Novel Functionalized Cannabinoid Receptor Probes: Development of Exceptionally Potent Agonists

Article abstract of DOI:10.1021/acs.jmedchem.0c02053

We report the development of novel cannabinergic probes that can stabilize the cannabinoid receptors (CBRs) through tight binding interactions. Ligand design involves the introduction of select groups at a judiciously chosen position within the classical

Full text of DOI:10.1021/acs.jmedchem.0c02053

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Article
Novel Functionalized Cannabinoid Receptor Probes: Development
of Exceptionally Potent Agonists
Shan Jiang,# Christos Iliopoulos-Tsoutsouvas,# Fei Tong, Christina A. Brust, Catherine M. Keenan,
Jimit Girish Raghav, Tian Hua, Simiao Wu, Jo-Hao Ho, Yiran Wu, Travis W. Grim, Nikolai Zvonok,
Ganesh A. Thakur, Zhi-Jie Liu, Keith A. Sharkey, Laura M. Bohn, Spyros P. Nikas,*
and Alexandros Makriyannis*
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ABSTRACT: We report the development of novel cannabinergic probes that can stabilize the cannabinoid receptors (CBRs)
through tight binding interactions. Ligand design involves the introduction of select groups at a judiciously chosen position within
the classical hexahydrocannabinol template (monofunctionalized probes). Such groups include the electrophilic isothiocyanato, the
photoactivatable azido, and the polar cyano moieties. These groups can also be combined to produce bifunctionalized probes
potentially capable of interacting at two distinct sites within the CBR-binding domains. These novel compounds display remarkably
high binding affinities for CBRs and are exceptionally potent agonists. A key ligand (27a, AM11245) exhibits exceptionally high
potency in both in vitro and in vivo assays and was designated as “megagonist,” a property attributed to its tight binding profile. By
acting both centrally and peripherally, 27a distinguishes itself from our previously reported “megagonist” AM841, whose functions
are restricted to the periphery.
selectivity” or “ligand bias.”.14,15 Functional selectivity derives
from a ligand-dependent stabilization of a receptor’s
conformation that favors interactions with an intracellular
INTRODUCTION
■
The two Gi/o-protein-coupled cannabinoid receptors (CBRs)
CB1 and CB2 have been recognized as important therapeutic
targets for CNS1 and cardiometabolic2 disorders, glaucoma,3
protein at the expense of others among the possible active
pain,4 cancer,5 as well as conditions related to the immune
system.6 An interesting feature of these two GPCRs is their
ability to be modulated by several structurally distinct classes
of compounds including endogenous lipid-like substances (e.g.,
anandamide and 2-arachidonoyl glycerol), as well as exogenous
synthetic (e.g., nabilone and rimonabant) and plant-derived
molecules [e.g., (−)-Δ9-tetrahydrocannabinol].7 For the
cannabinoid CB1 receptor, such ability to respond to a
plethora of ligands of considerably different sizes and shapes is
consistent with the remarkable plasticity of its orthosteric
binding pocket, as revealed by studies on the agonist- and
antagonist-bound crystal structures,8,9 as well as the cryo-
electron microscopy (cryo-EM) structures of agonist-bound
CB1 in complex with Gi.10,11 Furthermore, the ligand-
dependent activation of CB1 and CB2 is multifactorial and
can activate distinctly different signal transduction pathways
[e.g., G proteins (canonical Gαi/o or non-canonical Gαs, Gα16,
and Gαq/11) vs arrestins].12,13 This phenomenon is more
general in GPCRs, and it is referred to as “functional
receptor conformations. Additionally, the ligand−receptor
binding kinetics have emerged as an important concept in
GPCR drug development as they may influence the functional
potency and efficacy of both agonists and antagonists.16,17
Different ligands for a GPCR may have distinct association and
dissociation kinetics which determine the residence time of the
ligand on the receptor. Ligands with significantly slow
dissociation in a non-equilibrium system exhibit pseudo-
irreversible binding properties.18 In the case of CB1, we have
observed such effects with a ligand exhibiting irreversible
binding characteristics for the receptor. We designated this
Received: November 25, 2020
Published: March 24, 2021
https://dx.doi.org/10.1021/acs.jmedchem.0c02053
© 2021 American Chemical Society
J. Med. Chem. 2021, 64, 3870−3884
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Figure 1. Design of the novel mono and bifunctionalized HHC probes and structures of the natural product (−)-Δ9-THC and the marketed HHC
drug nabilone.
exceedingly potent agonist as “megagonist.”19,20 Moreover, the
lifetime of the ligand−receptor complex may also determine its
preferential signaling mechanisms and lead to functional
selectivity.21
chemical structures of the marketed drugs THC and
nabilone.7,34 One of the analogues developed in this project,
namely AM841,30 has distinguished itself by being a valuable
probe for LAPS studies,20,28,30,31 a unique ligand for structural
studies with the hCB1 receptor using X-ray crystallography and
cryo-EM8,11 and a peripherally acting cannabinergic “mega-
gonist.”19,20 We have also extended the scope of the groups
used in LAPS by adding moieties that may allow for the fine-
tuning of the ligand’s residence time within the receptors’ site.
One such group is the cyano moiety that albeit not being
covalently active, it can develop polar and/or hydrogen
bonding interactions with amino acid residues. Here, we
report the design, synthesis, and biochemical and pharmaco-
logical characterization of novel HHC probes for the CBRs.
Our key ligand AM11245 (27a) exhibits tight binding
properties and exceptionally high potency in inhibiting the
forskolin-stimulated cAMP accumulation and in various in vivo
assays and was designated as “megagonist.” Unlike the
peripherally restricted “megagonist” AM841, AM11245 ex-
hibited its “megagonist” profile both centrally and peripherally
when tested in vivo.
Deeper understanding of the molecular basis of the ligand−
CBR binding motif(s) and the kinetics through which ligands
block or enable signaling events and intracellular processes
offers the opportunity to develop more targeted cannabinoid-
based therapeutics. Toward this goal, our laboratory has
developed the ligand-assisted protein structure (LAPS), a
successful approach that is currently being used to explore the
binding motif(s) of cannabinergic ligands with their respective
native receptors with a focus on exploring how these motifs are
associated with distinct signaling profiles.22,23 LAPS is a
powerful tool that utilizes a combination of purpose-designed
cannabinergic ligands, CBR mutants, and mass spectrometric
proteomic analysis and allows for the characterization of
critical receptor residues that interact with a chemically and
functionally diverse set of ligands at the level of specific amino
acid residues.20,24−28 Design of such ligands is based on the
incorporation of reactive groups at carefully chosen sites within
the chemical structure of interest. The reactive groups, we have
generally chosen, include the electrophilic isothiocyanato and
the photoactivatable azido moieties.29,30 Over the past two
decades, our laboratory has developed such ligands that
represent different classes of cannabinergic compounds
including endogenous, plant-derived, and synthetic agonists
and antagonists at CB1 and CB2. These ligands allow us to
identify amino acid residues within or near the binding pocket
of the native receptor that are critical for ligand engagement
and receptor function.20,22−28,31−33 They have also been used
to structurally characterize in more detail the CBR−ligand and
CBR−Gi−ligand complexes using X-ray crystallography and
cryo-EM, respectively.8,9,11
RESULTS AND DISCUSSION
■
Design. Structure−activity relationship studies from our
own and other laboratories have suggested that presence of
gem-dimethyl or cyclopentyl groups at the C1′-position of the
classical cannabinoids (HHC, or Δ9/Δ8-THC, 2, 1, Figure 1)
enhances the ligand’s potency and efficacy.7,35−37 It has also
been demonstrated that incorporation of various groups
including heterocyclic rings at the ω-position of the side
chain can maintain, or even enhance, the affinity of the
compound for CBRs.37−40 Additionally, in the classical
cannabinoids, side chains consisting of six to eight linear
methylene groups are optimal for activity.7,36,39 Furthermore, it
has been demonstrated in both HHCs and THCs that the
presence of a hydroxyl at the northern region of the tricyclic
prototype enhances the ligand’s affinity for both CBRs.7,37,41,42
Finally, we have shown that the presence of an equatorial
hydroxymethyl group at C9 is optimal for activity.43,44
A considerable amount of our effort has been centered
around the tricyclic hexahydrocannabinol (HHC) prototype, a
chemical scaffold that: (a) exhibits the highest binding
affinities for CB1/CB2 and the most potent in vitro and in
vivo agonist properties, (b) encompasses three chiral centers,
thus reducing target promiscuity, and (c) resembles the
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a
Scheme 1
a
Reagents and conditions: (a) PPh3, benzene, reflux, 72 h, 98−99%; (b) isopropenyl acetate, p-toluenesulfonic acid, reflux, 6 h, 98%; (c)
Pb(OAc)4, benzene, reflux, 3 h; 90%; (d) CH3I, NaH, DMF, 0 °C to r t, 2 h, 98%; (e) (Me3Si)2N−K+, Br(CH2)4Br, THF, 0 °C to r t, 5 min, 95%;
(f) DIBAL-H, CH2Cl2, −78 °C, 0.5 h, 92%; (g) Br−P+Ph3(CH2)5OPh, KHMDS, THF, 0−10 °C, 30 min, then addition of 13a or 13b, 0 °C to r t,
20 min, 96% for 14a or 97% for 14b; (h) Br−P+Ph3(CH2)6OPh, KHMDS, THF, 0−10 °C, 30 min, then addition of 13b, 0 °C to r t, 20 min, 96%
for 14c; (i) H2, 10% Pd on C, AcOEt, r t, 2.5 h, 98%; (j) BBr3, CH2Cl2, −78 °C to r t, 3 h, 91−92%; (k) 10, p-TsOH, CHCl3, 0 °C to r t, 4 days,
59−64%; (l) TMSOTf, CH2Cl2/MeNO2, 0 °C to r t, 3 h, 70−75%; (m) TBSCl, imidazole, DMAP, DMF, 62 °C, 12 h, 91−93%; (n)
Cl−Ph3P+CH2OMe, KHMDS, THF, 0 °C to r t, 1 h, then addition to 19a−c, 0 °C to r t, 1.5 h, 70−73%; (o) Cl3CCOOH, CH2Cl2, r t, 50 min,
90−95%; (p) K2CO3, EtOH, r t, 3 h, 80−84%; (q) NaBH4, EtOH, 0 °C, 30 min, 97−98%.
