C3-Heteroaroyl cannabinoids as photolabeling ligands for the CB2 cannabinoid receptor

Article abstract of DOI:10.1016/j.bmcl.2012.06.013

A series of tricyclic cannabinoids incorporating a heteroaroyl group at C3 were prepared as probes to explore the binding site(s) of the CB1 and CB2 receptors. This relatively unexplored structural motif is shown to be CB2 selective with K_i values at low nanomolar concentrations when the heteroaromatic group is 3-benzothiophenyl (41) or 3-indolyl (50). When photoactivated, the lead compound 41 was shown to successfully label the CB2 receptor through covalent attachment at the active site while 50 failed to label. The benzothiophenone moiety may be a photoactivatable moiety suitable for selective labeling.

Full text of DOI:10.1016/j.bmcl.2012.06.013

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5322–5325
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
C3-Heteroaroyl cannabinoids as photolabeling ligands for the CB2
cannabinoid receptor
Darryl D. Dixon a, Marcus A. Tius a, , Ganesh A. Thakur b, Han Zhou b, Anna L. Bowman b,
⇑
Vidyanand G. Shukla b, Yan Peng b, Alexandros Makriyannis b,
⇑
a Department of Chemistry, University of Hawaii at Manoa, 2545 The Mall, Honolulu, HI 96822, USA
b Center for Drug Discovery, Northeastern University, 360 Huntington Avenue, 116 Mugar Life Sciences Building, Boston, MA 02115, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of tricyclic cannabinoids incorporating a heteroaroyl group at C3 were prepared as probes to
explore the binding site(s) of the CB1 and CB2 receptors. This relatively unexplored structural motif is
shown to be CB2 selective with Ki values at low nanomolar concentrations when the heteroaromatic
group is 3-benzothiophenyl (41) or 3-indolyl (50). When photoactivated, the lead compound 41 was
shown to successfully label the CB2 receptor through covalent attachment at the active site while 50
failed to label. The benzothiophenone moiety may be a photoactivatable moiety suitable for selective
labeling.
Received 14 April 2012
Revised 3 June 2012
Accepted 5 June 2012
Available online 15 June 2012
Keywords:
CB2
Cannabinoid
Ó 2012 Elsevier Ltd. All rights reserved.
Ligand-assisted protein structure
Photolabeling
Photoactivatable group
The known phytocannabinoids have long been known to exhibit
only moderate receptor binding affinities and signaling profiles
in vitro, yet they exhibit substantial potency in vivo.1,2 The best
sisted Protein Structure (LAPS).11 The present work describes our
efforts to develop a new class of photoaffinity labels thus extend-
ing current work in our laboratory aimed at characterizing
ligand–cannabinoid receptor binding motifs.
known of these classical cannabinoids,
D
9-tetrahydrocannabinol
(D
9-THC) binds with nearly equal affinity3–5 to the two known
Earlier work from our laboratories had shown that 3-naphthoyl
and 3-naphthylmethyl tricyclic cannabinoids have moderate affin-
ities for the CB1 receptor.12 More recently Moore and coworkers13
G-protein coupled cannabinoid receptors,6,7 CB18 and CB2.9 Sub-
stantial available SAR data for classical cannabinoids10 have shown
that the northern b-C9 hydroxyl, C1 phenolic hydroxyl and the C3
side chain are key pharmacophores in determining receptor affin-
ity and pharmacological potency for both CB1 and CB2. The design
of novel CB1/CB2 analogues possessing higher affinities and selec-
tivities can be based on structural information related to the inter-
action of cannabinergic ligands with their respective receptors. In
the absence of either X-ray crystallographic or NMR data, informa-
tion on the structural features of the ligand-cannabinoid receptor
binding motifs can be gained through the use of carefully designed
high-affinity electrophilic or photoactivatable probes. Such com-
pounds interact with the receptor at or near the binding site and
attach covalently to one or more amino acid residues. Identifica-
tion of the attachment site(s) can subsequently be accomplished
using targeted mutations within the receptor or by using LC/MS/
MS to characterize the ligand–receptor complex. This approach
that was developed in our laboratory combines the use of receptor
mutants and mass spectrometry and was designated as Ligand-As-
showed that the tricyclic
D
8-THC analogue 1 bearing a benzoyl unit
at C3 is CB2 selective while we have shown that the bicyclic ana-
logues such as 2 are also selective for CB2 (Fig. 1).14 We have
now designed and synthesized a series of tricyclic cannabinoids
bearing a heteroaromatic group with a carbonyl spacer at C3. De-
sign of our novel compounds incorporates the northern b-hydroxyl
pharmacophore as well as an arylphenone component, a photoac-
tivable group capable of transforming the ligand into a GPCR cova-
lent label.15 Earlier work from the laboratories of Martin and co-
workers16a and from our laboratory16b has shown that the northern
b-hydroxyl enhances affinity for both receptors while imparting
the molecule with enhanced polar properties and water solubility.
⇑
Corresponding author.
E-mail address: tius@hawaii.edu (M.A. Tius).
Figure 1. Aroyl cannabinoids.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.06.013

