Divalent cannabinoid-1 receptor ligands: A linker attachment point survey of SR141716A for development of high-affinity CB1R molecular probes

Article abstract of DOI:10.1016/j.bmcl.2019.126644

The cannabinoid-1 receptor (CB1R) inverse agonist SR141716A has proven useful for study of the endocannabinoid system, including development of divalent CB1R ligands possessing a second functional motif attached via a linker unit. These have predominantly employed the C3 position of the central pyrazole ring for linker attachment. Despite this precedent, a novel series of C3-linked CB1R-D2R divalent ligands exhibited extremely high affinity at the D2R, but only poor affinity for the CB1R. A systematic linker attachment point survey of the SR141716A pharmacophore was therefore undertaken, establishing the C5 position as the optimal site for linker conjugation. This linker attachment survey enabled the identification of a novel divalent ligand as a lead compound to inform ongoing development of high-affinity CB1R molecular probes.

Full text of DOI:10.1016/j.bmcl.2019.126644

Journal Pre-proofs
Divalent Cannabinoid-1 Receptor Ligands: A Linker Attachment Point Survey
of SR141716A for Development of High-Affinity CB1R Molecular Probes
Phillip S. Grant, Nils Kahlcke, Karan Govindpani, Morag Hunter, Christa
MacDonald, Margaret A. Brimble, Michelle Glass, Daniel P. Furkert
PII:
S0960-894X(19)30589-X
https://doi.org/10.1016/j.bmcl.2019.126644
BMCL 126644
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
7 June 2019
23 August 2019
27 August 2019
Please cite this article as: Grant, P.S., Kahlcke, N., Govindpani, K., Hunter, M., MacDonald, C., Brimble, M.A.,
Glass, M., Furkert, D.P., Divalent Cannabinoid-1 Receptor Ligands: A Linker Attachment Point Survey of
SR141716A for Development of High-Affinity CB1R Molecular Probes, Bioorganic & Medicinal Chemistry
Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.126644
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Graphical Abstract
Divalent Cannabinoid-1 Receptor Ligands: A Linker
Attachment Point Survey of SR141716A for
Development of High-Affinity CB1R Molecular
Probes
Phillip S. Granta , Nils Kahlckea, Christa MacDonaldb, Karan
Govindpanib, Morag Hunterb, Margaret A. Brimblea,c,
Michelle Glassb,c,d and Daniel P. Furkerta,c*
aSchool of Chemical Sciences, University of Auckland, 23 Symonds Street, Auckland 1142, New Zealand.
bDepartment of Pharmacology & Clinical Pharmacology, University of Auckland, 85 Park road, Auckland
1142, New Zealand
cMaurice Wilkins Centre for Molecular Biodiscovery
dCurrent address: Department of Pharmacology and Toxicology, The University of Otago, Dunedin, New
Zealand

