Synthesis and biological evaluation of substituted [18F] imidazo[1,2-a]pyridines and [18F]pyrazolo[1,5-a]pyrimidines for the study of the peripheral benzodiazepine receptor using positron emission tomography

Article abstract of DOI:10.1021/jm7014556

The fluoroethoxy and fluoropropoxy substituted 2-(6-chloro-2-phenyl) imidazo[1,2-a]pyridin-3-yl)-N,N-diethylacetamides 8 (PBR102) and 12 (PBR111) and 2-phenyl-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)-N,N-diethylacetamides 15 (PBR099) and 18 (PBR146) wer

Full text of DOI:10.1021/jm7014556

3700
J. Med. Chem. 2008, 51, 3700–3712
Articles
Synthesis and Biological Evaluation of Substituted [18F]Imidazo[1,2-a]pyridines and
[18F]Pyrazolo[1,5-a]pyrimidines for the Study of the Peripheral Benzodiazepine Receptor Using
Positron Emission Tomography
Christopher J. R. Fookes, Tien Q. Pham, Filomena Mattner, Ivan Greguric,* Christian Loc’h, Xiang Liu, Paula Berghofer,
Rachael Shepherd, Marie-Claude Gregoire, and Andrew Katsifis
Radiopharmaceuticals Research Institute, Australian Nuclear Science and Technology Organisation,
PMB 1 Menai NSW 2234, Sydney, Australia
ReceiVed NoVember 19, 2007
The fluoroethoxy and fluoropropoxy substituted 2-(6-chloro-2-phenyl)imidazo[1,2-a]pyridin-3-yl)-N,N-
diethylacetamides 8 (PBR102) and 12 (PBR111) and 2-phenyl-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)-
N,N-diethylacetamides 15 (PBR099) and 18 (PBR146) were synthesized and found to have high in vitro
affinity and selectivity for the peripheral benzodiazepine receptors (PBRs) when compared with the central
benzodiazepine receptors (CBRs). The corresponding radiolabeled compounds [18F]8 [18F]12, [18F]15, and
[18F]18 were prepared from their p-toluenesulfonyl precursors in 50-85% radiochemical yield. In
biodistribution studies in rats, the distribution of radioactivity of the [18F]PBR compounds paralleled the
known localization of PBRs. In the olfactory bulbs, where the uptake of radioactivity was higher than in the
rest of the brain, PK11195 and Ro 5-4864 were able to significantly inhibit [18F]12, while little or no
pharmacological action of these established PBR drugs were observed on the uptake of [18F]8, [18F]15, and
[18F]18 compared to control animals. Hence, [18F]12 appeared to be the best candidate for evaluation as an
imaging agent for PBR expression in neurodegenerative disorders.
Introduction
The peripheral benzodiazepine receptor (PBRa) is a trans-
membrane multimeric protein complex primarily located in the
outer mitochondrial membrane of cells. It is composed of an
18 kDa isoquinoline carboxamide-binding protein, a 32 kDa
voltage dependent anion channel (VDAC), and a 30 kDa adenine
nucleotide translocase (ANT).1 The PBR is predominantly
expressed in peripheral organs such as the kidney, heart, and
the steroid hormone producing cells of the adrenal cortex, testis,
and ovaries.2 In the CNS, the PBR is distinct from the central
benzodiazepine receptor (CBR). In rodents, PBR is found in
the ependymal lining of the ventricles, choroid plexus, and the
olfactory bulb,3 where it has been shown to modulate neuronal
activity through the production of neurosteroids.4
In humans, PBR density is considerably overexpressed in
neuroinflammation and neurodegeneration, suggesting that the
PBR could be a clinically useful marker for the detection of
such disorders. Consequently, radioligands based on the 1,4-
benzodiazepine 7-chloro-1,3-dihydro-1-methyl-5(4′-chlorophe-
nyl)-2H-1,4-benzodiazepine-2-one (Ro 5-4864)2,5 and the iso-
quinoline carboxamide 2-chlorophenyl-N-methylpropyl)-3-
Figure 1. Structures of some established PBR ligands.
isoquinolinecarboxamide (PK11195)3,6 (Figure 1), which can
target the PBR, have been shown to measure changes in PBR
receptor density in a number of pathological conditions such
as neurodegeneration,7 inflammation,8 Alzheimer’s,9,10 Park-
inson’s11,12 and Huntington’s diseases,13 and multiple sclerosis.8,14
In neoplastic tissue enhanced PBR, expression has been
observed in gliomas,15,16 breast,17 colon,18 ovarian carcinomas,19
and prostatic adenocarcinomas.20
Accordingly, analogues of PK11195 and Ro 5-4864 radio-
labeled with carbon-11 were able to image several pathological
conditions associated with altered PBR expression using the
noninvasive imaging modality positron emission tomography
(PET),21 while replacement of the chlorine in PK11195 by
* To whom correspondence should be addressed. Phone: +61 2 9717
3759. Fax: +61 2 9717 9262. E-mail: ivg@ansto.gov.au.
a Abbreviations: CNS, central nervous system; DMF, dimethylformamide;
EDCI, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide; DEA, diethy-
lamine; DIPEA, N,N′-diisopropylethylamine; HOBT, N-hydroxybenzotria-
zole; K2.2.2, 4,7,13,21,24-hexaoxa-1,10-diazabicyco[8.8.8]hexacosane; NMM,
N-methylmorpholine; RP-HPLC, reverse phase high pressure liquid chro-
matography; PBR, peripheral benzodiazepine receptor; PET, positron
emission tomography; RT, room temperature; PBSF, perfluoro-1-butane-
sulfonyl fluoride; SAR, structure-activity relationship; TEA.3HF, triethy-
lamine trihydrofluoride; TMHD,N,N,N′,N′-tetramethylhexane-1,6-diamine.
10.1021/jm7014556 CCC: $40.75
2008 American Chemical Society
Published on Web 06/17/2008

[
18F]Imidazo[1,2-a]pyridines and [18F]Pyrazolo[1,5-a]pyrimidines
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3701
fluorination from their p-toluenesulfonyl precursors and their
radiopharmacological properties assessed.
Results and Discussion
Chemistry. The synthesis of the four target molecules 8, 12,
15, and 18 and their corresponding p-toluenesulfonyl precursors
required for fluorine-18 labeling are shown in Schemes 1
and 2.
Figure 2. General structure of imidazo[1,2-a]pyridines.
iodine-123 enabled imaging with single photon emission
computer tomography (SPECT).22,23 More recently, new ligands,
The synthesis of the imidazo[1,2-a]pyridine scaffold 2 was
achieved by condensing 2-amino-5-chloropyridine with 2′-
bromo-4-methoxyacetophenone. The acetic acid side-chain was
introduced at position -3 by a four-step procedure starting with
Mannich condensation with formaldehyde and dimethylamine
to give 3, followed by quaternization with methyl iodide. The
resulting salt 4 underwent reaction with potassium cyanide in
refluxing ethanol/water to give the amide 5 resulting from
hydrolysis of the intermediate nitrile under the strongly alkaline
reaction conditions. This was demethylated and further hydro-
lyzed to the phenolic acid 6 using hydrobromic acid in acetic
acid. During the formation of the key diethylamide intermediate
7 (using EDCI, HOBT, and DIPEA), there was significant
reaction between the phenol and carboxyl groups, producing
30-50% of dimeric and trimeric species. This unwanted reaction
was reversed by treating the crude product with diethylamine
in DMF. Alkylation of the phenol group of 7 with 1-bromo-2-
fluoroethane in DMF, using potassium carbonate and potassium
iodide in the presence of the phase transfer catalyst tetra-
butylammonium iodide, produced 8. Similarly, 2-bromoethyl
acetate gave the acetoxyethyl derivative 9a, however, the
product was accompanied by considerable quantities of the
corresponding alcohol 10a. Subsequent treatment with cesium
carbonate in aqueous methanol40 cleanly converted the acetate
9a into the alcohol 10a, which was tosylated using N,N,N′,N′-
tetramethylhexane-1,6-diamine (THMD) as a base to give 11a.
The same 2-step procedure was followed to prepare the
homologous alcohol 10b. The product was converted into the
corresponding fluoride 12 by treatment with perfluorobutane-
sulfonyl fluoride and triethylamine trihydrofluoride in the
presence of DIPEA.41 Reaction of 10b with p-toluenesulfonyl
chloride and THMD gave the radiolabeling precursor 11b.
The pyrazolo[1,5-a]pyrimidine PBR ligands 15 and 18 were
synthesized from the known compound 13.14 Boron trichloride-
catalyzed demethylation by tetrabutylammonium iodide42 gave
the phenol 14, which was alkylated with 1-bromo-2-fluoroethane
to produce 15. Alkylation of 14 with either 2-bromoethyl acetate
or 3-bromopropyl acetate, followed by cesium carbonate hy-
drolysis (without purification of the intermediate acetates),
yielded the alcohols 16a and 16b, respectively. Compound 17b
was then converted into 18 by the same method used for 12.
Finally, tosylation of 16a and 16b gave the radiolabeling
precursors 17a and 17b.
including the imidazo[1,2-b]pyridines,24,25 123I]CLINDE,26
[
(Figure 1) [11C]CLINME,27
ally related imidazopyridazines29 the 2-phenylpyrazolo[1,5-
a]pyrimidineacetamides [11C]DPA-713,30 123I] N,N-dialkyl-2-
[
123I]CLINME,28 and the structur-
[
phenylindol-3-yl-glyoxylamides,31 N-benzyl-N-(2-phenoxyaryl)-
acetamide derivatives 1 ([11C]DAA1106)32,33 (Figure 1), and
their structural analogues [11C]pyridinylacetamides,34 have been
developed as potential probes for imaging PBR receptors in vivo
using PET or SPECT.
The increasing availability of PET, and the short half-life of
carbon-11 has prompted the development of novel [18F] ligands,
including the N-benzyl-N-(2-phenoxyaryl)acetamides21,35,36 and
2-(2-(4-tert-butylphenyl)-6-fluoroimidazo[1,2-b]pyridazin-3-yl)-
N,N-diethylacetamide (PBR132),37 with promising preclinical
data. Similarly, halogenated 2-phenylimidazopyridines, imida-
zopyridazines, and pyrazolopyrimidine-3-acetamides have been
prepared with high affinity and selectivity for the PBR
receptor.24,29 One aspect of the extensive structure affinity
studies carried out in our laboratory24,29 and elsewhere38 has
revealed that 6-chloro-2-phenylimidazo[1,2-a]pyridine-3-yl ac-
etamides (Figure 2), where R1 and R2 are small alkyl groups,
have high affinity for the PBR when the para-substituent X on
the 2-phenyl ring is moderately large. Consequently, a large
number of ligands based on this structure have been prepared
bearing the SPECT isotope I-123. For example, CLINDM (R1
and R2 ) Me, X ) I) is a potent PBR ligand, while the
incorporation of fluorine directly on the 2′-phenyl ring has
resulted in a marked increase in CBR affinity and concomitant
loss in PBR affinity and selectivity.24
However, the observation that a methoxy group in the 4′-
position is well tolerated and the associated difficulties in
introducing fluorine directly on to these aromatic heterocycles
suggested that a short side-chain incorporating a fluorine atom,
as in the fluoroethoxy or fluoropropoxy derivatives, could
provide compounds of suitable PBR affinity and selectivity as
well as providing a convenient route to the efficient incorpora-
tion of fluorine-18. Similarly, the methoxy group of the high
affinity and selective pyrazolo[1,5-a]pyrimidine 1339 could also
be replaced by the larger fluoroethoxy and fluoropropoxy
substituents, providing potentially higher affinity PBR com-
pounds for applications in PET imaging.
Radiochemistry. Radiofluorination was achieved via nucleo-
philic substitution of the p-toluenesulfonyl precursor (11a, 11b,
17a, or 17b) by [18F]fluoride in the presence of equimolar ratios
of K2.2.2 and K2CO3 as shown in Scheme 3. Azeotropic drying
in acetonitrile was performed twice, before the dried
K[18F]F-K2.2.2 complex was treated with the p-toluenesulfonyl
precursor in anhydrous acetonitrile. The reactions were heated
with stirring at 100 °C for 5 min (Table 1) to form the
radiofluorinated products and quenched with mobile phase
before injection onto a preparative HPLC column for purifica-
tion. The overall radiosynthesis, including [18F]fluorination,
HPLC purification, evaporation, and radiotracer formulation was
completed in a time range of 60-90 min with a radiochemical
In this work, the fluoroethoxy and fluoropropoxy substituted
2-(6-chloro-2-phenyl)imidazo[1,2-a]pyridin-3-yl)-N,N-diethyl-
acetamides 8 (PBR102)29 and 12 (PBR111)29 [Scheme 1] and
2-phenyl-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)-N,N-di-
ethylacetamides 15 (PBR099)29 and 18 (PBR146)29 [Scheme
2] were synthesized and their affinities for both the PBR and
the CBR evaluated. The corresponding [18F]fluoroalkoxy de-
rivatives were subsequently prepared via nucleophilic radio-

