Pyrazolo[1,5-a]pyrimidine acetamides: 4-Phenyl alkyl ether derivatives as potent ligands for the 18 kDa translocator protein (TSPO)

Article abstract of DOI:10.1016/j.bmcl.2010.07.135

Herein, we report the synthesis of four new phenyl alkyl ether derivatives (7, 9-11) of the pyrazolo[1,5-a]pyrimidine acetamide class, all of which showed high binding affinity and selectivity for the TSPO and, in the case of the propyl, propargyl, and bu

Full text of DOI:10.1016/j.bmcl.2010.07.135

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5799–5802
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pyrazolo[1,5-a]pyrimidine acetamides: 4-Phenyl alkyl ether derivatives as
potent ligands for the 18 kDa translocator protein (TSPO)
Aaron Reynolds a, Raphy Hanani a, David Hibbs b, Annelaure Damont c, Eleonora Da Pozzo d, Silvia Selleri e,
Frédéric Dollé c, Claudia Martini d, Michael Kassiou a,f,g,
*
a School of Chemistry, University of Sydney, Sydney, NSW 2006, Australia
b Faculty of Pharmacy, University of Sydney, Sydney, NSW 2006, Australia
c CEA, I2BM, Service Hospitalier Frédéric Joliot, F-91401 Orsay, France
d Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology, University of Pisa, Via Bonanno, 6 56126 Pisa, Italy
e Department of Pharmaceutical Sciences, University of Florence, Via U. Schiff, 650019 Polo Scientifico Sesto Fiorentino, Italy
f Brain and Mind Research Institute, Sydney, NSW 2050, Australia
g Discipline of Medical Radiation Sciences, University of Sydney, Sydney, NSW 2006, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein, we report the synthesis of four new phenyl alkyl ether derivatives (7, 9–11) of the pyrazolo[1,5-
a]pyrimidine acetamide class, all of which showed high binding affinity and selectivity for the TSPO and,
in the case of the propyl, propargyl, and butyl ether derivatives, the ability to increase pregnenolone bio-
synthesis by 80–175% over baseline in rat C6 glioma cells. While these compounds fit our in silico gen-
erated pharmacophore for TSPO binding the current model does not account for the observed functional
activity.
Received 23 June 2010
Revised 29 July 2010
Accepted 30 July 2010
Available online 4 August 2010
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
Peripheral-type benzodiazepine receptor
Translocator protein
TSPO
Steroidogenesis
Pyrazolo[1,5-a]pyrimidine
Neurodegeneration
Neuroprotection
Microglia
The translocator protein (TSPO), formerly known as the periphe-
ral-type benzodiazepine receptor (PBR),1 is ubiquitously expressed
in most peripheral organs, with the highest densities in steroido-
genic tissues2 as well as in heart, kidney, lung and testis,3 but only
minimally expressed in the healthy brain.4 Increased levels of TSPO
expression have been noted in a number of neuroinflammatory and
neurodegenerative conditions such as Alzheimer’s disease, Parkin-
son’s disease, and stroke, coinciding with the activation of microglia,
the principle immune effector cells in the central nervous system
(CNS).
The TSPO is a highly lipophilic 18 kDa protein comprising 169
amino acids.5 Primarily located on the outer mitochondrial mem-
brane6 with five trans-membrane domains, the TSPO is believed
to be closely associated with the voltage-dependant ion channel
(VDAC) and the adenine nucleotide transporter (ANT) as part of
the mitochondrial permeability transition pore (MPTP).7 While its
exact physiological role remains unknown, the TSPO is implicated
in several biochemical processes including cell proliferation,8
apoptosis,9 steroidogenesis,3,10–12 porphyrin transport and heme
synthesis,13 and immunomodulation.14 Among these, the rate-lim-
iting translocation of cholesterol from the outer to inner mitochon-
drial membrane and subsequent enzymatic side chain cleavage to
form pregnenolone, the precursor to all mammalian steroids, is
perhaps the best documented.
The relationship between neuroinflammation/microglial activa-
tion and TSPO expression has resulted in the development of a
number of structurally diverse TSPO ligands which have been de-
scribed in reviews by James et al.15 and Scarf et al.16 Some of these
ligands have been shown to mediate non-sedating anxiolytic-like
effects in both rodent models of anxiety17 and in humans18 related
to the production of neurosteroids which enhance
c-aminobutyric
acid-mediated neurotransmission.19
The synthesis and biological evaluation of the arylpyrazolo[1,5-
a]pyrimidine acetamides, a novel and selective class of high affinity
ligands for the TSPO, was first reported by Selleri et al.20 The
authors concluded that substitution on the pyrimidine ring is
important in determining selectivity for the TSPO over the central
* Corresponding author. Address: The Brain and Mind Research Institute, 100
Mallett Street, Camperdown, NSW 2050, Australia. Tel.: +61 2 9351 0799; fax: +61 2
9351 0652.
E-mail address: michael.kassiou@sydney.edu.au (M. Kassiou).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.135

