Synthesis and preliminary biological evaluation of indol-3-yl-oxoacetamides as potent cannabinoid receptor type 2 ligands

Article abstract of DOI:10.3390/molecules22010077

A small series of indol-3-yl-oxoacetamides was synthesized starting from the literature known N-(adamantan-1-yl)-2-(5-(furan-2-yl)-1-pentyl-1H-indol-3-yl)-2-oxoacetamide (5) by substituting the 1-pentyl-1H-indole subunit. Our preliminary biological evaluation showed that the fluorinated derivative 8 is a potent and selective CB2 ligand with K_i = 6.2 nM.

Full text of DOI:10.3390/molecules22010077

molecules
Communication
Synthesis and Preliminary Biological Evaluation of
Indol-3-yl-oxoacetamides as Potent Cannabinoid
Receptor Type 2 Ligands
Rare s¸ -Petru Moldovan *, Winnie Deuther-Conrad , Andrew G. Horti and Peter Brust 1
1
,
1
2
1
Helmholtz-Zentrum Dresden-Rossendorf e.V., Institute of Radiopharmaceutical Cancer Research,
Permoserstr. 15, 04318 Leipzig, Germany; w.deuther-conrad@hzdr.de (W.D.-C.); p.brust@hzdr.de (P.B.)
Johns Hopkins School of Medicine, Division of Nuclear Medicine and Molecular Imaging,
2
Department of Radiology, Baltimore, MD 21287, USA; ahorti1@jhmi.edu
*
Correspondence: r.moldovan@hzdr.de; Tel.: +49-341-234-179-4634
Academic Editor: Diego Muñoz-Torrero
Received: 21 October 2016; Accepted: 22 December 2016; Published: 4 January 2017
Abstract: A small series of indol-3-yl-oxoacetamides was synthesized starting from the
literature known N-(adamantan-1-yl)-2-(5-(furan-2-yl)-1-pentyl-1H-indol-3-yl)-2-oxoacetamide (5) by
substituting the 1-pentyl-1H-indole subunit. Our preliminary biological evaluation showed that the
fluorinated derivative 8 is a potent and selective CB ligand with K = 6.2 nM.
2
i
Keywords: positron emission tomography; cannabinoid receptor type 2; binding affinity; indole
1
. Introduction
For centuries, Cannabis sativa was used as medicinal plant for the treatment of pain and
9
inflammatory processes. The discovery by Gaoni and Mechoulam [
(
1] of
∆
-tetrahydrocannabinol
9
∆ -THC)—the primary psychoactive ingredient in Cannabis sativa—set the stage for the
identification of the endogenous cannabinoid (endocannabinoid) transmitter system in the
brain. The endocannabinoid system consists of cannabinoid (CB) receptors, endogenous ligands
(
e.g., anandamide, 2-O-arachidonoylglycerol) that activate the CB receptors, and the enzymes, which
are responsible for the biosynthesis (e.g., N-acyltransferase, diacylglycerol lipase) and deactivation
(e.g., fatty acid amide hydrolases, monoacylglycerol lipases) of the endogenous ligands [2,3].
Two types of specific Gi/o-protein-coupled CB receptors were cloned in the 1990s, termed CB1
and CB receptor [4,5]. CB receptors are abundantly expressed in the central nervous system, and
1
2
their function has been thoroughly investigated [
has been reported, providing a tool for a more accurate pharmacological investigation of this receptor
subtype [ ]. In contrast to CB receptors, the CB receptor was originally regarded as a peripheral
receptor predominantly expressed in cells of the immune system [
investigations proved the presence of CB receptors in glial cells (microglia and astrocytes) [11
6]. Recently, the crystal structure of the CB receptors
1
7,8
1
2
5,
9,
10]. However, more recent
12], and
,
2
oligodendroglial [13] progenitors in vitro, as well as in microglia [14] and neuronal progenitors [12] in
normal mouse brain in vivo [15].
