New resorcinol-anandamide "hybrids" as potent cannabinoid receptor ligands endowed with antinociceptive activity in vivo

Article abstract of DOI:10.1021/jm8016255

Bearing in mind the pharmacophoric requirements of both (-)-trans-Δ⁹-tetrahydrocannabinol (THC) and anandamide (AEA), we designed a novel pharmacophore consisting of both a rigid aromatic backbone and a flexible chain with the aim to develop a series of stable and potent ligands of cannabinoid receptors. In this paper we report the synthesis, docking studies, and structure-activity relationships of new resorcinol-anandamide "hybrids" differing in the side chain group. Compounds bearing a 2-methyloctan-2-yl group at position 5 showed a significantly higher affinity for cannabinoid (CB) receptors, in particular when an alkyloxy chain of 7 or 10 carbon atoms was also present at position 1. Derivative 32 was a potent CB ₁ and CB₂ ligand, with K_i values similar to that of WIN 55-212 and potent antinociceptive activity in vivo. Moreover, derivative 38, although less potent, proved to be the most selective ligand for CB₂ receptor (K_i(CB₁)) 1 μM, K _i(CB₂)) 35 nM).

Full text of DOI:10.1021/jm8016255

2506
J. Med. Chem. 2009, 52, 2506–2514
New Resorcinol-Anandamide “Hybrids” as Potent Cannabinoid Receptor Ligands Endowed
with Antinociceptive Activity in Vivo
Antonella Brizzi,*,§ Vittorio Brizzi,§ Maria Grazia Cascio,‡,# Federico Corelli,*,§ Francesca Guida,† Alessia Ligresti,‡
,|
Sabatino Maione,† Adriano Martinelli, Serena Pasquini,§ Tiziano Tuccinardi, and Vincenzo Di Marzo‡
Dipartimento Farmaco Chimico Tecnologico, UniVersita` degli Studi di Siena, Via A. Moro 2, 53100 Siena, Italy, Endocannabinoid Research
Group, Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Via Campi Flegrei 34, 80078 Pozzuoli, Napoli, Italy,
Dipartimento di Medicina Sperimentale, Sezione di Farmacologia “L. Donatelli”, Seconda UniVersita` di Napoli, Via Costantinopoli 16,
80138 Napoli, Italy, Dipartimento di Scienze Farmaceutiche, UniVersita` di Pisa, Via Bonanno 6, 56126 Pisa, Italy, and Sbarro Institute for
Cancer Research and Molecular Medicine, Center for Biotecnology, College of Science and Technology, Temple UniVersity,
Philadelphia, PennsylVania 19122
ReceiVed December 22, 2008
Bearing in mind the pharmacophoric requirements of both (-)-trans-∆9-tetrahydrocannabinol (THC) and
anandamide (AEA), we designed a novel pharmacophore consisting of both a rigid aromatic backbone and
a flexible chain with the aim to develop a series of stable and potent ligands of cannabinoid receptors. In
this paper we report the synthesis, docking studies, and structure-activity relationships of new
resorcinol-anandamide “hybrids” differing in the side chain group. Compounds bearing a 2-methyloctan-
2-yl group at position 5 showed a significantly higher affinity for cannabinoid (CB) receptors, in particular
when an alkyloxy chain of 7 or 10 carbon atoms was also present at position 1. Derivative 32 was a potent
CB1 and CB2 ligand, with Ki values similar to that of WIN 55-212 and potent antinociceptive activity in
vivo. Moreover, derivative 38, although less potent, proved to be the most selective ligand for CB2 receptor
(Ki(CB1) ) 1 µM, Ki(CB2) ) 35 nM).
Chart 1. Chemical Structures of Main Cannabinoid Receptor
Ligands
Introduction
The possible therapeutic applications of Cannabis satiVa L.
have triggered intensive research on cannabinoid receptors and
have aroused a considerable interest in the therapeutic potential
of cannabimimetic ligands.1 Cannabis mainly exerts its phar-
macological effects via a group of typical diterpene C21
compounds (now termed phytocannabinoids), and its most
important component, THCa (Chart 1), is rapidly absorbed and
converted in the liver and lungs into a centrally active
metabolite, 11-hydroxy-∆9-THC.2 Phytocannabinoids have been
shown to produce a typical syndrome of effects on the human
behavior via two G-protein-coupled receptors: CB1,3 expressed
at high levels in several brain areas but also present in peripheral
tissues,4 including ileum, testis, urinary bladder, and vas
deferens; CB2, predominantly detected in the periphery, mainly
in the immune system (spleen, tonsils, immune cells,5 and brain
microglia under inflammatory conditions6,7).
donylglycerol (2-AG, Chart 1),9-11 which are metabolically less
stable and structurally very dissimilar from the plant-derived
molecules.
