Design, synthesis, and binding studies of new potent ligands of cannabinoid receptors

Article abstract of DOI:10.1021/jm0501533

Despite their different chemical structures, Δ⁹- tetrahydrocannabinol (THC) and anandamide (AEA) have common pharmacological properties. This study was aimed at finding new cannabinoid receptor ligands that overcome the instability of AEA and i

Full text of DOI:10.1021/jm0501533

J. Med. Chem. 2005, 48, 7343-7350
7343
Design, Synthesis, and Binding Studies of New Potent Ligands of Cannabinoid
Receptors
Antonella Brizzi,†,* Vittorio Brizzi,† Maria Grazia Cascio,‡ Tiziana Bisogno,‡ Rossella Sirianni,† and
Vincenzo Di Marzo‡
Dipartimento Farmaco Chimico Tecnologico, Universita` degli Studi di Siena, Via A. Moro 2, 53100 Siena (Italy), and
Endocannabinoid Research Group, Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche,
Via Campi Flegrei 34, Comprensorio Olivetti, 80078 Pozzuoli, Napoli (Italy)
Received February 17, 2005
Despite their different chemical structures, ∆9-tetrahydrocannabinol (THC) and anandamide
(AEA) have common pharmacological properties. This study was aimed at finding new
cannabinoid receptor ligands that overcome the instability of AEA and its analogues. To this
end we planned the synthesis of a series of compounds which retained both a rigid structure,
like that of plant cannabinoids, and a flexible portion similar to that of anandamide. Binding
studies on CB1 and CB2 receptors, anandamide membrane transporter (AMT), and fatty acid
amide hydrolase (FAAH) showed that some of the newly developed compounds have high affinity
and specificity for cannabinoid CB1 and CB2 receptors. Compound 25 is a potent CB1 and CB2
ligand, with affinity constants significantly lower than AEA and similar to WIN 55-212,
compound 52 is a potent CB2 ligand, although not very selective over CB1 receptors, and
compound 43 is CB2 ligand, with at least a 26-fold selectivity over CB1 receptors. Compound
25 behaved as a inverse agonist at CB1 receptors as assessed in the cyclic AMP functional
assay.
Chart 1
Introduction
The great popularity of hashish and marihuana, both
derived from the Indian hemp Cannabis Sativa L., and
of their major psychoactive component, (-)-trans-∆9-
tetrahydrocannabinol (THC, Chart 1),1 is due to their
medicinal as well as their psychotomimetic effects.
Cannabinoids have been shown to produce a wide range
of pharmacological effects2 mediated by two receptors:
CB1, very abundant in the brain3 and cloned in 1990,4
and CB2, expressed predominantly in the periphery,5
mainly in the spleen and in immune cells. The presence
of these receptors in mammalian cells was indicative
of the existence of an endogenous cannabinoid system.
Several years ago the first natural ligand was isolated
from porcine brain6 and identified as an amide related
to arachidonic acid, the cis-5,8,11,14-eicosatetraenoyl-
ethanolamide, or anandamide (AEA), showing proper-
ties similar to the plant-derived agonist THC. Three
years later a well-known intermediate in phosphogly-
ceride metabolism, 2-arachidonoylglycerol (2-AG), was
found to act also as a potential ligand at cannabinoid
receptors.7-9
The molecular basis of the biological effects of these
endogenous compounds were elucidated also thanks to
the understanding of their metabolism, including bio-
chemical pathways that lead to their synthesis and
inactivation. Both AEA and 2-AG are produced from the
metabolism of membrane phospholipids and immedi-
ately released into extracellular space. Inactivation
occurs in two steps: cellular reuptake, facilitated by a
putative transporter protein known as the anandamide
membrane transporter (AMT)10 and enzymatic hydroly-
sis by a fatty acid amide hydrolase (FAAH)11 or by a
monoacylglycerol lipase, in the case of 2-AG. The
proteins of the endocannabinoid system, including CB1,
CB2, AMT, and FAAH, represent excellent targets for
the development of new therapeutic drugs to be em-
ployed in many pathologies, like pain, immunodepres-
sion, vascular perpherical disorders, increase or lower-
ing of appetite, and motor disorders. The therapeutic
value of these compounds as analgesic, anti-glaucoma,
and antiinflammatory agents, and in the chemotherapy-
induced nausea and vomiting and in appetite stimula-
tion of patients with AIDS, is nowadays of great
interest.12
Since these discoveries several groups have carried
out the synthesis and biological evaluation of AEA
analogues, obtained by modifications of the fatty acyl
chain or/and of the ethanolamide “head”.13 Unfortu-
nately, arachidonic acid and analogous polyunsaturated
fatty acids are very expensive and/or easily oxidized.
AEA itself is very unstable and in the body is rapidly
metabolized by FAAH and/or oxidation. To prevent in
part the degradation of AEA and of its synthetic
* Corresponding author. Fax: +39 577 234333; Phone: +39 577
234327; e-mail: brizzi3@unisi.it.
† Universita` degli Studi di Siena.
‡ Consiglio Nazionale delle Ricerche.
10.1021/jm0501533 CCC: $30.25 © 2005 American Chemical Society
Published on Web 10/13/2005

7344 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
Brizzi et al.
Figure 1.
Figure 3.
give the corresponding acids, which were used as such
and reacted with amines in the presence of 1-hydroxy-
benzotriazole (HOBt) and 1-cyclohexyl-3-(2-morpho-
linoethyl)carbodiimide methyl-p-toluenesulfonate (CMC)
in dichloromethane or acetonitrile.18 Method B: esters
were warmed with ethanolamine as solvent.
Figure 2.
Scheme 1a
The O-alkylation of phenols was carried out with or
without KF; as expected, in both conditions the reaction
afforded all possible alkylated products when the aro-
matic nucleus had two free hydroxyl groups, although
addition of KF improved the yields and specifically
increased those of monoalkylated over dialkylated prod-
ucts. Moreover, O-alkylation of 4-hexylresorcinol af-
forded two different monoalkylated regioisomers. The
reaction occurred mainly on the hydroxyl in C-1 irre-
spectively of the bromomethyl ester used and indepen-
dently from the length of its chain; in fact the C-1
position results less hindered when the hexyl chain is
present as para substituent. The structure of two
regioisomers has been definitively assigned by NOESY
experiments which showed a NOE effect between the
OCH2 and both the ortho aromatic hydrogens (H-2 and
H-6) in the isomer with the alkylated hydroxyl in C-1,
and a NOE effect between the OCH2 and the only ortho
aromatic hydrogen (H-2) in the isomer with the alkyl-
ated hydroxyl in C-3 (Figure 3).
Pharmacological Evaluation. Binding Assays.
For CB1 and CB2 receptor binding assays, the new
compounds were first subjected to a preliminary screen-
ing carried out using three concentrations (5, 10, and
25 µM), membranes from rat brain and spleen, and [3H]-
N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophen-
yl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride
(SR141716A, 2 nM) as the high affinity ligand.19 Com-
pounds which displaced [3H]-SR141716A by more than
50% at 10 µM were further analyzed by using P2
membranes from COS cells transfected with either the
human CB1 or CB2 receptor and [3H]-(-)-cis-3-[2-hy-
droxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxy-
propyl)cyclohexanol ([3H]CP-55,940) (Kd ) 690 pM) as
the high affinity ligand as described by the manufac-
turer (Perkin-Elmer, Italy). Displacement curves were
generated by incubating drugs with 0.5 nM of [3H]CP-
55,940. 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.
a Reagents: (i) Br-(CH2)nCOOCH3, CH3COCH3, K2CO3, KF,
reflux, 48-72 h; (ii) Method A: MeOH/NaOH, reflux, 3 h; amine,
HOBt, CMC, r.t., overnight; (iii) Method B: H2NCH2CH2OH, 120-
130 °C, 5 h.
analogues their activity at cannabinoid receptors is often
assessed by conducting receptor binding assays in the
presence of a serine protease inhibitor, phenylmethane-
sulfonyl fluoride (PMSF).14
While AEA is a very flexible molecule, as demon-
strated also by NMR studies,15 and can assume both
linear and nonlinear conformations, THC, and canna-
binoid analogues, including tricyclic and bicyclic can-
nabinoids, are rigid compounds. Nevertheless, AEA and
THC have been shown to share many, albeit not all,
pharmacological properties. Bearing in mind the phar-
macophore requirements16 of both AEA and THC,
necessary for the binding to cannabinoid CB1 and CB2
receptors, we have planned the synthesis of a series of
stable compounds which consist of both a rigid aromatic
portion, as in THC, and a flexible chain, as in AEA.