Based on the above, our probe design adopts the 11-
hydroxyl-HHC scaffold (2) with a seven or eight carbon long
side-chain carrying a gem-dimethyl or a cyclopentyl substituent
at the C1′. The site in which the reactive groups (−N3,
−NCS) are introduced is critical so that the ligands can
optimally interact with the amino acid residue(s) within the
receptor’s binding domains.7,23,27 Thus, incorporation of a
reactive group at the ω-position of the side chain led to
monofunctionalized probes (3) and the addition of a second
reactive group at the C11 generated the bifunctionalized
ligands (4). In addition to the C1′-gem-dimethyl-substituted
bifunctionalized probes that we reported earlier,28 we chose to
explore the C1′-cyclopentyl-substituted HHCs in an effort to
further refine the stability of the ligand−receptor complex. We
also explored the potential of the cyano group at the ω-
position of the side chain for its tight binding capability as this
polar group is eight times smaller than a methyl group and, as
reported, can develop polar interactions or hydrogen bonds
with polar amino acid residues (e.g., serine or arginine) in
sterically congested environments.45
that which produces the final compounds in substantial
quantities as required for subsequent in vitro, in vivo, and
structural studies. For example, the synthesis of the key
intermediate chlorides 23a−c was accomplished in excellent
overall yields (15−22%), and it requires 12 synthetic steps
starting from commercially available 11.
The optimized syntheses of the key intermediate chlorides
23a−c are summarized in Scheme 1. Thus, deprotonation of
11 with sodium hydride followed by geminal dimethylation
using methyl iodide gave nitrile 12a (98% yield).46 Sequential
treatment of 11 with KHMDS and 1,4-dibromobutane
afforded (3,5-dimethoxyphenyl) cyclopentane carbonitrile
12b in very good yield (95%).35 Reduction of the cyano
group in 12a and 12b with diisobutylaluminum hydride led to
the respective aldehydes 13a and 13b (92%) which upon
Wittig reaction with the ylide, derived from (5-
phenoxypentyl)triphenylphosphonium bromide (7a) or (6-
phenoxyhexyl)triphenylphosphonium bromide (7b) and
KHMDS, afforded exclusively the Z olefins 14a−c (JH2′−H3′
= 11.1 Hz) in excellent yields (96−97%).35,37 In turn, the
required phosphonium salts 7a or 7b were synthesized from
commercially available 5-phenoxypentylbromide (6a) or 6-
phenoxyhexylbromide (6b), respectively, and triphenylphos-
phine in refluxing benzene.37 Catalytic hydrogenation of the
double bond in 14a−c proceeded in excellent yields (98%) by
Chemistry. The monofunctionalized and the respective
homo- and hetero-bifunctionalized probes for each series of
analogues were prepared from a common advanced
intermediate chloride (23a−c, Scheme 1). It is worth noting
that the robustness of the multistep stereoselective approach is
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a
Scheme 2
a
Reagents and conditions: (a) n-Bu4NF, THF, −78 °C, 10 min, 95−98%; (b) TMG-N3, CHCl3/CH3NO2, 70 °C, 30 h, 84−88%; (c) CS2, PPh3,
THF, r t, 18 h, 76−87%; and (d) NaCN, DMSO, r t, 18 h, 77−85%.
a
Scheme 3
a
−
Reagents and conditions: (a) I2, imidazole, PPh3, benzene, 50 °C, 30 min, 95%; (b) (n-Bu)4N+N3 , CH2Cl2, r t, 30 h, 83%; (c) TMG-N3, CHCl3/
CH3NO2, 70 °C, 30 h, 74%; (d) n-Bu4NF, THF, −78 °C, 30 min, 92%; and (e) CS2, PPh3, THF r t, 18 h, 86%.
using 10% Pd/C in ethyl acetate. This was followed by
exposure of the intermediate alkanes 15a−c to boron
tribromide which cleaved the ether groups and introduced
the C7′ and C8′ bromo group for resorcinols 16a−c (91−92%
yields).37,39 Following our modifications of an original
procedure, the mixture of the chiral terpene diacetates 10
was produced in two steps from commercially available (1R)-
(+)-nopinone (8).43,47,48 Condensation of 10 with resorcinols
16a−c led to norpinanones 17a−c (59−64% yields) which
upon treatment with catalytic trimethylsilyl triflate gave the 9-
keto-HHCs 18a−c (70−75%) with the required (6aR, 10aR)
stereochemistry.37,48
At this point, we envisioned that ω-substituted chlorides
(i.e., 23a−c) could be very useful as common synthetic
intermediates for the preparation of the monofunctionalized, as
well as the bifunctionalized probes. This will significantly
reduce the synthetic effort required for the multi-step synthesis
of all analogues pursued in this study. Thus, exposure of 18a−c
to TBSCl, imidazole, and DMAP in the presence of
dimethylformamide at 62 °C effectively protects the free
phenolic hydroxyl group and displaces the bromine with
chlorine in a single step producing the key synthetic
intermediates 19a−c in high yields (91−93%).41,49 Treatment
of commercially available (methoxymethyl)-
triphenylphosphonium chloride with KHMDS and coupling
of the in situ formed ylide with the 9-keto-cannabinoids 19a−c
gave the respective enol ethers 20a−c (70−73% yields) as 2:1
mixtures of two isomers (see the Supporting Information)
based on 1H NMR analysis.41,44 Subsequently, the methyl vinyl
ethers 20a−c were hydrolyzed with wet trichloroacetic acid
(90−95% yields), and the resulting diastereomeric mixture of
1
C9 aldehydes 21a−c (2:1 ratio by H NMR) was epimerized
to give the β-equatorial isomers 22a−c in 80−84% yields.41,44
Finally, reduction with sodium borohydride afforded the key
11-hydroxy-chlorides 23a−c in high yields (97−98%).
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a
Scheme 4
a
Reagents and conditions: (a) NaCN, DMSO, r t, 15 h, 57% and (b) CS2, PPh3, THF, r t, 18 h, 78%.
Figure 2. (Α) Cell membranes were pretreated with 27a (4 nM) or buffer (control) for 1 h, washed three times, and then subjected to [3H]CP-
55,940 binding. (Β) Bmax values (maximal binding capacity) were calculated from (Α) where the incubation time was increased following the
washing. Data represent the mean values SEM of at least three independent experiments performed in duplicate.
Synthesis of the side-chain monofunctionalized probes was
accomplished, as shown in Scheme 2. Thus, 11-hydroxy-
intermediates 23a−c were treated with tetra-n-butylammo-
nium fluoride (TBAF) to give the C7′/C8′-chloro-cannabi-
noids 24a−c in high yields (95−98%).41,44 Displacement of
the chloride in HHC 24a−c by N,N,N′,N′-tetramethylguani-
dinium azide led to monofunctionalized C7′/C8′ azide probes
25a−c (84−88% yields).25 Exposure of these analogues first to
triphenylphosphine and then to carbon disulfide converted the
azide to isothiocyanate, leading to the respective probes 26a−c
(76−87% yields).25,50 Treatment of chlorides 24a−c with
sodium cyanide afforded the C7′ and C8′ cyano-probes 27a−c
in high yields (77−85%).37
The heterobifunctionalized cannabinoid ligands represent a
much greater challenge to the synthetic chemist. This
challenge is imposed by the need to introduce regioselectively
two reactive groups within the molecule. We have found an
efficient approach to accomplish this task which takes
advantage of the different reactivities of the alkyl chlorides
and iodides under conditions of nucleophilic substitution.
Thus, the synthesis of both homo- and heterobifunctionalized
probes proceeded in high overall yields from the common key
intermediate 8′-chloro-11-iodo-HHC 28c with complete
control of the halogen substitution under the experimental
conditions used. Synthesis of the homo-bifunctionalized azido
and isothiocyanato probes carrying a cyclopentyl ring at the
C1′ is depicted in Scheme 3. The 11-hydroxy-HHC chloride
23c was converted to the requisite 8′-chloro-11-iodo-HHC
analogue 28c in 95% yield by using the triphenylphosphine,
iodine, imidazole method.39 The first azide displacement
reaction occurs exclusively at C11 and in high yield (83%)
upon exposure of 28c to tetra-n-butylammonium azide at room
temperature. The second azide displacement at C8′ was
accomplished by treatment of the 8′-chloro-11-azido-HHC
analogue 29c with N,N,N′,N′-tetramethylguanidinium azide in
chloroform/nitromethane at 70 °C to give 30c (74% yield).
This was followed by cleavage of the silyl ether at C1 using
TBAF to produce the di-azido-probe 31c in 92% yield. The
subsequent Staudinger reactions worked well at both the C11
and the C8′ azido groups and afforded the homo-
bifunctionalized isothiocyanato ligand 32c in a single operation
and in high yield (86%). The intermediate C11 azide 29c
served as the starting point for the synthesis of the hetero-
bifunctionalized probes 33c and 34c (Scheme 4). Thus,
treatment of chloride 29c with sodium cyanide displaces the
chlorine atom at the side chain and cleaves the phenyl silyl
ether in one step, leading to compound 33c (57% yield). The
most reactive isothiocyanato group in the hetero-bifunction-
alized probe 34c was introduced in the last synthetic step by
exposure of the azide 33c to Staudinger conditions (78%
yield).
Binding Affinities on CB1 and CB2 Receptors. The
abilities of the ligands 25a−c, 26a−c, 27a−c, 31c, 32c, 33c,
and 34c to displace [3H]CP-55,940 from membranes prepared
from rat brain (source of CB1) and HEK 293 cells expressing
either mouse or human CB2 were determined, as described
earlier,37,38 and inhibition constant values (Ki) from the
respective competition binding curves are given in Table 1.
