High affinity electrophilic and photoactivatable covalent endocannabinoid probes for the CB1 receptor

Article abstract of DOI:10.1021/jm050272i

We have designed and synthesized the first two high affinity covalent anandamide probes for the CB1 receptor by introducing either an electrophilic isothiocyanato or a photoactivatable azido group at the terminal carbon of the arachidonic acid moiety. The headgroup of these anandamide analogues was optimized by using a cyclopropylamide substituent to impart optimal CB1 affinity. Both 20-isothiocyanato-eicosa-5,8,11,14-tetraenoic acid cyclopropylamide (1, AM3677) and 20-azido-eicosa-5,8,11,14-tetraenoic acid cyclopropylamide (2, AM3661) exhibited high selectivities for the CB1 receptor with K_i values of 1.3 and 0.9 nM, respectively. Using suitable experimental conditions, both ligands were shown to covalently label the CB1 receptor with high efficiency. These two covalent probes for the endocannabinoid CB1 binding site open the door for exploring the ligand binding motifs involved in the activation of the CB1 receptor by its endogenous ligand, anandamide.

Full text of DOI:10.1021/jm050272i

J. Med. Chem. 2005, 48, 6423-6429
6423
High Affinity Electrophilic and Photoactivatable Covalent Endocannabinoid
Probes for the CB1 Receptor
Chen Li, Wei Xu, Subramanian K. Vadivel, Pusheng Fan, and Alexandros Makriyannis*
Center for Drug Discovery, Northeastern University, 116 Mugar Hall, 360 Huntington Avenue, Boston, Massachusetts 02115
Received March 24, 2005
We have designed and synthesized the first two high affinity covalent anandamide probes for
the CB1 receptor by introducing either an electrophilic isothiocyanato or a photoactivatable
azido group at the terminal carbon of the arachidonic acid moiety. The headgroup of these
anandamide analogues was optimized by using a cyclopropylamide substituent to impart
optimal CB1 affinity. Both 20-isothiocyanato-eicosa-5,8,11,14-tetraenoic acid cyclopropylamide
(
1, AM3677) and 20-azido-eicosa-5,8,11,14-tetraenoic acid cyclopropylamide (2, AM3661)
exhibited high selectivities for the CB1 receptor with K values of 1.3 and 0.9 nM, respectively.
i
Using suitable experimental conditions, both ligands were shown to covalently label the CB1
receptor with high efficiency. These two covalent probes for the endocannabinoid CB1 binding
site open the door for exploring the ligand binding motifs involved in the activation of the CB1
receptor by its endogenous ligand, anandamide.
Introduction
is critical for understanding the structural requirements
involved in ligand-receptor binding and is of great
value in the design of novel, more selective, and more
potent drug candidates.
The main psychoactive ingredient of marijuana, (-)-
∆9
-tetrahydrocannabinol, produces most of its pharma-
cological effects by interacting with cannabinoid recep-
tors (CBs). To date, two subtypes of the CB receptor,
The structural analysis of GPCRs is severely limited
by the difficulties associated with their purification and
crystallization. An alternative method currently pur-
sued in our laboratory for obtaining such information
is based on the identification of key amino acid residues
1
-3
CB1 and CB2, have been identified,
both belonging
to the G-protein-coupled receptor (GPCR) superfamily,
which characteristically has seven transmembrane-
spanning helical domains connected by intervening
intracellular and extracellular loops. An important
development in cannabinoid research was the identifi-
cation of N-arachidonoylethanolamine (anandamide,
AEA) as an endogenous cannabinoid that was shown
to possess biochemical and pharmacological profiles
18
associated with the receptor’s binding domain. This
experimental approach involves the development of high
affinity selective ligands capable of forming a covalent
bond with an amino acid residue within the receptor
binding site or immediately adjacent to it. The identity
of the labeled amino acid residue is then revealed
through purification of the covalently linked ligand-
receptor complex, followed by digestion of the entire
protein, and analysis of the individual peptide frag-
ments using sequencing or mass spectral analysis.
4-7
similar to those of cannabinoid agonists.
The endog-
8
enous cannabinoid system is primarily responsible for
all of the effects mediated by cannabinoids and is
involved in the regulation of a wide variety of physio-
logical functions such as antinociception, brain develop-
ment, memory, retrograde neuronal communication,
control of movement, cardiovascular and immune regu-
lation, and cellular proliferation. Small molecules
capable of modulating the endocannabinoid system can
be of potential value as therapeutic agents for the
treatment of diverse pathologies, including neurode-
generative disorders, nociceptive conditions, obesity, and
malignant tumors.
The structure-activity relationship (SAR) of anan-
damide has been explored through the design and
synthesis of a considerable number of anandamide
analogues that helped to establish structural require-
Receptor covalent probes can be designed by incor-
porating potentially reactive groups into high affinity
reversible prototypic ligands followed by suitable struc-
tural modifications, if necessary, to ensure that the
novel ligands maintain a high affinity for the binding
site. Over the past decade, our laboratory has been
successful in designing a number of such covalent
9
1
0
18-21
probes, both photoactivatable and electrophilic.