3702 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
Fookes et al.
Scheme 1. Synthesis of 8 and 12 and the p-Toluenesulfonyl Radiolabelling Precursors 11a and 11ba
a (i) (CH3)2NH (aq), CH2O, CH3COOH, 60 °C, 20 h; (ii) CH3I, benzene, RT, 18 h; (iii) KCN, EtOH/H2O 1:1, reflux, 24 h; (iv) HBr, CH3COOH, reflux,
48 h; (v) EDCI, HOBT, DIPEA, DMF, DEA, RT, 4 days; (vi) K2CO3, KI, Bu4NI, 1-bromo-2-fluoroethane, 48 h; (vii) K2CO3, KI, 2-bromoethyl acetate (9a)
or 3-bromopropyl acetate (9b) Bu4NI at RT for 48 h; (viii) Cs2CO3, MeOH, H2O, RT, 48 h; (ix) TsCl, TMHD, CH3CN, RT, 15 h; (x) DIPEA, PBSF,
TEA.3HF CH3CN, 40 °C, 30 min then RT, 12 h.
Scheme 2. Synthesis of 15 and 18 and the Corresponding p-Toluenesulfonyl Precursors 17a and 17ba
a (i) Bu4NI, BCl3, DCM, -70 °C, 5 min, then 4 °C for 2.5 h; (ii) K2CO3, KI, Bu4NI, 1-bromo-2-fluoroethane, DMF, RT, 48 h; (iii) K2CO3, KI, 2-bromoethyl
acetate (16a) or 3-bromopropyl acetate (16b), Bu4NI, DMF, RT, 48 h; (vi) Cs2CO3, MeOH, H2O, RT, 48 h; (v) TsCl, TMHD, CH3CN at RT; (iv) DIPEA,
PBST, TEA.3HF, CH3CN, 40 °C, 30 min, then RT 12 h.
purity greater than 95%. The identity of the [18F]compounds
coinjection of their corresponding unlabeled compound using
analytical HPLC conditions (Table 1).
([18F]8, [18F]12, [18F]15, and [18F]18) was confirmed by

[
18F]Imidazo[1,2-a]pyridines and [18F]Pyrazolo[1,5-a]pyrimidines
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3703
Scheme 3. Radiolabelling of [18F]Imidazo[1,2-a]pyridines and [18F]Pyrazolo[1,5-a]pyrimidines
Table 1. Radiolabelling Data for the [18F]PBR Radiotracers
injection of the fluoropropoxy derivatives [18F]12 and [18F]18
were 70% higher than the fluoroethoxy compounds [18F]15 and
[18F]8 (4-5%ID/g). Similarly, after injection of [18F]12 and
[18F]18, the kidney radioactivity uptake (5%ID/g) was 45%
higher than that of the fluoroethoxy compounds [18F]15 and
[18F]8 (3.5%ID/g). Radioactivity uptake in the lungs of all
radiotracersdecreasedovertimefromaninitialvalue14-20%ID/g
at 0.25 h to 3-6%ID/g at 4 h. In the spleen, the radioactivity
uptake decreased over time from 5-6% to 2-4%ID/g for all
radiotracers except for [18F]15, where the uptake remained
constant (4.5%ID/g) over the course of 4 h. In the steroidogenic
organs, after injection of the [18F]PBR ligands, the radioactivity
uptake in adrenals and in testis was 5-8%ID/g and 0.2-0.3%ID/
g, respectively, at 0.25 h and increased slowly with time.
The initial blood radioactivity concentration following [18F]8
and [18F]15 injection decreased from an initial 0.2% to almost
0.1%ID/g in the first hour but was then followed by a slow
increase with time. This small incremental increase could be
related to the specific metabolism of the two compounds. The
blood radioactivity concentration for the fluoropropoxy deriva-
tives [18F]12 and [18F]18 were initially high (0.3-0.4%ID/g at
0.25 h), which also decreased rapidly, falling to 0.1%ID/g at
4 h (Figure 3). In the brain, the radioactive concentration
displayed the same kinetics as the blood irrespective of the
radiofluorinated tracer studied, so the displayed brain to blood
concentration ratios were constant over time, indicating an
equilibrium between brain and blood. Although no direct
relationship between lipophilicity and brain uptake could be
made in this study, all four compounds displayed lipophilicity
values (log P7.5) ranging from 2.5 to 3.2, which is in the range
favored for blood brain barrier permeability (Table 2).
At 0.25 h, the olfactory bulbs showed 50 and 70% higher
radioactivity concentration than the brain for the pyrazolopy-
rimidine derivatives [18F]18 and [18F]15, respectively, while the
radioactivity concentration in the olfactory bulbs for the
imidazopyridine derivatives [18F]8 and [18F]12 were, respec-
tively, 150 and 170% higher than the corresponding concentra-
tion in the brain. The activity in the olfactory bulbs remained
constant or slowly decreased during the study. The olfactory
bulb to blood concentration ratios were constant (∼2) over the
duration of the study for [18F]15 and [18F]8, but as a conse-
quence of the decrease in activity in the blood, the ratios
increased with time and were 3 and 5 at 4 h for [18F]18 and
[18F]12, respectively.
[
18F]
purification
solventb
retention
QC
compound precursora
time (min) RCY%e solvent
[
[
[
[
18F]15
18F]8
17a
11a
17b
11b
40/60a
45/55d
40/60c
45/55d
13
13
12
13
55-85
55-75
50-67
56-75
30/70f
45/55g
50/50g
30/70f
18F]18
18F]12
a Precursor mass of 2 mg, reacting at 100 °C, 5 min. b Acetonitrile/H2O
+ 0.1%TFA. c Waters X-Bridge C18 (10 µm, 30 mm × 100 mm), 15
mL/min. d Grace Alltima C18 (10 µm; 22 mm × 250 mm), 10 mL/min.
e Isolated yield (decay corrected), specific activity 110-150 GBq/µmol.
f Phenomenex Luna C18(2) (5 µm; 4.6 mm × 150 mm) 1 mL/min,
acetonitrile/H2O 0.1%TFA. g Phenomenex Gemini C18 (5 µm; 4.6 mm ×
150 mm), 1 mL/min acetonitrile/0.1 M ammonium acetate.
Table 2. Inhibition Constant (Ki) and log P7.5 of Compounds Studied
Kia PBR
Kia PBR
Kia CBR
([3H]PK11195)b ([3H]Ro 5-4864)b ([3H]flumazenil)c log P7.5
15
8
18
12
5.6 ( 0.9
5.8 ( 0.4
4.1 ( 0.4
3.7 ( 0.4
5.7 ( 0.1
3.3 ( 0.4
5.8 ( 0.5
4.2 ( 0.4
>5000
2.5 ( 0.1
2.7 ( 0.1
3.2 ( 0.1
3.2 ( 0.1
790 ( 90
>5000
800 ( 120
a Ki values, expressed in nM, were calculated from the experimental IC50
values using the Chen-Prussof equation. b [3H]PK11195 and [3H]Ro 5-4864
binding on rat kidney membranes. c [3H]Flumazenil binding on brain cortex
membranes.
Ligand Binding Assays. The inhibition constant (Ki) for
the CBRs as measured with [3H]ethyl 8-fluoro-5,6-dihydro-
5-methyl-6-oxo-4H-imidazo[1,5-a](1,4)benzodiazepine-3-car-
boxylate ([3H]flumazenil) were 800 nM for the imidazopy-
ridine derivatives 8 and 12 and >5000 nM for the
pyrazolopyrimidine compounds 15 and 18 (Table 2). The Ki
values for PBR as measured with [3H]PK11195 and [3H]Ro
5-4864 ranged from 3-6 nM. Interestingly, the fluoropropoxy
derivatives 12 and 18 were slightly more potent inhibitors
of PK11195 binding (Ki ∼ 4 nM) than the fluoroethoxy
derivatives 15 and 8 (Ki ∼ 6 nM). Similarly, the imidazopy-
ridine analogues, 8 and 12, were somewhat more potent in
inhibiting Ro 5-4864 binding (Ki ∼ 4 nM) than the pyrazol-
opyrimidine analogues, 15 and 18 (Ki ∼ 6 nM). According
to these assays, the compounds described here are potent PBR
inhibitors with low CBR affinity.
Biodistribution Studies. The time-course distribution of the
radiofluorinated tracers in rats is presented in Table 3. In the
kidney and in the heart, the radioactivity concentration peaked
between 0.25 and 1 h post injection and then decreased. In early
time points, the heart radioactivity uptake (8-9%ID/g) after
In the femur, the uptake of radioactivity increased over time
(Figure 4). This uptake was 0.4%ID/g at 0.25 h and reached

