giorgio Ortar,† Maria Grazia Cascio, Luciano De Petrocellis,§ Enrico Morera,† Francesca Rossi,‡ Aniello Schiano-Moriello,§
Marianna Nalli,† Vito de Novellis,‡ David F. Woodward,# Sabatino Maione,‡ and Vincenzo Di Marzo*,
Dipartimento di Studi Farmaceutici, UniVersity of Rome “La Sapienza”, Piazzale Aldo Moro 5, 00185 Rome, Italy, Endocannabinoid Research
Group, Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Via dei Campi Flegrei 34, 80078 Pozzuoli, Naples, Italy,
Endocannabinoid Research Group, Institute of Cybernetics, Consiglio Nazionale delle Ricerche, Via dei Campi Flegrei 34,
80078 Pozzuoli, Naples, Italy, Department of Biological Sciences, Allergan, Inc., 2525 Dupont DriVe (RD-2C), IrVine, California 92612, and
Department of Experimental MedicinesSection of Pharmacology “L. Donatelli”, Second UniVersity of Naples, Naples, Italy
ReceiVed June 11, 2007
N-Arachidonoylserotonin (AA-5-HT, 1a) is an inhibitor of fatty acid amide hydrolase (FAAH) that acts
also as an antagonist of transient receptor potential vanilloid-type 1 (TRPV1) channels and is analgesic in
rodents. We modified the chemical structure of 1a with the aim of developing “hybrid” FAAH/TRPV1
blockers more potent than the parent compound or obtaining analogues with single activity at either of the
two targets to study the mechanism of the analgesic action of 1a. Thirty-eight AA-5-HT analogues, containing
a serotonin “head” bound to a variety of lipophilic moieties via amide, urea, or carbamate functionalities,
were synthesized. Unlike 1a, most of the new compounds possessed activity at only one of the two considered
targets. The amides 1b and 1c of R- and γ-linolenic acid, however, showed “hybrid” activity similar to 1a.
The carbamate 3f (OMDM106), although unable to antagonize TRPV1 receptors, was the most potent FAAH
inhibitor in this study (IC50 ) 0.5 µM). Compounds 3f and 1m (OMDM129), which exhibited activity at
only FAAH or TRPV1, respectively, were 10-fold less potent than 1a at preventing formalin-induced
hyperalgesia in mice.
Introduction
strong inhibitory action on FAAH may act as analgesic and anti-
inflammatory agents.
The proteins of the endocannabinoid system, including CB1
and CB2 cannabinoid receptors, a putative and still elusive
endocannabinoid membrane transporter, and fatty acid amide
hydrolase (FAAHa), represent excellent targets for the develop-
ment of new therapeutic drugs to be employed in many
pathologies, including pain, inflammation, cancer, neuronal
excitotoxicity and eating and motor disorders.1 In particular,
FAAH, first identified and cloned from several mammalian
species in the mid- to late 1990s,2–5 might be considered a
promising target for the treatment of chronic pain conditions.
It catalyzes the hydrolysis of several bioactive fatty acid amides
and esters,6 including (1) the endogenous ligands of cannabinoid
receptors, anandamide (AEA) and 2-arachidonoylglycerol
(2-AG),7–9 (2) the anandamide congeners, N-palmitoylethano-
lamide (PEA) and N-oleoylethanolamide, which do not bind to
cannabinoid receptors,10–13 and (3) the sleep-inducing factor,
cis-9-octadecenamide14 (oleamide) and its congeners (Figure 1).
Since FAAH null mice15 exhibit less sensitivity to several pain
stimuli,16,17 this enzyme has been suggested to control tonically
the levels of endogenous analgesic fatty acid amides, such as
AEA and PEA, and this indicates that synthetic compounds with
The transient receptor potential vanilloid-type 1 (TRPV1)
channel is another potential new target for the development of
analgesic and anti-inflammatory drugs. It has been described
as a molecular integrator, expressed mostly in unmyelinated
sensory fiber afferents of the C-type, of various nociceptive
stimuli, and of both a physical (high temperature and low pH)
and chemical (plant toxins and endogenous fatty acid deriva-
tives) nature.18 Several studies using animal models have shown
altered levels of expression of TRPV1 in pain states. In
particular, in models of neuropathic pain,19–22 there is evidence
that TRPV1 expression is decreased in the injured nerve fibers
but increased in those proximal to the site damage. Both
agonists, through desensitization of TRPV1 receptors23,24 or
inhibition of voltage-activated Ca2+ channels,25 and antago-
nists26 have been proposed as new therapies for the treatment
of chronic and inflammatory pain.
In view of this background, a possible strategy for the
development of new analgesic drugs might be to design
molecules with the capability of both (1) inhibiting FAAH in
order to elevate the levels of the endogenous analgesic and anti-
inflammatory substrates of this enzyme (i.e., fatty acid amides
such as AEA and PEA, and the endocannabinoid 2-AG)27,28
and (2) inactivating TRPV1 receptors, e.g., through pharmaco-
logical antagonism. N-Arachidonoylserotonin (AA-5-HT, 1a,
Table 1) is a well-known FAAH inhibitor (IC50 ) 1–12
µM),29–31 and it was also recently reported to act as a potent
antagonist of both human and rat recombinant TRPV1 receptors
(IC50 ) 70–100 nM vs capsaicin 10-7 M).32 This prototypical
“hybrid” FAAH/TRPV1 blocker has been selected in the present
study as the lead compound for the synthesis of new analogues
with potential “dual” effects (FAAH inhibitors/TRPV1 antago-
nists). Our present aim was to investigate the structure-activity
* To whom correspondence should be addressed. Phone: +39-81-
8675093. Fax: +39-81-8041770. E-mail: vdimarzo@icmib.na.cnr.it.
†
University of Rome “La Sapienza”.
Institute of Biomolecular Chemistry, C.N.R.
Institute of Cybernetics, C.N.R.
Second University of Naples.
Allergan, Inc.
§
‡
#
a
Abbreviations: AA-5-HT, N-arachidonoylserotonin; AEA, anandamide;
2-AG, 2-arachidonoylglycerol; EDC, N-ethyl-N′-(3-dimethylaminopropyl)-
carbodiimide hydrochloride; HEK293, human embryonic kidney cells;
HOBt, 1-hydroxybenzotriazole; PEA, N-palmitoylethanolamide; FAAH,
fatty acid amide hydrolase; I-RTX, iodoresiniferatoxin; TRPV1, transient
receptor potential vanilloid-type 1.
10.1021/jm070678q CCC: $37.00
2007 American Chemical Society
Published on Web 11/21/2007

“Hybrid” FAAH/TRPV1 Blockers
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6555
Figure 1. Substrates of fatty acid amide hydrolase (FAAH).
relationships for the interaction of 1a with FAAH and TRPV1
in order to either develop “hybrid” FAAH/TRPV1 blockers more
potent than the parent compound or obtain analogues with single
activity at either of the two targets, to be used as leads for the
design of new therapeutic drugs. Thirty-eight structural ana-
logues of 1a containing the unmodified serotonin moiety bound
to a variety of lipophilic side chains were synthesized, thereby
systematically modulating two of three pharmacophoric regions
of 1a, i.e., the hydrophobic region and the amide template. In
fact, we have also evaluated the influence of the nature of the
link between serotonin and the side chain, by synthesizing urea,
amide, and carbamate derivatives. The structures of the new
compounds are reported in Table 1.
Chemistry. The analogues of 1a in Table 1 were prepared
as detailed below and as summarized in Schemes 1–3. The
synthesis of amides 1b-o has been carried out by condensation
between the appropriate carboxylic acids 4b-o and serotonin
using 1-hydroxybenzotriazole (HOBt)/N-ethyl-N′-(3-dimethyl-
aminopropyl)carbodiimide hydrochloride (EDC) as the car-
boxylate activator (Scheme 1). Ureas 2a-g were synthesized
by treatment of the appropriate primary amines 5a-g with
bis(trichloromethyl)carbonate, followed by coupling of the re-
sulting isocyanates with serotonin (Scheme 2). Finally, the
preparation of carbamates 3a-q involved conversion of the ap-
propriate alcohols or phenols 6a-q with phosgene to the
corresponding chloroformates and subsequent reaction with
serotonin (Scheme 3).
The preparation of the noncommercial reagents has been
carried out as follows. 9-Phenylnonanoic acid (4k)33 was
obtained by oxidation of 9-phenyl-1-nonanol with pyridinium
dichromate. (3-Pentylphenyl)acetic acid (4l) was synthesized
by the Sonogashira reaction of methyl (3-iodophenyl)acetate
(7) with 1-pentyne followed by catalytic reduction of arylalkyne
8 and alkaline hydrolysis (Scheme 4). [1,1′-Biphenyl]-3-acetic
acid (4m)34 was prepared by Suzuki cross-coupling of 7 with
phenylboronic acid followed by alkaline hydrolysis (Scheme
5). The synthesis of [3′-pentyl-1,1′-biphenyl]-4-butanoic acid
(4n) was carried out in six steps from 3-pentylphenol (9).35 After
triflation of 9, the triflate 10 was subjected to a Suzuki cross-
coupling with 3-hydroxyphenylboronic acid to give [3′-pentyl-
1,1′-biphenyl]-3-ol (11). Triflation of 11 followed by a Sono-
gashira reaction of the triflate 12 with 3-butyn-1-ol afforded
the alkynol 13. Catalytic reduction of 13 to give 14 followed
by oxidation of the alcoholic group with pyridinium dichromate
provided eventually the acid 4n (Scheme 6). For the preparation
of 7-pentyl-2-naphthalenebutanoic acid (4o), 7-hydroxy-2-
naphthalenyl triflate (15)36 was employed as the starting
material. Protection of the phenol function followed by Ni(0)-
catalyzed cross-coupling of triflate 16 with pentylmagnesium
chloride37 and deprotection of the MOM protecting group38 gave
7-pentyl-2-naphthalenol (18). Repetition of steps c-f of Scheme
6 on 18 afforded acid 4o (Scheme 7). Primary amines 5f,g were
obtained by catalytic reduction of the appropriate nitriles.
3-Pentylaniline 5b was prepared by Sonogashira coupling
between 3-iodonitrobenzene (22) and 1-pentyne followed by
catalytic reduction of the resulting arylalkyne 23 according to
Gaudreault and co-workers39 (Scheme 8). Alcohols 6a-d were
eventually obtained by reduction of the corresponding acids
4a-d, activated as mixed anhydrides, with NaBH4.
Biological Evaluation. In vitro assays were aimed at examin-
ing the effect of the new compounds on rat brain FAAH activity
and on TRPV1 by measuring the effect on [14C]anandamide
hydrolysis by rat brain membranes and on capsaicin-induced
intracellular Ca2+ elevation in HEK293 cells overexpressing
the human recombinant TRPV1 receptor, respectively. We also
evaluated the effect of different doses of 3f (OMDM106) and
1m (OMDM129), two structurally related serotonin derivatives
with high activity at only either FAAH or TRPV1, respectively,
compared to 1a, on the biphasic nociceptive response induced
by an intrapaw injection of a formalin-containing aqueous
solution in mice in vivo. This test was used for three reasons:
(1) it allows the determination of the effect of administered
compounds on both acute peripheral pain (especially during the
first phase of the nocifensive response to formalin) and
inflammatory-like pain (especially during the second phase of
the nocifensive response to formalin);32 (2) it has been previ-
ously used to study the analgesic effects of both FAAH and
TRPV1 blockers;46,47 (3) it was originally employed to establish
the analgesic effect of 1a.32 It is noteworthy that all pharma-
cological tools used in the present study to evaluate the
mechanism of the in vivo antinociceptive effects of compounds
1a, 3f, and 1m, i.e., AM251, AM639, and iodoresiniferatoxin
(I-RTX) were inactive at inhibiting [14C]anandamide hydrolysis
(less than 10% inhibition) up to 50 µM; AM251 and AM639
were also inactive on TRPV1 (less than 15% stimulation or
inhibition of capsaicin response) up to 10 µM (data not shown).
Results and Discussion
All newly synthesized compounds were evaluated in both
TRPV1 and FAAH assays, and the results are summarized in
Table 1. The most important points are as following. The urea
derivatives (2a-g) are generally inactive (IC50 g 50 µM) FAAH
inhibitors, while their TRPV1 antagonistic effects appear to be
highly dependent on the length of the linker between the urea
functionality and the aromatic portion and on the proper choice
of the lipophilic substituents on the phenyl ring, according to
structure–activity relationships developed for small-molecule
TRPV1 antagonists whose structural features can be traced back
to the prototypical TRPV1 antagonist capsazepine.40 Amide

6556 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26
Ortar et al.
Table 1. Structures and Results of FAAH (IC50) and TRPV1 (IC50) Assays on 1a and 1a Analoguesa

“Hybrid” FAAH/TRPV1 Blockers
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6557
Table 1. Continued

6558 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26
Table 1. Continued
Ortar et al.
a Data represent mean values ( SD for at least three separate experiments performed in duplicate and are expressed as IC50 (µM) for FAAH and for
TRPV1 assays.
Scheme 1a
Scheme 4a
a Reagents and conditions: HOBt/EDC, room temp, 1 h, then serotonin ·
HCl, Et3N, DMF, room temp, 16 h.
a Reagents and conditions: (a) 1-pentyne, Pd(OAc)2/2PPh3, CuI,
DMSO-iPr2NH, room temp, 4 h; (b) H2, Pd/C, 12 N HCl, AcOEt, room
temp, 4 h; (c) 1 N LiOH, THF-H2O, room temp, 16 h.
