Synthesis, labeling, and biological evaluation of halogenated 2-quinolinecarboxamides as potential radioligands for the visualization of peripheral benzodiazepine receptors

Article abstract of DOI:10.1016/j.bmc.2006.02.004

The previous exploration of the structure-affinity relationships concerning 4-phenyl-2-quinolinecarboxamide peripheral benzodiazepine receptor (PBR) ligands 6 showed as an interesting result the importance of the presence of a chlorine atom in the methyle

Full text of DOI:10.1016/j.bmc.2006.02.004

Bioorganic & Medicinal Chemistry 14 (2006) 4055–4066
Synthesis, labeling, and biological evaluation of halogenated
2-quinolinecarboxamides as potential radioligands for
the visualization of peripheral benzodiazepine receptors
Andrea Cappelli,a,* Mario Matarrese,b Rosa Maria Moresco,b Salvatore Valenti,a
Maurizio Anzini,a Salvatore Vomero,a Elia Anna Turolla,b Sara Belloli,b
Pasquale Simonelli,b Maria Azzurra Filannino,b Michela Lecchic and Ferruccio Faziob
aDipartimento Farmaco Chimico Tecnologico and European Research Centre for Drug Discovery and Development,
`
Universita degli Studi di Siena, Via A. Moro, 53100 Siena, Italy
`
b
Istituto di Bioimmagini e Fisiologia Molecolare, CNR, Universita di Milano/Bicocca, Istituto H. S. Raffaele,
Via Olgettina 60, 20132 Milano, Italy
c
` `
Istituto di Scienze Radiologiche, Universita degli Studi di Milano, Via Rudinı 8, 20142 Milano, Italy
Received 6 December 2005; revised 1 February 2006; accepted 3 February 2006
Available online 21 February 2006
Abstract—The previous exploration of the structure–affinity relationships concerning 4-phenyl-2-quinolinecarboxamide peripheral
benzodiazepine receptor (PBR) ligands 6 showed as an interesting result the importance of the presence of a chlorine atom in the
methylene carbon at position 3 of the quinoline nucleus. The subnanomolar PBR affinity shown by N-benzyl-3-chloromethyl-N-
methyl-4-phenyl-2-quinolinecarboxamide (6b) suggested its chlorine atom to be replaced with other halogens in order to optimize
the interaction of the quinolinecarboxamide derivatives with PBR and to develop suitable candidates for positron emission tomog-
raphy (PET) or single photon emission computed tomography (SPECT) studies. The binding studies led to the discovery of fluo-
romethyl derivative 6a, which showed an IC50 value of 0.11 nM and is, therefore, one of the most potent PBR ligands so far
described. Fluoromethyl derivative 6a has been labeled with 11C (t1/2 = 20.4 min, b+ = 99.8%) starting from the corresponding
des-methyl precursor (14) using [11C]CH3I in the presence of tetrabutylammonium hydroxide in DMF with a 35–40% radiochemical
yield (corrected for decay) and 1.5 Ci/lmol of specific radioactivity. Ex vivo rat biodistribution and inhibition (following intrave-
nous pre-administration of PK11195) studies showed that [11C]6a rapidly and specifically accumulated in PBR-rich tissues such
as heart, lung, kidney, spleen, and adrenal, and at a lower level in other peripheral organs and in the brain. The images obtained
in mouse with small animal YAP-(S)PET essentially confirmed the result of the ex vivo biodistribution experiments. The biological
data suggest that [11C]6a is a promising radioligand for peripheral benzodiazepine receptor PET imaging in vivo.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
kidney, heart, testis and at a lower level in the brain
parenchyma, ependyma, choroid plexus, and olfactory
neurons. Furthermore, it has been found that PBR is
overexpressed in a variety of tumors (e.g., certain brain
tumors, ovarian cancer, liver tumors, breast carcinoma,
colorectal cancer, etc.) and the PBR expression appears
to be related to the tumor malignancy grade.2 Increased
concentrations of PBR were observed in lesioned brain
areas in a variety of neuropathologies such as multiple
sclerosis, Alzheimer’s disease, and Huntington’s dis-
ease.3 At a subcellular level, it is mainly localized on
the outer mitochondrial membrane even if nonmito-
chondrial (nuclear or microsomal) localizations in some
cells have been suggested. The physiological role of PBR
is still unclear, but a wide range of pharmacological
The peripheral benzodiazepine receptor (PBR) was
originally discovered in the periphery by means of triti-
um-labeled diazepam and such an important finding
stimulated intensive research toward the characteriza-
tion of this receptor.1 PBR is mainly localized in periph-
eral tissues and glial cells. It is highly expressed in
steroidogenic tissues such as adrenal gland, but also in
Keywords: Peripheral benzodiazepine receptor; PET; Radiotracer;
Synthesis.
*
Corresponding author. Tel.: +39 0577 234320; fax: +39 0577
234333; e-mail: cappelli@unisi.it
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.02.004

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A. Cappelli et al. / Bioorg. Med. Chem. 14 (2006) 4055–4066
activities, such as anticonvulsant, anxiolytic, immuno-
modulating, and cardiovascular, have been related to
its activation.4 In particular, this receptor appears to be
involved in steroidogenesis, the regulation of which rep-
resents a potential clinical application of PBR ligands.5
PK11195 (1, Fig. 1) was the first non-benzodiazepine li-
gand which was found both to bind the PBR with nano-
molar affinity and to facilitate the transport of
cholesterol from the outer to the inner mitochondrial
membrane, increasing the rate of pregnenolone synthe-
sis. Isoquinolinecarboxamide derivative 1 is the most
widely used pharmacological tool for the study of the
expression and the function of PBR (e.g., steroidogenesis
and apoptosis). Compound 1 labeled with positron emit-
ter carbon-11 was used in the imaging of brain tumors,
multiple sclerosis, cerebral infarction, and abnormalities
of calcium channels in heart diseases by means of posi-
tron emission tomography (PET).6
R = 11CH3) and its fluoromethyl (4b: R = 18FCH2) and
fluoroethyl (4c: R = 18FCH2CH2) derivatives were de-
scribed and [18F]FEDAA1106 (4c) was proposed as a
useful PET ligand for PBR which is currently used for
the PBR imaging in human brain.7 Our work culminat-
ed in the design and synthesis of potent ligands both in
the class of alpidem-related compounds and in the class
of PK11195 derivatives8 as well as in the development of
theoretical models for the interaction of PBR with the
best known ligands.9 Again, the quinolinecarboxamide
derivatives 5a–c (5a: X = H, R1 = CH(CH3)CH2CH3,
R2 = CH3;
5b:
X = F,
R1 = CH(CH3)CH2CH3,
R2 = CH3; 5c: X = H, R1 = CH2C6H5, R2 = CH3) were
11C-labeled and the biodistribution studies suggested
that these compounds are promising PET tracers for
the in vivo imaging of PBR.10 The evaluation of 11C-la-
beled 5a-c in an excitotoxic model of Huntington’s dis-
ease in rats revealed that benzyl derivative 5c can be
considered an interesting candidate for the in vivo
PET monitoring of neurodegenerative processes.11 The
exploration of the structure–affinity relationships
(SAFIRs) concerning compounds 6 showed as an inter-
esting result the effect of the presence of a chlorine atom
in the methylene carbon at position 3 of the quinoline
nucleus.12 Based on the subnanomolar PBR affinity
shown by 3-chloromethyl derivative 6b, its chlorine
atom was replaced by other halogens [e.g., fluorine
(6a), bromine (6c), and iodine (6d)] with the aim of opti-
mizing the interaction of the quinolinecarboxamide
derivatives with PBR and developing once more better
candidates for PET studies. In this paper, we describe
the synthesis and the preliminary pharmacological eval-
uation of a short series of 3-halomethyl-2-quinolinecarb-
oxamide derivatives 6a,c,d showing subnanomolar
affinity for PBR as well as the radiolabeling with 11C,
the results of ex vivo biodistribution and inhibition
experiments in rats, and the in vivo PET studies in mice
performed on fluoromethyl derivative 6a to evaluate its
potential use as a PET radioligand.