Bioorganic & Medicinal Chemistry
Divalent Cannabinoid-1 Receptor Ligands: A Linker Attachment Point Survey of
SR141716A for Development of High-Affinity CB1R Molecular Probes
Phillip S. Granta , Nils Kahlckea, Karan Govindpanib, Morag Hunterb, Christa MacDonaldb, Margaret A.
Brimblea,c, Michelle Glassb,c,d and Daniel P. Furkerta,c*
aSchool of Chemical Sciences, University of Auckland, Auckland, New Zealand.
bDepartment of Pharmacology & Clinical Pharmacology, University of Auckland, Auckland, New Zealand.
cMaurice Wilkins Centre for Molecular Biodiscovery
d Current address: Department of Pharmacology and Toxicology, The University of Otago, Dunedin, New Zealand
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
The cannabinoid-1 receptor (CB1R) inverse agonist SR141716A has proven useful for study of
the endocannabinoid system, including development of divalent CB1R ligands possessing a
second functional motif attached via a linker unit. These have predominantly employed the C3
position of the central pyrazole ring for linker attachment. Despite this precedent, a novel series
of C3-linked CB1R-D2R divalent ligands exhibited extremely high affinity at the D2R, but only
poor affinity for the CB1R. A systematic linker attachment point survey of the SR141716A
pharmacophore was therefore undertaken, establishing the C5 position as the optimal site for
linker conjugation. This linker attachment survey enabled the identification of a novel divalent
ligand as a lead compound to inform ongoing development of high-affinity CB1R molecular
probes.
Available online
Keywords:
SR141716A
Cannabinoid-1 receptor
G-protein coupled receptor
Molecular probe
Divalent ligand
2009 Elsevier Ltd. All rights reserved.
The CB1 cannabinoid receptor is an important therapeutic
target, with involvement in the regulation of multiple
neurotransmitters.1 Unfortunately, undesired side-effects have
limited the use of some clinically approved therapeutics and
prevented approval of other candidates.2,3 There remains a need
for improved pharmacological tools to better characterize
pathways.10 Further, as these two GPCRs are predominantly co-
expressed in neurons of the striatum,11 a CB1R-D2R divalent
ligand could potentially be used to target or study CB1 receptors
in this particular region of the brain.12,13 In concept, divalent
molecular probes for GPCRs (Figure 1) are composed of a high
affinity receptor ligand (Ligand A, red), conjugated via a linker
(green) to a second GPCR ligand (Ligand B, blue) or functional
receptor binding and downstream signalling processes.4,5
A
6,7
growing body of evidence suggests that GPCRs not only function
individually but are also able to form homodimeric,
heterodimeric or higher-order oligomeric complexes that exhibit
unique signalling and functional behaviour.6,7 Interrogation of
these multi-receptor complexes using pharmacological tools is
important in order to understand their physiological significance.
Additionally, direct targeting of GPCR dimers appears to offer
potential for (i) selective modulation of specific signaling
pathways associated with GPCR dimers, to avoid undesired
therapeutic side effects, or (ii) for selective localisation of
molecular probes to specific tissues co-expressing both
constituent receptors in a GPCR heterodimer of interest.
tag (Tag, yellow). The ligand binds to the receptor, while the
linker extends out of the binding pocket to display either an
additional ligand for simultaneous binding to a second GPCR
(left), or a pharmacological tag for detection (right).14,15 It should
be noted that determination of the exact binding mode is non-
trivial and other binding modes are possible. To be suitable for
probe development, a ligand requires (a) high affinity and
selectivity for the target receptor, (b) a suitable molecular motif
that enables linker conjugation, and crucially (c) retention of the
original pharmacological profile after linker attachment.
The present study stemmed from a number of studies
indicating that CB1 and D2 GPCRs are capable of forming
hetereodimers that exhibit altered G-protein signaling with
respect to the individual receptors.8,9 Development of divalent
ligands able to selectively bind CB1R-D2R heterodimers would
provide a useful tool with which to investigate the complex
cross-talk between the cannabinoid and dopamine signalling
Figure 1. Design features for GPCR ligands binding divalently to
two GPCRs (left) or enabling detection via a functional tag (right).