Photoactivatable ligands carry chemically less reactive
groups, including azides, diazirines, and R-ketodiazo
groups, which can be transformed into highly reactive
1
1-17
ments for ligand-receptor interactions.
Many of
22
nitrenes or carbenes upon UV irradiation. The ensuing
these ligands exhibit superior binding affinities and
potencies than anandamide. Notwithstanding the sub-
stantial SAR data, there is only sketchy direct informa-
tion available regarding the binding motif of the endog-
enous ligands with the CB1 receptor. Such information
reactive intermediates are then capable of forming
covalent bonds with amino acid residues situated in
their immediate vicinity. Alternatively, electrophilic
affinity ligands incorporate intrinsically reactive groups
such as isothiocyanates, Michael acceptors, haloacet-
amides, aldol esters, or nitrogen mustards, which can
react with an amino acid residue carrying a nucleophilic
*
Corresponding author. Tel.: (617) 373-4200. Fax: (617) 373-7493.
E-mail: a.makriyannis@neu.edu.
1
0.1021/jm050272i CCC: $30.25 © 2005 American Chemical Society
Published on Web 09/13/2005

6
424 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Li et al.
Scheme 1a
Scheme 2a
a
Reagents and conditions: (a) TBDMSCl, imidazole, DMF, 0
C to room temperature, 90%; (b) sodium acetylide, DMF, 0 °C to
°
room temperature, 85%; and (c) TBAF, THF, rt, 85%.
6
with prop-2-yn-1-ol (7) in the presence of CuI, NaI,
and K2CO3 followed by bromination of 8 with CBr4/PPh3
afforded 13-bromo-trideca-5,8,11-triynoic acid methyl
ester (9). Coupling of 9 with hept-6-yn-1-ol (10) under
the same conditions described for 5 gave 20-hydroxy-
eicosa-5,8,11,14-tetraynoic acid methyl ester (11). Hy-
drogenation of 11 using Lindlar’s catalyst in the pres-
ence of quinoline gave (5Z,8Z,11Z,14Z)-20-hydroxy-
eicosa-5,8,11,14-tetraenoic acid methyl ester (12) in 83%
yield as the all cis product in high isomeric purity.27 20-
Azido-eicosa-5,8,11,14-tetraenoic acid (14) was obtained
by converting the hydroxyl group of 12 to an azido group
using zinc azide under the Mitsunobu reaction28 condi-
tions followed by hydrolysis of the methyl ester of 13
with LiOH. The desired photoactivatable covalent probe
(
5Z,8Z,11Z,14Z)-20-azido-eicosa-5,8,11,14-tetraenoic acid
cyclopropylamide (2) was prepared from acid 14 via the
corresponding acid chloride and cyclopropylamine in
a
Reagents and conditions: (a) hex-5-ynoic acid methyl ester (4),
CuI, NaI, K2CO3, DMF, rt, 85%; (b) PPh3, CBr4, 0 °C, 85%; (c)
9
1
0% yield. The electrophilic covalent probe, (5Z,8Z,11Z,-
4Z)-20-isothiocyanato-eicosa-5,8,11,14-tetraenoic acid
prop-2-yn-1-ol (7), CuI, NaI, K2CO3, DMF, rt, 80%; (d) PPh3, CBr4,
0
(
°C, 82%; (e) hept-6-yn-1-ol (10), CuI, NaI, K2CO3, DMF, rt, 85%;
cyclopropylamide (1), was obtained in 85% yield by
treatment of azide 2 with CS2/PPh3 in THF.29
The key intermediate hept-6-yn-1-ol (10) was synthe-
sized from 5-bromo-pentan-1-ol (15) (Scheme 2). Protec-
tion of the hydroxyl group of 15 followed by coupling
with sodium acetylide gave 1-(t-butyldimethylsilyloxy)-
hept-6-yne (17). Deprotection of 17 with TBAF in THF
afforded 10 in 85% yield.
f) H2, Lindlar’s catalyst, Et2O/THF, rt, 83%; (g) Zn(N3)2‚2Py,
DIAD, PPh3, toluene, 0 °C to room temperature, 87%; (h) 1 M
LiOH, THF, rt, 88%; (i) oxalyl chloride/DMF, cyclopropylamine,
CH2Cl2, 0 °C to room temperature, 90%; and (j) CS2, PPh3, THF,
8
5%.
group, such as imidazolyl, sulfhydryl, amino, or hydroxyl
groups, which are at or near the binding site.23
Here, we report the design, synthesis, and biochemical
characterization of the first two high affinity covalent
probes for the anandamide binding site on the CB1
receptor, 20-isothiocyanato-eicosa-5,8,11,14-tetraenoic
acid cyclopropylamide (1, AM3677) and 20-azido-eicosa-
Results and Discussion
Receptor Binding Studies. Affinity binding values
for 1 and 2 to the CB1 and CB2 receptors were
determined according to previously reported proce-
,13,15
5
,8,11,14-tetraenoic acid cyclopropylamide (2, AM3661).