The class of high affinity PBR ligands also comprises the
benzodiazepine Ro5-4864 (2) and the imidazopyridine
alpidem (3), capable of stimulating pregnenolone forma-
tion from mitochondria of C6-2B glioma cells.5 Very
recently, the properties of labeled DAA1106 (4a:
H3C
O
O
N
N
N
CH3
N
O
Cl
Cl
2
PK11195 (1)
Cl
H3C
OCH3
OR
F
N
O
N
N
Cl
2. Results
Cl
2.1. Chemistry
O
N
3
The previously described quinolinecarboxamide deriva-
tives 5c and 6b were re-prepared following the procedure
described elsewhere,12 while new 3-halomethyl-2-quino-
linecarboxamide derivatives 6a,c,d were synthesized by
means of a nucleophilic substitution of the chlorine
atom of 6b with fluoride, bromide, or iodide in the pres-
ence of 18-crown-6 (Scheme 1).13
4a R = 11CH3
4b R = 18FCH2
4c R = 18FCH2CH2
O
N
R1
O
N
In order to obtain the precursor of fluoromethyl deriva-
tive 6a to be used in [11C]CH3I labeling, a new synthetic
procedure was developed in which the fluorine atom was
inserted into the molecule in the early steps of the se-
quence to obtain the fluorinated acid 12 (Scheme 2).
N
R2
Me
N
CH3
X
X
5a X = H, R1 = i Bu, R2 = Me
5b X = F, R1 = i Bu, R2 = Me
5c X = H, R1 = Bn, R2 = Me
Acid 78b was converted into ester 9 which was brominat-
ed with NBS in the presence of dibenzoyl peroxide to
obtain bromomethyl derivative 10. Nucleophilic substi-
tution of 10 bromine atom with potassium fluoride in
6
Figure 1. Structure of PBR ligands 1–6.

A. Cappelli et al. / Bioorg. Med. Chem. 14 (2006) 4055–4066
4057
which was used for the preparation of the secondary
amide 14 or the tertiary amide 6a (Scheme 3). The latter
compound was obtained also from the former by meth-
ylation with methyl iodide in the presence of sodium hy-
dride as the base. This result demonstrated the feasibility
of the labeling of fluoromethyl derivative 6a with
[11C]CH3I.
O
F
O
N
N
i
N
N
CH3
CH3
Cl
6a
6b
2.2. Radiosynthesis
Compound [11C]6a was synthesized by N–[11C]methyl-
ation of the corresponding des-methyl precursor 14
with [11C]methyl iodide in the presence of tetrabuty-
lammonium hydroxide (TBAH) at 80ꢁC for 4 min
(Scheme 4) with a radioactivity incorporation yield of
80%. The overall radiosynthesis, including 11C-methyl-
ation, HPLC purification and radiopharmaceutical for-
mulation for intravenous administration was completed
in an average time of 35 min, from the end of bom-
bardment (EOB) with a 35–40% radiochemical yield
(corrected for decay) and 1.5 Ci/lmol of specific radio-
activity at the end of radiosynthesis (EOS). In a typical
experiment, starting from 900 mCi of [11C]CO2,
100 mCi of [11C]6a were obtained with a chemical
and radiochemical purity of 100% and over 99%,
respectively.
ii
iii
O
I
N
N
O
CH3
N
N
6d
CH3
Br
6c
Scheme 1. Reagents: (i) KF, 18-Crown-6, CH3CN; (ii) NaI, 18-
Crown-6, CH3CN; (iii) LiBr, 18-Crown-6, CH3CN.
The identity of the final radioactive 11C-radioligand was
confirmed by co-injection with the authentic sample of
non-radioactive 6a on reverse-phase high-performance
liquid chromatography (HPLC) (see Section 4).
the presence of 18-crown-6 gave ester 11, which was
cleaved with formic acid to afford acid 12. The acid
was converted into the corresponding acid chloride 13,
O
O
N
N
i
OH
Cl
CH3
CH3
7
8
ii
CH3
O
O
CH3
CH3
CH3
CH3
CH3
N
N
iii
iv
O
O
CH3
Br
9
10
CH3
O
F
O
O
CH3
CH3
N
N
v
OH
F
12
11
Scheme 2. Reagents: (i) SOCl2, CH2Cl2; (ii) t-BuOK, THF; (iii) NBS, dibenzoyl peroxide, CCl4; (iv) KF, 18-Crown-6, CH3CN; (v) HCOOH.

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A. Cappelli et al. / Bioorg. Med. Chem. 14 (2006) 4055–4066
Table 1. PBR binding affinities of compounds 5c and 6a–d
O
F
O
N
N
i
OH
Cl
O
X
O
N
N
F
N
N
12
13
CH3
CH3
CH3
5c
6a-d
ii
iv
Compound
X
IC50 (nM) SEMa
O
F
5c
6a
6b
6c
6d
1
H
F
2.1 0.3
0.11 0.02
0.30 0.032
0.61 0.05
0.40 0.023
2.5 0.4
O
N
N
iii
N
Cl
Br
I
N
H
CH3
F
6a
14
a Each value is the means SEM of three determinations and repre-
sents the concentration giving half the maximum inhibition of [3H]1
(final concentration 1 nM) specific binding to rat cortical
membranes.