Olivetol, or 5-pentyl-benzene-1,3-diol (5-pentylresor-
cinol), is involved in the biogenesis of THC in Cannabis17
(Figure 1), and it has been selected as the basic aromatic
scaffold of the rigid part of our new derivatives; other
modified phenols have also been considered in this work
in order to define a structure-activity relationship. As
flexible chains we have chosen saturated carboxylic
acids with chain lengths of 6 or 11 or 16 carbon atoms,
easily converted into the final amides. Finally, we have
evaluated the influence of several factors such as sub-
stitutions of the aromatic backbone, the length of the
chain carrying the amidic head, and nature of amide.
As a result, different series of new compounds were
designed and synthesized, whose general structure is
reported in Figure 2.
Chemistry. As shown in Scheme 1, the synthesis of
the new compounds starts from a substituted phenol
[3-pentadecylphenol, olivetol, resorcinol (or benzene-1,3-
diol), and 4-hexylresorcinol] which is reacted with a
bromoalkylmethyl ester (6-bromohexanoic acid methyl
ester or 11-bromoundecanoic acid methyl ester or 16-
bromo-hexadecanoic acid methyl ester) in dry acetone
in the presence of anhydrous K2CO3 and KF, giving
esters 1-14. The final amides are obtained by either of
two simple methods. Method A: esters were refluxed
with methanolic/aqueous sodium hydroxide solution to
Fatty Acid Amide Hydrolase Assays. The effect
of compounds on the enzymatic hydrolysis of [14C]-
anandamide (6 µM) was studied by using membranes
prepared from rat brain incubated with increasing
concentrations of compounds in 50 mM Tris-HCl, pH 9,
for 30 min at 37 °C.19 [14C]Ethanolamine produced from
[14C]anandamide hydrolysis was measured by scintilla-

Potent Ligands of Cannabinoid Receptors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23 7345
Table 1. Radioligand Binding Assays and Selectivity over Other Proteins of the Endocannabinoid System of the Synthesized
Compoundsa
compd
R
R1
n
R2
CB1
CB2
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.a
0.96
n.t.
0.16
0.013
1.4
n.a.
n.a.
n.a.
n.t.
n.t.
n.t.
n.a.
5.4
FAAH
AMT
15
H
H
H
H
H
H
3-CH2(CH2)13CH3
3-CH2(CH2)13CH3
3-CH2(CH2)13CH3
3-CH2(CH2)13CH3
3-CH2(CH2)13CH3
3-CH2(CH2)13CH3
5-CH2(CH2)3CH3
5-CH2(CH2)3CH3
5-CH2(CH2)3CH3
5-CH2(CH2)3CH3
5-CH2(CH2)3CH3
5-CH2(CH2)3CH3
5-CH2(CH2)3CH3
5-CH2(CH2)3CH3
5-CH2(CH2)3CH3
H
5
5
5
10
10
10
5
5
5
CH2CH2OH
c.C3H5
p.OH-C6H4
CH2CH2OH
c.C3H5
p.OH-C6H4
CH2CH2OH
c.C3H5
p.OH-C6H4
CH2CH2OH
c.C3H5
p.OH-C6H4
CH2CH2OH
c.C3H5
p.OH-C6H4
CH2CH2OH
c.C3H5
p.OH-C6H4
CH2CH2OH
c.C3H5
p.OH-C6H4
CH2CH2OH
c.C3H5
p.OH-C6H4
CH2CH2OH
c.C3H5
p.OH-C6H4
CH2CH2OH
c.C3H5
p.OH-C6H4
CH2CH2OH
c.C3H5
p.OH-C6H4
CH2CH2OH
c.C3H5
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
1.3
n.a.
n.a.
n.a.
n.t.
n.t.
n.t.
n.a.
n.a
>25
n.t.
>25
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
25
16
17
18
19
20
21
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
5-OH
5-OH
5-OH
5-OH
5-OH
5-OH
5-OH
5-OH
5-OH
22
23
n.t.
0.8
n.a.
n.a
n.a
24
10
10
10
15
15
15
5
25
0.0052
3
17
26
n.a.
n.t.
n.t.
n.t.
n.a.
n.a.
n.t.
n.a.
n.a.
n.t.
n.t.
n.t.
n.t.
n.a.
n.a.
n.t.
n.a.
n.a.
50
n.t.
n.t.
n.t.
n.a.
n.a.
n.t.
n.a.
>25
18.0
n.t.
n.t.
n.t.
n.a.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.a.
>25
>25
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
>25
13
27
12.5
28
>10
29
>10
30
n.t.
n.t.
n.t.
31
H
5
5
32
H
33
H
10
10
10
15
15
15
5
>10
>10
n.a.
>10
4.25
5
34
H
35
H
>10
36
H
n.a.
n.a.
37
H
38
H
10
39
4-CH2(CH2)4CH3
4-CH2(CH2)4CH3
4-CH2(CH2)4CH3
4-CH2(CH2)4CH3
4-CH2(CH2)4CH3
4-CH2(CH2)4CH3
4-CH2(CH2)4CH3
4-CH2(CH2)4CH3
4-CH2(CH2)4CH3
2-CH2(CH2)4CH3
2-CH2(CH2)4CH3
2-CH2(CH2)4CH3
2-CH2(CH2)4CH3
2-CH2(CH2)4CH3
2-CH2(CH2)4CH3
2-CH2(CH2)4CH3
2-CH2(CH2)4CH3
2-CH2(CH2)4CH3
n.t.
0.18
n.t.
6.43
0.54
n.t.
40
5
5
41
42
10
10
10
15
15
15
5
>10
>10
2.7
0.35
43
44
n.a.
n.a.
n.a.
>10
45
n.a.
n.a.
n.a.
46
47
>10
48
n.t.
>10
49
5
5
n.t.
3.57
50
p.OH-C6H4
CH2CH2OH
c.C3H5
n.t.
n.t.
51
10
10
10
15
15
15
1.13
0.42
52
0.21
0.03
53
p.OH-C6H4
CH2CH2OH
c.C3H5
3.3
2.11
>25
n.t.
n.t.
n.t.
54
n.a
n.a.
55
5.5
2.49
56
p.OH-C6H4
3
n.a.
anandamide
WIN, 55-212
HU-210
72 ( 3.1 n M
21 ( 1.1 nM
-
-
2.1 ( 0.1 nM
0.15 ( 0.03
a Data are means ( SEM of n ) 3 separate experiments and are expressed as Ki (µM), for CB1 and CB2 binding assays, and in IC50
(µM) for FAAH and AMT assays. Reference compounds were tested under the same conditions in this study. Anandamide was tested in
the presence of PMSF (100 nM). n.a. ) IC50 > 10 in the preliminary screening carried out with rat brain and spleen membranes; n.t. )
not tested; insol. ) insoluble in any of the solvents normally used in binding assays (DMSO, ethanol, or methanol). Binding affinity
costants of the most potent compounds (Ki e 1 µM) are highlighted in bold.
tion counting of the aqueous phase after extraction of
the incubation mixture with 2 volumes of CHCl3/CH3-
OH (2:1 by volume). In most cases, only the most potent
compounds in the binding assays were subjected to this
assay.
well dishes and stimulated for 10 min at 37 °C with
forskolin 1 µM in 400 µL of serum-free Dulbecco’s
modified Eagle’s Medium containing 20 mM HEPES,
0.1 mg/mL BSA, 0.1 mM 1-methyl-3-isobutylxanthine.20
Cells were treated with vehicle (methanol, 0.1%) or
compounds, or WIN55,212-2 at various concentrations,
or with SR141716A (100 nM). After incubation, 800 µL
of ethanol was added, cells were extracted and cyclic
AMP was determined by means of a cyclic AMP assay
kit (Amersham, UK), as advised by the manufacturer.
Anandamide Membrane Transporter (AMT) As-
says. The effect of compounds on the uptake of anan-
damide by RBL-2H3 cells was studied as described pre-
viously.19 Cells were incubated with [14C]anandamide
(4 µM) for 5 min at 37 °C, in the presence or absence of
varying concentrations of the inhibitors. Residual [14C]-
anandamide in the incubation media after extraction
with CHCl3/CH3OH (2:1 by volume) was used as a mea-
sure of the anandamide that was taken up by cells. Data
are expressed as the concentration exerting 50% inhibi-
tion of anandamide uptake (IC50) calculated with Graph-
Pad. In most cases, only the most potent compounds in
the binding assays were subjected to this assay.