3704 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
Table 3. Biodistribution of [18F]PBR Compounds in Rats
Fookes et al.
time (h)
kidneya
hearta
lungsa
spleena
livera
adrenalsa
testisa
[
[
[
[
18F]15
0.25
0.5
1
3.8 ( 0.2
3.6 ( 0.5
3.5 ( 0.2
2.4 ( 0.4
3.3 ( 0.37
3.2 ( 0.5
2.7 ( 0.2
1.3 ( 0.1
5.5 ( 1.2
4.9 ( 0.2
4.7 ( 0.2
3.2 ( 0.3
4.3 ( 0.6
5.0 ( 0.3
3.7 ( 0.2
1.8 ( 0.3
5.8 ( 0.4
5.6 ( 0.3
6.4 ( 0.4
4.3 ( 0.9
4.3 ( 0.3
4.4 ( 0.3
4.6 ( 0.3
2.8 ( 0.2
9.0 ( 0.7
9.0 ( 0.5
8.7 ( 1.0
5.8 ( 0.9
7.8 ( 0.5
8.3 ( 0.2
6.4 ( 0.2
2.9 ( 0.3
14.6 ( 1.3
8.8 ( 0.5
5.7 ( 0.6
2.8 ( 0.5
14.3 ( 1.4
12.1 ( 2.0
11.6 ( 1.5
8.1 ( 1.1
20 ( 4
12.4 ( 1.3
7.1 ( 0.9
3.3 ( 0.2
12.3 ( 0.5
7.7 ( 0.5
4.1 ( 0.3
2.2 ( 0.3
4.6 ( 0.3
4.7 ( 0.6
4.5 ( 0.7
3.9 ( 0.6
4.5 ( 0.5
3.9 ( 0.4
3.4 ( 0.2
1.9 ( 0.2
5.5 ( 0.9
5.5 ( 0.4
5.1 ( 0.4
3.6 ( 0.2
5.5 ( 0.5
6.0 ( 0.4
4.5 ( 0.3
2.5 ( 0.4
0.63 ( 0.06
0.54 ( 0.18
0.40 ( 0.05
0.29 ( 0.04
0.63 ( 0.15
0.52 ( 0.14
0.35 ( 0.01
0.29 ( 0.02
1.08 ( 0.05
0.98 ( 0.21
0.69 ( 0.26
0.38 ( 0.09
1.08 ( 0.09
0.79 ( 0.07
0.61 ( 0.11
0.30 ( 0.07
5.6 ( 0.5
6. Six ( 1.2
7.8 ( 1.7
9.7 ( 1.5
6.7 ( 1.3
7.9 ( 1.7
7.3 ( 1.1
7.1 ( 1.0
8.0 ( 0.7
7.9 ( 1.5
11.0 ( 2.0
12.2 ( 1.6
8.5 ( 1.4
11.8 ( 2.5
9.6 ( 0.5
11.4 ( 1.8
0.18 ( 0.01
0.21 ( 0.01
0.27 ( 0.02
0.35 ( 0.01
0.17 ( 0.01
0.23 ( 0.03
0.31 ( 0.03
0.42 ( 0.01
0.27 ( 0.02
0.28 ( 0.02
0.32 ( 0.03
0.43 ( 0.04
0.33 ( 0.02
0.43 ( 0.02
0.41 ( 0.02
0.53 ( 0.01
4
18F]8
0.25
0.5
1
4
18F]18
18F]12
0.25
0.5
1
4
0.25
0.5
1
4
a Results are the mean of %ID/g of tissue ( SD, n ) 4.
Figure 3. Time course distribution of [18F]15, [18F]8, [18F]18, and [18F]12 in olfactory bulbs (0), brain ([), and blood (4). The radioactive
concentration is expressed in %ID/g and the error bars represent the standard deviation of the mean value (n ) 4).
0.7%ID/g at 4 h for both [18F]15 and [18F]8. For [18F]18 and
[18F]12, the femur uptake was 0.6 and 2.2%ID/g at 0.25 and
4 h post injection, respectively. The higher radioactive concen-
tration in bone after injection of the fluoropropoxy compounds
versus the fluoroethoxy derivatives was investigated. The
radioactivity concentration in femur bone marrow, in femur,
and in the skull frontal bone (bone with less marrow than femur)
were followed after injection of [18F]12 in rats. In bone marrow,
the radioactivity concentration was 2.4 and 1.2 times higher
than those of skull at 0.25 and 4 h, respectively. In the skull,
the radioactivity concentration was half of that in the femur
during the time course of the study. Hence, after injection of
[18F]12, the bone marrow radioactivity, reflecting PBR expres-
sion in the marrow, amounted to 60 and 40% of the total activity
of the femur at 0.25 and 4 h, respectively. The majority of the
remaining activity is likely to be specific [18F]fluoride adsorption
in the bone apatite.
Competition Studies. Drug saturation and competition
experiments were carried out to test the ability of central and
peripheral drugs to inhibit [18F]PBR radiotracer uptake in
peripheral organs and in the brain. The saturating effect of the
unlabeled compounds 15, 8, 18, and 12 (1 mg/kg) on the
radioactivity uptake of the corresponding [18F]labeled com-
pounds in the heart was observed, with very significant decreases
greater than 82% (p < 0.01) compared to the heart radioactivity
uptake of untreated animals (Figure 5). This saturating effect
was also observed in the kidney, with decreases in uptake
exceeding 76% (p < 0.01), and in the spleen (80%, p < 0.01)
but not in adrenals.
The administration of the unlabeled fluoroethoxy compounds
prior to the radiotracer increased significantly the brain (15 and
8) and the olfactory bulb radioactivity uptake (8) for the
corresponding F-18 compounds that paralleled the increase in
blood radioactivity concentration. No effect was observed with

[
18F]Imidazo[1,2-a]pyridines and [18F]Pyrazolo[1,5-a]pyrimidines
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3705
of unchanged tracer was carried out by radio-HPLC. After
ultrasonic disruption and protein elimination, at least 85% of
the radioactivity in the olfactory bulbs and 75% in the cortex
was recovered in the supernatant of 0.25 and 0.5 h samples for
HPLC analysis. For later time points, the recovery was still
greater than 70% and 60%, respectively, in these tissues. Further
extraction of protein precipitate using pure acetonitrile removed
almost all radioactivity. Analysis of this extract using radio TLC
and solid phase extraction showed it contained only radioactive
metabolites.
In plasma, rapid decrease of unchanged radiofluorinated
tracers was observed. The amount of parent compounds was
approximately 10% for [18F]18 and [18F]12 at 0.25 h post
injection and less than 1% for [18F]15 and [18F]8 at 4 h (Figure
6). In the cortex, 55-80% of the radioactivity represented
[18F]PBR compounds at 0.25 h. These values dropped to 10%
for [18F]15 and [18F]8 and to 30% for [18F]18 and [18F]12 at
4 h. In olfactory bulbs, the fraction of unchanged tracer was
higher than in the cortical region. For [18F]15 and [18F]8, the
percentage of unchanged ligand diminished from 80% at 0.25 h
to 40% at 4 h post injection. For [18F]18 and [18F]12, more
than 80% of the radioactivity still represented unchanged
radiotracer at 4 h pi.
Considering the chemical structures of the studied com-
pounds, it appeared that the amount of unchanged fluoropropoxy
derivatives [18F]18 and [18F]12 in the brain were much higher
than the fluoroethoxy derivatives[18F]15 and [18F]8. Moreover,
in the olfactory bulbs, the percentage of unmetabolized fluo-
ropropoxy derivatives were almost constant versus time and
higher than 86% for [18F]12. By using water as eluent in the
first step of HPLC analysis, it was shown that the main
radioactive metabolites in plasma, cortex, and olfactory bulbs
were highly polar products as they eluted with the solvent front.
The remaining radioactivity was analyzed using a gradient
system in the second HPLC step; no compounds more lipophilic
than the parent compound were observed.
The metabolite and bone uptake results are consistent with
the expected fate of the [18F]-containing moiety of the four PBR
ligands in vivo. Alkyl aryl ethers are known to dealkylate in
mammals by a monooxygenase-mediated reaction that occurs
by hydroxylation of the carbon R to the oxygen.43,44 Cleavage
of the resulting hemiacetal generates an aliphatic aldehyde,
which in the case of the fluoroethoxy compounds [18F]15 and
[18F]8 would be [18F]fluoroacetaldehyde. In related studies, using
the PET imaging agent 18F-FECNT, [18F]fluoroacetaldehyde (or
its oxidation product [18F]fluoroacetic acid) was proposed to
be the radiometabolite that accumulates in rat, human, and
nonhuman primate brain and was implicated in confounding
radioligand measurements.45 These metabolites would not
compete with PK11195 or Ro 5-4864 in the present competition
study. In contrast, 3-[18F]fluoropropionic acid, an expected
metabolite from [18F]18 and [18F]12, is known to be completely
catabolized to inorganic fluoride,46 which is rapidly sequestered
by bone. However, the resulting higher bone uptake of activity
observed with [18F]18 and [18F]12 versus [18F]15 and [18F]8 is
still acceptable for PET imaging.
Figure 4. Time course uptake in rat femur of [18F]15(4), [18F]8 (2),
[18F]18 (9), and [18F]12 (0). The radioactive concentration is expressed
in %ID/g and the error bars represent the standard deviation of the
mean value (n ) 4).
18 and 12 on the brain radioactivity uptake for [18F]18 and
[18F]12. In the olfactory bulbs, the administration of the 12
decreased [18F]12 uptake by 45%, (p < 0.01) despite increases
in blood radioactive concentration of 33%.
The competitive effect of PK11195 on the radioactivity uptake
in the kidney, heart, and the spleen following [18F]PBR injection
was higher than the effect of Ro 5-4864, with decreases of 70
to 85% (p < 0.01) for PK11195 and decreases of 30 to 70%
for Ro 5-4864 observed. In these three organs, the highest
inhibition was observed for the radiotracer [18F]12.
In the adrenals, pretreatment of the rats with PK11195 or Ro
5-4864 significantly increased the radioactivity concentrations
of [18F]15, [18F]8, and [18F]18 by 50-100%. These particular
effects of the PBR drugs could be explained by an increased
concentration of the respective radiotracers [18F]15, [18F]8, and
[18F]18 made available in the blood after displacement from
other tissue or that a component of the radioactivity in the
adrenals could be due to nonspecific binding. For [18F]12, no
significant increase of radioactivity concentration was seen in
rats that received PK11195.
The effect of PK11195 and Ro 5-4864 in the brain were
similar, as these two PBR drugs were unable to displace the
radioactivity (Figure 5). Increases, probably due to the blood
were observed for [18F]15 and [18F]8. In the olfactory bulbs,
however, PK11195 and Ro 5-4864 were able to inhibit
significantly (p < 0.01) the uptake of [18F]12 by 55 and 36%,
respectively, while no or minimal pharmacological action of
the two PBR drugs were observed on the radioactivity uptake
of [18F]8, [18F]15, and [18F]18 compared to control animals.
When the animals received flumazenil, a CBR drug, the uptake
of the radiofluorinated PBR ligands were not significantly
different to controls in all the organs and the brain structures
studied.
In competition studies, we were unable to significantly inhibit
the radioactivity uptake of the fluoroethoxy compounds [18F]15
and [18F]8 in the olfactory bulbs, while a very significant
inhibition was observed for [18F]12. On the other hand, the
radioactivity uptake of fluoropropoxy compounds [18F]18 and
[18F]12 in the bone was higher than that for the fluoroethoxy
derivatives. These results may be due to the different in vivo
metabolic pathways of the respective radiotracers.
Summary
The PBR compounds described here have nanomolar inhibi-
tion constants (Ki) for the PBR specific ligands PK11195 and
Ro 5-4864 and micromolar Ki values for the CBR ligand
flumazenil. The pyrazolopyrimidine derivatives 15 and 18
bearing the fluoroethoxy and fluoropropoxy substituent on the
phenyl group displayed similar affinity and selectivity to the
Metabolite Studies. The stability of the [18F]PBR compounds
in vivo was studied over 4 h after iv injection. The determination