Scheme 2a
Scheme 5a
a Reagents and conditions: (a) Cl3COCOOCCl3, Et3N, dry CH2Cl2, reflux,
5 h; (b) serotonin ·HCl, Et3N, dry CH2Cl2-pyridine, room temp, 16 h.
a Reagents and conditions: (a) phenylboronic acid, Pd(PPh3)4, K3PO4,
DMF, 90 °C, 16 h; (b) 1 N LiOH, THF-H2O, 16 h.
Scheme 3a
1k IC50 >50, 34 ( 3, and 30 ( 2 µM, respectively).
Remarkably, in the acyl heterocycle series of competitive FAAH
inhibitors described by Boger and co-workers, the introduction
of ω-phenylalkyl moieties led to the most potent inhibitors.41
FAAH inhibitory activity of aliphatic amides increases signifi-
cantly with increasing number of double bonds in the chain
(1a-e). Most amides are able to antagonize TRPV1 receptors
(9.12 > IC50 > 0.24), and in particular, 1b,l,m exhibit equipotent
antagonistic activities compared to 1a. The nature of the
lipophilic portion does not seem to influence critically the
a Reagents and conditions: (a) COCl2, Et3N, dry toluene, room temp,
2 h; (b) serotonin ·HCl, Et3N, dry DMF, room temp, 16 h.
compounds with saturated aliphatic or arylaliphatic chains
(1f-o) are essentially inactive as FAAH inhibitors, although
the activity exhibits a modest increase with increasing alkyl
chain length in the case of ω-phenylalkylamides (1i, 1j, and

“Hybrid” FAAH/TRPV1 Blockers
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6559
Scheme 6a
a Reagents and conditions: (a) (CF3SO2)2O, 2,6-di-tert-butyl-4-methylpyridine, dry CH2Cl2, room temp, 3.5 h; (b) 3-hydroxyphenylboronic acid, Pd(PPh3)4,
K3PO4, KBr, dioxane, 85 °C, 5 h; (c) (CF3SO2)2O, 2,6-di-tert-butyl-4-methylpyridine, dry CH2Cl2, room temp, 16 h; (d) 3-butynol, Pd(OAc)2/2PPh3, CuI,
DMSO-iPr2NH, room temp, 48 h; (e) H2, Pd/C, AcOEt, room temp, 3 h; (f) PDC, DMF, room temp, 16 h.
Scheme 7a
a Reagents and conditions: (a) MeOCH2Cl, Et3N, dry THF, room temp, 16 h; (b) 2 M C5H11MgCl, NiCl2(dppp), dry THF, room temp, 1 h; (c) CBr4, dry
iPrOH, reflux, 1.5 h; (d) (CF3SO2)2O, 2,6-di-tert-butyl-4-methylpyridine, dry CH2Cl2, room temp, 2.5 h; (e) 3-butynol, Pd(OAc)2/2PPh3, CuI, DMSO-iPr2NH,
room temp, 4 h; (f) H2, Pd/C, AcOEt, room temp, 1.5 h; (g) PDC, dry DMF, room temp, 16 h.
Scheme 8a
aryl ester series of FAAH inhibitors.43 Carbamates are generally
unable to antagonize TRPV1 receptors, with the exception of
3a, 3e, and 3h (5.25 > IC50 > 2.40). Some of the compounds
of this series (3e, 3o, 3f, and 3q) were also evaluated as
inhibitors of anandamide cellular uptake, but they were inactive
up to 25 µM (data not shown). A comparison between amides
and carbamates with analogous lipophilic moieties (e.g., 1a-3a,
1b-3b, 1c-3c, 1d-3d, and 1l-3e) indicates a 10-fold differ-
ence of the TRPV1 antagonistic effects between the two classes
of compounds. In summary, (a) the urea motif might be suitable
for the interaction with TRPV1 as a central hydrogen bond
acceptor/donor motif, but none of our ureidic compounds were
able to efficaciously inhibit FAAH. (b) The amide functionality
seems to serve as a better template for the “dual” action than
the carbamate functionality in compounds with unsaturated
aliphatic chains (see compounds 1a-d and 3a-d). (c) The
carbamate moiety appears to be superior to the other two
templates for FAAH inhibitory activity, provided that O-
aromatic substituents are present, as suggested by the compari-
son between compounds 1l and 3e, 1m and 3f, 2b and 3e, and
2e and 3f. In contrast, O-alkyl substituted carbamates are worse
than the corresponding amides as FAAH inhibitors (see
compounds 1a-d and 3a-d). Some compounds of the series
a Reagents and conditions: (a) 1-pentyne, Pd(OAc)2/2PPh3, CuI,
DMSO-iPr2NH, room temp, 4 h; (b) H2, Pd/C, EtOH, 60 °C, 6 h.
activity of amides on TRPV1. Special attention should be paid
to compounds 1b and 1c, which behave as the lead compound
1a as both FAAH inhibitors (IC50 ) 10 vs 8 µM for 1a) and
TRPV1 antagonists (IC50 ) 0.47 ( 0.16 and 0.95 ( 0.33 vs
0.27 ( 0.07 for 1a). All carbamates with aromatic O-substituents
show good FAAH inhibitory activities, while the presence of
an unsaturated alkyl chain is detrimental to the inhibitory
activity, according to the notion that the O-substituted moiety
of carbamates acts as the leaving group in the reaction leading
to enzyme carbamoylation.42 Compounds with meta substituted
aromatic rings are invariably more potent as FAAH inhibitors
than the para substituted ones, independent of the nature of the
substituent. The strongest FAAH inhibition is observed with
the m-biphenyl derivative 3f, a result similar to that previously
described by Mor and co-workers for their alkylcarbamic acid

6560 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26
Ortar et al.
3 are also TRPV1 antagonists. (d) The serotonin template may
afford compounds endowed with dual activity against FAAH
and TRPV1.
In order to support the hypothesis that the previously
described high efficacy of 1a as an analgesic compound was
due to its unique capability of inhibiting both FAAH and
TRPV1, we compared the potency of this compound to two of
its analogues with “single” activity as either FAAH inhibitors
or TRPV1 antagonists. We selected two structurally related
analogues, 3f and 1m, which were among the most potent
FAAH or TRPV1 blockers, respectively, out of the 38 new
compounds synthesized here (Table 1). These compounds were
also selected because, among those belonging to the amide/
carbamate “couples” synthesized here, they were the only ones
to be active at inhibiting only either FAAH or TRPV1.
Therefore, 3f and 1m, by being as structurally similar as possible
for two compounds belonging to the 1 and 3 series, represented
ideal candidates to test the role of FAAH and TRPV1 in the
analgesic effects of 1a analogues in vivo. These compounds
are also likely to have pharmacokinetic profiles similar to each
other and to most of the other serotonin derivatives studied here,
including 1a. The biphasic nocifensive response of mice treated
with formalin was selected as the in vivo assay to compare the
analgesic activity of the three compounds (Figures 2 and 3). In
fact, it has been reported that formalin injection in mice causes
a late phase (15–60 min after injection) of hyperalgesia, probably
through activation of the PKA/PKC pathway and sensitization
of the TRPV1 receptors,44,45 and this late response is blocked
by TRPV1 antagonists46 and also by FAAH inhibitors.47,48 As
shown in Figure 2 (upper panel), we found that the systemic
administration of 1a (0.3, 1, 2.5, and 5 mg/kg) dose-dependently
inhibited the late phase of formalin-induced hyperalgesia, with
IC50 ) 0.5 mg/kg (estimated at 40 min from formalin injection).
The effect of 1a (5 mg/kg) was antagonized by the CB1
antagonist AM251 (but not by the CB2 antagonist AM630), in
agreement with FAAH inhibition by 1a and subsequent elevation
of endocannabinoid levels and activation of CB1 receptors
(Figure 2, upper middle panel). The TRPV1 antagonist, I-RTX,
exerted a strong antihyperalgesic effect per se (Figure 2, lower
middle panel) and did not antagonize the effect of 1a (Figure
2, lower panel). Therefore, although this experiment confirms
that TRPV1 antagonists are antihyperlgesic in the second phase
of the formalin test, it cannot be used to demonstrate that
compound 1a acts in this assay by also blocking TRPV1
receptors. The administration of compounds 3f (FAAH inhibitor)
and 1m (TRPV1 antagonist) also prevented the second phase
of formalin-induced hyperalgesia, although with significantly
less potency than 1a, with IC50 values of 4 and 5 mg/kg,
respectively (estimated at 40 min from formalin injection)
(Figure 3). Furthermore, the antihyperalgesic effect of 3f was
prevented only by the CB1 antagonist AM251, as one would
expect from a compound with inhibitory action at FAAH. By
contrast, the antihyperalgesic effect of 1m was not significantly
attenuated by AM251 (except for a small reduction observed
only at two time-points, which might have been due to some
nonspecific activity of 1m at FAAH or cannabinoid receptor at
the high systemic dose tested), in agreement with the present
finding that this compound is not a FAAH inhibitor, nor, as
expected, by I-RTX (Figure 3). The relative potencies of 1m,
3f, and 1a in the formalin assay do not disagree with our
hypothesis that 1a might exert its antihyperalgesic effects by
inhibiting both FAAH and TRPV1 receptors. In fact, if only
FAAH had been involved in these effects of 1a, 3f, which is
>10-fold more potent than 1a as a FAAH inhibitor and is likely
Figure 2. Analgesic effect of 1a in the formalin test in mice. Each
point represents the mean ( standard error of the mean (SEM) of 8–10
animals per group. Full symbols denote effects that are significantly
different from the respective control as assessed using two-way
ANOVA followed by the Bonferroni’s test. For the lower two panels,
the 1a dose was 5 mg/kg.
to have similar pharmacokinetics (and, by being a carbamate
and not an amide, should possess even higher metabolic stability
to enzymatic hydrolysis), should have also been more potent
in vivo. On the other hand, if only TRPV1 had been involved
in the actions of 1a, its effects in the formalin test should not
have been blocked by the CB1 antagonist and 1m should have
been equipotent at inhibiting formalin-induced nociception.
However, only specific experiments, analyzing the activity in
vivo of all the new serotonin derivatives synthesized in this study

“Hybrid” FAAH/TRPV1 Blockers
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6561
Cascio and Di Marzo, unpublished data) and hence are unlikely
to act as “direct” CB1 agonists in vivo. Independent of their
circulating concentrations after systemic administration, it has
been suggested16 that compounds that exhibit affinity constants
higher than 5 µM in binding assays are very unlikely to exert
any direct CB1-mediated effect in vivo, whereas FAAH inhibi-
tors with IC50 ≈ 5 µM can still exert pharmacological actions
in vivo via FAAH blockade, enhancement of AEA levels, and
indirect activation of CB1 receptors. This is possibly due to the
fact that AEA has micromolar affinity for FAAH and nanomolar
affinity for CB1 receptors, and hence, compounds that inhibit
FAAH with IC50 ≈ 5 µM can still efficaciously reduce AEA
degradation, whereas those that bind to CB1 with micromolar
affinity cannot compete with, or add to, endogenous AEA
activity at this receptor.
Conclusions
In the present study, we have described the synthesis and
the pharmacological evaluation of the new structural analogues
of 1a. Our goal was to obtain new molecules able, like the
starting compound, to target simultaneously both FAAH and
TRPV1 receptors in order to have new analgesic drugs. Of the
new compounds, 1b and 1c, i.e., the R- and γ-linolenic acid
amides of serotonin, showed high dual efficacy as inhibitors
for the two considered targets, a result that could have been
expected from the chemical similarity of these two compounds
with 1a. Also, compound 3e exhibited the capability of
inhibiting both FAAH and TRPV1, although it was relatively
less potent than 1a at the latter target. However, we identified
several compounds with potent “single” activity at either FAAH
or TRPV1, which might be used as templates for the future
development of novel FAAH and TRPV1 blockers. Furthermore,
compounds 3f and 1m, two analogues of 1a with activity at
only FAAH and TRPV1, respectively, were found here to be
∼10-fold less potent than 1a at preventing the second phase of
formalin-induced hyperalgesia. New studies are now necessary
to understand the structural requirements involved in the
simultaneous recognition of the two targets, FAAH and TRPV1,
in order to direct our future research toward the design and the
synthesis of new and ever more potent analgesic drugs.
Experimental Section
Chemistry. 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 Perkin-Elmer 1000 FT-IR spectrophotometer as KBr
1
disks unless otherwise indicated. H and 13C NMR spectra were
obtained on a Varian Mercury 300 spectrometer using CDCl3 as
solvent unless otherwise indicated and using TMS as the internal
standard. Satisfactory elemental analysis results were obtained for
the newly synthesized compounds (C, H, N, (0.4%).