Cannabinoid receptors, their endogenous ligands, and en-
docannabinoid proteins responsible for cellular uptake and
inactivation, i.e., the putative anandamide membrane transporter
(AMT)12 and the fatty acid amide hydrolase (FAAH),13 or the
monoacylglycerol lipase (MAGL)14 in the case of 2-AG,
constitute the “endocannabinoid (EC) system” and represent
potentially interesting targets15 for the development of new
therapeutic agents to be employed in many pathologies, such
as pain, loss of appetite in patients with AIDS, obesity,
chemotherapy-induced nausea and vomiting, immune and
inflammatory disorders, cardiovascular and gastrointestinal
disorders, and neurodegenerative diseases.16 In fact, the EC
system plays a role in a variety of physiological processes and
in the past 2 decades it became clear that at least in mammals,
the functions of this signaling system are not limited to the brain
but are exerted in the whole organism. Moreover, during
pathological states, the levels of these mediators in tissues
change and their effects vary from those of protective endog-
enous compounds to those of dysregulated signals. These
The discovery of cannabinoid receptors was followed by the
discovery of a family of endogenous lipid modulators, the most
studied members of which are AEA (Chart 1)8 and 2-arachi-
* To whom correspondence should be addressed. For A.B.: phone, +39
577 234327; fax, +39 577 234333; e-mail, brizzi3@unisi.it. For F.C.: phone,
+39 577 234308; fax, +39 577 234333; e-mail, corelli@unisi.it.
§
Universita` degli Studi di Siena.
Istituto di Chimica Biomolecolare.
Present address: University of Aberdeen Medical School, Aberdeen,
‡
#
Scotland, U.K.
†
Universita` di Napoli.
Universita` di Pisa.
Temple University.
|
a Abbreviations: THC, (-)-trans-∆9-tetrahydrocannabinol; AEA, anan-
damide; CB, cannabinoid; SI, selectivity index; AG, arachidonylglycerol;
AMT, anandamide membrane transporter; FAAH, fatty acid amide hydro-
lase; MAGL, monoacylglycerol lipase; EC, endocannabinoid; CBD, can-
nabidiol; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; HOBt,
1-hydroxybenzotriazole; CMC, 1-cyclohexyl-3-(2-morpholinoethyl)carbo-
diimide methyl-p-toluenesulfonate.
10.1021/jm8016255 CCC: $40.75
2009 American Chemical Society
Published on Web 03/30/2009

Resorcinol-Anandamide Hybrids
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2507
phytocannabinoid affinity and selectivity toward cannabinoid
receptors, as well as their pharmacological potency,27 suggesting
that similar changes in the side chain of THC and the terminal
alkyl position of AEA might lead to parallel changes in their
biological activity.28 Moreover, modifications of the side chain
may give rise to high affinity compounds with either antagonist,
partial agonist, or full agonist effects, while it is well-known
that the branching of the side chain can increase functional
potency.29
Figure 1. General structure of the synthesized compounds.
Chemistry
Chart 2. Proposed Pharmacophore Model for the
Resorcinol-Anandamide “Hybrids”
As shown in Scheme 1, the synthetic pathway started from
phenols 1-4, which were alkylated with 11-bromoundecanoic
acid methyl ester in dry acetone in the presence of anhydrous
potassium carbonate and potassium fluoride to furnish esters
5-8 in low yields (30-38%). Under the same conditions, 5-(2-
methyloctan-2-yl)benzene-1,3-diol 9 reacted with the corre-
sponding bromoalkyl methyl esters (8-bromooctanoic acid
methyl ester, 11-bromoundecanoic acid methyl ester, and 12-
bromododecanoic acid methyl ester) to give esters 10-12 in
34-40% yield.
5-Alkyloxybenzene-1,3-diols 2-4 were obtained from com-
mercial phloroglucinol (or 1,3,5-trihydroxybenzene) 1, which
reacted in dry dimethyl sulfoxide (DMSO) with an alkyl bromide
(1-bromobutane, 1-bromoheptane, and 1-bromo-3-methylbutane)
in the presence of anhydrous potassium carbonate and potassium
fluoride, while compound 9 was obtained with 85% yield by
cleavage of the methoxy groups from 1,3-dimethoxy-5-(2-
methyloctan-2-yl)benzene 13 using boron tribromide in dry
dichloromethane.30 As already described in a previous work,23
O-alkylation of phenols afforded after column chromatography
both monoalkylated and dialkylated products, although addition
of potassium fluoride increased yields of the former over the
latter, yields that were usually between 10% and 20%. Starting
phenols were always recovered, too, by chromatographic
purification of the crude reaction mixture. With the aim to
improve the reaction yields, alkylation reactions were carried
out using other solvents (N,N-dimethylformamide (DMF),
DMSO, and acetone) and at different temperatures (reflux in
acetone or at room temperature in DMF and DMSO) or by
protracting the reaction time up to 120 h. However, regarding
workup, reaction times, and yields, the best results were obtained
using refluxing acetone for 48-72 h; because of its very low
solubility, only phloroglucinol 1 was alkylated using DMSO
as solvent.
