Synthesis and in vivo evaluation of a novel peripheral benzodiazepine receptor PET radioligand

Article abstract of DOI:10.1016/j.bmc.2005.06.030

The novel pyrazolopyrimidine ligand, N,N-diethyl-2-[2-(4-methoxyphenyl)-5, 7-dimethyl-pyrazolo[1,5-a]pyrimidin-3-yl]-acetamide 1 (DPA-713), has been reported as a potent ligand for the peripheral benzodiazepine receptor (PBR) displaying an affinity of K

Full text of DOI:10.1016/j.bmc.2005.06.030

Bioorganic & Medicinal Chemistry 13 (2005) 6188–6194
Synthesis and in vivo evaluation of a novel
peripheral benzodiazepine receptor PET radioligand
Michelle L. James,a Roger R. Fulton,b,c David J. Henderson,b Stefan Eberl,b
Steven R. Meikle,b Sally Thomson,d Robin D. Allan,a Frederic Dolle,e
Michael J. Fulhamb,f and Michael Kassioua,b,
*
aDepartment of Pharmacology, University of Sydney, NSW 2006, Australia
bDepartment of PET and Nuclear Medicine, Royal Prince Alfred Hospital, Missenden Road, Camperdown, NSW 2050, Australia
cSchool of Physics, University of Sydney, NSW 2006, Australia
dDepartment of Renal Medicine, Royal Prince Alfred Hospital, Missenden Road, Camperdown, NSW 2050, Australia
e
´ ´
´
´
Service Hospitalier Frederic Joliot, Departement de Recherche Medicale, CEA/DSV,
´ ´
4 Place du General Leclerc, F-91401 Orsay, France
fFaculty of Medicine, University of Sydney, NSW 2006, Australia
Received 31 May 2005; revised 16 June 2005; accepted 17 June 2005
Available online 20 July 2005
Abstract—The novel pyrazolopyrimidine ligand, N,N-diethyl-2-[2-(4-methoxyphenyl)-5,7-dimethyl-pyrazolo[1,5-a]pyrimidin-3-yl]-
acetamide 1 (DPA-713), has been reported as a potent ligand for the peripheral benzodiazepine receptor (PBR) displaying an affinity
of Ki = 4.7 nM. In this study, 1 was successfully synthesised and demethylated to form the phenolic derivative 6 as precursor for
labelling with carbon-11 (t1/2 = 20.4 min). [11C]1 was prepared by O-alkylation of 6 with [11C]methyl iodide. The radiochemical yield
of [11C]1 was 9% (non-decay corrected) with a specific activity of 36 GBq/lmol at the end of synthesis. The average time of synthesis
including formulation was 13.2 min with a radiochemical purity >98%. In vivo assessment of [11C]1 was performed in a healthy
Papio hamadryas baboon using positron emission tomography (PET). Following iv administration of [11C]1, significant accumula-
tion was observed in the baboon brain and peripheral organs. In the brain, the radioactivity peaked at 20 min and remained con-
stant for the duration of the imaging experiment. Pre-treatment with the PBR-specific ligand, PK 11195 (5 mg/kg), effectively
reduced the binding of [11C]1 at 60 min by 70% in the whole brain, whereas pre-treatment with the central benzodiazepine receptor
ligand, flumazenil (1 mg/kg), had no inhibitory effect on [11C]1 uptake. These results indicate that accumulation of [11C]1 in the
baboon represents selective binding to the PBR. These exceptional in vivo binding properties suggest that [11C]1 may be useful
for imaging the PBR in disease states. Furthermore, [11C]1 represents the first ligand of its pharmacological class to be labelled
for PET studies and therefore has the potential to generate new information on the pathological role of the PBR in vivo.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
brane of the mitochondria.2 Currently, it is understood
that the PBR forms a trimeric complex with the adenine
nucleotide carrier (ANC) (30 kDa) and the voltage-de-
pendent anion channel (VDAC) (32 kDa) to constitute
the mitochondrial permeability transition pore
(MPTP).2 Although the PBR has been implicated in
numerous biological processes, its exact physiological
role remains unclear. Studies implicate the importance
of the PBR in the rate-limiting step of steroid biosynthe-
sis,3 immunomodulation,4 porphyrin transport,5 calci-
um homeostasis and programmed cell death.6
The peripheral benzodiazepine receptor (PBR) was orig-
inally characterised as an alternative binding site for the
benzodiazepine, diazepam,1 and was thought to be a
subtype of the central benzodiazepine receptor (CBR).
