Synthesis and structure-activity relationships of 5,6,7-substituted pyrazolopyrimidines: Discovery of a novel TSPO PET ligand for cancer imaging

Article abstract of DOI:10.1021/jm4001874

Focused library synthesis and structure-activity relationship development of 5,6,7-substituted pyrazolopyrimidines led to the discovery of 2-(5,7-diethyl-2-(4-(2-fluoroethoxy)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)-N, N-diethylacetamide (6b), a novel transl

Full text of DOI:10.1021/jm4001874

Brief Article
pubs.acs.org/jmc
Synthesis and Structure−Activity Relationships of 5,6,7-Substituted
Pyrazolopyrimidines: Discovery of a Novel TSPO PET Ligand for
Cancer Imaging
Dewei Tang,†,‡ Eliot T. McKinley,†,§ Matthew R. Hight,†,∥ Md. Imam Uddin,†,▽ Joel M. Harp,⊥,#
Allie Fu,† Michael L. Nickels,†,▽ Jason R. Buck,†,▽ and H. Charles Manning*,†,‡,§,▽,○,◆
†Vanderbilt University Institute of Imaging Science (VUIIS), Vanderbilt University Medical Center, Nashville, Tennessee, United
States
‡Program in Chemical and Physical Biology, Vanderbilt University Medical Center, Nashville, Tennessee, United States
§Department of Biomedical Engineering, Vanderbilt University, Nashville, Tennessee, United States
∥Interdisciplinary Materials Science Program, Department of Physics & Astronomy, Vanderbilt University, Nashville, Tennessee,
United States
⊥Department of Biochemistry, Vanderbilt University Medical Center, Nashville, Tennessee, United States
#Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee, United States
▽Department of Radiology and Radiological Sciences, Vanderbilt University Medical Center, Nashville, Tennessee, United States
○Vanderbilt Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, Tennessee, United States
◆Department of Neurosurgery, Vanderbilt University Medical Center, Nashville, Tennessee, United States
S
* Supporting Information
ABSTRACT: Focused library synthesis and structure−activity relationship development of 5,6,7-substituted pyrazolopyr-
imidines led to the discovery of 2-(5,7-diethyl-2-(4-(2-fluoroethoxy)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)-N,N-diethylaceta-
mide (6b), a novel translocator protein (TSPO) ligand exhibiting a 36-fold enhancement in affinity compared to another
pyrazolopyrimidine-based TSPO ligand, 6a (DPA-714). Radiolabeling with fluorine-18 (18F) facilitated production of 2-(5,7-
diethyl-2-(4-(2-[18F]fluoroethoxy)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)-N,N-diethylacetamide (18F-6b) in high radiochemical
yield and specific activity. In vivo studies of 18F-6b were performed which illuminated this agent as an improved probe for
molecular imaging of TSPO-expressing cancers.
Our laboratory has explored translocator protein (TSPO)
expression as a target for molecular imaging of cancer.1−5
Formerly referred to as peripheral benzodiazepine receptor
(PBR), TSPO is an 18 kDa protein typically localized to the
outer mitochondria membrane. TSPO participates in the
regulation of numerous cellular processes, including steroid
biosynthesis, cholesterol metabolism, apoptosis, and cellular
proliferation.6 In normal tissues, TSPO tends to be expressed in
those that produce steroids and those that are mitochondrial-
enriched such as myocardium, skeletal muscle, and renal tissue.