N-[2-(5-Hydroxy-1H-indol-3-yl)ethyl]-r-linolenamide (1b). To
a stirred solution of 4b (160 mg, 0.58 mmol) in DMF (3 mL) were
added at 0 °C HOBT (102 mg, 0.60 mmol) and EDC (116 mg,
0.60 mmol). The mixture was stirred for 15 min at 0 °C and for
1 h at room temperature. Then serotonin hydrochloride (146 mg,
0.68 mmol) and Et3N (0.080 mL, 0.68 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, saturated NaHCO3, and brine,
dried (Na2SO4), and evaporated under vacuum. The residue (265
mg) was chromatographed on silica gel (14 g) using CH2Cl2/AcOEt
) 85/15 as eluent to give 220 mg (88%) of 1b as an oil. IR (CHCl3)
Figure 3. Analgesic effect of 3f (OMDM106) and 1m (OMDM129)
in the formalin test in mice. Each point represents the mean ( standard
error of the mean (SEM) of 8–10 animals per group. Full symbols
denote effects that are significantly different from the respective control
as assessed using two-way ANOVA followed by the Bonferroni’s test.
and using also FAAH and TRPV1 null mice in these tests, need
to be carried out in order to demonstrate the mechanism of action
in vivo of 1a. It is emphasized that 1a, 3f, and 1m have little,
if any, affinity (Ki > 5 µM) for CB1 receptors in binding assays
previously carried out in our laboratory (refs 29 and 32 and
3481, 3009, 2931, 2856, 1657, 1518, 1465, 1234, 1170, 1088 cm-1
;
1H NMR (300 MHz) δ 0.96 (3H, t, J ) 7.4 Hz), 1.26 (8H, m),
1.54 (2H, m), 1.98–2.10 (6H, m), 2.77 (6H, m), 3.46 (2H, m), 5.35

6562 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26
Ortar et al.
(6H, m), 5.85 (1H, t, J ) 5.8 Hz), 6.77–7.12 (4H, m), 7.24 (1H, br
s), 8.24 (1H, m); 13C NMR (75 MHz) δ 14.27, 20.56, 25.34, 25.55,
25.64, 25.78, 27.23, 29.16, 29.25, 29.61, 36.78, 39.80, 103.18,
111.96, 112.28, 123.06, 127.13, 127.71, 128.02, 128.28, 128.34,
130.33, 131.47, 132.01, 150.11, 174.08. Anal. (C28H40N2O2) C,
H, N.
N-[2-(5-Hydroxy-1H-indol-3-yl)ethyl]lauramide (1h). The title
compound was prepared from 4h following the same procedure
that was used for the synthesis of 1b and using hexane/AcOEt )
1/1 as eluent for the chromatographic purification. Yield 87%; mp
103–105 °C; IR 3499, 3415, 3307, 2918, 2849, 1635, 1542, 1487,
1
1469, 1417, 1247, 1218, 1193, 1093 cm-1; H NMR (300 MHz,
CDCl3/CD3OD ) 5/1) δ 0.87 (3H, t, J ) 6.2 Hz), 1.24 (16H, m),
1.58 (2H, m), 2.10 (2H, t, J ) 7.6 Hz), 2.84 (2H, t, J ) 7.0 Hz),
3.49 (2H, m), 6.75–7.20 (4H, m); 13C NMR (75 MHz, CDCl3/
CD3OD ) 5/1) δ 14.11, 22.73, 25.45, 25.85, 29.37, 29.56, 29.64,
31.96, 36.77, 36.82, 39.93, 40.05, 102.87, 111.77, 111.84, 111.95,
111.99, 112.11, 123.02, 123.18, 128.12, 131.43, 131.57, 150.24,
174.40, 174.48. Anal. (C22H34N2O2) C, H, N.
N-[2-(5-Hydroxy-1H-indol-3-yl)ethyl]-γ-linolenamide (1c).
The title compound was prepared from 4c following the same
procedure that was used for the synthesis of 1b and using CH2Cl2/
AcOEt )85/15 as eluent for the chromatographic purification. Yield
81%; oil; IR (CHCl3) 3481, 3009, 2930, 2858, 1659, 1518, 1466,
1
1231, 1170, 1088 cm-1; H NMR (300 MHz) δ 0.87 (3H, t, J )
7.4 Hz), 1.28 (8H, m), 1.58 (2H, m), 2.01–2.12 (6H, m), 2.80 (6H,
m), 3.51 (2H, m), 5.36 (6H, m), 5.73 (1H, t, J ) 5.6 Hz), 6.73–7.26
(5H, m), 8.12 (1H, m); 13C NMR (75 MHz) δ 14.08, 22.57, 25.37,
25.61, 25.64, 26.90, 27.22, 29.20, 29.32, 31.51, 36.70, 39.73,
103.17, 111.92, 112.09, 112.28, 123.02, 127.58, 128.01, 128.13,
128.15, 128.44, 129.69, 130.52, 131.45, 150.09, 173.65. Anal.
(C28H40N2O2) C, H, N.
N-[2-(5-Hydroxy-1H-indol-3-yl)ethyl]-7-phenylheptanamide
(1i). The title compound was prepared from 4i following the same
procedure that was used for the synthesis of 1b and using hexane/
AcOEt ) 4/6 as eluent for the chromatographic purification. Yield
92%; oil; IR (CHCl3) 3480, 3336, 3029, 2933, 2858, 1657, 1519,
1
1495, 1454, 1356, 1234, 1170, 1088 cm-1; H NMR (300 MHz)
δ 1.16 (4H, m), 1.45 (4H, m), 1.96 (2H, t, J ) 7.4 Hz), 2.46 (2H,
t, J ) 7.5 Hz), 2.66 (2H, m), 3.34 (2H, m), 5.81 (1H, t, J ) 5.5
Hz), 6.71–7.24 (9H, m), 7.89 (1H, br s), 8.28 (1H, s); 13C NMR
(75 MHz) δ 25.17, 25.65, 28.88, 29.00, 31.22, 35.79, 36.55, 39.81,
103.07, 111.65, 112.02, 112.12, 123.13, 125.57, 127.92, 128.22,
128.39, 131.36, 142.74, 149.96, 174.30. Anal. (C23H28N2O2) C,
H, N.
N-[2-(5-Hydroxy-1H-indol-3-yl)ethyl]linoleamide (1d). The
title compound was prepared from 4d following the same procedure
that was used for the synthesis of 1b and using CH2Cl2/AcOEt )
85/15 as eluent for the chromatographic purification. Yield 85%;
mp 65–66 °C; IR (CHCl3) 3687, 3481, 3009, 2929, 2857, 1657,
1
1602, 1518, 1466, 1234, 1170, 1088 cm-1; H NMR (300 MHz)
δ 0.88 (3H, t, J ) 6.6 Hz), 1.22–1.36 (14H, m), 1.55 (2H, m),
1.99–2.10 (6H, m), 2.78 (4H, m), 3.47 (2H, m), 5.33 (4H, m), 5.79
(1H, t, J ) 5.5 Hz), 6.78–7.15 (5H, m), 8.20 (1H, m); 13C NMR
(75 MHz) δ 14.09, 22.58, 25.35, 25.64, 25.77, 27.21, 29.17, 29.27,
29.34, 29.63, 31.52, 36.80, 39.75, 103.16, 111.94, 112.00, 112.28,
123.03, 127.92, 128.02, 130.11, 130.28, 131.44, 150.10, 174.00.
Anal. (C28H42N2O2) C, H, N.
N-[2-(5-Hydroxy-1H-indol-3-yl)ethyl]-8-phenyloctana-
mide (1j). The title compound was prepared from 4j following
the same procedure that was used for the synthesis of 1b and using
hexane/AcOEt ) 4/6 as eluent for the chromatographic purification.
Yield 92%; oil; IR (CHCl3) 3480, 3011, 2932, 2857, 1657, 1519,
1
1454, 1209, 1170, 1088 cm-1; H NMR (300 MHz) δ 1.18 (6H,
m), 1.50 (4H, m), 2.01 (2H, t, J ) 7.8 Hz), 2.51 (2H, t, J ) 7.6
Hz), 2.72 (2H, t, J ) 6.7 Hz), 3.41 (2H, m), 5.81 (2H, m), 5.81
(1H, t, J ) 5.8 Hz), 6.75–7.26 (9H, m), 7.43 (1H, br s), 8.16 (1H,
m); 13C NMR (75 MHz) δ 25.27, 25.74, 29.09, 29.14, 29.16, 31.39,
35.89, 36.71, 39.78, 103.13, 111.87, 111.98, 112.23, 123.08, 125.58,
127.98, 128.24, 128.42, 131.41, 142.88, 150.07, 174.10. Anal.
(C24H30N2O2) C, H, N.
(Z)-N-[2-(5-Hydroxy-1H-indol-3-yl)ethyl]-9-octadecenamide
(1e). The title compound was prepared from 4e following the same
procedure that was used for the synthesis of 1b and using hexane/
AcOEt ) 1/1 as eluent for the chromatographic purification. Yield
82%; mp 67–68 °C; IR (CHCl3) 3480, 3011, 2928, 2855, 1657,
1518, 1466, 1234, 1170, 1088 cm-1; 1H NMR (300 MHz) δ 0.87
(3H, t, J ) 6.8 Hz), 1.26 (20H, m), 1.54 (2H, m), 1.98 (4H, m),
2.08 (2H, t, J ) 7.6 Hz), 2.79 (2H, t, J ) 6.8 Hz), 3.46 (2H, m),
5.33 (2H, m), 5.81 (1H, t, J ) 5.7 Hz), 6.78–7.25 (5H, m), 8.20
(1H, m); 13C NMR (75 MHz) δ 14.13, 22.69, 25.34, 25.79, 27.21,
27.24, 29.19, 29.28, 29.34, 29.55, 29.74, 29.79, 31.91, 36.80, 39.75,
103.16, 111.95, 112.00, 112.29, 123.04, 127.99, 129.80, 129.99,
131.44, 150.11, 174.03. Anal. (C28H44N2O2) C, H, N.
9-Phenylnonanoic Acid (4k).33 A suspension of 9-phenylnonan-
1-ol (500 mg, 2.27 mmol) and pyridinium dichromate (5.124 g,
13.67 mmol) in DMF (20 mL) was stirred at room temperature for
20 h. The mixture was then diluted with water and extracted with
AcOEt. The organic phase was washed twice with brine, dried
(Na2SO4), and evaporated under vacuum. The residue (652 mg)
was chromatographed on silica gel (20 g) using CH2Cl2/AcOEt )
94/6 as eluent to give pure 4k (420 mg, 79%).
(()-N-[2-(5-Hydroxy-1H-indol-3-yl)ethyl]-5-(1,2-dithiolan-
3-yl)pentanamide (1f). The title compound was prepared from
4f following the same procedure that was used for the synthesis of
1b and using CH2Cl2/AcOEt ) 3/7 as eluent for the chromato-
graphic purification. Yield 86%; oil; IR (CHCl3) 3301, 2923, 1624,
N-[2-(5-Hydroxy-1H-indol-3-yl)ethyl]-9-phenylnonanamide
(1k). The title compound was prepared from 4k following the same
procedure that was used for the synthesis of 1b and using CH2Cl2/
AcOEt ) 65/35 as eluent for the chromatographic purification. Yield
94%; oil; IR (CHCl3) 3480, 3009, 2931, 2856, 1657, 1519, 1466,
1234, 1199, 1089 cm-1; 1H NMR (300 MHz) δ 1.16 (8H, m), 1.51
(4H, m), 2.02 (2H, t, J ) 6.8 Hz), 2.54 (2H, t, J ) 7.6 Hz), 2.72
(2H, t, J ) 6.8 Hz), 3.40 (2H, m), 5.84 (1H, t, J ) 5.4 Hz),
6.77–7.27 (9H, m), 7.53 (1H, br s), 8.19 (1H, m); 13C NMR (75
MHz) δ 25.26, 25.73, 29.23, 29.31, 31.44, 35.92, 36.69, 39.78,
103.11, 111.82, 111.96, 112.20, 123.06, 125.55, 127.95, 128.22,
128.40, 131.39, 142.90, 150.04, 174.17. Anal. (C25H32N2O2) C,
H, N.
Methyl [3-(1-Pentynyl)phenyl]acetate (8). A stirred suspension
of methyl (3-iodophenyl)acetate (7) (276 mg, 1 mmol), Pd(OAc)2
(11 mg, 0.05 mmol), PPh3 (26 mg, 0.1 mmol), and CuI (5 mg,
0.025 mmol) in DMSO/iPr2NH ) 1/1 (2.8 mL) was purged with
N2 for 15 min at room temperature. 1-Pentyne (0.40 mL, 4 mmol)
was then added, and the mixture was then stirred at room
temperature for 2 h under N2. The mixture was diluted with water
and extracted with AcOEt. The organic phase was washed with 2
N HCl solution, saturated NaHCO3, and brine, dried (Na2SO4), and
evaporated under vacuum. The residue (324 mg) was chromato-
graphed on silica gel (10 g) using hexane/AcOEt ) 95/5 as eluent
1
1456, 1362, 1188 cm-1; H NMR (300 MHz, CDCl3/CD3OD )
5/1) δ 1.33 (2H, m), 1.58 (4H, m), 1.84 (1H, m), 2.07 (2H, m),
2.38 (1H, m), 2.84 (2H, t, J ) 6.9 Hz), 3.11 (2H, m), 3.46 (3H,
m), 6.49 (1H, t, J ) 5.6 Hz), 6.74–7.19 (4H, m), 9.01 (1H, m);
13C NMR (75 MHz, CDCl3/CD3OD ) 5/1) δ 25.33, 25.50, 28.83,
34.57, 36.40, 38.49, 40.07, 40.31, 56.53, 102.78, 111.65, 111.96,
112.07, 123.35, 128.14, 131.59, 150.08, 174.20. Anal.
(C18H24N2O2S2) C, H, N.
