Evaluation of (Z)-2-((1-benzyl-1H-indol-3-yl)methylene)-quinuclidin-3-one analogues as novel, high affinity ligands for CB1 and CB2 cannabinoid receptors

Article abstract of DOI:10.1016/j.bmcl.2013.02.025

A small library of N-benzyl indolequinuclidinone (IQD) analogs has been identified as a novel class of cannabinoid ligands. The affinity and selectivity of these IQDs for the two established cannabinoid receptor subtypes, CB1 and CB2, was evaluated. Compounds 8 (R = R² = H, R¹ = F) and 13 (R = COOCH₃, R¹ = R² = H) exhibited high affinity for CB2 receptors with K_i values of 1.33 and 2.50 nM, respectively, and had lower affinities for the CB1 receptor (K_i values of 9.23 and 85.7 nM, respectively). Compound 13 had the highest selectivity of all the compounds examined, and represents a potent cannabinoid ligand with 34-times greater selectivity for CB2R over CB1R. These findings are significant for future drug development, given recent reports demonstrating beneficial use of cannabinoid ligands in a wide variety of human disease states including drug abuse, depression, schizophrenia, inflammation, chronic pain, obesity, osteoporosis and cancer.

Full text of DOI:10.1016/j.bmcl.2013.02.025

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2019–2021
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Evaluation of (Z)-2-((1-benzyl-1H-indol-3-yl)methylene)-quinucli-
din-3-one analogues as novel, high affinity ligands for CB1 and CB2
cannabinoid receptors
Nikhil Reddy Madadi a, Narsimha Reddy Penthala a, Lisa K. Brents b, Benjamin M. Ford b,
Paul L. Prather b, Peter A. Crooks a,
⇑
a Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
b Department of Pharmacology and Toxicology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A small library of N-benzyl indolequinuclidinone (IQD) analogs has been identified as a novel class of can-
nabinoid ligands. The affinity and selectivity of these IQDs for the two established cannabinoid receptor
subtypes, CB1 and CB2, was evaluated. Compounds 8 (R = R2 = H, R1 = F) and 13 (R = COOCH3, R1 = R2 = H)
exhibited high affinity for CB2 receptors with Ki values of 1.33 and 2.50 nM, respectively, and had lower
affinities for the CB1 receptor (Ki values of 9.23 and 85.7 nM, respectively). Compound 13 had the highest
selectivity of all the compounds examined, and represents a potent cannabinoid ligand with 34-times
greater selectivity for CB2R over CB1R. These findings are significant for future drug development, given
recent reports demonstrating beneficial use of cannabinoid ligands in a wide variety of human disease
states including drug abuse, depression, schizophrenia, inflammation, chronic pain, obesity, osteoporosis
and cancer.
Received 3 January 2013
Accepted 1 February 2013
Available online 14 February 2013
Keywords:
N-Benzyl indolequinuclidinone
N-Benzyl indolequinuclidinol
N-Benzyl indolequinuclidin-3-one oxime
Cannabinoid ligands
CB1 receptors
Ó 2013 Elsevier Ltd. All rights reserved.
CB2 receptors
D
9-Tetrahydrocannabinol ( 9-THC, Fig. 1), the well-known
D
primary psychoactive constituent of marijuana, exhibits a variety
of potential therapeutic effects, including anti-convulsive, anti-
emetic, analgesic, lowering of intraocular pressure and immune
modulation.1 However, undesirable central nervous system (CNS)
effects and potential addictive properties of
D
9-THC has limited
its medical use, and raised the necessity for identifying the recep-
tors responsible for cannabinoid activity in order to develop drugs
with fewer adverse side-effects. CB1 and CB2 are the two major
cannabinoid-specific receptors which have been characterized
from mammalian tissues.2,3 CB1 receptors (CB1R) are abundantly
expressed in the CNS. These receptors are also expressed in periph-
eral nerve terminals and in many extra-neuronal sites. CB1R acti-
vation is responsible for most of the pharmacological effects of
cannabinoids in the nervous system. In contrast, CB2 receptors
(CB2R) are known to be expressed in other tissues,4,5 predomi-
nantly in cells of the immune system.6 Interestingly, many types
of cancer cells express relatively high densities of CB1 and CB2
receptors, and most recent studies report that cannabinoids can
reduce tumor growth and progression in several animal models
of cancer.7–9 There are three main structural classes of cannabinoid
ligands. These are the ‘classical’ cannabinoid analogues of
⇑
Corresponding author. Tel.: +1 501 686 6495; fax: +1 501 686 6057.