3706 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
Fookes et al.
Figure 5. Relative uptake of the [18F]PBR compounds in the kidney, heart, spleen, adrenals, brain, and in the olfactory bulbs in animals treated
with the corresponding unlabeled PBR ligand, PK11195, Ro 5-4864, and flumazenil in comparison to untreated animals. For an organ, the relative
uptake value is the ratio of the mean radioactive concentrations of treated animals divided by the mean radioactive concentrations of control
animals and is expressed in %. *At least p < 0.05 calculated using ANOVA test.
methoxy compound 13. The imidazopyridines 8 and 12 pre-
sented similar affinity for PBR but were found to be at least 50
times more selective for PBR versus CBR than the parent
compound alpidem.47
In peripheral organs, the distribution of radioactivity after
injection of the [18F]PBR compounds paralleled the localization
of PBRs that are reported to be present in high density in
endocrine tissues such as adrenal glands and in organs such as
kidney and heart.
5-methoxybenzyl- acetamide and N-(5-fluoro-2-phenoxyphenyl)-
N-(2-[18F]fluoroethyl-5-methoxybenzylacetamide. The specific
to nonspecific uptake, calculated as the ratio of the radioactive
concentration in olfactory bulbs to the brain, was 4 at 1 h post
injection for [18F]12 while the ratios were ∼2 for the other
radiotracers. The inability to significantly inhibit the uptake of
the fluoroethoxy compounds [18F]15 and [18F]8 in the olfactory
bulbs while a very significant inhibition was observed for
[18F]12 using the PBR drugs PK11195 and Ro 5-4864 demon-
strates that the uptake of [18F]12 is related to a specific binding
on the PBRs. This and the observation that the uptake of
radioactivity in the bone after injection of the fluoropropoxy
compounds [18F]18 and [18F]12 was higher than the fluoroethoxy
derivatives suggests that it may be due to the different in vivo
metabolic pathways of the respective radiotracers.
In the brain, after injection of [18F]PBR compounds, the
uptake in the olfactory bulbs was higher than in the other brain
structures throughout the experiment. This is likely related to
PBRs, as this region is known to have a higher density of
peripheral binding sites than other parts of the brain as measured
in vitro with the benzodiazepine [3H]Ro 5-4864 and with the
imidazopyridine [3H]CB 34,48 and in ex vivo autoradiography
with the N-(5-fluoro-2-phenoxyphenyl)-N-(2-[18F]fluoromethyl-
In conclusion, among the two series of compounds, [18F]12
appears to be the most promising radiofluorinated ligand as it

[
18F]Imidazo[1,2-a]pyridines and [18F]Pyrazolo[1,5-a]pyrimidines
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3707
mass spectrometry (HRMS) was performed using a Bruker Dal-
tonics BioApex-II 7T FTICR mass spectrometer equipped with an
off-axis analytical ESI source. Preparative HPLC was performed
on a Waters Empower system, equipped with a 600 gradient pump
and a 996 PDA detector, 717 autosampler, and fraction collector
III. Purification of the radiotracers was performed on a radio-HPLC
system equipped with Waters 486 UV, set at 254 nm, and an Ortec
Acemate Scint 925 γ-detector with a sodium iodide crystal. For
radiochemical purity and stability measurements, the radiofluori-
nated tracer was ascertained on an analytical HPLC system equipped
with a Varian 9002 pump, linear UV detector set at 254 nm, and
an Ortec Acemate Scint 925 γ-detector with a sodium iodide crystal
using Laura version 3 software package. The radiochemical and
chemical purity and specific activity measurements were confirmed
on an analytical HPLC using a C18 analytical column (Table 1).
Metabolite studies were performed on a system consisting of a
(Waters) 600 gradient pump, a 486 UV detector set at 254 nm, a
717 autosampler and an in line detector for measurement of
radioactivity (ꢀ-Ram IN/US) operated by Waters Empower software.
6-Chloro-2-(4-methoxyphenyl)imidazo[1,2-a]pyridine (2). A
mixture of 2-bromo-4′-methoxyacetophenone (25 g, 110 mmol),
2-amino-5-chloropyridine (14.0 g, 109 mmol), and absolute ethanol
(220 mL) was stirred at reflux for 2 h. NaHCO3 (5.5 g, 65 mmol)
was then added and heating continued for another 6 h. Additional
NaHCO3 (3.65 g, 43 mmol) was then added and stirring continued
for a further 15 min. The mixture was cooled to 4 °C for 12 h and
the solid was filtered, washed with cold ethanol (50 mL), water
(100 mL), then ethanol (30 mL). The solid was then boiled with
ethanol (100 mL) for 10 min, cooled, and filtered. The solid was
washed with ethanol (30 mL) again and dried in vacuo to give a
pale-yellow powder (21 g, 83%); mp 220-222 °C. 1H NMR
(DMSO-d6) δ: 3.80 (s, 3H), 7.01 (d, J ) 8.8 Hz, 2H), 7.26 (dd, J
) 9.6, 2.0 Hz, 1H), 7.59 (d, J ) 9.6 Hz, 1H), 7.89 (d, J ) 8.8 Hz,
2H), 8.26 (s, 1H), 8.78 (dd, J ) 2.0, 0.8 Hz, 1H). 13C NMR
(DMSO-d6) δ: 55.1, 108.7, 114.2, 117.2, 118.7, 124.6, 125.5, 126.0,
127.0, 143.2, 145.5, 159.2. MS ES(+ve) m/z 259 (M + 1).
1-(6-Chloro-2-(4-methoxyphenyl)imidazo[1,2-a]pyridin-3-yl)-
N,N-dimethylmethanamine (3). Compound (2) (20.7 g, 80 mmol)
in acetic acid (250 mL) was slowly treated with aqueous dimethyl-
amine (40%, 127 mL) followed by aqueous formaldehyde (37%,
51 mL) and the mixture stirred at 55 °C for 20 h. After cooling,
the solution was concentrated, and the syrupy residue was dissolved
in a mixture of water (100 mL) and chloroform (200 mL). Aqueous
NaOH (10%) was added; until the pH was 12, the chloroform layer
separated and the aqueous phase was further extracted with
chloroform (2 × 50 mL). The combined chloroform solutions were
then extracted with 2N HCl (3 × 100 mL) and the extract partially
neutralized with NaOH (15 g) to pH 3. After standing at RT for
12 h, the precipitate of hydroxymethyl byproduct (2.0 g) was filtered
off. The filtrate was then basified to pH 12 with NaOH to afford
the product as a thick oil, which slowly solidified. The solid was
recrystallized from ethanol/water (18.9 g, 75%); mp 106-107 °C.
1H NMR (DMSO-d6) δ: 2.16 (br s, 6H), 3.33 (s, 2H), 3.81 (s, 3H),
3.90 (s, 2H), 7.03 (d, J ) 8.8 Hz, 2H), 7.31 (dd, J ) 9.6, 1.8 Hz,
1H), 7.62 (d, J ) 9.6 Hz, 1H), 7.79 (d, J ) 8.8 Hz, 2H), 8.63 (d,
J ) 1.8 Hz, 1H). 13C NMR (DMSO-d6) δ: 44.4, 51.3, 55.0, 113.8,
117.1, 117.7, 118.4, 123.4, 125.1, 126.4, 129.4, 142.2, 144.4, 158.8.
MS ES(+ve) m/z 316 (M + 1). Anal. (C17H18ClN3O) C, H, N.
1-(6-Chloro-2-(4-methoxyphenyl)imidazo[1,2-a]pyridin-3-yl)-
N,N,N-trimethylmethanammonium iodide (4). A solution of (3)
(16.5 g, 52 mmol), methyl iodide (24.5 g, 0.17 mol), and benzene
(200 mL) was stirred at RT for 48 h in darkness. The precipitated
solid was filtered, washed with benzene (20 mL), and dried in vacuo
to give a light-yellow solid (21.4 g, 89%); mp 164 °C (decomp).
1H NMR (DMSO-d6) δ: 2.89 (s, 9H), 3.84 (s, 3H), 5.21 (s, 2H),
7.08 (d, J ) 8.6 Hz, 2H), 7.50 (m, 1H), 7.77 (m, 1H), 7.79 (d, 2H,
J ) 8.6 Hz), 9.24 (br s, 1H). 13C NMR (DMSO-d6) δ: 51.4, 55.2,
56.4, 109.8, 114.3, 118.0, 120.5, 123.3, 125.8, 127.3, 130.0, 144.2,
149.0, 159.5. MS ES(+ve) m/z 330 (M). Anal. (C18H21ClIN3O) C,
H, N.
Figure 6. Percentage of unmetabolized [18F]15, [18F]8, [18F]18, and
18F]12 in plasma, brain cortex, and olfactory bulbs 0.25, 0.5, 1, and
4 h after injection, as measured using radio-HPLC.
[
possesses the requisite pharmacological profile for brain imaging
PBR expression in neurodegenerative disorders.
Experimental Section
General. N,N-Diethyl-2-(2-(4-methoxyphenyl)-5,7-dimethylpyra-
zolo[1,5-a]pyrimidin-3-yl)acetamide49 and 3-bromopropyl acetate50
were synthesized using literature procedures. 1-Bromo-2-fluoro-
ethane was purchased from Matrix Scientific (USA). Flumazenil,
PK11195, and Ro 5-4864 were obtained from Sigma-RBI. [3H]Flu-
mazenil (3.2 MBq/nmol) and [3H]PK11195 (2.8 MBq/nmol) were
obtained from Perkin-Elmer. [3H]Ro 5-4864 (3.1 MBq/nmol) was
obtained from American Radiolabeled Chemicals. [18F]HF was
produced by Cyclotek, Melbourne, Australia, via the 18O(p, n)
reaction. All other chemicals were purchased from Sigma-Aldrich
and used without further purification. Melting points were deter-
mined in open capillary tubes using a Gallenkamp melting point
apparatus and are uncorrected. NMR spectroscopy was performed
on a Bruker Advance DPX 400 operating at 400 MHz for 1H NMR
spectra and 100 MHz for 13C NMR spectra. Elemental analyses
were determined at the Campbell Microanalytical Laboratory,
Department of Chemistry, University of Otago, New Zealand. Low
resolution mass spectrometry (LRMS) was performed on a Micro-
mass ZMD quadrupole mass spectrometer, while high resolution