Tissues such as liver and brain exhibit comparatively modest
expression.6 While classically exploited as a target in neuro-
science, elevated TSPO expression is also observed in many
cancers.7 In oncology, TSPO expression is typically linked with
disease progression and diminished survival and is a hallmark of
aggressive and potentially metastatic tumors.7 For this reason,
our laboratory has explored the use of TSPO imaging ligands
within the context of colon cancer,1 breast cancer,2 and
glioma,4,5 as these agents could potentially serve as useful
cancer imaging biomarkers. We recently reported the first
INTRODUCTION
■
There remains a critical need to develop and rigorously validate
molecular imaging biomarkers that aid tumor diagnosis, predict
clinical outcome, and quantify response to therapeutic
interventions. Imaging techniques routinely used in clinical
oncology include magnetic resonance imaging (MRI), X-ray
computed tomography (CT), ultrasound imaging (US), and
positron emission tomography (PET). Of these, the sensitivity
and quantitative nature of PET, coupled with the ability to
readily produce biologically active compounds bearing
positron-emitting isotopes (e.g., 11C, 18F), renders PET imaging
as one of the most attractive techniques for detecting tumors
and profiling their molecular features. By far, the most widely
used PET tracer in clinical oncology is 2-deoxy-2-[18F]fluoro-D-
glucose (18F-FDG), a probe that accumulates in tissue as a
function of glucose utilization. PET using 18F-FDG is a
powerful approach for tumor detection in many organ sites.
However, not all tumors exhibit elevated glucose avidity, and
18F-FDG uptake can be affected by a plethora of normal
metabolic processes. Furthermore, tumor imaging can be
confounded by 18F-FDG uptake in normal tissues such as
healthy brain. These issues highlight an unmet need to explore
and validate additional molecular targets for cancer imaging.
Received: February 5, 2013
© XXXX American Chemical Society
A
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Brief Article
Table 1. Affinity, Liphopilicity, and Molecular Weight of Pyraxolopyrimidines
d
compd
R1
R2
R3
R4
−OMe
−OMe
−OMe
−OMe
−OMe
−OMe
−OMe
−OCH2CH2F
−OCH2CH2F
−OCH2CH2F
−OCH2CH2F
−OCH2CH2F
−OCH2CH2F
−OCH2CH2F
MW
log P7.5
Ki (nM)
a
5a
−Me
−Et
−H
−H
−H
−Me
−Et
−Me
−Et
366.21
394.51
422.56
380.48
394.51
408.49
400.9
2.40
2.84
2.98
2.45
2.78
2.57
2.99
2.12
2.50
2.73
2.47
2.53
2.07
2.55
12.23
0.18
5b
b
5c
−iPr
−Me
−Me
−Me
−Me
−Me
−Et
−iPr
−Me
−Me
−Me
−Me
−iPr
−Me
−Me
−Me
−Me
−Me
−Et
−iPr
−Me
−Me
−Me
−Me
>500
93.75
157.14
200.89
55.36
9.73
5d
5e
5f
−CH2C(O)CH3
5g
−Cl
−H
−H
−H
−Me
−Et
c
6a
398.47
426.53
454.58
412.5
e
6b
6c
6d
6e
6f
0.27
>500
83.04
426.53
440.51
432.92
276.79
251.79
−CH2C(O)CH3
6g
−Cl
c
67.86
e
a
b
d
3
DPA-713, see ref 8−11. See ref 12. DPA-714, see refs 8−10,13. Competitive binding against H-PK 11195 in C6 glioma cell lysate. Ki versus
3H-flunitrazepam >10000 nM.
utilizations of the PET agents N-[18F]fluoroacetyl-N-(2,5-
dimethoxybenzyl)-2-phenoxyaniline (18F-PBR06)5 and N,N-
diethyl-2-(2-(4-(2- [18F]fluoroethoxy)phenyl)-5,7-
dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide (18F-DPA-
714)4 for quantitative assessment of TSPO expression in
preclinical glioma. In these proof-of-principle PET imaging
studies, tumors were detectable among surrounding normal
brain and, importantly, TSPO levels could be quantitatively
assayed in tumors using compartmental analysis of the PET
data.4,5 However, drawbacks were observed with both agents in
this context, including tracer accumulation in normal brain that
reached levels potentially sufficient to prevent detection of
gliomas with modest TSPO expression. Both tracers also
exhibited significant metabolism in vivo, which required
correction of plasma input functions for quantitative analysis.
While illustrating the potential of TSPO PET to detect tumors
in brain, these studies prompted our desire to develop novel
TSPO PET ligands with improved properties for cancer
imaging.