dures.1
For the CB1 receptor, binding data were
The design of these ligands was based on the attach-
ment of azido or isothiocyanato moieties to the terminal
arachidonoyl carbon of (5Z,8Z,11Z,14Z)-eicosa-5,8,11,-
obtained using a rat brain membrane in the presence
3
0,31
of phenylmethanesulfonyl fluoride (PMSF),
a gen-
eral serine protease inhibitor to protect the tested
1
4-tetraenoic acid cyclopropylamide (N-arachidonoyl-
analogues from the hydrolytic activity of fatty acid
amide hydrolase.1
4,31-33
CB2 data were obtained using
cyclopropylamine, ACPA), a high affinity and selective
2
4
ligand for the CB1 receptor. The availability of en-
docannabinoid covalent probes for the CB1 receptor
should prove to be useful for obtaining direct informa-
tion about the structural features involved in the
activation of the CB1 receptor by its endogenous ligand,
anandamide.
Chemistry. Both anandamide analogues, 1 and 2,
were synthesized starting with cross-coupling of the
easily accessible 4-chlorobut-2-yn-1-ol (3), which was
prepared from commercially available but-2-yne-1,4-diol
and hex-5-ynoic acid methyl ester (4) in the presence of
a membrane preparation from mouse spleen known to
3
be rich in CB2. [ H]CP-55,940 was chosen as a compet-
ing ligand for the assays as it has high affinity for both
CB1 and CB2 receptors and is nonselective. It is one of
the most widely used radioligands for characterizing
both CB1 and CB2 cannabinoid receptors.34 SAR, muta-
tion, and computer modeling study results indicate that
nonclassical cannbinoids (e.g., CP-55940), classical can-
nabinoids, and anandamide share some binding motifs,
while aminoalkylindole cannabinoids (e.g., WIN55212-
2) might have a different binding site.35
The affinities of 1 and 2 for the CB1 and CB2
receptors are summarized in Table 1, where we also
include data for N-arachidonoylcyclopropylamine (ACPA),
the parent compound, for comparison. The Ki values of
2
5
2
6
copper(I) iodide (Scheme 1). Subsequent bromination
of 10-hydroxy-deca-5,8-diynoic acid methyl ester (5) with
CBr4 in the presence of PPh3 gave 10-bromo-deca-5,8-
diynoic acid methyl ester (6) in 85% yield. Coupling of

Covalent Endocannabinoid Probes for CB1 Receptor
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6425
a
Table 1. Affinity (Ki) of 1 and 2 for CB1 and CB2 Receptors
(95% Confidence Limits)
compound
R
CB1 (Ki, nM) (PMSF)
CB2 (Ki, nM)
1
-NCS
-N3
-H
1.3 ( 0.2
0.9 ( 0.2
2.7 ( 0.4 (2.2)
48.5 ( 13
2
57.6 ( 19
b
b
ACPA
157 ( 37 (715)
a
CB1 affinities were determined using rat brain membranes
3
and 0.76 nM [ H]CP-55,940 as the radioligand following previously
described procedures.
1
,13,15
Mouse spleen was used as the source
of CB2 receptor. Data were analyzed using nonlinear regression
analysis. Ki values were obtained from a minimum of two
independent experiments run in duplicate and are expressed as
b
the mean ( standard error. Data from ref 24. ACPA: N-
arachidonoylcyclopropylamine
Figure 1. 20-Isothiocyanato-eicosa-5,8,11,14-tetraenoic acid
cyclopropylamide (1) in rat forebrain membranes inhibits the
1
and 2 for the CB1 receptor in the presence of PMSF
3
specific binding of [ H]CP-55940 to the cannabinoid receptor
are 1.3 and 0.9 nM, respectively, both ligands exhibiting
higher CB1 affinities than ACPA. Our results indicate
that substitution of the terminal C-20 carbon with an
azido or isothiocyanato group leads to enhanced binding
affinities for the CB1 receptor, while both 1 and 2
exhibited over 50-fold selectivities for CB1 versus CB2.
Irreversible Binding Studies. The two novel af-
finity ligands (1 and 2) were subsequently evaluated for
their abilities to irreversibly label the CB1 receptor
through formation of a covalent bond following previ-
(
CB1). The membranes were pretreated with 0.15 mM PMSF
for 10 min on ice twice and then were washed 3 times with
0.1% BSA in TME to remove excess PMSF. Heterologous
displacements are shown for membranes preequilibrated for
1
h in the absence or presence of 3.9, 13, and 26 nM
3
isothiocyanate 1. The [ H]CP-55,940 specific bindings were
3
6
determined by the reported saturation binding method, and
the reductions in binding as percentages of control are given
in parentheses.
1
9-21
ously reported procedures.
To minimize potential
hydrolysis of the ligands by endogenous FAAH, the
membranes were pretreated with PMSF before the
labeling experiments, and excess PMSF was subse-
quently removed by extensive washing.