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A. Reynolds et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5799–5802
benzodiazepine receptor (CBR) and that substituents at the 40-po-
sition of the phenyl ring had an effect on TSPO binding affinity.
Some of these compounds were shown to increase pregnenolone
biosynthesis in rat C6 glioma cells with similar potency to that of
known TSPO ligand, PK11195.
CH3
N
CH3
N
N
N
a or b
OR
OH
H3C
N
O
H3C
N
O
We aimed to explore the effect on TSPO binding affinity and
pregnenolone biosynthesis of various length 40-phenyl alkyl ethers
derived from the arylpyrazolo[1,5-a]pyrimidine acetamide 6,
which we have previously reported.21
NEt2
NEt2
6
07 R = CH2CH3
08 R = CH2CH2F
09 R = (CH2)2CH3
10 R = (CH2)3CH3
11 R = CH2C CH
The synthetic route (Scheme 1) commenced with commercially
available methyl 4-methoxybenzoate (1) which underwent nucle-
ophilic attack in the presence of the stabilized carbanion derived
from acetonitrile to form a tetrahedral intermediate which, upon
loss of methoxide, gave the 3-oxopropanenitrile 2 in modest yield
(58%). C-alkylation of 2 occurred upon treatment with sodium
hydroxide to form the corresponding enolate followed by addition
of commercially available N,N-diethylchloroacetamide and sodium
iodide to afford 3 (80%). Aminopyrazole 4 was obtained in 80%
yield following cyclization of a hydrazone intermediate derived
from 3 and hydrazine hydrate. Treatment of 4 with 2,4-pentanedi-
one in refluxing ethanol generated the pyrazolo[1,5-a]pyrimidine 5
in 93% yield via a double condensation. Cleavage of the methyl
ether 5 to give the corresponding phenol 6 was achieved in the
presence of refluxing 48% v/v aqueous hydrobromic acid and cata-
lytic tetra-n-butylphosphonium bromide (0.10 equiv) in 54% yield.
The phenol 6 was elaborated to give the desired 40-phenyl alkyl
ethers (7–11) by Mitsunobu alkylation or by simple O-alkylation of
the corresponding in situ generated phenoxide (Scheme 2). We
have previously reported the binding of compounds 5 and 8 and
their labeling with the short-lived positron-emitters carbon-11
and fluorine-18, respectively, as radioligands22 for in vivo positron
emission tomography (PET) imaging studies.21,23–30
Scheme 2. Reagents and conditions: (a) DIAD (2.2 equiv), PPh3 (2.2 equiv), NEt3 (0
or 2.2 equiv), ROH (2.2 equiv), THF or DMF, 0 °C ? RT, 72 h; (b) NaH or K2CO3, DMF,
0 °C, then ROTs or RBr, rt.
Table 1
Binding affinity of compounds 5, 7–11 and PK11195 for the TSPO and CBR
CH3
N
N
OR
N
H3C
O
NEt2
Ki (TSPO) (nM)
Compound
R
Ki (CBR) (nM)
PK11195
5
7
8
9
9.3 0.5
4.7 0.2
5.7 0.5
7.0 0.4
1.4 0.2
1.1 0.1
4.8 0.5
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
CH3
CH2CH3
CH2CH2F
(CH2)2CH3
(CH2)3CH3
CH2C„CH
10
11
The TSPO binding of compounds 5, 7–11 and PK11195 was eval-
uated in a membrane binding assay using [3H]PK11195 as the radi-
oligand and mitochondrial fractions from rat kidney as the
receptor source. To determine the selectivity for the TSPO, binding
to the CBR was also assessed using [3H]Ro15,1788 and rat brain tis-
sue. The results are summarized in Table 1.
Each of the newly synthesized compounds was assessed, along-
side PK11195, for its ability to increase pregnenolone biosynthesis
in rat C6 glioma cells using a well developed steroidogenic assay.20
Each TSPO ligand was used at the same concentration (40 lM) and
All of the new compounds (7, 9–11) displayed high affinity and
complete selectivity for the TSPO over the CBR as well as a large
range of CNS receptors and transporters (see Supplementary data).
Increasing the length of the alkyl ether had little effect on TSPO bind-
ing affinity, suggesting the possible existence of a lipophilic binding
pocket that can accommodate the steric bulk of an n-butyl group.
at the end of the incubation period (2 h) the amount of pregneno-
lone was quantified by radio-immunoassay (RIA). The results are
shown in Figure 1 (the values are the mean of three determina-
tions). The known 40-phenyl (200-fluoroethyl) ether 8 stimulated
pregnenolone biosynthesis at levels 80% above baseline, displaying
significantly greater potency than PK11195 whereas the known
O
O
O
a
b
OCH3
OCH3
OCH3
H3CO
NC
NC
O
1
2
3
NEt2
c
CH3
N
H
N
N
d
N
OCH3
OR
H2N
H3C
N
O
O
NEt2
NEt2
5 R = CH3
6 R = H
4
e
Scheme 1. Reagents and conditions: (a) NaOMe, MeCN, reflux, 32 h, 58%; (b) N,N-diethylchloroacetamide, NaI, NaOH, EtOH/H2O (80:20), rt, 7 h, 80%; (c) NH2NH2ꢀH2O, AcOH,
EtOH, reflux, 4 h, 86%; (d) 2,4-pentanedione, EtOH, reflux, 12 h, 93%; (e) 48% v/v HBr, tetra-n-butylphosphonium bromide (0.10 equiv), 100 °C, 7 h, 54%.