observations have shown that both direct or indirect agonists
and antagonists of cannabinoid receptors can produce beneficial
effects, sometimes even in the same conditions, in agreement
with the pleiotropic homeostatic function of this system.17
The use of marijuana preparations and phytocannabinoids for
medicinal purposes has been limited because of their psycho-
active properties, but in recent years a combination of THC
and cannabidiol (CBD) from Cannabis extracts has been
developed as an oromucosal spray for the treatment of neuro-
pathic pain in multiple sclerosis and cancer. Several CB1 receptor
antagonists, i.e., SR14171618 (rimonabant19), have been devel-
oped to be used therapeutically for the treatment of obesity and
associated metabolic disorders,20 and they represent another
example of endocannabinoid system-based drugs, although not
without complications, since they seem to interfere with
endocannabinoid-mediated adaptation to new stressful condi-
tions. In fact, their chronic use in obese patients was associated
with increased risk of developing signs of depression.21
On the basis of the pharmacophoric requirements22 of both AEA
and THC for binding to cannabinoid CB1 and CB2 receptors, we
have designed a novel pharmacophore (Chart 2) containing both a
rigid backbone, as in THC, and a flexible chain, as in AEA, and
developed a series of stable and potent ligands of cannabinoid
receptors.23,24 Intriguingly, more recent studies25 have shown that
these compounds act as partial CB1 agonists and CB2 neutral
antagonists. The results of cannabinoid receptor binding assays
obtained with these derivatives, which can be considered as a new
class of compounds with great affinity for cannabinoid receptors,26
supplied us with useful information on their structure-activity
relationships. The most critical requirements for the observation
of high affinity appeared to be (a) the presence of a free phenolic
hydroxy group, (b) the presence of an aliphatic chain of appropriate
length on the aromatic ring, (c) the length of the alkyloxy chain
carrying the amidic “head”, and (d) the nature of the amidic “head”
(Chart 2).
Final amides 21-41 were synthesized according to two
methods (Scheme 2): (i) esters 5-8 and 10-12 were heated
with redistilled ethanolamine as solvent to give the correspond-
ing ethanolamides 21-27 in low (30%) to good (78%) yields
(method A); (ii) acids 14-20, obtained from hydrolysis of esters
5-8 and 10-12 with methanolic/aqueous sodium hydroxide
solution in 85-93% yields, were reacted with the appropriate
amines (cyclopropylamine, cyclopropanemethylamine, 3,4-di-
hydroxyphenethylamine hydrochloride, or 4-hydroxy-3-meth-
oxybenzylamine hydrochloride) in the presence of 1-hydroxy-
benzotriazole
(HOBt)
and
1-cyclohexyl-3-(2-
On the ground of this pharmacophore model, in this paper
we report the design and synthesis of twenty-one new deriva-
tives, which maintain a free phenolic hydroxy group, an amidic
“head” and a linear alkyloxy chain of 8, 11, or 12 carbon atoms,
but contain modifications in the C portion, where an hydroxy
group, a linear/branched alkyloxy chain, or a 1,1-dimethylheptyl
aliphatic chain were introduced (Figure 1). These modifications
were planned on the basis of the numerous reports showing that
the lipophilic pentyl “tail” plays a pivotal role in determining
morpholinoethyl)carbodiimide methyl-p-toluenesulfonate (CMC)
in dry dichloromethane or acetonitrile31 to provide the corre-
sponding amides 28-41 in good yield (50-87%) (method B).
Biology
All the newly synthesized compounds that exhibited IC50 e
10 µM in the preliminary screening in radioligand binding assays
for cannabinoid receptor were further evaluated for their affinity

2508 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Scheme 1. Synthesis of Intermediate Compounds 2-12a
Brizzi et al.
a Reagents and conditions: (i) alkyl bromide, DMSO, K2CO3, KF, room temp, 48-72 h; (ii) appropriate bromoacid methyl ester, acetone, K2CO3, KF,
room temp, 48-72 h; (iii) BBr3, CH2Cl2, 0 °C to room temp.
Scheme 2. Synthesis of Final Amides 21-41a
hydroxypropyl)cyclohexanol ([3H]CP-55,940) (Kd ) 0.31 nM
for CB2 and 0.18 nM for CB1 receptors) as the high affinity
ligand as described by the manufacturer (Perkin-Elmer, Italia).32
Compounds that displaced [3H]CP-55,940 by more than 50%
at 10 µM were further analyzed by carrying out a complete
dose-response experiment. Displacement curves were generated
by incubating drugs with [3H]CP-55,940 (0.084 µM for CB2
and 0.14 nM for CB1 binding assay). In all cases, Ki values
were calculated by applying the Cheng-Prusoff equation to the
IC50 values (obtained by GraphPad) for the displacement of the
bound radioligand by increasing concentrations of the test
compounds. The antinociceptive activity of the compound with
the highest affinity for cannabinoid receptors, i.e., 32, was tested
in mice treated with formalin, evaluating the effect of compound
alone and in combination with SR141716A (rimonabant, a CB1
antagonist) or AM630 (a CB2 antagonist). A good agreement
was observed between the cannabinoid receptor affinity of 32
and its effects in this in vivo assay.33
Results and Discussion
CB1 and CB2 Receptors Affinity. With the exception of
phloroglucinol derivatives (21, 28, and 35), that proved to be
devoid of affinity for both the receptor subtypes, all the new
compounds showed in the radioligand binding assays Ki values
for either CB1 or CB2 receptor in the micromolar or submicro-
molar range. Analysis of the binding assay results for com-
pounds 21-41 allowed us to assess with more accuracy the
structure-activity relationships of this class of compounds,
which can be summarized as follows. (a) In agreement with
data from our previous study23,24 and the literature,34 the
cyclopropylamides (29-34) proved to be more potent than the
corresponding ethanolamides (22-27), even though the insertion
of a methylene group in the alkyl moiety of the amide head
negatively affected affinity, the cyclopropylmethylamides
(36-39) being less potent than the corresponding cyclopropy-
lamides (29-34). (b) In agreement again with our previous data,
an aromatic group with a polar H-bond donor substituent in
the amidic head caused a decrease in receptor affinity (40, 41).