Cyclic AMP Assay. Cyclic AMP assays were per-
formed on intact confluent N18TG2 cells plated in six-
Results and Discussion
All the newly synthesized compounds that exibited
IC50 e 10 µM in the preliminary screening for canna-
binoid receptor binding activity were evaluated in
radioligand binding assays for affinity at recombinant
human CB1, CB2 overexpressed in COS cells, or for their
inhibitory actions on FAAH and AMT, and the results
are summarized in Table 1. Our preliminary goal was
to evaluate the hypothesis that putting together in a

7346 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
Brizzi et al.
Figure 4.
single structure the chemical stability of THC with the
flexibility of AEA might lead to compounds active on
the endocannabinoid systhem. To this end we have
introduced an aliphatic chain carrying an amidic “head”
in the aromatic structure of 3-pentadecylphenol, olivetol,
resorcinol, and 4-hexylresorcinol, achieving five series
of O-alkylated derivatives from which we could get
useful information on structure-activity relationships.
In fact, the binding assay results of the compounds
described here allow us to establish the effect of several
factors on the affinity for CB1 and CB2 receptors, and
the most important points can be summarized as
follows: (a) the presence of a phenolic hydroxyl group
seems to play an essential role, since without it the
3-pentadecyphenol derivatives 15-17 are unable to bind
to cannabinoid receptors with high affinity; (b) the
length of aliphatic chain on the aromatic ring has a
crucial influence on the affinity of our compounds, since
a chain of five or six carbon atoms, such as in olivetol
and 4-hexylresorcinol, is required, while a longer chain,
as in 3-pentadecylphenol, or its absence, as in resorcinol,
leads respectively to insoluble (18-20) or inactive (30-
38) compounds; (c) regarding the alkyloxy chain carry-
ing the amidic “head”, its length also turned out to be
of great importance: in fact, the short, five-carbon atom
derivatives (15-17, 21-23, 30-32, 39-41, and 48-50)
or the long fifteen carbon atom derivatives (27-29, 36-
38, 45-47, and 54-56) are inactive independently of
their aromatic structure. In support to this analysis one
can observe that the most potent compounds are olivetol
(24, 25, and 26) and 4-hexylresorcinol derivatives (51
and 52), all of them with a chain of 10 carbon atoms;
one notable exception to this rule was compound 40
which was more potent than compound 43 at CB1, and
equipotent at CB2, receptors; (d) in accordance with
literature data,13 cyclopropylamides (25, 43, and 52) are
more potent than the respective ethanolamides (24, 42,
and 51), and in particular special attention should be
given to compound 25 (Figure 4), which exhibits the
lowest Ki values and behaves as a very potent CB1 (5.2
nM) and CB2 ligand (13 nM), with affinities similar to
WIN55-212-2 (CB1 21 ( 1.1 nM and CB2 2.1 ( 0.1 nM);
(e) furthermore, it appears interesting to compare two
isomers of 4-hexylresorcinol: while compound 52 is a
potent CB2 ligand (30 nM), although not very selective
over CB1 receptors (210 nM), its regioisomer 43, even
though less potent, resulted in a CB2 selective ligand
with at least a 26-fold selectivity over CB1 receptors
(CB2 0.35 µM and CB1 > 10 µM); (f) regarding the
interaction of the most potent cannabinoid receptor
ligands with FAAH and the AMT, the data obtained
here show that all such compounds are essentially
unable to bind to either the enzyme or the putative
transporter.
Figure 5.
for its activity at CB1 receptors. We assessed the effect
of this compound on forskolin-induced adenylate cyclase
activity in mouse neuroblastoma N18TG2 cells, which
constitutively and selectively express cannabinoid CB1
receptors. As shown in Figure 5, similar to the well-
known CB1 inverse agonist/antagonist, SR141716A,
compound 25 significantly stimulated forskolin-induced
cyclic AMP formation (half-maximal effect observed at
a concentration of 13 nM similar to the Ki for this
compound at CB1 receptors). As expected, WIN55,212-2
behaved as an agonist since it inhibited forskolin-
induced cyclic AMP formation (half-maximal effect
observed at a concentration of 15 nM). The effect of
compound 25 at the doses of 20 and 100 nM was blocked
by a per se inactive dose (5 nM) of WIN55,212 and hence
was mediated by CB1 receptors (Figure 5, higher doses
of 25 could not be tested as they would have required
non inactive doses of WIN55,212-2 to be counteracted).
Therefore, this compound behaved as a inverse agonist/
antagonist at CB1 receptors. Also another compound
that exhibited affinity for CB1 receptors, albeit lower
than compound 25, i.e., compound 40, also behaved as
a inverse agonists/antagonist (half-maximal effect ob-
served at a concentration of 150 nM, data not shown).
We also attempted at performing functional assays for
CB2 receptors in cell lines constitutively expressing this
receptor but could not get a response even with WIN55,-
212-2 and, therefore, did not go further.
Conclusions
Our hypothesis that compounds active at cannabinoid
receptors can be obtained by linking a stable and rigid
structure typical of aromatic compounds, such THC,
olivetol, and its analogues, to a flexible chain carrying
an amidic “head”, as in AEA, appeared to be correct and
resulted in the synthesis of a new class of compounds.
Some of the new compounds exhibited potent activity
at CB1 and CB2 receptors and exhibited antagonist/
inverse agonist activity. As a result of these studies we
are currently pursuing new experiments in order to
further evaluate the functional activity of these mol-
ecules, to elucidate the requirements involved in the
recognition and binding of their biological targets, and
to evaluate their pharmacological activity in vivo, to
direct our research toward the design and synthesis of
ever more potent and selective compounds.
To gain some insight into the possible use as a
therapeutic agent of the most potent ligand described
here, i.e., compound 25, we carried out functional assays

Potent Ligands of Cannabinoid Receptors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23 7347
J ) 7.5 Hz), 1.69 (t, 2H, J ) 7.3 Hz), 1.36-1.03 (mm, 8H),
1.0-0.79 (mm, 24H), 0.40 (t, 3H, J ) 6.3 Hz), 0.24-0.15 (m,
2H), 0.11-0.02 (m, 2H). MS m/z: 458 [M + 1]+ (100). Anal.
(C30H51NO2) C, H, N.
6-(3-Pentadecylphenoxy)hexanoic Acid (4-Hydroxy-
phenyl)amide (17). White solid (CHCl3/MeOH ) 47/3) (70%
yield): mp 103.2 °C (M). 1H NMR (CDCl3) δ (ppm): 7.31 (half
of ABq, 2H, J ) 8.7 Hz), 7.20-7.12 (m, 2H), 7.00 (s br, 1H),
6.79-6.68 (m, 4H), 3.95 (t, 2H, J ) 7.1 Hz), 3.92 (s br, 1H),
2.55 (t, 2H, J ) 7.6 Hz), 2.36 (t, 2H, J ) 7.0 Hz), 1.80-1.69
(m, 4H), 1.63-1.53 (m, 4H), 1.25-1.18 (mm, 24H), 0.87 (t, 3H,
J ) 6.6 Hz). MS m/z: 510 [M + 1]+, 1019 [2M + 1]+ (100).
Anal. (C33H51NO3) C, H, N.
11-(3-Pentadecylphenoxy)undecanoic Acid (2-Hydroxy-
ethyl)amide (18). White solid (CHCl3/MeOH ) 47/3) (75%
yield): mp 83-85 °C (K). 1H NMR (CDCl3) δ (ppm): 7.18-
7.11 (m, 1H), 6.74-6.67 (m, 3H), 5.85 (s br, 1H), 3.92 (t, 2H,
J ) 6.4 Hz), 3.71 (t, 2H, J ) 4.7 Hz), 3.44-3.36 (m, 2H), 2.60
(s br, 1H), 2.55 (t, 2H, J ) 7.5 Hz), 2.18 (t, 2H, J ) 7.5 Hz),
1.78-1.51 (mm, 6H), 1.28-1.24 (mm, 36H), 0.86 (t, 3H, J )
6.3 Hz). Anal. (C34H61NO3) C, H, N.
Experimental Section
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 in a
Perkin-Elmer elemental apparatus Mod. 240 for C, H, N and
the data are within (0.4% of the theoretical values. 1H NMR
spectra were recorded at 25 °C on a Brucker AC200F employ-
ing TMS as internal standard, while NOESY experiments have
been performed with a Varian VRX 300 MHz and chemical
shifts are expressed as δ (ppm). Mass spectral data were
determined by direct insertion at 70 eV with a VG70 spec-
trometer. 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
columns 12.25 mm, packed with KP-Sil, 60A, 32-63 µM.