The goal of this study was to determine whether
optimization of the pyrazolopyrimidine scaffold, specifically at
the 5-, 6-, and 7-positions, would yield TSPO ligands with
greater affinity and potentially serve as more robust PET
imaging ligands in vivo. These experiments led to the discovery
of 2-(5,7-diethyl-2-(4-(2-fluoroethoxy)phenyl)pyrazolo[1,5-a]-
pyrimidin-3-yl)-N,N-diethylacetamide (6b), a novel TSPO-
selective ligand that exhibits a surprising 36-fold enhancement
in affinity compared to another pyrazolopyrimidine, 6a (DPA-
714). An appropriate analogue of 6b, compound 7, could be
radiolabeled with fluorine-18 (18F) to yield the novel TSPO
PET ligand 2-(5,7-diethyl-2-(4-(2-[18F]fluoroethoxy)phenyl)-
pyrazolo[1,5-a]pyrimidin-3-yl)-N,N-diethylacetamide (18F-6b),
which was subsequently evaluated in vivo in healthy rats and a
preclinical model of glioma. 18F-6b exhibited negligible
accumulation in normal brain, yet robust accumulation in
tumor tissue, which facilitated excellent imaging contrast.
Overall, these studies illuminate 18F-6b as a promising, novel
PET ligand for evaluating TSPO expression in tumors and
potentially other diseases.
RESULTS AND DISCUSSION
■
Chemistry. MAOS of 5,6,7-Substituted Pyrazolopyrimi-
dine Library. A library of structurally diverse pyrazolopyr-
imidines (Table 1) was assembled using a microwave-assisted
organic synthesis (MAOS) method that prioritized points of
divergence at the 5-R3, 6-R2, and 7-R1 positions on the core
pyrazolopyrimidine scaffold. Members of this library were
formed through condensation of a pyrazole core (4) with
various substituted diones.12 Entries in Table 1 fall into two
distinct series (5 and 6), with seven compounds (a−g) per
series. Each compound within the series results from one of
seven diones utilized, while each series is differentiated by the
installation of a unique surrogate imaging handle at the 4-
position (R4) of the 2-phenyl group pendant to the core
scaffold. Series 5 entries (5a−5g) feature a methoxy group at R4
and include a previously reported pyrazolopyrimidine, 5a
(DPA-713),8−11 while series 6 entries (6a−6g) feature a 2-
fluoroethoxy group at R4 and include 6a.8−10,13 Given our
ultimate goal of developing novel PET imaging ligands, coupled
with an interest in expanding structure−activity relationships
(SAR) around the 5,6,7-substituted pyrazolopyrimidine core,
we rationalized that maintaining the surrogate imaging handles
of future representative imaging probes would accelerate the
development of novel agents.
The overall synthetic methodology is presented in Scheme
1.12 Here, compound 4, bearing a 3-amino-1H-pyrazole core,
was accessible in two steps using MAOS from commercially
available phenylpropanenitrile 1 and chloroacetamide 2. From
4, the synthesis diverges at the condensation step with a series
of unique diones to yield the “five” series of pyrazolopyr-
imidines (5a−5g) that mimic analogous 11C-based PET probes
with an R4-OMe. Subsequently, compounds 6a−6g were
achieved by cleavage of methoxy group followed by micro-
wave-assisted ether synthesis with 2-fluoroethyl-4-methylben-
zenesulfonate which yielded an R4-2-fluoroethoxy surrogate
B
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Table 1. Encompassing modifications at R1−R4, both series
exhibited a spectrum of Ki values that spanned micromolar to
subnanomolar affinity. As expected, replacing the 4-methoxy
with 4-(2-fluoroethoxy) at the R4 position had minimal impact
upon TSPO affinity, as similar SAR trends emerged within
series 5 and 6. In contrast, R1, R2, and R3 modification led to
major impacts upon affinity. Although the R1 and R3 positions
showed intolerance to steric bulk beyond ethyl, a surprising
level of improvement in affinity could be realized by replacing
methyl with ethyl at the R1 and R3 positions. For example,