The use of two CB2 receptor preparations was aimed at
addressing species differences that we have observed earlier.51
The compounds of this study are HHC analogues with one
group at the ω-position (C7′/C8′) of the C3 side-chain
(monofunctionalized probes) or two groups (bifunctionalized
probes) at both the C8′ and the C11 of the HHC structure.
The seven or eight carbon long side chains of the
monofunctionalized ligands carry a gem-dimethyl or a cyclo-
pentyl substituent at C1′ while the bifunctionalized ligands
bear a cyclopentyloctyl chain at C3. In agreement with our
rational design, the monofunctionalized ligands have remark-
ably high binding affinities for both CB1 and CB2 receptors
with most of their Ki values being in the picomolar range and
they do not exhibit species differences. The monofunctional-
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Table 1. Affinities (Ki) and Reductions in the Specific Binding (Bmax) of [3H]CP-55,940 after Ligand Pretreatment of Mono-
and Bifunctionalized Classical Cannabinoid Probes for the CB1 and CB2 CBRs
a
Affinities for CB1 and CB2 were determined using rat brain membranes (CB1) or membranes from HEK293 cells expressing mouse or human
CB2 and [3H]CP-55,940 as the radioligand following previously described procedures. Data were analyzed using nonlinear regression analysis. Ki
values were obtained from three independent experiments performed in triplicate and are expressed as the mean of the three values with SEM.
b
Data are presented as mean with SEM of at least three experiments performed in duplicate. The receptor membranes were pretreated with
concentrations equal to 10-fold the compound’s Ki value. Percentage was calculated as: {[Bmax(control) − Bmax(ligand)]/Bmax(control)}F × 100.
B
max(ligand), as described under the Experimental Section. The Bmax(control) for [3H]-CP-55,940 was determined by conducting the assay as
c
described but in the absence of the test ligand. NR: no reduction.
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structural motif: (a) both seven and eight carbon long-chain
analogues substituted at C1′ with the gem-dimethyl or with the
bulkier cyclopentyl groups can fit well within the binding
pocket of CB1 and CB2, and (b) the presence of select groups
(i.e., N3, NCS, and CN) at the ω-position of the chain in most
cases increases the binding affinity of the monofunctionalized
ligands compared to their non-functionalized surrogate.
A cursory examination of the binding affinity data of the
side-chain monofunctionalized ligands 25c and 27c with their
bifunctionalized counterparts 31c, and 33c, 34c, respectively,
indicates that for ligands carrying azido and cyano ω-
substituted side chains, the replacement of the C11 hydroxy
group with the azido or isothiocyanate moiety maintains the
high binding affinity of the bifunctionalized ligand for both the
CB1 and CB2 receptors. However, comparisons of the data of
32c with those of 26c and 34c suggest that simultaneous
incorporation of the electrophilic isothiocyanate at C8′ and
C11 results in a reduction of the binding affinity of the ligand
for both CB1 and CB2 receptors.
Figure 3. Concentration-dependent inhibition of forskolin-stimulated
cAMP accumulation in cells expressing either hCB1 (left) or hCB2
(right) receptors by representative ligands. Data are the mean with
SEM; CP-55,940 was assessed in parallel for all assays and data were
normalized to 100 nM CP-55,930 maximum response (n = 12 for
CB1 and 11 for CB2); all other agonists represent the mean SEM
of 4−8 experiments performed in duplicate.
Tight Binding on CB1 and CB2 Receptors. The most
prominent feature of the tight binding probes which
differentiates them from other high-affinity binding ligands is
their ability to bind insurmountably in a wash-resistant manner
in each of the two receptors.8,9 The ability of the mono- and
bifunctionalized probes carrying isothiocyanato, azido, and
cyano moieties to bind insurmountably in a wash-resistant
manner on each of the two receptors was determined by
measuring reductions in the saturation curve profile produced
for the standard radioligand [3H]CP-55,940, when the
preparation was pretreated with the ligand being tested, and
comparing it to the untreated sample. Following earlier work
from our laboratory,27 the experiment was conducted by
pretreating the sample with concentrations equal to 10-fold the
compound’s Ki value for the receptor in question, washing the
preparation with buffer three times, and subsequently
measuring the decrease of Bmax (maximal binding capacity)
obtained from a saturation curve using [3H]CP-55,940.
Testing results are summarized in Table 1, while saturation
binding curves for all analogues are provided under the
Supporting Information. We expect that the effectiveness for
tight binding for each ligand is determined by its ability to
accurately fit within the receptors’ orthosteric-binding pocket
and being further enhanced by additional interactions (e.g.,
hydrogen bonding, electrostatic, π−π interactions, etc.). Our
data clearly show that all the side-chain monofunctionalized
probes (25a−c, 26a−c, and 27a−c) reduce the Bmax of
[3H]CP-55,940 for both CB1 and CB2 receptors after
extensive washing when compared to control. The non-
functionalized analogue 2a exhibits no reductions in the levels
a
Table 2. Functional Data of Selected Probes for the hCB1
and hCB2 Receptors in the Forskolin-Stimulated Adenylyl
Cyclase Assay
Emax
%
nM
EC50
CB1 cAMP
100 nM CP
95% CI
95% CI nM
CP-55,940
25a
26a
26b
26c
100
99
96
104
105
103
112
1.13
0.098
5.8
0.52
0.43
0.088
0.31
(0.87−1.46)
(0.068−0.141)
(3.8−8.7)
(0.37−0.72)
(0.30−0.60)
(0.063−0.124)
(0.22−0.44)
(93−104)
(86−107)
(98−110)
(98−111)
(98−108)
(105−118)
95% CI
27a
27b
CB2 cAMP Emax %100 nM CP
nM EC50 95% CI nM
CP-55,940
25a
26a
26b
26c
100
72
107
57
74
70
1.03
0.77
5.5
(0.74−1.43)
(0.45−1.30)
(3.6−8.2)
(65−79)
(96−118)
(51−63)
(68−80)
(63−77)
(53−70)
2.7
(1.6−4.4)
0.39
0.38
0.96
(0.24−0.62)
(0.22−0.66)
(0.45−1.95)
27a
27b
61
a
Data presented as mean with 95% confidence intervals are shown:
EC50, half-maximum effective concentration; Emax, percentage of
maximum response relative to 100 nM CP-55.940, curves are
presented in Figure 3.
ized probes show significant increase in binding affinity up to
8-fold for CB1 and 5-fold for CB2, when compared to the
nonfunctionalized analogue 2a. This suggests that within this
Figure 4. AM841 (26a) binding pocket analysis and molecular docking of 26b and 27a based on the hCB1 crystal structure (PDB: 5XR8). (A)
Key residues involved in AM841 (pink sticks) binding. (B,C) Docking pose of 26b (B, blue sticks) and 27a (C, yellow sticks).
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□
○
Figure 5. (A) Effects of 27a ( ), or vehicle ( ) on antinociception using male CD-1 mice. Abscissa: dose in mg/kg. Ordinate: percentage of the
maximum possible antinociceptive effect. Symbols represent the group mean SEM (n = 6/group). Asterisks indicate antinociceptive effects that
are significantly different from vehicle, *p < 0.001. (B) Tail-flick latencies in a hot water bath (52 °C) after administration of four doses of 27a at
four time points (20, 60, 180, and 360 min post-administration) using male CD-1 mice (n = 6/dose). Abscissa: time (min) after injection.
Ordinate: tail-flick withdrawal latencies expressed as a percentage of the maximum possible effect (% MPE; group mean SEM). For clarity in data
presentation, data of vehicle group is not shown. The average antinociceptive effect of vehicle did not exceed 20% MPE at any of the four time
points examined.
Figure 6. Effects of 27a on spontaneous locomotion (A), thermoregulation (B), and colonic bead expulsion (C). Compound 27a (0.1−1 mg/kg)
dose-dependently reduced locomotion (A) and reduced core body temperature (B) in a CB1 receptor-specific manner and slowed colonic motility
(C) at a dose of 0.1 mg/kg. Inset in (B) shows the maximum change in body temperature from mean baseline (−100 min before injection) over
the time monitored post-injection (400 min). Note the very long-lasting effects of 27a in both thermoregulation and bead expulsion. wt, wild-type
mice; ko, CB1 receptor knockout mice. **P < 0.01, ***P < 0.001.
of Bmax of [3H]CP-55,940. Within the azido- and the cyano-
substituted ligands, the highest reduction can be seen with
those carrying the seven carbon long side chains. The C1′-gem-
dimethyl-substituted isothiocyanato (NCS, 26a) and cyano
(CN, 27a) probes exhibit the highest reduction for both CB1
and CB2, while the C1′-cyclopentyl-substituted azido (N3,
25b) ligand seems to be the best probe for photoirradiation
experiments.27,29 Moreover, a cursory examination of the data
in Table 1 reveals that, in general, the synthesized probes
exhibit slightly reduced results for the CB2 receptor when
compared to CB1. The preference of the CB2 receptor for
optimal side chains parallel those of CB1 for the azido (N3,
25b), isothiocyanato (NCS, 26a), and cyano (CN, 27a)
probes.
In contrast to the electrophilic isothiocyanato group that can
interact with cysteines20 and the azido moiety that can interact
with aminoacid residues after UV irradiation,29 the aliphatic
cyano group has not been shown to interact covalently with
aminoacid residues. For this reason, in order to explore the
time-dependent binding characteristics of the cyano-bearing
cannabinergic probes, we sought to determine whether they
can retain their tight binding properties over time. We selected
probe 27a as it showed the optimal results in the tight binding
assay among the cyano probes 27a−c. We followed a protocol,
as described in our previous work,8 where we pretreat CB1
membrane preparations with a concentration equal to 10-fold
the Ki value of 27a, then washed the preparation with buffer
and subsequently measured the decrease of Bmax obtained from
a saturation curve using [3H]CP-55,940 in different time
points (60 and 180 min after the washout step). Based on the
data summarized in Figure 2, cyano probe 27a retains its tight
binding profile by reducing the Bmax of [3H]CP-55,940 for at
least 180 min after the washout step.