(c) Replacement of the alkyl chain of olivetol compounds23,24
with a hydroxy group led to inactive compounds (21, 28, 35),
a Reagents and conditions. Method A: redistilled ethanolamine, 120-130
°C, 5 h. Method B: (i) MeOH/NaOH, reflux, 3 h; (ii) amine, HOBt, CMC,
CH3CN or CH2Cl2, room temp, overnight.
at recombinant human CB1 and CB2 receptors overexpressed
in COS cells, and the results are summarized in Table 1. The
preliminary screening was carried out using three concentrations
(5, 10, and 25 µM) of each compound, membranes from HEK
cells transfected with the human CB1 or CB2 receptor and [3H]-
(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-

Resorcinol-Anandamide Hybrids
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2509
Table 1. Structure, CB1 and CB2 Receptor Affinity (Ki Values), and Selectivity of Derivatives 21-41 and Reference Compounds CB25, AEA, WIN
55,212-2, and HU-210a
compd
n
R′
R′′
CB1 Ki (µM)
CB2 Ki (µM)
CB1/CB2 SI
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
10
10
10
10
7
10
11
10
10
10
10
7
10
11
10
10
7
10
11
10
10
10
OH
O(CH2)3CH3
O(CH2)6CH3
O(CH2)2CH(CH3)2
C(CH3)2(CH2)5CH3
C(CH3)2(CH2)5CH3
C(CH3)2(CH2)5CH3
OH
CH2CH2OH
CH2CH2OH
CH2CH2OH
CH2CH2OH
CH2CH2OH
CH2CH2OH
CH2CH2OH
c-C3H5
c-C3H5
c-C3H5
c-C3H5
c.C3H5
na
na
2.41
3.07
2.88
0.056
0.31
0.82
na
0.28
2.22
0.22
0.0056
0.021
0.17
na
2.30
0.10
1.00
5.40
3.4
2.82
0.0052
0.072
0.021
1.62
2.42
0.57
0.16
0.03
0.08
na
0.12
0.21
0.13
0.0079
0.0079
0.02
na
1.5
1.3
5.1
0.4
10.3
10.3
O(CH2)3CH3
O(CH2)6CH3
2.3
10.6
1.7
0.7
2.7
O(CH2)2CH(CH3)2
C(CH3)2(CH2)5CH3
C(CH3)2(CH2)5CH3
C(CH3)2(CH2)5CH3
OH
O(CH2)2CH(CH3)2
C(CH3)2(CH2)5CH3
C(CH3)2(CH2)5CH3
C(CH3)2(CH2)5CH3
C(CH3)2(CH2)5CH3
C(CH3)2(CH2)5CH3
n.(CH2)4CH3
c-C3H5
c-C3H5
8.5
CH2c-C3H5
CH2c-C3H5
CH2c-C3H5
CH2c-C3H5
CH2c-C3H5
3,4-OH-phenethyl
3-OCH3-4-OH-benzyl
c-C3H5
0.20
0.034
0.035
0.60
5.0
11.5
2.9
28.6
9.0
0.7
5.5
0.51
0.013
CB25
AEA
WIN 55,212-2
HU-210
0.4
0.0021
10.0
0.15 × 10-3
a Data are mean values of n ) 3 separate experiments and are expressed as Ki (µM) for CB1 and CB2 binding assays. Reference compounds were tested
under the same conditions in this study. Anandamide was tested in the presence of PMSF (100 nM). na ) IC50 > 10 in the preliminary screening carried out
with rat brain and spleen membranes. Binding affinity costants of the most potent compounds (Ki e 1 µM) are in bold as well as the most selective
compounds for CB1 and CB2. Standard errors are not shown for the sake of simplicity and were never higher than 10% of the mean values.
further supporting the importance of the chain in determining
cannabinoid receptor affinity. (d) Accordingly, introduction of
a short alkyloxy chain led to still active but less potent
compounds (22-24, 29-31, and 36). (e) Very potent com-
pounds were obtained by introduction of a 1,1-dimethylheptyl
chain, which improved the CB2 binding affinity compared to
that of the olivetol analogues. Particularly, compounds 32 and
33 proved to be the most potent CB1 (Ki of 5.6 and 21 nM,
respectively) and CB2 ligands (Ki ) 7.9 nM for both com-
pounds), with affinity constants comparable to those of WIN
55,212-2 (Ki(CB1) ) 21 nM and Ki(CB2) ) 2.1 nM) and our
“lead compound” CB25 (Ki(CB1) ) 5.2 nM and Ki(CB2) ) 13
nM). (f) Some compounds, although less potent, showed
selectivity for CB2 receptors, in particular, compound 38
(Ki(CB1) ) 1000 nM and Ki(CB2) ) 35 nM), which still elicited
a nanomolar affinity for CB2 receptor and a selectivity index
(SI), calculated as Ki(CB1)/Ki(CB2), of 28.6. (g) Moreover, in
the 1,1-dimethylheptyl series, receptor subtype selectivity was
only exhibited when the methylene linker length was 10 or 11
carbon atoms.
Figure 2. Antinociceptive effect of vehicle (10% DMSO in 0.9% NaCl)
or of compound 32, here denoted as CB86, in the formalin test in mice.
Each point represents the mean ( standard error of the mean (SEM)
of 8-10 animals per group. Data were analyzed using the one-way
ANOVA followed by the Bonferroni’s test, and statistical significance
was taken as P < 0.05. The effects of the CB1 antagonist rimonabant
(SR) and the CB2 antagonist AM630 are also shown. Rimonabant
reversed in a statistically significant manner the effect of the new
compound on the second phase of the nocifensive response to formalin
at all times between 30 and 45 min.