N-[2-(5-Hydroxy-1H-indol-3-yl)ethyl]undecanamide (1g). The
title compound was prepared from 4g following the same procedure
that was used for the synthesis of 1b and using hexane/AcOEt )
45/55 as eluent for the chromatographic purification. Yield 85%;
mp 95 °C; IR (CHCl3) 3480, 3017, 2928, 2855, 1657, 1518, 1467,
1
1200, 1170, 1088 cm-1; H NMR (300 MHz, CDCl3/CD3OD )
5/1) δ 0.88 (3H, t, J ) 6.3 Hz), 1.24 (14H, m), 1.57 (2H, m), 2.11
(2H, t, J ) 7.6), 2.86 (2H, t, J ) 7.0 Hz), 3.48 (2H, t, J ) 7.0 Hz),
6.74–7.21 (4H, m); 13C NMR (75 MHz, CDCl3/CD3OD ) 5/1) δ
14.12, 22.75, 25.45, 25.87, 29.35, 29.42, 29.58, 29.70, 31.99, 36.77,
39.95, 102.82, 111.72, 111.96, 112.06, 123.06, 128.11, 131.47,
150.21, 174.48. Anal. (C21H32N2O2) C, H, N.

“Hybrid” FAAH/TRPV1 Blockers
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6563
to give 204 mg (94%) of 8 as an oil. IR (CHCl3) 3025, 2966, 2228,
5.1 Hz), 6.52–7.79 (14H, m), 7.79 (1H, m); 13C NMR (75 MHz)
δ 24.93, 39.82, 43.74, 103.18, 111.71, 111.94, 112.28, 123.11,
125.95, 127.14, 127.51, 127.89, 128.16, 128.36, 128.83, 129.35,
131.46, 135.31, 140.65, 141.71, 150.02, 171.59. Anal. (C24H22N2O2)
C, H, N.
1
1735, 1580, 1484, 1437, 1339, 1159, 1017 cm-1; H NMR (300
MHz) δ 1.04 (3H, t, J ) 7.2 Hz), 1.62 (2H, m), 2.37 (2H, t, J )
6.9 Hz), 3.57 (2H, s), 3.67 (3H, s), 7.16–7.35 (4H, m); 13C NMR
(75 MHz) δ 13.56, 21.39, 22.22, 40.93, 52.06, 80.48, 90.52, 124.44,
128.45, 128.47, 130.30, 132.41, 134.00, 171.71. Anal. (C14H16O2)
C, H.
3-Pentylphenyl Trifluoromethanesulfonate (10). A stirred
solution of 3-pentylphenol (9)35 (1.376 g, 8.4 mmol) and 2,6-di-
tert-butyl-4-methylpyridine (2.414 g, 11.8 mmol) in dry CH2Cl2
(42 mL) was treated with triflic anhydride (1.65 mL, 10.1 mmol)
at 0 °C, and the mixture was stirred at room temperature for 3.5 h.
The resulting suspension was filtered, and the filtrate was diluted
with AcOEt, washed with 2 N HCl solution, saturated NaHCO3
and brine, dried (Na2SO4), and evaporated under vacuum. The
residue (3.10 g) was chromatographed on silica gel (100 g) using
hexane as eluent to give 2.205 g (89%) of 10 as an oil. IR (CHCl3)
3038, 2932, 2860, 1614, 1580, 1486, 1423, 1249, 1223, 1142, 939
cm-1; 1H NMR (300 MHz) δ 0.90 (3H, t, J ) 6.9 Hz), 1.32 (4H,
m), 1.62 (2H, m), 2.64 (2H, t, J ) 7.8 Hz), 7.07–7.36 (4H, m);
13C NMR (75 MHz) δ 13.94, 22.42, 30.71, 31.25, 35.53, 118.18,
118.58 (q, J ) 318 Hz), 120.91, 128.23, 129.67, 145.74, 149.41.
Anal. (C12H15F3O3S) C, H.
Methyl (3-Pentylphenyl)acetate. A stirred solution of 8 (100
mg, 0.46 mmol) in AcOEt (2 mL) was hydrogenated in the presence
of 10% Pd/C (11 mg) at room temperature and atmospheric pressure
for 4 h. The suspension was filtered through a short pad of silica
gel, and the filtrate was evaporated under vacuum to leave the title
compound (108 mg, 99%) as an oil; IR (CHCl3) 3031, 2956, 2931,
1
2858, 1733, 1607, 1488, 1437, 1266, 1153, 1016 cm-1; H NMR
(300 MHz) δ 0.95 (3H, t, J ) 7.0 Hz), 1.30 (4H, m), 1.58 (2H, m),
2.54 (2H, t, J ) 7.6 Hz), 3.60 (2H, s), 3.70 (3H, s), 7.07–7.27 (4H,
m); 13C NMR (75 MHz) δ 14.01, 22.54, 31.10, 31.58, 35.87, 41.24,
51.94, 126.47, 127.19, 128.45, 129.36, 133.87, 143.32, 172.10.
Anal. (C14H20O2) C, H.
(3-Pentylphenyl)acetic Acid (4l). Aqueous LiOH (1 N, 0.65 mL)
was added to a stirred solution of methyl (3-pentylphenyl)acetate
(101 mg, 0.43 mmol) in THF/H2O ) 5/1 (7 mL), and the solution
was stirred at room temperature overnight. After acidification to
pH 4 with 2 N HCl, the mixture was concentrated under vacuum
and extracted with AcOEt. The organic phase was washed with
brine until neutral, dried (Na2SO4), and evaporated under vacuum
to give the title compound (89 mg, 100%), which was used in the
next step without further purification.
[3′-Pentyl-1,1′-biphenyl]-3-ol (11). A suspension of 10 (1.464
g, 4.94 mmol), 3-hydroxyphenylboronic acid (954 mg, 6.92 mmol),
K3PO4 (1.573 g, 7.41 mmol), KBr (647 mg, 5.43 mmol), and
Pd(PPh3)4 (173 mg, 0.15 mmol) in dioxane (25 mL) was stirred at
85 °C for 5 h under N2. The mixture was cooled, diluted with water,
and extracted with AcOEt. The organic phase was washed with
brine until neutral, dried (Na2SO4), and evaporated under vacuum.
The residue (2.204 g) was chromatographed on silica gel (110 g)
using hexane/CH2Cl2 ) 88/12 as eluent to give 834 mg (70%) of
the title compound as an oil. IR (CHCl3) 3596, 3320, 2959, 2931,
N-[2-(5-Hydroxy-1H-indol-3-yl)ethyl]-2-(3-pentylphenyl)-
acetamide (1l). The title compound was prepared from 4l following
the same procedure that was used for the synthesis of 1b and using
CH2Cl2/AcOEt ) 75/25 as eluent for the chromatographic purifica-
tion. Yield 77%; oil; IR (CHCl3) 3481, 3424, 3029, 2931, 2858,
1
2859, 1596, 1578, 1471, 1309, 1205, 1176, 1038 cm-1; H NMR
(300 MHz) δ 0.88 (3H, t, J ) 6.9 Hz), 1.32 (4H, m), 1.63 (2H, m),
2.62 (2H, t, J ) 7.8 Hz), 5.60 (1H, br s), 6.77–7.36 (8H, m); 13C
NMR (75 MHz) δ 13.99, 22.53, 31.17, 31.55, 36.01, 114.14, 114.18,
119.86, 124.44, 127.25, 127.61, 128.63, 129.92, 140.66, 143.27,
143.44, 155.70. Anal. (C17H20O) C, H.
1
1655, 1522, 1487, 1467, 1234, 1170, 1088 cm-1; H NMR (300
MHz) δ 0.85 (3H, t, J ) 6.7 Hz), 1.24 (4H, m), 1.51 (2H, m), 2.47
(2H, t, J ) 7.7 Hz), 2.70 (2H, t, J ) 6.7 Hz), 3.38 (2H, m), 3.45
(2H, s), 5.72 (1H, t, J ) 5.8 Hz), 6.62–7.17 (8H, m), 7.26 (1H, br
s), 8.12 (1H, m); 13C NMR (75 MHz) δ 14.02, 22.50, 25.05, 31.06,
31.56, 35.76, 40.02, 43.74, 103.21, 111.85, 111.90, 112.28, 123.01,
126.66, 127.28, 128.02, 128.78, 129.58, 131.48, 134.64, 143.75,
150.11, 171.96. Anal. (C23H28N2O2) C, H, N.
3′-Pentyl-[1,1′-biphenyl]-3-yl Trifluoromethanesulfonate (12).
A stirred solution of 11 (1.002 g, 4.17 mmol) and 2,6-di-tert-butyl-
4-methylpyridine (1.191 g, 5.8 mmol) in dry CH2Cl2 (21 mL) was
treated with triflic anhydride (0.82 mL, 5.0 mmol) at 0 °C, and the
mixture was stirred at room temperature overnight. The resulting
suspension was filtered, and the filtrate was diluted with AcOEt,
washed with 2 N HCl solution, saturated NaHCO3 and brine, dried
(Na2SO4), and evaporated under vacuum. The residue (1.64 g) was
chromatographed on silica gel (60 g) using hexane as eluent to
give 1.343 g (86%) of 12 as an oil. IR (CHCl3) 3008, 2932, 2859,
Methyl [1,1′-Biphenyl]-3-acetate. A suspension of methyl (3-
iodophenyl)acetate (7) (138 mg, 0.50 mmol), phenylboronic acid
(67 mg, 0.55 mmol), K3PO4 (265 mg, 1.25 mmol), and Pd(PPh3)4
(17 mg, 0.015 mmol) in dry DMF (2.0 mL) was stirred at 90 °C
for 16 h under N2. The mixture was cooled, diluted with water,
and extracted with AcOEt. The organic phase was washed twice
with brine until neutral, dried (Na2SO4), and evaporated under
vacuum. The residue (123 mg) was chromatographed on silica gel
(4 g) using hexane/CH2Cl2 ) 2/1 as eluent to give 56 mg (50%) of
the title compound as an oil.34 IR (CHCl3) 3025, 2954, 1735, 1600,
1
1607, 1573, 1474, 1424, 1248, 1141, 932 cm-1; H NMR (300
MHz) δ 0.90 (3H, t, J ) 6.5 Hz), 1.37 (4H, m), 1.70 (2H, m), 2.68
(2H, t, J ) 7.7 Hz), 7.22–7.62 (8H, m); 13C NMR (75 MHz) δ
14.01, 22.56, 31.22, 31.59, 36.04, 118.58 (q, J ) 318 Hz), 119.66,
120.04, 124.56, 127.11, 127.30, 128.50, 128.98, 130.40, 139.07,
143.92, 144.36, 150.06. Anal. (C18H19F3O3S) C, H.
1
1479, 1437, 1342, 1260, 1217, 1160, 1014 cm-1; H NMR (300
MHz) δ 3.70 (5H, br s), 7.25–7.61 (9H, m); 13C NMR (75 MHz)
δ 41.28, 52.04, 125.99, 127.21, 127.37, 128.16, 128.74, 128.99,
134.49, 140.96, 141.66, 171.91.
4-(3′-Pentyl[1,1′-biphenyl]-3-yl)-3-butyn-1-ol (13). A stirred
suspension of 12 (559 mg, 1.5 mmol), Pd(OAc)2 (17 mg, 0.075
mmol), PPh3 (39 mg, 0.15 mmol), and CuI (7 mg, 0.038 mmol) in
DMSO/iPr2NH ) 1/1 (8.4 mL) was purged with N2 for 15 min at
room temperature. 3-Butyn-1-ol (0.23 mL, 3 mmol) was then added,
and the mixture was stirred at room temperature for 48 h under
N2. The mixture was diluted with water and extracted with AcOEt.
The organic phase was washed with 2 N HCl solution, saturated
NaHCO3 and brine, dried (Na2SO4), and evaporated under vacuum.
The residue (714 mg) was chromatographed on silica gel (22 g)
using hexane/AcOEt ) 9/1 as eluent to give 272 mg (62%) of 13
as an oil. IR (CHCl3) 3591, 3030, 2931, 2859, 1597, 1472, 1220,
[1,1′-Biphenyl]-3-acetic Acid (4m). Aqueous LiOH (1 N, 2.1
mL) was added to a stirred solution of methyl [1,1′-biphenyl]-3-
acetate (312 mg, 1.38 mmol) in THF/H2O ) 5/1 (23 mL), and the
solution was stirred at room temperature overnight. After acidifica-
tion to pH 4 with 2 N HCl, the mixture was concentrated under
vacuum and extracted with AcOEt. The organic phase was washed
with brine until neutral, dried (Na2SO4), and evaporated under
vacuum to give the title compound34 (289 mg, 99%), which was
used in the next step without further purification.
N-[2-(5-Hydroxy-1H-indol-3-yl)ethyl]-[1,1′-biphenyl]-3-aceta-
mide (1m). The title compound was prepared from 4m following
the same procedure that was used for the synthesis of 1b and using
CH2Cl2/AcOEt ) 6/4 as eluent for the chromatographic purification.
Yield 93%; mp 67–70 °C; IR 3406, 3300, 2919, 2850, 1638, 1528,
1455, 1365, 1213, 1187, 1093 cm-1; 1H NMR (300 MHz) δ 2.66
(2H, t, J ) 6.2 Hz), 3.36 (2H, m), 3.46 (2H, s), 5.72 (1H, t, J )
1
1051 cm-1; H NMR (300 MHz) δ 0.89 (3H, m), 1.33 (4H, m),
1.65 (2H, m), 2.23 (1H, br s), 2.67 (4H, m), 3.81 (2H, t, J ) 6.3
Hz), 7.14–7.65 (8H, m); 13C NMR (75 MHz) δ 14.01, 22.52, 23.78,
31.20, 31.51, 35.97, 61.04, 82.32, 86.34, 123.49, 124.17, 126.60,
126.97, 127.45, 128.41, 128.45, 130.06, 130.18, 140.03, 141.28,
143.24, 162.40. Anal. (C21H24O) C, H.

6564 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26
Ortar et al.