The predominant expression of the CB receptor in cells of the immune system suggests a
2
modulation of diverse immune functions, including cytokine production, lymphocyte proliferation,
and humoral and cell-mediated immune responses [16,17]. With respect to neuroinflammation,
microglia adopt a key function. Under healthy conditions, the expression of CB receptors in the brain
2
is rather low [18]. However, inflammatory effects result in considerably increased expression levels
of CB receptors [19
,20]. It was found that neurodegenerative and neuroinflammatory processes in
2
the brain related to, for example, depression, Alzheimer’s disease, multiple sclerosis, amyotrophic
Molecules 2017, 22, 77; doi:10.3390/molecules22010077
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Molecules 2017, 22, 77
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lateral sclerosis, or brain tumors such as glioblastoma are associated with an upregulation of CB2
receptor expression [18 21 23]. CB receptor agonists lead to a reduction of neuroinflammation
and stimulation of neurogenesis. Therefore, the therapeutic potential of CB agonists is related
,
–
2
2
to neurodegenerative and neuroinflammatory processes [19,24]. Moreover, the availability of CB2
receptors as measured with positron emission tomography (PET) [25] could potentially be used as a
biomarker for neurodegenerative and neuroinflammatory processes in the brain [26–29].
Indole has been a very popular key building block for the medicinal chemistry of cannabinoid
receptor ligands, and a large number of indole-derived CB2 selective ligands have been
developed [30–33]. However, most of these ligands do not comply with the most important
needs of a ligand suitable for the development of a tracer for CB brain imaging with PET;
2
namely, high affinity towards CB (K (CB ) < 1 nM) and high selectivity over CB (K (CB )/(CB )
2
i
2
1
i
2
1
>
500) [26
,
34].
Recently, we reported the development of a highly affine and selective
1
8
F-labeled CB radiotracer (K (CB ) = 0.4 nM, K (CB ) = 380 nM), and proved its applicability
2
i
2
i
1
in a mouse model of neuroinflammation [35]. However, this radioligand suffers from low
metabolic stability in vivo, and therefore we redirected our focus on the structure of the literature
known, highly affine N-(adamantan-1-yl)-2-(5-(furan-2-yl)-1-pentyl-1H-indol-3-yl)-2-oxoacetamide
(5, K (CB ) = 0.37 nM and K (CB ) = 345 nM) [32] for the development of a CB radiotracer. In the
i 2 i 1 2
present work, we show the synthesis and preliminary biological evaluation of fluorine-containing
indol-3-yl-oxoacetamide derivatives.
2
. Results and Discussion
The structure–affinity relationship study which lead to the identification of compound 5 [32,36]
(
Scheme 1) and the work performed on the structurally related quinolinone-3-carboxamides as CB2
ligands [37] has proven the importance of the substitution pattern at the 5-indole position, and also
the need of the lipophilic adamantane subunit. Herein, we investigate the possibility of introducing a
fluorine atom by modifying the alkyl chain at the indole 1-position.
Scheme 1. Synthesis of the key building block
3
, lead compound
5
, and ether derivatives
) amantadine,
) 2-furanboronic acid,
tetrakis(triphenylphosphine)palladium(0) (Pd(PPh ) ), Na CO , EtOH, reflux, 14 h, 60%;
6,
◦
7
, and ; (
8
a
) oxalyl chloride, diethyl ether (Et O), 0 C to r.t., 1.5 h, 60%; (
b
2
triethylamine (Et N), dichloromethane (DCM), r.t., 16 h, 82%;
(c
3
3
4
2
3
(
7
d
2%; (
) 1-bromobutane (n-BuBr), tetrabutylammonium bromide (TBAB), 20% aq. NaOH, DCM, r.t., 14 h,
◦
e
) bromo-alkyl reagent, KOH, N,N-dimethylformamide (DMF), 90 C, 6 h, 67%–72%; (
f
) NaH,
◦
methyl iodide (MeI) for 7 and Br(CH ) F for 8, DMF, 0 C to r.t., 6 h, 85%–90% [32].
2
2

Molecules 2017, 22, 77
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The synthesis of the key building block
and depicted in Scheme 1. Treatment of 5-bromoindole (
corresponding indole-3-oxoacetyl chloride in ~60% yield, which was further reacted with amantadine
adamantan-1-amine) in presence of triethylamine (Et N) to give compound Next, Suzuki
coupling was performed using the commercially available 2-furanboronic acid in presence of
Pd(PPh ) and Na CO to give . The lead molecule 32] was synthesized by treating with
-bromobutane in basic reaction condition in ~25% yield over four steps starting from , as a light
3
was performed as described in the literature [32
]
1) with oxalyl chloride delivered the
(
2.