Comparisons of the side-chain cyclopentyloctyl di-azido 31c
and di-isothiocyanato 32c ligands with their monofunctional-
ized counterparts 25c and 26c, respectively, shows a reduction
in the tight binding properties of these homo-bifunctionalized
probes. Within the cyclopentyloctyl side-chain structural motif,
the hetero-bifunctionalized probes 33c and 34c exhibited no
ability to reduce the specific binding of the radioligand. This
indicates that the stereoelectronic requirements are more
stringent for the bifunctionalized probes when compared to
their monofunctionalized counterparts. This may be due to the
presence of an 11-OH group in the monofunctionalized
probes, as observed in the molecular docking process.
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Functional Characterization. The long-term goal of this
project was to develop mono- and bifunctionalized classical
cannabinoid probes exhibiting tight binding characteristics and
distinct agonist profiles. Thus, the monofunctionalized probes
25a, 26b, 26c, 27a, and 27b were selected for further
evaluation in assays assessing the regulation of adenylyl cyclase
using overexpression systems of hCB1 and human CB2
(hCB2) receptors, as described in the Experimental
Section.8,9,11,52 In an earlier work, we have already reported
that 26a (AM841) behaves as a potent agonist at both the CB1
and CB2 receptors, which we designated as “megagon-
ist.”8,19,20,30,32 In this study, we are focused on the above key
analogues not only because they possess high CB1/CB2
receptor binding affinities and abilities to bind tightly in CB1
and/or CB2 binding sites but also because these ligands are
representatives of all side-chain structural motifs that we
explored in our study. The dose−response curves of the
analogues for both the CB1 and CB2 are depicted in Figure 3
where data for the standard cannabinoid agonist CP-55,940 are
also shown for comparison. Functional potencies (EC50) and
efficacies (Emax) of the ligands are presented in Table 2. Our
testing results indicate that in the cyclase assay, all
representative probes behave as highly potent and full efficacy
agonists at the CB1 receptor as they all decrease the levels of
forskolin-stimulated cAMP to the same extent as CP-55,940.
Notably, among the compounds tested, the C7′-cyano-
substituted analogue 27a has distinguished itself as the most
potent CB1 agonist with a potency (EC50 = 88 pM for CB1)
much higher than that of the standard CB1/CB2 agonist CP-
55,940. The exceedingly high potency of this compound leaves
us to designate it as a “megagonist.” Arguably, this property
may be related to its ability to form a very tight complex with
the CB1 receptor.
Molecular Docking. Based on the AM841 (26a)-bound
hCB1 crystal structure (PDB: 5XR8),8 we studied the
interactions of representative side-chain agonists with the
CB1 receptor through molecular docking (Figure 4A−C). We
observe that all agonists adopt an L-shape conformation in the
orthosteric-binding pocket. The interactions between the
ligands and CB1 are mainly hydrophobic and aromatic. The
tricyclic HHC ring system of all ligands forms π−π interactions
with Phe268ECL2, Phe3797.35, Phe1893.25, and Phe1772.64
(superscripts denote Ballesteros−Weinstein numbering),53
while the phenolic hydroxyl at C1 of all analogues forms a
hydrogen bond with Ser3837.39. The hydroxy group at the C11
position of the ligands forms an additional hydrogen bond with
the backbone carbonyl oxygen atom of Ile267ECL2. The C3
alkyl chain of the three analogues extends into a long channel
as we have reported earlier.8 We also observe that the C3 alkyl
chain of the most potent compound 27a is in all-anti
conformation with the lowest energy, while 26a and 26b are
in mixed anti and gauche conformation.
peripherally mediated in vivo effects of our newly identified
C1′-dimethyl-C7′-cyano counterpart, 27a, which also behaves
as an exceptionally potent CB1 agonist in vitro (cAMP assay).
Thus, cyano probe 27a was studied in the centrally mediated
CB1 receptor-characteristic nociception (Figure 5A,B) and
locomotion and thermoregulation (Figure 6A,B, respectively)
tests in mice. We also examined the magnitude and the
duration of the effects of 27a in the periphery using the colonic
bead expulsion test (Figure 6C) to compare its effects with the
peripherally restricted “megagonist” AM841.
The tail-flick latency procedure was used to measure the
analgesic effects of 27a. Animals were observed over a period
of 6 h following drug or vehicle administration. The baseline
latency for all the treatment conditions was 1.57
0.5 s.
Compound 27a produced dose-dependent antinociceptive
effects (Figure 5A), with 1 mg/kg producing maximal effects.
Doses of 0.1−1 mg/kg 27a had significant antinociceptive
effects, as compared to vehicle (p < 0.001), with mean ( 95%
CI) ED50 value of 0.12 mg/kg (0.0859−0.1676). As shown in
Figure 5B, 27a has a fast onset of action and its antinociceptive
effects lasted over 6 h. Two-way repeated measures ANOVA
showed significant effects for dose (D) [F(4,99) = 40.441; p <
0.001] and time (T) [F(3,99) = 7.102; p = 0.005], with no
significant interaction between D × T. Compound 27a had a
quick onset of action (Figure 5B), at 20 min post-injection
with 0.3 and 1 mg/kg producing significant antinociceptive
effect, as compared to vehicle (p < 0.001). At 60 min post-
injection, all the doses except 0.03 mg/kg produced peak
antinociceptive effects, as compared to vehicle p < 0.001.
We then assessed the cyano probe 27a for its effects in two
additional assays of the cannabinoid tetrad,54 hypolocomotion,
and thermoregulation (Figure 6A,B, respectively). Here, we
demonstrate that 27a was highly potent as it reduced
locomotion significantly (Figure 6A) and behaved as a CB1
receptor specific ligand in reducing body temperature (Figure
6B). Conversely, we had shown that compound 26a was
unable to produce neither hypolocomotion nor hypothermia.19
Having established that the cyano probe 27a shows centrally
mediated CB1 effects, unlike its isothiocyanate congener 26a,
we compared the two ligands for their peripheral effects in
reducing gastrointestinal motility. 27a slowed colonic
expulsion of a bead (Figure 6C) to the same extent as we
have previously reported for 26a.19 Of note, the effect of the
cyano probe 27a barely waned after 4 h, whereas the
isothiocyanate probe 26a’s action was short lived.19
An overall comparison of the in vivo effects of the C1′-
dimethyl-C7′-isothiocyanato analogue 26a with the C1′-
dimethyl-C7′-cyano counterpart 27a indicates that although
both compounds exhibit exceptionally high potency in the
peripheral assay of colonic bead expulsion, only the cyano
analogue 27a induces potent centrally mediated cannabinergic
effects. These data suggest that 27a behaves as a globally
(centrally and peripherally) acting CB1 “megagonist,” while
26a behaves as a peripherally restricted CB1 “megagonist.”19
In Vivo Behavioral Characterization. In an earlier work,
we have shown that the C1′-dimethyl-C7′-isothiocyanato-
substituted analogue 26a (AM841) behaved as an irreversible
and remarkably potent cannabinergic agonist in vitro.20,30,31
The ligand powerfully reduced gastrointestinal motility by
acting on CB1 receptors in the small and large intestine under
physiological conditions. However, AM841 did not exhibit
characteristic centrally CB1 receptor-mediated effects (an-
algesia, hypothermia, or hypolocomotion) and behaved as a
peripherally restricted ligand, showing very little brain
penetration.19 We have now explored key centrally and
CONCLUSIONS
■
We report the development of novel cannabinergic ligands
with remarkably high affinities for the CB1 and CB2 CBRs,
which are powerful CB1/CB2 agonists with tight binding
profiles. Our ligand design is based on the incorporation of
select chemical groups at carefully chosen sites within the
classical cannabinoid structure. These groups include the
electrophilic isothiocyanato and the photoactivatable aliphatic
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C-ring), 1.87−1.78 (m, 2H, the cyclopentyl ring), 1.76−1.58 (m, 9H,
6H of the cyclopentyl ring, 2H of the side chain, 1H of C-ring), 1.52−
1.44 (m, 3H, 2′-H, C-ring), 1.38 (s, 3H, 6-Me), 1.36−1.29 (m, 2H,
side chain), 1.22−1.10 (m, 6H, 4H of the side chain, 2H of C-ring),
1.05 (s, 3H, 6-Me), 1.02−0.93 (s and m overlapping, 11H, 2H of the
side chain, Si(Me)2CMe3, especially, 1.00, s, 9H, Si(Me)2CMe3),
0.89−0.82 (m, 1H, C-ring), 0.22 (s, 3H, Si(Me)2CMe3), 0.13 (s, 3H,
Si(Me)2CMe3). 13C NMR (100 MHz CDCl3): δ 154.3, 154.1, 148.5,
113.0, 110.8, 109.4, 50.6, 49.2, 45.1, 41.8, 49.7, 37.6, 37.0, 35.5, 33.5,
32.6, 30.1, 29.9, 28.7, 27.6, 27.5, 26.8, 26.0, 25.2, 23.3, 18.7, 18.3, 15.1
(−CH2I), −3.8, −4.6. Mass spectrum (ESI) m/z (relative intensity):
687 (M+ + H, 100). HPLC analysis showed a retention time of 8.0
min for the title compound.
azido moieties, as well as the polar cyano. Our experiments
indicate that all groups when introduced in the HHC template
lead to tight binding interactions with the CBRs. Further
evaluation of the wash-resistant binding profile of the C1′-
dimethyl-C7′-cyano-substituted analogue 27a, namely
AM11245, showed that it can retain its tight binding profile.
Representative probes behave as potent CB1 and CB2 agonists
in the inhibition of the adenylyl cyclase assay. A striking finding
is the exceptionally high potency of analogue 27a (EC50 = 88
pM for CB1), as reflected by the cAMP assay, which we
designated as “megagonist,” a property that may be related to
the tight binding interactions with the receptor.