4-(3′-Pentyl[1,1′-biphenyl]-3-yl)butan-1-ol (14). A stirred solu-
tion of 13 (272 mg, 0.93 mmol) in AcOEt (2 mL) was hydrogenated
in presence of 10% Pd/C (27 mg) at room temperature and
atmospheric pressure for 3 h. The suspension was filtered through
a short pad of silica gel, and the filtrate was evaporated under
vacuum to leave the title compound (273 mg, 99%) as an oil. IR
(CHCl3) 3588, 3032, 2931, 2859, 1602, 1472, 1410, 1230, 1058
δ 13.72, 22.25, 30.68, 31.18, 35.73, 55.59, 94.05, 109.14, 117.49,
125.04, 125.14, 126.90, 127.44, 128.56, 134.11, 140.48, 154.52.
Anal. (C17H22O2) C, H.
7-Pentyl-2-naphthalenol (18). A solution of 17 (628 mg, 2.3
mmol) and CBr4 (76 mg, 0.23 mmol) in dry iPrOH (11.5 mL) was
refluxed for 1.5 h.38 The solution was cooled to room temperature,
and the organic solvent was removed under reduced pressure. The
residue (553 mg) was chromatographed on silica gel (17 g) using
hexane/AcOEt ) 8/2 as eluent to give 468 mg (95%) of 18 as an
oil. IR (CHCl3) 3284, 3052, 2960, 2931, 2859, 1632, 1605, 1510,
1450, 1239, 1141, 1047 cm-1; 1H NMR (300 MHz) δ 0.88 (3H, t,
J ) 6.8 Hz), 1.32 (4H, m), 1.66 (2H, m), 2.69 (2H, t, J ) 7.8 Hz),
6.97–7.66 (6H, m); 13C NMR (75 MHz) δ 14.02, 22.53, 30.96,
31.50, 35.98, 109.00, 116.60, 124.65, 125.03, 127.17, 127.33,
129.28, 134.56, 140.98, 153.03. Anal. (C15H18O) C, H.
1
cm-1; H NMR (300 MHz) δ 0.91 (3H, m), 1.32 (4H, m), 1.66
(6H, m), 2.68 (4H, m), 3.62 (2H, t, J ) 6.5 Hz), 7.12–7.41 (8H,
m); 13C NMR (75 MHz) δ 14.02, 22.26, 27.57, 31.23, 31.52, 32.27,
35.68, 36.00, 62.60, 124.28, 124.48, 126.99, 127.06, 127.09, 128.34,
128.40, 141.04, 141.24, 142.47, 143.10. Anal. (C21H28O) C, H.
4-(3′-Pentyl[1,1′-biphenyl]-3-yl)butanoic Acid (4n). To a stirred
solution of 14 (273 mg, 0.92 mmol) in DMF (18 mL) was added
portionwise pyridinium dichromate (2.077 g, 5.52 mmol). The
mixture was stirred overnight at room temperature and then was
diluted with brine and extracted with AcOEt. The organic phase
was washed twice with brine, dried (Na2SO4), and evaporated under
vacuum. The residue (308 mg) was chromatographed on silica gel
(10 g) using hexane/AcOEt ) 8/2 as eluent to give 226 mg (79%)
of 4n. Mp 50–51 °C; IR (CHCl3) 3035, 2931, 2859, 1709, 1601,
1474, 1408, 1270, 1223, 1127 cm-1; 1H NMR (300 MHz) δ 0.90
(3H, m), 1.34 (4H, m), 1.66 (2H, m), 2.01 (2H, m), 2.40 (2H, t, J
) 7.5 Hz), 2.66 (2H, t, J ) 7.8 Hz), 2.73 (2H, t, J ) 7.5 Hz),
7.16–7.41 (8H, m); 13C NMR (75 MHz) δ 14.02, 22.53, 26.20,
31.23, 31.54, 33.35, 35.04, 36.01, 124.30, 124.80, 127.06, 127.11,
127.12, 127.17, 128.36, 128.54, 140.94, 141.34, 141.42, 143.14,
179.72. Anal. (C21H26O2) C, H, N.
7-Pentyl-2-naphthalenyl Trifluoromethanesulfonate (19). A
stirred solution of 18 (705 mg, 3.3 mmol) and 2,6-di-tert-butyl-4-
methylpyridine (950 mg, 4.62 mmol) in dry CH2Cl2 (16.5 mL) was
treated with triflic anhydride (0.65 mL, 4.0 mmol) at 0 °C, and the
mixture was stirred at room temperature for 2.5 h. The resulting
suspension was filtered, and the filtrate was diluted with AcOEt,
washed with 2 N HCl solution, saturated NaHCO3, and brine, dried
(Na2SO4), and evaporated under vacuum. The residue (1.17 g) was
chromatographed on silica gel (35 g) using hexane as eluent to
give 873 mg (76%) of 19 as an oil. IR (CHCl3) 3052, 2959, 2931,
1
2859, 1635, 1604, 1509, 1422, 1239, 1141, 1110, 955 cm-1; H
NMR (300 MHz) δ 0.91 (3H, m), 1.34 (4H, m), 1.70 (2H, m),
2.77 (2H, t, J ) 7.8 Hz), 7.27–7.86 (6H, m); 13C NMR (75 MHz)
δ 14.00, 22.53, 30.84, 31.46, 35.98, 118.40, 118.52, 118.66 (q, J
) 320 Hz), 126.21, 127.53, 128.67, 130.03, 130.65, 133.42, 142.34,
147.01. Anal. (C16H17F3O3S) C, H.
N-[2-(5-Hydroxy-1H-indol-3-yl)ethyl]-4-(3′-pentyl[1,1′-biphe-
nyl]-3-yl)butanamide (1n). The title compound was prepared from
4n following the same procedure that was used for the synthesis
of 1b and using hexane/AcOEt ) 1/1 as eluent for the chromato-
graphic purification. Yield 80%; oil; IR (CHCl3) 3405, 2925, 2853,
4-(7-Pentyl-2-naphthalenyl)-3-butyn-1-ol (20). A stirred sus-
pension of 19 (522 mg, 1.5 mmol), Pd(OAc)2 (17 mg, 0.075 mmol),
PPh3 (39 mg, 0.15 mmol), and CuI (7 mg, 0.038 mmol) in DMSO/
iPr2NH ) 1/1 (8.4 mL) was purged with N2 for 15 min at room
temperature. 3-Butyn-1-ol (0.158 mg, 2.25 mmol) was then added,
and the mixture was stirred at room temperature for 4 h under N2.
The mixture was diluted with water and extracted with AcOEt. The
organic phase was washed with 2 N HCl solution, saturated
NaHCO3, and brine, dried (Na2SO4), and evaporated under vacuum.
The residue (500 mg) was chromatographed on silica gel (20 g)
using CH2Cl2/AcOEt ) 98/2 as eluent to give 333 mg (83%) of
20. Mp 103–104.5 °C; IR (CHCl3) 3587, 3028, 2931, 2859, 1603,
1
1645, 1529, 1456, 1365, 1213, 1186, 1093 cm-1; H NMR (300
MHz) δ 0.87 (3H, m), 1.26 (4H, m), 1.61 (2H, m), 2.02 (4H, m),
2.58 (6H, m), 3.36 (2H, m), 5.73 (1H, m), 6.72–7.36 (13H, m),
8.10 (1H, br s); 13C NMR (75 MHz) δ 14.13, 14.16, 22.53, 25.24,
27.11, 29.69, 31.25, 31.57, 35.19, 35.88, 36.04, 39.79, 103.16,
111.87, 111.98, 112.25, 123.09, 124.50, 124.83, 127.29, 127.99,
128.64, 128.74, 131.43, 141.16, 141.45, 141.96, 143.46, 150.06.
Anal. (C31H36N2O2) C, H, N.
7-(Methoxymethoxy)-2-naphthalenyl Trifluoromethanesulfonate
(16). To a stirred solution of 7-hydroxy-2-naphthalenyl trifluo-
romethanesulfonate (15)36 (3.17 g, 10.85 mmol) and Et3N (9.1 mL,
65.1 mmol) in dry THF (40 mL) was added dropwise at 0 °C
chloromethyl methyl ether (3.3 mL, 43 mmol). After being stirred
at room temperature overnight, the solution was concentrated under
reduced pressure, diluted with water, and extracted with AcOEt.
The organic phase was washed with 2 N HCl solution, saturated
NaHCO3 and brine, dried (Na2SO4), and evaporated under vacuum.
The residue (3.19 g) was chromatographed on silica gel (100 g)
using hexane/AcOEt ) 92/8 as eluent hexane to give 1.94 g (53%)
of 16 as an oil. IR (CHCl3) 3052, 2962, 2931, 2859, 1634, 1606,
1
1510, 1468, 1228, 1058 cm-1; H NMR (300 MHz) δ 0.89 (3H,
m), 1.33 (4H, m), 1.65 (2H, m), 2.23 (1H, br s), 2.67 (4H, m),
3.81 (2H, t, J ) 6.3 Hz), 7.14–7.65 (8H, m); 13C NMR (75 MHz)
δ 14.01, 22.52, 23.78, 31.20, 31.51, 35.97, 61.04, 82.32, 86.34,
123.49, 124.17, 126.60, 126.97, 127.45, 128.41, 128.45, 130.06,
130.18, 140.03, 141.28, 143.24, 162.40. Anal. (C19H22O) C, H.
4-(7-Pentyl-2-naphthalenyl)butan-1-ol (21). A stirred solution
of 20 (390 mg, 1.46 mmol) in AcOEt (3 mL) was hydrogenated in
the presence of 10% Pd/C (40 mg) at room temperature and
atmospheric pressure for 1.5 h. The suspension was filtered through
a short pad of silica gel, and the filtrate was evaporated under
vacuum to leave the title compound (391 mg, 99%). Mp 43.5–46
°C; IR (CHCl3) 3627, 3050, 3007, 2933, 2859, 1636, 1605, 1512,
1458, 1237, 1199, 1061, 1020 cm-1; 1H NMR (300 MHz) δ 0.89
(3H, m), 1.34 (4H, m), 1.68 (2H, m), 2.52 (1H, br s), 2.70 (4H,
m), 3.83 (2H, t, J ) 6.3 Hz), 7.28–7.85 (6H, m); 13C NMR (75
MHz) δ 13.99, 22.50, 23.85, 30.88, 31.43, 35.95, 61.06, 82.74,
86.43, 120.36, 125.76, 127.30, 127.34, 127.53, 127.86, 130.67,
130.87, 132.94, 140.91. Anal. (C19H26O) C, H.
1
1511, 1421, 1385, 1250, 1141, 1110 cm-1; H NMR (300 MHz)
δ 3.51 (3H, s), 5.29 (3H, s), 7.20–7.82 (6H, m); 13C NMR (75
MHz) δ 56.09, 94.31, 109.72, 117.31, 118.12, 118.66 (q, J ) 318
Hz), 120.23, 128.13, 129.24, 130.02, 134.53, 147.52, 156.12. Anal.
(C13H11F3O5S) C, H.
2-(Methoxymethoxy)-7-pentylnaphthalene (17). To a stirred
solution of 16 (1.100 g, 3.27 mmol) and NiCl2(dppp)49 (89 mg,
0.16 mmol) in dry THF (6.5 mL) was added dropwise at 0 °C a 2
M solution of pentylmagnesium chloride in dry THF (3.3 mL). The
mixture was stirred at room temperature for 1 h under N2 and then
diluted with water and extracted with AcOEt. The organic phase
was washed with brine, dried (Na2SO4), and evaporated under
vacuum. The residue (996 mg) was chromatographed on silica gel
(30 g) using hexane/AcOEt ) 98/2 as eluent to give 628 mg (70%)
of 17 as an oil. IR (CHCl3) 3009, 2962, 2931, 1633, 1607, 1513,
1463, 1385, 1250, 1152, 1125 cm-1; 1H NMR (300 MHz) δ 0.89
(3H, m), 1.33 (4H, m), 1.68 (2H, m), 2.72 (2H, t, J ) 7.6 Hz),
3.50 (3H, s), 5.27 (3H, s), 7.12–7.71 (6H, m); 13C NMR (75 MHz)
4-(7-Pentyl-2-naphthalenyl)butanoic Acid (4o). To a stirred
solution of 21 (391 mg, 1.44 mmol) in dry DMF (30 mL) was
added portionwise pyridinium dichromate (3.250 g, 8.64 mmol).
The mixture was stirred overnight at room temperature and then
was diluted with brine and extracted with AcOEt. The organic phase
was washed with brine, dried (Na2SO4), and evaporated under
vacuum. The residue (556 mg) was chromatographed on silica gel
(23 g) using hexane/AcOEt ) 8/2 as eluent to give 250 mg (61%)
of 4o. Mp 85–87 °C; IR 3048, 2927, 2854, 1690, 1511, 1459, 1436,

“Hybrid” FAAH/TRPV1 Blockers
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6565
1
1408, 1283, 1213, 904 cm-1; H NMR (300 MHz) δ 0.89 (3H, t,
6.8 Hz), 1.31 (4H, m), 1.58 (2H, m), 2.50 (2H, t, J ) 7.6 Hz), 3.43
(2H, br s), 6.49–7.07 (4H, m); 13C NMR (75 MHz) δ 14.04, 22.58,
31.07, 31.58, 35.96, 112.52, 115.29, 118.86, 129.10, 144.25, 146.29.
N-[2-(5-Hydroxy-1H-indol-3-yl)ethyl]-N′-(3-pentylphenyl)-
urea (2b). The title compound was prepared from 5b following
the same procedure that was used for the synthesis of 2a and using
CH2Cl2/AcOEt ) 7/3 as eluent for the chromatographic purification.