Antinociceptive Activity in Mice. As further evidence that
the new compounds likely behave in vivo as CB1 receptor
agonists, we found that the most potent compound described
in the present study, i.e., 32 (denoted as CB86), exhibited potent
antinociceptive effects against the second phase of the nocif-
ensive response to formalin in mice (Figure 2). The compound
was already maximally active at 1 mg/kg, ip, and its effect was
fully antagonized by the CB1 receptor antagonist rimonabant
but not by the CB2 receptor antagonist AM630. These observa-
tions are in agreement with our previous findings that potent
compounds of similar structure act as CB1 agonists or partial
agonists and as neutral CB2 antagonists in vivo.25 Compound
32, however, appeared to be more efficacious than previously
reported compounds, possibly because of pharmacokinetic
factors.
Molecular Modeling. A homology model of CB1 and CB2
receptors35 and the hypothetical binding interactions of AEA
into both receptors have been already described. In the CB1
receptor model, AEA adopted a U-shaped molecular conforma-
tion placed among TM2-3-6-7 with the aliphatic chain directed
toward the intracellular side of the receptor. In this disposition

2510 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Brizzi et al.
the 1,1-dimethylheptyl substituent was stabilized by the interac-
tion with F3.25, M6.55, V6.59, and F7.35. In the CB2 receptor
the ligand formed the three H-bonds with S3.31, S4.57, and
S6.58 and the 1,1-dimethylheptyl group interacted with W5.43
and F5.46. Thus, with both cannabinoid receptors, compound
33 showed the same interactions as compound 32, in agreement
with the experimental data, which highlighted an identical (CB2)
or very similar (CB1) affinity of the two compounds for the
two receptors.
Figure 3. Compound 32 docked into the CB1 (a) and CB2 (b) receptor
models (extracellular point of view).
Compound 38 was one of the most interesting compounds
among those reported herein. It differed from compound 33 for
the methylcyclopropylamide substitution and it possessed a good
CB2 affinity and a micromolar CB1 affinity, resulting in a high
CB2 selectivity (CB1/CB2 ) 28.6). In agreement with these data,
in the CB2 receptor this compound possessed all the main
interactions showed also by compounds 32 and 33 (see Figure
4f), including the three H-bonds and the main lipophilic
interactions. In the CB1 receptor the methylcyclopropyl group
was not able to interact with the small lipophilic pocket
delimited by G1.43, V2.58, and L7.43. As shown in Figure 4c,
the different arrangement of this substituent determined the loss
of the two H-bonds of the carboxamide group with T1.46 and
S2.54, thus explaining the low CB1 affinity of this compound.
Taken together, all these results suggest that lipophilicity may
play an important role in the CB affinity of analyzed compounds.
To further investigate this aspect, lipophilic properties of the
R′ side chain and R′′ amidic group, expressed as octanol/water
partition coefficients (QPlogPo/w), have been correlated with
ligand potencies, expressed as pKi(CB1) or pKi(CB2) (see Table
6 and Figure 1 of Supporting Information). Results indicate some
interesting correlations between CB1 and CB2 receptor affinities
and the lipophilic nature of the R′ aliphatic side chain, whereas
affinity at either cannabinoid receptor does not seem to be
affected by the more or less lipophilic character of the R′′ amidic
head.
the amide oxygen atom of AEA interacted with K3.28(192) and
the hydroxy group formed a H bond with S7.39(383). In the
CB2 receptor, AEA was placed among TM3-4-5-6 with the
aliphatic chain that interacted principally with W5.43 and
W6.48. In this disposition the ligand did not interact with K3.28;
it formed a H-bond with S3.31 through the amide oxygen atom,
and the hydroxy group interacted with the oxygen backbone of
L3.27.35 The hypothetical binding disposition of AEA into both
receptors was supported by the main mutagenesis data, which
suggested an important role for K3.28 in the CB1 and S3.31 in
the CB2 for the AEA interaction.36
The direct interaction of AEA with K3.28 is supported by
the work of Song and Bonner36b who found that the endocan-
nabinoid was unable to compete for [3H]WIN55,212-2 binding
in a human CB1 K3.28(192)A mutant and that its potency in
inhibiting cAMP accumulation was reduced by more than 100-
fold in this mutant. Moreover, the direct interaction of AEA
with K3.28 is also supported by the CB1-AEA binding hypoth-
esis of McAllister and co-workers.37
Figure 3 shows from an extracellular point of view the
disposition of compound 32 into both CB1 and CB2 receptors.
This compound is the most CB1 and CB2 active ligand among
those reported.
In agreement with our previous studies,24 in the CB1 receptor
model compound 32 was placed between TM1-2-3-6-7 with the
carboxamide group directed towards TM1-2 and the phenolic
group directed toward the extracellular side of TM3-6. As shown
in Figure 4a, in the CB1 receptor the phenolic substituent of
compound 32 formed a H-bond with K3.28 and the 1,1-
dimethylheptyl group showed lipophilic interactions with F3.25,
M6.55, V6.59, and F7.35. The heptamethylene chain at position
1 was stabilized through the interaction with F2.57, V3.32,
F3.36, and L7.43, the carboxamide group formed two H-bonds
with T1.46 and S2.54, and the cyclopropyl substituent was
inserted in a small lipophilic pocket mainly delimited by G1.43,
V2.58, and L7.43. In the CB2 receptor compound 32 showed a
completely different binding mode; it was placed between TM3-
4-5-6-7 with the phenolic group directed toward TM4-5 and
the carboxamide group directed toward TM7 (see Figure 3b).