{[(6aR,9R,10aR)-9-(Azidomethyl)-3-[1-(7-chloroheptyl)-
cyclopentyl]-6,6-dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]-
chromen-1-yl]oxy}(tert-butyl)dimethylsilane (29c). To a solution of
28c (528 mg, 0.77 mmol) in anhydrous CH2Cl2 (15.5 mL) at r t,
under an argon atmosphere, was added tetrabutylammonium azide
(1.6 g, 7.7 mmol). The reaction mixture was stirred for 30 h at the
same temperature and then quenched by brine. Extractive isolation
with diethyl ether and purification by flash column chromatography
on silica gel (0−10% diethyl ether in hexane) gave 29c (384 mg, 83%
yield) as a white foam. IR (neat): 2929, 2858, 2097 (s, N3), 1612,
The C1′-dimethyl-C7′-cyano- and C1′-dimethyl-C7′-iso-
thiocyanato-substituted analogues 27a (AM11245) and 26a
(AM841), respectively, were found to exhibit exceptionally
high in vivo potency. Our studies indicate that 27a is a globally
acting CBR “megagonist” while 26a is a “megagonist”
restricted to the periphery.
EXPERIMENTAL SECTION
■
Materials. All reagents and solvents were purchased from Sigma-
Aldrich Chemical Company, unless otherwise specified, and used
without further purification. All anhydrous reactions were performed
under a static argon atmosphere in the flame-dried glassware using
scrupulously dry solvents. Flash column chromatography employed
silica gel 60 (230−400 mesh). All compounds were demonstrated to
be homogeneous by analytical TLC on pre-coated silica gel TLC
plates (Merck, 60 F245 on glass, layer thickness 250 μm), and
chromatograms were visualized by phosphomolybdic acid staining.
Melting points were determined on a micro-melting point apparatus
and are uncorrected. IR spectra were recorded on a PerkinElmer
Spectrum One FT-IR spectrometer. Peak strength is indicated as w
(weak) and s (strong). NMR spectra were recorded on a Bruker Ultra
Shield 400 WB plus (1H at 400 MHz, 13C at 101 MHz) or on a Varian
INOVA-500 (1H at 500 MHz, 13C at 126 MHz) spectrometer, and
chemical shifts are reported in units of δ relative to internal TMS.
Multiplicities are indicated as br (broadened), s (singlet), d (doublet),
t (triplet), q (quartet), and m (multiplet), and coupling constants (J)
are reported in hertz (Hz). Low- and high-resolution mass spectra
were performed in the School of Chemical Sciences, University of
Illinois at Urbana-Champaign. Mass spectral data are reported in the
form of m/z (intensity relative to base = 100). Purities of compounds
were determined by HPLC/MS analysis using a Waters MicroMass
ZQ system [electrospray-ionization (ESI)] with Waters-2525 binary
gradient module coupled to a Photodiode Array Detector (Waters-
2996) and ELS detector (Waters-2424) using a XTerra MS C18, 5
μm, 4.6 mm × 50 mm column and acetonitrile/water; all tested
compounds were >95% pure.
1
1563, 1463, 1411, 1342, 1254 1138, 1062, 873 cm−1; H NMR (500
MHz, CDCl3): δ 6.33 (d, J = 1.9 Hz, 1H, Ar-H), 6.26 (d, J = 1.9 Hz,
1H, Ar-H), 3.48 (t, J = 6.9 Hz, 8′-H) 3.23−3.17 (dd and m as d
overlapping, 2H, 1H of C-ring, 1H of −CH2N3, especially, m, 1H, C-
ring, 3.21, dd, J = 12.1, 6.1 Hz, half of an AB system, 1H, −CH2N3)
3.14 (dd, J = 12.1, 7.1 Hz, half of an AB system, 1H, −CH2N3), 2.38−
2.31 (m as td, J = 11.1, 2.7 Hz, 1H, C-ring), 2.02−1.97 (m, 1H, C-
ring), 1.92−1.88 (m, 1H, C-ring), 1.87−1.73 (m, 3H, 2H of the
cyclopentyl ring, 1H of C-ring), 1.76−1.58 (m, 8H, 6H of the
cyclopentyl ring, 2H, side chain), 1.52−1.44 (m, 3H, 2′-H, C-ring),
1.38 (s, 3H, 6-Me), 1.35−1.28 (m, 2H, side chain), 1.24−1.10 (m,
6H, 4H of the side chain, 2H of C-ring), 1.05 (s, 3H, 6-Me), 1.02−
0.91 (s and m overlapping, 11H, 2H of the side chain, Si(Me)2CMe3,
especially, 1.00, s, 9H, Si(Me)2CMe3), 0.86−0.79 (m, 1H, C-ring),
0.22 (s, 3H, Si(Me)2CMe3), 0.13 (s, 3H, Si(Me)2CMe3). 13C NMR
(100 MHz CDCl3): δ 154.4, 154.2, 149.3, 113.5, 110.8, 109.5, 57.8,
51.5, 50.4, 49.1, 41.6, 40.5, 37.7, 37.5, 34.9, 34.1, 30.6, 29.9, 28.8,
28.7, 27.6, 27.3, 26.6, 25.9, 25.0, 23.2, 18.6, 18.1, −3.6, −4.3. Mass
spectrum (ESI) m/z (relative intensity): 602 (M+ + H, 100). HPLC
analysis showed a retention time of 9.1 min for the title compound.
{[(6aR,9R,10aR)-3-[1-(7-Azidoheptyl)cyclopentyl]-9-(azidometh-
yl)-6,6-dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-
1-yl]oxy}(tert-butyl)dimethylsilane (30c). To a solution of 29c (186
mg, 0.31 mmol) in anhydrous CH2Cl2 (5.2 mL) at r t, under an argon
atmosphere, was added N,N,N′,N′-tetramethylguanidinium azide (1.4
g, 9.1 mmol), and the reaction mixture was heated at 70 °C for 30 h.
On completion, the reaction was quenched with water and diluted
with CH2Cl2. The organic phase was washed with brine, dried over
MgSO4, and concentrated in vacuo. Purification by flash column
chromatography on silica gel (3−8% diethyl ether in hexanes) gave
151 mg of 30c as a white foam in 74% yield. IR (neat): 2928, 2857,
2097 (s, N3), 1756, 1612, 1411, 1342, 1254, 1138, 1062, 839 cm−1;
1H NMR (500 MHz, CDCl3): δ 6.33 (d, J = 1.9 Hz, 1H, Ar-H), 6.26
(d, J = 1.9 Hz, 1H, Ar-H), 3.24−3.15 (dd, t and m as d overlapping,
3H, C-ring, −CH2N3, 8′-H, especially, 3.21, t, J = 6.9 Hz, 8′-H, m as
br d, J = 12.8 Hz, 1H, C-ring, 3.21, dd, J = 121, 6.1 Hz, half of an AB
system, 1H, −CH2N3) 3.14 (dd, J = 12.1, 7.1 Hz, half of an AB
system, 1H, −CH2N3), 2.38−2.31 (m as td, J = 11.1, 2.7 Hz, 1H, C-
ring), 2.02−1.97 (m, 1H, C-ring), 1.92−1.88 (m, 1H, C-ring), 1.87−
1.73 (m, 3H, 2H of the cyclopentyl ring, 1H of C-ring), 1.71−1.58
(m, 6H, cyclopentyl ring), 1.54−1.43 (m, 5H, 4H of the side-chain
group, 1H of C-ring), 1.38 (s, 3H, 6-Me), 1.31−1.24 (m, 2H, side
chain), 1.21−1.10 (m, 6H, 4H of the side chain, 2H of C-ring), 1.06
(s, 3H, 6-Me), 1.02−0.91 (s and m overlapping, 11H, 2H of the side
chain, Si(Me)2CMe3, especially, 1.00, s, 9H, Si(Me)2CMe3), 0.84−
0.76 (m, 1H, C-ring), 0.24 (s, 3H, Si(Me)2CMe3), 0.12 (s, 3H,
Si(Me)2CMe3). 13C NMR (100 MHz CDCl3): δ 154.3, 154.1, 149.1,
{[(6aR,9R,10aR)-3-[1-(7-Chloroheptyl)cyclopentyl)]-9-(iodometh-
yl)-6,6-dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-
1-yl]oxy}(tert-butyl)dimethylsilane (28c). To a solution of 23c (461
mg, 0.8 mmol) in anhydrous benzene (16 mL), under an argon
atmosphere, were added imidazole (217 mg, 3.2 mmol) and
triphenylphosphine (419.7 mg, 1.6 mmol), then the reaction mixture
was heated to 50 °C, followed by a dropwise addition of a solution of
iodine (406 mg, 1.6 mmol) in benzene. The reaction mixture was
stirred for 30 min at 50 °C. The reaction was quenched by an aqueous
sodium sulfite solution. Extractive isolation with diethyl ether and
purification by flash column chromatography on silica gel (0−15%
diethyl ether in hexane) gave 28c (521 mg, 95% yield) as a white
viscous oil. IR (neat) 2929, 2857, 1612, 1562, 1411, 1361, 1324,
1253, 1058, 835, 779 cm−1; 1H NMR (500 MHz, CDCl3): δ 6.33 (d, J
= 1.9 Hz, 1H, Ar-H), 6.26 (d, J = 1.9 Hz, 1H, Ar-H), 3.48(t, J = 6.9
Hz, 8′-H) 3.26−3.19 (dd and m as d overlapping, 2H, 1H of C-ring,
1H of −CH2I, especially, m as br d, J = 12.8 Hz, 1H, C-ring, 3.21, dd,
J = 9.7,4.8 Hz, half of an AB system, 1H, −CH2I) 3.08 (dd, J = 9.7, 7.1
Hz, half of an AB system, 1H, −CH2I), 2.40−2.33 (m as td, J = 11.0,
2.6 Hz, 1H, C-ring), 2.12−2.05 (m, 1H, C-ring), 1.92−1.88 (m, 1H,
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113.6, 110.8, 109.5, 57.8, 51.5, 50.5, 49.0, 41.6, 38.2, 37.7, 37.5, 34.9,
34.1, 30.6, 29.8, 28.9, 28.7, 27.7, 27.4, 26.6, 25.9, 25.0, 23.2, 18.6,
18.1, −3.6, −4.3. Mass spectrum (ESI) m/z (relative intensity): 609
(M+ + H, 100). HPLC analysis showed a retention time of 8.8 min for
the title compound.
