Toward the development of bivalent ligand probes of cannabinoid CB1 and Orexin OX1 receptor heterodimers

Article abstract of DOI:10.1021/ml4004759

Cannabinoid CB1 and orexin OX1 receptors have been suggested to form heterodimers and oligomers. Aimed at studying these complexes, a series of bivalent CB1 and OX1 ligands combining SR141716 and ACT-078573 pharmacophores were designed, synthesized, and tested for activity against CB1 and OX1 individually and in cell lines that coexpress both receptors. Compound 20 showed a robust enhancement in potency at both receptors when coexpressed as compared to individually expressed, suggesting possible interaction with CB1-OX1 dimers. Bivalent ligands targeting CB1-OX1 receptor dimers could be potentially useful as a tool for further exploring the roles of such heterodimers in vitro and in vivo.

Full text of DOI:10.1021/ml4004759

Letter
pubs.acs.org/acsmedchemlett
Toward the Development of Bivalent Ligand Probes of Cannabinoid
CB1 and Orexin OX1 Receptor Heterodimers
David A. Perrey, Brian P. Gilmour, Brian F. Thomas, and Yanan Zhang*
Research Triangle Institute, Research Triangle Park, North Carolina 27709, United States
S
* Supporting Information
ABSTRACT: Cannabinoid CB1 and orexin OX1 receptors have been suggested to form heterodimers and oligomers. Aimed at
studying these complexes, a series of bivalent CB1 and OX1 ligands combining SR141716 and ACT-078573 pharmacophores
were designed, synthesized, and tested for activity against CB1 and OX1 individually and in cell lines that coexpress both
receptors. Compound 20 showed a robust enhancement in potency at both receptors when coexpressed as compared to
individually expressed, suggesting possible interaction with CB1-OX1 dimers. Bivalent ligands targeting CB1-OX1 receptor
dimers could be potentially useful as a tool for further exploring the roles of such heterodimers in vitro and in vivo.
KEYWORDS: Cannabinoid, orexin, heterodimer, bivalent ligands
anticipated that these bivalent ligands, provided that they have
growing list of biochemical and functional evidence
Asuggests that G protein-coupled receptors (GPCRs), the appropriate linker length, will bind to the receptor with
high affinity due to the small containment volume for the
second pharmacophore, resulting in thermodynamically more
favorable binding interactions than the monovalent binding of
two molecules. Such an approach may result in enhanced
selectivity and efficacy and improved safety relative to drugs
that address only a single target.28 For example, Portoghese et
al. have reported a range of homo- and heterodimeric opioid
ligands with varying linker length, some of which displayed
significantly greater potency and selectivity compared to their
monomeric congeners.29 In addition, bivalent ligands are
capable of inducing the formation of heterodimers, thereby
shifting the equilibrium between monomers, homomers, and
heteromers as demonstrated in recent studies on MOP and
CCK2 receptors.30
In this study we present our efforts in developing bivalent
ligands targeting CB1 and OX1 heterodimers. The key
elements of ligand design are the selection of the individual
ligands, the identification of the point of attachment for the
linker, and the nature and length of the linker. The CB1
pharmacophore selected is 1,5-diarylpyrazole, based on the
CB1 antagonist/inverse agonist SR141716 (1). We have
previously demonstrated the efficacy of symmetric CB1 bivalent
historically considered monomers, form and function as
homo- and heterodimers, or even higher-ordered oligo-
mers.1−5 These receptor dimers/oligomers often display unique
ligand binding, distinct phenotypic trafficking, and altered
signaling properties compared to their individual mono-
mers.3,6,7 A wide variety of GPCRs, including cannabinoid,8−11
opioid,12−16 dopamine,17−19 and serotonin receptors,20 have
been demonstrated to form dimers or oligomers both within
and across receptor types. In particular, the CB1 and OX1
receptors were shown to dimerize in cotransfected cells by
coexpression, coimmunoprecipitation, and resonance energy
transfer studies.21,22 Moreover, these two receptors have
overlapping distribution in certain brain areas such as the
lateral hippocampus, and hypothalamic CB1 mRNA was found
to be coexpressed with prepro-orexin.23,24 Finally, the in vivo
functional cross-talk between these two receptors has been
demonstrated in the finding that pretreatment with subeffective
doses of the CB1 antagonist SR141716 attenuated the
orexigenic actions of orexin A in rats.25
GPCR dimerization has been widely reported in primary
cultures and, more recently, has also been demonstrated in
native tissues.26,27 However, their in vivo existence and
functional significance have yet to be fully understood.4 One
approach to study these complexes is to develop bivalent
ligands that preferentially interact with the receptor hetero-
dimers to probe receptor heterodimerization in vivo.28 It is
Received: November 19, 2013
Accepted: March 25, 2014
© XXXX American Chemical Society
A
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a
ligands based on this scaffold targeting the homodimeric CB1
receptor (2),31 in which linkers of 15 atoms seemed to be
optimal within the series tested. The 1,2,3,4-tetrahydroisoqui-
noline scaffold derived from the orexin antagonist almorexant
(ACT-078573, 3) was selected as the OX1 pharmacophore.
