Development of endocannabinoid-based chemical probes for the study of cannabinoid receptors

Article abstract of DOI:10.1021/jm2004392

We report the synthesis of new chemical probes (1a,b, 2a-c, 3a-c) based on the structure of the main endocannabinoids for their use in biological systems directly or via click chemistry. As proof of concept, 2-arachidonyl glyceryl ether based biotinylated

Full text of DOI:10.1021/jm2004392

BRIEF ARTICLE
pubs.acs.org/jmc
Development of Endocannabinoid-Based Chemical Probes for the
Study of Cannabinoid Receptors†
Lidia Martín-Couce, Mar Martín-Fontecha, Samanta Capolicchio, María L. Lꢀopez-Rodríguez,* and
Silvia Ortega-Gutiꢀerrez*
Departamento de Química Orgꢀanica I, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, E-28040 Madrid, Spain
S Supporting Information
b
ABSTRACT: We report the synthesis of new chemical probes (1a,b, 2aꢀc, 3aꢀc) based on the structure of the main
endocannabinoids for their use in biological systems directly or via click chemistry. As proof of concept, 2-arachidonyl glyceryl
ether based biotinylated 3b enables direct visualization of CB1 receptor in cells. These results represent the starting point for the
development of advanced small molecule chemical probes able to generate valuable information about the cannabinoid receptors.
’ INTRODUCTION
As an initial proof of concept, in our research group we have
already reported the first efficacious fluorescent probes for the
specific labeling of human serotonin receptors 5-HT1A and
5-HT6 in transfected cells.11,12 This fact has led us to extend
these efforts to CBRs, where development of tagged small-
molecule probes would greatly improve our understanding of
the endogenous cannabinoid system, its physiology and its
therapeutic potential. Some previous attempts have addressed
the development of fluorescent ligands directed toward CB2R.
Among them, the attempts made to modify the CB2 agonist
(2-methyl-1-propyl-1H-indol-3-yl)(1-naphthyl)methanone
(JWH-015) with a fluorophore (although this modification leads
to a complete loss of affinity13) or the CB2 antagonist-based
probe 1-(4-{[(6-aminohexyl)amino]methyl}benzyl)-5-(4-chloro-
3-methylphenyl)-N-(1,3,3-trimethylbicyclo[2.2.1]hept-2-yl)-
1H-pyrazole-3-carboxamide (MBC94) coupled to a near-
infrared dye, which exhibits a Ki for this receptor of 260 nM
and enables cell visualization, deserve special attention.14,15
In this work we report the synthesis of a set of new chemical
probes 1a,b, 2aꢀc, and 3aꢀc that bear different tags (biotin,
benzophenone, terminal alkyne) and are based on the structures
of the main endocannabinoids anandamide (AEA), 2-arachido-
noylglycerol (2-AG), and 2-arachidonyl glyceryl ether (13,
2-AGE, noladin ether) (Figure 1). Among the synthesized
compounds, 3b stands out as a CB1 ligand (Ki = 221 nM)
suitable for direct visualization of this receptor in cell systems in a
comparable manner to available commercial antibodies. These
results provide the basis for further development of these or
other derivatives as probes for endocannabinoid-target proteins
that enable not only visualization in native systems but also to
identify other target protein(s) of endocannabinoids, studies that
are underway in our laboratory.
Endocannabinoids are lipid signaling molecules that regulate a
wide range of physiological functions in mammals including pain,
inflammation, neurodegeneration, feeding, and cognitive and
emotional states.1ꢀ3 Although the pharmacology and bioactivity
of these molecules have been extensively studied during the past
decade, many basic questions remain unanswered. For instance,
while most of their effects are thought to be mediated through
CB1 and CB2 cannabinoid receptors (CBRs), thorough pharma-
cological studies indicate that cannabinoids regulate cell func-
tions independently of these two receptors.4 However, the
evidence is rather indirect and the molecular characterization
of these proposed receptors is still lacking. Therefore, tools that
enable acquisition of direct information about the location, levels,
and any other relevant aspect of the CBRs would be of utmost
importance. Currently such tools are basically limited to the use
of antibodies that recognize CB1 and CB2 receptors. None-
theless, they exhibit some shortcomings such as limited sensitiv-
ity, specificity, or selectivity in certain biological settings that
make difficult the unequivocal interpretation of results;5 they are
not suitable for in vivo or ex vivo applications, and the availability
of antibodies requires the molecular characterization of the
receptor under study. In this context, small molecule ligands
with appropriate affinity and selectivity can greatly contribute to
the study of a given receptor or target protein even if it has not
been molecularly characterized by their conversion into chemical
probes.6 This strategy has been successfully applied to the study
of enzymes with the development of activity-based probes7
(ABPs) that take advantage of the inherent chemical activity of
the enzymes. Hence, extension of this approach to other super-
families of proteins such as G-protein-coupled receptors
(GPCRs), main drug targets in medicinal chemistry programs,8
currently constitutes an important challenge. In general, efforts
have been made toward the development of noninvasive imaging
probes,9 although in the case of GPCRs this goal normally
requires the expression of tagged receptors,10 a fact that hinders
direct observation of GPCRs in native systems. In this context,
we have recently started a project aimed at the development of
small molecule probes suitable for the labeling of native GPCRs.