3
3
5
[
3
3
4
2
3
1
1
yellow solid. To overcome the high lipophilicity of the indole-N-alkyl chain [38], ether groups
were introduced at this site of the molecule, and simultaneously, the influence of its length on the
CB binding affinity was investigated. Thus, the glycol ether compound
6
,
was synthesized from
40] as alkylating reagent
was synthesized by reacting compound with 2-bromethanol.
3
2
by using 2-(2-(2-fluoroethoxy)ethoxy)ethyl-4-methylbenzenesulfonate [39
in 72% yield. Similarly, alcohol
4
3
Alcohol
4
was further etherified via Williamson ether synthesis [41] by using methyl iodide and
1
2
-fluoro-1-bromoethane to give compounds
and 8 see Supplementary Materials).
The binding affinity towards CB receptors was determined in vitro by radioligand inhibition
7
and
8, respectively (for H-NMR of compounds
3,
5,
6
,
7
2
binding assays according to a recently published protocol [42,43] using cell membranes from CHO
cells stably transfected with the human CB (Prof Paul L. Prather, University Arkansas for Medical
2
3
Sciences, Little Rock, AR, USA), [ H]WIN55.212-2 as competitive radioligand (KD = 2.1 nM) and
increasing concentrations (100 pM to 10
µM) of compounds
5
,
6,
7
, and
8 added in triplicate for
each experiment. Non-specific binding was determined using 10
µM WIN55.212-2. The individual
IC50 values were determined by non-linear curve fitting using GraphPad Prism software (version 3.0,
GraphPhad, San Diego, CA, USA), and the corresponding K values calculated using the Cheng–Prusoff
i
equation [44]. As shown by the K values in Figure 1b, we could not reproduce the reported
i
sub-nanomolar CB affinity of the lead molecule (compound 5) [32], but recorded a K value of
i
2
8
.5 nM instead. In addition, these values and the individual binding curves in Figure 1a illustrate that
a similar low-nanomolar affinity was observed for the fluorinated derivative (K = 6.2 nM), while
slightly lower affinities were determined for compounds and (K = 27.3 nM, and K = 16.1 nM,
8
i
6
7
i
i
respectively). Additionally, the binding affinities towards the CB receptor were investigated for
1
3
compounds
6
and
8
by using [ H]CP55.40 as competitive radioligand. None of the two derivatives
3
could displace [ H]CP55.40 from the human CB receptors up to a concentration of 100
µ
M. Thus,
is most suitable
to obtain high affinity binding at the CB receptor, combined with an excellent selectivity against the
1
within this series of fluoro-substituted compounds, the N-alkyl chain of compound
8
2
CB receptor subtype.
1
a
Cpd.
Ki
(CB
2
) (nM)
b
5
6
7
8
8.5 ± 1.2 (0.37 )
27.3 ± 12
16.1 ± 6
6.2 ± 0.02
(
a)
(b)
Figure 1.
(
a
) Individual competition binding curves of compounds
5
,
6
,
7
, and
8
. Inhibition of
3
[
H]WIN55.212-2 binding by increasing concentrations (100 pM to 10
µM) of compounds
5,
6
,
7, and
8
to membrane homogenates of CHO cells stably transfected with human CB . Each value represents the
2
mean
±
standard deviation of a triplicate in a single experiment; (
b
) Binding affinity (K ) of compounds
i
a
5,
6,
7
, and
8
for the human cannabinoid receptor type 2 (CB ) receptor. Values are means
±
standard
2
b
deviations of two to three experiments run in triplicate; K of compound 5 as reported in [23].