Reaction of Electrophilic Ligand 1 with CB1. As
depicted in Figure 1, equilibration of rat forebrain
membranes for 1 h with 3.9 nM 1 (3 times its Ki value
of 1.3 nM), followed by the removal of excess unbound
3
ligand, lowered the [ H]CP-55940 specific binding to the
CB1 receptor by 23%. When the membranes were
equilibrated with 13 and 26 nM 1 (10 and 20 times its
3
Ki value), the [ H]CP-55940 specific binding was lowered
by 32 and 58%, respectively. In the control experiments,
the membranes were treated according to the same
procedures but in the absence of the electrophilic affinity
ligand 1 and fully retained their ability to bind the [ H]-
CP-55940.
Figure 2. Time course for cannabinoid receptor labeling with
20-isothiocyanato-eicosa-5,8,11,14-tetraenoic acid cyclopropyl-
amide (1). The membranes were pretreated with 0.15 mM
PMSF for 10 min on ice twice and then were washed 3 times
with 0.1% BSA in TME to remove excess PMSF. Then, rat
forebrain membranes were preincubated with 3.9 nM isothio-
cyanate 1 for 15, 30, and 60 min. Membranes in the control
experiment were preincubated for 60 min without isothiocy-
3
To better understand the receptor labeling charac-
teristics of 1, we carried out a time-dependent study
using a 3.9 nM concentration of 1 and varied the
incubation time (Figure 2). Our studies showed that
with 15 min of incubation, 15% of the receptor sites was
occupied irreversibly. By increasing the incubation time
to 30 min, covalent binding increased to 25% with no
further increase seen if the incubation time was ex-
tended to 60 min. Thus, at the ligand concentration
used, covalent binding had reached its plateau at 30 min
incubation time. The control experiment was a 60 min
incubation with no added 1. However, we note that the
true kinetic behavior of the covalent reaction between
3
anate 1. The [ H]CP-55,940 specific bindings were determined
3
6
by the reported saturation binding method.
the termination of the ligand-receptor covalent reaction
is accomplished by centrifugation. Unreacted ligand was
removed by repeatedly pelleting and washing the mem-
brane preparation after the incubation period was
completed. The observation that full receptor labeling
is not observed at lower concentrations, even if the
incubation period is increased, is probably a reflection
of the slow inactivation of the isothiocyanato group of
the probe through nonspecific reactions. In this regard,
the isothiocyanate analogue 1 was found to be stable
1
and the cannabinoid receptor is not necessarily
reflected by this time-dependent receptor labeling since
the incubation time does not represent the precise
reaction time. According to our experimental conditions,

6
426 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Li et al.
end of the hydrophobic tail of an anandamide analogue,
led to high affinity ligands capable of covalently binding
to the receptor. We plan to utilize probes 1 and 2 in more
detailed experiments aimed at identifying and charac-
terizing the endocannabinoid binding domain within the
CB1 receptor. Such information will assist in the design
of improved cannabinoid receptor probes and enhance
our understanding of the structural features involved
in CB1 activation.
Experimental Procedures
General. Unless otherwise noted, starting materials were
obtained from commercial suppliers and were used without
further purification. All final compounds were oils. Tetraenoic
compounds were very oxygen sensitive. Final compounds were
shown to be homogeneous by HPLC employing two diverse
elution solvent mixtures. HPLC experiment A was performed
using a Waters 510 single pump chromatograph with a Waters
2487 dual λ absorbance detector, UV detection at 254 nm,
normal phase Altech 5 µ silica, 4.6 mm × 25 cm column,
eluting with ethyl acetate at 0.75 mL/min. HPLC experiment
B was performed on a Waters 717 plus autosampler dual pump
chromatograph with a Waters 2487 dual λ absorbance detector,
UV detection at 204 nm, reversed-phase Ultrasphere ODS, 4.6
mm × 4.5 cm column, eluting with acetonitrile/8.5% aqueous
phosphoric acid, 70:30 at 1.0 mL/min. HPLC experiment C was
performed on a Beckman 126 solvent module dual pump
chromatograph with a model 168 detector, UV detection at 220
nm, normal phase Phenosphere 5 µm silica, 10 mm × 25 cm
column, eluting with 2-propanol/hexane, 5:95 at 2.0 mL/min.
The final products were fully characterized by high-resolution
Figure 3. Photoirradiation of 20-azido-eicosa-5,8,11,14-tet-
raenoic acid cyclopropylamide (2) in rat forebrain membranes
3
inhibits the specific binding of [ H]CP-55940 to the cannab-
inoid receptor (CB1). The membranes were pretreated with
0
3
.15 mM PMSF for 10 min on ice twice and then were washed
times with 0.1% BSA in TME to remove the excess PMSF.
Heterologous displacements are shown for membranes irradi-
ated in the absence or presence of 2.7, 9, and 18 nM azide 2.
The [ H]CP-55,940 specific bindings were determined by the
reported saturation binding method, and the reductions in
binding as percentages of control are given in parentheses.
3
3
6
when tested under the buffer conditions utilized in our
experiments. We thus speculate that its slow inactiva-
tion is due to other nucleophiles (e.g., cysteines) present
in the membrane preparation. Notwithstanding these
experimental limitations, our data clearly demonstrate
that time-dependent receptor labeling does occur.