Later studies identified the PBR to be a separate class
of receptor due to its distinct structure, physiological
functions and subcellular location on the outer mem-
Keywords: Pyrazolopyrimidine; Peripheral benzodiazepine receptor;
Carbon-11; PET.
The PBR is densely distributed in most peripheral or-
gans including the lungs, heart and kidneys, yet it is only
minimally expressed in the normal brain parenchyma.7
*
Corresponding author. Tel.: +612 9515 5150; fax: +612 9515 6381;
e-mail: mkassiou@med.usyd.edu.au
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.06.030

M. L. James et al. / Bioorg. Med. Chem. 13 (2005) 6188–6194
6189
Following neuronal injury, PBR expression is dramati-
cally increased.8 In vivo studies in patients suffering
from Alzheimerꢀs disease (AD) and multiple sclerosis
(MS) confirmed that elevation of PBR binding is pri-
marily localised on activated microglial cells.9 Microg-
lia, the resident macrophages of the CNS, are
extremely sensitive to alterations to their microenviron-
ment and hence readily become activated due to injury
or infection.10 For this reason, the PBR are believed to
be intimately associated with initial inflammatory pro-
cesses in the early stages of several neurological diseas-
es.11 However, the exact role of microglia in the
pathology of these diseases has not been completely elu-
cidated due to the lack of specific and selective PBR
ligands.
The development of PBR ligands with improved brain
kinetics is vital to understand the cellular processes under-
lying numerous disease states. Selective and specific PBR
ligands may also have the potential to serve as diagnostic
and therapeutic tools. The recently synthesised
[11C]DAA1106 has demonstrated a high specificity for
the PBR in rodent16 and primate brains17 and is an attrac-
tive candidate for PET imaging of the PBR in humans.
Another class of PBR ligands, the pyrazolopyrimidines,
have been reported and although displaying high affinity
are yet to be radiolabelled for use in PET. One of the lead
compounds from this series, 1 (DPA-713) (shown in
Scheme 1), has a higher affinity (Ki = 4.7 nM) for the
PBR than PK 11195 (Ki = 9.3 nM) and is notably more
selective for the PBR over the CBR (Ki > 10,000 nM for
CBR).18 The aim of the present study was to label 1
(DPA-713) with carbon-11 and perform preliminary in
vivo assessment in a healthy baboon using PET.
The development of novel PBR radioligands for non-
invasive imaging with positron emission tomography
(PET) has enabled study of microglial activation in the
living brain. The isoquinoline carboxamide [11C](R)-
PK 11195 has been extensively used as a pharmacologi-
cal probe for studying the function and expression of
PBRs.12 A number of PET studies conducted in patients
with AD,13 MS14 and multiple system atrophy (MSA)15
have shown that measurement of PBRs in vivo with
[11C](R)-PK 11195 is feasible in the living brain. Howev-
er, [11C](R)-PK 11195 displays a poor signal-to-noise ra-
tio and has demonstrated low brain permeability which
ultimately decreases its sensitivity in detecting areas of
microglial activation. These unfavourable characteristics
may be attributable to the tracerꢀs high lipophilicity and
low bioavailability (88% bound to plasma protein).7
2. Results and discussion
2.1. Chemistry
N,N-Diethyl-2-[2-(4-methoxy-phenyl)-5,7-dimethylpy-
razolo[1,5-a]pyrimidin-3-yl]-acetamide (1, as reference)
was synthesised in four chemical steps with some modi-
fications to literature procedures18–20 (Scheme 1).
The aroylacetonitrile, 3, was formed in 17% yield by
reaction of commercially available methyl-4-methoxy
Scheme 1. Reagents and conditions: Synthesis of N,N-diethyl-2-[2-(4-methoxy-phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl]-acetamide (1)
(DPA-713) and the radiolabelling precursor N,N-diethyl-2-[2-(4-hydroxy-phenyl)-5,7-dimethyl-pyrazolo[1,5-a]pyrimidin-3-yl]-acetamide (6);
(i) acetonitrile, sodium methoxide, 100 ꢁC, 24 h; (iia) N,N-diethylchloroacetamide, sodium iodide, NaOH/80% EtOH, reflux 7 h; (ii) N,N-
diethylchloroacetamide, sodium iodide, NaOH/80% EtOH, rt, 7 h; (iii) hydrazine hydrate, EtOH, acetic acid, reflux 4 h; (iv) 2,4-pentadione, EtOH,
reflux 12 h; (v) 48% HBr, tributylhexadecylphosphonium bromide, 100 ꢁC, 7 h.