anhydrous DMSO (4 mL) at room temperature, was added sodium
azide (100 mg, 2.1 mmol). The reaction mixture was stirred for an
additional 15 h at the same temperature. The reaction mixture was
quenched by the addition of ice-cold water and diluted with diethyl
ether and was stirred for 30 min. The two phases were separated, and
the aqueous layer was extracted with diethyl ether. The combined
organic layer was washed with brine, dried over MgSO4, and
evaporated under reduced pressure. Purification by flash column
chromatography on silica gel (20−60% diethyl ether in hexane) gave
33c (55 mg, 57% yield) as light-yellow foam. IR (neat): 3394 (OH),
2929, 2859, 2252 (w, CN), 2096 (s, N3), 1621, 1573, 1455, 1414,
(6aR,9R,10aR)-3-[1-(7-Azidoheptyl)cyclopentyl]-9-(azidometh-
yl)-6,6-dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-
1-ol (31c). To a solution of 30c (150 mg, 0.24 mmol) in anhydrous
THF (6.1 mL) at −78 °C, under an argon atmosphere, was added
TBAF (0.3 mL, 0.3 mmol, 1 M solution in anhydrous THF). The
reaction mixture was stirred for 30 min at the same temperature and
then quenched using a saturated aqueous ammonium chloride
solution. Extractive isolation with diethyl ether and purification by
flash column chromatography on silica gel (8−25% ethyl acetate in
hexane) gave 31c (114 mg, 92% yield) as a white foam. IR (neat):
2931, 2861, 2097 (s, N3), 1756, 1622, 1413, 1331, 1251, 1138, 1039,
1
1137, 1037, 838 cm−1; H NMR (500 MHz, CDCl3): δ 6.29 (d, J =
1.8 Hz, 1H, Ar-H), 6.15 (d, J = 1.8 Hz, 1H, Ar-H), 4.87 (br s, 1H, Ar-
OH), 3.28−3.13 (m and dd overlapping, 3H, 1H of C-ring, 2H of
−CH2N3, especially, m as br d, J = 12.4 Hz, 1H, C-ring, 3.21, dd, J =
12.0, 6.0 Hz, half of an AB system, 1H, (−CH2N3) 3.14, dd, J = 12.0,
7.0 Hz, half of an AB system, 1H, (−CH2N3), 2.47 (m as td, J = 11.2
Hz, J = 2.7 Hz, 1H, C-ring), 2.31 (t, J = 7.1 Hz, 2H, H-8′), 2.02−1.97
(m, 1H, C-ring), 1.95−1.88 (m, 1H, C-ring), 1.87−1.75 (m, 3H, 2H
of the cyclopentyl ring, 1H of C-ring), 1.73−1.58 (m, 6H, cyclopentyl
ring), 1.55−1.42 (m, 5H, 4H of the side chain, 1H of C-ring), 1.38 (s,
3H, 6-Me), 1.35 (quintet, J = 7.7 Hz, 2H, the side chain), 1.28−1.12
(m, 8H, 6H of the side chain, 2H of C-ring), 1.08 (s, 3H, 6-Me),
1.03−0.96 (m, 2H, side chain), 0.89−0.82 (m, 1H, C-ring). 13C NMR
(100 MHz CDCl3): δ 154.6, 154.5, 149.2, 120.0, 109.5, 108.9, 106.6,
58.0, 50.7, 49.3, 41.6, 38.3, 37.9, 37.6, 35.2, 34.3, 31.0, 29.8 (2 C
overlapping), 28.5, 27.9, 27.5, 25.4, 24.9, 23.5, 19.2, 17.2. Mass
spectrum (ESI) m/z (relative intensity): 479 (M+ + H, 100). Exact
mass (ESI): calcd for C29H43N4O2 (M+ + H), 479.3386; found,
479.3382. HPLC analysis showed a retention time of 5.9 min for the
title compound.
1
839 cm−1; H NMR (500 MHz, CDCl3): δ 6.29 (d, J = 1.9 Hz, 1H,
Ar-H), 6.14 (d, J = 1.9 Hz, 1H, Ar-H), 5.12 (br s, 1H, OH) 3.27−3.19
(dd, t and m as d overlapping, 3H, C-ring, −CH2N3, 8′-H, especially,
3.21, t, J = 6.9 Hz, 8′-H, m as br d, J = 12.8 Hz, 1H, C-ring, 3.21, dd, J
= 12.1, 6.1 Hz, half of an AB system, 1H, −CH2N3) 3.15 (dd, J = 12.1,
7.1 Hz, half of an AB system, 1H, −CH2N3), 2.51−2.43 (m as td, J =
11.1, 2.7 Hz, 1H, C-ring), 2.02−1.97 (m, 1H, C-ring), 1.95−1.88 (m,
1H, C-ring), 1.87−1.75 (m, 3H, 2H of the cyclopentyl ring, 1H of C-
ring), 1.73−1.58 (m, 6H, cyclopentyl ring), 1.55−1.42 (m, 5H, 4H of
−CH2− of the side-chain group, 1H of C-ring), 1.38 (s, 3H, 6-Me),
1.31−1.24 (m, 2H, the side chain), 1.21−1.10 (m, 6H, 4H of the side
chain, 2H of C-ring), 1.08 (s, 3H, 6-Me), 1.04−0.93 (m, 2H side
chain), 0.85−0.81 (m as q, J = 11.9 Hz, 1H, C-ring). 13C NMR (100
MHz CDCl3): δ 154.4, 154.1, 149.1, 109.2, 108.8, 106.3, 57.8, 51.4,
50.6, 49.1, 41.7, 38.1, 37.5, 37.4, 35.0, 34.2, 30.8, 30.0, 28.9, 28.7,
27.7, 27.3, 26.6, 25.0, 23.3, 19.0. Mass spectrum (ESI) m/z (relative
intensity): 495 (M+ + H, 100). Exact mass (ESI) calculated for
C28H43N6O2 (M+ + H), 495.3447; found 495.3440. HPLC analysis
showed a retention time of 6.1 min for the title compound.
(6aR,9R,10aR)-3-[1-(7-Isothiocyanatoheptyl)cyclopentyl]-9-(iso-
thiocyanatomethyl)-6,6-dimethyl-6a,7,8,9,10,10a-hexahydro-6H-
benzo[c]chromen-1-ol (32c). To a solution of 31c (65 mg, 0.13
mmol), in anhydrous THF (4.3 mL) at room temperature, was added
triphenyl phosphine (340 mg, 1.3 mmol). Anhydrous carbon disulfide
(0.48 mL, 8.0 mmol) was then added dropwise, and the reaction
mixture was stirred for an additional 18 h at the same temperature.
Upon completion, the reaction mixture was concentrated under
reduced pressure and purified by flash column chromatography on
silica gel (50−80% diethyl ether in hexanes) to give 59 mg of 32c in
86% yield as light-yellow foam. IR (neat): 2923, 2859, 2106 (s, NCS),
1728, 1618, 1574, 1413, 1372, 1339, 1251, 1139, 1036, 863 cm−1; 1H
NMR (500 MHz, CDCl3): δ 6.30 (d, J = 1.9 Hz, 1H, Ar-H), 6.14 (d, J
= 1.9 Hz, 1H, Ar-H), 4.90 (br s, 1H, OH) 3.51−3.44 (dd and t, 2H,
−CH2NCS, 8′-H, especially, 3.49, t, J = 6.9 Hz, 8′-H, m as br d, J =
12.6 Hz, 1H, C-ring, 3.21, dd, J = 14.2, 5.5 Hz, half of an AB system,
1H, −CH2NCS), 3.41 (dd, J = 14.2, 6.4 Hz, half of an AB system, 1H,
−CH2NCS), 3.29−3.23(m as br d, J = 12.6 Hz, 1H, C-ring), 2.53−
2.45 (m as td, J = 11.2, 2.7 Hz, 1H, C-ring), 2.02−1.90 (m, 2H, C-
ring), 1.86−1.78 (m, 2H, the cyclopentyl ring), 1.73−1.58 (m, 9H,
6H of cyclopentyl ring, 1H of C-ring, 2H of the side chain), 1.53−
1.44 (m, 3H, 2H of the side chain, 1H of C-ring), 1.39 (s, 3H, 6-Me),
1.34−1.26 (m, 2H, of the side chain), 1.23−1.11 (m, 6H, 4H of the
side chain, 2H of C-ring), 1.09 (s, 3H, 6-Me), 1.04−0.93 (m, 2H, side
chain), 0.92−0.89 (m as q, J = 11.9 Hz, 1H, C-ring). 13C NMR (100
MHz CDCl3): δ 154.4, 154.1, 149.2, 130.1 (NCS), 108.9, 108.7,
106.4, 50.9, 50.6, 48.8, 45.0, 41.6, 38.5, 37.5, 37.4, 34.9, 33.8, 30.4,
29.9, 29.8, 28.5, 27.7, 27.2, 26.4, 24.9, 23.3, 19.0. Mass spectrum
(ESI) m/z (relative intensity): 527 (M+ + H, 100). Exact mass (ESI):
calcd for C30H43N2O2S2 (M+ + H), 527.2766; found, 527.2776.
HPLC analysis showed a retention time of 6.4 min for the title
compound.