Reaction of Photoactivatable Ligand 2 with
CB1. Photolabeling experiments with the azidoen-
docannabinoid 2 initially followed similar procedures as
with 1 (Figure 3). Rat forebrain membranes were
equilibrated with 2 at a concentration that is 3 times
its Ki value for 1 h. After equilibration, membranes were
centrifuged and resuspended in ice-cold TME buffer.
Then, membranes were photoirradiated with ultraviolet
light (254 nm), washed extensively to remove unbound
1
1
H NMR, IR, and HRMS. H NMR spectra were recorded on
either a Bruker DRX 400 or 500 MHz spectrometer using
CDCl as the solvent with TMS as the internal standard. All
3
chemical shifts are reported in ppm. Multiplicities of the
signals are indicated as follows: s for singlet, d for doublet, t
for triplet, q for quartet, p (pentet) for quintet, m for multiplet,
and any combinations as appropriate. IR spectra were per-
formed on a Perkin-Elmer Spectrum One FT-IR spectrometer.
High-resolution mass spectra (HRMS) for all final compounds
were obtained on a ZAB-SE mass spectrometer and performed
by the School of Chemical Sciences, University of Illinois at
Urbana-Champaign, Urbana, IL. TME buffer contains 25 mM
Tris base, 5 mM MgCl
to pH 7.4 with HCl. All solvent ratios are by volume.
0-Hydroxy-deca-5,8-diynoic Acid Methyl Ester (5).
2
, and 1 mM EDTA, and was adjusted
1
2
5
4
-Chloro-but-2-ny-1-ol (3) (3.0 g, 28.7 mmol) and hex-5-ynoic
acid methyl ester (4) (3.62 g, 28.7 mmol) were added to a
2, and then tested for cannabinoid receptor binding. As
suspension of CuI (11.0 g, 57.4 mmol), NaI (8.61 g, 57.4 mmol),
shown in Figure 3, 23% of the CB1 receptor was
irreversibly labeled by 2. In control experiments, mem-
branes were irradiated in the absence of 2. Furthermore,
2 3
and K CO (5.94 g, 43 mmol) in 10 mL of anhydrous DMF
under Ar. The mixture was stirred overnight at room temper-
4
ature and then quenched with saturated aqueous NH Cl, and
3
the lipophilic products were extracted with Et O. The combined
using [ H]CP-55,940 to determine the fraction of unoc-
2
organic extracts were washed with water and brine, and dried
cupied CB1 receptor demonstrated that the equilibra-
tion of the membrane preparation with higher concen-
trations of 2 led to progressively larger percentages of
CB1 within the membrane preparation being irrevers-
ibly labeled. Thus, when 2 was used at concentrations
of 9 and 18 nM, the covalent receptor occupancy was
2 4
with Na SO . After rotary evaporation of solvents, the residue
was chromatographed on silica gel to afford 5 (4.73 g, 85%) as
1
an oil: R
MHz, CDCl
f
) 0.51 (petroleum ether/Et
) δ 4.26 (t, J ) 2.3 Hz, 2H), 3.68 (s, 3H), 3.18 (p,
2
O, 1:2). H NMR (500
3
J ) 2.3 Hz, 2H), 2.42 (t, J ) 7.5 Hz, 2H), 2.25 (tt, J ) 6.9, 2.3
Hz, 2H), 1.81 (p, J ) 7.2 Hz, 2H).
4
2 and 68%, respectively (Figure 3). These experiments
10-Bromo-deca-5,8-diynoic Acid Methyl Ester (6). A
solution of PPh (7.03 g, 26.8 mmol) in 15 mL of dry CH Cl
3 2 2
demonstrate that the azido analogue 2 can be used as
an efficient and high-yield photoaffinity probe for co-
valent labeling of the CB1 cannabinoid receptor.
was added dropwise to a stirred solution of 5 (4.73 g, 24.4
mmol) and CBr (8.9 g, 26.8 mmol) in 15 mL of dry CH Cl at
°C. Then, the mixture was stirred for another 1.5 h at 0 °C.
The solvent was evaporated, and the residue was diluted with
Et O and filtered through a short pad of Celite. The filtrate
was concentrated and then chromatographed on silica gel to
provide 6 (5.33 g, 85%) as an oil: R ) 0.41 (petroleum ether/
O, 5:1). H NMR (500 MHz, CDCl ) δ 3.69 (t, J ) 2.3 Hz,
4
2
2
0
Conclusions
2
Novel anandamide analogues 1 and 2, which can be
used as covalent probes for the CB1 receptor, were
designed and synthesized. Our data demonstrate that
the isothiocyanato or azido groups, when placed at the
f
1
Et
2
3
2H), 3.68 (s, 3H), 3.20 (p, J ) 2.3 Hz, 2H), 2.42 (t, J ) 7.5 Hz,
2H), 2.24 (tt, J ) 6.9, 2.3 Hz, 2H), 1.82 (p, J ) 7.2 Hz, 2H).