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M. L. James et al. / Bioorg. Med. Chem. 13 (2005) 6188–6194
benzoate 2 with acetonitrile in the presence of sodium
methoxide at 100 ꢁC. Rearrangement of purification
steps reported in the literature allowed the benzoic acid
(structure not shown) by-product to be removed more
efficiently leading to improved purity and yield of the fi-
nal product. The reactivity of the methylene group in 3
was exploited by reacting it in an alkaline solution with
N,N-diethylchloroacetamide and sodium iodide. How-
ever, the product (4a) isolated after 7 h reflux displayed
1H NMR and mass spectrum indicative of an ethyl ester
functional group instead of the desired diethyl amide
moiety. Repeating the same reaction under milder con-
ditions at room temperature afforded 4 in 64% yield
Formulation of labelled product for iv injection was
achieved by: (1) evaporation of HPLC solvent; (2)
reconstitution of residue in physiological saline (2 mL)
and (3) sterile filtration through a 0.22 lm filter. The fi-
nal injectable solution was clear and colourless with a
pH of 5.0. The preparation was free from starting label-
ling precursor. Administration to the animal was per-
formed within 10 min following end of synthesis in
each study.
2.3. Pharmacology
2.3.1. Evaluation using PET. The uptake of [11C]1 was
examined using PET. Dynamic PET brain imaging com-
menced just prior to iv administration of [11C]1
(200 MBq in 2 mL saline) and was terminated 60 min
post-injection. This was immediately followed by a
whole body acquisition for 2 min at each of six bed posi-
tions. Figure 1a shows a 60-min PET summation image
of a 3.4 mm thick transaxial baboon brain slice from the
dynamic scan. The PET image provided visual evidence
for the ability of [11C]1 to traverse the blood–brain bar-
rier (BBB) with considerable accumulation of radioac-
tivity in the baboon brain.
1
as confirmed by H NMR and mass spectrum. In the
third step, 4 was converted to the pyrazole 5 by heating
at reflux in ethanol with hydrazine hydrate, in the pres-
ence of acetic acid. The condensation of 5 with the elec-
trophilic reagent, 2,4-pentadione, led to closure of the
pyrimidine ring to yield the title compound 1 as pale yel-
low crystals in 93% yield. Compound 1 was subsequent-
ly demethylated by heating in a solution of hexadecyl
tributyl phosphonium bromide in 45% HBr to form
the phenolic derivative 6 (N,N-diethyl-2-[2-(4-hydroxy-
phenyl)-5,7-dimethyl-pyrazolo[1,5-a]pyrimidin-3-yl]-
acetamide), as precursor for labelling with carbon-11 in
54% yield.
The whole brain uptake of [11C]1 as a function of time is
represented graphically in Figure 2. [11C]1 reached maxi-
mal uptake after 20 min and stayed at approximately the
same uptake level for the remaining 40 min of imaging.
2.2. Radiochemistry
The pyrazolopyrimidine 1 was labelled with carbon-11
(t1/2 = 20.4 min) using [11C]methyl iodide ([11C]CH3I)
and the phenolic precursor 6 (Scheme 2).
In an earlier reported study using Papio anubis baboons,
time–activity curves (TACs) of [11C](R)-PK 11195 brain
uptake provided evidence of rapid BBB penetration and
maximal brain activity within 3–5 min.21 Directly fol-
lowing maximum brain uptake, [11C](R)-PK 11195
activity steeply declined to the plasma level. This dra-
matic washout of [11C](R)-PK 11195 between 15 and
20 min was later revealed to be a consequence of binding
to plasma proteins.22 Because the uptake of [11C](R)-PK
11195 closely resembled that of blood flow, PET images
generated were not indicative of specific binding to the
PBR. In contrast, the slower kinetics of [11C]1 enabled
longer imaging in which signals at later time points were
attributable to specific PBR binding. For this reason,
[11C]1 may provide a more accurate assessment of
PBR density than the widely used [11C](R)-PK 11195.
[11C]CH3I was synthesised from cyclotron-produced
[11C]carbon dioxide ([11C]CO2), trapped in a DMF solu-
tion containing 6 and tetrabutyl ammonium hydroxide
(TBAH) and allowed to react at room temperature for
3 min. The reaction mixture was purified via reverse-
phase semi-preparative high-performance liquid chroma-
tography (HPLC). This afforded [11C]1 in 9% (n = 6) non-
decay corrected radiochemical yield based on starting
[11C]CH3I in an average synthesis time of 13.2 min
(including HPLC purification and formulation). Co-in-
jection of the non-radioactive 1 was performed using ana-
lytical HPLC to confirm the identity of the product. In the
final product solution, radiochemical and chemical purity
was greater than 98% with a specific activity of 36
GBq/lmol. No attempts were made to optimise these con-
ditions as sufficient quantities of the radioligand were pro-
duced to enable pharmacological evaluation.