Yield 76%; mp 172–174 °C; IR 3425, 3331, 2928, 2855, 1643,
1611, 1596, 1489, 1457, 1366, 1315, 1268, 1245, 1208, 1187, 1092,
J ) 6.3 Hz), 1.34 (4H, m), 1.69 (2H, m), 2.04 (2H, m), 2.39 (2H,
t, J ) 7.5 Hz), 2.74 (2H, t, J ) 7.5 Hz), 2.81 (2H, t, J ) 7.5 Hz),
7.23–7.73 (6H, m); 13C NMR (75 MHz) δ 14.00, 22.53, 26.03,
30.99, 31.47, 33.25, 35.08, 36.03, 125.65, 125.96, 126.08, 126.65,
127.20, 127.51, 130.34, 133.55, 138.34, 140.36, 179.52. Anal.
(C19H24O2) C, H, N.
N-[2-(5-Hydroxy-1H-indol-3-yl)ethyl]-4-(7-pentyl-2-naphtha-
lenyl)butanamide (1o). The title compound was prepared from 4o
following the same procedure that was used for the synthesis of
1b and using hexane/AcOEt ) 1/1 as eluent for the chromatographic
purification. Yield 97%; mp 141–142 °C; IR 3522, 3386, 2925,
1
1037 cm-1; H NMR (300 MHz, CD3OD) δ 0.89 (3H, t, J ) 6.8
Hz), 1.31 (4H, m), 1.57 (2H, m), 2.51 (2H, t, J ) 7.6 Hz), 2.88
(2H, t, J ) 7.0 Hz), 3.48 (2H, t, J ) 7.0 Hz), 6.68–7.17 (8H, m);
13C NMR (75 MHz, CD3OD) δ 14.41, 23.58, 27.07, 32.30, 32.62,
36.96, 41.45, 103.60, 112.44, 112.49, 112.71, 117.73, 120.37,
123.63, 124.42, 129.44, 129.61, 133.19, 140.78, 144.78, 151.13,
158.47. Anal. (C22H27N3O2) C, H, N.
1630, 1567, 1485, 1463, 1312, 1202, 1157, 1056 cm-1; H NMR
1
(300 MHz, CDCl3/CD3OD ) 5/1) δ 0.89 (3H, m), 1.34 (4H, m),
1.68 (2H, m), 1.97 (2H, m), 2.10 (2H, m), 2.78 (4H, m), 2.96 (2H,
m), 3.46 (2H, m), 6.06 (1H, t, J ) 5.2 Hz), 6.64–7.71 (10H, m);
13C NMR (75 MHz, CDCl3/CD3OD ) 5/1) δ 14.06, 22.61, 25.39,
26.99, 31.14, 31.57, 35.25, 35.86, 36.12, 39.96, 102.81, 111.70,
112.11, 123.09, 125.98, 126.13, 126.39, 126.91, 127.08, 127.46,
127.70, 128.04, 130.53, 131.29, 133.78, 138.94, 140.71, 150.22,
173.86. Anal. (C29H34N2O2) C, H, N.
N-[2-(5-Hydroxy-1H-indol-3-yl)ethyl]-N′-(2-phenylethyl)-
urea (2c). The title compound was prepared from 5c following
the same procedure that was used for the synthesis of 2a and using
preparative layer chromatography (silica gel, 0.5 cm thick) and
CH2Cl2/AcOEt ) 3/7 as eluent for the purification of 2c. Yield
32%; mp 148–150 °C; IR 3361, 3317, 2936, 2870, 1607, 1588,
1
1520, 1495, 1458, 1384, 1258, 1235, 1209, 1192, 1072 cm-1; H
N-(4-tert-Butylbenzyl)-N′-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-
urea (2a). To a stirred solution of 4-tert-butylbenzylamine (49 mg,
0.30 mmol) and Et3N (0.084 mL, 0.60 mmol) in dry CH2Cl2 (2
mL) was added at 0 °C under N2 bis(trichloromethyl) carbonate
(89 mg, 0.30 mmol), and the reaction mixture was then gradually
brought to reflux. After 5 h at reflux, the reaction mixture was
cooled and filtered through a short pad of silica, and the filtrate
was evaporated under vacuum to leave the crude isocyanate
intermediate (55 mg), monitored by IR analysis for the strong NCO
stretching band (2269 cm-1). A solution of serotonin hydrochloride
(64 mg, 0.30 mmol) and Et3N (0.126 mL, 0.90 mmol) in dry
pyridine (0.5 mL) was added to a cooled solution of the crude
isocyanate in dry CH2Cl2 (2 mL). The reaction mixture was stirred
at room temperature for 18 h and then diluted with water and
extracted with AcOEt. The organic phase was washed with water
until neutral, dried (Na2SO4), and evaporated under vacuum to leave
a residue (101 mg), which was chromatographed on silica gel using
CH2Cl2/AcOEt ) 1/1 as eluant to give 47 mg of pure 2a (44%).
Mp 136–138 °C; IR 3379, 2965, 2870, 1609, 1564, 1478, 1435,
1362, 1270, 1185 cm-1; 1H NMR (300 MHz, CD3OD) δ 1.28 (9H,
s), 2.84 (2H, t, J ) 7.0 Hz), 3.30 (1H, s), 3.42 (2H, t, J ) 7.0 Hz),
4.24 (2H, s), 6.65–7.33 (8H, m); 13C NMR (75 MHz, CD3OD) δ
27.23, 31.86, 35.30, 41.81, 44.48, 103.63, 112.41, 112.66, 112.68,
124.39, 126.35, 127.96, 129.54, 133.20, 138.21, 151.00, 151.16,
161.24. Anal. (C22H27N3O2) C, H, N.
NMR (300 MHz, CD3OD) δ 2.71 (2H, t, J ) 7.1 Hz), 2.82 (2H, t,
J ) 7.1 Hz), 3.31 (2H, t, J ) 7.1 Hz), 3.38 (2H, t, J ) 7.1 Hz),
6.65–7.26 (9H, m); 13C NMR (75 MHz, CD3OD) δ 27.19, 37.54,
41.70, 42.63, 103.62, 112.40, 112.67, 124.35, 127.22, 129.46,
129.86, 133.16, 140.80, 151.13, 161.19. Anal. (C19H21N3O2) C,
H, N.
N-[2-(4-Chlorophenyl)ethyl]-N′-[2-(5-hydroxy-1H-indol-3-
yl)ethyl]urea (2d). The title compound was prepared from 5d
following the same procedure that was used for the synthesis of
2a and using CH2Cl2/AcOEt ) 4/6 as eluent for the chromato-
graphic purification. Yield 38%; mp 114–116 °C; IR 3424, 3322,
1
2927, 1626, 1581, 1459, 1367, 1273, 1132, 1054 cm-1; H NMR
(300 MHz, CD3OD) δ 2.85 (4H, m), 3.36 (4H, m), 6.65–7.31 (8H,
m); 13C NMR (75 MHz, CD3OD) δ 27.19, 35.22, 40.80, 41.69,
103.63, 112.41, 112.65, 124.36, 128.11, 129.01, 129.51, 130.46,
132.35, 133.18, 135.12, 138.29, 151.13. Anal. (C19H20ClN3O2) C,
H, N.
N-[1,1′-Biphenyl]-3-yl-N′-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-
urea (2e). The title compound was prepared from 5e following
the same procedure that was used for the synthesis of 2a and using
CH2Cl2/AcOEt ) 6/4 as eluent for the chromatographic purification.
Yield 68%; mp 191–193 °C; IR 3423, 3313, 2923, 1636, 1560,
1
1480, 1457, 1365, 1207, 1181, 1091, 1075 cm-1; H NMR (300
MHz, CD3OD) δ 2.89 (2H, t, J ) 7.1 Hz), 3.50 (2H, t, J ) 7.1
Hz), 6.69–7.64 (13H, m); 13C NMR (75 MHz, CD3OD) δ 27.02,
41.46, 103.62, 112.45, 112.76, 118.79, 119.08, 122.06, 124.44,
124.61, 127.95, 128.32, 129.43, 129.74, 130.24, 133.16, 141.36,
143.32, 143.12, 151.10, 158.38. Anal. (C23H21N3O2) C, H, N.
2-[1,1′-Biphenyl]-4-ethanamine Hydrochloride (5f). A stirred
solution of 4-biphenylacetonitrile (500 mg, 2.6 mmol) in MeOH
(20 mL) containing a few drops of concentrated hydrochloric acid
was hydrogenated in the presence of 10% Pd/Cl (370 mg) at room
temperature and atmospheric pressure for 16 h. The suspension was
filtered through a short pad of Celite, and the filtrate was evaporated
under vacuum to leave the crude title compound (600 mg, 99%).
Mp 243–245 °C (lit.50 245 °C); IR 3422, 3026, 1582, 1511, 1487,
1409, 1125, 1075, 1006 cm-1; 1H NMR (300 MHz, DMSO-d6) δ
3.03 (2H, m), 3.08 (2H, m), 7.35–7.66 (9H, m), 8.37 (3H, br s);
13C NMR (75 MHz, DMSO-d6) δ 32.49, 39.82, 126.54, 126.87,
127.36, 128.95, 129.28, 136.81, 138.57, 139.87.
1-Nitro-3-(1-pentynyl)benzene (23).39 A stirred suspension of
3-iodonitrobenzene (22) (996 mg, 4 mmol), Pd(OAc)2 (45 mg, 0.2
mmol), PPh3 (105 mg, 0.4 mmol), and CuI (19 mg, 0.1 mmol) in
DMSO/iPr2NH ) 1/1 (22 mL) was purged with N2 for 15 min at
room temperature. 1-Pentyne (0.79 mL, 8 mmol) was then added,
and the mixture was stirred at room temperature for 4 h under N2.
The mixture was diluted with water and extracted with AcOEt. The
organic phase was washed with 2 N HCl solution, saturated
NaHCO3, and brine, dried (Na2SO4), and evaporated under vacuum.
The residue (976 mg) was chromatographed on silica gel (30 g)
using hexane as eluent to give 695 mg (92%) of 2339 as an oil. IR
(CHCl3) 3085, 2967, 2242, 1531, 1352, 1215, 1096 cm-1; 1H NMR
(300 MHz) δ 1.06 (3H, t, J ) 7.0 Hz), 1.64 (2H, m), 2.41(2H, t,
J ) 7.2 Hz) 7.43–8.27 (4H, m); 13C NMR (75 MHz) δ 13.56, 21.35,
21.97, 78.67, 93.43, 122.23, 125.96, 126.40, 129.14, 137.32, 148.09.
3-Pentylaniline (5b).39 A stirred solution of 23 (673 mg, 3.56
mmol) in EtOH (10 mL) was hydrogenated in the presence of 10%
Pd/C (182 mg) at 60 °C and atmospheric pressure for 4 h. The
suspension was filtered through a short pad of silica gel and the
filtrate was evaporated under vacuum to leave a residue (563 mg),
which was chromatographed on silica gel (30 g) using hexane/
AcOEt ) 85/15 as eluent to give 523 mg (90%) of 5b39 as an oil.
IR (CHCl3) 3451, 3375, 3038, 3009, 2959, 2931, 2859, 1619, 1493,
N-(2-[1,1′-Biphenyl]-4-ylethyl)-N′-[2-(5-hydroxy-1H-indol-3-
yl)ethyl]urea (2f). The title compound was prepared from 5f
following the same procedure that was used for the synthesis of
2a and using CH2Cl2/AcOEt ) 4/6 as eluent for the chromato-
graphic purification. Yield 40%; mp 159–161 °C; IR 3436, 3395,
1
3311, 1629, 1557, 1488, 1250, 1218, 1189 cm-1; H NMR (300
MHz, CD3OD) δ 2.75 (2H, t, J ) 6.9 Hz), 2.83 (2H, t, J ) 6.9
Hz), 3.33 (2H, t, J ) 7.2 Hz), 3.39 (2H, t, J ) 7.2 Hz), 6.96–7.55
1460, 1288, 1167 cm-1; H NMR (300 MHz) δ 0.89 (3H, t, J )
1

6566 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26
Ortar et al.
(13H, m); 13C NMR (75 MHz, CD3OD) δ 27.17, 37.11, 41.68,
42.55, 103.63, 112.41, 112.66, 124.35, 127.82, 128.02, 128.11,
129.54, 129.80, 130.39, 133.16, 139.94, 140.40, 142.27, 151.14,
161.19. Anal. (C25H25N3O2) C, H, N.
(all-Z)-Octadeca-9,12,15-trienyl [2-(5-Hydroxy-1H-indol-3-
yl)ethyl]carbamate (3b). The title compound was prepared from
6b following the same procedure that was used for the synthesis
of 3a and using CH2Cl2/AcOEt ) 85/15 as eluent for the
chromatographic purification. Yield 61%; oil; IR (CHCl3) 3481,
3344, 2931, 2856, 1704, 1628, 1586, 1516, 1465, 1340, 1236, 1203,
2-(4-Phenoxyphenyl)ethanamine Hydrochloride (5g). A stirred
solution of (4-phenoxyphenyl)acetonitrile (209 mg, 1 mmol) in
MeOH (10 mL) containing a few drops of concentrated hydrochloric
acid was hydrogenated in the presence of 10% Pd/C (140 mg) at
room temperature and atmospheric pressure for 16 h. The suspen-
sion was filtered through a short pad of Celite, and the filtrate was
evaporated under vacuum to leave the crude title compound (245
mg, 98%). Mp 195–197 °C (lit.51 209–210 °C); IR 3423, 3001,
1170, 1072 cm-1 1H NMR (300 MHz) δ 0.97 (3H, t, J ) 7.3
;
Hz), 1.26 (10H, m), 1.56 (2H, m), 2.06 (4H, m), 2.80 (6H, m),
3.36 (2H, m), 4.04 (2H, m), 4.93 (1H, m), 5.36 (6H, m), 6.77–7.11
(5H, m), 8.19 (1H, br s); 13C NMR (75 MHz, CDCl3) δ 14.27,
20.55, 25.53, 25.62, 25.85, 27.23, 29.00, 29.24, 29.27, 29.44, 29.62,
41.02, 65.27, 103.20, 111.84, 111.92, 112.13, 123.16, 127.13,
127.68, 127.92, 128.28, 130.34, 131.53, 131.97, 149.77, 157.29.