Covalent Endocannabinoid Probes for CB1 Receptor
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6427
1
3-Hydroxy-trideca-5,8,11-triynoic Acid Methyl Ester
8). Coupling of 6 (5.33 g, 20.7 mmol) with prop-2-yn-1-ol (7)
1.4 g, 24.9 mmol) following the procedure described for
compound 5 gave 13-hydroxy-trideca-5,8,11-triynoic acid meth-
imidazole (1.23 g, 18 mmol) in 10 mL of DMF was added 1 M
TBDMSCl in THF (15.6 mL, 15.6 mmol) at 0 °C under Ar.
Stirring was continued at room temperature overnight. The
(
(
reaction mixture was diluted with Et
a short pad of Celite. The filtrate was washed with 1 N HCl,
water, and brine, dried with Na SO , and concentrated. The
residue was purified by column chromatography to afford 16
(3.03 g, 90%) as a colorless oil: R ) 0.43 (petroleum ether/
) δ 3.61 (t, J ) 6.1 Hz,
2
O and filtered through
yl ester (8) (3.84 g, 80%) as a pale yellow oil:
petroleum ether/Et
.25 (t, J ) 2.3 Hz, 2H), 3.68 (s, 3H), 3.23 (p, J ) 2.3 Hz, 2H),
.12 (p, J ) 2.3 Hz, 2H), 2.43 (t, J ) 7.5 Hz, 2H), 2.24 (tt, J )
.9, 2.3 Hz, 2H), 1.82 (p, J ) 7.2 Hz, 2H).
R
f
) 0.43
1
(
2
O, 1:1.5). H NMR (500 MHz, CDCl ) δ
3
2
4
4
3
6
f
1
2 3
Et O, 50:1). H NMR (500 MHz, CDCl
1
3-Bromo-trideca-5,8,11-triynoic Acid Methyl Ester
9). The bromide 9 was prepared from alcohol 8 (3.8 g, 16.6
mmol), CBr (6.06 g, 18.3 mmol), and PPh (4.8 g, 18.3 mmol)
following the procedure described for 6 to give 9 (4.02 g, 82%)
2H), 3.41 (t, J ) 6.7 Hz, 2H), 1.88 (p, J ) 6.9 Hz, 2H), 1.53
(m, 4H), 0.89 (s, 9H), 0.05 (s, 6H).
(
4
3
1-(t-Butyldimethylsilyloxy)-hept-6-yne (17). To a solu-
tion of 16 (3.03 g, 10.8 mmol) in 10 mL of DMF was added an
18 wt % slurry of sodium acetylide (4.32 g, 16.2 mmol) in
xylene/light mineral oil at 0 °C under Ar. Stirring was
continued at room temperature overnight. The reaction mix-
1
as a pale yellow oil: R
NMR (400 MHz, CDCl
f
) 0.33 (petroleum ether/Et
3
2
O, 5:1). H
) δ 3.91 (t, J ) 2.3 Hz, 2H), 3.68 (s,
3
H), 3.23 (p, J ) 2.3 Hz, 2H), 3.14 (p, J ) 2.3 Hz, 2H), 2.43 (t,
J ) 7.4 Hz, 2H), 2.25 (tt, J ) 6.9, 2.3 Hz, 2H), 1.84 (p, J ) 7.2
ture was diluted with Et
the reaction mixture was washed with 1 N HCl, water, and
brine, dried with Na SO , and concentrated. Chromatography
on silica gel gave 17 (2.07 g, 85%) as a colorless oil: R
2
O and quenched with water. Then,
Hz, 2H).
2
0-Hydroxy-eicosa-5,8,11,14-tetraynoic Acid Methyl
Ester (11). The coupling of 9 (4.02 g, 13.6 mmol) with hept-
-yn-1-ol (10) (1.83 g, 16.3 mmol) following the procedure
2
4
f
) 0.27
2 3
(petroleum ether/Et O, 60:1). H NMR (500 MHz, CDCl ) δ 3.61
1
6
described for 5 gave 11 (3.78 g, 85%) as a pale yellow oil: R
f
(t, J ) 6.3 Hz, 2H), 2.20 (td, J ) 7.0, 2.6 Hz, 2H), 1.93 (t, J )
2.6 Hz, 1H), 1.53 (m, 4H), 1.45 (m, 2H), 0.89 (s, 9H), 0.05 (s,
6H).
Hept-6-yn-1-ol (10). To a solution of 17 (2.07 g, 9.18 mmol)
in 10 mL of THF was added 1 M TBAF in THF (18.4 mL, 18.4
mmol). Stirring was continued for 2 h. The reaction was
quenched with MeOH, washed with water and brine, dried
1
)
0.35 (petroleum ether/acetone, 2:1). H NMR (500 MHz,
CDCl
3
) δ 3.68 (s, 3H), 3.65 (t, J ) 6.1 Hz, 2H), 3.14 (m, 6H),
2
.43 (t, J ) 7.5 Hz, 2H), 2.24 (m, 2H), 2.17 (m, 4H), 1.83 (p, J
)
7.2 Hz, 2H), 1.58 (p, J ) 7.2 Hz, 2H), 1.54 (p, J ) 7.2 Hz,
2
H), 1.45 (p, J ) 7.2 Hz, 2H).