’ RESULTS AND DISCUSSION
Visualization and profiling of endocannabinoid binding sites,
including the known cannabinoid receptors CB1 and CB2, have
Received: April 13, 2011
Published: June 15, 2011
r
2011 American Chemical Society
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Journal of Medicinal Chemistry
BRIEF ARTICLE
raised much attention. To aid this objective, we envisioned the
possibility of developing biotinylated derivatives that could be
tracked using the adequate avidin or streptavidin conjugate (such
as a fluorophore for visualization or solid support for enrichment
and identification). Therefore, our first objective was to synthe-
size a set of AEA-, 2-AG-, and 2-AGE-based biotinylated probes.
On the basis of the AEA structure, we envisaged the possibility of
introducing the different tags in the tail region or in the polar
headgroup (Figure 1). Reported docking studies proposed
different possibilities for the AEA bioactive conformation in
the CB1 binding site.16 Moreover, previous structureꢀaffinity
relationship studies on AEA analogues provided some clues on
the requirements involved in ligandꢀreceptor recognition.17 In
this regard, replacement of the ethanolamine moiety of AEA for
cyclopropylamine or (R)-2-hydroxy-1-methylethylamine leads
to an increase of CB1 affinity and metabolic stability (as in the
case of (R)-methanandamide).18,19 Therefore, we chose these
two moieties as headgroups when biotin was introduced as tag in
the tail region (probes 1a,b, Figure 1). Conversely, to assess
whether the head position would tolerate the incorporation
of chemical tags, we synthesized probes 2aꢀc (Figure 1). In
this case, besides biotin, a photoactivable cross-linker (benzo-
phenone) and a bioorthogonal chemical reporter (terminal
alkyne) were also used as chemical tags. The terminal alkyne
would be useful for performing click chemistry, while the
presence of a benzophenone moiety introduces the possibility
of covalent binding between the probe and the receptor after UV
irradiation.
Synthesis of probes 1a,b and 2aꢀc is depicted in Scheme 1.
Direct amidation of methyl ester 4 with the appropriate amine in
the presence of trimethylaluminium yielded the corresponding
hydroxyamides 5a,b, whose esterification with N-(+)-biotinyl-6-
aminohexanoic acid, followed by deprotection in the case of 6,
yielded target compounds 1a,b. Because of the lack of significant
affinity of these compounds for the CBRs, we next focused our
efforts on the AEA-based probes substituted in the polar head
(2aꢀc) to determine whether the tag attachment at this position
could keep the affinity of the probe for the CBRs. For this
purpose (R)-methanandamide (7)18 was derivatized with biotin
(2a) and a terminal alkyne (2b) by coupling reaction with the
corresponding commercial carboxylic acids, whereas 2c was
prepared by esterification of 7 with Boc-4-benzoyl-L-phenylala-
nine, followed by Boc cleavage and further condensation with
Figure 1. Structures of the main endocannabinoids anandamide, 2-ara-
chidonoylglycerol, and 2-arachidonyl glyceryl ether and designed probes
1ꢀ3.
Scheme 1. Synthesis of Compounds 1a,b and 2aꢀca
a Reagents and conditions: (a) R-NH2, Al(CH3)3, CH2Cl2, 0 °C to rt, 39ꢀ79%; (b) N-(+)-biotinyl-6-aminohexanoic acid, EDC, DMAP, DMSO,
CH2Cl2, ꢀ10 °C to rt, 17ꢀ48%; (c) (Bu)4NF, THF, rt, 46%; (d) 5-hexynoic acid, DCC, DMAP, CH2Cl2, 0 °C to rt, 98%; (e) Boc-4-benzoyl-L-
phenylalanine, DCC, DMAP, CH2Cl2, 0 °C to rt, 47%; (f) TFA, CH2Cl2, rt, 85%; (g) 5-hexynoic acid, EDC, HOBt, DMF, CH2Cl2, rt, 26%.