Reagents were purchased from Sigma Aldrich Srl (Italy) and
used as received unless otherwise stated.
General Method for Esters. A mixture of phenolic com-
pound (5.0 mmol), anhydrous potassium carbonate (2.5 mmol),
and potassium fluoride (5.0 mmol) in dry acetone was refluxed
under nitrogen atmosphere and continuously stirred for half
hour, and then a solution of the corresponding bromoalkyl-
methyl ester (5.0 mmol) in dry acetone was added, refluxing
for another 48-72 h and checking the reaction by TLC.
Afterward the reaction mixture was concentrated, diluted with
water, and extracted with chloroform. The extracts were
collected, dried, and evaporated under reduced pressure, and
the residue was purified by column chromatography on silica
gel. Yields, melting points, and chemical and spectroscopic data
of all esters are available in the Supporting Information.
General Methods for the Final Products. Method A.
Each ester (1.0 mmol) was refluxed in a methanolic/aqueous
sodium hydroxide solution (0.1 M, 3 equiv) for 3 h. Then the
reaction mixture was allowed to get room temperature, made
acidic (pH 3-4) by adding diluted HCl, and finally extracted
with ethyl acetate. The organic layer was dried and the solvent
evaporated to yield the crude acid, which was thoroughly dried
under vacuum before being subjected to the successive reaction
without further manipulation. To a mixture of the crude acid
(1.0 mmol), the appropriate amine (1.5 mmol), and 1-hydroxy-
benzotriazole (HOBt, 1.2 mmol) in dry dichloromethane or in
dry acetonitrile, kept in an ice bath, was added dropwise a
solution, in the same solvent, of 1-cyclohexyl-3-(2-morpholi-
noethyl)carbodiimide methyl p-toluensulfonate (CMC, 1.5
mmol) under nitrogen atmosphere and continuous stirring.
The reaction mixture was left to room temperature, and
stirring was continued 24 h. The solvent was removed under
reduced pressure, and the residue was diluted with chloroform.
The organic layer was washed with 5% aqueous NaHCO3 and
then with 1 N HCl and dried. After drying and evaporation of
the solvent, a crude residue was obtained and purified by
column chromatography on silica gel.
Method B. Each ester (0.150 g) was dissolved, under
nitrogen atmosphere and stirring, in ethanolamine (4 mL) and
warmed at 120-130 °C for 4-6 h. The resulting mixture was
diluted with water and extracted with chloroform, the extracts
were washed with a solution of ammonium chloride, dried,
evaporated, and the raw material was purified by column
chromatography on silica gel.
6-(3-Pentadecylphenoxy)hexanoic Acid (2-Hydroxy-
ethyl)amide (15).21 White solid (CHCl3/MeOH ) 47/3) (80%
yield): mp 62.7 °C (M). 1H NMR (CDCl3) δ (ppm): 7.14-7.10
(m, 1H), 6.75-6.66 (m, 3H), 5.95 (s br, 1H), 3.93 (t, 2H, J )
6.3 Hz), 3.70 (t, 2H, J ) 4.9 Hz), 3.44-3.36 (m, 2H), 2.65 (s
br, 1H), 2.54 (t, 2H, J ) 7.7 Hz), 2.22 (t, 2H, J ) 7.3 Hz), 1.81-
1.46 (mm, 8H), 1.44-1.24 (mm, 24H) 0.86 (t, 3H, J ) 6.2 Hz).
Anal. (C29H51NO3) C, H, N.
11-(3-Pentadecylphenoxy)undecanoic Acid Cyclopro-
pylamide (19). White needles (CHCl3, recrystallized from
acetone) (91% yield): mp 82.0 °C (K). 1H NMR (CDCl3) δ
(ppm): 7.15 (t, 1H, J ) 8.1 Hz), 6.75-6.67 (m, 3H), 5.46 (s br,
1H), 3.92 (t, 2H, J ) 6.5 Hz), 2.71-2.64 (m, 1H), 2.55 (t, 2H,
J ) 7.6 Hz), 2.09 (t, 2H, J ) 7.5 Hz), 1.78-1.41 (mm, 10H),
1.27-1.24 (mm, 32H), 0.86 (t, 3H, J ) 6.2 Hz), 0.80-0.70 (m,
2H), 0.55-0.46 (m, 2H). MS m/z: 1077 [2M + Na]+ (100). Anal.
(C35H61NO2) C, H, N.
11-(3-Pentadecylphenoxy)undecanoic Acid (4-Hydroxy-
phenyl)amide (20). White solid (CHCl3/MeOH ) 47/3) (86%
yield): mp 107.0 °C (M). 1H NMR (CDCl3) δ (ppm): 7.33 (half
of ABq, 2H, J ) 7.5 Hz), 7.21-7.12 (m, 2H), 7.01 (s br, 1H),
6.97-6.54 (m, 4H), 3.93 (t, 2H, J ) 6.5 Hz), 2.56 (t, 2H, J )
7.7 Hz), 2.35 (t, 2H, J ) 7.4 Hz), 1.79-1.60 (m, 8H), 1.30-
1.22 (mm, 34H), 0.87 (t, 3H, J ) 6.6 Hz). MS: 579 [M]+, 1159
[2M + 1]+ (100). Anal. (C38H61NO3) C, H, N.
6-(3-Hydroxy-5-pentylphenoxy)hexanoic Acid (2-Hy-
droxyethyl)amide (21). Pink oil (ethyl acetate/MeOH ) 46/
4) (65% yield). 1H NMR (Acetone-d6) δ (ppm): 8.43 (s, 1H),
7.33 (s br, 1H), 6.25-6.21 (m, 3H), 4.18 (s br, 1H), 3.86 (t, 2H,
J ) 6.3 Hz), 3.60-3.57 (m, 2H), 3.37-3.28 (m, 2H), 2.44 (t,
2H, J ) 7.6 Hz), 2.22 (t, 2H, J ) 7.3 Hz), 1.70-1.29 (mm, 12H),
0.85 (t, 3H, J ) 6.5 Hz). Anal. (C19H31NO4) C, H, N.
6-(3-Hydroxy-5-pentylphenoxy)hexanoic Acid Cyclo-
propylamide (22). Pale yellow oil (CHCl3/MeOH ) 48/2)
1
(60%). H NMR (CDCl3) δ (ppm): 7.04 (s br, 1H), 6.29-6.24
(m, 3H), 5.87 (s br, 1H), 3.86 (t, 2H, J ) 6.2 Hz), 2.74-2.65
(m, 1H), 2.46 (t, 2H, J ) 7.6 Hz), 2.14 (t, 2H, J ) 7.3 Hz),
1.75-1.24 (mm, 12H), 0.86 (t, 3H, J ) 6.5 Hz), 0.78-0.69 (m,
2H), 0.56-0.47 (m, 2H). Anal. (C20H31NO3) C, H, N.
6-(3-Hydroxy-5-pentylphenoxy)hexanoic Acid (4-Hy-
droxyphenyl)amide (23). White solid (CHCl3/MeOH ) 45/
5) (75% yield): mp 69.2 °C (M). 1H NMR (CDCl3) δ (ppm): 7.33
(half of ABq, 2H, J ) 8.7 Hz), 7.02 (s br, 1H), 6.80 (half of ABq,
2H, J ) 8.7 Hz), 6.29-6.21 (m, 3H), 4.95 (s br, 1H), 3.92 (t,
2H, J ) 6.2 Hz), 2.49 (t, 2H, J ) 7.6 Hz), 2.36 (t, 2H, J ) 7.2
Hz), 1.83-1.76 (m, 4H), 1.61-1.53 (m, 4H), 1.33-1.25 (mm,
4H), 0.88 (t, 3H, J ) 6.5 Hz). MS m/z: 386 [M + 1]+ (100), 771
[2M + 1]+, 793 [2M + Na]+. Anal. (C23H31NO4) C, H, N.