8-(1-((6aR,9R,10aR)-1-Hydroxy-9-(isothiocyanatomethyl)-6,6-di-
methyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-3-yl)-
cyclopentyl)octanenitrile (34c). To a solution of 33c (20 mg, 0.04
mmol), in anhydrous THF (0.8 mL) at room temperature, was added
triphenyl phosphine (52 mg, 0.2 mmol). Carbon disulfide (70 μL, 1.2
mmol) was then added, and the reaction mixture was stirred for an
additional 18 h at the same temperature. Upon completion, the
reaction mixture was concentrated under reduced pressure and
purified by flash column chromatography on silica gel (10−40%
diethyl ether in hexanes) to give 16 mg of 34c in 78% yield as light-
yellow foam. IR (neat): 3394 (OH), 2929, 2858, 2253 (w, CN), 2100
1
(s, NCS), 1621, 1574, 1451, 1414, 1140, 1037, 838 cm−1; H NMR
(500 MHz, CDCl3): δ 6.29 (d, J = 1.8 Hz, 1H, Ar-H), 6.16 (d, J = 1.8
Hz, 1H, Ar-H), 5.06 (br s, 1H, Ar-OH), 3.47 (dd, J = 14.2, 5.4 Hz,
1H, half of an AB system, −CH2NCS), 3.40 (dd, J = 14.2, 6.4 Hz, 1H,
half of an AB system, −CH2NCS), 3.25 (m as d, J = 12.6 Hz, 1H of
C-ring), 2.47 (m as td, J = 11.2 Hz, J = 2.7 Hz, 1H, C-ring), 2.31 (t, J
= 7.1 Hz, 2H, H-8′), 2.02−1.88 (m, 3H, 2H of the cyclopentyl ring,
1H of C-ring), 1.87−1.77 (m, 2H, C-ring), 1.73−1.58 (m, 6H,
cyclopentyl ring), 1.55−1.42 (m, 5H, 4H of the side-chain group, 1H
of C-ring), 1.39 (s, 3H, 6-Me), 1.34 (quintet, J = 7.7 Hz, 2H, side
chain), 1.28−1.12 (m, 8H, 6H of the side chain, 2H of C-ring), 1.08
(s, 3H, 6-Me), 1.03−0.97 (m, 2H, side chain), 0.96−0.87 (m as q, J =
11.9 Hz, 1H, C-ring). 13C NMR (100 MHz CDCl3): δ 154.6, 154.4,
149.3, 130.0, 120.0, 109.2, 109.0, 106.6, 51.2, 50.7, 49.0, 41.6, 38.7,
37.9, 37.6, 35.1, 34.0, 30.6, 29.8, 29.7, 28.5, 27.9, 27.4, 25.4, 24.9,
23.5, 19.2, 17.2. Mass spectrum (ESI) m/z (relative intensity): 495
(M+ + H, 100). Exact mass (ESI): calcd for C30H43N2O2S (M+ + H),
497.3045; found, 495.3040. HPLC analysis showed a retention time
of 6.0 min for the title compound.
Radioligand-Binding Assays. The binding affinities (Ki) of the
new compounds were obtained using membrane preparations from
rat brain (source of rCB1) or HEK293 cells expressing either mCB2
or hCB2 receptors and [3H]CP-55,940 as the radioligand, as
previously described.37,38 The results from the competition binding
assays were analyzed using nonlinear regression to determine the IC50
values for the ligand; Ki values were calculated using the IC50 (Prism
by GraphPad Software, Inc.). Each experiment was performed in
8-(1-((6aR,9R,10aR)-9-(Azidomethyl)-1-hydroxy-6,6-dimethyl-
6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-3-yl)cyclopentyl)-
octanenitrile (33c). To a solution of 29c (120 mg, 0.2 mmol) in
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triplicate, and Ki values were determined from three independent
experiments and are expressed as the mean of these values.
Methods for Characterization of In Vivo Effects. Tail-Flick
Assay. Subjects. Male CD-1 mice (n = 6/dose) weighing between 25
and 30 g (Charles River, Wilmington, MA) were used. Mice were
housed 4/cage in a climate-controlled vivarium with unrestricted
access to food and water. Mice were acclimatized to these vivarium
conditions for at least one week before any experimental procedure.
Analgesia testing took place at the same time between 9 am and 4 pm.
Mice were used once. The experimental protocol for the studies was
approved by the Northeastern University Institutional Animal Care
and Use Committee. Subjects were maintained in a facility licensed by
the Committee on Care and Use of Laboratory Animals of the
Institute of Laboratory Animal Resources, National Institutes of
Health, 2011.
Photoaffinity Covalent Labeling. Rat brain membranes (rCB1,
Pel-Freez Biologicals, Rogers, AR) were prepared following the
previously described and appropriately modified procedures.27
Membranes from hCB2 expressed in HEK293 were incubated with
the azido ligands in concentrations of 10-fold of their Ki values for 30
min at 37 °C in a water bath with gentle agitation and then exposed to
UV (254 nm) for 1 min to activate the ligand.55 The unbound excess
ligand was washed out twice with 1% BSA in TME buffer (25 mM
Tris base, 5 mM MgCl2, 1 mM EDTA, pH 7.4). This was followed by
an additional washing to remove residual BSA, and the membranes
were isolated by centrifugation (Beckman Coulter, JA 20, 17 000 rpm,
10 min, 25 °C). A blank membrane sample was treated in parallel
using the same procedure and used as a control.
Electrophilic Covalent Labeling. CB1 and CB2 membranes
were incubated with the test ligand in a concentration of 10-fold of
their Ki values at 37 °C in a water bath with gentle agitation for 1 h
and treated as mentioned above without the photoirradiation step.
The unbound excess ligand was washed out twice with 1% BSA in
TME and once with TME alone to remove BSA, and the membranes
were isolated by centrifugation.
Saturation Binding Assay. Protein concentrations were
determined by using a Bio-Rad Bradford protein assay kit,56 and
saturation binding assays were performed in a 96-well format.30
Membrane pellets were resuspended in TME containing 0.1% BSA. A
total of 25 μg of protein was added to each well, and [3H]CP-55,940
was diluted in 0.1% BSA/TME buffer to yield ligand concentrations
ranging from 0.5 to 23.8 nM. Nonspecific binding was determined in
the presence of 4 μM unlabeled CP-55,940. The assay plates were
incubated at 30 °C with gentle agitation for 1 h. The resultant mixture
was then transferred to unifilter GF/B filter plates, and the bound
ligand was separated from unbound using a Packard Filtermate-96 cell
harvester (PerkinElmer Packard, Shelton, CT). Filter plates were
washed five times with ice-cold wash buffer (50 mM Tris-base, 5 mM
magnesium chloride with 0.5% BSA, pH 7.4). Bound radioactivity was
quantitated in a Packard TopCount scintillation counter. Nonspecific
binding was subtracted from the total bound radioactivity to calculate
the specific binding of [3H]CP-55,940 (measured as pmol/g in
saturation curves). Saturation assays were performed in triplicate, and
data points were presented as the mean SEM. Bmax and Kd values
were calculated by nonlinear regression using GraphPad Prism 4.0
(one-site binding analysis equation Y = BmaxX/(Kd + X), GraphPad
Software, San Diego, CA).
Functional Analysis Studies. The inhibition of forskolin-
stimulated cAMP was determined using CISBIO cAMP homogeneous
time-resolved fluorescence resonance energy transfer HiRange assay,
according to the manufacturer’s instructions and as described
previously.8,11 CHO-K1 cells stably expressing 3xHA tagged CB1 or
CB2 receptors (3xHA-CB1-CHO or 3xHA-CB2-CHO cells) were
plated at 4000 cells per well in white low-volume 384-well plates in
Opti-MEM with 1% FBS for 3 h at 37 °C. Cells were then cotreated
with 25 μM RO-20-1724 (an inhibitor of phosphodiesterase), 20 μM
forskolin (to directly stimulate adenylyl cyclase), and the indicated
concentrations of agonists for 30 min at 37 °C. Cells were lysed by the
addition of reagents to detect cAMP levels which were incubated at
room temperature for 1 h in the dark. Fluorescence was measured at
665 nm/620 nm using a Biotek Synergy Neo Multi-Mode Reader.
Data are plotted as the mean SEM of four or more experiments
performed in duplicate, as noted in the legend. The data were
normalized to vehicle and 100 nM CP-55,940 to determine the
maximum response (% CP); pharmacological parameters are
presented in the tables and were derived from three parameter
nonlinear regression curve fitting using GraphPad Prism 8.0.
Molecular Docking. Prediction of ligand binding to CB1 was
carried out with Schrodinger Suite 2015−4. Processing of the protein
structure (PDB: 5XR8)8 was performed with the Protein Preparation
Wizard. Converting of ligands from 2D to 3D structures was
performed using LigPrep. Molecular docking in standard precision
was performed with Glide 6.9.57−59
Drugs. For tail-flick analgesia testing, compound 27a was initially
dissolved in 2% dimethyl sulfoxide, 4% Tween 80, and 4% propylene
glycol before saline was slowly added just prior to the 10 mL/kg ip
administration. All drug suspensions were prepared just prior to
analgesia testing.
Analgesia Procedure. The tail-flick latency test was performed
using a thermostatically controlled 2L water bath commercially
available from VWR international. The temperature of the water bath
for the test day was set at 52 °C. For testing the analgesic effects of
compound 27a, the tip of the tail (2 cm) was immersed in the water
bath and withdrawal latency was recorded using a commercially
available stopwatch (Fisher Scientific) permitting measurement within
1/100 s. Injections occurred immediately following baseline record-
ing, and five recordings occurred after drug administration at 20, 60,
180, and 360 min, respectively, to complete the analgesia testing.
Animals were acclimatized for 3 days prior to the test day (4th day),
where tails were immersed in water bath held at room temperature.
The tail-flick withdrawal latencies are expressed as a percentage of
maximum possible effect (% MPE), based on the formula % MPE =
[(test latency minus baseline latency) divided by (10 minus baseline
latency)] times 100.