Design of a CB1R-D2R divalent ligand required the selection
of appropriate ligands meeting the criteria above. In the case of
the D2R, derivatives of the selective agonist 2-(N-phenylethyl-N-
propyl)-amino-5-hydroxytetralin (PPHT) had been reported
bearing both a fluorescent label16 or an adenosine (A2AR)
receptor antagonist,17 that both demonstrated high affinity at the
D2R. For CB1, the selective inverse agonist SR141617A (Figure
2, 1) that had received clinical approval for the treatment of
obesity in 2006 as Rimonabant appeared to be an excellent
candidate. Despite being later removed from the market due to
psychological side-effects, the SR141716A diarylpyrazole
scaffold has remained highly useful as a research platform. A
wealth of structure-activity data exists for derivatives of 1 in both
the scientific and patent literature, including different substituents
at all positions of the pyrazole core (highlighted in red) and
substitution of alternative (hetero)aromatic cores.18,19
Figure 2. CB1R Ligand SR141716A (1). The pyrazole core
(red) showing linker attachment positions N1, C3 and C5.
Scheme 1. CB1R probes based on SR141716A (1). Previously reported ligands20–22 (A, above) and the current study (B, below).
Ligands based on this framework have been successfully
applied towards the long-elusive goal of obtaining X-ray crystal-
lographic data for the CB1R.18–20 Importantly for our purposes, a
number of studies towards divalent ligands based on 1 have been
reported (Scheme 1A, 2-4), dominated by linker conjugation to
the SR141716A core via the C3 position.21–26
The initial goal of the current study was the preparation of a
series of divalent CB1R-D2R ligands based on conjugation at the
C3 position of 1, to investigate CB1R-D2R heterodimerisation
and signalling modulation. On the basis of previously reported
work towards CB1R divalent ligands it appeared reasonable to
expect that C3 linker attachment should deliver divalent ligands
with good affinity for the CB1R. In the event however, all
efforts to design C3-conjugated CB1R-D2 divalent ligands
possessing useful CB1R affinity proved futile. In order to build a
foundation of data to enable eventual design of high-affinity
a novel CB1R ligand (5) incorporating a fluorophore as a lead
compound to inform ongoing development of high-affinity
hCB1R visualization tools.
To prepare the initial series of proposed divalent CB1R-D2R
ligands (Table 1), the C3 carboxylic acid chloride derivative of 1
was prepared by slightly modified literature methods23 (see
Supporting Information) and conjugated with a range of amine
linkers, broadly following the earlier work of Zhang, Thomas,
Fernandez-Fernandez and Portoghese.20–22,24,25 The D2R ligand
was incorporated through preparation of (±)-PPHT-NH2
according to the synthesis described by Neumeyer16 and
subsequent coupling with a carboxylic acid terminated linker
segment. The compounds prepared varied in linker length (22-50
atoms) and composition (lipophilic, hydrophilic, aromatic) in an
effort to accommodate the differing chemical environments
potentially encountered in accessing the receptor binding pocket.
To our disappointment, given the strong literature precedent
for high affinity C3 derivatives, no compounds in the series were
capable of reaching the minimum 75% radioligand displacement
threshold at hCB1R to warrant further investigation ([3H]-
CP55,940 competition binding assay at 10 µM) indicating very
poor affinity.
bivalent CB1R ligands,
a systematic survey of possible
alternative linker conjugation sites for the tetrazole core of
SR141717A (1) at N1, C3, and C5 was undertaken (Scheme 1B),
incorporating linkers varying in length and composition. This
foundation eventually led to the identification of position C5 as
the optimal conjugation point for 1, and enabled the discovery of

Table 1. C3-Conjugated heterodivalent CB1R-D2R, homodivalent CB1R-CB1R ligands and monovalent acetate controls.
Displacement
% (hCB1R)
hD2R Ki
(nM)
CB1R ligand
Linker
Secondary ligand
5
6
62
11
41
33
29
23
44
23
37
33
29
23
0
0.84
2.2
2.4
1.1
4.2
22 atoms
7
8
28 atoms
9
10
11
12
13
14
15
16
17
18
31 atoms
40 atoms
0
50 atoms
19
0