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Journal of Medicinal Chemistry
BRIEF ARTICLE
Scheme 2. Synthesis of Compounds 3aꢀca
a Reagents and conditions: (a) N-(+)-biotinyl-6-aminohexanoic acid, DCC, DMAP, HOBt, DMF, CH2Cl2, 60 °C to rt, 20ꢀ90%; (b) arachidonic acid,
DCC, DMAP, DMF, CH2Cl2, ꢀ20 °C to rt, 15%; (c) TFA, CH2Cl2, rt, 60%; (d) 5-hexynoic acid, DCC, DMAP, CH2Cl2, 0 °C to rt, 44%.
(13)22 with N-(+)-biotinyl-6-aminohexanoic acid or 5-hexynoic
Table 1. CB1 and CB2 Binding Affinities for Compounds
3aꢀc
acid afforded the desired 3b or 3c, respectively.
Notably, these probes exhibited moderate to good affinities,
with Ki in the nanomolar range (Table 1), and derivative 3c
receptor affinitya (Ki ( SEM) (nM)
showed the best affinity values for both CBRs [Ki(CB1) = 84.7
compd
CB1
CB2
nM; Ki(CB2) = 84.9 nM], a feature that makes it a promising
candidate for click chemistry attachment of more complex tags
within biological samples. Also of note, the 2-AG derived probed
3a is selective for CB2 [Ki(CB1) > 5000 nM; Ki(CB2) = 379 nM]
while the 2-AGE-based probe 3b maintains a moderate affinity
for both CBRs [Ki(CB1) = 221 nM; Ki(CB2) = 450 nM]. Given
that the highest affinity was obtained for 3b at the CB1R, we
chose this probe to assess its suitability for in vitro cell visualiza-
tion of the CB1 receptor. The mouse hippocampal cell line HT-
22 was transiently transfected with CB1 receptor. Cells were
incubated with 3b and streptavidin-Alexa 488 was used to detect
the probe and visualized by fluorescence microscopy. Figure 2
shows CB1 labeling (green). To evaluate the specificity of the
labeling, parallel experiments were carried out under the same
conditions in nontransfected cells (Figure 2C) and in the
presence of an excess of the high affinity (Ki = 0.06 nM) CBR
ligand (6aR,10aR)-3-(1,1-dimethylheptyl)-9-(hydroxymethyl)-
6,6-dimethyl-6a,7,10,10a-tetrahydro-6H-benzo[c]chromen-1-ol
(HU210)23 (Figure 2D), showing only low background levels of
staining. Although in this model it does not seem to be an issue,
we cannot rule out that this staining could represent nonspecific
binding or other binding sites different from CB1 or CB2. To
confirm that the labeling was indeed due to CB1 binding and to
assess the sensitivity of this method, we compared it to antibody-
based detection. In this case, transfected HT-22 cells were
sequentially treated with 3b, streptavidin-Alexa 488, anti-CB1
antibody, and anti-rabbit Alexa 633 conjugate. The fluorescence
image shows that labeling with a small molecule chemical probe
can be as sensitive as antibody labeling for in vitro detection of
CB1 (Figure 2E,F).
3a
>5000
379 ( 90
450 ( 11
84.9 ( 0.6
1300 ( 215
>1000
3b
221 ( 8
84.7 ( 0.8
480 ( 12
25 ( 7
3c
2-AG
2-AGE
a Affinity of compounds was evaluated using HEK293EBNA cells
transfected with human CB1 or CB2 receptors, respectively, and
[3H]CP55940. Ki values are the mean ( SEM from two to four
independent experiments performed in triplicate.
5-hexynoic acid. For the different coupling reactions, conditions
and reagents were optimized for each case.
Affinity of 1a,b and 2aꢀc for CBRs was evaluated by
radioligand competitive binding assays using membranes of
HEK-293-EBNA cells transfected with human CB1 or CB2 re-
ceptors and [3H]CP55940. The affinity constant Ki was cal-
culated from the inhibitory concentration 50 (IC50), using the
ChengꢀPrusoff equation.20 All these derivatives exhibited very
poor affinity toward both receptors (Ki > 2000 nM), results that
indicate that AEA does not admit tags without a concomitant
decrease of affinity for CBRs.