Scheme 3. Reagents: (i) SOCl2, CH2Cl2; (ii) C6H5CH2NH2, TEA,
CH2Cl2; (iii) CH3I, NaH, DMF; (iv) C6H5CH2N(CH3)H, TEA,
CH2Cl2.
series of highly congeneric PBR ligands confirmed the
positive effect of the presence of an halogen atom on
the PBR affinity. This ‘halogen effect’ is particularly evi-
dent in the case of fluoromethyl derivative 6a and ap-
pears to correlate with the electron-withdrawing
properties of the halogen atom only in the case of com-
pounds 6a,b. If the electron-withdrawing properties are
believed to play a role, they may be assumed to activate
the methylene hydrogen atoms, which could interact
with D1579 by establishing a non-classical hydrogen
bond.12b This assumption is considered an interesting
working hypothesis for the molecular modeling work,
which is in progress in our laboratories.
O
O
F
N
N
i
N
N
H
11CH3
F
[11C]6a
14
Scheme 4. Reagents: (i) [11C]CH3I, TBAH, DMF.
2.4. Kinetic studies
2.4.1. [11C]6a Biodistribution. The tissue biodistribution
of [11C]6a ([11C]VC701) in albino male CD rats as a
function of time is presented in Table 2. As expected,
high radioactivity accumulation was observed in periph-
eral tissues known to be rich in PBR, such as heart, lung,
kidney, spleen, and adrenal gland (see Gavish et al.14 for
a review). A lower uptake was observed in other periph-
eral organs such as intestine, stomach, and liver.
2.3. Binding studies
Compounds 6a–d were tested for their potential activity
in inhibiting the specific binding of [3H]1 to rat cortical
membrane in comparison with reference compounds 5c
and 1. The results of the binding studies (Table 1) show
that all the 3-halomethyl-2-quinolinecarboxamide deriv-
atives 6a–d display subnanomolar affinity for PBR (IC50
values ranging from 0.11 to 0.61 nM). The most potent
3-fluoromethyl derivative 6a shows an IC50 value of
0.11 nM and is about 20 times more potent than refer-
ence compound 1. Thus, 3-fluoromethyl derivative 6a,
being one of the most potent PBR ligands so far
described, represents an interesting candidate for radio-
labeling with carbon-11 or with fluorine-18 and PET
studies. Although slightly less potent, bromomethyl
derivative 6c (IC50 = 0.61 nM) can be considered a can-
didate for radiolabeling with bromine-76 and iodometh-
yl derivative 6d (IC50 = 0.40 nM) may become a suitable
candidate of a SPECT ligand for imaging PBR. The
structure–affinity relationship analysis in this short
In the remaining tissues, including brain, radioactivity
concentration was very low or negligible. In most of
the tissues examined, the maximum uptake was reached
at 30 min from the injection. At this time, tissue to plas-
ma radioactivity concentration ratios were 2–7 times
higher (blood, lung, liver, kidney, spleen, and brain)
than the ones previously observed with [11C]5c, another
PBR ligand developed by our groups (Table 3).10a Tis-
sue or blood to plasma ratios of [11C]6a remained in
general higher than the [11C]5c ones also at later times
except for blood, liver, adrenal, and testis. A lower inter-
action with plasma protein may contribute to the higher
plasma clearance and tissue to plasma ratios.

A. Cappelli et al. / Bioorg. Med. Chem. 14 (2006) 4055–4066
Table 2. Cerebral and peripheral distribution of [11C]6a in ratsa
4059
tomograph YAP-(S)PET are shown in Figures 3 and
4. In particular, Figure 3 shows a montage of images
of radioactivity distribution obtained in a CD-1 mouse
injected with [11C]6a during the bed 2 of the first acqui-
sition of the study 1 [from 10 to 20 min after the injec-
tion, abdomen centered in the field of view (FOV)].
High levels of radioactivity accumulation were observed
in the lungs, in the first descending intestinal tract (duo-
denum), and in the kidneys. Tracer retention was also
present in liver, heart, and spleen. No or negligible levels
of radioactivity were observed in the brain (bed 1 of the
first acquisition in study 1, data not shown) and in the
urinary bladder. Thirty minutes after (second acquisi-
tion in study 1), no modification in radioactivity distri-
bution was observed. Results of PET images have
been confirmed by tissue sampling experiment per-
formed at the end of PET scan in one CD-1 mouse that
showed high levels of radioactivity in lungs, intestine
and kidney followed by heart, spleen, and liver.
Tissue
10 min
30 min
60 min
Blood
0.038 0.009
0.008 0.002
0.459 0.025
1.612 0.131
0.100 0.029
0.144 0.025
0.390 0.098
0.375 0.045
0.321 0.088
0.019 0.002
0.137 0.022
0.062 0.005
0.049 0.005
0.062 0.004
0.062 0.004
0.020 0.007
0.004 0.001
0.489 0.048
1.023 0.039
0.139 0.004
0.103 0.023
0.523 0.000
0.358 0.009
0.487 0.006
0.018 0.000
0.174 0.034
0.052 0.009
0.041 0.021
0.039 0.005
0.032 0.007
0.009 0.003
0.008 0.002
0.454 0.048
0.797 0.017
0.173 0.017
0.066 0.002
0.674 0.116
0.334 0.038
0.486 0.025
0.020 0.001
0.148 0.035
0.051 0.009
0.011 0.007
0.021 0.006
0.015 0.005
Plasma
Heart
Lung
Stomach
Liver
Adrenal gland
Kidney
Spleen
Testis
Intestine
Muscle
Striatum
Cortex
Cerebellum
a Radioactivity concentration is expressed as % of injected dose per
gram of tissue (%ID/g). Values are expressed as means SD of three
rats for each time point.
The effect of PK11195 on [11C]6a distribution was also
evaluated by PET (study 2). Figure 4 shows the radioac-
tivity distribution images obtained at the level of thorax
in two CD-1 mice co-injected with vehicle (panel A) or
PK11195 (panel B), respectively. Co-injection with
PK11195 did not reduce radioactivity accumulation in
the small intestine, indicating that tracer retention was
not specific (panel B) in this region; on the contrary in
agreement with the results of ex vivo inhibition experi-
ments performed in rats, radioactivity uptake was clear-
ly reduced in regions expressing PBRs such as lung,
heart, and spleen (panel B).