D. D. Dixon et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5322–5325
5323
Scheme 1. Reagents and conditions: (a) pTsOH, CHCl3/acetone (4/1), 0 °C, 1 h; rt,
1 h; (b) CH2Cl2, cat. DMAP, pyr, Ac2O, 0 °C to rt, 12 h; (c) KOH, MeOH, 0 °C, 1.5 h;
68% from 4 to 5; (d) TMSOTf, MeNO2, 0 °C, 2.5 h; (e) PhNTf2, Et3N, CH2Cl2, 0 °C to rt;
57% from 7; (f) NaBH4, MeOH, rt, 1 h; b/
a ca. 95/5, 97%; (g) MeOCH2Cl, iPr2NEt,
CH2Cl2, 0 °C to rt, 2.5 h; 93%.
Scheme 2. Reagents and conditions: (a) DMF, CO, LiCl, BHT, 4 Å MS, 110 °C,
ArSnBu3, PdCl2(dppf)ꢀCH2Cl2, 24 h.
Our SAR approach involves the attachment of different aryl groups
to the 3-keto group of the tricyclic cannabinoid moiety.
Chemistry. Utilizing a strategy that has been developed in our
group,17,18 we prepared bicyclic intermediate 7 via the acid cata-
lyzed condensation between persilylated phloroglucinol 6 and a
mixture of diacetates 4 and 5 (Scheme 1) following a general ap-
proach that was applied to the synthesis of nabilone by the Eli Lilly
group.19 It should be noted that persilylating phloroglucinol was
essential to improve solubility in the reaction medium so as to en-
sure a high yield of 7. Ketone 7 was subsequently treated with
TMSOTf to promote the rearrangement-cyclization to yield tricy-
clic compound 8. Selective conversion of the C3 phenolic hydroxyl
group to the corresponding triflate led to 9 in 57% overall yield
from 7. Reduction of ketone 9 with NaBH4 led to a 95/5 mixture
of C9 diastereoisomers in 97% yield. Simultaneous protection of
the phenolic and aliphatic hydroxy groups in 10 as methoxymethyl
ether groups (MOM) led to 11 in 93% yield.
Scheme 3. Reagents and conditions: (a) CH2Cl2, DIBAL, ꢁ78 °C; 96%; (b) ArBr, n-
BuLi, THF, ꢁ78 °C; (c) MnO2, CH2Cl2; 30, Ar = 3-fluorophenyl, 74%; 31, Ar = 3-
(trifluoromethyl)phenyl, 81%; 32, 52% (2 steps); (d) KOH, MeOH, indole; (e) DMF,
NaH, MeI; 97%.
As in earlier work,18 we wanted to prepare all compounds from
11, a common advanced intermediate, utilizing a cross coupling
procedure. The carbonylative Stille coupling was an attractive op-
tion for the installation of the heteroaroyl unit due to the large
variety of commercially available heteroaryl stannanes and their
relative ease of preparation from their corresponding aryl bromide
or iodide. Treatment of triflate 11 with a slight excess of heteroaryl
stannane, LiCl, 4 Å molecular sieves (MS), 2,6-di-tert-butyl-4-
methylphenol (BHT) and catalytic 1,10-bis(diphenylphosphino)fer-
rocene palladium(II) dichloride dichloromethane complex
(PdCl2(dppf)ꢀDCM) under an atmosphere of CO at 110 °C in N,N-
dimethylformamide (DMF) for 24 h yielded heteroaroyl cannabi-
noids 12-26 in moderate to excellent yields (Scheme 2). Under
these reaction conditions none of the direct coupling of triflate
with stannane was observed.
In cases in which the stannane was either not commercially
available or was unreactive, a slightly modified synthetic proce-
dure was used in order to prepare the heteroaroyl cannabinoids.
We have shown in earlier work that triflate 11 can be converted
to nitrile 27 (Scheme 3) in 96% yield.18 Reduction of 27 to alde-
hyde 28 with diisobutylaluminum hydride (DIBAL) took place in
96% yield. Nucleophilic addition of the aryllithium reagents de-
rived from 3-bromofluorobenzene and 3-bromobenzotrifluoride
to 28 followed by oxidation of the respective products with ac-
tive manganese dioxide led to phenones 30 and 31 in 74% and
81% yield, respectively. Direct addition of various aryllithium or
arylmagnesium compounds to nitrile 27 failed to produce the de-
sired phenones, necessitating the two-step procedure. Exposure
of 28 to indole in methanolic KOH followed by oxidation of the
resulting alcohol led to 32 in 52% yield for the two steps. N-
Methylation of 32 with NaH and CH3I in DMF furnished 33 in
97% yield.
Scheme 4. Reagents and conditions: (a) TMSBr, CH2Cl2, ꢁ40 °C, 1.5 h; 0 °C, 1 h.
Removal of the methoxymethyl ether protecting groups from
12 to 26 and 30 to 33 with TMSBr led to 3 and 34–51 in moderate
to good yields (Scheme 4). The low yield for deprotection of the
furyl and thiophenyl compounds can be attributed to the high
nucleophilicity of the electron rich aromatic ring. Reaction with
the methoxymethyl bromide that is generated during deprotection
may be responsible for the appearance of byproducts. Since poor
yields were also observed in these cases in the presence of
poly(4-vinylpyridine), it is unlikely that the poor yields of depro-
tected products can be attributed to the presence of strong acid.