Compound 4, a lead compound in the discovery of 3, showed
reasonable potency at and selectivity for the OX1 receptor.32,33
There is limited structure−activity relationship (SAR)
information on this tetrahydroisoquinoline scaffold, and
determining a suitable point of attachment was required.
Therefore, we performed focused SAR studies on compound 4
at three locations (Figure 1).
Scheme 2. Modifications at Three Locations
a
Reagents and conditions: (a) butyric acid, BOP, iPr2EtN, CH2Cl2;
(b) 1-bromohexane, K2CO3, Bu4NI, DMF; (c) (i) ethyl bromophe-
nylacetate, iPr2EtN, Bu4NI, DMF; (ii) 2 N NaOH, EtOH; (d) 1-
aminoheptane, BOP, iPr2EtN, CH2Cl2.
hydroxyl of the 1-benzyl group (9b) gave the esters 10 and 12,
respectively. Similarly, alkylation of 9a or 9b with 1-
bromohexane provided respectively the ethers 11 and 13. For
the α-phenyl analogue (15), a slightly different route than that
of 9c was followed. N-Alkylation of 8c with the α-bromoester
followed by hydrolysis of resulting ester provided the acid 14. A
final amide coupling gave 15.
Figure 1. CB1 and OX1 ligands and a CB1 bivalent ligand.
Activity of these substituted tetrahydroisoquinolines at OX1
was determined in a calcium mobilization based functional
assay using RD-HGA16 cell lines overexpressing the OX1
receptor, as previously described.33,34 The apparent dissociation
constant Ke was calculated, and the values are the mean of at
least three independent experiments performed in duplicate
(Table 1). The results were then used to determine the optimal
linker attachment positions to construct bivalent ligands.
The tetrahydroisoquinoline-based orexin antagonists were
prepared following procedures in the literature and developed
in our laboratory (Scheme 1).32,33 Thus, the phenethylamines
a
Scheme 1. Synthesis of OX1 Antagonists
Table 1. OX1 Antagonist Activity
number
Ke (nM) SEM
4
200 50
590 230
78 24
9c
10
11
12
13
15
310 80
43
4
120 20
22 10
a
Reagents and conditions: (a) HBTU, iPr2EtN, DMF; (b) (i) POCl3,
The 7-position of the tetrahydroisoquinoline has been shown
to favor substitutions with more steric demanding groups.32,33
Indeed, replacement of the methoxyl group with a butyl ester
gave 10, which displayed increased potency over 4. The 7-
hexylether 11 was slightly less potent than 4. These results
confirm tolerance of steric bulk at this position. For the 4-
position on the 1-benzyl group, both ester 12 and the 4′-
hexylether 13 were more potent than the parent compound,
suggesting this position as a viable point for linker attachment.
Finally, the N-heptyl derivative 9c was lower in potency, but in
general, the change was tolerated. Further encouraging results
came from the α-phenyl compound 15, which showed the
greatest potency of the series despite a large heptyl group.
Together, the results showed that all three positions could
potentially be suitable for ligand attachment.
toluene; (ii) NaBH4, MeOH; (c) R3NHCOCH2Br, iPr2EtN, Bu4NI,
DMF.
5a−b and phenylacetic acid derivatives 6a−b were coupled
using HBTU to give the amides 7a−c. Bischler−Napieralski
reaction using phosphorus oxychloride gave the cyclization to
the dihydroisoquinoline, which could be readily reduced to the
tetrahydroisoquinoline 8a−c using sodium borohydride. The
amine was then alkylated with the α-bromoacetamide, prepared
by coupling of bromoacetylbromide and the corresponding
amine,33 to give 9a−c.
Alterations at the three proposed locations (9a−c) were
achieved as shown in Scheme 2. Thus, acylation with butyric
acid of the 7-hydroxyl of the tetrahydroisoquinoline (9a) or the
B
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Synthesis of the bivalent ligands with linkers at the three
respective positions was accomplished in an analogous fashion
to the monomer synthesis (Schemes 3−5). For bivalent ligands
acid 23. Coupling between 23 and 16d using BOP as coupling
agent provided bivalent ligand 24.