11-(3-Hydroxy-5-pentylphenoxy)undecanoic Acid (2-
Hydroxyethyl)amide (24). Pasty pink solid (ethyl acetate/
MeOH ) 44/6) (70% yield): mp 55-56 °C (K). 1H NMR
(DMSO) δ (ppm): 9.34 (s, 1H), 7.68 (s br, 1H), 6.13-6.09 (m,
3H), 4.57 (t, 1H, J ) 5.3 Hz), 3.82 (t, 2H, J ) 6.3 Hz), 3.39-
3.30 (m, 2H), 3.11-3.03 (m, 2H), 2.39 (t, 2H, J ) 7.5 Hz), 2.02
(t, 2H, J ) 7.2 Hz), 1.63-1.45 (mm, 6H), 1.41-1.23 (mm, 16H),
0.83 (t, 3H, J ) 6.4 Hz). Anal. (C24H41NO4) C, H, N.
6-(3-Pentadecylphenoxy)hexanoic Acid Cyclopropyl-
11-(3-Hydroxy-5-pentylphenoxy)undecanoic Acid Cy-
amide (16). White solid (CHCl3, recrystallized from acetone/
clopropylamide (25). White solid (CHCl3/MeOH ) 47/3) (61%
1
1
ethyl ether) (50% yield): mp 67.8 °C (M). H NMR (CDCl3) δ
yield): mp 70.7 °C (M). H NMR (CDCl3) δ (ppm): 6.98 (s br,
(ppm): 6.65 (t, 1H, J ) 7.7 Hz), 6.50 (s br, 1H), 6.26-6.17 (m,
3H), 3.46 (t, 2H, J ) 6.3 Hz), 2.28-2.18 (m, 1H), 2.08 (t, 2H,
1H), 6.28-6.26 (m, 3H), 5.79 (s br, 1H), 3.88 (t, 2H, J ) 6.3
Hz), 2.72-2.67 (m, 1H), 2.47 (t, 2H, J ) 7.6 Hz), 2.12 (t, 2H,

7348 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
Brizzi et al.
J ) 7.5 Hz), 1.75-1.49 (mm, 8H), 1.28-1.26 (mm, 14H), 0.87
(t, 3H, J ) 6.4 Hz), 0.79-0.70 (m, 2H), 0.55-0.43 (m, 2H).
Anal. (C25H41NO3) C, H, N.
J ) 6.3 Hz), 2.76-2.66 (m, 1H), 2.11 (t, 2H, J ) 7.2 Hz), 1.79-
1.60 (mm, 4H), 1.46-1.27 (mm, 12H), 0.80-0.70 (m, 2H),
0.48-0.42 (m, 2H). MS m/z: 334 [M + 1]+, 667 [2M + 1]+ (100),
689 [2M + Na]+. Anal. (C20H31NO3) C, H, N.
11-(3-Hydroxy-5-pentylphenoxy)undecanoic Acid (4-
Hydroxyphenyl)amide (26). Pasty white solid (CHCl3/
MeOH ) 47/3) (65% yield). 1H NMR (CDCl3) δ (ppm): 7.28
(half of ABq, 2H, J ) 9.0 Hz), 7.15 (s br, 1H), 6.74 (half of ABq,
2H, J ) 8.8 Hz), 6.29-6.25 (m, 3H), 3.90 (t, 2H, J ) 6.4 Hz),
2.50 (t, 2H, J ) 7.6 Hz), 2.32 (t, 2H, J ) 7.4 Hz), 1.73-1.53
(m, 8H), 1.33-1.29 (m, 14H), 0.87 (t, 3H, J ) 6.6 Hz). MS
m/z: 456 [M + 1]+ (100), 911 [2M + 1]+, 933 [2M + Na]+. Anal.
(C28H41NO4) C, H, N.
11-(3-Hydroxyphenoxy)undecanoic Acid (4-Hydroxy-
phenyl)amide (35). White solid (CHCl3/MeOH ) 45/5) (79%
1
yield): mp 121.2 °C (M). H NMR (DMSO) δ (ppm): 9.50 (s,
1H), 9.26 (s, 1H), 9.05 (s, 1H), 7.30 (half of ABq, 2H, J ) 8.7
Hz), 6.98 (t, 1H, J ) 8.0 Hz), 6.63 (half of ABq, 2H, J ) 8.6
Hz), 6.31-6.26 (m, 3H), 3.83 (t, 2H, J ) 6.4 Hz), 2.19 (t, 2H,
J ) 7.3 Hz), 1.62-1.56 (m, 4H), 1.53-1.24 (mm, 12H). Anal.
(C23H31NO4) C, H, N.
16-(3-Hydroxy-5-pentylphenoxy)hexadecanoic Acid (2-
Hydroxyethyl)amide (27). White solid (ethyl acetate/MeOH
) 50/5; recrystallized from ethyl acetate) (57% yield): mp 84-
16-(3-Hydroxyphenoxy)hexadecanoic Acid (2-Hydroxy-
ethyl)amide (36). White solid (Ethyl acetate/MeOH ) 50/1)
(35% yield): mp 94.5 °C (M). 1H NMR (DMSO) δ (ppm): 9.51
(s, 1H), 7.91 (s br, 1H), 7.44 (t, 1H, J ) 8.2 Hz), 6.81-6.75 (m,
3H), 4.80 (t, 1H, J ) 5.5 Hz), 4.33 (t, 2H, J ) 6.4 Hz), 3.94-
3.85 (m, 2H), 3.66-3.57 (m, 2H), 2.54 (t, 2H, J ) 7.0 Hz), 2.18-
2.10 (m, 4H), 2.00-1.70 (mm, 22H). Anal. (C24H41NO4) C, H,
N.
16-(3-Hydroxyphenoxy)hexadecanoic Acid Cyclopro-
pylamide (37). White bright solid (Ethyl acetate/MeOH ) 50/
4; recrystallized Ethyl acetate) (78% yield): mp 105.6 °C (M).
1H NMR (DMSO) δ (ppm): 9.25 (s, 1H), 7.73 (s br, 1H), 6.98
(t, 1H, J ) 7.9 Hz), 6.31-6.26 (m, 3H), 3.83 (t, 2H, J ) 6.4
Hz), 2.58-2.51 (m, 1H), 1.94 (t, 2H, J ) 7.3 Hz), 1.64-1.59
(m, 2H), 1.41-1.20 (mm, 24H), 0.58-0.49 (m, 2H), 0.40-0.26
(m, 2H). Anal. (C25H41NO3) C, H, N.
16-(3-Hydroxyphenoxy)hexadecanoic Acid (4-Hydroxy-
phenyl)amide (38). White solid (CHCl3/MeOH ) 50/5) (71.5%
yield): 123.6 °C (M). 1H NMR (DMSO) δ (ppm): 9.57 (s, 1H),
9.32 (s,1H), 9.05 (s, 1H), 7.36 (half of ABq, 2H, J ) 8.6 Hz),
7.04 (t, 1H, J ) 7.9 Hz), 6.69 (half of ABq, 2H, J ) 8.6 Hz),
6.37-6.32 (m, 3H), 3.89 (t, 2H, J ) 6.3 Hz), 2.24 (t, 2H, J )
7.3 Hz), 1.69-1.57 (m, 4H), 1.50-1.27 (mm, 22H). Anal.
(C28H41NO4) C, H, N.
6-(3-Hydroxy-4-hexylphenoxy)hexanoic Acid (2-Hy-
droxyethyl)amide (39). Pale yellow solid (CHCl3/MeOH )
46/4) (55% yield): mp 71.2 °C (M). 1H NMR (Acetone-d6) δ
(ppm): 8.42 (s, 1H), 7.42 (s br, 1H), 6.93 (d, 1H, J ) 8.0 Hz),
6.45 (d, 1H, J ) 2.0 Hz), 6.32 (dd, 1H, J ) 2.0 Hz, J ) 8.0 Hz),
4.30 (t, 1H, J ) 5.2 Hz), 3.87 (t, 2H, J ) 6.3 Hz), 3.67-3.59
(m, 2H), 3.40-3.31 (m, 2H), 2.54 (t, 2H, J ) 7.5 Hz), 2.27 (t,
2H, J ) 7.3 Hz), 1.76-1.65 (m, 4H), 1.61-1.42 (mm, 4H),
1.32-1.30 (m, 6H), 0.88 (t, 3H, J ) 6.1 Hz). MS m/z: 352 [M
+ 1]+, 374 [M + Na]+ (100), 725 [2M + Na]+. Anal. (C20H33-
NO4) C, H, N.