3708 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
Fookes et al.
2-(6-Chloro-2-(4-methoxyphenyl)imidazo[1,2-a]pyridin-3-yl)ac-
etamide (5). A solution of (4) (21.4 g, 47 mmol), KCN (15.2 g,
0.23 mol), and ethanol/H2O (1/1, 300 mL) was heated to reflux for
24 h. The solution was then cooled to RT and evaporated to dryness
under a stream of nitrogen. Water (50 mL) was added and the solid
filtered to collect a brown solid, which was dried in vacuo (13.9 g,
94%); mp 232-234 °C. 1H NMR (DMSO-d6) δ: 3.82 (s, 3H), 4.0
(s, 2H), 7.05 (d, J ) 8.6 Hz, 2H), 7.24 (br s, 1H), 7.30 (dd, J )
9.5, 1.9 Hz, 1H), 7.53 (d, J ) 9.5 Hz, 1H), 7.74 (d, 2H, J ) 8.6
Hz), 7.80 (br s, 1H), 8.61 (d, J ) 1.9 Hz, 1H). 13C NMR (DMSO-
d6) δ: 32.1, 56.8, 115.7, 117.7, 118.9, 120.3, 124.5, 126.5, 128.2,
130.8, 143.8, 145.2, 160.6, 172.0. MS ES(+ve) m/z 316 (M + 1).
Anal. (C16H14ClN3O2) C, H, N.
2-(6-Chloro-2-(4-(2-hydroxyethoxy)phenyl)imidazo[1,2-a]py-
ridin-3-yl)-N,N-diethylacetamide (10a). A mixture of (7) (300 mg,
0.8 mmol), K2CO3 (442 mg, 3.2 mmol), KI (20 mg, 0.12 mmol),
Bu4NI (6 mg, 0.016 mmol), and anhydrous DMF (2 mL) was
vigorously stirred for 2 min before 2-bromoethyl acetate (160 mg,
0.96 mmol) was added. The reaction was stirred at RT for 12 h,
diluted with ethyl acetate (10 mL) and washed with water (3 × 10
mL), followed by brine (10 mL). The organic extract was dried
(Na2SO4), filtered, and then concentrated in vacuo to give 2-(4-(6-
chloro-3-(2-(diethylamino)-2-oxoethyl)imidazo[1,2-a]pyridin-2-
yl)phenoxy)ethyl acetate (9a) as an orange solid (311 mg), which
was hydrolyzed without further purification. 1H NMR (DMSO-d6)
δ: 1.05 (t, J ) 7.0 Hz, 3H), 1.16 (t, J ) 7.0 Hz, 3H), 2.04 (s, 3H),
3.31 (q, J ) 7.0 Hz, 2H), 3.44 (q, J ) 7.0 Hz, 2H), 4.19 (s, 2H),
4.22 (m, 2H), 4.33 (m, 2H), 7.05 (d, J ) 8.8 Hz, 2H), 7.28 (dd, J
) 9.6, 2.0 Hz, 1H), 7.52 (d, J ) 8.8 Hz, 2H), 7.60 (d, J ) 9.6, 2.0
Hz, 1H), 8.50 (d, J ) 2.0 Hz, 1H). The orange solid (9a) was
dissolved in methanol (2 mL), and Cs2CO3 (443 mg, 1.36 mmol)
in H2O (1 mL) was added. After 48 h of stirring, at RT a precipitate
had formed, which was collected by filtration and washed with
water. The solid was recrystallized from ethyl acetate/hexane (1:
1) to give the product as fine colorless needles (241 mg, 79%); mp
2-(6-Chloro-2-(4-hydroxyphenyl)imidazo[1,2-a]pyridin-3-yl)ace-
tic acid hydrobromide hydrate (6). Compound (5) (13.7 g, 43
mol) was added to a solution containing 48% HBr (200 mL) and
acetic acid (100 mL) and heated to 110 °C for 48 h. The solution
was cooled to RT forming a light-brown precipitate. The solid was
filtered, washed with acetic acid, and dried in vacuo (10.8 g, 63%);
1
mp 260 °C (decomp). H NMR (DMSO-d6) δ: 4.26 (s, 2H), 7.03
(d, J ) 8.0 Hz, 2H), 7.54 (d, J ) 8.0 Hz, 2H), 7.94 (m, 2H), 9.28
(s, 1H), 10.15 (br s, 1H). 13C NMR (DMSO-d6) δ: 30.9, 114.9,
118.0, 118.3, 118.8, 125.1, 127.0, 131.6, 134.8, 136.8, 139.6 × 2,
1
201-202 °C. H NMR (DMSO-d6) δ: 1.07 (t, J ) 7.1 Hz, 3H),
1.17 (t, J ) 7.1 Hz, 3H), 3.34 (q, J ) 7.1 Hz, 2H), 3.45 (q, J ) 7.1
Hz, 2H), 3.73 (dt, J ) 5.3, 5.0 Hz, 2H), 4.03 (t, J ) 5.0 Hz, 2H),
4.20 (s, 2H), 4.87 (t, J ) 5.3 Hz, 1H), 7.03 (d, J ) 8.8 Hz, 2H),
7.29 (dd, J ) 9.5, 2.0 Hz, 1H), 7.52 (d, J ) 8.8 Hz, 2H), 7.61 (dd,
J ) 9.5, 0.7 Hz, 1H), 8.50 (dd, J ) 2.0, 0.7 Hz, 1H). 13C NMR
(DMSO-d6) δ: 13.1, 14.1, 28.7, 39.9, 41.7, 59.6, 69.6, 114.6, 116.4,
117.1, 118.4, 123.0, 124.7, 126.5, 129.0, 142.2, 143.5, 158.4, 167.0.
MS ES(+ve) m/z 402 (M + 1). Anal. (C21H24ClN3O3) C, H, N.
2-(6-Chloro-2-(4-(3-hydroxypropoxy)phenyl)imidazo[1,2-a]py-
ridin-3-yl)-N,N-diethylacetamide (10b). Compound (7) (268 mg,
0.75 mmol) was treated with 3-bromopropyl acetate (204 mg, 1.13
mmol), Bu4NI (7 mg, 0.019 mmol), KI (19 mg, 0.11 mmol), and
K2CO3 (206 mg, 1.5 mmol) in anhydrous DMF (1.5 mL) as
described in the synthesis of (10a) to give 3-(4-(6-chloro-3-(2-
(diethylamino)-2-oxoethyl)imidazo[1,2-a]pyridin-2-yl)phenoxy)pro-
pyl acetate (9b) as a pale-yellow solid (346 mg) that was used
161.1, 171.5. MS ES(+ve) m/z 303 (M
(C15H11ClN2O3 ·HBr.H2O) C, H, N.
+ 1). Anal.
2-(6-Chloro-2-(4-hydroxyphenyl)imidazo[1,2-a]pyridin-3-yl)-
N,N-diethylacetamide (7). A mixture of (6) (1.15 g, 3.0 mmol),
HOBT (405 mg, 3.0 mmol), DEA (372 µL, 3.6 mmol), and DIPEA
(2.09 mL, 12.0 mmol) was dissolved with stirring in anhydrous
DMF (8 mL). After 1 min, EDCI (748 mg, 3.90 mmol) was added
and stirred for a further 18 h at RT. Water (60 mL) and acetic acid