2.3.2. Blocking studies. The in vivo specificity and selec-
tivity of [11C]1 in the baboon brain was assessed via two
blocking studies which were performed 3 weeks apart:
Scheme 2. Radiosynthesis of N,N-diethyl-2-[2-(4-[11C]methoxy-phenyl)-5,7-dimethyl-pyrazolo[1,5-a]pyrimidin-3-yl]-acetamide, ([11C]1).

M. L. James et al. / Bioorg. Med. Chem. 13 (2005) 6188–6194
6191
Figure 1. PET summation images of a single transaxial brain slice over 60 min: (a) [11C]1, (b) [11C]1 + PK 11195 (5 mg/kg) and (c) [11C]1 + flumazenil
(1 mg/kg). Images (a) and (c) are slightly over saturated to allow visualization of image (b).
with the PBR-specific ligand [11C]DAA1106 and 0.1 mg/
kg flumazenil.17 Pharmacological challenge using PK
11195 resulted in TACs symptomatic of radioligand
inhibition. Consequently, [11C]1 was washed out of the
brain within 10 min, denoted by the rapid decline in
the TAC. Similar results have been reported for PK
11195 blocking studies with both [11C]DAA110617 and
[11C](R)-PK 11195.21
Figure 3a–c show the whole body images of the baboon,
which were acquired at the conclusion of dynamic scan-
ning in all three studies. These images demonstrate the
significant inhibition of [11C]1 uptake in the brain, heart
and kidneys following pre-treatment with PK 11195,
Figure 2. Whole brain TACs in baseline study with [11C]1 and
thus providing evidence that [11C]1 was specifically
blocking studies performed with PK 11195 and flumazenil.
bound to the PBR in peripheral organs. No inhibitory
effects were observed in the flumazenil blocking study,
which indicated the selectivity of [11C]1 for the PBR over
the CBR in peripheral organs.
one using the PBR-specific ligand, PK 11195, and the
other using the CBR-specific ligand, flumazenil. Pre-
treatment of the baboon with PK 11195 (5 mg/kg, iv,
5 min prior to radioligand injection) resulted in marked-
ly decreased uptake of [11C]1 (Fig. 1b) compared to the
baseline study (Fig. 1a). In contrast, pre-treatment with
flumazenil (1 mg/kg, iv, 5 min prior to radioligand injec-
tion) displayed no inhibitory effect on [11C]1 uptake
(Fig. 1c). These results clearly demonstrated that reten-
tion of [11C]1 in the baboon brain represents selective
binding to the PBR and not the CBR.
LogP values for 1 and PK 11195 using HPLC analysis
were found to be 2.4 and 3.4, respectively. It is recom-
mended that the log of the octanol–water partition coef-
ficient (logP) for CNS radioligands be between 2 and 3
in order to achieve a high brain uptake relative to weak
non-specific binding.23 The logP of compound 1 is thus
within the optimum range which is supported by its ra-
pid penetration of the BBB and high specific binding to
the PBR. Compound 1 is clearly less lipophilic than PK
11195 which may partly explain the different degrees of
specific binding to the PBR of the two ligands. There
may be other factors that contribute to the overall in
vivo kinetics and binding of PBR radioligands. Hence,
The TACs (Fig. 2) for the baseline study and pharmaco-
logical challenge with flumazenil in the whole brain were
similar, indicating no inhibitory effect on [11C]1 uptake.
These results compare well to those generated in studies
Figure 3. PET images of the body 1 h after iv injection of (a) [11C]1 (baseline), (b) [11C]1 + PK 11195 (5 mg/kg) and (c) [11C]1 + flumazenil (1 mg/kg).