subnanomolar affinities (Ki) were observed for analogues 5b
(0.18 nM) and 6b (0.27 nM), which represent major
improvements over their respective leads, 5a (12.23 nM) and
6a (9.73 nM) (Table 1), as well as another high-affinity TSPO
ligand, N-(2-methoxybenzyl)-N-(4-phenoxypyridin-3-yl)-
acetamide (PBR28) (4.0 nM).16 Increasing the R1 and R3 to
a bulkier isopropyl group proved detrimental, with Ki values for
both 5c and 6c greater than 500 nM. Overall, the R2 position
was less tolerant to steric or electron density deviations from
hydrogen in these studies. Substitution of the nascent R2
hydrogen (5a, 6a) with methyl (5d, 93.75 nM; 6d, 83.04
nM) decreased affinity approximately 10-fold with respect to
their parent compounds, 5a and 6b, respectively; this trend was
more dramatic with ethyl substitution (5e, 157.14 nM; 6e,
276.79 nM). Introduction of polarity at the R2 position with 2-
propanone (5f, 200.89 nM; 6f, 251.79 nM) bore similar adverse
effects upon affinity. Somewhat less dramatic decreases in
affinity were observed upon halogen substitution at R2 with
chlorine (5g, 55.36 nM; 6g, 67.86 nM). Given the size of
chlorine atom, lying between methyl and ethyl, it is reasonable
that the Ki of entries 5g and 6g are only slightly worse than
those with alkyl substituents at the R2 position (Table 1).
Overall, these experiments illuminated 5,7-diethyl, 6-hydrogen
substitution as the optimal pattern in this series to impart
improved affinity. Additionally, the studies also suggested that
an analogue could be produced for PET imaging using either
fluorine-18 (6b) or carbon-11 (5a) substitution on the R4
position with minimal effect on TSPO affinity and only minor
effect on lipophilicity. Because fluorine-18 has a significantly
longer half-life than carbon-11 (109.8 min vs 20.4 min), we
elected to carry compound 6b forward for further character-
ization. The selectivity of compound 6b for TSPO over the
central benzodiazepine receptor (CBR) was evaluated using
Scheme 1. Synthetic Methodology Utilized to Generate a
Focused Library of 5,6,7-Substituted Pyrazolopyrimidines
α
and 18F-6b
α
Reagents and conditions: (a) NaOH, NaI, microwave, 80 °C, 40 min,
80% EtOH in H2O, 70%; (b) hydrazine, AcOH, microwave, 90 °C, 40
min, EtOH, 42%; (c) microwave, 140−190 °C, 45 min, EtOH, 10−
90%; (d) HTPB, microwave, 110 °C, 40 min, aqueous HBr; (e) NaH,
2-fluoroethyl-4-methylbenzenesulfonate, microwave, 120 °C, 30 min,
dry THF; (f) ethylene di(p-toluenesulfonate), NaH, microwave, 120
°C, 30 min, dry THF, 45%; (g) fluoride-18 ion, K+-K2.2.2/K2CO3, 99
°C, 20 min, dry DMSO.
imaging handle to mimic 18F-labeled PET probes (Table 1).
The library developed primarily explored the effects of R1, R2,
and R3 substituents that varied in steric bulk. Generally,
substituents at R1 and R3 mirrored one another and consisted
of linear and branched alkyl substituents such as methyl, ethyl,
and isopropyl moieties. These substituents were cross-matched
with similar groups at the R2 position, which included
hydrogen, methyl, ethyl, chloro, and 2-propanone.
Lipophilicity. Lipophilicity of series 5 and 6 were evaluated
at pH = 7.5 (log P7.5) using reversed phase HPLC14 and is
tabulated in Table 1. The values for library entries varied from
2.0 to 3.0 for both series and correlated predictably with dione
structure, where polarity and hydrocarbon content of the R1−
R3 substituents appeared to be key determinants. In both series,
analogues a, d, and f, with R1−R3 combinations of hydrogen,
methyl, and 2-propanone, proved the least lipophilic.
Introduction of ethyl groups at R1, R2, or R3 increased log
3
radioligand displacement of the CBR ligand H-flunitrazepam
in rat cerebral cortex membranes (Table 1). Unlike the
displacement studies with 3H-PK 11195, compound 6b
demonstrated poor affinity for CBR (Ki > 10000 nM),
indicating TSPO selectivity.