5Z,8Z,11Z,14Z)-20-Hydroxy-eicosa-5,8,11,14-tetraeno-
ic Acid Methyl Ester (12). To a 250 mL flask containing 5.67
g of Lindlar’s catalyst was added 20 mL of Et O. The mixture
was saturated with H at room temperature, a solution of 11
3.78 g, 11.6 mmol) and quinoline (200 mg) in 15 mL of THF
was added, and a stream of H was bubbled through the
(
with Na
2 4
SO , and concentrated. The residue was purified by
column chromatography to give 10 (0.87 g, 85%) as a colorless
2
1
oil: R
f
) 0.33 (petroleum ether/ethyl acetate, 3:1). H NMR
2
(
(500 MHz, CDCl ) δ 3.66 (t, J ) 6.3 Hz, 2H), 2.21 (td, J ) 6.9,
3
2.6 Hz, 2H), 1.93 (t, J ) 2.6 Hz, 1H), 1.56 (m, 4H), 1.48 (m,
2
1
mixture. The reaction was monitored by H NMR. After the
hydrogenation was finished, the mixture was filtered, washed
2H), 1.34 (br, 1H).
(5Z,8Z,11Z,14Z)-20-Azido-eicosa-5,8,11,14-tetraenoic
Acid Cyclopropylamide (2). To a solution of acid 14 (0.10
g, 0.29 mmol) and DMF (0.02 mL) in 5 mL of CH Cl at 0 °C
with 2 N HCl and brine, and dried with Na
2 4
SO . The solvent
was evaporated, and the residue was chromatographed on
2
2
silica gel to give 12 (3.21 g, 83%) as a colorless oil: R
f
) 0.34
2 2
was added 2 M oxalyl chloride in CH Cl (0.29 mL, 0.58 mmol)
1
(
petroleum ether/acetone, 5:1). H NMR (500 MHz, CDCl ) δ
3
in a dropwise manner. The reaction mixture was stirred
further at 0 °C for an additional 1.5 h. Then cyclopropylamine
(166 mg, 2.9 mmol) was added to the reaction mixture very
slowly at 0 °C. Stirring was continued at room temperature
5
.38 (m, 8H), 3.67 (s, 3H), 3.63 (t, J ) 6.1 Hz, 2H), 2.84 (m,
H), 2.33 (t, J ) 7.5 Hz, 2H), 2.10 (m, 4H), 1.71 (p, J ) 7.2
6
Hz, 2H), 1.58 (p, J ) 6.8 Hz, 2H), 1.39 (m, 4H).
(
5Z,8Z,11Z,14Z)-20-Azido-eicosa-5,8,11,14-tetraenoic
overnight. Et
water and brine, and dried over Na
removed to give crude product. Chromatography on silica gel
gave pure 2 (100 mg, 90%) as an oil: R ) 0.37 (petroleum
ether/acetone, 3:1). H NMR (500 MHz, CDCl ) δ 5.62 (br, 1H),
2
O was added, and the mixture was washed with
2
8
Acid Methyl Ester (13). A zinc azide/bis-pyridine complex
2
SO . The solvent was
4
(
9
2.36 g, 7.7 mmol) was suspended in a solution of 12 (3.22 g,
.6 mmol) and PPh
3
(5.03 g, 19.2 mmol) in 20 mL of anhydrous
f
1
toluene. To this stirred mixture at room temperature was
added diisopropyl azodicarboxylate (3.88 g, 19.2 mmol) drop-
wise. Stirring was continued until complete consumption (TLC
monitoring) of 12 was observed. The heterogeneous mixture
was filtered through a Celite pad, concentrated, and purified
3
5.39 (m, 8H), 3.26 (t, J ) 7.5 Hz, 2H), 2.84 (m, 6H), 2.70 (m,
1H), 2.11 (m, 6H), 1.70 (p, J ) 7.4 Hz, 2H), 1.61 (p, J ) 6.8
Hz, 2H), 1.40 (m, 4H), 0.76 (m, 2H), 0.47 (m, 2H); IR (neat)
-
1
+
cm 2092, 1643. HRMS for C23
37 4
H N O (MH ) 385.2966. Calcd.
by column chromatography to afford pure 13 (3.0 g, 87%) as a
385.2967. HPLC A, 5.85 min (99%); HPLC B, 3.15 min (99%).
1
colorless oil: R
500 MHz, CDCl
f
) 0.48 (petroleum ether/Et
3
2
O, 10:1). H NMR
(5Z,8Z,11Z,14Z)-20-Isothiocyanato-eicosa-5,8,11,14-tet-
raenoic Acid Cyclopropylamide (1). To a solution of 2 (50
mg, 0.13 mmol) in 2 mL of anhydrous THF was added PPh3
(52 mg, 0.2 mmol) in a single portion at room temperature.