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M. L. James et al. / Bioorg. Med. Chem. 13 (2005) 6188–6194
more extensive studies are required to gain further in-
sight into the PBR and its interaction with various li-
gands. This will ultimately lead to the design and
synthesis of superior PBR ligands.
dilute H2SO4. Following the addition of diethyl ether,
the organic layer was extracted, dried over anhydrous
Na2SO4 and evaporated to dryness. The resulting yellow
solid was dissolved in CHCl3 and washed with saturated
NaHCO3 aqueous solution (5 · 100 mL) to remove ben-
zoic acid. The organic layer was dried over anhydrous
Na2SO4 and evaporated to dryness. The solid was puri-
fied by washing with petroleum ether which yielded
319,20 (5.16 g, 17%) as fine, light yellow crystals; mp:
132–137 ꢁC; 1H NMR (CDCl3, 300 MHz) d 3.89 (s,
3H, OCH3), 4.03 (s, 2H, CH2), 6.97 (d, J = 9.0 Hz,
2H, Ph), 7.90 (d, J = 9.0 Hz, 2H, Ph).
3. Conclusion
In this study, the pyrazolopyrimidine 1 (DPA-713) was
synthesised, radiolabelled with carbon-11 and evaluated
in a baboon using PET. [11C]1 was prepared by O-alkyl-
ation of the phenolic derivative 6 with [11C]CH3I in
reproducible yields and specific activity. The results
from the PET imaging studies were consistent with
retention of [11C]1 in the baboon brain due to specific
binding to the PBR. We therefore believe that this radi-
oligand may be useful in detecting changes in PBR den-
sity in disease states and warrants further investigation.
Because it is the first PBR ligand from the pyrazolopyr-
imidine class to be radiolabelled, it may generate novel
information on the structure and physiological roles of
the PBR in vivo.
4.2.2. 3-Cyano-N,N-diethyl-4-(4-methoxy-phenyl)-4-oxo-
butyramide (4). A mixture of 319,20 (2.0 g, 11.4 mmol),
N,N-diethylchloroacetamide (1.7 g, 11.4 mmol) and NaI
(5.1 g, 34 mmol) were added to a solution of NaOH
(0.5 g, 12.5 mmol) in 80% EtOH (80 mL) while being stir-
red continuously. The mixture was stirred at room tem-
perature for 7 h and monitored by TLC. Once the
reaction was complete, it was allowed to cool and was fil-
tered to remove the inorganic material. The filtrate was
concentrated and the residue was purified by column
chromatography (CH2Cl2 as eluent) to yield 418 (2.1 g,
1
4. Experimental
4.1. General
64%) as a dark yellow oil; H NMR (CDCl3, 300 MHz)
d 1.06–1.30 (m, 6H, N(CH2CH3)2), 2.85 (dd, J = 4.5,
16.2 Hz, 1H, CH2), 3.21–3.43 (m, 5H: 4H, N(CH2CH3)2:
1H, CH2), 3.90 (s, 3H, OCH3), 4.89–5.02 (m, 1H, CH),
6.98 (d, J = 8.7 Hz, 2H, Ph), 8.05 (d, J = 9.0 Hz, 2H,
Ph). Mass Spectrum: CI, m/z 289 (M + 1).
4.1.1. Chemicals and TLCs. Chemicals were purchased
from Aldrich and used with no further purification. PK
11195 and flumazenil were purchased from Tocris, UK.
Melting points were performed in a sealed capillary using
a Stuart Melting Point Apparatus SMP3 and are uncor-
rected. Column chromatography was performed with
Merck silica gel 60 (230–400 mesh). Thin layer chroma-
tography (TLC) was carried out using aluminium-backed
plates pre-coated with silica gel (60 F254) and was devel-
oped using UV fluorescence (254 nm and/or 365 nm).
4.2.3. 2-[3-Amino-5-(4-methoxy-phenyl)-1H-pyrazol-4-
yl]-N,N-diethylacetamide (5). Hydrazine hydrate (0.73 g,
14.6 mmol) and acetic acid (0.73 mL) were added to a
solution of 418 (2.1 g, 7.3 mmol) in EtOH (37 mL). The
mixture was heated at reflux for 4 h and monitored by
TLC. Once the reaction was complete, it was allowed to
cool to room temperature. The solution was evaporated
to dryness and the residue was purified by silica gel col-
umn chromatography (CH2Cl2/MeOH, 10:1 (v/v), as elu-
ent). The purified product was re-dissolved in CH2Cl2 and
washed with saturated NaHCO3 aqueous solution
(4 · 20 mL) to remove acetic acid. This afforded 518
(1.52 g, 68%) as yellow crystals; mp: 154.5–157.5 ꢁC; 1H
4.1.2. Spectroscopies. Proton nuclear magnetic reso-
nance (1H NMR) spectra were recorded using
a 300 MHz Varian Gemini instrument. Chemical shifts
(d) are reported in parts per million (ppm) downfield
from internal tetramethylsilane (TMS). Multiplicities
are reported as s (singlet), d (doublet), dd (doublet of
doublets), m (multiplet) and the observed coupling con-
stant (J). Mass spec was carried out on a Thermo Finn-
igan Polaris Q GCMS instrument.