i

Molecules 2017, 22, 77
4 of 8
3
. Materials and Methods
3
.1. General Methods
All reagents were used directly as obtained commercially, unless otherwise noted. Reaction
progress was monitored by thin-layer chromatography (TLC) using silica gel 60 F254 (0.040–0.063 mm)
with detection by UV. All moisture-sensitive reactions were performed under an argon atmosphere
using oven-dried glassware and anhydrous solvents. Column flash chromatography was carried out
using E. Merck silica gel 60F (230–400 mesh) (Merck Millipore, Darmstadt, Germany). Analytical
TLC was performed on aluminum sheets coated with silica gel 60 F254 (0.25 mm thickness, E. Merck,
1
Darmstadt, Germany). H-NMR spectra were recorded with a Bruker-400 NMR spectrometer (Bruker,
Billerica, MA, USA) at nominal resonance frequencies of 400 MHz, in CDCl or DMSO-d (referenced
3
6
to internal Me Si at δH 0 ppm). The chemical shifts (δ) were expressed in parts per million (ppm).
4
High resolution mass spectra were recorded utilizing electrospray ionization (ESI) at the University of
Notre Dame Mass Spectrometry facility.
3
.2. Procedures and Compound Characterization
N-(Adamantan-1-yl)-2-(5-(furan-2-yl)-1-(2-hydroxyethyl)-1H-indol-3-yl)-2-oxoacetamide (
55 L, 1.2 eq, 0.77 mmol) was added to a solution of (300 mg, 1 eq, 0.64 mmol) in 5 mL
4). 2-bromethanol
(
µ
3
N,N-dimethylformamide (DMF) at room temperature, followed by powder KOH (116 mg, 3 eq,
◦
1
.93 mmol), and the reaction mixture was warmed to 90 C for 6 h. Saturated aqueous NaHCO solution
3
(
15 mL) and ethyl acetate (EA, 15 mL) were added, and the phases were separated. The aqueous phase
was washed 2 EA (10 mL), the combined organic solutions were dried over MgSO , filtered, and
concentrated by rotary evaporation. The residue was purified by column chromatography (silica,
EA:Hex, 1/1) to give alcohol
(224 mg, 67% yield) as yellow solid. 1H-NMR (400 MHz, CDCl3):
(ppm) = 1.67–1.76 (m, 6H), 2.05–2.12 (m, 6H), 2.07–2.11 (m, 6H), 2.14 (br s, 3H), 2.41 (br s, 1H),
×
4
4
δ
3
0
.96–4.06 (t, J = 5.18 Hz, 2H), 4.31 (t, J = 5.18 Hz, 2H), 6.51 (dd, J = 3.28, 1.77 Hz, 1H), 6.71 (dd, J = 3.28,
.76 Hz, 1H), 7.35 (s, 1H), 7.40 (dd, J = 8.59, 0.76 Hz, 1H), 7.50 (dd, J = 1.77, 0.76 Hz, 1H), 7.65 (dd,
13
J = 8.59, 1.52 Hz, 1H), 8.71 (dd, J = 1.77, 0.51 Hz, 1H), 8.99–9.10 (m, 1H). C-NMR (100 MHz, CDCl3):
(ppm) = 29.36 (3C), 36.29 (3C), 41.15 (3C), 49.58, 51.84, 61.26, 104.42, 110.40, 111.72, 112.16, 118.02,
20.29, 126.77, 128.24, 135.74, 141.77, 142.03, 154.59, 161.48, 181.14.
δ
1
N-(Adamantan-1-yl)-2-(5-(furan-2-yl)-1-pentyl-1H-indol-3-yl)-2-oxoacetamide (
5
). This compound was
1
obtained according to literature. H-NMR (400 MHz, CDCl ):
δ (ppm) = 0.86–0.94 (m, 3H), 1.28–1.44
3
(
m, 4H), 1.69–1.81 (m, 6H), 1.93 (quin, J = 7.33 Hz, 2H), 2.10–2.20 (m, 9H), 4.16 (t, J = 7.33 Hz, 2H), 6.51
(
1
dd, J = 3.28, 1.77 Hz, 1H), 6.73 (dd, J = 3.28, 0.76 Hz, 1H), 7.37–7.44 (m, 2 H), 7.50 (dd, J = 1.89, 0.63 Hz,
H), 7.69 (dd, J = 8.59, 1.77 Hz, 1H), 8.73–8.78 (m, 1H), 9.04 (s, 1H). C-NMR (100 MHz, CDCl3): δ
13
(
1
ppm) = 13.94, 22.29, 28.99, 29.38 (3C), 29.60, 36.32 (3C), 41.21 (3C), 47.58, 51.78, 104.35, 110.43, 111.70,
11.91, 118.10, 120.13, 126.67, 128.36, 135.60, 141.62, 141.74, 154.70, 161.58, 180.97.