E-mail address: pacrooks@uams.edu (P.A. Crooks).
Figure 1. High affinity cannabinoid receptor ligands.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.02.025

2020
N. R. Madadi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2019–2021
D
9-THC, the ‘non-classical’ bicyclic and tricyclic cannabinoid ana-
(PTC) conditions using triethylbenzylammonium chloride in 50%
w/v aqueous NaOH solution in dichloromethane.16 Reduction of
compounds and with NaBH4 in methanol afforded the
logues of
D
9-THC, the aminoalkylindoles (e.g., WIN 55212-2,
Fig. 1)10 and other diverse heterocyclic structures such as the pyr-
azoles (e.g., SR141716A, Fig. 1)11–13 and indolopyridones (e.g.,
IPDN, Fig. 1).14 In the evaluation of synthetic or non-classical can-
nabinoids, the amino alkylindole WIN 55212-2 was reported as a
potent non-selective cannabinoid receptor agonist with Ki values
of 1.9 nM for CB1R and 0.3 nM for CB2R.14
7
9
corresponding (Z)-( )-2-(N-benzylindol-3-yl methylene)-quinucl-
idin-3-ol derivatives 14 and 15 in 82–88% yield.16 Reaction of com-
pounds 7 and 9 with hydroxyl amine hydrochloride in the presence
of sodium acetate/methanol afforded the (2Z,3E)-2-((1-benzyl-1H-
indol-3-yl)methylene)-quinuclidin-3-one oximes 16 and 17.17
Confirmation of the structure and purity of these analogs was
obtained from 1H and 13C NMR spectroscopic analysis.18 The
geometry of the double bond in these molecules was established
from X-ray crystallographic data.17,19,20
In contrast, the pyrazole compound SR141716A was reported to
selectively bind to CB1R where it acts as an antagonist/inverse ago-
nist without producing cannabimimetic activity in vivo.12,13 The
indolopyridone analogue IPDN exhibits higher affinity for CB2R
(Ki = 1.0 nM) than for CB1R (Ki = 16 nM).14
The indole nucleus has played an important role in the activity
of the cannabinoid compound WIN 55212-2, and indolopyridone
(IPDN) analogues and other analogs that incorporate a variety of
different bicyclic ring systems as subunits have emerged as potent
cannabinoid ligands.14,15 These observations prompted us to
undertake a more detailed investigation into the structure–activity
relationships of various indole analogs that incorporate a quinuc-
lidinone bicyclic ring system as a structural subunit. The present
study focuses on the synthesis and evaluation of some (Z)-2-((1-
Membrane homogenates of either whole mouse brain (50
(CB1R) or Chinese hamster ovary (CHO)-hCB2 cells (25
l
l
g)
g)
(CB2R) were incubated for 90 min at room temperature with
0.2 nM [3H]CP-55, 940, 5 mM MgCl2, and either increasing IQD
concentrations (10 fM–10 lM), 1 lM WIN 55212-2 (to define
non-specific binding) or vehicle (to determine total binding). Each
ligand concentration was examined in triplicate, in a final volume
of 1 mL of buffer containing 50 mM Tris, 0.05% bovine serum albu-
min (BSA) and 0.1% ethanol/0.1% dimethyl sulfoxide vehicle. Reac-
tions were terminated by rapid vacuum filtration through
Whatman GF/B glass fiber filters, followed by five washes with
ice-cold buffer (50 mM Tris, 0.05% BSA). Filters were immediately
placed into 7 mL scintillation vials to which 4 mL of scintillation
fluid was added. Bound radioactivity was determined after
overnight incubation at room temperature by liquid scintillation
spectrophotometry. Specific binding was expressed as total minus
non-specific binding and graphed for each data point as a percent-
age of specific binding occurring in the absence of any competitor.