Table 3. Tissue to plasma ratio of [11C]6a and [11C]5c in rat at 30 min
after the injectiona
Tissue
[
11C]6a
4.49 0.57
[
11C]5cb
Blood
Heart
1.06 0.09
112.04 16.15
236.12 47.78
23.28 0.49
121.24 29.06
82.71 17.86
113.15 28.45
4.27 0.95
41.28 11.24
33.51 4.32
7.29 0.63
44.70 41.71
17.35 2.84
24.16 3.63
2.08 0.56
9.52 3.87
4.17 1.37
1.98 0.42
1.57 0.28
Lung
Liver
Adrenal gland
Kidney
Spleen
Testis
Intestine
Muscle
Cerebellum
Cortex
39.28 1.71
11.88 0.74
8.84 1.10
3. Discussion
7.15 0.07
The exploration of the structure–affinity relationships of
PBR ligands 6 revealed the importance of the presence of
a chlorine atom in the methylene carbon at position 3 of
the quinoline nucleus.12 Based on the subnanomolar
PBR affinity shown by 3-chloromethyl derivative 6b, its
chlorine atom has been replaced by other halogens
(e.g., fluorine, bromine, and iodine) in order to optimize
the interaction of the quinolinecarboxamide derivatives
with PBR and to develop candidates for PET studies bet-
ter than the previously described [11C]5c.10a These stud-
ies led to the discovery of fluoromethyl derivative 6a,
which show an IC50 value of 0.11 nM (about 20-fold
more potent than 1 in the same test system) and can
therefore be considered one of the most potent PBR
ligands so far described. The properties of fluoromethyl
derivative [11C]6a as a potential PET radioligand for
peripheral benzodiazepine receptor were evaluated
ex vivo in rats with biodistribution and inhibition exper-
iments and in vivo in mice with small animal YAP-
(S)PET. As expected, in the rat biodistribution, the
radioligand rapidly accumulated in PBR-rich tissues
such as heart, lung, kidney, spleen, and adrenal, and at
a lower level in other peripheral organs and in the brain.
The maximum uptake was achieved at 30 min after the iv
administration of the tracer, and at this time the specific-
ity of the binding by pre-injecting the isoquinolinecarb-
oxamide derivative PK11195 was investigated. The
a The ratio was calculated %ID/g of tissue divided per %ID/g of
plasma.
b Data of [11C]5c were calculated from the results published by
Matarrese et al.10a Values are expressed as means SD of three rats
for each time point.
2.4.2. Inhibition study. The specificity of [11C]6a in vivo
binding to PBR was evaluated in rats pretreated with
PK11195 (5 mg/kg, iv, n = 5) or with the vehicle (iv,
n = 3), injected immediately before the radioligand
administration. Results of the inhibition experiment at
30 min after [11C]6a injection are presented in Figure 2.
The highest effect of PK11195 pre-administration was
observed in the heart, lung, kidney, spleen, and stomach
(reduction of 86%, 92%, 74%, 87%, and 75%, respective-
ly; p 6 0.001). A lower effect was observed in the intestine
and in the muscle (52% and 61%, respectively; 60.05),
while no significant inhibitions were observed in blood
and brain. Despite the high levels of radioactivity con-
centration observed in biodistribution experiments, the
uptake in the adrenal gland was found to be non-specific
as indicated by the absence of PK11195 effect.
2.4.3. PET images. Biodistribution and inhibition imag-
es obtained by means of the small animal dedicated

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A. Cappelli et al. / Bioorg. Med. Chem. 14 (2006) 4055–4066
Figure 2. Effect of PK11195 pretreatment on [11C]6a radioactivity uptake in cerebral and peripheral tissues of rat measured 30 min after [11C]6a
injection. Radioactivity concentration is expressed as % of injected dose per gram of tissue (%ID/g); 5 mg/kg of PK11195 (pretreated) or vehicle
(basal) was administered immediately before [11C]6a injection. Values are expressed as means SD of three rats for the basal group and as
means SD of five rats for pretreated group. Differences between the two groups were examined with Student t-test: NS, not significant (p > 0.05);
*significant (p 6 0.05); **very significant (p 6 0.001).
Figure 3. Small animal YAP-(S)PET axial images of CD-1 mice-injected with [11C]6a. The animal was acquired prone with the abdomen centred in
the FOV starting from 10 min after the tracer injection (bed 2 of the first acquisition of study 1). Numbers refer to the sections and L indicates the left
side.

A. Cappelli et al. / Bioorg. Med. Chem. 14 (2006) 4055–4066
4061
6a will be labeled with the longer-living 18F (t1/2
=
109.7 min) and evaluated in both metabolism studies
and suitable preclinical models.
4. Experimental
4.1. Chemistry
Figure 4. Small animal YAP-(S)PET images (thorax) of two CD-1
mouse injected with [11C]6a. One animal was co-injected with the
vehicle and acquired after 20 min (A); the other animal was co-injected
with the cold compound PK11195 and acquired after 20 min (B).
All chemicals used were of reagent grade. Yields refer to
purified products and are not optimized. Melting points
were determined in open capillaries on a Gallenkamp
apparatus and are uncorrected. Microanalyses were car-
ried out by means of a Perkin-Elmer 240C or a Perkin-
Elmer Series II CHNS/O Analyzer 2400. Merck silica gel
60 (230–400 mesh) was used for column chromatogra-
phy. Merck TLC plates, silica gel 60 F254 were used
for TLC. 1H NMR spectra were recorded with a Bruker
AC 200 spectrometer in the indicated solvents (TMS as
internal standard); the values of the chemical shifts are
expressed in ppm and the coupling constants (J) in
hertz. Mass spectra were recorded on either a Varian
Saturn 3 spectrometer or a ThermoFinnigan LCQ-Deca.
inhibition experiment demonstrated that the tracer
[11C]6a specifically binds to PBR in heart, lung, kidney,
and spleen, but not in the adrenal. These data disagree
with the result obtained for other quinolinecarboxamide
derivatives (5a–c) previously tested,10a in which the bind-
ing to adrenals was found to be specifically inhibited by
pre-injection of PK11195 at the same experimental dose
applied. The result can be explained by the higher affinity
of 6a with respect to 5c and PK11195, that in a region
with a very high and clusterized expression of PBR
may preclude the observation of any inhibitory effect of
PK11195 pre-treatment. Therefore, further inhibition
studies with doses of PK11195 or with the cold com-
pound 6a will be performed to better understand the
binding of the tracer in the adrenals. At 30 min after
the tracer injection, compound [11C]5c (that among the
group 5a–c had proved to possess the best pharmacoki-
netic characteristics) showed a biodistribution profile
similar to that of [11C]6a in the majority of the organs
(Table 3). However, interestingly, the tissue-to-plasma
ratios observed with [11C]6a were definitely higher than
those obtained with [11C]5c, suggesting a lower interac-
tion of this compound with plasma proteins, a fact which
could be of particular relevance in brain studies on neu-
rodegeneration where new PET ligands with higher sig-
nal-to-noise ratios are needed. The value of this tracer
for brain studies will be further evaluated and possibly
confirmed in selected animal models.