In dramatic contrast however, hD2R competition binding curves
generated for divalent CB1R-D2R ligands 5, 8, 11, 14 and 17
revealed that these ligands exhibited uniformly excellent affinity
for the D2R, exceeding that of the parent agonist (±)-PPHT
(hD2R Ki 13.3 nM).16
Prompted by our preliminary results, the focus of the study was
therefore adjusted to encompass a systematic survey of the
SR141716A inverse agonist scaffold, in order to identify
candidates suitable for further development of high-affinity
divalent CB1R ligands.
These data demonstrated that the linkers and D2R ligand used
in this study were effective choices for preparation of high
affinity divalent D2R ligands. Crucially however, the broad lack
of useful CB1R affinity demonstrated by the more complex
divalent ligands (5, 6, 8, 9, 11, 12, 14, 15, 17 and 18) highlighted
the challenge of designing strongly-binding divalent ligands that
incorporate a CB1R ligand. These latter findings may be due in
part to direct entry of the D2R ligand into the receptor from the
extracellular matrix, as opposed to entry via the hydrophobic
lipid bilayer that is proposed to occur at CB1R.26,27
Two alternative sites for linker conjugation to 1 were
identified as synthetically tractable for library preparation; the N1
and C5 positions of the pyrazole core (Scheme 1B). A series of
simple ligands conjugated at C3 were also included for
comparison. Synthetic intermediates adapted to bear a functional
handle to enable linker conjugation were prepared using
modifications of reported procedures (Schemes 2-4). Linker
fragments of varying length and composition were then
conjugated at the respective positions. The resultant SR141617A
conjugates were again screened in an hCB1R radioligand
competition binding assay at a single concentration of 10 µM.
Concentration response curves were obtained only for those
compounds capable of >75% radioligand displacement in the
initial screen.
Although many SR141716A derivatives with differing
substitution are known, to our knowledge there has been no
systematic comparison of linker attachment sites, specifically
directed towards the design of high-affinity divalent ligands.
Scheme 2. C3-Conjugated CB1R ligand series. General preparative route (top) and derivative series prepared (below).
Reagents and conditions: a) LiHMDS (1.0 M in hexanes), Et2O, 45 min, rt then diethyloxalate, rt, 16 h, 38%, b) 2,4-
dichlorophenylhydrazine.HCl, EtOH, reflux, 16 h, 81%, c) KOH, MeOH/H2O, reflux, 16 h, quant., d) [for 23a (87%), 23b (79%)
and 23d (20%)]: oxalyl chloride, DMF (cat.), CH2Cl2, rt, 1 h then amine, DIPEA, CH2Cl2, rt, 16 h; [for 23c (77%)]: EDCI.HCl,
HOAt, DIPEA, 30 min, rt, then amine.
Scheme 3. N1-Conjugated CB1R ligand series. General preparative route (top) and derivative series prepared (below).
Reagents and conditions: e) LiHMDS (1.0 M in hexanes), Et2O, 45 min, rt then diethyloxalate, rt, 16 h then N2H4.2HCl, EtOH,
rt, 20 h, 78%, f) KOH, MeOH/H2O, reflux, 5 h, quant., g) oxalyl chloride, DMF (cat.), CH2Cl2, rt, 0.5 h then 1-aminopiperidine,
DIPEA, CH2Cl2, rt, 16 h, h) tert-butyl-6-bromohexanoate, K2CO3, DMF, rt, 24 h, 68%, i) TFA, CH2Cl2, rt, 0.5 h, 25b (quant.), j)
[for 25c (89%) and 25f (91%)]: HATU, amine, DMF, rt, 16 h; [for 25d (65%)]: EDCI.HCl, HOAt, DIPEA, CH2Cl2, rt, 16 h; then
[for 25e (35%)]: KOH, MeOH/H2O, rt, 16 h.
The C3-conjugated series of ligands prepared for the
systematic survey (Scheme 2) were selected to probe in particular
the effect of linker length on CB1R affinity. Although a large
number of high- affinity C3 variants of this scaffold have been