5324
D. D. Dixon et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5322–5325
Table 1
Binding Affinities (Ki) for CB1 and CB2 cannabinoid receptors
Compound
Ki (nM)a
rCB1
mCB2
hCB2
3
968
247
587
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
>1000
>1000
1356
2880
>1000
1037
156.6
1254
>1000
>1000
>1000
>1000
>1000
>1000
460
>1000
>1000
163.3
169.2
>1000
525
—
—
209.4
118.2
—
1551
113.1
124.8
>1000
—
>1000
>1000
>1000
—
152.1
34.2
>1000
>1000
>1000
>1000
>1000
>1000
370
45.8
60.4
406
—
61.7
1045
3270
37.3
158.6
3006
a
Binding affinities for CB1 and CB2 were determined using rat brain (CB1) or
membranes from HEK293 cells expressing mouse or human CB2 and [3H]CP-55,940
as the radioligand following previously described procedures.20 Ki values for these
compounds were obtained from one experiment (8 point) run in triplicate when
experiments using the two point data (in triplicate) showed Ki values below
1000 nM.
Other common conditions to remove methoxymethyl ethers such
as methanolic HCl or ZnBr2/n-BuSH, which served us well in the
past, also failed to improve the yields.
Structure–Activity Relationships. Earlier work from our labo-
ratory12 as well as from the Moore and co-workers13 explored
the role of aroyl groups as substitutions at the C-3 position in the
classical cannabinoid in lieu of the traditionally used alkyl side
chain. It was shown13 that introduction of a 3-benzoyl substituent
Figure 2. Accessible conformers within 6 kcal molꢁ1 of the global energy minimum
for 40 (magenta), 41 (orange), and AM755 (blue). Analogues are shown superim-
posed at their aromatic rings. The global minimum energy conformer for each
compound is shown in stick representation.21
in a
D
8-THC tricyclic structure results in a compound (1) with high
affinity for CB2. We have now extended the limited available SAR
in this class of cannabinoids with a series of analogues carrying
the cannabinoid receptor-favorable 9b-OH group10,15as well as dif-
ferent heteroaryl groups at the 3-position. These structural modifi-
cations were aimed at identifying novel ligands and photoaffinity
probes for the CB2 cannabinoid receptor with improved overall
profiles. Our work has led to the discovery of a novel effective
covalent probe for this receptor. The SAR of all novel arylphenone
analogues was evaluated by measuring their respective affinities
for the rat CB1 (rCB1), mouse CB2 (mCB2) and human CB2
(hCB2) receptors (Table 1). All synthesized novel analogues exhib-
ited reduced affinities for both CB1 and CB2 receptors when com-
pared with the earlier synthesized benzophenone analogue 1.13
All novel 9b-OH analogues were shown to have reduced binding
poration of a meta fluorine substituent in the aromatic ring (48)
improved binding at CB1 with a slight loss in binding affinity at
mCB2. Replacement of the fluorine group with a lipophilic trifluo-
romethyl group (49) gave encouraging results. This compound
exhibited significantly improved affinity for both CB receptors
(Ki = 61.7 nM at rCB1, 45.8 nM at mCB2 and 37.3 nM at hCB2).
Our most promising results with regard to CB2 affinities and
selectivities were observed with analogues carrying fused bicyclic
3-heteroaroyl substitutions. The best were those carrying 3-benzo-
furan (37; Ki mCB2 169.2 nM rCB1/mCB2 = 17-fold), 3-benzothio-
pheno (41; Ki mCB2 34.2 nM rCB1/mCB2 = 37-fold) and 3-indole
(50; Ki mCB2 60.4 nM rCB1/mCB2 = 17-fold) substituents while
those with 2-benzothiopheno (40) or 3-(N-methylindole; (51)
had somewhat reduced affinities or CB2 selectivities. The binding
data of the analogues included in this study point to steric factors
playing a key role in determining the effectiveness of the 3-aroyl
pharmacophore’s ability to interact with each of the two receptors.
An interesting observation in our SAR is the significant differ-
ence in mCB2 affinities between the lead 3-benzothiophene ana-
logue 41 and its 2-regioisomer 40. To better interpret these
interesting results we have explored the rotational space available
by these two substituents. We also included in our comparison re-
sults for the earlier published 3-vinyladamantyl analogue AM755
that also exhibited CB2 selectivity.22 A comparison of the computa-
tional data (Fig. 2) points out the steric similarities between the
respective pharmacophores for the two analogues with favorable
CB2 affinities and selectivities (41, AM755) and the distinct
differences when their conformational spaces are compared with
affinities for both receptors when compared to the
D
8-THC ana-
logue 1. The reason for this observation is unclear. Arguably, the
additional 9b-OH group of analogue 3 may be orienting the planar
benzophenone side chain differently in the receptor hydrophobic
pocket to cause an overall unfavorable interaction. Our SAR data
show that all analogues containing 10-five-membered heteroaro-
matic ring (34, 35, 38, 39, 42, and 43) exhibited significantly dimin-
ished affinities for both CB1 and CB2 receptors. This is presumably
due to lack of sufficient (hydrophobic) interaction of this ring with
the hydrophobic pocket of the receptor which, in general, is a
determining factor for the affinity, potency and selectivity of
classical cannabinoids.10 To further probe the interaction of this
3-benzophenone group, we incorporated a nitrogen atom into var-
ious positions of the aromatic ring (44–47). All of these compounds
exhibited further reduction in affinity for both CB receptors. Incor-