4.2. 3-Bromomethyl-N-methyl-4-phenyl-N-(phenylmeth-
yl)quinoline-2-carboxamide (6c)
To a solution of 18-crown-6 (0.023 g, 0.087 mmol) in
dry acetonitrile (8.0 mL) was added LiBr (0.22 g,
2.5 mmol) and the resulting mixture was stirred at room
temperature for 30 min. To the reaction mixture was
added a solution of compound 6b12 (0.10 g, 0.25 mmol)
in dry acetonitrile (6.0 mL) and the resulting mixture
was refluxed under argon for 24 h, poured into ice-
water, and extracted with CHCl3. The organic layer
was dried over sodium sulfate, and concentrated under
reduced pressure. Purification of the residue by flash
chromatography with dichloromethane/ethyl acetate
(9:1) as the eluent gave pure 6c as a pale yellow oil
(0.070 g, yield 63%). The 1H NMR spectrum of this
amide shows the presence of two different rotamers in
equilibrium. For the sake of simplification, the integral
intensities have not been given. 1H NMR (CDCl3):
2.96 (s), 3.08 (s), 4.47 (s), 4.68 (s), 4.90 (s), 7.30-7.76
(m), 8.10 (m). MS (ESI): m/z 445 (M+H+). Anal. Calcd
for (C25H21BrN2O): C, 67.42; H, 4.75; N, 6.29. Found:
C, 67.37; H, 5.04; N, 6.08. Rf, SiO2 (dichloromethane/
ethyl acetate (9:1)): 0.48.
The images obtained in mouse with small animal YAP-
(S)PET show that the radiotracer accumulates in lungs,
heart, small intestine, and kidneys, and at a lower con-
centration in spleen and liver. This distribution of the
radioactivity was confirmed by our preliminary tissue
sampling experiment performed on one mouse at
20 min after the tracer injection (data not shown) and
overlapped with the results obtained by Hashimoto
et al. in mice injected with [3H]PK11195.15 The radioac-
tivity uptake in heart, spleen, and liver was masked in
the image by the higher signal of the lung and small
intestine. The higher uptake found in duodenum was
found to be unspecific because it was not inhibited by
cold PK11195. No evident binding of the tracer in brain
and bladder was detected.
4.3. 3-Iodomethyl-N-methyl-4-phenyl-N-(phenylmeth-
yl)quinoline-2-carboxamide (6d)
To a solution of 18-crown-6 (0.013 g, 0.049 mmol) in
dry acetonitrile (10 mL) was added NaI (0.38 g,
2.5 mmol) and the resulting mixture was stirred at room
temperature for 30 min. To the reaction mixture was
added a solution of compound 6b12 (0.10 g, 0.25 mmol)
in dry acetonitrile (8.0 mL) and the resulting mixture
was stirred at room temperature under argon for 24 h,
poured into ice-water, and extracted with CHCl3. The
organic layer was dried over sodium sulfate and concen-
trated under reduced pressure. Purification of the resi-
due by flash chromatography with dichloromethane/
In conclusion, the biological data suggest that [11C]6a is
a promising radioligand for PBR imaging in vivo with
PET showing a higher PBR affinity and better pharma-
cokinetics features with respect to the previously validat-
ed [11C]5c.11 On the basis of these results, compound

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A. Cappelli et al. / Bioorg. Med. Chem. 14 (2006) 4055–4066
ethyl acetate (9:1) as the eluent gave pure 6d as a yellow
oil, which slowly crystallized on standing (0.064 g, yield
52%, mp 112–113 ꢁC). The H NMR spectrum of this
amide shows the presence of two different rotamers in
equilibrium. For the sake of simplification, the integral
intensities have not been given. 1H NMR (CDCl3):
2.98 (s), 3.09 (s), 4.48 (s), 4.64 (s), 4.90 (s), 7.29–7.74
(m), 8.03 (d, J = 8.3), 8.10 (d, J = 8.3). MS (ESI): m/z
493 (M+H+). Anal. Calcd for (C25H21IN2O): C, 60.99;
H, 4.30; N, 5.69. Found: C, 60.80; H, 4.24; N, 5.58.
Rf, SiO2 (dichloromethane/ethyl acetate (9:1)): 0.49.
CH2Cl2. The organic layer was washed with water, dried
over sodium sulfate, and concentrated under reduced
pressure. Purification of the residue by flash chromatog-
raphy with n-hexane/ethyl acetate (8:2) as the eluent
gave pure 6a as a yellow oil (0.036 mg, yield 96%), which
crystallized on standing.
1
4.5. tert-Butyl 3-methyl-4-phenylquinolin-2-carboxylate
(9)
A mixture of compound 78b (0.50 g, 1.9 mmol) in
CH2Cl2 (1.0 mL) and SOCl2 (2.0 mL) was refluxed for
3 h. Most of SOCl2 was distilled under reduced pressure
and the remainder was removed by azeotropic distilla-
tion with toluene. The residue was treated with a solu-
tion of t-BuOK (0.30 g, 2.67 mmol) in dry THF
(10 mL). The reaction mixture was stirred at room tem-
perature for 1 h, poured into ice-water, and extracted
with CH2Cl2. The organic layer was dried over sodium
sulfate and concentrated under reduced pressure. Purifi-
cation of the residue by flash chromatography with
dichloromethane/ethyl acetate (95:5) as the eluent gave
pure 9 as a colorless solid. (0.31 g, yield 51%, mp 146–
4.4. 3-Fluoromethyl-N-methyl-4-phenyl-N-(phenylmeth-
yl)quinoline-2-carboxamide (6a)
4.4.1. Method A. To a solution of 18-crown-6 (5.3 mg,
0.020 mmol) in dry acetonitrile (5.0 mL) was added
KF (0.075 g, 1.3 mmol) and the resulting mixture was
stirred at room temperature for 30 min. To the reaction
mixture was added a solution of compound 6b12
(0.090 g, 0.224 mmol) in dry acetonitrile (6.0 mL) and
the resulting mixture was refluxed for 14 h, poured into
ice-water, and extracted with CHCl3. The organic layer
was dried over sodium sulfate and concentrated under
reduced pressure. Purification of the residue by flash
chromatography with dichloromethane/ethyl acetate
(9:1) as the eluent gave pure 6a as a pale yellow oil,
which crystallized on standing (0.035 g, yield 41%).