The affinity of IQDs for CB1R and CB2Rs was derived by employing
the Cheng–Prusoff equation21 to convert the observed IC50 values
to Ki values, from 3 to 4 separate competition receptor binding
curves for each ligand.
All the synthesized IQDs were evaluated against the two can-
nabinoid receptor subtypes, CB1 and CB2. Compounds 7–9, 11
and 13 displayed high affinity (<150 nM) for both CB1 and CB2
receptors, with Ki values ranging from 9.23 to 135 nM for CB1R
and 1.33 to 114 nM for CB2R. Compounds 7 (Ki = 11.7 nM), 8
(Ki = 1.33 nM), 13 (Ki = 2.50 nM), 14 (Ki = 55.3 nM) and 16
(Ki = 68.6 nM) demonstrated some degree of CB2R selectivity
benzyl-1H-indol-3-yl)methylene)-quinuclidin-3-one
(IQD) as novel potent ligands for CB1 and CB2 cannabinoid
receptors.
analogues
The synthetic strategy for the preparation of IQDs 7–17 is sum-
marized in Scheme 1. The appropriate 1-acetylindole-3-carboxal-
dehyde (1 and 2) was prepared by N-acetylation of the
corresponding indole-3-carboxaldehyde with acetic anhydride/ tri-
ethylamine in dichloromethane under reflux conditions. Aldol con-
densation of the appropriate 1-acetylindole-3-carboxaldehyde
with quinuclidin-3-one hydrochloride in the presence of lithium
diisopropylamide in tetrahydrofuran at À78 °C afforded the (Z)-
2-(N-acetylindol-3-ylmethylene)quinuclidin-3-one derivative (3
and 4). Subsequent N-deacetylation in refluxing 1 N aqueous NaOH
solution afforded the appropriate (Z)-2-(1H-indol-3-ylmethylene)-
quinuclidin-3-ones (5 and 6).
The
(Z)-2-(N-benzylindol-3-ylmethylene)-quinuclidin-3-one
derivatives (7–13) were prepared in 81–87% yield by treatment
of the (Z)-2-(1H-indol-3-yl methylene)quinuclidin-3-ones 5 and 6
with a variety of benzyl halides under phase-transfer catalytic
Scheme 1. Synthesis of indole quinuclidine analogs (IQDS) (7–17). Reagents and conditions: (a) Ac2O, Et3N, DCM, reflux; (b) quinuclidin-3-one hydrochloride, THF, LDA,
À78 °C; (c) 1 N NaOH (aq), reflux; (d) substituted benzyl halide, 50% NaOH (aq), triethylbenzylammonium chloride, DCM, rt; (e) NaBH4, methanol, rt. (f) hydroxylamine
hydrochloride, NaOAc, methanol, rt.