P
7.5 values accordingly, as with analogues b and e, which ranged
from 2.50 to 2.84. The most lipophilic analogues proved to be c
and g (log P7.5 = 2.55−2.99), with isopropyl groups at the R1
and R3 positions or chlorine at R2.
Interestingly, of the two series, series 5 (R4 = OMe) tended
to be more lipophilic than series 6 (R4 = OCH2CH2F). Overall,
the library entries exhibited lipophilicities (2.07−2.99) suitable
for in vivo imaging,15 where potential agents must be
sufficiently membrane penetrant to bind intracellular targets
such as TSPO.
Biological Testing. Binding Affinity in Cancer Cell Lysate
and SAR Analysis. To evaluate binding of the library entries to
TSPO, radioligand displacement was carried out in rat glioma
cell lysate (C6) using N-(sec-butyl)-1-(2-chlorophenyl)-N-
methyl-3H-isoquinoline-3-carboxamide (3H-PK 11195).4,5 Af-
finities of library members are expressed as Ki (nM) values in
Radiochemistry. Radioligand Precursor Preparation and
Radiosynthesis. To produce 18F-6b, we synthesized precursor
(7), a tosylated intermediate prepared from 5b in two steps
with an overall yield of approximately 45% (Scheme 1).
Nucleophilic fluorination of 7 with fluorine-18 was then
performed (Scheme 1) in anhydrous dimethyl sulfoxide at
165 °C for 5 min. Purification of 18F-6b was carried out with
preparative HPLC using 10 mM sodium phosphate buffer (pH
6.7) in ethanol (47.5/52.5, v/v). The retention time of 18F-6b
was 12 min according to gamma detection and corresponded to
the UV retention time of nonradioactive 6b. Radiochemical
purity was consistently greater than 99%, with specific activity
consistently greater than 4203 Ci/mmol (156 TBq/mmol) (n
= 33).
C
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Imaging Studies. Uptake of 18F-6b in C6 Glioma. The in
vivo performance of 18F-6b was evaluated in glioma-bearing
Wistar rats using microPET imaging, with a typical study shown
in Figure 1. MRI (T2-weighted) was used to localize tumors
clearance of 18F-6b from plasma, rendering detection of low
levels of metabolites challenging with HPLC.
Biodistribution and Specific Binding of 18F-6b in Rats. The
biodistribution and TSPO-specificity of 18F-6b was evaluated in
normal tissues. Tissue samples were harvested 60 min after
infusion of 18F-6b. The radioactivity of harvested tissue was
measured using standard analytical methods and recorded as
percentage of the injected dose per gram of tissue (%ID/g) (SI
Figure 2A). Consistent with known TSPO densities,7 we
observed elevated accumulation of 18F-6b in spleen, heart,
kidney, and lung.17 Moderate uptake was also observed in liver,
colon, small bowel, and stomach. Very minor accumulation was
observed in the brain, testes, skull, and skeletal muscle. At 60
min postadministration of tracer, very little radioactivity was
observed in biological fluids such as whole blood or urine.
To evaluate the specific binding of 18F-6b in normal tissue,
we carried out a similar 60 min biodistribution assay that
included a bolus infusion of nonradioactive 6b (10 mg/kg) at
30 min (SI Figure 2B). With displacement, we observed that
activity in the organs exhibiting the greatest accumulation of
18F-6b, such as spleen, heart, kidney, and lung, was reduced to
background levels, indicating near complete displacement and
reversible binding in healthy tissue. Organs that exhibited low
to moderate accumulation of 18F-6b without displacement, such
as muscle or liver, were essentially unchanged with displace-
ment, suggesting that the low to moderate tracer accumulation
in these tissues may reflect nonspecificity. Interestingly, activity
found in the urine did not follow this trend and was elevated
with displacement, suggesting that blocking TSPO with
nonradioactive 6b altered the excretion profile of the agent.
Overall, the results indicate a high degree of specific binding
and reversibility of 18F-6b to TSPO in healthy tissues.