NMR (CDCl3, 300 MHz)
d 0.90–1.10 (m, 6H,
N(CH2CH3)2), 3.04–3.33 (m, 4H, N(CH2CH3)2), 3.50 (s,
2H, CH2), 3.85 (s, 3H, OCH3), 6.98 (d, J = 8.7 Hz, 2H,
Ph), 7.32 (d, J = 9.0 Hz, 2H, Ph).
4.2. Chemistry
4.2.4. N,N-Diethyl-2-[2-(4-methoxy-phenyl)-5,7-dimeth-
ylpyrazolo[1,5-a]pyrimidin-3-yl]-acetamide (1). 2,4-Pen-
tanedione (0.4 g, 4 mmol) was added to a solution of 518
(1.2 g, 4 mmol) in EtOH (20 mL). The mixture was heated
at reflux for 12 h. The reaction mixture was allowed to
cool and the solvent was evaporated to dryness. The resi-
due was purified by silica gel column chromatography
(CHCl3/MeOH, 40:1 (v/v), as eluent) which yielded 118
(1.37 g, 93%) as pale yellow crystals; mp: 120.5–
4.2.1. 3-(4-Methoxy-phenyl)-3-oxo-propionitrile (3). A
mixture of methyl-4-methoxybenzoate (30 g, 181 mmol)
and sodium methoxide (9.75 g, 181 mmol) was heated at
80 ꢁC under an argon atmosphere with continuous stir-
ring until homogeneous. Acetonitrile (16.5 mL,
313 mmol) and chlorobenzene (19 mL) were added
dropwise to the mixture. The reaction was heated at
90–100 ꢁC for 24 h with continuous stirring. After the
mixture was cooled to 0 ꢁC and treated with ice water
(50 mL) and diethyl ether (200 mL), it was shaken until
the solid material dissolved. The aqueous layer was sep-
arated from the organic layer and acidified to pH 2 with
1
123.5 ꢁC; H NMR (CDCl3, 300 MHz) d 1.09–1.22 (m,
6H, N(CH2CH3)2), 2.54 (s, 3H, 5-CH3), 2.74 (s, 3H, 7-
CH3), 3.39–3.51 (m, 4H, N(CH2CH3)2), 3.85 (s, 3H,
OCH3), 3.91 (s, 2H, CH2), 6.51 (s, 1H, H-6), 6.98 (d,
J = 9.0 Hz, 2 H, Ph), 7.76 (d, J = 9.0 Hz, 2H, Ph).

M. L. James et al. / Bioorg. Med. Chem. 13 (2005) 6188–6194
6193
4.2.5. N,N-Diethyl-2-[2-(4-hydroxy-phenyl)-5,7-dimethyl-
pyrazolo[1,5-a]pyrimidin-3-yl]-acetamide (6). A solution
of 118 (0.43 g, 1.16 mmol), hexadecyl tributyl phospho-
nium bromide (0.06 g, 0.116 mmol) and 45% HBr
(6 mL) was heated at 100 ꢁC for 7 h under constant stir-
ring. The reaction mixture was basified to pH 8–9 using
NaHCO3 and extracted with CH2Cl2. The organic layer
was collected and dried over anhydrous Na2SO4. The
solvent was removed under vacuum and the residue
was purified by column chromatography (CHCl3/
MeOH, 40:1 (v/v), as eluent) to yield 6 (220 mg, 54%)
measured at 254 nm corresponding to the carrier prod-
uct was measured (integrated) on the HPLC chromato-
gram and compared to a standard curve relating mass to
UV absorbance.
4.4. PET studies
4.4.1. Animals. A male Papio hamadryas baboon aged 13
and weighing 26.5 kg was selected for PET scanning.
The baboon was maintained and handled in accordance
with the NHMRC code of practice for the care and use
of non-human primates for scientific purposes. The
project application was approved by the Central
Sydney Area Health Service (CSAHS) Animal Ethics
Committee.