The purification of the residue also gave a mixture of
compounds 6b [Rf, SiO2 (dichloromethane/ethyl acetate
(9:1)): 0.46] and 6a [Rf, SiO2 (dichloromethane/ethyl ace-
tate (9:1)): 0.31] on which the above-cited procedure was
repeated to obtain further 0.030 g of 6a (total yield 75%,
1
147 ꢁC). H NMR (CDCl3): 1.69 (s, 9H), 2.27 (s, 3H),
7.23–7.67 (m, 8H), 8.17 (d, J = 8.4, 1H). MS (ESI): m/
z 342 (M+Na+).
4.6. tert-Butyl 3-bromomethyl-4-phenylquinolin-2-car-
boxylate (10)
A mixture of compound 9 (0.55 g, 1.7 mmol) in CCl4
(30 mL) with N-bromosuccinimide (0.34 g, 1.9 mmol)
and dibenzoyl peroxide (0.040 g, 0.165 mmol) was re-
fluxed for 2 h. The reaction mixture was then cooled
to room temperature, filtered, and concentrated under
reduced pressure. The residue was purified by flash chro-
matography with dichloromethane as the eluent to give
pure 10 as a colorless oil (0.62 g, yield 92%), which crys-
tallized from diethyl ether (colorless prisms, mp 125–
1
mp 94–96 ꢁC). The H NMR spectrum of this amide
shows the presence of two different rotamers in equilib-
rium. For the sake of simplification, the integral intensi-
ties have not been given. H NMR (CDCl3): 2.88 (s),
1
3.08 (s), 4.48 (s), 4.89 (s), 5.44 (d, J = 46.6), 7.29–7.60
(m), 7.74 (m), 8.16 (m). MS (ESI): m/z 407 (M+Na+).
Anal. Calcd for (C25H21FN2O): C, 78.10; H, 5.51; N,
7.29. Found: C, 78.23; H, 5.43; N, 7.02.
1
127 ꢁC). H NMR (CDCl3): 1.77 (s, 9H), 4.68 (s, 2H),
7.32–7.61 (m, 7H) 7.70 (m, 1H), 8.21 (d, J = 8.4,1H).
MS (ESI): m/z 420 (M+Na+).
4.4.2. Method B. A mixture of compound 12 (0.18 g,
0.64 mmol) in CH2Cl2 (4 mL) and SOCl2 (4.0 mL) was
refluxed for 3 h. Most of SOCl2 was distilled under re-
duced pressure and the remainder was removed by aze-
otropic distillation with toluene. The residue was diluted
with CH2Cl2 (10 mL) and treated in sequence with
N-benzylmethylamine (0.17 mL, 1.32 mmol) and a solu-
tion of triethylamine (0.18 mL, 1.3 mmol) in CH2Cl2
(1.0 mL). The reaction mixture was stirred at room tem-
perature for 10 min, diluted with CH2Cl2, washed with
water, dried over sodium sulfate and concentrated under
reduced pressure. Purification of the residue by flash
chromatography with petroleum ether/ethyl acetate
(8:2) as the eluent gave pure 6a as a pale yellow, oil
which crystallized on standing (0.19 g, yield 77%).
4.7. tert-Butyl 3-fluoromethyl-4-phenylquinolin-2-carbox-
ylate (11)
The title compound was obtained from compound 10
(2.1 g, 5.3 mmol) following the procedure described for
the preparation of compound 6a (Method A). Purifica-
tion of crude 11 by flash chromatography with n-hex-
ane/ethyl acetate (8:2) as the eluent gave pure 11 as a
white solid (0.70 g, yield 39%, mp 121–123 ꢁC). 1H
NMR (CDCl3): 1.71 (s, 9H), 5.43 (d, J = 47.2, 2H),
7.28–7.33 (m,2H), 7.46–7.56 (m, 5H), 7.74 (m, 1H),
8.25 (d, J = 8.3, 1H). MS (ESI): m/z 360 (M+Na+).
4.8. 3-Fluoromethyl-4-phenylquinolin-2-carboxylic acid
(12)
4.4.3. Method C. To a solution of compound 14 (0.036 g,
0.097 mmol) in dry DMF (2.0 mL) with CH3I (0.30 mL,
4.85 mmol) was added NaH (3.6 mg, 0.15 mmol). The
resulting mixture was stirred at room temperature for
1 h, then poured into ice-water and extracted with
A mixture of compound 11 (0.70 g, 2.1 mmol) in formic
acid (15 mL) was stirred at room temperature for 20 h.
Most of the formic acid was evaporated under reduced
pressure and the remainder was removed by azeotropic

A. Cappelli et al. / Bioorg. Med. Chem. 14 (2006) 4055–4066
4063
distillation with toluene. The resulting residue was treat-
ed with diethyl ether to obtain pure 12 as off-white crys-
tals (0.57 g, yield 96%, mp 123–125 ꢁC). 1H NMR
(CDCl3): 5.85 (d, J = 46.2, 2H), 7.34 (m, 2H), 7.48-
7.66 (m, 5H), 7.86 (t, J = 7.4, 1H), 8.21 (d, J = 8.4,
1H). MS (ESI, negative ions): m/z 280 (MÀH+).
the Limulus Amebocyte Lysate (LAL) test (BioWhittak-
er, Inc.).
4.11. No-carrier added radiosynthesis of [11C]6a
[11C]Methyl iodide was transported by a stream of ar-
gon into the reaction vessel containing 1 mg
(0.0027 mmol) of des-methyl precursor 14 in 100 lL
DMF containing 1 lL (2 lmol) of tetrabutylammonium
hydroxide (60% aqueous solution, Fluka) at 80 ꢁC.
After 4 min, the reaction mixture was diluted with
0.8 mL of a solution of acetonitrile/water (1:1; v:v) and
injected into the HPLC semi-preparative reverse-phase
column. The purification of the tracer was performed
by means of an HPLC X-Terra RP18, 250 · 10 mm,
5 lm (Waters) column with CH3CN:50 mM sodium
dihydrogen orthophosphate-1-hydrate (60:40; v:v) at
5 mL/min flow rate with UV detection set at 254 nm.