A. Reynolds et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5799–5802
5801
offer no common insight into the possible ligand-based mecha-
nism of action of the compounds presented here. Using a number
of the standard properties used in chemoinformatics, no noticeable
trends were observed, once again, presumably due to the small
number of ligands available. We are currently developing a com-
parative model of the human TSPO, which will provide a much
clearer picture of the intermolecular interactions of this class of
compounds with the protein.
In summary, we have synthesized four new pyrazolo[1,5-
a]pyrimidine acetamides substituted with various length alkyl
ethers at the 40-position of the phenyl ring. All of these compounds
displayed high affinity for the TSPO with complete selectivity over
the CBR as well as a large range CNS receptors and transporters.
Compounds 9, 10, and 11 represent the most potent modulators
of steroidogenesis within this class to date and have potential ther-
apeutic applications as anxiolytics or neuroprotective agents.
Figure 1. Results of steroidogenesis assay. Complete details can be found in the
Supplementary data.
40-phenyl methyl ether derivative 5 and 40-phenyl ethyl ether
derivative 7 showed no effect on steroidogenesis in the same assay.
Compounds 9, 10, and 11, corresponding to the propyl, butyl and
propargyl ethers, respectively, displayed the greatest increase in
pregnenolone biosynthesis (140–175% above baseline) of any high
affinity TSPO ligand within this class assessed using this assay.
In an effort to understand the structural dependence on the rel-
ative activities of the TSPO ligands 5, 7–11, it was decided to deter-
mine the likely pharmacophore hypothesis responsible for activity.
A number of ligands with high binding affinity for the TSPO have
been previously reported,15,16 and these were used in the construc-
tion of the initial hypotheses.
Acknowledgments
Ki determinations for targets included in the SI were generously
provided by the National Institute of Mental Health0s Psychoactive
Drug Screening Program, Contract #NO1MH32004 (NIMH PDSP).
The NIMH PDSP is Directed by Bryan L. Roth MD, PhD at the Uni-
versity of North Carolina at Chapel Hill and Project Officer Jamie
Driscol at NIMH, Bethesda MD, USA. For experimental details
please refer to the PDSP web site http://pdsp.med.unc.edu/.
Supplementary data
A collection of 44 compounds, representing a variety of classes of
TSPO ligands, that are available in our laboratory were built using
MAESTRO 9.0,31 and energy minimized using the conjugate gradient
method as implemented in Macromodel,32 with a convergence crite-
rion of 0.05 kcal Aꢁ1, with the OPLS 2005 potential. Solvation effects
were modeled using a continuum field, with a dielectric constant of
1.0. Each molecule then underwent a conformational analysis,
retaining only those conformers with a relative energy value within
10 kcal molꢁ1 of the minimized parent molecule.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.07.135.
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