Since the low affinity could be an important shortcoming in
terms of specificity and background noise for the intended
application of these probes, we decided to explore whether the
scaffolds of the other endocannabinoids 2-AG and 2-AGE
(Figure 1) could incorporate a tag without dramatically affecting
their affinity. These ligands contain two equivalent hydroxyl
groups that could potentially be used for introducing the
biotin tag. 2-AG-biotinylated probe 3a was synthesized by
consecutive esterifications of monoprotected glycerol 1021 with
N-(+)-biotinyl-6-aminohexanoic acid and arachidonic acid, fol-
lowed by further cleavage of the p-methoxybenzyl (PMB)
protecting group of intermediate 12 with trifluoroacetic acid
(TFA) (Scheme 2). On the other hand, condensation of 2-AGE
Taken together, these results confirm that the synthesized
probe 3b is a useful visualization tool able to label CB1 receptor in
vitro in a comparable manner to existing antibodies. Considering
that antibodies sometimes have important problems of specificity
and background, this new probe complements the available tools
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BRIEF ARTICLE
4-(dimethylamino)pyridine (DMAP, 1.5 equiv) was added, and the
mixture was stirred at room temperature (rt) for 48 h. Then the solution
was washed sequentially with saturated NaHCO3 and NH4Cl solutions.
The organic layer was dried (Na2SO4), filtered, and evaporated under
reduced pressure. The residue was purified by column chromatography
(DCM/MeOH, 9:1) to afford 1a (48% yield from 0.10 mmol of alcohol
5a) and 2a (17% yield from 0.10 mmol of alcohol 7).
Synthesis of 1b. To a solution of protected alcohol 6 (29 mg,
30 μmol) in anhydrous tetrahydrofuran (THF, 1.1 mL), tetrabutylam-
monium fluoride (10 mg, 3 μmol) was added. The mixture was stirred at
rt for 2 h. Then the solvent was evaporated under reduced pressure and
the residue was purified by column chromatography (DCM/MeOH,
9:1) to afford 1b (10 mg, 46%).
General Procedure for the Synthesis of 2b and 3c. To a
solution of 5-hexynoic acid (1.1 equiv) in dry DCM (2 mL/mmol), a
solution of N,N0-dicyclohexylcarbodiimide (DCC, 1.1 equiv) and
DMAP (0.2 equiv) in dry DCM (1.9 mL/mmol) was added at 0 °C
and under an argon atmosphere. The mixture was stirred at this
temperature for 30 min before a solution of alcohol 7 or 13 (1 equiv)
in dry DCM (0.3 mL/mmol) was added. The mixture was stirred at rt for
10 h, and the solvent was evaporated under reduced pressure. The
residue was dissolved in DCM and washed with 5% NaHCO3 solution.
The aqueous layer was extracted with DCM, and the organic layers were
dried (Na2SO4), filtered, and evaporated under reduced pressure. The
residue was purified by column chromatography (hexane/EtOAc, 6:4)
to afford 2b (98% yield from 0.28 mmol of alcohol 7) and 3c (44% yield
from 0.10 mmol of alcohol 13).
Synthesis of 2c. A mixture of 5-hexynoic acid (7 mg, 0.06 mmol),
1-hydroxybenzotriazole (HOBt, 8 mg, 0.06 mmol), and EDC (11 mg,
0.06 mmol) in anhydrous DMF (0.25 mL) was stirred at rt and under
an argon atmosphere for 1 h. Then a solution of amine 9 (30 mg,
0.05 mmol) in anhydrous DMF (0.5 mL) was added. The mixture was
stirred for 10 h. The solvent was evaporated under reduced pressure, and
the residue was dissolved in EtOAc and washed with 10% NaHCO3
solution. The organic layer was dried (Na2SO4), filtered, and evaporated
under reduced pressure. The residue was purified by column chroma-
tography (hexane/EtOAc, 1:1) to yield 2c (9 mg, 26%).
Synthesis of 3a. A solution of PMB protected alcohol 12 (40 mg,
0.05 mmol) in 2.7 mL of 10% TFA in DCM was stirred at rt for 5 min.
The reaction was quenched by pouring the mixture into 50 mL of
saturated NaHCO3 (pH ≈ 8). The aqueous layer was extracted with
DCM, the combined organic layers were dried (Na2SO4), and the
solvent was removed under reduced pressure. The residue was purified
by column chromatography (DCM/MeOH, 95:5 to 8:2) to afford 3a
(22 mg, 60%).