(0.5 mL) were then added, stirred for 1 min, and then placed at 4
°C for 6 h to give a precipitate which was filtered, washed with
H2O (20 mL), and dried in vacuo. Anhydrous DMF (3 mL) and
DEA (0.5 mL) were then added to the crude product and heated to
100 °C for 2 h until the DEA was evaporated from the solution.
After cooling to 80 °C, ethyl acetate (10 mL) was added rapidly
and the mixture cooled to 5 °C for 4 h to form a white crystalline
solid, which was washed with ethyl acetate (3 mL) and dried in
vacuo (816 mg, 76%) as white needles; mp 226-228 °C. 1H NMR
(DMSO-d6) δ: 1.06 (t, J ) 7.1 Hz, 3H), 1.16 (t, J ) 7.1 Hz, 3H),
3.32 (q, J ) 7.1 Hz, 2H), 3.44 (q, J ) 7.1 Hz, 2H), 4.18 (s, 2H),
6.83 (d, J ) 8.8 Hz, 2H), 7.26 (dd, J ) 9.4, 1.8 Hz, 1H), 7.41 (d,
J ) 8.8 Hz, 2H), 7.57 (d, J ) 9.4 Hz, 1H), 8.49 (d, J ) 1.8 Hz,
1H), 9.61 (br s, 1H). 13C NMR (DMSO-d6) δ: 13.0, 14.1, 28.7,
39.9, 41.6, 115.4, 116.1, 117.0, 118.3, 122.9, 124.5, 125.0, 129.1,
142.1, 143.9, 157.2, 167.1. MS ES(+ve) m/z 358 (M + 1). Anal.
(C19H20ClN3O2) C, H, N.
1
without further purification. H NMR (DMSO-d6) δ: 1.06 (t, J )
7.0 Hz, 3H), 1.17 (t, J ) 7.0 Hz, 3H), 2.01 (s, 3H), 2.04 (m, 2H),
3.32 (q, J ) 7.0 Hz, 2H), 3.44 (q, J ) 7.0 Hz, 2H), 4.09 (t, J ) 6.4
Hz, 2H), 4.17 (t, J ) 6.4 Hz, 2H), 4.21 (s, 2H), 7.03 (d, J ) 8.8
Hz, 2H), 7.28 (dd, J ) 9.6, 2.0 Hz, 1H), 7.53 (d, J ) 8.8 Hz, 2H),
7.60 (d, J ) 9.6 Hz, 1H), 8.52 (d, J ) 2.0 Hz, 1H). Compound
(9b) was treated with Cs2CO3 (534 mg, 1.64 mmol) as described
in the synthesis of (10a). Recrystallization of the resulting solid
from ethanol/water gave colorless flakes of the title compound (277
mg, 89%); mp 119-120 °C. 1H NMR (DMSO-d6) δ: 1.07 (t, J )
7.0 Hz, 3H), 1.17 (t, J ) 7.0 Hz, 3H), 1.88 (m, 2H), 3.33 (q, J )
7.0 Hz, 2H), 3.45 (q, J ) 7.0 Hz, 2H), 3.59 (t, J ) 6.0 Hz, 2H),
4.09 (t, J ) 6.0 Hz, 2H), 4.22 (s, 2H), 4.58 (br s, 1H), 7.03 (d, J
) 8.8 Hz, 2H), 7.29 (dd, J ) 9.6, 2.0 Hz, 1H), 7.53 (d, J ) 8.8
Hz, 2H), 7.61 (d, J ) 9.6 Hz, 1H), 8.52 (d, J ) 2.0 Hz, 1H). 13C
NMR (DMSO-d6) δ: 13.0, 14.1, 28.6, 32.0, 39.7, 41.6, 57.2, 64.6,
114.4, 116.3, 117.0, 118.3, 122.9, 124.6, 126.4, 128.9, 142.1, 143.4,
158.3, 166.7. MS ES(+ve) m/z 416 (M + 1).
2-(6-Chloro-2-(4-(2-fluoroethoxy)phenyl)imidazo[1,2-a]pyri-
din-3-yl)-N,N-diethylacetamide (8). A mixture of (7) (357 mg,
1.0 mmol), 1-bromo-2-fluoroethane (190 mg, 1.5 mmol), Bu4NI
(7.5 mg, 0.02 mmol), KI (25 mg, 0.15 mmol), and K2CO3 (207
mg, 1.5 mmol) in anhydrous DMF (2 mL) was stirred at 55 °C for
48 h before it was diluted with ethyl acetate (20 mL) and partitioned
with water (10 mL). The organic layer was washed with water (3
× 5 mL), cold 2N NaOH (5 mL), water (3 × 5 mL), brine (5 mL),
and then dried over Na2SO4. The dried organic solution was passed
through a silica plug (1 × 0.5 cm) and elution continued with more
ethyl acetate (10 mL). Evaporation of the solvent gave a yellow
residue that was crystallized from ethyl acetate/hexane to give the
title compound as pale-yellow flakes (287 mg, 71%); mp 140-142
°C. 1H NMR (DMSO-d6) δ: 1.06 (t, J ) 7.0 Hz, 3H), 1.18 (t, J )
7.0 Hz, 3H), 3.34 (q, J ) 7.0 Hz, 2H), 3.46 (q, J ) 7.0 Hz, 2H),
4.22 (s, 2H), 4.30 (dm, J ) 30.1 Hz, 2H), 4.78 (dm, J ) 47.6 Hz,
2H), 7.08 (d, J ) 9.1 Hz, 2H), 7.29 (dd, J ) 9.4, 2.0 Hz, 1H), 7.55
(d, J ) 9.1 Hz, 2H), 7.62 (d, J ) 9.4 Hz, 1H), 8.52 (d, J ) 2.0 Hz,
1H). 13C NMR (DMSO-d6) δ: 13.0, 14.1, 28.7, 39.9, 41.7, 67.1 (d,
J ) 18.8 Hz), 82.3 (d, J ) 166.8 Hz), 114.7, 116.5, 117.1, 118.4,
123.0, 124.7, 127.0, 129.0, 142.2, 143.3, 157.9, 167.0. MS ES(+ve)
m/z 404 (M + 1), 442 (M + K). Anal. (C21H23ClFN3O2) C, H, N.
2-(4-(6-Chloro-3-(2-(diethylamino)-2-oxoethyl)imidazo[1,2-
a]pyridin-2-yl)phenoxy)ethyl 4-methylbenzenesulfonate (11a).
To a solution of (10a) (120 mg, 0.3 mmoL) in ethanol-free CHCl3
(5 mL) at 40 °C was added TMHD (206 mg, 1.2 mmol), followed
by p-toluenesulfonyl chloride (114 mg, 0.6 mmol). The solution
was then cooled to RT and stirring continued for 15 h before it
was washed with water (3 × 1 mL), aqueous tartaric acid (10%, 1
mL), and brine (5 mL). The organic solution was dried (Na2SO4),
filtered, and concentrated in vacuo to give a light-yellow foam (102
mg, 60%). 1H NMR (CD3CN) δ: 1.15 (t, J ) 7.0 Hz, 3H), 1.20 (t,
J ) 7.0 Hz, 3H), 2.46 (s, 3H), 3.43 (m, 4H), 4.13 (br s, 2H), 4.23
(m, 2H), 4.39 (m, 2H), 6.94 (d, J ) 8.8 Hz, 2H), 7.26 (dd, J ) 9.6,
1.8 Hz, 1H), 7.47 (d, J ) 8.8 Hz, 2H), 7.55 (d, J ) 9.6 Hz, 1H),
7.57 (d, J ) 8.5 Hz, 2H), 7.84 (d, J ) 8.5 Hz, 2H), 8.52 (d, J )