6-(3-Hydroxy-4-hexylphenoxy)hexanoic Acid Cyclo-
propylamide (40). White bright solid (Ethyl acetate; recrys-
tallized from n.hexane) (70% yield): mp 134.2 °C (M). 1H NMR
300 MHz (DMSO) δ (ppm): 9.11 (s, 1H), 7.80 (d, 1H, J ) 3.6
Hz)), 6.85 (d, 1H, J ) 8.4 Hz), 6.31 (d, 1H, J ) 2.4 Hz), 6.24
(dd, 1H, J ) 2.4 Hz, J ) 8.4 Hz), 3.80 (t, 2H, J ) 6.6 Hz),
2.61-2.53 (m, 1H), 2.42 (t, 2H, J ) 7.3 Hz), 2.00 (t, 2H, J )
7.1 Hz), 1.68-1.60 (m, 2H), 1.55-1.41 (m, 4H), 1.37-1.23 (mm,
8H), 0.83 (t, 3H, J ) 6.6 Hz), 0.60-0.53 (m, 2H), 0.36-0.31
(m, 2H). MS m/z: 348 [M + 1]+ (100), 695 [2M + 1]+. Anal.
(C21H33NO3) C, H, N.
6-(3-Hydroxy-4-hexylphenoxy)hexanoic Acid (4-Hy-
droxyphenyl)amide (41). White solid (CHCl3/MeOH ) 47/
3) (79.5% yield): mp 128.4 °C. 1H NMR (Acetone-d6) δ (ppm):
8.82 (s, 1H), 8.00 (s br, 1H), 7.43 (half of ABq, 2H, J ) 8.6 Hz),
6.92 (half of ABq, 2H, J ) 8.6 Hz), 6.73 (d, 1H, J ) 8.8 Hz),
6.40 (d, 1H, J ) 2.7 Hz), 6.31 (dd, 1H, J ) 2.5 Hz, J ) 8.7 Hz),
3.87 (t, 2H, J ) 6.4 Hz), 2.51 (t, 2H, J ) 7.4 Hz), 2.32 (t, 2H,
J ) 7.2 Hz), 1.77-1.64 (m, 6H), 1.55-1.44 (m, 4H), 1.40-1.27
(m, 4H), 0.85 (t, 3H, J ) 6.6 Hz). Anal. (C24H33NO4) C, H, N.
11-(3-Hydroxy-4-hexylphenoxy)undecanoic Acid (2-
Hydroxyethyl)amide (42). White solid (Ethyl acetate/MeOH
) 50/4) (43% yield): mp 85.3 °C (M). 1H NMR (CDCl3) δ
(ppm): 6.96 (d, 1H, J ) 9.0 Hz), 6.40-6.35 (m, 2H), 5.96 (s br,
1H), 3.89 (t, 2H, J ) 6.2 Hz), 3.73 (t, 2H, J ) 5.0 Hz), 3.46-
3.39 (m, 2H), 2.51 (t, 2H, J ) 7.6 Hz), 2.21 (t, 2H, J ) 7.5 Hz),
1.76-1.52 (mm, 8H), 1.45-1.22 (mm, 16H), 0.87 (t, 3H, J )
1
85 °C (K). H NMR (DMSO) δ (ppm): 9.17 (s, 1H), 7.72 (s br,
1H), 6.17-6.13 (m, 3H), 4.61 (t, 1H, J ) 5.1 Hz), 3.86 (t, 2H,
J ) 6.1 Hz), 3.40-3.33 (m, 2H), 3.15-3.09 (m, 2H), 2.42 (t,
2H, J ) 7.3 Hz), 2.06 (t, 2H, J ) 7.2 Hz), 1.66-1.52 (mm, 8H),
1.48-1.25 (mm, 24H), 0.87 (t, 3H, J ) 6.3 Hz). Anal. (C29H51-
NO4) C, H, N.
16-(3-Hydroxy-5-pentylphenoxy)hexadecanoic Acid Cy-
clopropylamide (28). White solid (CHCl3/MeOH ) 48/2)
(88.5% yield): mp 91.0 °C (M). 1H NMR (CDCl3) δ (ppm):
6.29-6.24 (m, 3H), 5.52 (s br, 1H), 3.89 (t, 2H, J ) 6.4 Hz),
2.72-2.67 (m, 1H), 2.48 (t, 2H, J ) 7.6 Hz), 2.11 (t, 2H, J )
7.5 Hz), 1.76-1.66 (mm, 8H), 1.57-1.24 (mm, 24H), 0.87 (t,
3H, J ) 6.5 Hz), 0.80-0.71 (m, 2H), 0.55-0.42 (m, 2H). Anal.
(C30H51NO3) C, H, N.
16-(3-Hydroxy-5-pentylphenoxy)hexadecanoic Acid (4-
Hydroxyphenyl)amide (29). White solid (CHCl3/MeOH )
46/4) (83.0% yield): mp 89.5 °C (M). 1H NMR (DMSO) δ
(ppm): 9.50 (s, 1H), 9.30 (s, 1H), 9.12 (s br, 1H), 7.29 (half of
ABq, 2H, J ) 8.4 Hz), 6.63 (half of ABq, 2H, J ) 8.6 Hz), 6.12-
6.07 (m, 3H), 3.80 (t, 2H, J ) 6.3 Hz), 2.37 (t, 2H, J ) 7.4 Hz),
2.14 (t, 2H, J ) 7.6 Hz), 1.70-1.15 (mm, 32H), 0.81 (t, 3H, J
) 6.4 Hz). Anal. (C33H51NO4) C, H, N.
6-(3-Hydroxyphenoxy)hexanoic Acid (2-Hydroxyethyl)-
amide (30). Pale pink oil (ethyl acetate/MeOH ) 50/2) (50.0%
1
yield). H NMR (CDCl3) δ (ppm): 8.70 (s br, 1H), 7.41 (s br,
1H), 7.02 (t, 1H, J ) 8.3 Hz), 6.97-6.33 (m, 3H), 4.31 (s br,
1H), 3.87 (t, 2H, J ) 6.3 Hz), 3.62-3.57 (m, 2H), 3.35-3.27
(m, 2H), 2.23 (t, 2H, J ) 7.2 Hz), 1.74-1.58 (m, 4H), 1.50-
1.43 (m, 2H). MS m/z: 268 [M + 1]+ (100), 290 [M + Na]+.
Anal. (C14H21NO4) C, H, N.
6-(3-Hydroxyphenoxy)hexanoic Acid Cyclopropylam-
ide (31). White needles (CHCl3/MeOH ) 47/3) (86% yield):
1
102.5 °C (M). H NMR (CDCl3) δ (ppm): 7.12-7.04 (m, 1H),
6.43-6.40 (m, 3H), 6.11 (s, 1H), 5.63 (s br, 1H), 3.90 (t, 2H, J
) 6.1 Hz), 2.74-2.67 (m, 1H), 2.14 (t, 2H, J ) 7.3 Hz), 1.82-
1.62 (mm, 4H), 1.55-1.37 (m, 2H), 0.80-0.70 (m, 2H), 0.50-
0.47 (m, 2H). MS: 264 [M + 1]+, 527 [2M + 1]+, 549 [2M +
Na]+ (100). Anal. (C15H21NO3) C, H, N.
6-(3-Hydroxyphenoxy)hexanoic Acid (4-Hydroxyphen-
yl)amide (32). Pale pink solid (CHCl3/MeOH ) 45/5; recrys-
tallized from ethyl acetate) (88.0% yield): 123.1 °C (M). 1H
NMR (DMSO) δ (ppm): 9.53 (s, 1H), 9.26 (s, 1H), 9.05 (s, 1H),
7.31 (half of ABq, 2H, J ) 8.8 Hz), 6.95 (t, 1H, J ) 8.1 Hz),
6.64 (half of ABq, 2H, J ) 8.7 Hz), 6.31-6.27 (m, 3H), 3.85 (t,
2H, J ) 6.3 Hz), 2.23 (t, 2H, J ) 7.2 Hz), 1.74-1.53 (m, 4H),
1.46-1.36 (m, 2H). Anal. (C18H21NO4) C, H, N.