N-(Adamantan-1-yl)-2-(1-(2-(2-(2-fluoroethoxy)ethoxy)ethyl)-5-(furan-2-yl)-1H-indol-3-yl)-2-oxoacetamide (
-(2-(2-fluoroethoxy)ethoxy)ethyl-4-methylbenzenesulfonate (40 mg, 1.5 eq, 0.15 mmol) and KOH
25 mg, 5 eq, 0.5 mmol) were added at room temperature to a solution of compound (46 mg, 1 eq,
.1 mmol) in 3 mL DMF, and the reaction mixture was warmed to 90 C for 6 h. To the cooled
6).
2
(
0
3
◦
down solution, 15 mL saturated aqueous NaHCO solution was added followed by 15 mL EA.
3
The phases were separated, and the aqueous phase was separated 2
×
10 mL EA. The combined
organic phases were washed with 20 mL brine, dried over MgSO , and concentrated under reduced
4
pressure. The resulting residue was purified by column chromatography (silica, EA:Hex, 1/1) to give
6
1
(
(
(
44 mg, 72% yield) as yellow solid. H-NMR (400 MHz, CDCl ):
δ
(ppm) = 1.68–1.79 (m, 6H), 2.08–2.19
3
m, 9H), 3.51–3.71 (m, 6H), 3.65–3.70 (m, 2H), 3.90 (t, J = 5.43 Hz, 2H), 4.37 (t, J = 5.43 Hz, 2H), 4.40–4.45
m, 1H), 4.49–4.68 (m, 1 H), 6.51 (dd, J = 3.28, 1.77 Hz, 1H), 6.72 (dd, J = 3.28, 0.76 Hz, 1H), 7.38 (s, 1H),
7
.41–7.54 (m, 2H), 7.69 (dd, J = 8.59, 1.77 Hz, 1H), 8.69–8.88 (m, 1H), 9.08 (s, 1H). 13C-NMR (100 MHz,

Molecules 2017, 22, 77
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CDCl3):
δ
(ppm) = 29.38 (3C), 36.32 (3C), 41.20 (3C), 47.43, 51.76, 69.66 (2C), 70.43, 70.62, 70.81, 71.02
(
(
2C), 83.95, 104.37, 110.64, 111.70, 117.98, 120.15, 126.69, 128.21, 135.84, 141.76, 142.29, 181.21. HRMS
ESI+): m/z (%) = 523.6150 (ccd. 523.6151) [M + H] .
+
N-(Adamantan-1-yl)-2-(5-(furan-2-yl)-1-(2-methoxyethyl)-1H-indol-3-yl)-2-oxoacetamide (
suspension in mineral oil, 10 mg, 2 eq, 0.25 mmol) was added to a solution of (65 mg, 1 eq, 1.12 mmol)
in 2 mL DMF at 0 C, and the mixture was stirred at 0 C for 15 min. MeI (64 L, 8 eq, 1.02 mmol)
7). NaH (60%
4
◦
◦
µ
was added, the ice bath was removed, and the reaction was magnetically stirred at room temperature.
After 5 h, the reaction was quenched by slow addition of 2 mL cold water followed by 10 mL saturated
aqueous NaHCO solution and 15 mL EA. The phases were separated, and the aqueous phase was
3
washed three times with 10 mL EA. The combined organic phases were washed with brine, dried over
MgSO , and concentrated under reduced pressure. The resulting material was purified by column
4
1
chromatography (silica, EA:Hex, 1/1) to give
CDCl3):
7
(55 mg, 84% yield), yellow solid. H-NMR (400 MHz,
δ
(ppm) = 1.75 (m., 6H), 2.10–2.20 (m, 9H), 3.25–3.39 (s, 3H), 3.77 (t, J = 5.43 Hz, 2H), 4.34
(
1
t, J = 5.43 Hz, 2H), 6.46–6.55 (m, 1H), 6.68–6.78 (m, 1H), 7.39 (s, 1H), 7.40–7.45 (m, 1H), 7.47–7.54 (m,
H), 7.69 (dd, J = 8.59, 1.77 Hz, 1H), 8.74 (d, J = 1.77 Hz, 1H), 9.01–9.10 (m, 1H). C-NMR (100 MHz,
13
CDCl3):
δ
(ppm) = 30.00 (3C), 31.81, 36.43 (3C), 39.23 (3C), 47.15, 59.15, 70.73, 104.46, 110.37, 111.71,
+
1
20.26, 138.52, 141.71, 161.32, 181.22. HRMS (ESI+): m/z (%) = 447.5457 (ccd. 447.5455) [M + H] .