[
18F]Imidazo[1,2-a]pyridines and [18F]Pyrazolo[1,5-a]pyrimidines
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3709
1.8 Hz, 1H). 13C NMR (DMSO-d6) δ: 13.0, 14.0, 21.0, 28.5, 39.8,
41.6, 65.3, 69.0, 114.6, 116.6, 116.7, 118.9, 123.2, 125.4, 126.1,
127.6, 128.9, 130.0, 132.2, 141.6, 142.2, 144.9, 157.5, 166.8. MS
ES(+ve) m/z 556 (M + 1).
108.0, 115.1, 124.2, 129.2, 144.4, 147.1, 153.8, 157.1, 157.6, 169.0.
MS ES(+ve) m/z 353 (M + 1). Anal. (C20H24N4O2) C, H, N.
2-(2-(4-(2-Fluoroethoxy)phenyl)-5,7-dimethylpyrazolo[1,5-
a]pyrimidin-3-yl)-N,N-diethylacetamide (15). A mixture of (14)
(176 mg, 0.5 mmol), 1-bromo-2-fluoroethane (49 µL, 0.65 mmol),
Bu4NI (3.7 mg, 0.01 mmol), K2CO3 (76 mg, 0.55 mmol), KI (12.5
mg 0.075 mmol), and anhydrous DMF (1 mL) was treated as
described in the synthesis of (8). The product was recrystallized
from ethyl acetate/hexane to give the title compound as a white
3-(4-(6-Chloro-3-(2-(diethylamino)-2-oxoethyl)imidazo[1,2-
a]pyridin-2-yl)phenoxy)propyl 4-methylbenzenesulfonate (11b).
. Compound (10b) (100 mg, 0.24 mmol) was treated with TMHD
(86 mg, 0.5 mmol) and p-toluenesulfonyl chloride (92 mg, 0.48
mmol) as described for the synthesis of (11a). The resulting orange
solid was recrystallized from ethyl acetate/hexane to give colorless
needles (81 mg, 59%); mp 150-152 °C. 1H NMR (CD3CN) δ:
1.15 (t, J ) 7.0 Hz, 3H), 1.23 (t, J ) 7.0 Hz, 3H), 2.08 (m, 2H),
2.38 (s, 3H), 3.38 (q, J ) 7.0 Hz, 2H), 3.42 (q, J ) 7.0 Hz, 2H),
3.96 (t, J ) 5.8 Hz, 2H), 4.10 (s, 2H), 4.22 (t, J ) 5.8 Hz, 2H),
6.88 (d, J ) 8.8 Hz, 2H), 7.25 (dd, J ) 9.6, 1.8 Hz, 1H), 7.33 (d,
J ) 8.0 Hz, 2H), 7.53 (d, J ) 8.8 Hz, 2H), 7.54 (d, J ) 9.6 Hz,
1H), 7.75 (d, J ) 8.0 Hz, 2H), 8.24 (d, J ) 1.8 Hz, 1H). 13C NMR
(DMSO-d6) δ: 13.0, 14.1, 21.0, 28.1, 28.6, 37.8, 41.6, 63.1, 67.5,
114.5, 116.4, 117.0, 118.6, 123.0, 125.4, 126.5, 127.5, 128.9, 130.1,
132.1, 142.3, 143.2, 144.8, 157.8, 167.0. MS ES(+ve) m/z 570 (M
+ 1).
2-(6-Chloro-2-(4-(3-fluoropropoxy)phenyl)imidazo[1,2-a]py-
ridin-3-yl)-N,N-diethylacetamide (12). To a stirred suspension of
(10b) (62.3 mg, 0.15 mmol) in anhydrous acetonitrile (0.5 mL)
was added DIPEA (158 µL, 0.9 mmol), PBSF (52 µL, 0.3 mmol),
and TEA ·3HF (50 µL, 0.3 mmol). The mixture was warmed to 50
°C to dissolve all the solids, and the solution was stirred at 40 °C
for 2 h and RT for 12 h. Ethyl acetate (3 mL) was then added and
the mixture washed with water (2 × 1 mL), brine (2 × 1 mL),
dried over Na2SO4, and evaporated to give a glassy residue, which
was purified by preparative HPLC on a Grace Alltima C18 column
(10 µm, 22 mm × 250 mm), eluting with acetonitrile/H2O +
0.1%TFA (40/60) at 10 mL/min. The major peak at 21.5 min was
collected, evaporated to dryness, and redissolved in ethyl acetate
(3 mL), washed with saturated NaHCO3 solution, and dried
(Na2SO4). The organic phase was concentrated in vacuo to give a
white solid that was recrystallized from ethyl acetate/hexane to give
the title compound as colorless needles (27.3 mg, 44%); mp
160-162 °C. 1H NMR (CD3CN) δ: 1.13 (t, J ) 7.1 Hz, 3H), 1.18
(t, J ) 7.1 Hz, 3H), 2.20 (m, 2H), 3.40 (q, J ) 7.1 Hz, 2H), 3.44
(q, J ) 7.1 Hz, 2H), 4.11 (s, 2H), 4.17 (t, J ) 6.2 Hz, 2H), 4.67
(dt, J ) 47.3, 5.9 Hz, 2H), 7.05 (d, J ) 8.8 Hz, 2H), 7.24 (dd, J
) 9.5, 2.0 Hz, 1H), 7.54 (dd, J ) 9.5, 0.8 Hz, 1H), 7.58 (d, J )
8.8 Hz, 2H), 8.24 (dd, J ) 2.0, 0.8 Hz, 1H). 13C NMR (CD3CN)
δ: 13.3, 14.5, 30.2, 29.9 (d, J ) 19.9 Hz), 41.1, 42.9, 64.7 (d, J )
5.4 Hz), 80.9 (d, J ) 161.4 Hz), 115.6, 117.3, 118.1, 120.1, 123.6,
125.6, 128.0, 130.3, 143.8, 145.1, 159.6, 168.0. MS ES(+ve) m/z
418 (M + 1). Anal. (C22H25ClFN3O2) C, H, N.
1
solid (139 mg, 70%); mp 148-150 °C. H NMR (DMSO-d6) δ:
1.02 (t, J ) 7.0 Hz, 3H), 1.18 (t, J ) 7.0 Hz, 3H), 2.48 (s, 3H),
2.69 (s, 3H), 3.28 (q, J ) 7.0 Hz, 1H), 3.52 (q, J ) 7.0 Hz, 1H),
3.83 (s, 2H), 4.29 (dm J ) 30.1 Hz, 2H), 4.76 (dm, J ) 47.6 Hz,
2H), 6.84 (s, 1H), 7.06 (d, J ) 8.8 Hz, 2H), 7.72 (d, J ) 8.8 Hz,
2H). 13C NMR (DMSO-d6) δ: 13.1, 14.2, 16.3, 24.2, 27.5, 39.8,
41.6, 67.1 (d, J ) 18.9 Hz), 82.1 (d, J ) 166.0 Hz), 100.6, 108.3,
114.5, 126.3, 129.3, 144.5, 147.2, 153.4, 157.3, 158.3, 169.0. MS
ES(+ve) m/z 399 (M + 1). Anal. (C22H27FN4O2) C, H, N.
N,N-Diethyl-2-(2-(4-(2-hydroxyethoxy)phenyl)-5,7-dimeth-
ylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide (16a). A mixture of
(14) (264 mg, 0.75 mmol), 2-bromoethyl acetate (124 µL, 1.3
mmol), K2CO3 (310 mg, 2.25 mmol), KI (19 mg, 0.11 mmol),
Bu4NI (7 mg), and anhydrous DMF (1.5 mL) was treated as
described for the synthesis of (10a). The resulting solid was
recrystallized from ethanol/water to give white flakes of the title
1
compound. (202 mg, 68%); mp 148-150 °C H NMR (DMSO-
d6) δ: 1.02 (t, J ) 7.0 Hz, 3H), 1.18 (t, J ) 7.0 Hz, 3H), 2.49 (s,
3H), 2.70 (d, J ) 0.8 Hz, 3H), 3.31 (q, J ) 7.0 Hz, 2H), 3.52 (q,
J ) 7.0 Hz, 2H), 3.74 (dt, J ) 5.5, 5.0 Hz, 2H), 3.84 (s, 2H), 4.04
(t, J ) 5.0 Hz, 2H), 4.88 (t, J ) 5.5 Hz, 1H), 6.84 (q, J ) 0.8 Hz,
1H), 7.06 (d, J ) 9.0 Hz, 2H), 7.72 (d, J ) 9.0 Hz, 2H). 13C NMR
(DMSO-d6) δ: 13.0, 14.1, 16.2, 24.1, 27.4, 39.8, 41.5, 59.5, 69.5,
100.3, 108.2, 114.3, 125.7, 129.1, 144.4, 147.1, 153.4, 157.2, 158.8,
168.9. MS ES(+ve) m/z 397 (M + 1). Anal. (C22H28N4O3) C,
H, N.
N,N-Diethyl-2-(2-(4-(3-hydroxypropoxy)phenyl)-5,7-dimeth-
ylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide (16b). A mixture of
(14) (352 mg, 1.0 mmol), 3-bromopropyl acetate (185 µL, 1.5
mmol), K2CO3 (414 mg, 3.0 mmol), KI (25 mg, 0.15 mmol), Bu4NI
(9 mg), and anhydrous DMF was treated as described for the
synthesis of (10a). The resulting solid was recrystallized from
ethanol/water to give pale-yellow needles of the title compound
(282 mg, 69%); mp 116-118 °C. 1H NMR (DMSO-d6) δ: 1.02 (t,
J ) 7.0 Hz, 3H), 1.18 (t, J ) 7.0 Hz, 3H), 1.88 (m, 2H), 2.49 (s,
3H), 2.70 (d, J ) 0.9 Hz, 3H), 3.29 (q, J ) 7.0 Hz, 2H), 3.52 (q,
J ) 7.0 Hz, 2H), 3.57 (td, J ) 6.4, 5.3 Hz, 2H), 3.84 (s, 2H), 4.09
(t, J ) 6.4 Hz, 2H), 4.56 (t, J ) 5.3 Hz, 1H), 6.84 (q, J ) 0.8 Hz,
1H), 7.02 (d, J ) 8.8 Hz, 2H), 7.69 (d, J ) 8.8 Hz, 2H). 13C NMR
(DMSO-d6) δ: 13.0, 14.1, 16.2, 24.1, 27.4, 32.0, 39.7, 41.5, 57.2,
64.6, 100.4, 108.2, 114.3, 125.7, 129.2, 144.4, 147.1, 153.4, 157.2,
158.8, 169.0. MS ES(+ve) m/z 411 (M + 1). Anal. (C23H30N4O3)
C, H, N.
N,N-Diethyl-2-(2-(4-hydroxyphenyl)-5,7-dimethylpyrazolo[1,5-
a]pyrimidin-3-yl)acetamide (14). N,N-Diethyl-2-(2-(4-methox-
yphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide48
(13) (730 mg, 2.0 mmol) and Bu4NI (810 mg, 2.2 mmol) were
dissolved in anhydrous dichloromethane (10 mL) under N2. The
mixture was cooled in an ethanol/liquid N2 bath (-70 °C) and
treated with BCl3 in hexanes (9.0 mL, 9.0 mmol) over 5 min with
vigorous stirring. After 5 min, the reaction was placed in an ice
bath, and the mixture stirred for a further 2.5 h. Ice and water were
added to quench the reaction and the mixture shaken vigorously
with water (50 mL), chloroform (50 mL), and ethanol (5 mL). Solid
NaHCO3 was added in small portions to neutralize the acids from
the hydrolysis of borate complexes. The organic layer was separated
and the aqueous phase was further extracted with additional
chloroform (2 × 20 mL). The combined organic extracts were
washed with brine (50 mL), dried over Na2SO4, and evaporated to
give a crystalline semisolid. This was slurried with acetonitrile (4
mL) and the colorless crystals collected by filtration (620 mg,
2-(4-(3-(2-(Diethylamino)-2-oxoethyl)-5,7-dimethylpyrazolo[1,5-
a]pyrimidin-2-yl)phenoxy)ethyl 4-methylbenzenesulfonate (17a).
Compound (16a) (100 mg, 0.25 mmol) was treated with TMHD
(150 µL, 0.7 mmol) and p-toluenesulfonyl chloride (96 mg, 0.5
mmol) in anhydrous acetonitrile (3 mL) as described in the synthesis
of (11a). The resulting residue was evaporated in vacuo to give a
light-brown foam of the title compound (120 mg, 88%). 1H NMR
(CD3CN) δ: 1.11 (t, J ) 7.0 Hz, 3H), 1.20 (t, J ) 7.0 Hz, 3H),
2.45 (s, 3H), 2.54 (s, 3H), 2.75 (d, J ) 0.9 Hz, 3H), 3.40 (q, J )
7.0 Hz, 2H), 3.51 (q, J ) 7.0 Hz, 2H), 3.90 (s, 2H), 4.19 (m, 2H),
4.39 (m, 2H), 6.51 (q, J ) 0.9 Hz, 1H), 6.86 (d, J ) 8.8 Hz, 2H),
7.35 (d, J ) 8.2 Hz, 2H), 7.75 (d, J ) 8.8 Hz, 2H), 7.83 (d, J )
8.2 Hz, 2H). 13C NMR (DMSO-d6) δ: 13.0, 14.1, 16.1, 21.0, 24.1,
27.4, 40.1, 41.5, 65.3, 69.0, 100.5, 108.2, 114.3, 126.3, 127.6, 129.1,
130.0, 132.2, 144.4, 144.9, 147.1, 153.3, 157.2, 157.8, 168.9. MS
ES(+ve) m/z 551 (M + 1). Anal. (C29H34N4O5S) C, H, N.
1
88%);mp 235-238 °C. H NMR (DMSO-d6) δ: 1.02 (t, J ) 7.0
Hz, 3H), 1.17 (t, J ) 7.0 Hz, 3H), 2.48 (s, 3H), 2.69 (s, 3H), 3.30
(q, J ) 7.0 Hz, 2H), 3.51 (q, J ) 7.0 Hz, 2H), 3.82 (s, 2H), 6.82
(s, 1H), 6.83 (d, J ) 8.8 Hz, 2H), 7.58 (d, J ) 8.8 Hz, 2H). 13C
NMR (DMSO-d6) δ: 13.0, 14.1, 16.2, 24.1, 27.4, 40.4, 14.5, 100.3,
3-(4-(3-(2-(Diethylamino)-2-oxoethyl)-5,7-dimethylpyrazolo[1,5-
a]pyrimidin-2-yl)phenoxy)propyl 4-methylbenzenesulfonate