As shown in Figure 4d, the phenolic substituent formed two
H-bonds with S3.31 and the backbone of S4.57 and the 1,1-
dimethylheptyl group established lipophilic interactions with
W5.43 and F5.46. The alkyloxy chain at position 1 was
stabilized through the interaction with L3.27, M6.55, and L6.59,
the nitrogen atom of the carboxamide group formed a H-bond
with S6.58 and the cyclopropyl substituent was stabilized by
lipophilic interactions with I3.29 and L6.54.
Conclusions
In this study, 21 novel resorcinol-anandamide “hybrids” were
designed and synthesized with the aim to better identify the
structural modifications that influence either affinity or selectivity
for cannabinoid receptors within this class of compounds,
obtained by linking a rigid aromatic structure such as that of
THC to a flexible chain carrying an amidic “head”, mimicking
that present in AEA. Many of the synthesized compounds
exhibited high affinity for CB1 and CB2 receptors, and some of
them, with the exception of those with the lowest Ki values for
both receptors, exhibited also a reasonable CB2 selectivity.
Moreover, our results confirmed the pivotal role played by the
aliphatic chain of the C region; its removal or shortening led to
compounds that are inactive or less potent in the binding assays,
whereas introduction of a 1,1-dimethylheptyl tail led to some
compounds that are strong CB2 ligands, still retaining high CB1
affinity, confirming that changing the length and branching of
the side pentyl chain can increase both potency and selectivity.
Also, the nature and size of the amidic “head” significantly
affected the ability of these hybrids to bind cannabinoid
receptors. In fact, introduction of a polar H-bond donor
substituent in both aliphatic and aromatic amines negatively
affected the cannabinoid receptor affinities. These results are a
significant starting point to define the structural requirements
for the design of a second generation series of highly CB1 or
CB2 selective ligands.
The replacement of the heptamethylene chain with the
decamethylene chain (compound 33) caused only a slight
decrease of CB1 affinity, and the CB2 affinity remained the same.
As shown in Figure 4b and Figure 4e, all the main interactions
already reported for compound 32 were maintained into both
receptors. In particular, in the CB1 receptor model compound
33 formed the three H-bonds with T1.46, S2.54, and K3.28 and
Interestingly, these resorcinol-anandamide “hybrids” acted
in vivo as CB1 agonists or partial CB1 agonists, showing a potent

Resorcinol-Anandamide Hybrids
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2511
Figure 4. Compounds 32 (a, d), 33 (b, e), and 38 (c, f) docked into the CB1 (a-c) and CB2 (d-f) receptor models.
elemental apparatus model 240 for C, H, N, and the data are within
antinociceptive effect fully antagonized only by the CB1 receptor
antagonist/inverse agonist rimonabant but not by the CB2
receptor antagonist AM630. This is in agreement with our
previous report that compounds in this series behave in vitro
mostly as partial CB1 agonists and neutral CB2 antagonists.25
Furthermore, molecular modeling studies on the new com-
pounds provided a reasonable hypothesis of their interaction
mode with both CB1 and CB2 receptor binding sites. As already
observed for AEA, the docking study suggested a completely
different disposition of the compounds within the two recep-
tors.35 In the CB1 receptor model, the carboxamide group of
the most active compounds was directed toward TM1-2, the
phenolic group was directed toward the extracellular side of
TM3-6, and residues T1.46, S2.54, and K3.28 seemed to possess
a key role because they formed H-bonds with the most active
ligands. In the CB2 receptor model, the analyzed compounds
were placed between TM3-4-5-6-7 with the phenolic group
directed toward TM4-5 and the carboxamide group directed
toward TM7 and the nonconserved S3.31 and the backbone of
S4.57 that formed two H-bonds with the ligands. Finally, an
interesting correlation between the lipophilic nature of the
aliphatic side chain and cannabinoid receptor potencies of the
analyzed compounds was evidenced.
(0.4% of the theoretical values. Proton nuclear magnetic resonance
(1H NMR) spectra were recorded in the indicated solvent at 25 °C
on a Bruker AC200F employing TMS as internal standard, and
chemical shifts are expressed as δ (ppm). Mass spectral data were
determined by direct insertion at 70 eV with a VG70 spectrometer.
All compounds were checked for purity by TLC on Merck 60 F254
silica plates. For column chromatography, Merck 60 silica gel,
230-400 mesh, was used. Final products were purified by a Biotage
flash chromatography system with 12.25 mm columns, packed with
KP-Sil, 60A, 32-63 µM.
General Procedure for the Synthesis of Ethers 2-4. A mixture
of phloroglucinol 1 (0.63 g, 5.0 mmol), anhydrous potassium
carbonate (0.35 g, 2.5 mmol), and potassium fluoride (0.29 g, 5.0
mmol) in dry DMSO (4 mL) was stirred for 30 min at room
temperature under nitrogen atmosphere, and then a solution of the
appropriate alkyl halide (5.0 mmol) in dry DMSO (2 mL) was
added. After being stirred for a further 48-72 h, the reaction
mixture was diluted with water (10 mL), neutralized by addition
of diluted HCl, and extracted with chloroform. The organic layer
was washed with brine, dried, and concentrated, and the residue
was purified by silica gel column chromatography.