1
as ivory crystals; mp: 242.5–247 ꢁC; H NMR (CDCl3,
300 MHz) d 1.06–1.18 (m, 6H, N(CH2CH3)2), 2.54 (s,
3H, 5-CH3), 2.73 (s, 3H, 7-CH3), 3.34–3.51 (m, 4H,
N(CH2CH3)2), 3.96 (s, 2H, CH2), 6.49 (s, 1H, H-6),
6.79–6.82 (d, J = 8.7 Hz, 2H, Ph), 7.61–7.64 (d,
J = 8.4 Hz, 2H, Ph); (Found, C, 66.35; H, 6.71; N,
15.38. C20H24N4O2 Æ 1/2H2O requires C, 66.48; H,
6.93; N, 15.51%). Mass Spectrum: CI, m/z 353 (M + 1).
4.4.2. Baboon PET imaging. All PET data were acquired
using a Siemens Biograph LSO PET-CT scanner in the
Department of PET and Nuclear Medicine at Royal
Prince Alfred Hospital. This dual modality device has
a fully 3D PET scanner with 24 crystal rings and a dual
slice CT scanner in the same gantry. It yields a recon-
structed PET spatial resolution of 6.3 mm FWHM (full
width at half maximum) at the centre of the field of view.
A CT scan of the head was completed prior to radioli-
gand injection. The baboon was initially anaesthetised
with ketamine (4 mg/kg, im) in addition to medetomi-
dine hydrochloride (Domitor, 30 lg/kg, im) which is
an a2-adrenoceptor agonist. Anaesthesia was main-
tained with the use of an iv infusion of ketamine in
saline at a dose rate of 0.2 mg ketamine/kg/min. The ba-
boon also received MgSO4 (2 mL) given over half an
hour and atropine (1 mg) plus maxolon (5 mg). The
head of the baboon was immobilized with plastic tape
to minimise motion artefacts. Acquisition of dynamic
PET data (20 · 30 s, 30 · 60 s and 4 · 300 s frames)
was commenced just prior to radioligand injection and
yielded a total of 54 frames over a period of 60 min.
4.3. Radiochemistry
4.3.1. Preparation of [11C]CH3I. The target gas
(14N + 0.5% 16O2) was bombarded with protons using
a
16 MeV cyclotron to produce [11C]CO2. The
[11C]CO2 was transferred to a GE Microlab automated
module and concentrated onto molecular sieves. The
sieves were heated to release [11C]CO2 which was re-
duced by hydrogen on a nickel catalyst to form
[11C]CH4. The [11C]CH4 was released into the CH3I con-
version part of the module where it was recirculated
through a quartz column (packed with ascarite and
iodine crystals) by helium carrier gas. The [11C]CH3I
formed was trapped in ascarite while any unconverted
[11C]CH4 was transferred to waste.
4.3.2. Preparation and formulation of N,N-diethyl-2-[2-(4-
[11C]methoxy-phenyl)-5,7-dimethyl-pyrazolo[1,5-a]pyrim-
idin-3-yl]-acetamide, ([11C]1). Under helium gas flow, the
synthesised [11C]CH3I was delivered to a 1 mL reaction
vessel containing 6 (0.5 mg, 0.0014 mmol) in DMF
(300 lL) and tetrabutylammonium hydroxide (2 lL)
and allowed to stand at room temperature for 3 min.
The reaction mixture was diluted with 0.5 mL of a solu-
tion of 0.1 M NaH2PO4–CH3CN (70:30, v/v) and injected
onto a HPLC XTerra RP C-18 (100 · 7.8 mm, 5 lm)
semi-preparative reverse-phase column. Using a mobile
phase of 0.1 M NaH2PO4–CH3CN (70:30, v/v) and a flow
rate of 6.0 mL/min, the retention time (tR) of [11C]1 was
6.5 min. The radioactive fraction corresponding to
[11C]1 was collected and evaporated under vacuum. The
residue was reconstituted in sterile saline (2 mL) and fil-
tered through a sterile Millipore GS 0.22-lm filter into a
sterile pyrogen free evacuated vial.
The dynamic 3D PET data were rebinned using FORE
(Fourier rebinning) and reconstructed into 47 transaxial
slices with filtered backprojection and CT data based cor-
rections for photon attenuation and scatter. Reconstruct-
ed voxel dimensions were 0.206 · 0.206 · 0.337 cm. The
radioligand uptake was converted to units of percent
injected dose per volume of brain tissue (% dose/mL)
and plotted against time. An automated 3D registration
algorithm24 was used to co-register the three reconstruct-
ed scans prior to ROI definition. Decay corrected time–
activity curves representing the variation in radioligand
concentration versus time were constructed from selected
slices for regions of interest over the whole brain.
After dynamic acquisition, a whole body PET-CT scan
was performed to determine other sites of uptake of
the radioligand.