N. R. Madadi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2019–2021
2021
Table 1
abuse, depression, schizophrenia, inflammation, chronic pain,
obesity, osteoporosis and cancer.22
Ki values for (Z)-2-(N-benzylindol-3-ylmethylene) quinuclidin-3-ones (7–13) at CB1
and CB2 receptors
Compound
Ki (nM)
Acknowledgements
CB1
CB2
We are grateful to NCI/NIH (Grant number CA 140409) and to
the Arkansas Research Alliance (ARA) for financial support
7
8
9
10
11
12
13
135 44.1
9.23 0.64
31.7 9.25
629 201
55.3 8.89
953 162
85.7 15.9
11.7 5.55
1.33 0.45
20.3 7.43
333 157
114 36.2
588 237
2.50 0.49
References and notes
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Table 2
Effects of simple and p-chloro substituted N-benzyl indole quinuclidin-3-ol analogs
(14 and 15) the quinuclidin-3-one oxime analogs (16 and 17) on binding affinity
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Ki (nM)
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14
15
16
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540 64.2
85.3 42.5
357 52.2
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compared to CB1R. Compound 13, which contains a COOCH3 moi-
ety at the indolic 6-position, was a potent CB2 receptor ligand and
exhibited 34-fold selectivity for CB2R (Ki = 2.50 nM) when com-
pared to CB1R (Ki = 85.7 nM) (Table 1). The N-4-fluorobenzyl ana-
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The (Z)-( )-2-(N-benzylindol-3-ylmethylene)quinuclidin-3-ols
(14 and 15) and (2Z,3E)-2-((1-benzyl-1H-indol-3-yl) methylene)-
quinuclidin-3-one oximes (16 and 17) exhibited lower affinity for
both CB1 and CB2 receptors when compared to their parent com-
pounds (7 and 9) and were generally less selective. The overall
affinities of quinuclidin-3-ol and quinuclidin-3-one oxime deriva-
tives for CB1R and CB2R were reduced when compared to those
of the corresponding quinuclidin-3-one derivatives (Table 2).
In summary N-benzylindolequinuclidinone (IQD) analogs have
been identified as a novel class of cannabinoid ligands. These IQDs
were evaluated against the two cannabinoid receptor subtypes,
CB1 and CB2. Compounds 8 and 13 exhibited high affinity for
CB2 receptors with Ki values of 1.33 and 2.50 nM, respectively,
and had lower affinities for the CB1 receptor (Ki values of 9.23
and 85.7 nM, respectively). Compound 13 had the highest selectiv-
ity of all the compounds examined, and represents a potent can-
nabinoid ligand with 34-times greater selectivity for CB2R over
CB1R. These findings are significant for future drug development,
given recent reports demonstrating beneficial use of cannabinoid
ligands in a wide variety of human disease states including drug
18. Analytical data and yields for the most active compounds: (8): 1H NMR (300 MHz,
CDCl3): d 2.01–2.03 (m, 4H), 2.62 (p, J = 3.0 Hz, 1H), 2.92–3.02 (m, 2H), 3.08–
3.18 (m, 2H), 5.34 (s, 2H), 6.96–7.25 (m, 7H), 7.46 (s, 1H), 7.87–7.90 (m, 1H),
8.36 (s, 1H); 13C NMR (75 MHz, CDCl3): d 26.4, 40.5, 47.5, 50.0, 110.1, 111.4,
115.6, 118.8, 121.1, 121.8, 122.8, 124.3, 127.7, 128.27, 130.7, 134.3, 135.5,
135.8, 135.9, 185.2; yield: 87%: (13): 1H NMR (300 MHz, CDCl3): d 1.98–2.04
(m, 4H), 2.62–2.63 (p, J = 2.7 Hz, 1H), 2.91–3.01 (m, 2H), 3.08–3.17 (m, 2H),
3.90 (s, 3H, OCH3), 5.44 (s, 2H), 7.10–7.13 (m, 2H), 7.28–7.43 (m, 3H), 7.43 (s,
1H), 7.81–7.89 (m, 2H), 8.03 (s, 1H), 8.51 (s, 1H); 13C NMR (75 MHz, CDCl3): d
26.3, 40.4, 47.5, 50.6, 52.0, 110.6, 112.4, 117.1, 118.7, 122.0, 124.4, 126.5, 127.9,
128.9, 132.2, 135.5, 136.2, 136.9, 141.3, 167.7, 205.3; yield: 91%.
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