Finally, for bivalent ligand with the α-phenylacetamide
linkage (26), previously prepared amine 2531 was coupled to
the acid 14 with BOP as the coupling agent to give bivalent
ligand 26 (Scheme 5).
a
Scheme 3. Synthesis of Bivalent Ligand 18
a
Scheme 5. Synthesis of Bivalent Ligand 26
a
Reagents and conditions: (a) 14, BOP, iPr2EtN, DMF.
a
Reagents and conditions: (a) (i) Br-(CH2)6-CO2Et, Et3N, toluene;
The synthesized bivalent ligands were first evaluated for their
activity at the CB1 and OX1 receptors, respectively, in calcium
functional assays in RD-HGA16 cell lines stably expressing
either CB1 or OX1 receptors (Table 2). CP55,940 was
employed as the agonist for activity at the CB1 receptor and
orexin A at the OX1 receptor. The bivalent ligands showed a
significant drop in activity over the individual components at
either CB1 or OX1 receptors. In the CB1 cell lines, all the
bivalent ligands were active but showed reduced activity at the
CB1 receptor. Whereas SR141716 shows a Ke of 1 nM, the best
activity shown in this series is 1100 nM (21). Surprisingly, they
were mostly inactive at the OX1 receptor in cells singly
expressing the OX1 receptor.
Cells coexpressing both CB1 and OX1 receptors were
obtained by stably expressing OX1 receptors in RD-HGA16
cells that already stably express CB1 receptors. The expression
level of the newly transfected OX1 receptors was determined in
radioligand binding studies using [125I]-orexin A, which
demonstrated 293.6 pmol OX1 receptors/106 cells. These
results are in accordance with other dual-expressing systems
that have been shown to readily form CB1-OX1 receptor
heterodimers, where Hilairet et al. reported Bmax of 338 fmol/
106 cells for the OX1 receptor in their cells coexpressing CB1
and OX1 receptors.22
(ii) HCHO, Na(AcO)3BH, 1,2-DCE; (iii) 2 N NaOH, EtOH; (b) 9a,
BOP, iPr2EtN, CH2Cl2.
with the 7-position ester linker (18, Scheme 3), synthesis began
with amines 16a−d, which were readily synthesized following
procedures previously described by us.31 Alkylation of 16a−d
with ethyl 7-bromoheptanoate, followed by N-methylation via
reductive amination and ester hydrolysis gave the acids 17a−d.
Finally, reaction of 17a with intermediate 9a via a BOP
mediated coupling gave ester-linked bivalent ligand 18.
Bivalent ligands linked at the 1-benzyl position (19−22 and
24) were prepared as shown in Scheme 4. Similar to the
preparation of 18, bivalent ligands 19−22 with the ester linkage
were synthesized by amide coupling of 17a−d to 9b. The
synthesis of 24 started with alkylation of phenol 9b with ethyl
7-bromoheptanoate in the presence of potassium carbonate in
DMF, followed by the hydrolysis of the resulting ester to give
a
Scheme 4. Synthesis of Bivalent Ligands 19−22 and 24
In these dual-expressing cells, SR141716 showed CB1
potency that is comparable to that in the cells singly expressing
the CB1 receptor (Table 2). Similarly, SB334867, an OX1
selective antagonist, showed almost identical potency in these
cells and cells singly expressing the OX1 receptors. As a control,
SR141716 did not block orexin A activity at the OX1 receptor,
and conversely, SB334867 did not block CP55940 activity at up
to 10 μM in these cells (Table 2). These data confirm that both
the CB1 and OX1 receptors function properly in the dual-
transfected cells and that there is no change in the receptor
intrinsic pharmacological properties.
The bivalent ligands were then evaluated in the dual-
expressing cells for activity at the CB1 or OX1 receptors. In
general, the bivalent ligands were active at the CB1 receptor,
but little or no activity was observed at the OX1 receptor,
showing a trend similar to that seen in cells singly expressing
these receptors. This suggests that the OX1 receptor, unlike the
CB1 receptor, may have limited tolerance of steric bulk.
Alternatively, since almorexant has been shown to be a
noncompetitive antagonist at the OX2 receptor,35 an allosteric
binding site could also be present at the OX1 receptor with
these bivalent ligands that negatively impact receptor binding.
a
Reagents and conditions: (a) 9b, BOP, iPr2EtN, CH2Cl2; (b) (i) Br-
(CH2)6-CO2Et, K2CO3, DMF; (ii) 2 N NaOH, EtOH; (c) 16d, BOP,
iPr2EtN, DMF.