Figure 1. PET imaging of preclinical glioma using 18F-6b.
and for registration of anatomical features with PET4,5 (Figure
1A). Dynamic PET imaging with 18F-6b illustrated that the
majority of tracer uptake in the brain was localized to the
tumor, with very modest accumulation in the adjacent, normal
brain (Figure 1B,C). The tumor-selective uptake characteristics
of 18F-6b afforded excellent imaging contrast between tumor
and normal tissue. In addition to the tumor, accumulation of
18F-6b was observed outside of the brain in olfactory epithelium
and the Harderian glands, as well as the tongue, where TSPO
expression is elevated.7 Figure 1D illustrates time−activity
curves (TACs) that were typical of eight representative studies
for tumor, normal brain, and plasma over a 90 min dynamic
acquisition. We found that 18F-6b was rapidly delivered to
tumor and normal brain but cleared tumor tissues at a much
slower rate than normal brain. After an initial spike in
radioactivity consistent with tracer injection and rapid
distribution, 18F-6b quickly cleared from the plasma. To
validate the PET, imaging-matched brains were processed for
post-mortem staining and immunohistochemistry. We observed
close agreement between tumor tissue (H&E, Figure 1E),
elevated TSPO expression by immunohistochemistry (Figure
1F), and tumor accumulation of 18F-6b accumulation (Figure
1C). To more quantitatively compare the performance of 18F-
6b to 18F-6a, we conducted dynamic PET using 18F-6b in a
cohort of tumor-bearing rats (n = 5) and carried out
pharmacokinetic modeling of the PET data, similar to our
previous studies.4,5 We found that 18F-6b demonstrated a
higher ratio of total distribution volume (VT) between tumor
and normal brain (6.0, n = 5) when compared to 18F-6a (3.9, n
= 11),4 which resulted in greater signal-to-noise between tumor
and surround normal brain.
Crystallography. 6b was recrystallized from hot acetoni-
trile and ethanol (95%) by slow evaporation at room
temperature and characterized by diffractometry. 6b demon-
strated two ethyl branches at the 5- and 7-positions of the
pyrazolopyrimidine ring, corresponding to the structure
elucidated from NMR spectroscopy. The crystal structure
also revealed three distinct planar entities, the pyrazolopyr-
imidine ring, the phenyl ring, and the amide group (SI Figure
3).
CONCLUSION
■
In this study, we found that the 5,6,7-substitution pattern is a
critical determinant of TSPO affinity among pyrazolopyrimi-
dines. Experiments shown here led to the discovery of 6b, a
TSPO ligand exhibiting significantly increased affinity com-
pared to 6a and TSPO ligands derived from other chemical
entities.18 Furthermore, in addition to improved TSPO affinity,
compound 6b was TSPO-selective, exhibiting negligible
binding affinity to CBR. Analogue 7 of 6b could be radiolabeled
with fluorine-18 to yield 18F-6b. Suggesting its potential as a
probe for cancer imaging, preclinical imaging studies
demonstrated robust accumulation of 18F-6b in tumor tissue
and negligible accumulation in normal brain. Overall, these
studies illuminate 18F-6b as a promising, novel PET ligand for
evaluating TSPO expression in tumors and potentially other
diseases.
Characterization of 18F-6b Radiometabolites in Plasma.
Analogous to our previous work,4 radio-HPLC was used to
evaluate metabolism of 18F-6b in plasma at multiple time points
during the PET scanning period (2, 12, 30, 60, 90 min)
(Supporting Information (SI) Figure 1). Parent compound
(18F-6b) was the only radioactive species detected in plasma at
all time points, suggesting a lack of circulating 18F-6b
metabolites observed over a 90 min period. This behavior
contrasted the in vivo performance of 18F-6a, where we
previously observed up to 30% metabolism within the first 12
min post-administration.4 Potentially, the apparent improved
stability of 18F-6b over 18F-6a may stem from the rapid
D
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DPA-713, 18F-DPA-714, and 11C-PK11195 in a rat model of acute
neuroinflammation. J. Nucl. Med. 2009, 50, 468−476.