N-(Adamantan-1-yl)-2-(1-(2-(2-fluoroethoxy)ethyl)-5-(furan-2-yl)-1H-indol-3-yl)-2-oxoacetamide (
8). This
compound was synthesized by employing the same procedure as for compound
7, and it was obtained
1
in 75% yield as yellow solid. H-NMR (400 MHz, CDCl ):
δ (ppm) = 1.75 (m, 6H), 2.07–2.21 (m, 9H),
3
3
4
0
1
6
.58–3.66 (m, 1H), 3.66–3.72 (m, 1H), 3.92 (t, J = 5.56 Hz, 2H), 4.38 (t, J = 5.43 Hz, 2H), 4.42–4.48 (m, 1H),
.52–4.60 (m, 1H), 6.46–6.55 (m, 1H), 6.73 (dd, J = 3.28, 0.76 Hz, 1H), 7.37 (s, 1H), 7.46 (dd, J = 8.59,
.51 Hz, 1H), 7.50 (dd, J = 1.77, 0.76 Hz, 1H), 7.70 (dd, J = 8.59, 1.77 Hz, 1H), 8.75 (dd, J = 1.64, 0.63 Hz,
H), 9.07 (s, 1H). 13C-NMR (100 MHz, CDCl3):
δ
(ppm) = 29.38 (3C), 36.32 (3C), 41.20 (3C), 47.44, 51.79,
9.88, 70.47, 70.67, 82.31, 83.99, 104.39, 110.59, 111.70, 112.25, 118.00, 120.23, 128.21, 135.82, 141.76,
+
1
42.14, 154.65, 161.45. HRMS (ESI+): m/z (%) = 479.5627 (ccd. 479.5625) [M + H] .
4
. Conclusions
In this study, the N-alkyl chain of the high affinity and selective CB ligand
5
was modified for
the possibility of introducing a fluorine atom. The herein developed fluorinated compound retained
the high affinity of the lead compound, and will be considered for the development of an 18F-labeled
2
8
radiotracer for CB receptors imaging with PET.
2
1
Supplementary Materials: The H NMR spectra of compounds
3,
5,
6
,
7
and
8
are available online at http:
/
/www.mdpi.com/1420-3049/22/1/77/s1.
Acknowledgments: This research was supported by DFG fellowship MO 2677/1-1 (R.-P. Moldovan), NIH grant
AG037298 (A.G. Horti) and, in part, by Division of Nuclear Medicine of The Johns Hopkins University School of
Medicine. We thank Mrs. Tina Spalholz for help with radioligand binding studies, and Dr. Rodrigo Teodoro for
scientific discussions.
Author Contributions: Rare s¸ -Petru Moldovan and Andrew G. Horti conceived and performed the chemical
syntheses. Winnie Deuther-Conrad and Peter Brust planned and performed the radioligand binding studies.
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2017, 22, 77
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Abbreviations
CB2
cannabinoid receptors type 2
CB1
cannabinoid receptors type 1
positron emission tomography
N,N-dimethylformamide
tetrabutylammonium bromide
triethylamine
PET
DMF
TBAB
Et N
3
CHO
EA
2
Chinese Hamster Ovary
ethyl acetate
Et O
diethyl ether
DCM
dichloromethane
Pd(PPh3)4
n-BuBr
MeI
tetrakis(triphenylphosphine)palladium(0)
1-bromobutane
methyl iodide
Hex
hexane
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