The retention times were 10 min and 8 min for des-
methyl precursor 14 and [11C]6a, respectively. The
retention time of [11C]6a was confirmed before each
radiosynthesis by comparison with authentic standard
of 6a. The effluent from the column corresponding to
[11C]6a was collected in 30 mL of sterile water, and the
radioligand was recovered by solid-phase extraction
(SPE) on pre-activated Sep-Pak C-18 cartridge (Milli-
pore). The Sep-Pak was washed with water (10 mL) be-
fore eluting with ethanol (0.5 mL) in a vial containing
9.5 mL of saline solution which was sterilized through
a sterile 0.22 lm filter (Gelman Acrodisc). The pH of
the final solution was neutral. The quality control of
[11C]6a was carried out on an analytical HPLC
reverse-phase (X-Terra RP18, 5 lm, 250 · 4.6 mm) col-
umn (Waters) by means of CH3CN/25 mM sodium
dihydrogen orthophosphate-1-hydrate (75:25; v:v) at
1 mL/min flow rate with monitors for radioactivity
and UV detector set at 254 nm. The retention time for
[11C]6a was 5.4 min. The amount of carrier was calculat-
ed from the UV absorbance peak by means of the exter-
nal standard calibration plot. The minimal detectable
concentration of [11C]6a was 1.75 nmol/mL.
4.9. 3-Fluoromethyl-4-phenyl-N-(phenylmethyl)quinoline-
2-carboxamide (14)
A mixture of compound 12 (0.20 g, 0.71 mmol) in
CH2Cl2 (4.0 mL) with SOCl2 (4.0 mL) was refluxed for
3 h. Most of SOCl2 was distilled under reduced pressure
and the remainder was removed by azeotropic distilla-
tion with toluene. The residue was diluted with CH2Cl2
(10 mL) and treated in sequence with benzylamine
(0.15 mL, 1.4 mmol) and a solution of triethylamine
(0.20 mL, 1.4 mmol) in CH2Cl2 (1.0 mL). The reaction
mixture was stirred at room temperature for 10 min,
diluted with CH2Cl2, washed with water, dried over
sodium sulfate and concentrated under reduced pres-
sure. Purification of the residue by flash chromatogra-
phy with petroleum ether/ethyl acetate (8:2) as eluent
gave pure 14 as a colorless oil (0.18 g, yield 68%), which
crystallized from diethyl ether–n-hexane to give white
crystals melting at 130–132 ꢁC. 1H NMR (CDCl3):
4.75 (d, J = 6.0, 2H), 5.88 (d, J = 46.6, 2H), 7.29–7.55
(m, 12H), 7.75 (m, 1H), 8.11 (d, J = 8.5, 1H), 8.45 (br
t, 1H). MS (ESI): m/z 371 (M+H+).
4.10. Radiosynthesis
[11C]Carbon dioxide was produced by the 14N(p,a)11C
reaction on a IBA Cyclone 18/9 cyclotron, using
18 MeV proton beam at currents 15–30 lA, and trapped
in a hollow stainless steel loop, cooled with liquid nitro-
gen. [11C]Methyl iodide was substantially synthesized as
described by Crouzel et al.16 involving the reduction of
[11C]CO2 with 0.07 M LiAlH4 to lithium aluminum-
[11C]methylate, hydrolysis of this intermediate
organometallic complex, iodination of the formed
[11C]methanol with hydriodic acid, and distillation
through an Ascarite–Sicapent purification column.
Radiochemical synthesis and purification of [11C]6a
were performed on the partially modified fully automat-
ed synthesis module (PET Tracer Synthesizer, Nuclear
4.12. In vitro binding assays
Male Sprague–Dawley CD rats (Charles River Italia,
Calco, CO, Italy) with body masses of 200–250 g were
used. Rats were acclimatized to the new housing condi-
tions for at least one week. They were housed six per
cage under an artificial 12-h-light, 12-h-dark cycle at a
constant temperature of 22 2 ꢁC, and a relative
humidity of 65%. They had free access to water and
standard laboratory food at all times. Animal care and
handling throughout the experimental procedures were
in accordance with the European Communities Council
Directive of 24 November, 1986 (86/609/EEC).
Interface Datentechnik GmbH, Munster, Germany)
¨
for [11C]-methylation described in detail elsewhere.17
High-performance liquid chromatography (HPLC) was
performed with a Waters 515 isocratic pump and a
Waters 2487 variable-wavelength UV detector in series
with a b+-Bioscan Flow Count detector. Data collection
and HPLC control were performed with the use of a
Waters Millennium 32 chromatography software pack-
age. The course of the 11C-methylation and the quality
control of the final radioligand were performed on a re-
verse-phase analytical HPLC X-Terra RP18, 5 lm,
250 · 4.6 mm, column (Waters). In the analysis of the
11C-labeled compound, unlabeled reference standard
6a was used for comparison in all the HPLC runs. The
pH of the final solution was measured on a Schott Ger-
a¨te pH-meter. The pyrogenity test was performed using
Rats were sacrificed by decapitation and their brains
were rapidly dissected into the various areas which were
stored at À80 ꢁC until the day of the assay. The binding
assays were performed as described in the literature.18
The cerebral cortex was subsequently thawed and then
homogenized in 50 volumes of ice-cold Dulbecco’s
phosphate-buffered saline (PBS), pH 7.4, at 4 ꢁC with

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A. Cappelli et al. / Bioorg. Med. Chem. 14 (2006) 4055–4066
a Polytron PT 10 disrupter (setting 5 for 20 s). The
homogenate was centrifuged at 40,000g and 4 ꢁC for
30 min and the resulting pellet was resuspended in the
same volume of fresh buffer and recentrifuged. The
new pellet was resuspended in 10 volumes of the incuba-
tion buffer (PBS) and used for the binding assay.
gamma counter and its concentration calculated as the
percentage of injected dose per gram of tissue (%ID/g).
4.13.3. Inhibition study. The specificity of [11C]6a uptake
(injected dose: 100 9 lCi; specific activity at the time
of injection: 2.35 Ci/lmol) was studied in rats pretreated
with PK11195 (5 mg/kg dissolved into DMSO/EtOH
1:1, iv, n = 5) or vehicle (DMSO/EtOH 1:1, iv, n = 3)
immediately before the radioligand injection. The inhib-
itory effect of cold PK11195 was evaluated 30 min after
the tracer injection on the basis of our previous experi-
ence10a and because at this time a maximum radioactiv-
ity uptake was found in peripheral organs with high
PBR density20 and was compared to the pre-injection
of the vehicle. After this time, the animals were sacri-
ficed by decapitation under a light anesthesia (ether)
and processed as described above for the biodistribution
to obtain the %ID/g for cerebral and peripheral tissues.
The effect of the pretreatment with PK11195 on the
[11C]6a uptake was expressed as the percentage of inhi-
bition for all the tissues examined and only the tissue
with positive percentage is reported.