Anal. (C29H42N2O3) C, H, N.
(all-Z)-Octadeca-6,9,12-trienyl [2-(5-Hydroxy-1H-indol-3-yl)-
ethyl]carbamate (3c). The title compound was prepared from 6c
following the same procedure that was used for the synthesis of
3a and using CH2Cl2/AcOEt ) 8/2 as eluent for the chromato-
graphic purification. Yield 59%; oil; IR (CHCl3) 3481, 3349, 2931,
2858, 1705, 1627, 1588, 1515, 1466, 1340, 1236, 1206, 1170, 1088,
1073 cm-1; 1H NMR (300 MHz) δ 0.88 (3H, t, J ) 7.3 Hz), 1.33
(10H, m), 1.58 (2H, m), 2.04 (4H, m), 2.79 (6H, m), 3.39 (2H, m),
4.05 (2H, m), 4.89 (1H, m), 5.38 (6H, m), 6.60 (1H, br s), 6.77–7.13
(4H, m), 8.15 (1H, br s); 13C NMR (75 MHz, CDCl3) δ 14.07,
22.57, 25.41, 25.53, 25.64, 25.81, 27.10, 27.22, 28.94, 29.28, 29.32,
31.51, 41.04, 65.16, 103.23, 111.91, 112.15, 123.14, 127.62, 127.95,
127.98, 128.18, 128.39, 129.97, 130.47, 131.55, 149.80, 157.21.
Anal. (C29H42N2O3) C, H, N.
1
1592, 1507, 1489, 1251, 1113, 1068, 1017 cm-1; H NMR (300
MHz, DMSO-d6) δ 2.89 (2H, m), 3.03 (2H, m), 6.99–7.43 (9H,
m), 8.00 (3H, br s); 13C NMR (75 MHz, DMSO-d6) δ 32.12, 40.01,
118.39, 118.95, 123.32, 130.03, 130.30, 132.62, 155.28, 156.88.
N-[2-(5-Hydroxy-1H-indol-3-yl)ethyl]-N′-[2-(4-phenoxyphe-
nyl)ethyl]urea (2g). The title compound was prepared from 5g
following the same procedure that was used for the synthesis of
2a and using CH2Cl2/AcOEt ) 4/6 as eluent for the chromato-
graphic purification. Yield 41%; mp 164–166 °C; IR 3394, 3293,
1
2920, 1622, 1573, 1505, 1489, 1250, 1194 cm-1; H NMR (300
MHz, CD3OD) δ 2.68 (2H, t, J ) 7.0 Hz), 2.82 (2H, t, J ) 7.0
Hz), 3.30 (2H, t, J ) 7.1 Hz), 3.39 (2H, t, J ) 7.1 Hz), 6.65–7.05
(13H, m); 13C NMR (75 MHz, CD3OD) δ 27.17, 36.77, 41.71,
42.64, 103.61, 112.39, 112.68, 119.53, 120.01, 124.10, 124.35,
129.51, 130.72, 131.23, 133.14, 135.88, 151.11, 156.97, 159.00,
161.16. Anal. (C25H25N3O3) C, H, N.
(all-Z)-Octadeca-9,12-dienyl [2-(5-Hydroxy-1H-indol-3-yl)-
ethyl]carbamate (3d). The title compound was prepared from 6b
following the same procedure that was used for the synthesis of
3a and using CH2Cl2/AcOEt )85/15 as eluent for the chromato-
graphic purification. Yield 40%; oil; IR (CHCl3) 3481, 3349, 2929,
2856, 1705, 1627, 1586, 1517, 1466, 1340, 1236, 1200, 1170, 1087,
1074 cm-1; 1H NMR (300 MHz) δ 0.88 (3H, t, J ) 6.7 Hz), 1.28
(16H, m), 1.58 (2H, m), 2.04 (4H, m), 2.80 (4H, m), 3.43 (2H, m),
4.05 (2H, t, J ) 6.6 Hz), 4.86 (1H, t, J ) 5.2 Hz), 5.34 (4H, m),
6.25 (1H, br s), 6.76–7.18 (4H, m), 8.08 (1H, m); 13C NMR (75
MHz) δ 14.05, 22.57, 25.68, 25.89, 27.24, 29.06, 29.26, 29.36,
29.45, 29.67, 31.54, 41.15, 41.19, 65.27, 103.31, 111.86, 112.08,
112.18, 123.13, 127.97, 128.05, 130.14, 130.25, 131.64, 149.83,
157.17. Anal. (C29H44N2O3) C, H, N.
(all-Z)-Eicosan-5,8,11,14-tetraenol (6a). To a stirred solution
of arachidonic acid (4a) (82 mg, 0.27 mmol) and N-methylmor-
pholine (0.025 mL, 0.27 mmol) in dry THF (0.5 mL) was added
dropwise at -15 °C isobutyl chloroformate (0.035 mL, 0.27 mmol),
and the mixture was stirred at -15 °C for 15 min. The suspension
was filtered, and NaBH4 (15 mg, 0.40 mmol) was added at 0 °C to
the filtrate, followed by 0.020 mL of water. The reaction mixture
was stirred in an ice bath for 1 h and then diluted with water and
extracted with AcOEt. The organic phase was washed with water
until neutral, dried (Na2SO4), and evaporated under vacuum to leave
the title alcohol (78 mg), pure from TLC, which was used in the
next step without further purification.
(all-Z)-Octadeca-9,12,15-trienol (6b). Prepared as above from
R-linolenic acid.
(all-Z)-Octadeca-6,9,12-trienol (6c). Prepared as above from
γ-linolenic acid.
(all-Z)-Octadeca-9,12-dienol (6d). Prepared as above from
linoleic acid.
3-Pentylphenyl [2-(5-Hydroxy-1H-indol-3-yl)ethyl]carbam-
ate (3e). The title compound was prepared from 6e following the
same procedure that was used for the synthesis of 3a and using
CH2Cl2/AcOEt ) 87/13 as eluent for the chromatographic purifica-
tion. Yield 58%; oil; IR (CHCl3) 3480, 3009, 2931, 2859, 1729,
(all-Z)-Eicosan-5,8,11,14-tetraenyl [2-(5-Hydroxy-1H-indol-
3-yl)ethyl]carbamate (3a). To a stirred solution of 6a (78 mg, 0.27
mmol) and Et3N (0.045 mL, 0.32 mmol) in dry toluene (2.7 mL)
was added dropwise at 0 °C a 20% phosgene solution in toluene
(0.56 mL, 1.08 mmol). After being stirred at room temperature for
2 h, the solution was evaporated under reduced pressure to give
the crude chloroformate, which was dissolved in dry CH2Cl2 (1.4
mL) and to which was added dropwise a solution of serotonin
hydrochloride (63 mg, 0.30 mmol) and Et3N (0.083 mL, 0.59 mmol)
in dry DMF (1.3 mL). The mixture was stirred at room temperature
overnight and then diluted with water, neutralized with 2 N HCl,
and extracted with AcOEt. The organic phase was washed with
water, dried (Na2SO4), and evaporated under vacuum to leave a
residue (141 mg), which was chromatographed on silica gel (7 g)
using CH2Cl2/AcOEt ) 85/15 as eluent to give the title compound
(73 mg, 55% yield) as an oil. IR (CHCl3) 3481, 3349, 2930, 2858,
1706, 1627, 1586, 1516, 1467, 1340, 1236, 1170, 1140, 1088, 1042
cm-1; 1H NMR (300 MHz) δ 0.88 (3H, t, J ) 6.6 Hz), 1.29 (8H,
m), 1.60 (2H, m), 2.06 (4H, m), 2.81 (8H, m), 3.43 (2H, m), 4.07
(2H, t, J ) 6.4 Hz), 4.83 (1H, m), 5.36 (8H, m), 6.15 (1H, br s),
6.76–7.25 (4H, m) 8.06 (1H, m); 13C NMR (75 MHz) δ 14.05,
22.56, 25.67, 25.92, 26.85, 27.24, 28.68, 29.32, 31.52, 41.10, 41.13,
41.17, 51.50, 65.09, 103.28, 111.86, 112.10, 112.18, 123.14, 127.58,
127.93, 128.02, 128.17, 128.23, 128.32, 128.60, 129.73, 130.51,
131.62, 149.82, 157.13. Anal. (C31H44N2O3) C, H, N.
1
1588, 1509, 1484, 1376, 1238, 1156, 1090, 1041 cm-1; H NMR
(300 MHz) δ 0.85 (3H, t, J ) 6.5 Hz), 1.26 (4H, m), 1.56 (2H, m),
2.53 (2H, t, J ) 7.7 Hz), 2.82 (2H, t, J ) 6.7 Hz), 3.43 (2H, m),
5.22 (1H, t, J ) 5.8 Hz), 6.29 (1H, br s), 6.68–7.24 (8H, m), 8.08
(1H, m); 13C NMR (75 MHz) δ 13.99, 22.48, 25.59, 30.85, 31.49,
35.69, 41.39, 103.24, 111.79, 111.95, 112.14, 118.79, 121.54,
123.23, 125.47, 127.98, 129.00, 131.62, 144.62, 149.69, 150.97,
155.17. Anal. (C22H26N2O3) C, H, N.
[1,1′-Biphenyl]-3-yl [2-(5-Hydroxy-1H-indol-3-yl)ethyl]car-
bamate (3f). The title compound was prepared from 6f following
the same procedure that was used for the synthesis of 3a and using
CH2Cl2/AcOEt ) 85/15 as eluent for the chromatographic purifica-
tion. Yield 63%; oil; IR (CHCl3) 3480, 3014, 1731, 1598, 1506,
1
1477, 1420, 1236, 1189, 1089, 1045 cm-1; H NMR (300 MHz)
δ 2.81 (2H, t, J ) 6.6 Hz), 3.42 (2H, m), 5.23 (1H, t, J ) 5.9 Hz),
6.13 (1H, br s), 6.67–7.51 (13H, m), 8.02 (1H, m); 13C NMR (75
MHz) δ 25.57, 41.43, 103.26, 111.83, 111.96, 112.16, 120.38,
120.45, 123.24, 124.10, 127.17, 127.58, 128.01, 128.76, 129.59,
131.63, 140.24, 142.67, 149.68, 151.41, 154.99. Anal. (C23H20N2O3)
C, H, N.
3-tert-Butylphenyl [2-(5-Hydroxy-1H-indol-3-yl)ethyl]car-
bamate (3g). The title compound was prepared from 6g following
the same procedure that was used for the synthesis of 3a and using
CH2Cl2/AcOEt ) 9/1 as eluent for the chromatographic purification.

“Hybrid” FAAH/TRPV1 Blockers
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6567
Yield 41%; oil; IR (CHCl3) 3480, 3029, 2967, 1731, 1606, 1509,
1487, 1374, 1236, 1197, 1044 cm-1; 1H NMR (300 MHz) δ 1.26
(9H, s), 2.86 (2H, t, J ) 6.8 Hz), 3.47 (2H, m), 5.21 (1H, t, J )
5.7 Hz), 5.99 (1H, br s), 6.69–7.30 (8H, m), 8.05 (1H, br s); 13C
NMR (75 MHz) δ 25.63, 31.24, 34.74, 41.38, 103.26, 111.88,
111.94, 112.15, 118.69, 122.40, 123.22, 127.99, 128.80, 131.64,
149.68, 150.88, 152.97, 155.15, 155.19. Anal. (C21H24N2O3) C,
H, N.
Hz), 3.49 (2H, m), 5.68 (1H, br s), 6.76–7.55 (8H, m), 8.59 (1H,
br s); 13C NMR (75 MHz, CDCl3/CD3OD ) 2/1) δ 25.72, 41.67,
93.33, 102.94, 111.55, 112.01, 112.20, 121.29, 123.26, 128.12,
130.60, 130.91, 131.60, 134.50, 150.20, 151.37, 154.66. Anal.
(C17H15IN2O3) C, H, N.
4-Iodophenyl [2-(5-Hydroxy-1H-indol-3-yl)ethyl]carbamate
(3n). The title compound was prepared from 6n following the same
procedure that was used for the synthesis of 3a and using CH2Cl2/
AcOEt ) 87/13 as eluent for the chromatographic purification. Yield
43%; mp 146–149 °C; IR 3383, 2922, 2848, 1717, 1579, 1478,
4-tert-Butylphenyl [2-(5-Hydroxy-1H-indol-3-yl)ethyl]car-
bamate (3h). The title compound was prepared from 6h following
the same procedure that was used for the synthesis of 3a and using
CH2Cl2/AcOEt ) 9/1 as eluent for the chromatographic purification.