reported,28-33 few have been prepared to specifically explore the
influence of substituents significantly longer than six atoms for
use as covalent linker units. Target analogues were readily
accessed via carboxylic acid 22, prepared in three steps from
4-chloropropiophenone (20) by slightly modified literature
methods.23 Conversion of 22 to the acid chloride followed by
coupling with aliphatic amines then afforded C3-conjugated
derivatives 23a-d.
affinity N1 CB1R ligands have been described in the patent
literature.34 To access a series of N1-derivatives for this study,
tert-butyl ester 24 (Scheme 3) was prepared by condensation of
-keto ethyl glyoxalate 21 with hydrazine, followed by alkylation
with 6-bromo tert-butylhexanoate. Removal of the tert-butyl
group to give the corresponding acid 25b then allowed the
installation of linkers of varying length and composition, to give
a series of N1-conjugated derivatives 25a-f.
Only a small number of reports describing N1-substituted
analogues of 1 have been published, although a series of high-
Scheme 4. C5-Conjugated CB1R ligand series. General preparative route (above) and derivatives prepared (below).
Reagents and conditions: k) 4-methoxyphenylboronic acid, Pd(OAc)2 (10 mol %), PPh3 (20 mol %), K2CO3, DME/H2O (1:1), 80 °C, 16 h,
67%, l) LiOH, THF/H2O, rt-reflux, 9 d, 87%, m) EDCI.HCl, DMAP (10 mol %), amine, CH2Cl2, rt, 16 h, n) 4-butyn-1-ol (31), Pd(OAc)2 (10
mol %), PPh3 (20 mol %), CuI (10 mol %), NEt3, 16 h, 80 °C, 89%, o) [for 33a]: Ac2O, DMAP (10 mol %), CH2Cl2, rt, 1.5 h, 91%; [for
33b]: EDCI.HCl, N-Boc 6-aminocaproic acid, CH2Cl2, rt, 16 h, 50%; then [for 33c]: TFA, CH2Cl2, rt, 30 min; 33c (quant.); [for 33d]: tert-
butyl-6-bromohexanoate, TBAI (10 mol %), NaH, DMF, 16 h, 80°C, 17%; [for 33e]: EDCI.HCl, N-Boc 11-aminoundecanoic acid, CH2Cl2,
rt, 16 h then TFA, CH2Cl2, rt, 30 min; 33e (50% from 32).
At the outset of this investigation, very few C5-conjugated
derivatives were known.35 The ligand Tocrifluor, derived from 1,
possesses a fluorescent C5-conjugated rhodamine tag,37 although
it has been reported to exhibit only moderate to low affinity for
the CB1R.5,36 The Makriyannis group have developed a C5-
modified variant (AM6538) that enabled the determination of the
first crystal structure of the CB1R,37 and has been shown to
exhibit long-lasting in vivo effects.30
Functionalization of the C5 substituent of SR141716A for
linker conjugation was envisaged to be achieved through
palladium-mediated cross-coupling of 27, the brominated
analogue of 1. (Scheme 4). Aryl bromide 27 was accordingly
prepared from 4’-bromopropiophenone, analogously to 1,
followed by coupling with either boronic acid 28, or 3-butyn-1-ol
(31), affording ester 29 or alcohol 32, respectively. Linkers of
varying length and composition were subsequently coupled to
carboxylic acid 30a, derived from hydrolysis of 29, and alcohol
31, affording a diverse range of C5-conjugated SR141716A
derivatives.All SR141716A-linker conjugates prepared were
initially screened for affinity at the hCB1R in a radioligand
competition binding assay at a single concentration of 10 µM.
Full concentration-response curves were then generated for those
compounds capable of >75% radioligand displacement in the
initial screen, for determination of the influence of the linker
attachment position, length and composition on CB1R affinity
(Table 2). Of the C3 SR141716A-linker conjugates 23a-d, only
23a and 23b (Ki 8.2 and 13 nM, respectively) demonstrated high
affinity at CB1R. Further extension of the C3 linker substituent
to give 23c and 23d lowered CB1R affinity to the extent that
these compounds failed to meet the 75% radioligand
displacement threshold. It is possible that the high affinity of 23a
and 23b is accounted for by the short linker acting as an effective
isostere for the aminopiperidyl moiety of SR141716A (1). It
should be noted that the low affinity of 23c-23d is in contrast
with the modest to high affinities previously reported for C3 long
chain amide congeners of SR141716A (2, Scheme 1).29

Table 2. Systematic survey of C3, N1 and C5 linker conjugation sites.
Displacementa
% (hCB1)
hCB1R Ki
Compound
C3 Series
R
Linker length
(nM)
(atoms)
ndb
nd
8.2 ± 5.5
13 ± 3
23a
9
8
23b
23c
23d
14
13
5
43
N1 Series
25a
25b
25c
OtBu
OH
-
-
-
37
10
3
NHMe
25d
25e
25f
8
7
9
33
13
13
C5 Series
29
7
6
nd
66
55
49
0
131 ± 22
30a
30b
30c
30d
32
7
14
19
5
98
100
79
59
97
87
180 ± 76
11 ± 5
33a
33b
33c
33d
33f
7
16
12
14
21
106 ± 79
1810 ± 210
45 ± 33
97 ± 80
a Radioligand [3H]-CP 55,940 displacement at 10 µM, n=3; b nd = not determined