11-(3-Hydroxyphenoxy)undecanoic Acid (2-Hydroxy-
ethyl)amide (33). Cream solid (Ethyl acetate/MeOH ) 50/4)
1
(34% yield): mp 72.1 °C (M). H NMR (CDCl3) δ (ppm): 7.10
(t, 1H, J ) 8.0 Hz), 6.44-6.38 (m, 3H), 6.15 (s br, 1H), 5.96 (s
br, 1H), 3.92 (t, 2H, J ) 6.2 Hz), 3.74-3.70 (m, 2H), 3.46-
3.38 (m, 2H), 2.58 (s br, 1H), 2.19 (t, 2H, J ) 7.5 Hz), 2.02-
1.56 (mm, 6H), 1.43-1.24 (mm, 10H). MS m/z: 338 [M + 1]+,
360 [M + Na]+, 697 [2M + Na]+ (100). Anal. (C19H31NO4) C,
H, N.
11-(3-Hydroxyphenoxy)undecanoic Acid Cyclopropil-
amide (34). White solid (Ethyl acetate) (90% yield): mp 92.8
1
°C (M). H NMR (CDCl3) δ (ppm): 7.08 (t, 1H, J ) 8.2 Hz),
6.50 (s br, 1H), 6.47-6.39 (m, 3H), 5.60 (s br, 1H), 3.91 (t, 2H,

Potent Ligands of Cannabinoid Receptors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23 7349
6.4 Hz). MS m/z: 422 [M + 1]+, 444 [M + Na]+ (100). Anal.
(C25H43NO4) C, H, N.
(ppm): 9.52 (s, 1H), 9.04 (s, 1H), 9.02 (s, 1H), 7.30 (half of ABq,
2H, J ) 8.8 Hz), 6.80 (d, 1H, J ) 8.1 Hz), 6.62 (half of ABq,
2H, J ) 8.7 Hz), 6.28 (s, 1H), 6.20 (d, 1H, J ) 8.0 Hz), 3.83 (t,
2H, J ) 6.0 Hz), 2.36 (t, 2H, J ) 7.3 Hz), 2.23 (t, 2H, J ) 7.1
Hz), 1.70-1.57 (mm, 10H), 1.53-1.20 (m, 4H), 0.81 (t, 3H, J
) 6.7 Hz). Anal. (C24H33NO4) C, H, N.
11-(2-Hexyl-5-hydroxyphenoxy)undecanoic Acid (2-
Hydroxyethyl)amide (51). Pale yellow solid (Ethyl acetate/
MeOH ) 50/4) (25% yield): mp 75.4 °C (M). 1H NMR (CDCl3)
δ (ppm): 6.90 (d, 1H, J ) 7.9 Hz), 6.38-6.36 (d, 1H, J ) 2.0
Hz), 6.31 (dd, 1H, J ) 7.9 Hz, J ) 2.0 Hz), 6.08 (s br, 1H),
3.87 (t, 2H, J ) 6.3 Hz), 3.70 (t, 2H, J ) 4.9 Hz), 3.43-3.36
(m, 2H), 2.48 (t, 2H, J ) 7.5 Hz), 2.17 (t, 2H, J ) 7.6 Hz),
1.81-1.68 (m, 2H), 1.63-1.43 (mm, 4H), 1.40-1.27 (mm, 18H),
0.86 (t, 3H, J ) 6.3 Hz). MS m/z: 422 [M + 1]+ (100), 444 [M
+ Na]+, 843 [2M + 1]+. Anal. (C25H43NO4) C, H, N.
11-(3-Hydroxy-4-hexylphenoxy)undecanoic Acid Cy-
clopropylamide (43). White bright solid (Ethyl acetate;
recrystallized from ethyl acetate/n.hexane) (81% yield): 112.3
°C (M). 1H NMR 300 MHz (DMSO) δ (ppm): 9.10 (s, 1H), 7.77
(s br, 1H), 6.85 (d, 1H, J ) 8.4 Hz), 6.30 (d, 1H, J ) 2.4 Hz),
6.24 (dd, 1H, J ) 2.4 Hz, J ) 8.0 Hz), 3.81 (t, 2H, J ) 6.4 Hz),
2.59-2.53 (m, 1H), 2.39 (t, 2H, J ) 7.4 Hz), 1.96 (t, 2H, J )
7.4 Hz), 1.65-1.58 (m, 2H), 1.46-1.38 (m, 4H), 1.34-1.23 (mm,
18H), 0.83 (t, 3H, J ) 6.4 Hz), 0.57-0.53 (m, 2H), 0.34-0.31
(m, 2H). MS m/z: 418 [M + 1]+, 440 [M + Na]+ (100). Anal.
(C26H43NO3) C, H, N.
11-(3-Hydroxy-4-hexylphenoxy)undecanoic Acid (4-
Hydroxyphenyl)amide (44). White solid (CHCl3/MeOH )
48/2) (88.0% yield): mp 121.2 °C (M). 1H NMR (DMSO) δ
(ppm): 9.48 (s, 1H), 9.04 (s, 1H), 9.02 (s, 1H), 7.30 (half of ABq,
2H, J ) 8.8 Hz), 6.84 (d, 1H, J ) 8.1 Hz), 6.63 (half of ABq,
2H, J ) 8.8 Hz), 6.29 (d, 1H, J ) 2.1 Hz), 6.23 (dd, 1H, J )
2.3 Hz, J ) 8.2 Hz), 3.80 (t, 2H, J ) 6.4 Hz), 2.38 (t, 2H, J )
7.4 Hz), 2.20 (t, 2H, J ) 7.4 Hz), 1.65-1.40 (mm, 10H), 1.37-
1.22 (mm, 14H), 0.82 (t, 3H, CH3, J ) 6.4 Hz). Anal. (C29H43-
NO4) C, H, N.
11-(2-Hexyl-5-hydroxyphenoxy)undecanoic Acid Cy-
clopropylamide (52). White solid (CHCl3/MeOH ) 47/3) (90%
1
yield): mp 57.3 °C (M). H NMR 300 MHz (DMSO) δ (ppm):
9.05 (s, 1H), 7.76 (s br, 1H), 6.81 (d, 1H, J ) 7.7 Hz), 6.29 (s,
1H), 6.20 (d, 1H, J ) 8.1 Hz), 3.83 (t, 2H, J ) 5.5 Hz), 2.58-
2.54 (m, 1H), 2.38 (t, 2H, J ) 7.3 Hz), 1.95 (t, 2H, J ) 6.9 Hz),
1.69-1.64 (m, 2H), 1.43-1.38 (m, 4H), 1.26-1.14 (mm, 18H),
0.82 (t, 3H, J ) 6.1 Hz), 0.57-0.53 (m, 2H), 0.33-0.31 (m, 2H).
MS m/z: 418 [M + 1]+, 440 [M + Na]+ (100). Anal. (C26H43-
NO3) C, H, N.
16-(3-Hydroxy-4-hexylphenoxy)hexadecanoic Acid (2-
Hydroxyethyl)amide (45). White solid (Ethyl acetate/MeOH
1
) 50/1) (50% yield): mp 96.4 °C. H NMR (DMSO) δ (ppm):
9.14 (s, 1H), 7.73 (s br, 1H), 6.88 (d, 1H, J ) 8.3 Hz), 6.34 (d,
1H, J ) 1.7 Hz), 6.27 (dd, 1H, J ) 8.3 Hz, J ) 1.7 Hz), 4.63 (t,
1H, J ) 5.2 Hz), 3.84 (t, 2H, J ) 6.2 Hz), 3.57-3.40 (m, 2H),
3.15-3.06 (m, 2H), 2.43 (t, 2H, J ) 7.2 Hz), 2.06 (t, 2H, J )
7.3 Hz), 1.70-1.48 (mm, 8H), 1.44-1.25 (mm, 26H), 0.87 (t,
3H, J ) 5.8 Hz). Anal. (C30H53NO4) C, H, N.