Then, carbon disulfide (99 mg, 1.3 mmol) was added. The
reaction mixture was stirred at room temperature for 48 h and
was then concentrated. The residue was purified by column
(
) δ 5.41 (m, 8H), 3.67 (s, 3H), 3.26 (t, J ) 7.5
Hz, 2H), 2.82 (m, 6H), 2.32 (t, J ) 7.5 Hz, 2H), 2.10 (m, 4H),
1
4
.71 (p, J ) 7.4 Hz, 2H), 1.60 (p, J ) 6.9 Hz, 2H), 1.39 (m,
H); IR (neat) cm-1 2094, 1737.
(
5Z,8Z,11Z,14Z)-20-Azido-eicosa-5,8,11,14-tetraenoic
Acid (14). To a solution of 13 (1.0 g, 2.8 mmol) in 10 mL of
THF was added 1 M LiOH (5.6 mL) at room temperature
under Ar. Stirring was continued for 48 h, and then the
reaction mixture was acidified with 2 N HCl to pH 5.5, and
f
chromatography to give pure 1 (44 mg, 85%) as an oil: R )
1
2 3
0.32 (petroleum ether/Et O, 4:1). H NMR (500 MHz, CDCl )
δ 5.58 (br s, 1H), 5.40 (m, 8H), 3.51 (t, J ) 7.5 Hz, 2H), 2.81
lipophilic products were extracted with Et
organic extracts were washed with water and brine, and dried
with Na SO . The solvent was evaporated to give a yellowish
oily residue. Chromatography on silica gel gave 14 (0.85 g, 88%
yield) as a colorless oil: R ) 0.39 (petroleum ether/Et O, 3:1).
H NMR (500 MHz, CDCl ) δ 5.40 (m, 8H), 3.26 (t, J ) 7.5
Hz, 2H), 2.84 (m, 6H), 2.37 (t, J ) 7.5 Hz, 2H), 2.15 (q, J )
2
O. The combined
(m, 6H), 2.70 (m, 1H), 2.10 (m, 6H), 1.72 (m, 4H), 1.42 (m,
-
1
4H), 0.77 (m, 2H), 0.47 (m, 2H); IR (neat) cm 2089, 1643.
+
2
4
37 2
HRMS for C24H N OS (MH ) 401.2628. Calcd. 401.2627.
HPLC B, 2.83 min (98%); HPLC C, 8.09 min (97%).
f
2
Radioligand Binding Assay. For CB1, rat forebrain
1
3
membranes were prepared according to the procedure of Dodd
et al.36 The binding of the novel anandamide analogues to the
7
1
1
.0 Hz, 2H), 2.07 (q, J ) 7.0 Hz, 2H), 1.72 (p, J ) 7.4 Hz, 2H),
.62 (p, J ) 6.9 Hz, 2H), 1.39 (m, 4H); IR (neat) cm 2092,
cannabinoid receptor was assessed as previously described,1,13,15
-
1
except that the membranes were pretreated with PMSF.
Membranes, previously frozen at -80 °C, were thawed on ice.
To the stirred suspension was added 3 volumes of 25 mM Tris-
706.
5
-Bromo-1-(t-butyldimethylsilyloxy)-pentane (16). To
a solution of 5-bromo-pentan-1-ol (15) (2.0 g, 12 mmol) and
2
HCl buffer, 5 mM MgCl , and 1 mM EDTA, pH 7.4 (TME

6
428 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Li et al.
buffer) containing 150 µM PMSF (made fresh in 2-propanol
as a 100 mM stock). The suspension was incubated at 4 °C,
after 15 min, a second addition of PMSF stock brought the
concentration to 300 µM PMSF, and then the mixture was
incubated for another 15 min. At the end of the second 15 min
incubation, the membranes were pelleted and washed 3 times
with TME to remove unreacted PMSF. The pretreated mem-
branes were subsequently used in the binding assay described
next. Approximately 30 µg of PMSF-treated membranes was
incubated in a silanized 96-well microtiter plate with TME
containing 0.1% essentially fatty acid-free bovine serum
using GraphPad Prism version 3.03 (one site binding analysis
d
equation Y ) BmaxX/(K + X); GraphPad Software, San Diego,
CA) on a Windows platform.
Acknowledgment. This work was supported by
Grants DA-3801, DA-152, DA-9158, and DA-7215 from
the National Institute on Drug Abuse. We also thank
Jerzy Golik for useful discussions; Dai Lu, Ying Pei, and
JodiAnne Wood for HPLC experiments; and Richard
Duclos for assistance with the manuscript.
3
albumin (BSA), 0.76 nM [ H]CP-55,940, and various concen-
trations of anandamide analogues in a final volume of 200 µL.
The binding assay was performed at 30 °C for 1 h with gentle
agitation. The resultant material was transferred to Unifilter
GF/B filter plates, and unbound ligand was removed using a
Packard Filtermate-96 Cell Harvester (Perkin-Elmer Packard,
Shelton, CT). Filter plates were washed 4 times with ice-cold
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(
of epimeric hepoxilins (-)-(10R)-B
3
and (+)-(10S)-B
3
. Tetrahe-
JM050272I