D. D. Dixon et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5322–5325
5325
will be used in future studies to obtain information on the binding
motifs of the arylphenone cannabinoid analogues for the CB2
receptor. Additionally, our results suggest that the 3-benzothio-
pheno group is an excellent moiety to be incorporated in cannabin-
ergic photolabeling probes.
Acknowledgments
Acknowledgment is made to The National Institute on Drug
Abuse (DA07215, 2P01 DA09158) for generous support of this
research.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.06.
013.
Figure 3. Compound 41 inhibits the specific binding of [3H]CP-55,940 to mCB2
receptor. HEK293 cell membranes expressing wild type mouse cannabinoid
receptor 2 (mCB2) were suspended in TME buffer (25 mM Tris-Base, 5 mM MgCl2,
1 mM EDTA, pH 7.4) with 0.1% BSA, containing 0.34 lM 41 (i.e., 10-fold Ki of 41 for
References and notes
WT mCB2). A membrane devoid of 41 was used as a parallel control. Incubations of
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wavelength ultraviolet lamp at 365 nm in ice-cold silanized Petri dishes.24 The
membranes were washed once with 1% BSA TME buffer to remove unbound ligands,
and once with TME buffer (no BSA) to remove BSA. Saturation binding assays were
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the analogue (40) with lower affinity and selectivity for mCB2. The
limited binding data included here did not allow us to carry out a
full exploration of the arylphenone pharmacophore for the CB2
receptor. This will be attempted in future work when a larger data-
base becomes available. However, our computational exercise
underscores the steric factors associated with mCB2 binding and
provide a basis for the design of higher affinity analogues.
Photolabeling of mCB2. To explore the value of this class of
cannabinoid analogues as photolabeling reagents for the CB recep-
tors we tested some of our compounds for their abilities to interact
covalently with the mCB2 receptor. The experiment was carried
out using membrane preparations obtained from a HEK293 cell
line expressing mCB2. We used methodology developed in our lab-
oratory with cannabinergic ligands carrying different photoactivat-
able groups23 while employing conditions reported earlier for
labeling the NK-1 receptor with ligands incorporating a benzophe-
none moiety.24 Of the heteroaryl benzophenones tested, the two
benzothiophenones (40 and 41) exhibited the highest ability to
photolabel the mCB2 receptor (77% and 67% respectively; Fig. 3).
The meta-trifluoro analogue 49 also labeled the receptor, however,
less effectively.25 Conversely, the 3-indolyl analogue (50) failed to
label mCB2. These very successful results confirmed the value of
the 3-arylphenone moieties as useful photolabels for the CB2
receptors.
Conclusions. In this SAR study we explored the value of can-
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the 3-alkyl chains found in the phytocannabinoid structures as po-
tential photoaffinity ligands for the mCB2 receptor. The lead com-
pound 41 provided evidence that the 3-benzothiophene analogue
had the highest affinity and selectivity for mCB2. Our results
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pharmacophoric moiety for the CB2 receptor and provide the basis
for the design of later generation analogues with improved affinity
profiles. Importantly, we demonstrated that 41 is capable of phot-
olabeling the mCB2 receptor in excellent yields. This compound
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25. Analogue 49 was used to label hCB2. The extent of labeling was 30%.