4.3.3. Quality control of [11C]1. For determination of
specific radioactivity and radiochemical purity, an ali-
quot of the final solution of known volume and radioac-
tivity was injected onto an analytical reverse-phase
HPLC column (XTerra RP C-18, 150 · 4.6 mm). A
mobile phase of 0.1 M NaH2PO4–CH3CN (50:50, v/v)
at a flow rate of 1.0 mL/min was used to elute [11C]1
(tR = 2.3 min). The area of the UV absorbance peak
4.4.3. Blocking studies. Both PK 11195 (5 mg/kg) and
flumazenil (1 mg/kg) were dissolved in saline with a
small quantity of propylene glycol and acetic acid. Both
blocking drugs were injected intravenously 5 min prior
to tracer injection into the cephalic vein. Drug treat-
ments were separated by at least 3-week intervals.

6194
M. L. James et al. / Bioorg. Med. Chem. 13 (2005) 6188–6194
Abbracchio, M. P.; Gremigni, V.; Martini, C. Biochem.
Pharmacol. 2004, 68, 125.
4.5. Lipophilicity measurements
7. Belloli, S.; Moresco, R. M.; Matrarres, M.; Biella, G.;
Sanvito, F.; Simonelli, P.; Turolla, E.; Olivieri, S.; Capelli,
A.; Vomera, S.; Galli-kienle, M.; Fazio, F. Neurochem.
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8. Galiegue, S.; Tinel, N.; Casellas, P. Curr. Med. Chem.
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9. Banati, R. B. Glia 2002, 40, 206.
The logP7.4 was calculated (for derivative 1 and PK
11195) by employing a HPLC method previously de-
scribed.25 Phosphate buffer (0.1 M) was prepared by dis-
solving weighed amounts of potassium dihydrogen
orthophosphate in HPLC water and the pH was adjusted
to 7.5 with sodium hydroxide solution (0.1 M). Samples
were analysed using a C-18 column (XTerra, 150 · 4.6
mm, 5 lm) and a mobile phase of MeOH and phosphate
buffer (60:40 (v/v), pH 7.4) with a flow rate of 1 mL/min.
The lipophilicity of each compound was estimated by
comparing its retention time to that of standards having
known logP values. The standards used to generate a
general calibration equation were aniline, benzene,
bromobenzene, ethyl benzene, trimethyl benzene and
hexachlorobenzene dissolved in mobile phase. All sample
injections were performed three times and the results were
averaged to yield the final values. A calibration curve of
logP versus retention time was produced which resulted
in an experimental calibration equation (y = 1.0791
10. Gonzalez-Scarano, F.; Baltuch, G. Annu. Rev. Neurosci.
1999, 22, 219.
11. Versijpt, J. J.; Dumont, F.; Van Laere, K. J.; Decoo, D.;
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Dierchx, R. A.; Korf, J. Eur. Neurol. 2003, 50, 39.
12. Bribes, E.; Carriere, D.; Goubet, C.; Galiegue, S.; Casel-
las, P.; Simony-Lafontaine, J. J. Histochem. Cytochem.
2004, 52, 19.
13. Cagnin, A.; Brooks, D. J.; Kennedy, A. M.; Gunn, R. N.;
Myers, R.; Turkheimer, F. E.; Jones, T.; Banati, R. B.
Lancet 2001, 358, 461.
14. Banati, R. B.; Newcombe, J.; Gunn, R. N.; Cagnin, A.;
Turkheimer, F.; Heppner, F.; Price, G.; Wegner, F.;
Giovannoni, G.; Miller, D. H.; Perkin, G. D.; Smith, T.;
Hewson, A. K.; Bydder, G.; Kreutzberg, G. W.; Jones, T.;
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15. Gerhard, A.; Banati, R. B.; Goerres, G. B.; Cabnin, A.;
Myers, R.; Gunn, R. N.; Turkheimer, F.; Good, C. D.;
Mathias, C. J.; Quinn, N.; Schwarz, J.; Brooks, D. J.
Neurology 2003, 61, 686.
e
the trendline function from the calibration graph and
0.7527x) with r2 of 0.995. The exponential equation of
ExcelTM allowed the logP values to be calculated.
Acknowledgments
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J.; Suhara, T.; Suzuki, K. Nucl. Med. Biol. 2003, 30, 513.
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The authors thank Ms. Jodie Brackenreg for technical
assistance during imaging studies and Dr. Ken Mewett
from the Adrien Albert Laboratory of Medicinal Chem-
istry for helpful discussions. This work was supported
by DEST and the French Embassy in Australia under
the FAST programme (FR040051).
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