C
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ACS Medicinal Chemistry Letters
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Table 2. Activity of Bivalent Ligands against CB1, OX1, and Coexpressed Receptors
Ke (nM) SEM
No.
linker length (atoms)
CB1 vs CP55,940
CB1-OX1 vs CP55,940
CB1-OX1 vs Orexin-A
OX1 vs Orexin-A
1
1.1 0.1
1.0 0.6
>10000
>10000
>10000
SB334867
>10000
32 20
>10000
45 12
18
19
20
21
22
24
26
15
15
18
20
19
19
15
2100 140
1800 710
1800 1300
1100 500
1800 680
8000 2800
3000 1100
1200 270
610 16
110 45
410 46
420 280
580 260
230 11
8800 2300
8460 680
>10000
>10000
5900 1300
>10000
>10000
4500 2200
>10000
3500 1300
8600 1200
5500 1300
>10000
Interestingly, an enhancement of CB1 activity was observed
with all bivalent ligands compared to cells singly expressing the
CB1 receptor. Specifically, the CB1 potency is enhanced in all
cases by approximately 2−5-fold, which is similar to the
responses seen in our CB1 homodimer study.31 On the
contrary, compound 20 was the only compound that showed a
slight potency enhancement at the OX1 receptor.
CB1 and OX1 in a cell line coexpressing both receptors
suggesting possible interaction with both receptors. Clearly,
bivalent ligands with improved potency, particularly at the OX1
binding site, are needed for further studies. Once available,
these bivalent ligands can potentially be exploited to further
probe the interaction of these two receptors and may lead to
novel therapeutic approaches to neurological disorders
mediated by these receptors.
During bivalent ligand studies, normally a decrease and then
increase in affinity/potency with the elongation of the linker is
present when these bivalent ligands simultaneously bind to
both receptors. Such a trend was present in our CB1
homobivalent ligands and other studies.29,31,36,37 The effect of
linker length on activity was investigated in the 4′-ester series
(19−21). Interestingly, compound 20 with an 18-atom linker
was more potent than 19 (15 atoms) and 21 (20 atoms) at the
CB1 receptor. At the OX1 receptor, 20 was the only compound
that showed activity, though in the micromolar range.
Moreover, 20 in fact showed potency enhancement at both
the CB1 and the OX1 receptor, though the effect is minimal at
the OX1 receptor. While these results may suggest binding of
20 to both receptors, more potent bivalent ligands are clearly
needed before a more definitive answer can be obtained.
The nature and position of the linker should also be
considered. We have previously observed in homodimers of
CB1 antagonists that hydrophobic linkers are generally
preferred and peptide-based linkers show poor activity. In
this case, the glycol linkers (22 and 24) showed comparable
activity to those with hydrophobic linkers at the CB1 receptor.
The 4-position of the pyrazole on SR141716 was successfully
employed in building homobivalent ligands. These CB1-OX1
bivalent ligands were linked via this position and again showed
reasonable activity at the CB1 receptor. On the orexin
pharmacophore, three different positions of attachment for
the linker were identified that tolerated steric bulk prior to the
synthesis of the bivalent ligands. However, the fact that all the
bivalent ligands have significantly reduced or abolished activity
at the OX1 receptor indicates that all of these positions only
tolerate limited size. While the α-phenyl acetamide (15) was
the most active monomer at the OX1 receptor, the resulting
bivalent ligand 26 showed diminished activity in coexpressing
CB1-OX1 cells. In general, the measured activity was low so it
is difficult to see any appreciable potency enhancement.
ASSOCIATED CONTENT
* Supporting Information
Synthesis and characterization of target compounds and
radioligand binding studies. This material is available free of
charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
* (Y.Z.) E-mail: yzhang@rti.org. Tel: 919-541-1235. Fax: 919-
541-6499.
■
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
This work was supported by National Institute on Drug Abuse,
National Institutes of Health, USA (grant nos DA026582 and
DA032837).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Tiffany Langston, Keith Warner, Dr. Ann Decker,
and Dr. Elaine Gay for their valuable technical assistance.
ABBREVIATIONS
■
BOP, (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium
hexafluorophosphate; CB1, cannabinoid receptor type 1;
HBTU, O-benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexa-
fluoro-phosphate; HPLC, high performance liquid chromatog-
raphy; OX1, orexin 1 receptor; OX2, orexin 2 receptor; SAR,
structure−activity relationship
In summary, we have explored the tetrahydroisoquinoline
scaffold with regard to finding potential sites for linkers and,
based on the findings, developed a series of bivalent CB1-OX1
agents. While all the bivalent ligands showed reasonable
potency at the CB1 receptor, most of them were inactive up
to 10 μM at the OX1 receptor. One compound (20) with an
18-atom linker showed enhancement in activity against both
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