Example. 5-Butoxy-1,3-dihydroxybenzene (2). Eluent: CHCl3/
MeOH, 50/1. Yield: 30% (pasty yellow solid). 1H NMR (CDCl3):
δ 6.84 (br s, 2H, disappears on treatment with D2O), 5.98-5.94
(m, 3H), 3.67 (t, 2H, J ) 6.4 Hz), 1.70-1.56 (m, 2H), 1.45-1.30
(m, 2H), 0.88 (t, 3H, J ) 7.3 Hz). Anal. (C10H14O3 (182.22)) C, H,
N.
Experimental Section
Chemistry. All starting materials, reagents, and solvents were
purchased from common commercial suppliers and were used as
received unless otherwise indicated. Organic solutions were dried
over anhydrous sodium sulfate and concentrated with a Bu¨chi rotary
evaporator R-110 equipped with a KNF N 820 FT 18 vacuum pump.
Melting points were determined on a Kofler hot stage apparatus
(K) or using a Mettler FPI apparatus (2 °C/min, M) and are
uncorrected. Elemental analyses of all synthesized compounds were
performed by our analytical laboratory using a Perkin-Elmer
General Procedure for the Synthesis of Esters 5-8, 10-12.
A mixture of the appropriate diphenol derivatives 2-4, 9 (5.0
mmol), anhydrous potassium carbonate (0.35 g, 2.5 mmol), and
potassium fluoride (0.29 g, 5.0 mmol) in dry acetone (20 mL) was
refluxed under stirring and nitrogen atmosphere for 30 min. Then
a solution of the appropriate bromoalkanoic acid methyl ester (5.0
mmol) in dry acetone (10 mL) was added, and the reaction mixture
was kept at reflux temperature for a further 48-72 h. Afterward,

2512 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Brizzi et al.
the reaction mixture was concentrated, diluted with saturated
ammonium chloride solution (20 mL), and extracted with chloro-
form. The organic layers were collected, dried, and evaporated under
reduced pressure to afford a residue, which was purified by silica
gel column chromatography.
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.38 The total time of the
nociceptive response was measured every 5 min and expressed as
the total time of the nociceptive responses in minutes (mean (
SEM). Recording of nociceptive behavior commenced immediately
after formalin injection and was continued for 60 min. The version
of the formalin test we applied is based on the fact that a
correlational analysis showed that no single behavioral measure
can be a strong predictor of formalin or drug concentrations on
spontaneous behaviors.38,39 Consistently, we considered that a
simple sum of time spent licking plus elevating the paw, or the
weighted pain score,33 is in fact superior to any single (lifting,
favoring, licking, shaking, and flinching) measure (r ranging from
0.75 to 0.8637). Treatments were as follows: groups of 8-10
animals per treatment were used, with each animal being used for
one treatment only. Mice received intraperitoneal administration
of vehicle (10% DMSO in saline) or two doses of compound 32 (1
or 3 mg/kg, ip) alone or compound 32 (1 mg/kg, ip) in combination
with rimonabant (1 mg/kg, ip), a selective CB1 receptor antagonist,
or AM630 (1 mg/kg, ip), a selective CB2 receptor antagonist.
Compound 32 was administered 15 min before the peripheral
injection of formalin. The CB1 or CB2 antagonists were administered
5 min before compound 32.
Molecular Modeling. The ligands were submitted to a confor-
mational search of 1000 steps. The algorithm used was the Monte
Carlo method with MMFFs as the force field and a distance-
dependent dielectric constant of 1.0. The ligands were then
minimized using the conjugated gradient method until a conver-
gence value of 0.05 kcal/(Å mol) was obtained, using the same
force field and dielectric constant used for the conformational
search.40 Then the ligand was docked into CB1 and CB2 receptor
using the AUTODOCK 3.0 program.41 AUTODOCK TOOLS42
was used to identify the torsion angles in the ligands, to add the
solvent model, and to assign partial atomic charges (Gasteiger for
the ligands and Kollman for the receptors). The regions of interest
used by AUTODOCK were defined by considering for both CB1
and CB2 receptors T3.33 as the central residue of a grid of 60, 46,
and 50 points in the x, y, and z directions. A grid spacing of 0.375
Å and a distance-dependent function of the dielectric constant were
used for the energetic map calculations.
Example. 8-[3-Hydroxy-5-(2-methyloctan-2-yl)phenoxy]oc-
tanoic Acid Methyl Ester (10). Eluent: CHCl3/MeOH, 100/0.5.
1
Yield: 40% (pale-yellow oil). H NMR (CDCl3): δ 6.43 (s, 1H),
6.36 (d, 1H, J ) 1.5 Hz), 6.21 (d, 1H, J ) 1.9 Hz), 4.83 (s, 1H,
disappears on treatment with D2O), 3.89 (t, 2H, J ) 6.3 Hz), 3.65
(s, 3H), 2.29 (t, 2H, J ) 7.3 Hz), 1.77-1.45 (mm, 8H), 1.29-1.06
(mm, 24H), 0.82 (t, 3H, J ) 5.9 Hz). MS m/z: 415 [M + Na]+
(100). Anal. (C24H40O4 (392.57)) C, H, N.