[3H]PK11195 binding was measured in a final volume of
500 lL, consisting of 50 lL membrane suspension
(0.15–0.20 mg of protein), 50 lL of [3H]PK11195 (spe-
cific activity 85.5 Ci/mmol, New England Nuclear; final
assay concentration of 1 nM), 5 lL of drug solution or
solvent, and 395 lL PBS. The binding reaction was per-
formed at 25 ꢁC for 90 min and began with the addition
of membranes. The incubation was terminated by rapid
filtration through glass-fiber filters (Whatman GF/B)
which had been presoaked with 0.3% polyethyleneimine
and placed in a cell harvester filtration manifold (Bran-
del). The filters were washed five times with 4 mL of ice-
cold PBS, after which filter-bound radioactivity was
quantified by liquid scintillation spectrometry. Nonspe-
cific binding was defined as binding in the presence of
10 lM of unlabeled PK11195 (Sigma). Specific binding
was determined by subtracting the nonspecific from
the total binding and was about 80% of the total bind-
ing. The concentration of the test compounds that inhib-
ited [3H]ligand binding by 50% (IC50) was determined by
means of Jandel Sigmaplot19 program with 6–10 concen-
trations of the displacers, each performed in triplicate.
4.13.4. PET studies. Imaging studies were performed
using the YAP-(S)PET scanner (ISE, Pisa, Italy). This
small animal tomograph is made up of four detector
heads, composed each of a 4 · 4 cm2 of YAlO3:Ce ma-
trix of 20 · 20 elements, 2 · 2 · 25 mm3 each, coupled
to PS-PMT (Hamamatsu R2486). The four modules
are positioned on a rotating gantry; opposite detectors
are 15 cm apart and in time coincidence when used in
PET mode. The scanner has a field of view (FOV) of
4 · 4 cm of diameter and provides a set of 20 slices of
2 mm thickness. In PET mode, the volume resolution
is below 8 mm3 and is nearly constant over the whole
FOV. The maximum absolute sensitivity measured at
the center of the FOV is 1.9% (19 cps/KBq) for 50–
850 keV energy window.
4.13. Kinetic experiments
4.13.1. Animals. Male albino CD rats (225–250 g;
Charles River, Italy) were used for biodistribution and
inhibition studies. Male CD-1 mice (35–40 g; Charles
River, Italy) were used for small-animal PET imaging.
Animal experiments were approved by the Ethical Com-
mittee for animal care of the San Raffaele Hospital and
carried out in agreement with the Italian and EEC rec-
ommendations for the care and use of laboratory
animals.
Two studies were performed on healthy CD-1 mice: in
the first study, one animal was acquired in three beds
at two different times after the tracer injection; in the
second study, two animals were acquired at the level
of the thorax starting 20 min after the tracer injection:
one animal co-injected with a vehicle (control) and the
other co-injected with the inhibitor (inhibited).
4.13.2. [11C]6a Biodistribution. Compound [11C]6a
(specific activity at the time of injection: 1.5 Ci/lmol)
was diluted to a final volume of 100 lL in saline (0.9%
NaCl) and injected into a rat tail vein using a
0.5 · 9.5 mm syringe needle. The cerebral and peripheral
distribution of the radiotracer was assayed at 10, 30, and
60 min (n = 3 at each time point) after the injection of
approximately 6.3 0.3 MBq of [11C]6a (corresponding
to 278 12 pmol of cold compound). At the times indi-
cated the animals were sacrificed by decapitation under
a light anesthesia (ether) and a blood sample was collect-
ed into heparinized tubes, one part of which was counted
in a gamma counter and the remaining part was centri-
fuged for the plasma radioactivity determination. The
brain was rapidly removed from the skull and discrete
cerebral areas (striatum, cortex, and cerebellum) were
dissected out and placed in pre-weighed tubes. The body
of the animal was opened and a series of peripheral or-
gans (heart, lung, stomach, liver, adrenal, kidney, spleen,
testis, intestine, and muscle) were collected and placed in
pre-weighed tubes. The radioactivity was counted in a
Before every study, the mice were anesthetized ip with
800 lL of 1.7% tribromoethanol and positioned supine
on the YAP-(S)PET bed.
4.13.5. Study 1. In the first acquisition, the animal was
positioned with its head centered in the FOV and was
injected into the tail vein with 128 lCi (4.74 MBq) of
[11C]6a. The acquisition started immediately after the
tracer injection. The specific activity calculated at the
end of the synthesis was 1.50 Ci/lmol. The first scan
was focused on the head (2 time frames of 5 min each),
the second scan was focused 4 cm below, at the level of
the thorax (2 time frames of 5 min each), and the third
scan was focused 4 cm below again, at the level of the
abdomen (2 time frames of 5 min each). The data were
acquired in list mode using the full axial acceptance

A. Cappelli et al. / Bioorg. Med. Chem. 14 (2006) 4055–4066
4065
A.; Hauw, J. J.; MacKenzie, E. T.; Sazdovitch, V.;
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Schoemaker, H.; Morelli, M.; Deshmukh, P. Brain Res.
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angle of the scanner (3D mode) and then reconstructed
with the Expectation Maximization (EM) algorithm.21
4.13.6. Study 2. Both the control and the inhibited ani-
mals were positioned with the thorax centered in the
FOV and acquired 20 min after the tracer injection.
The control mouse was injected into the tail vein with
57 lCi (2.11 MBq) of [11C]6a and co-injected ip with
saline 5% ethanol (vehicle). The inhibited mouse was
injected into the tail vein with 25 lCi (0.93 MBq) of
[11C]6a and co-injected i.p. with 5 mg/kg of the cold
compound PK11195 dissolved in DMSO/EtOH (1:1).
The specific activity calculated at the end of the syn-
thesis was 6.38 Ci/lmol. For each mouse the thorax
was acquired in four time frames of 5 min each. The
data were acquired in list mode using the full axial
acceptance angle of the scanner (3D mode) and then
reconstructed with Expectation Maximization (EM)
algorithm.21
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Acknowledgments
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Maeda, J.; Suhara, T.; Suzuki, K. Nucl. Med. Biol. 2003,
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The authors are grateful to Prof. Stefania D’Agata
D’Ottavi for the careful reading of the manuscript and
to Prof. Alberto Del Guerra and Dr. Antonietta Bartoli
`
(Universita degli Studi di Pisa) for assistance in PET
acquisition. This work was financially supported by
8. (a) Anzini, M.; Cappelli, A.; Vomero, S.; Giorgi, G.;
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