(9) Doorduin, J.; Klein, H. C.; Dierckx, R. A.; James, M.; Kassiou, M.;
de Vries, E. F. [11C]-DPA-713 and [18F]-DPA-714 as new PET tracers
for TSPO: a comparison with [11C]-(R)-PK11195 in a rat model of
herpes encephalitis. Mol. Imaging Biol. 2009, 11, 386−398.
ASSOCIATED CONTENT
* Supporting Information
Synthetic and analytical results for all compounds; methods;
supplemental figures. This material is available free of charge via
the Internet at http://pubs.acs.org.
■
S
(10) James, M. L.; Fulton, R. R.; Henderson, D. J.; Eberl, S.; Meikle,
S. R.; Thomson, S.; Allan, R. D.; Dolle, F.; Fulham, M. J.; Kassiou, M.
Synthesis and in vivo evaluation of a novel peripheral benzodiazepine
receptor PET radioligand. Bioorg. Med. Chem. 2005, 13, 6188−6194.
(11) Selleri, S.; Bruni, F.; Costagli, C.; Costanzo, A.; Guerrini, G.;
Ciciani, G.; Costa, B.; Martini, C. 2-Arylpyrazolo[1,5-a]pyrimidin-3-yl
acetamides. New potent and selective peripheral benzodiazepine
receptor ligands. Bioorg. Med. Chem. 2001, 9, 2661−2671.
AUTHOR INFORMATION
Corresponding Author
*Phone: 615-322-3793; Fax: 615-322-0734; E-mail: henry.c.
manning@vanderbilt.edu. Address: Vanderbilt University In-
stitute of Imaging Science (VUIIS), 1161 21st Avenue South,
AA1105 MCN, Nashville, TN 37232-2310.
■
(12) Tang, D.; Buck, J. R.; Hight, M. R.; Manning, H. C. Microwave-
Assisted Organic Synthesis of a High-Affinity Pyrazolo-pyrimidinyl
TSPO Ligand. Tetrahedron Lett. 2010, 51, 4595−4598.
Author Contributions
Dr. Manning directed and designed the study. Mr. Tang
performed the synthetic chemistry and, along with Mr.
McKinley, Mr. Hight, and Dr. Buck, performed the imaging
experiments. Dr. Nickels performed the radiochemistry, Dr.
Uddin and Dr. Harp carried out the crystallography. All authors
participated in writing or editing the manuscript.
(13) Martin, A.; Boisgard, R.; Theze, B.; Van Camp, N.; Kuhnast, B.;
Damont, A.; Kassiou, M.; Dolle, F.; Tavitian, B. Evaluation of the
PBR/TSPO radioligand [(18)F]DPA-714 in a rat model of focal
cerebral ischemia. J. Cereb Blood Flow Metab. 2010, 30, 230−241.
(14) Waterhouse, R. N.; Mardon, K.; Giles, K. M.; Collier, T. L.;
O’Brien, J. C. Halogenated 4-(phenoxymethyl)piperidines as potential
radiolabeled probes for sigma-1 receptors: in vivo evaluation of [123I]-
1-(iodopropen-2-yl)-4-[(4-cyanophenoxy)methyl]piperidine. J. Med.
Chem. 1997, 40, 1657−1667.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(15) Pike, V. W. PET radiotracers: crossing the blood−brain barrier
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(16) Owen, D. R.; Howell, O. W.; Tang, S. P.; Wells, L. A.; Bennacef,
I.; Bergstrom, M.; Gunn, R. N.; Rabiner, E. A.; Wilkins, M. R.;
Reynolds, R.; Matthews, P. M.; Parker, C. A. Two binding sites for
[3H]PBR28 in human brain: implications for TSPO PET imaging of
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V. W.; Innis, R. B.; Fujita, M. Biodistribution and radiation dosimetry
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We acknowledge funding from the National Institutes of
Health (K25 CA127349, P50 CA128323, S10 RR17858, U24
CA126588, 1R01 CA163806), The Kleberg Foundation, and
The Lustgarten Foundation. M. Noor Tantawy, George H.
Wilson, Dan Colvin, and Yiu-Yin Cheung are acknowledged.
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