General Procedure for the Synthesis of Acids 14-20. A
solution of the appropriate methyl esters 5-8, 10-12 (2.0 mmol)
in methanol (6 mL) and 3 N aqueous NaOH (2 mL, 6 mmol) was
refluxed for 3 h. Then the reaction mixture was cooled in an ice
bath and acidified to pH 3 with diluted HCl. The aqueous layer
was extracted several times with ethyl acetate, and the collected
organic solution was dried and evaporated to yield the crude acid,
which was purified by silica gel column chromatography.
Example. 8-[5-(2-Methyloctan-2-yl)phenoxy]octanoic Acid
(18). Eluent: CHCl3/MeOH, 50/1. Yield: 80% (white solid), mp 111.1
°C (M). 1H NMR (CDCl3): δ 6.45 (s, 1H), 6.37 (d, 1H, J ) 1.6 Hz),
6.21 (d, 1H, J ) 1.7 Hz), 3.90 (t, 2H, J ) 6.6 Hz), 2.36 (t, 2H, J )
7.1 Hz), 1.76-1.61 (mm, 4H), 1.56-1.40 (mm, 8H), 1.37-1.09 (mm,
14H), 0.82 (t, 3H, J ) 6.1 Hz). MS m/z: 401 [M + Na]+, 779 [2M +
Na]+ (100). Anal. (C23H38O4 (378.55)) C, H, N.
General Procedure for the Synthesis of Ethanolamides
21-27. Method A. Esters 5-8, 10-12 (2.0 mmol) were dissolved
under nitrogen atmosphere and continuous stirring in redistilled
ethanolamine (4 mL), and the solution was heated at 120-130 °C
for 4 h. The reaction mixture was diluted with water, neutralized
with diluted HCl, and extracted with chloroform. The collected
extracts were washed with a solution of saturated ammonium
chloride, dried, and evaporated, and the raw material so obtained
was purified by silica gel column chromatography.
Example. 11-(3,5-Dihydroxyphenoxy)undecanoic Acid (2-
Hydroxyethyl)amide (21). Eluent: CHCl3/MeOH, 50/1. Yield 30%
1
(pale-yellow solid), mp 125-130 °C (K). H NMR (CDCl3): δ
5.98-5.91 (m, 3H), 3.85 (t, 2H, J ) 6.2 Hz), 3.54 (t, 2H, J ) 5.6
Hz), 3.31-3.22 (m, 2H), 2.16 (t, 2H, J ) 7.8 Hz), 1.69-1.54 (m,
4H), 1.48-1.29 (mm, 12H). MS m/z: 376 [M + Na]+ (100). Anal.
(C19H31NO5 (353.45)) C, H, N.
By use of the Lamarckian genetic algorithm, all docked
compounds were subjected to 250 runs of the AUTODOCK search
in which the default values of the other parameters were used.
Cluster analysis was performed on the docked results, using an rms
tolerance of 1.0 Å.
General Procedure for the Synthesis of Amides 28-41.
Method B. To a mixture of acids 14-20 (1.0 mmol), the
appropriate amine (1.5 mmol), and 1-hydroxybenzotriazole (HOBt,
1.2 mmol) in dry dichloromethane (10 mL) kept at 0 °C in an ice
bath, a solution in the same solvent (5 mL) of 1-cyclohexyl-3-(2-
morpholinoethyl)carbodiimide methyl-p-toluensulfonate (CMC, 1.5
mmol) was added dropwise under nitrogen atmosphere and continu-
ous stirring. The solution was allowed to warm to room temperature,
and stirring was continued for 24 h. The organic solution was
washed with 5% aqueous NaHCO3, then with 1 N HCl, and dried.
After evaporation of solvent, the crude product was purified by
silica gel column chromatography.
Example. 8-[5-(2-Methyloctan-2-yl)phenoxy]octanoic Acid
Cyclopropylamide (32). Eluent: CHCl3/MeOH, 50/1. Yield 81%
(pale-yellow oil). 1H NMR (CDCl3): δ 6.48-6.41 (m, 2H),
6.29-6.27 (m, 1H), 6.06 (br s, 1H), 5.61 (br s, 1H), 3.92 (t, 2H, J
) 6.7 Hz), 2.75-2.66 (m, 1H), 2.14 (t, 2H, J ) 7.2 Hz), 1.78-1.52
(mm, 6H), 1.48-1.09 (mm, 20H), 0.83 (t, 3H, J ) 6.2 Hz),
0.79-0.73 (m, 2H), 0.57-0.43 (m, 2H). MS m/z: 440 [M + Na]+,
857 [2M + Na]+ (100). Anal. (C26H43NO3 (417.62)) C, H, N.
Evaluation of Antinociceptive Activity in Mice. Formalin
injection induces a biphasic stereotypical nocifensive behavior.33
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 saline, 30 µL) in the dorsal surface of one side of the
Acknowledgment. The authors from the University of Siena
thank the Ministero dell’Universita` e della Ricerca (PRIN 2006,
Prot. n 2006030948_002) for financial support. Molecular graphics
images were produced using the UCSF Chimera package from the
Resource for Biocomputing, Visualization, and Informatics at the
University of California, San Francisco (supported by NIH Grant
P41 RR-01081). The authors are grateful to Marco Allara` for
technical assistance with the binding assays.
Supporting Information Available: Physical and spectral data
for compounds 3-12, 14-20, and 22-41 and elemental analysis
data for compounds 21-41. This material is available free of charge
via the Internet at http://pubs.acs.org.
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