Yield 24%; mp 159–160 °C; IR 3421, 3320, 2961, 1708, 1582,
1
1208, 1007 cm-1; H NMR (300 MHz, CDCl3/CD3OD ) 2/1) δ
2.94 (2H, t, J ) 7.1 Hz), 3.49 (2H, t, J ) 7.1 Hz), 6.69–7.67 (8H,
m); 13C NMR (75 MHz, CDCl3/CD3OD ) 2/1) δ 25.72, 41.74,
89.09, 102.87, 111.42, 112.03, 112.09, 123.41, 124.07, 128.23,
131.73, 138.44, 150.19, 151.20, 155.15. Anal. (C17H15IN2O3) C,
H, N.
1
1540, 1508, 1460, 1394, 1275, 1225, 1178, 1029 cm-1; H NMR
(300 MHz, CDCl3/CD3OD ) 3/1) δ 1.29 (9H, s), 2.91 (2H, t, J )
7.0 Hz), 3.48 (2H, m), 5.54 (1H, t, J ) 5.8 Hz), 6.75–7.38 (8H,
m), 8.52 (1H, br s); 13C NMR (75 MHz, CDCl3/CD3OD ) 3/1) δ
25.78, 31.45, 34.48, 41.63, 102.98, 111.73, 112.00, 112.16, 121.09,
123.23, 126.23, 128.14, 131.60, 148.32, 148.70, 150.17, 155.52.
Anal. (C21H24N2O3) C, H, N.
2-Naphthalenyl [2-(5-Hydroxy-1H-indol-3-yl)ethyl]carbam-
ate (3o). The title compound was prepared from 6o following the
same procedure that was used for the synthesis of 3a and using
CH2Cl2/AcOEt ) 9/1 as eluent for the chromatographic purification.
Yield 58%. Mp 158–159 °C; IR 3392, 3265, 3050, 2911, 1702,
3-(Trifluoromethyl)phenyl [2-(5-Hydroxy-1H-indol-3-yl)ethyl]-
carbamate (3i). The title compound was prepared from 6i following
the same procedure that was used for the synthesis of 3a and using
CH2Cl2/AcOEt ) 8/2 as eluent for the chromatographic purification.
Yield 68%; mp 91–93 °C; IR 3419, 2927, 1709, 1528, 1452, 1338,
1
1557, 1508, 1462, 1360, 1279, 1240, 1210, 1068 cm-1; H NMR
(300 MHz) δ 2.96 (2H, t, J ) 7.0 Hz), 3.52 (2H, m), 6.76–7.82
(11H, m); 13C NMR (75 MHz) δ 25.76, 41.69, 102.82, 111.38,
111.96, 112.09, 118.58, 121.59, 123.38, 123.54, 125.64, 126.62,
127.65, 127.82, 128.18, 129.34, 131.38, 133.94, 148.78, 150.12,
155.77. Anal. (C21H18N2O3) C, H, N.
1
1328, 1253, 1208, 1173, 1117, 1066, 1030 cm-1; H NMR (300
MHz, CDCl3/CD3OD ) 2/1) δ 2.98 (2H, t, J ) 7.2 Hz), 3.52 (2H,
t, J ) 7.2 Hz), 6.74–7.46 (8H, m); 13C NMR (75 MHz, CDCl3/
CD3OD ) 2/1) δ 25.97, 42.09, 102.95, 111.48, 111.97, 112.27,
119.25, 122.29, 123.70, 125.80, 128.44, 130.29, 132.04, 150.25,
151.75, 155.42. Anal. (C18H15F3N2O3) C, H, N.
4-Methoxyphenyl [2-(5-Hydroxy-1H-indol-3-yl)ethyl]carbam-
ate (3p). The title compound was prepared from 6p following the
same procedure that was used for the synthesis of 3a and using
CH2Cl2/AcOEt ) 8/2 as eluent for the chromatographic purification.
Yield 39%; mp 151–153 °C; IR (CHCl3) 3480, 3031, 2938, 2838,
4-(Trifluoromethyl)phenyl [2-(5-Hydroxy-1H-indol-3-yl)ethyl]-
carbamate (3j). The title compound was prepared from 6j
following the same procedure that was used for the synthesis of
3a and using CH2Cl2/AcOEt ) 8/2 as eluent for the chromato-
graphic purification. Yield 30%; mp 130–133 °C; IR (CHCl3) 3480,
3029, 2944, 1735, 1615, 1484, 1415, 1325, 1238, 1170, 1130, 1065
1
1731, 1601, 1496, 1374, 1249, 1197, 1180, 1042 cm-1; H NMR
(300 MHz, DMSO-d6) δ 2.84 (2H, t, J ) 7.4 Hz), 3.34 (2H, m),
3.74 (3H, s), 6.64–7.21 (8H, m), 7.70 (1H, t, J ) 5.4 Hz), 8.90
(1H, s), 10.47 (1H, s); 13C NMR (75 MHz, DMSO-d6) δ 25.71,
41.77, 55.80, 102.76, 111.18, 111.79, 112.30, 114.67, 123.20,
123.76, 128.42, 131.32, 144.88, 150.44, 155.52, 156.76. Anal.
(C18H18N2O4) C, H, N.
1
cm-1; H NMR (300 MHz, CDCl3/CD3OD ) 1/1) δ 2.97 (2H, t,
J ) 7.0 Hz), 3.51 (2H, t, J ) 7.0 Hz), 6.75–7.62 (8H, m); 13C
NMR (75 MHz, CDCl3/CD3OD ) 1/1) δ 25.86, 42.00, 102.93,
111.45, 111.98, 112.25, 122.43, 123.65, 126.82, 126.86, 128.40,
131.96, 150.22, 154.16, 155.14. Anal. (C18H15F3N2O3) C, H, N.
3-Chlorophenyl [2-(5-Hydroxy-1H-indol-3-yl)ethyl]carbam-
ate (3k). The title compound was prepared from 6k following the
same procedure that was used for the synthesis of 3a and using
CH2Cl2/AcOEt ) 8/2 as eluent for the chromatographic purification.
Yield 37%; mp 120–123 °C; IR 3395, 3286, 2913, 2863, 1706,
4-(Benzyloxy)phenyl [2-(5-Hydroxy-1H-indol-3-yl)ethyl]car-
bamate (3q). The title compound was prepared from 6q following
the same procedure that was used for the synthesis of 3a and using
CH2Cl2/AcOEt ) 85/15 as eluent for the chromatographic purifica-
tion. Yield 39%; mp 120–121 °C; IR 3394, 3287, 2916, 2865, 1702,
1552, 1507, 1453, 1382, 1267, 1246, 1215, 1101, 1069, 1042, 1028
cm-1; 1H NMR (300 MHz) δ 2.90 (2H, t, J ) 6.9 Hz), 3.46 (2H,
m), 4.99 (2H, s), 5.63 (1H, t, J ) 5.6 Hz), 6.74–7.42 (13H, m);
13C NMR (75 MHz) δ 25.73, 41.67, 70.58, 102.95, 111.64, 112.01,
112.12, 115.51, 122.61, 123.29, 127.54, 128.06, 128.16, 128.64,
131.64, 136.99, 144.87, 150.17, 155.74, 156.26. Anal. (C24H22N2O4)
C, H, N.
1
1589, 1549, 1470, 1255, 1211, 1068 cm-1; H NMR (300 MHz,
CDCl3/CD3OD ) 1/1) δ 2.96 (2H, t, J ) 7.4 Hz), 3.48 (2H, t, J )
7.4 Hz), 6.71–7.67 (8H, m); 13C NMR (75 MHz, CDCl3/CD3OD
) 1/1) δ 26.22, 42.42, 103.16, 111.78, 112.08, 112.43, 120.83,
122.92, 123.92, 125.97, 128.81, 130.69, 132.42, 134.97, 150.50,
152.58. Anal. (C17H15ClN2O3) C, H, N.
FAAH Assays. The effect of increasing concentrations of the
new synthetic compounds on the enzymatic hydrolysis of [14C]anan-
damide was studied as described previously52 by using membranes
prepared from rat brain. In brief, the entire rat brain was
homogenized at 4 °C in 50 mM Tris-HCl buffer, pH 7.0, by using
an Ultraturrax and a Dounce homogenizer. Homogenates were first
centrifuged at 800g to rid the debris, and the supernatant was
centrifuged at 10000g. The pellet from this latter centrifugation
was used for the assay. Membranes (70–100 µg) were incubated
with increasing concentrations (up to 50 µM) of the test compounds
and [14C]AEA (10 000 cpm, 1.8 µM) in 50 mM Tris-HCl, pH 9,
for 30 min at 37 °C. [14C]Ethanolamine produced from [14C]AEA
hydrolysis was used to calculate FAAH activity and was measured
by scintillation counting of the aqueous phase after extraction of
the incubation mixture with 2 volumes of CHCl3/CH3OH (1:1 by
volume). Data are expressed as the concentration exerting a half-
maximal inhibition (IC50). The effect of the compounds on the
uptake of anandamide by RBL-2H3 cells was studied as described
previously.53
4-Chlorophenyl [2-(5-Hydroxy-1H-indol-3-yl)ethyl]carbam-
ate (3l). The title compound was prepared from 6l following the
same procedure that was used for the synthesis of 3a and using
CH2Cl2/AcOEt ) 8/2 as eluent for the chromatographic purification.
Yield 36%; mp 130–133 °C; IR 3524, 3410, 3319, 2927, 1701,
1487, 1365, 1216, 1166, 1088 cm-1; 1H NMR (300 MHz, CDCl3/
CD3OD ) 1/1) δ 2.95 (2H, t, J ) 7.2 Hz), 3.48 (2H, t, J ) 7.2
Hz), 6.74–7.51 (8H, m); 13C NMR (75 MHz, CDCl3/CD3OD )
1/1) δ 25.95, 42.08, 102.99, 111.54, 111.98, 112.30, 123.56, 123.71,
128.50, 129.58, 130.89, 132.06, 150.15, 150.24, 155.77. Anal.
(C17H15ClN2O3) C, H, N.
3-Iodophenyl [2-(5-Hydroxy-1H-indol-3-yl)ethyl]carbamate
(3m). The title compound was prepared from 6m following the
same procedure that was used for the synthesis of 3a and using
CH2Cl2/AcOEt ) 87/13 as eluent for the chromatographic purifica-
tion. Yield 37%; mp 130–131 °C; IR (CHCl3) 3480, 3025, 3009,
1
2929, 1734, 1578, 1506, 1466, 1234, 1197, 1089, 1043 cm-1; H
NMR (300 MHz, CDCl3/CD3OD ) 2/1) δ 2.93 (2H, t, J ) 7.0

6568 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26
Ortar et al.
(8) Mechoulam, R.; Ben-Shabat, S.; Hanus, L.; Ligumsky, M.; Kaminski,
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Assays for Activity at Human Recombinant TRPV1. Human
embryonic kidney (HEK) 293 cells stably overexpressing human
recombinant TRPV1 cDNA were grown as monolayers in minimum
essential medium supplemented with nonessential amino acids, 10%
fetal calf serum, and 0.2 mM glutamine and maintained under 95%/
5% O2/CO2 at 37 °C. The effect of the new compounds on [Ca2+
]
I
was determined by using Fluo-3, a selective intracellular fluorescent
probe for Ca2+. One day prior to experiments, cells were transferred
into six-well dishes coated with poly-L-lysine (Sigma) and grown
in the culture medium mentioned above. On the day of the
experiment, the cells (50000–60000 per well) were loaded for 2 h
at 25 °C with 4 µM Fluo-3 methyl ester (Molecular Probes) in
DMSO. After the loading, cells were washed with Tyrode, pH 7.4,
trypsinized, resuspended in Tyrode, and transferred into the cuvette
of the fluorescence detector (Perkin-Elmer LS50B) under continuous
stirring. Experiments were carried out by measuring cell fluores-
cence at 25 °C (λEX ) 488 nm, λEM ) 540 nm) before and after
the addition of the test compounds at various concentrations.
Potency data are expressed as the concentrations exerting a half-
maximal inhibition (IC50).
Test of Antinociceptive Activity in Mice Treated with
Formalin. Formalin injection induces a biphasic stereotypical
nocifensive behavior. Nociceptive responses are divided into an
early, short-lasting first phase (0–7 min) caused by a primary
afferent discharge produced by the stimulus, followed by a quiescent
period and then a second, prolonged phase (15–60 min) of tonic
pain. Mice received formalin (1.25%) in the dorsal surface of one
side of the hind paw. Each mouse was randomly assigned to one
of the experimental groups (n ) 8–10) and placed in a Plexiglas
cage and allowed to move freely for 15–20 min. A mirror was
placed at a 45° angle under the cage to allow full view of the hind
paws. Lifting, favoring, licking, shaking, and flinching of the
injected paw were recorded as nociceptive responses. Mice received
intraperitoneal administration of 1a (0.3–5 mg/kg, ip), 3f (1–10
mg/kg, ip), or 1m (1–10 mg/kg, ip), alone or in combination with
the selective cannabinoid CB1 receptor antagonist AM251 (3 mg/
kg, ip), the selective cannabinoid CB2 receptor antagonist AM630
(3 mg/kg, ip), or the selective TRPV1 receptor antagonist iodor-
esiniferatoxin (I-RTX, 0.1–0.2 mg/kg, ip). 1a, 3f, or 1m were
administered 15 min before peripheral injection of formalin. The
CB1 or TRPV1 antagonists were administered 5 min before 1a, 3f,
or 1m. The total time of the nociceptive response was measured
every 5 min and expressed as the total time of the nociceptive
responses in min (mean ( SEM). Recording of nociceptive behavior
commenced immediately after formalin injection and was continued
for 60 min.
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Supporting Information Available: Elemental analysis data.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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