11-(2-Hexyl-5-hydroxyphenoxy)undecanoic Acid (4-
Hydroxyphenyl)amide (53). White solid (CHCl3/MeOH )
47/3; recrystallized diethyl ether) (83% yield): mp 108.7 °C
(M). 1H NMR (Acetone-d6) δ (ppm): 9.05 (s, 1H), 9.00 (s, 1H),
8.80 (s, 1H), 7.43 (half of ABq, 2H, J ) 8.8 Hz), 6.86 (d, 1H, J
) 7.9 Hz), 6.73 (half of ABq, 2H, J ) 8.8 Hz), 6.40 (d, 1H, J )
2.5 Hz), 6.30 (dd, 1H, J ) 2.5 Hz, J ) 7.9 Hz), 3.90 (t, 2H, J
) 6.1 Hz), 2.48 (t, 2H, J ) 7.4 Hz), 2.28 (t, 2H, J ) 7.2 Hz),
1.80-1.60 (mm, 14H), 1.50-1.32 (mm, 10H), 0.86 (t, 3H, J )
6.3 Hz). Anal. (C29H43NO4) C, H, N.
16-(2-Hexyl-5-hydroxyphenoxy)hexadecanoic Acid (2-
Hydroxyethyl)amide (54). White solid (Ethyl acetate/MeOH
) 50/1) (60% yield): mp 77.2 °C (M). 1H NMR (DMSO) δ
(ppm): 9.00 (s, 1H), 7.64 (s br, 1H), 6.80 (d, 1H, J ) 8.0 Hz),
6.28 (d, 1H, J ) 1.7 Hz), 6.20 (dd, 1H, J ) 8.0 Hz, J ) 1.7 Hz),
4.53 (t, 1H, J ) 5.2 Hz), 3.83 (t, 2H, J ) 5.8 Hz), 3.40-3.33
(m, 2H), 3.10-3.05 (m, 2H), 2.38 (t, 2H, J ) 7.3 Hz), 2.00 (t,
2H, J ) 7.4 Hz), 1.66-1.63 (m, 2H), 1.41-1.15 (mm, 32H),
0.82 (t, 3H, J ) 5.9 Hz). Anal. (C30H53NO4) C, H, N.
16-(3-Hydroxy-4-hexylphenoxy)hexadecanoic Acid Cy-
clopropylamide (46). White bright solid (Ethyl acetate/
MeOH ) 50/2; recrystallized from ethyl acetate) (94.5%
1
yield): mp 114.3 °C (M). H NMR (DMSO) δ (ppm): 9.15 (s,
1H), 7.80 (s br, 1H), 6.82 (d, 1H, J ) 8.1 Hz), 6.34 (d, 1H, J )
2.6 Hz), 6.20 (dd, 1H, J ) 2.4 Hz, J ) 8.3 Hz), 3.79 (t, 2H, J
) 6.3 Hz), 2.55-2.48 (m, 1H), 2.37 (t, 2H, J ) 7.4 Hz), 1.94 (t,
2H, J ) 7.3 Hz), 1.62-1.55 (m, 4H), 1.41-1.20 (mm, 30H),
0.82 (t, 3H, J ) 6.0 Hz), 0.58-0.49 (m, 2H), 0.34-0.27 (m, 2H).
Anal. (C31H53NO3) C, H, N.
16-(3-Hydroxy-4-hexylphenoxy)hexadecanoic Acid (4-
Hydroxyphenyl)amide (47). White solid (CHCl3/MeOH )
45/5) (81% yield): mp 133.2 °C (M). 1H NMR (DMSO) δ
(ppm): 9.50 (s, 1H), 9.06 (s, 1H), 9.04 (s, 1H), 7.30 (half of ABq,
2H, J ) 8.5 Hz), 6.83 (d, 1H, J ) 8.0 Hz), 6.62 (half of ABq,
2H, J ) 8.5 Hz), 6.28 (s, 1H), 6.22 (d, 1H, J ) 8.1 Hz), 3.80 (t,
2H, J ) 6.2 Hz), 2.38 (t, 2H, J ) 7.3 Hz), 2.18 (t, 2H, J ) 7.1
Hz), 1.61-1.21 (mm, 34H), 0.81 (t, 3H, J ) 6.4 Hz). Anal.
(C34H53NO4) C, H, N.
6-(2-Hexyl-5-hydroxyphenoxy)hexanoic Acid (2-Hy-
droxyethyl)amide (48). Pale yellow thick oil (CHCl3/MeOH
) 46/4) (50% yield). 1H NMR (Acetone-d6) δ (ppm): 8.23 (s,
1H), 7.27 (s br, 1H), 6.88 (d, 1H, J ) 8.0 Hz), 6.42 (d, 1H, J )
2.0 Hz), 6.35-6.31 (m, 1H), 4.08 (s br, 1H), 3.91 (t, 2H, J )
6.3 Hz), 3.61-3.58 (m, 2H), 3.36-3.28 (m, 2H), 2.50 (t, 2H, J
) 7.4 Hz), 2.25 (t, 2H, J ) 7.2 Hz), 1.85-1.67 (mm, 4H), 1.63-
1.52 (m, 4H), 1.49-1.31 (mm, 6H), 0.88 (t, 3H, J ) 6.4 Hz).
MS m/z: 353 [M + D]+, 374 [M + Na]+, 725 [2M + Na]+ (100).
Anal. (C20H33NO4) C, H, N.
6-(2-Hexyl-5-hydroxyphenoxy)hexanoic Acid Cyclo-
propylamide (49). Pale yellow thick oil (Ethyl acetate) (80%
yield). 1H NMR 300 MHz (DMSO) δ (ppm): 9.06 (s, 1H), 7.80
(d, 1H, J ) 3.9 Hz)), 6.81 (d, 1H, J ) 8.1 Hz), 6.30 (d, 1H, J )
2.3 Hz), 6.21 (dd, 1H, J ) 2.2 Hz, J ) 8.1 Hz), 3.82 (t, 2H, J
) 6.2 Hz), 2.61-2.52 (m, 1H), 2.38 (t, 2H, J ) 7.1 Hz), 2.00 (t,
2H, J ) 7.1 Hz), 1.71-1.62 (m, 2H), 1.56-1.47 (m, 2H), 1.42-
1.34 (m, 4H), 1.27-1.23 (mm, 6H), 0.82 (t, 3H, J ) 6.6 Hz),
0.58-0.52 (m, 2H), 0.35-0.30 (m, 2H). MS m/z: 348 [M + 1]+
(100), 695 [2M + 1]+. Anal. (C21H33NO3) C, H, N.
16-(2-Hexyl-5-hydroxyphenoxy)hexadecanoic Acid Cy-
clopropylamide (55). White solid (CHCl3/MeOH ) 47/3) (80%
1
yield): mp 72.8 °C (M). H NMR 300 MHz (DMSO) δ (ppm):
9.05 (s, 1H), 7.75 (s br, 1H), 6.81 (d, 1H, J ) 8.1 Hz), 6.29 (d,
1H, J ) 2.4 Hz), 6.20 (dd, 1H, J ) 2.4 Hz, J ) 8.1 Hz), 3.83 (t,
2H, J ) 6.3 Hz), 2.56-2.51 (m, 1H), 2.38 (t, 2H, J ) 7.2 Hz),
1.95 (t, 2H, J ) 7.2 Hz), 1.69-1.62 (m, 2H), 1.42-1.20 (mm,
32H), 0.82 (t, 3H, J ) 6.3 Hz), 0.56-0.52 (m, 2H), 0.34-0.31
(m, 2H). Anal. (C31H53NO3) C, H, N.
16-(2-Hexyl-5-hydroxyphenoxy)hexadecanoic Acid (4-
Hydroxyphenyl)amide (56). White solid (CHCl3/MeOH )
46/4) (70% yield): mp 82.4 °C (M). 1H NMR (DMSO) δ (ppm):
9.45 (s, 1H), 9.05 (s, 1H), 9.02 (s, 1H), 7.30 (half of ABq, 2H, J
) 8.7 Hz), 6.91 (d, 1H, J ) 8.0 Hz), 6.75 (half of ABq, 2H, J )
8.7 Hz), 6.35 (d, 1H, J ) 2.2 Hz), 6.29 (dd, 1H, J ) 2.2 Hz, J
) 8.0 Hz), 3.88 (t, 2H, J ) 6.3 Hz), 2.40 (t, 2H, J ) 7.5 Hz),
2.33 (t, 2H, J ) 7.5 Hz), 1.80-1.62 (m, 4H), 1.58-1.25 (mm,
30H), 0.86 (t, 3H, J ) 6.5 Hz). Anal. (C34H53NO4) C, H, N.
Acknowledgment. We thank the Universita` degli
Studi di Siena, Italy, for financial support. Dr. Alessia
Ligresti and Mr. Paolo Cavallo for technical assistance
with the assays. Dr. Luisa Chiasserini for NOESY
spectra.
6-(2-Hexyl-5-hydroxyphenoxy)hexanoic Acid (4-Hy-
droxyphenyl)amide (50). White solid (CHCl3/MeOH ) 47/
3) (63% yield): mp 106.0 °C (M). 1H NMR (Acetone-d6) δ
Supporting Information Available: Yields, melting
points, and chemical and spectroscopic data of all synthe-

7350 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
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