3710 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
Fookes et al.
(17b). Compound (16b) (100 mg, 0.24 mmol) was treated with
TMHD (204 µL, 0.96 mmol) and p-toluenesulfonyl chloride (92
mg, 0.48 mmol) in anhydrous acetonitrile (5 mL) as described in
the synthesis of (11a). The resulting solid was recrystallized from
ethyl acetate to give the title compound as yellow crystals (114
mg, 84%); mp 141-143 °C. 1H NMR (CDCl3) δ: 1.11 (t, J ) 7.0
Hz, 3H), 1.26 (t, J ) 7.0 Hz, 3H), 2.12 (m, 2H), 2.39 (s, 3H), 2.49
(s, 3H), 2.74 (d, J ) 0.9 Hz, 3H), 3.39 (q, J ) 7.0 Hz, 2H), 3.57
(q, J ) 7.0 Hz, 2H), 3.92 (s, 2H), 4.01 (t, J ) 5.8 Hz, 2H), 4.26
(t, J ) 6.1 Hz, 2H), 6.71 (q, J ) 0.9 Hz, 1H), 6.90 (d, J ) 8.8 Hz,
2H), 7.37 (d, J ) 8.2 Hz, 2H), 7.74 (d, J ) 8.8 Hz, 2H), 7.79 (d,
J ) 8.2 Hz, 2H). 13C NMR (DMSO-d6) δ: 13.0, 14.1, 16.2, 21.0,
24.1, 27.4, 28.0, 40.1, 41.5, 63.1, 67.5, 100.4, 108.2, 114.3, 126.0,
127.4, 129.1, 130.0, 132.1, 144.4, 144.8, 147.1, 153.4, 157.2, 158.2,
168.9. MS ES(+ve) m/z 565 (M + 1). Anal. (C30H36N4O5S) C,
H, N.
Nonspecific binding was determined with flumazenil (20 µM). In
all cases, incubations were terminated by rapid filtration through
Whatman GF/B glass fiber using a Brandel cell harvester. Filters
were washed rapidly 3 times with 5 mL of ice-cold 50 mM Tris/
HCl at pH 7.4. Filters were counted in a ꢀ-scintillation counter
(Packard) to measure the amount of bound radioactivity. The IC50
of the compounds for the PBR and CBR were calculated using
Kell 6 software. The IC50 values were converted to the inhibition
constant, Ki, usingthe Cheng-Prussof equation and radioligand
affinity values, [3H]PK11195 (Kd ) 2 nM), [3H]Ro 5-4864 (Kd )
8 nM), and [3H]flumazenil (Kd )1 nM).6,53
Biodistribution Studies. Animal experiments were performed
in compliance with the NHMRC Australian Code of Practice for
the care and use of animals for scientific purposes. [18F]PBR
radiotracers (1 MBq) were administered to 8-10 week old male
SD rats via tail vein injection in a volume of 0.1 mL. At 0.25, 0.5,
1, and 4 h postinjection of the radiotracer, groups of rats (n ) 4)
were sacrificed by CO2 administration followed by cervical fracture.
Peripheral organs, (including liver, spleen, femur, thyroid, pancreas,
lung, heart, kidney, and adrenals), blood, olfactory bulbs, and the
remainder of the brain were removed. To investigate the uptake of
radioactivity in the bone, bone marrow was also extracted from
the medullar cavity of the femur and was analyzed in comparison
to frontal skull bone. Samples of organs and tissues were weighed
and the radioactivity was measured using an automated gamma
counter. The percent injected dose (%ID) was calculated by
comparison to a diluted standard solution derived from the initial
injected solution. Radioactivity concentrations were expressed as
percent of injected dose per gram of wet tissue (%ID/g).
Competition Studies. In saturation experiments, rats were
injected intravenously with 1 mg/kg of unlabeled PBR compounds
5 min prior to injection of the respective [18F]PBR compound (1
MBq). In competition experiments, the specificity of the [18F]PBR
was investigated by injecting in rats 5 min prior to the F-18
radioligands, 1 mg/kg flumazenil, Ro 5-4864, or PK11195 dissolved
in dimethylsulfoxide/saline (1/1). The groups of rats (n ) 4) were
sacrificed at 1 h post injection and tissues were handled as described
for the biodistribution studies. Radioactive concentrations in organs
of treated animals were compared to the controls. Statistical
significance was evaluated using one-way ANOVA. The criteria
for significance were p < 0.01 and p < 0.05.
Metabolite Studies. The determination of unchanged radiotracer
in the plasma, frontal cortex, and olfactory bulbs was performed
by radio HPLC analysis, at 0.25, 0.5, 1, and 4 h after injection of
30 MBq of [18F]PBR compounds. Plasma (100 µL) was mixed with
200 µL of water containing the PBR compound (10 nmol in 10 µL
of CH3OH) and KF (200 nmol) for direct injection onto the HPLC.
Samples of brain tissue (100 mg) in 0.4 mL acetonitrile/water (1:
1) containing unlabeled PBR compound and KF was exposed for
2 min to an ultrasonic probe and the mixture centrifuged at 3000
rpm for 10 min. The supernatant was collected and diluted with
1.5 mL of H2O for HPLC analysis. The radioactivity of the
precipitate was measured to quantify the acetonitrile/water extraction
efficiency.
N,N-Diethyl-2-(2-(4-(3-fluoropropoxy)phenyl)-5,7-dimeth-
ylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide (18). Compound (16b)
(82 mg, 0.2 mmol) in anhydrous acetonitrile (0.8 mL) was treated
with DIPEA (210 µL, 1.2 mmol), TEA.3HF (67 µL, 0.4 mmol),
and PBSF (67 µL, 0.4 mmol) as described in the synthesis of (12).
The resulting product was purified by preparative HPLC using a
Grace Alltima C18 column (10 µm, 22 mm × 250 mm), eluting
with acetonitrile/H2O + 0.1%TFA (70/30), at 10 mL/min. The
major peak at 15 min was collected and treated as described in the
synthesis of (12) to give large white needles (48.6 mg, 59%); mp
1
106-108 °C. H NMR (DMSO-d6) δ: 1.02 (t, J ) 7.1 Hz, 3H),
1.18 (t, J ) 7.1 Hz, 3H), 2.14 (dm, J ) 25.7 Hz, 2H), 2.49 (s, 3H),
2.69 (d, J ) 0.8 Hz, 3H), 3.29 (q, J ) 7.1 Hz, 2H), 3.52 (q, J )
7.1 Hz, 2H), 3.84 (s, 2H), 4.13 (t, J ) 6.3 Hz, 2H), 4.58 (dm, J )
47.3 Hz, 2H), 6.84 (q, J ) 0.8 Hz, 1H), 7.05 (d, J ) 8.9 Hz, 2H),
7.70 (d, J ) 8.9 Hz, 2H). 13C NMR (DMSO-d6) δ: 13.1, 14.2,
16.2, 24.2, 27.5, 29.7 (d, J ) 19.6 Hz), 39.8, 41.6, 63.6 (d, J ) 5.6
Hz), 80.8 (d, J ) 161.6 Hz), 100.6, 108.3, 114.4, 126.1, 129.3,
144.6, 147.2, 153.5, 157.3, 158.6, 169.1. MS ES(+ve) m/z 413 (M
+ 1). Anal. (C23H29FN4O2) C, H, N.
Radiochemistry General Procedure. An aqueous [18F]fluoride
solution (6-7 GBq) was added to a 10 mL vial containing
anhydrous acetonitrile (1 mL), K2.2.2 and K2CO3. The solvent was
evaporated under a stream of nitrogen at 100 °C under vacuum.
This azeotropic drying was repeated twice by further addition of
anhydrous acetonitrile (2 × 1 mL). The p-toluenesulfonyl precursor
(11a), (11b), (17a), or (17b) (2 mg) was dissolved in anhydrous
acetonitrile (1 mL) and added to the dried K[18F]-K2.2.2 ·K2CO3.
complex. The reaction was stirred and heated at 100 °C for 5 min
before the reaction mixture was diluted with mobile phase (500
µL) and purified by preparative reverse phase chromatography
(Table 1). The collected radioactive peak was evaporated in vacuo
and formulated to a concentration of 1 MBq/100 µL of saline
containing less than 1% ethanol for biological studies.
Lipophilicity. The lipophilicity of 15, 8, 12, and 18 was assessed
using RP-HPLC by determining the log P7.5 value using literature
procedures.51 Samples, dissolved in methanol, were analyzed using
a Waters, Xterra C18 column (5 µm, 4.6 mm × 150 mm) with a
mobile phase consisting of methanol/phosphate buffer (0.1 M, pH
7.5), 60/40 and a flow rate of 1 mL/min. The log P7.5 of a studied
compound was estimated by a comparison of its retention time to
that of compounds of known log P value.
In Vitro Binding Assays. Kidney and brain cortical membranes
were prepared as described previously.52 The inhibition constant
(IC50) of the ligands for PBRs was determined by incubating, in
triplicate, aliquots (0.3 mL) of diluted kidney membrane preparation
(100-150 µg, protein) at 4 °C for 1 h with 7 concentrations of
each studied compound (10-10 to 10-6 M) and [3H]PK11195 (2
nM) or [3H]Ro 5-4864 (1 nM) in a final volume of 0.5 mL.
Nonspecific binding was determined with PK11195 (10 µM).
Similarly, the IC50 of the ligands for CBR was determined by
incubating aliquots (0.3 mL), in triplicate, of diluted brain cortex
membrane preparation (100-150 µg, protein) at 25 °C for 45 min
with 7 concentrations of each studied compound (10-10 to 10-5
M) and of [3H]flumazenil (2 nM) in a final volume of 0.5 mL.
Radio-HPLC analysis was performed by adaptation of a literature
procedure54,55 using a precolumn (Waters Oasis HLB, 25 µm; 3.9
× 20 mm) and a reverse phase HPLC column (Waters X-terra,
C18, 5 µm; 3.0 mm × 100 mm) in series, with a switching valve
between columns. The precolumn was used to wash protein residue
while trapping all nonpolar activity for subsequent gradient separa-
tion using the HPLC column. In the first step, the precolumn was
washed with water for 5 min at 1 mL/min, and the fraction
containing protein residue and nonlipophilic compounds bypassed
the HPLC column by a switching valve and was sent through the
UV and radioactivity detectors using a T-piece that was fitted post
HPLC column. In the second step, the solvent direction was changed
at 5 min to the HPLC column. Both columns in series were then
eluted by gradient (10/90 to 90/10, CH3CN/sodium acetate 0.05
M, pH 5.5) over 8 min at 1 mL/min. The radioactivity peak
corresponding to the authentic PBR compound was compared to

[
18F]Imidazo[1,2-a]pyridines and [18F]Pyrazolo[1,5-a]pyrimidines
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3711
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the total activity registered in the radiochromatogram to give the
fraction of unchanged ligand in the sample.
Acknowledgment. We gratefully acknowledge Donghai
Jiang and Allira Schuster-Woodhouse for their support in the
HPLC purification of the compounds and Tim Jackson for his
QC support and log P measurements. We thank Thomas
Bourdier for his excellent assistance in automated radiosynthesis
and metabolite studies.
Supporting Information Available: Table of microanalytical
data and HPLC purity test of target compounds 3-18. This material
is available free of charge via the Internet at http://pubs.acs.org.
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