Data Analysis. Nonlinear regression analysis of % MPE data was
performed after log-X transformation using Prism Software (v. 5,
GraphPad Software, San Diego, CA) to provide estimates of the
independent variable when the co-ordinates of X intersected with Y =
50 and their 95% confidence limits (ED50 95% CL; regression
model: log dose or log time vs response-variable slope with the top
and bottom of the curves constrained to 100 and 0). The results are
presented as the mean ( SEM).
Locomotor Assay, Thermoregulation Assay, and Colonic
Expulsion Assay. Drugs. WIN 55,212−2 was obtained from Tocris
(Tocris, Ellisville, MO, USA). Compound 27a was synthesized, as
described above. All drugs were dissolved in Tween 80 (1%) and
DMSO (2%) and then further diluted in 0.9% saline up to the final
concentration.
Animals for Behavioral and GI Studies. 9−16 week old male
CB1+/+ and CB1−/− mice (25−32 g) on a CB57BL/6 background, as
well as 6−8 week old C57BL/6 wild-type mice, were used. The
C57BL/6 mice were obtained from Charles River (Montreal, PQ,
−/−
Canada), and the CB1+/+ and CB1
were bred at the University of
Calgary mouse breeding facility. Mice were housed at a constant
temperature of 22 °C and kept at a constant photoperiod (12:12 h
light−dark cycle) in plastic cages on sawdust with access to standard
laboratory chow and tap water ad libitum. The mice were used after at
least 1 week of acclimation. These experiments were approved by the
University of Calgary Animal Care Committee and were performed in
accordance with guidelines established by the Canadian Council of
Animal Care.
Spontaneous Locomotor Activity Assay. Locomotor activity,
assessed in C57Bl/6 mice, was measured using an infrared beam
activity monitor (Columbus Instruments, Columbus, OH, USA).
Sequential breaking of the invisible infrared beams by the movement
of the mouse is recorded, by the monitor, as the ambulatory and
horizontal activity counts. Mice were individually placed in the
apparatus (at the same location), and the counts were recorded over a
10 min period. The activity apparatus was cleaned with Virkon spray
between subjects. Mice underwent a locomotor activity trial in the
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morning (between 9 am and 12 pm) to acclimatize them to the
locomotor activity box, and the experiment was conducted in the
afternoon (between 1 and 4 pm) of the same day. Mice (n = 6−7/
treatment) were injected ip with either 27a (0.1−1.0 mg/kg),
WIN55,122 (1 mg/kg), or vehicle (2% DMSO, 1% Tween 80 in
physiological saline). 20 min later, mice were placed in the locomotor
activity box where activity was recorded for 10 min.
Thermoregulation Assay. Body temperature was measured using
silicone-coated data loggers (SubCue, Calgary, Canada) that were
implanted surgically into the abdomen of CB1−/− and CB1+/+ mice (n
= 7−8/treatment) under halothane anesthesia. Following implanta-
tion, mice were allowed to recover for 1 week. On day 8−11, mice
were injected with 27a (0.1 mg/kg, ip). Mice were sacrificed 24 h
later, and the data logger was removed and data were retrieved and
analyzed using SubCue Analyzer software. Core body temperature
was taken every 5 min from 6 to 12 days post-implantation, and the
data for −100 to 400 post agonist injection were analyzed. The
maximum decrease in body temperature from baseline (−100 to 0
min) was determined.
Colonic Expulsion Assay. Distal colonic propulsion was measured,
as we have reported previously. After 1-week acclimation, C57BL/7
mice (n = 6−7/treatment) were brought to the laboratory and,
following a 1-h acclimation, injected with vehicle (DMSO/Tween 80/
saline 2:1:17; ip) or 27a (0.1 mg/kg ip). After 20, 120, and 220 min,
mice were lightly anesthetized with isofluorane and a 2.5 mm glass
bead was inserted 2 cm into the rectum with a silicone pusher. Mice
were then placed in a glass beaker, and the time to expulsion of the
glass bead was monitored. Mice were returned to their home cage
between bead insertions. The bead expulsion time of the three trials
was determined and reported as the colonic expulsion time in
seconds. Data were analyzed by two-way ANOVA with Sidak’s
multiple comparison.
Authors
Shan Jiang − Center for Drug Discovery and Departments of
Chemistry and Chemical Biology, Northeastern University,
Boston, Massachusetts 02115, United States
Christos Iliopoulos-Tsoutsouvas − Center for Drug
Discovery and Department of Pharmaceutical Sciences,
Northeastern University, Boston, Massachusetts 02115,
United States
Fei Tong − Center for Drug Discovery and Department of
Pharmaceutical Sciences, Northeastern University, Boston,
Massachusetts 02115, United States
Christina A. Brust − Department of Molecular Medicine, The
Scripps Research Institute, Jupiter, Florida 33458, United
States
Catherine M. Keenan − Hotchkiss Brain Institute and Snyder
Institute for Chronic Diseases, Department of Physiology and
Pharmacology, University of Calgary, Calgary, Alberta T2N
4N1, Canada
Jimit Girish Raghav − Center for Drug Discovery and
Department of Pharmaceutical Sciences, Northeastern
University, Boston, Massachusetts 02115, United States
Tian Hua − iHuman Institute, ShanghaiTech University,
Shanghai 201210, China
Simiao Wu − Center for Drug Discovery and Department of
Pharmaceutical Sciences, Northeastern University, Boston,
Massachusetts 02115, United States
Jo-Hao Ho − Department of Molecular Medicine, The Scripps
Research Institute, Jupiter, Florida 33458, United States
Yiran Wu − iHuman Institute, ShanghaiTech University,
Shanghai 201210, China
Travis W. Grim − Department of Molecular Medicine, The
Scripps Research Institute, Jupiter, Florida 33458, United
States
Nikolai Zvonok − Center for Drug Discovery and Department
of Pharmaceutical Sciences, Northeastern University, Boston,
Massachusetts 02115, United States
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02053.
Experimental procedures, spectroscopic, analytical, and
physical data for 12a−b, 13a−b, 14a−c, 15a−c, 16a−c,
17a−c, 18a−c, 19a−c, 20a−c, 21a−c, 22a−c, 23a−c,
24a−c, 25a−c, 26a−c, and 27a−c; saturation binding
curves using [3H]CP-55,940 for CB1 and CB2 receptors
pre-incubated with the 25a−c, 26a−c, 27a−c, 31c, 32c,
33c, and 34c; and HPLC traces of 25a, 26c, 27b, and
31c (PDF).
Ganesh A. Thakur − Center for Drug Discovery and
Department of Pharmaceutical Sciences, Northeastern
University, Boston, Massachusetts 02115, United States;
orcid.org/0000-0002-7468-8819
Zhi-Jie Liu − iHuman Institute, ShanghaiTech University,
Shanghai 201210, China
Keith A. Sharkey − Hotchkiss Brain Institute and Snyder
Institute for Chronic Diseases, Department of Physiology and
Pharmacology, University of Calgary, Calgary, Alberta T2N
4N1, Canada
Laura M. Bohn − Department of Molecular Medicine, The
Scripps Research Institute, Jupiter, Florida 33458, United
States
Molecular formula strings and biochemical data of 25a−
c, 26a−c, 27a−c, 31c, 32c, 33c, and 34c (CSV)
AUTHOR INFORMATION
■
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c02053
Corresponding Authors
Spyros P. Nikas − Center for Drug Discovery and Department
of Pharmaceutical Sciences, Northeastern University, Boston,
Massachusetts 02115, United States; orcid.org/0000-
0001-5052-5292; Phone: +1-617-373-7620;
Email: s.nikas@northeastern.edu; Fax: +1-617-373-7493
Alexandros Makriyannis − Center for Drug Discovery,
Department of Pharmaceutical Sciences, and Departments of
Chemistry and Chemical Biology, Northeastern University,
Boston, Massachusetts 02115, United States; orcid.org/
0000-0003-3272-3687; Phone: +1-617-373-4200;
Email: a.makriyannis@northeastern.edu; Fax: +1-617-
373-7493
Author Contributions
#S.J. and C.I.-T. contributed equally
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by grants from the National Institute
on Drug Abuse to A.M. (DA026795, DA041435, DA045020
DA009158, and DA007215) and from the Canadian Institutes
of Health Research to K.A.S. K.A.S. holds the Crohn’s and
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modulation at the CB1 cannabinoid receptor. Mol. Pharmacol. 2015,
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Colitis Canada Chair in Inflammatory Bowel Disease Research
at the University of Calgary.
(13) Mallipeddi, S.; Janero, D. R.; Zvonok, N.; Makriyannis, A.
Functional selectivity at G-protein coupled receptors: Advancing
cannabinoid receptors as drug targets. Biochem. Pharmacol. 2017, 128,
1−11.
ABBREVIATIONS
■
BSA, bovine serum albumin; cAMP, 3′,5′-cyclic adenosine
monophosphate; CHO, Chinese hamster ovary cell line;
(−)-Δ9-THC, (−)-Δ9-tetrahydrocannabinol; DIBAL-H, diiso-
butylaluminum hydride; DMAP, N,N-dimethyl-4-aminopyr-
idine; DMF, N,N-dimethylformamide; DMSO, dimethylsulf-
oxide; ESI, electron spray ionization; GPCR, G protein-
coupled receptor; HHC, hexahydrocannabinol; HEK293,
human embryonic kidney cell line; KHMDS, potassium
bis(trimethylsilyl)amide; LAPS, ligand assisted protein struc-
ture; MPE, maximum possible effect; NMR, nuclear magnetic
resonance; rt, room temperature; SEM, standard error of
mean; TBSCl, tert-butyldimethylsilyl chloride; TBAF, tetra-n-
butylammonium fluoride; THC, tetrahydrocannabinol; THF,
tetrahydrofuran; TMG-N3, N,N,N′,N′-tetramethylguanidinium
azide; TMS, tetramethylsilane; TMSOTf, trimethylsilyl tri-
fluoromethanesulfonate
(14) Rankovic, Z.; Brust, T. F.; Bohn, L. M. Biased agonism: An
emerging paradigm in GPCR drug discovery. Bioorg. Med. Chem. Lett.
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