None of the N1 SR141716A-linker conjugates 25a-25e met
the displacement threshold in the initial screen. This prevented a
meaningful assessment of N1 as a position for linker attachment
and suggests that alkyl N1-conjugated analogues of SR141716A
are poor candidates for molecular probe development.
With the exception of control 29, C5-biaryl SR141716A-
linker conjugates 30a-d possessed poor CB1 affinity, in line with
data reported for the biaryl Tocrifluor T117.5,37 In contrast, we
were pleased to discover that all derivatives of alcohol 32
retained appreciable affinity upon conjugation of a linker. CB1R
affinity of the parent alcohol 32 (Ki 180 nM) was found to be
improved by either acetylation (33a, Ki 11 nM) or etherification
(33d, Ki 45 nM), suggesting that use of these functional groups
for subsequent linker attachment might allow retention of good
CB1 affinity. 33b bearing an N-Boc protected aminocaproic acid
linker fragment, coupled via an ester linkage, corroborated this
hypothesis (Ki 106 nM). Removal of the Boc group to give 33c
saw a dramatic 20-fold loss of affinity (Ki 1810 nM) indicating
that the amine group, positively charged at physiological pH,
could not be accommodated at this position. In contrast,
extension of the linker to 16 atoms to give 33f proved to be a
decisive modification, as amine 33f (Ki 97 nM) displayed similar
high CB1 affinity to 33b. Although the ester linkage could
potentially be labile to hydrolysis in aqueous media, it was
anticipated, and borne out subsequent affinity assays, that this
functionality would be robust enough for SAR development,
especially in the context of likely localisation to the lipid bilayer
prior to engagement with the membrane-bound CB1R.
Table 3. Synthesis and biological characterization of fluorescent CB1R probes 34 and 35.
Linker length
Compound
hCB1R Ki (nM) ± SEM
(atoms)
12
260 ± 20
34
35
21
2110 ± 1000
These data suggested that conjugation of a linker to the C5
position of the SR141716A pharmacophore via an ester bond was
a suitable basis for the further development of CB1 molecular
probes. Informed by the completed linker attachment survey, two
CB1 fluorescent probes based on C5 conjugation of SR141716A
(1) were prepared, by coupling of fluorescein isothiocyanate
(FITC) to the 5 and 10-carbon C5 linker conjugates 33c and 33f
(Table 3). The affinity of the fluorescent probes 34 and 35 were
then determined through radioligand competition binding assays
at hCB1. The affinity of the 17 atom linker congener 35
(Ki 2110 nM) was modest. However compound 34 bearing an
12-atom linker was found to display a useful level of affinity for
CB1 (Ki 260 nM) incurring only a small loss of affinity from
precursor 33b (Ki 97 nM) despite the addition of the large
fluorophore. We anticipate that 34 or closely related derivatives
will have potential to become useful fluorescent probes for
ongoing study of the CB1R.
subunit, based on linker conjugation at C5. We anticipate that the
findings of this study will have broad utility in informing future
design of divalent or multifunctional high-affinity molecular
probes for the cannabinoid CB1R.
ASSOCIATED CONTENT
Supporting Information
The supporting information is available free of charge at ACS
websites under the DOI blank.
AUTHOR INFORMATION
Email: d.furkert@auckland.ac.nz
ORCID
In conclusion, investigation into the synthesis of divalent
CB1R ligands based on the pharmacophore of SR141716A (1)
was carried out. Despite strong literature precedent, linker
conjugation at the C3 position did not lead to divalent
compounds with useful levels of CB1R affinity. A systematic
survey of alternative linker attachment positions on the
SR141716A pharmacophore was therefore undertaken.
Derivatives conjugated at the C5 position via a hydrophobic ester
linkage were identified as the optimal foundation for ongoing
development of divalent or multifunctional molecular probes for
the CB1R. These findings enabled the discovery of a novel high
affinity CB1R molecular probe 34 incorporating a fluorescein
Daniel P. Furkert 0000-0001-6286-9105
Notes
The authors declare no competing financial interest
Acknowledgements
The authors wish to thank Dr Jack Flanagan and Dr Dow
Hurst for helpful discussions, the Royal Society of New Zealand
Marsden Fund for funding support (Marsden Grant 11-UOA-
201) and the University of Auckland for the award of a doctoral
scholarship to PSG.

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Antagonists: Structure-Activity Relationships and Potential
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Lange, J. H. M.; Kruse, C. G. Keynote Review: Medicinal
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Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.bmcl.xxxx.xx.xxx.
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