Synthesis of 3b. A suspension of N-(+)-biotinyl-6-aminohexanoic
acid (39 mg, 0.11 mmol) and HOBt (3 mg, 0.02 mmol) in anhydrous
DMF (1 mL) with activated 4 Å molecular sieves was heated until a clear
solution was obtained (60 °C, 20 min). Once the mixture was cooled to
rt, a solution of DCC (25 mg, 0.12 mmol) in DCM (0.2 mL) was added
dropwise. The mixture was stirred at rt for 3 h. Then a solution of 13
(80 mg, 0.22 mmol) and DMAP (0.12 mg, 0.001 mmol) in DMF was
added, and the mixture was stirred at 60 °C for 4 h and at rt for 24 h. The
mixture was filtered and washed with DCM/MeOH (1:1), and the
solvents were removed under reduced pressure. The residue was purified
by column chromatography (DCM/MeOH, 95:5 to 8:2) to afford 3b
(15 mg, 20%).
Figure 2. Labeling of HT-22 cells transiently transfected with CB1
receptor with probe 3b. Cells were incubated in the presence of 3b
(1 μM) for 1 h. Excess of the probe was removed, and the bound probe
was detected with streptavidin-Alexa Fluor 488 (green). Nuclei were
stained with H€oechst 33258 (blue), and cell samples were mounted and
imaged using a Zeiss inverted fluorescence microscope (A, B). To assess
specificity, the same experiment was carried out using nontransfected
cells (C) or in the presence of an excess of HU210 (D). Colabeling
studies of 3b (green) and anti-CB1 antibody detected with the
corresponding secondary antibody conjugated to Alexa 633 (red): red
emission channel (E) and merge (F). Bars: 25 μm.
for the study of the CBRs. In addition, this approach could be
extended to other GPCRs, superfamily that lacks available
antibodies for many of its members, and therefore, information
about expression of protein must be indirectly inferred from
mRNA expression and autoradiography images.
We are currently extending the number of probes for CBRs
using different synthetic cannabinoid ligands aimed at obtaining
selective probes with higher affinities. All these results will be
reported in due course.
’ EXPERIMENTAL SECTION
Chemistry. Full synthetic details and characterization data, includ-
ing HPLCꢀMS purity analysis of final compounds 1a,b, 2aꢀc, and 3a-c,
and synthesis of all intermediates are described in the Supporting
Information. All compounds tested were >95% pure by HPLC.
General Procedure for the Synthesis of 1a and 2a. To a
solution of the corresponding alcohol (1 equiv) in dry dichloromethane
(DCM, 90 mL/mmol) with activated 4 Å molecular sieves (30 mg), a
mixture of N-(+)-biotinyl-6-aminohexanoic acid (2 equiv) in DMSO
(10 mL/mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDC, 3.5 equiv) in dry DCM (10 mL/mmol) was added at ꢀ10 °C and
under an argon atmosphere. The mixture was stirred for 5 min before
’ ASSOCIATED CONTENT
S
Supporting Information. Additional synthetic proce-
b
dures, analytical and spectral data for all intermediates, spectral
data and HPLCꢀMS results for all final compounds (1a,b, 2aꢀc,
3aꢀc), binding assays, and cell visualization experiments.
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BRIEF ARTICLE
This material is available free of charge via the Internet at http://
pubs.acs.org.
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’ AUTHOR INFORMATION
Corresponding Author
*For M.L.L.-R.: phone, +34 913944239; fax, +34 913944103; e-mail,
mluzlr@quim.ucm.es. For S.O.-G.: phone, +34 913944894; fax, +34
913944103; e-mail, siortega@quim.ucm.es.
’ DEDICATION
†Dedicated to the memory of Professor Rafael Suau.
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’ ACKNOWLEDGMENT
This work was supported by grants from the Spanish Minis-
terio de Ciencia e Innovaciꢀon (MICINN, Grant SAF2010-
22198-C02-01, and predoctoral fellowship to L.M.-C.) and
Comunidad Autꢀonoma de Madrid (Grant S-SAL-249-2006).
The authors thank the Spanish Society of Medicinal Chemistry
(SEQT) and GlaxoSmithKline for the Young Researcher Award
to L.M.-C. S.O.-G. is a Ramon y Cajal Scholar funded by
MICINN and the European Social Fund.
’ ABBREVIATIONS USED
ABP, activity-based probe; AEA, anandamide; 2-AG, 2-arachidonoyl-
glycerol; 2-AGE, 2-arachidonyl glyceryl ether; CBR, cannabinoid
receptor; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide;
GPCR, G-protein-coupled receptor;HOBt, 1-hydroxybenzotria-
zole;SEM, standard error of the mean; rt, room temperature
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